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1.
Int. j. morphol ; 42(1): 216-224, feb. 2024. ilus
Article in English | LILACS | ID: biblio-1528818

ABSTRACT

SUMMARY: Senile osteoporosis is mainly caused by reduced osteoblast differentiation and has become the leading cause of fractures in the elderly worldwide. Natural organics are emerging as a potential option for the prevention and treatment of osteoporosis. This study was designed to study the effect of resveratrol on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in osteoporosis mice. A mouse model of osteoporosis was established by subcutaneous injection of dexamethasone and treated with resveratrol administered by gavage. In vivo and in vitro, we used western blot to detect protein expression, and evaluated osteogenic differentiation of BMSCs by detecting the expression of osteogenic differentiation related proteins, calcium deposition, ALP activity and osteocalcin content. Resveratrol treatment significantly increased the body weight of mice, the level of serum Ca2+, 25(OH)D and osteocalcin, ration of bone weight, bone volume/total volume, trabecular thickness, trabecular number, trabecular spacing and cortical thickness in osteoporosis mice. In BMSCs of osteoporosis mice, resveratrol treatment significantly increased the expression of Runx2, osterix (OSX) and osteocalcin (OCN) protein, the level of calcium deposition, ALP activity and osteocalcin content. In addition, resveratrol treatment also significantly increased the expression of SIRT1, p-PI3K / PI3K and p-AKT / AKT in BMSCs of osteoporosis mice. In vitro, resveratrol increased the expression of SIRT1, p-PI3K / PI3K and p-AKT / AKT, Runx2, OSX and OCN protein, the level of calcium deposition, ALP activity and osteocalcin content in BMSCs in a concentration-dependent manner, while SIRT1 knockdown significantly reversed the effect of resveratrol. Resveratrol can attenuate osteoporosis by promoting osteogenic differentiation of bone marrow mesenchymal stem cells, and the mechanism may be related to the regulation of SIRT1/PI3K/AKT pathway.


La osteoporosis senil es causada principalmente por una diferenciación reducida de osteoblastos y se ha convertido en la principal causa de fracturas en las personas mayores en todo el mundo. Los productos orgánicos naturales están surgiendo como una opción potencial para la prevención y el tratamiento de la osteoporosis. Este estudio fue diseñado para estudiar el efecto del resveratrol en la diferenciación osteogénica de las células madre mesenquimales de la médula ósea (BMSC) en ratones con osteoporosis. Se estableció un modelo de osteoporosis en ratones mediante inyección subcutánea de dexametasona y se trató con resveratrol administrado por sonda. In vivo e in vitro, utilizamos Western blot para detectar la expresión de proteínas y evaluamos la diferenciación osteogénica de BMSC detectando la expresión de proteínas relacionadas con la diferenciación osteogénica, la deposición de calcio, la actividad de ALP y el contenido de osteocalcina. El tratamiento con resveratrol aumentó significativamente el peso corporal de los ratones, el nivel sérico de Ca2+, 25(OH)D y osteocalcina, la proporción de peso óseo, el volumen óseo/ volumen total, el espesor trabecular, el número trabecular, el espaciado trabecular y el espesor cortical en ratones con osteoporosis. En BMSC de ratones con osteoporosis, el tratamiento con resveratrol aumentó significativamente la expresión de las proteínas Runx2, osterix (OSX) y osteocalcina (OCN), el nivel de deposición de calcio, la actividad de ALP y el contenido de osteocalcina. Además, el tratamiento con resveratrol también aumentó significativamente la expresión de SIRT1, p-PI3K/PI3K y p-AKT/AKT en BMSC de ratones con osteoporosis. In vitro, el resveratrol aumentó la expresión de las proteínas SIRT1, p-PI3K/PI3K y p- AKT/AKT, Runx2, OSX y OCN, el nivel de deposición de calcio, la actividad de ALP y el contenido de osteocalcina en BMSC de manera dependiente de la concentración, mientras que La caída de SIRT1 revirtió significativamente el efecto del resveratrol. El resveratrol puede atenuar la osteoporosis al promover la diferenciación osteogénica de las células madre mesenquimales de la médula ósea, y el mecanismo puede estar relacionado con la regulación de la vía SIRT1/PI3K/AKT.


Subject(s)
Animals , Male , Mice , Osteoporosis/drug therapy , Resveratrol/administration & dosage , Osteogenesis/drug effects , Cell Differentiation/drug effects , Blotting, Western , Disease Models, Animal , Sirtuin 1 , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Resveratrol/pharmacology , Mice, Inbred C57BL
2.
Braz. j. biol ; 84: e250151, 2024. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1350306

ABSTRACT

Abstract Mammals have a limited capacity to regenerate their tissues and organs. One of the mechanisms associated with natural regeneration is dedifferentiation. Several small molecules such as vitamin C and growth factors could improve reprogramming efficiency. In this study, the NTERA2-D1 (NT2) cells were induced towards differentiation (NT2-RA) with 10-5 M retinoic acid (RA) for three days and then subjected to various amounts of vitreous humor (VH). Results show that the growth rate of these cells was reduced, while this rate was partly restored upon treatment with VH (NT2-RA-VH). Cell cycle analysis with PI method also showed that the numbers of cells at the S phase of the cell cycle in these cells were increased. The levels of SSEA3 and TRA-1-81 antigens in NT2-RA were dropped but they increased in NT2- RA-VH to a level similar to the NT2 cells. The level of SSEA1 had an opposite pattern. Expression of OCT4 gene dropped after RA treatment, but it was recovered in NT2-RA-VH cells. In conclusion, we suggest VH as a potent mixture for improving the cellular reprogramming leading to dedifferentiation.


Resumo Os mamíferos têm uma capacidade limitada de regenerar seus tecidos e órgãos. Um dos mecanismos associados à regeneração natural é a desdiferenciação. Várias moléculas pequenas, como vitamina C e fatores de crescimento, podem melhorar a eficiência da reprogramação. Neste estudo, as células NTERA2-D1 (NT2) foram induzidas à diferenciação (NT2-RA) com ácido retinóico (RA) 10-5 M por três dias e depois submetidas a várias quantidades de humor vítreo (VH). Os resultados mostram que a taxa de crescimento dessas células foi reduzida, enquanto essa taxa foi parcialmente restaurada após o tratamento com VH (NT2-RA-VH). A análise do ciclo celular com o método PI também mostrou que o número de células na fase S do ciclo celular nessas células estava aumentado. Os níveis de antígenos SSEA3 e TRA-1-81 em NT2-RA diminuíram, mas aumentaram em NT2-RA-VH a um nível semelhante ao das células NT2. O nível de SSEA1 teve um padrão oposto. A expressão do gene OCT4 diminuiu após o tratamento com AR, mas foi recuperado em células NT2-RA-VH. Em conclusão, sugerimos o VH como uma mistura potente para melhorar a reprogramação celular levando à desdiferenciação.


Subject(s)
Humans , Vitreous Body , Cell Proliferation , Cell Dedifferentiation , Tretinoin , Tumor Cells, Cultured , Cell Differentiation , Cell Division , Cell Line
3.
Actual. osteol ; 19(2): 128-143, sept. 2023. ilus, tab
Article in Spanish | LILACS, UNISALUD, BINACIS | ID: biblio-1523882

ABSTRACT

El presente trabajo muestra la obtención de un material a partir de un polímero sintético (TerP) y otro natural, mediante entrecruzamiento físico y su caracterización fisicoquímica y biológica, con el fin de emplearlos para regeneración de tejido óseo. Las membranas fueron obtenidas por la técnica de evaporación del solvente y caracterizadas por espectroscopia FTIR, ensayos de hinchamiento, medidas de ángulo de contacto y microscopia electrónica de barrido (SEM). Se encontró que la compatibilidad entre los polímeros que la constituyen es estable a pH fisiológico y que, al incorporar mayor cantidad del TerP a la matriz, esta se vuelve más hidrofóbica y porosa. Además, teniendo en cuenta la aplicación prevista para dichos materiales, se realizaron estudios de biocompatibilidad y citotoxicidad con células progenitoras de médula ósea (CPMO) y células RAW264.7, respectivamente. Se evaluó la proliferación celular, la producción y liberación de óxido nítrico (NO) al medio de cultivo durante 24 y 48 horas y la expresión de citoquinas proinflamatorias IL-1ß y TNF-α de las células crecidas sobre los biomateriales variando la cantidad del polímero sintético. Se encontró mayor proliferación celular y menor producción de NO sobre las matrices que contienen menos proporción del TerP, además de poseer una mejor biocompatibilidad. Los resultados de este estudio muestran que el terpolímero obtenido y su combinación con un polímero natural es una estrategia muy interesante para obtener un biomaterial con posibles aplicaciones en medicina regenerativa y que podría extenderse a otros sistemas estructuralmente relacionados. (AU)


In the present work, the preparation of a biomaterial from a synthetic terpolymer (TerP) and a natural polymer, physically crosslinked, is shown. In order to evaluate the new material for bone tissue regeneration, physicochemical and biological characterizations were performed. The membranes were obtained by solvent casting and characterized using FTIR spectroscopy, swelling tests, contact angle measurements, and scanning electron microscopy (SEM). It was found that the compatibility between the polymers is stable at physiological pH and the incorporation of a higher amount of TerP into the matrix increases hydrophobicity and porosity.Furthermore, considering the intended application of these materials, studies of biocompatibility and cytotoxicity were conducted with Bone Marrow Progenitor Cells (BMPCs) and RAW264.7 cells, respectively. Cell proliferation, NO production and release into the culture medium for 24 and 48 hours, and proinflammatory cytokine expression of IL-1ß and TNF-α from cells grown on the biomaterials while varying the amount of the synthetic polymer were evaluated. Greater cell proliferation and lower NO production were found on matrices containing a lower proportion of TerP, in addition to better biocompatibility. The results of this study demonstrate that the obtained terpolymer and its combination with a natural polymer is a highly interesting strategy for biomaterial preparation with potential applications in regenerative medicine. This approach could be extended to other structurally related systems. (AU)


Subject(s)
Animals , Rats , Osteogenesis , Polymers/chemistry , Biocompatible Materials/chemical synthesis , Bone and Bones/chemistry , Bone Regeneration , Chitosan/chemistry , Polymers/toxicity , Biocompatible Materials/toxicity , Materials Testing , Cell Differentiation , Chromatography, Gel , Spectroscopy, Fourier Transform Infrared , Cell Culture Techniques , Nuclear Magnetic Resonance, Biomolecular , Chitosan/toxicity
4.
Chinese Journal of Preventive Medicine ; (12): 923-928, 2023.
Article in Chinese | WPRIM | ID: wpr-985497

ABSTRACT

To establish and identify induced pluripotent stem cells (iPSCs) derived from patients with Aicardi-Goutières syndrome (AGS) with TREX1 gene 667G>A mutation, and obtain a specific induced pluripotent stem cell model for Aicardi-Goutières syndrome (AGS-iPSCs). A 3-year-old male child with Aicardi-Goutieres syndrome was admitted to Zhongshan People's Hospital in December 2020. After obtaining the informed consent of the patient's family members, 5 ml peripheral blood samples from the patient were collected, and mononuclear cells were isolated. Then,the peripheral blood mononuclear cells(PBMCs) were transduced with OCT3/4, SOX2, c-Myc and Klf4 by using Sendai virus, and PBMCs were reprogrammed into iPSCs. The pluripotency and differentiation ability of the cells were identified by cellular morphological analysis, real-time PCR, alkaline phosphatase staining (AP), immunofluorescence, teratoma formation experiments in mice. The results showed that the induced pluripotent stem cell line of Aicardi-Goutieres syndrome was successfully constructed and showed typical embryonic stem-like morphology after stable passage, RT-PCR showed mRNA expression of stem cell markers, AP staining was positive, OCT4, SOX2, NANOG, SSEA4, TRA-1-81 and TRA-1-60 pluripotency marker proteins were strongly expressed. In vivo teratoma formation experiments showed that iPSCs differentiate into the ectoderm (neural tube like tissue), mesoderm (vascular wall tissue) and endoderm (glandular tissue). Karyotype analysis also confirmed that iPSCs still maintained the original karyotype (46, XY). In conclusion, induced pluripotent stem cell line for Aicardi-Goutières syndrome was successfully established using Sendai virus, which provided an important model platform for studying the pathogenesis of the disease and for drug screening.


Subject(s)
Animals , Male , Mice , Child, Preschool , Cell Differentiation , Induced Pluripotent Stem Cells/pathology , Leukocytes, Mononuclear , Teratoma/pathology
5.
Chinese Journal of Hepatology ; (12): 781-784, 2023.
Article in Chinese | WPRIM | ID: wpr-986212

ABSTRACT

Hepatic parenchymal cells are a type of liver cells that performs important functions such as metabolism and detoxification. The contribution of hepatic parenchymal cells, bile duct cells, and hepatic stem/progenitor cells to new hepatic parenchymal cells in the process of liver injury repair has become a controversial issue due to their strong proliferation ability. Lineage tracing technology, which has emerged in the past decade as a new method for exploring the origin of cells, can trace specific type of cells and their daughter cells by labeling cells that express the specific gene and their progeny. The article reviews the current literature on the origin and contribution of hepatic parenchymal cells by this technique. About 98% of new hepatic parenchymal cells originate from the existing hepatic parenchymal cells during liver homeostasis and after acute injury. However, under conditions of severe liver injury, such as inhibition of hepatic parenchymal cell proliferation, bile duct cells (mainly liver stem/progenitor cells) become the predominant source of hepatic parenchymal cells, contributing a steady increased hepatocyte regeneration with the extension of time.


Subject(s)
Hepatocytes/metabolism , Liver/metabolism , Bile Ducts , Stem Cells , Liver Regeneration/physiology , Cell Differentiation
6.
Chinese Journal of Hepatology ; (12): 776-780, 2023.
Article in Chinese | WPRIM | ID: wpr-986211

ABSTRACT

Type II innate lymphoid cell (ILC2) is a newly identified innate immunological cell that belongs to the lymphocyte lineage in cell morphology, resides in the body's mucosal tissues, and has the dual functions of innate and adaptive immunity to promote tissue remodeling and repair after injury. Additionally, it is involved in the occurrence and development of a variety of liver diseases and plays an important role in maintaining the immunological homeostasis of the liver region. This article reviews the differentiation, development, and biological functions of ILC2, with particular attention to the research progress in liver diseases.


Subject(s)
Humans , Immunity, Innate , Lymphocytes , Cell Differentiation , Liver Diseases
7.
Chinese Journal of Pathology ; (12): 447-453, 2023.
Article in Chinese | WPRIM | ID: wpr-985699

ABSTRACT

Objective: To investigate the clinicopathological features and immunohistochemical phenotypes of gastric SMARCA4-deficient undifferentiated carcinoma, and to discuss the daily diagnostics of this entity and analyze its prognosis. Methods: The cases of gastric SMARCA4-deficient undifferentiated carcinoma diagnosed at the Department of Pathology, Peking University Cancer Hospital, China from January 2010 to August 2022 were collected. The histological sections were reviewed, the immunohistochemical results and clinicopathological features were analyzed, and relevant literature was reviewed. Results: Pure foci of undifferentiated carcinoma were seen in 7 cases, and 1 case was accompanied by a moderately differentiated tubular adenocarcinoma component. Undifferentiated carcinoma foci showed similar sheet-like or solid diffuse growth pattern, medium-sized tumor cells characterized by 1-2 nucleoli, and abundant cytoplasm and rhabdoid appearance. The average patient age was 65±8 years. Six patients were male and 2 were female. Immunohistochemical staining showed that undifferentiated carcinoma of all 8 tumors were negative for SMARCA4 (BRG1). Among 7 patients who underwent SMARCA2 (BRM) and SMARCB1 (INI1) staining, 4 cases showed loss of BRM expression, 2 cases showed weakly positive staining, and 1 case was diffusely positive, but all 7 cases were diffusely strong positive for INI1. The neuroendocrine marker, synaptophysin, was weakly positive in 5 cases, while CgA and CD56 were negative in 8 cases. Ki-67 index was more than 70%. Two cases were mismatch repair deficient and showed the loss of MLH1/PMS2 expression, while 1 case showed only MSH2 loss. PD-L1 staining showed that combined positive score (CPS)≥1 in 4 cases (CPS ranging from 1 to 55) and CPS<1 in the other 3 cases. Four patients had clinical stage Ⅳ disease. Two of them died within 3 months after diagnosis. Conclusions: Gastric SMARCA4-deficient undifferentiated carcinoma/rhabdoid carcinoma is a rare group of highly malignant tumors with a poor prognosis. Loss of the core subunit of SWI/SNF complex may be associated with the development of dedifferentiated histological pattern and aggressive tumor progression, which may be more frequently accompanied with mismatch repair deficiency.


Subject(s)
Male , Female , Humans , Carcinoma/pathology , Adenocarcinoma , Colorectal Neoplasms , Cell Differentiation , Stomach Neoplasms , Biomarkers, Tumor , DNA Helicases , Nuclear Proteins , Transcription Factors
8.
Chinese Journal of Reparative and Reconstructive Surgery ; (12): 615-621, 2023.
Article in Chinese | WPRIM | ID: wpr-981641

ABSTRACT

OBJECTIVE@#To investigate the regulatory effects of miR-26a-5p on the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) by regulating cAMP response element binding protein 1 (CREB1).@*METHODS@#The adipose tissues of four 3-4 weeks old female C57BL/6 mice were collected and the cells were isolated and cultured by digestion separation method. After morphological observation and identification by flow cytometry, the 3rd-generation cells were subjected to osteogenic differentiation induction. At 0, 3, 7, and 14 days after osteogenic differentiation induction, the calcium deposition was observed by alizarin red staining, ALP activity was detected, miR- 26a-5p and CREB1 mRNA expressions were examined by real-time fluorescence quantitative PCR, and CREB1 protein and its phosphorylation (phospho-CREB1, p-CREB1) level were measured by Western blot. After the binding sites between miR-26a-5p and CREB1 was predicted by the starBase database, HEK-293T cells were used to conduct a dual-luciferase reporter gene experiment to verify the targeting relationship (represented as luciferase activity after 48 hours of culture). Finally, miR-26a-p inhibitor (experimental group) and the corresponding negative control (control group) were transfected into ADSCs. Alizarin red staining, ALP activity, real-time fluorescent quantitative PCR (miR-26a-5p) and Western blot [CREB1, p-CREB1, Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN)] were performed at 7 and 14 days after osteogenic induction culture.@*RESULTS@#The cultured cells were identified as ADSCs. With the prolongation of osteogenic induction culture, the number of calcified nodules and ALP activity significantly increased ( P<0.05). The relative expression of miR-26a-5p in the cells gradually decreased, while the relative expressions of CREB1 mRNA and protein, as well as the relative expression of p-CREB1 protein were increased. The differences were significant between 7, 14 days and 0 day ( P<0.05). There was no significant difference in p-CREB1/CREB1 between different time points ( P>0.05). The starBase database predicted that miR-26a-5p and CREB1 had targeted binding sequences, and the dual-luciferase reporter gene experiment revealed that overexpression of miR-26a-5p significantly suppressed CREB1 wild-type luciferase activity ( P<0.05). After 7 and 14 days of osteogenic induction, compared with the control group, the number of calcified nodules, ALP activity, and relative expressions of CREB1, p-CREB1, OCN, and RUNX2 proteins in the experimental group significantly increased ( P<0.05). There was no significant difference in p-CREB1/CREB1 between the two groups ( P>0.05).@*CONCLUSION@#Knocking down miR-26a-5p promoted the osteogenic differentiation of ADSCs by up-regulating CREB1 and its phosphorylation.


Subject(s)
Animals , Female , Mice , Cell Differentiation , Cells, Cultured , Core Binding Factor Alpha 1 Subunit/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Mesenchymal Stem Cells , Mice, Inbred C57BL , MicroRNAs/metabolism , Osteocalcin/metabolism , Osteogenesis/genetics , RNA, Messenger/genetics
9.
China Journal of Chinese Materia Medica ; (24): 2522-2529, 2023.
Article in Chinese | WPRIM | ID: wpr-981328

ABSTRACT

This study aimed to investigate the effects of Erxian Decoction(EXD)-containing serum on the proliferation and osteogenic differentiation of MC3T3-E1 cells under oxidative stress through BK channels. The oxidative stress model was induced in MC3T3-E1 cells by H_2O_2, and 3 mmol·L~(-1) tetraethylammonium(TEA) chloride was used to block the BK channels in MC3T3-E1 cells. MC3T3-E1 cells were divided into a control group, a model group, an EXD group, a TEA group, and a TEA+EXD group. After MC3T3-E1 cells were treated with corresponding drugs for 2 days, 700 μmol·L~(-1) H_2O_2 was added for treatment for another 2 hours. CCK-8 assay was used to detect cell proliferation activity. The alkaline phosphatase(ALP) assay kit was used to detect the ALP activity of cells. Western blot and real-time fluorescence-based quantitative PCR(RT-qPCR) were used to detect protein and mRNA expression, respectively. Alizarin red staining was used to detect the mineralization area of osteoblasts. The results showed that compared with the control group, the model group showed significantly blunted cell proliferation activity and ALP activity, reduced expression of BK channel α subunit(BKα), collagen Ⅰ(COL1), bone morphogenetic protein 2(BMP2), osteoprotegerin(OPG), and phosphorylated Akt, decreased mRNA expression levels of Runt-related transcription factor 2(RUNX2), BMP2, and OPG, and declining area of calcium nodules. EXD-containing serum could significantly potentiate the cell proliferation activity and ALP activity, up-regulate the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt, and forkhead box protein O1(FoxO1), promote the mRNA expression of RUNX2, BMP2, and OPG, and enlarge the area of calcium nodules. However, BK channel blockage by TEA reversed the effects of EXD-containing serum in promoting the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt and FoxO1, increasing the mRNA expression of RUNX2, BMP2, and OPG, and enlarging the area of calcium nodules. EXD-containing serum could improve the proliferation activity, osteogenic differentiation, and mineralization ability of MC3T3-E1 cells under oxidative stress, which might be related to the regulation of BK channels and downstream Akt/FoxO1 signaling pathway.


Subject(s)
Osteogenesis , Core Binding Factor Alpha 1 Subunit/pharmacology , Large-Conductance Calcium-Activated Potassium Channels/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Calcium/metabolism , Cell Differentiation , RNA, Messenger/metabolism , Cell Proliferation , Osteoblasts
10.
Chinese Journal of Biotechnology ; (12): 2695-2705, 2023.
Article in Chinese | WPRIM | ID: wpr-981226

ABSTRACT

The aim of this study was to clone the goat RPL29 gene and analyze its effect on lipogenesis in intramuscular adipocytes. Using Jianzhou big-eared goats as the object, the goat RPL29 gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR), the gene structure and expressed protein sequence were analyzed by bioinformatics, and the mRNA expression levels of RPL29 in various tissues and different differentiation stages of intramuscular adipocytes of goats were detected by quantitative real-time PCR (qRT-PCR). The RPL29 overexpression vector pEGFP-N1-RPL29 constructed by gene recombination was used to transfect into goat intramuscular preadipocytes and induce differentiation. Subsequently, the effect of overexpression of RPL29 on fat droplet accumulation was revealed morphologically by oil red O and Bodipy staining, and changes in the expression levels of genes related to lipid metabolism were detected by qRT-PCR. The results showed that the length of the goat RPL29 was 507 bp, including a coding sequence (CDS) region of 471 bp which encodes 156 amino acid residues. It is a positively charged and stable hydrophilic protein mainly distributed in the nucleus of cells. Tissue expression profiling showed that the expression level of this gene was much higher in subcutaneous adipose tissue and inter-abdominal adipose tissue of goats than in other tissues (P < 0.05). The temporal expression profile showed that the gene was expressed at the highest level at 84 h of differentiation in goat intramuscular adipocytes, which was highly significantly higher than that in the undifferentiated period (P < 0.01). Overexpression of RPL29 promoted lipid accumulation in intramuscular adipocytes, and the optical density values of oil red O staining were significantly increased (P < 0.05). In addition, overexpression of RPL29 was followed by a highly significant increase in ATGL and ACC gene expression (P < 0.01) and a significant increase in FASN gene expression (P < 0.05). In conclusion, the goat RPL29 may promote intra-muscular adipocyte deposition in goats by up-regulating FASN, ACC and ATGL.


Subject(s)
Animals , Lipogenesis/genetics , Adipogenesis/genetics , Goats/genetics , Adipocytes , Cell Differentiation/genetics , Sequence Analysis , Cloning, Molecular
11.
Chinese Journal of Biotechnology ; (12): 1773-1788, 2023.
Article in Chinese | WPRIM | ID: wpr-981169

ABSTRACT

A triple-transgenic (tyrosine hydroxylase/dopamine decarboxylase/GTP cyclohydrolase 1, TH/DDC/GCH1) bone marrow mesenchymal stem cell line (BMSCs) capable of stably synthesizing dopamine (DA) transmitters were established to provide experimental evidence for the clinical treatment of Parkinson's disease (PD) by using this cell line. The DA-BMSCs cell line that could stably synthesize and secrete DA transmitters was established by using the triple transgenic recombinant lentivirus. The triple transgenes (TH/DDC/GCH1) expression in DA-BMSCs was detected using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. Moreover, the secretion of DA was tested by enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC). Chromosome G-banding analysis was used to detect the genetic stability of DA-BMSCs. Subsequently, the DA-BMSCs were stereotactically transplanted into the right medial forebrain bundle (MFB) of Parkinson's rat models to detect their survival and differentiation in the intracerebral microenvironment of PD rats. Apomorphine (APO)-induced rotation test was used to detect the improvement of motor dysfunction in PD rat models with cell transplantation. The TH, DDC and GCH1 were expressed stably and efficiently in the DA-BMSCs cell line, but not expressed in the normal rat BMSCs. The concentration of DA in the cell culture supernatant of the triple transgenic group (DA-BMSCs) and the LV-TH group was extremely significantly higher than that of the standard BMSCs control group (P < 0.000 1). After passage, DA-BMSCs stably produced DA. Karyotype G-banding analysis showed that the vast majority of DA-BMSCs maintained normal diploid karyotypes (94.5%). Moreover, after 4 weeks of transplantation into the brain of PD rats, DA-BMSCs significantly improved the movement disorder of PD rat models, survived in a large amount in the brain microenvironment, differentiated into TH-positive and GFAP-positive cells, and upregulated the DA level in the injured area of the brain. The triple-transgenic DA-BMSCs cell line that stably produced DA, survived in large numbers, and differentiated in the rat brain was successfully established, laying a foundation for the treatment of PD using engineered culture and transplantation of DA-BMSCs.


Subject(s)
Rats , Animals , Dopamine , Parkinson Disease/metabolism , Mesenchymal Stem Cells/metabolism , Cell Line , Brain/metabolism , Cell Differentiation , Mesenchymal Stem Cell Transplantation
12.
Chinese Journal of Biotechnology ; (12): 1684-1695, 2023.
Article in Chinese | WPRIM | ID: wpr-981163

ABSTRACT

C-fos is a transcription factor that plays an important role in cell proliferation, differentiation and tumor formation. The aim of this study was to clone the goat c-fos gene, clarify its biological characteristics, and further reveal its regulatory role in the differentiation of goat subcutaneous adipocytes. We cloned the c-fos gene from subcutaneous adipose tissue of Jianzhou big-eared goats by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed its biological characteristics. Using real-time quantitative PCR (qPCR), we detected the expression of c-fos gene in the heart, liver, spleen, lung, kidney, subcutaneous fat, longissimus dorsi and subcutaneous adipocytes of goat upon induced differentiation for 0 h to 120 h. The goat overexpression vector pEGFP-c-fos was constructed and transfected into the subcutaneous preadipocytes for induced differentiation. The morphological changes of lipid droplet accumulation were observed by oil red O staining and bodipy staining. Furthermore, qPCR was used to test the relative mRNA level of the c-fos overexpression on adipogenic differentiation marker genes. The results showed that the cloned goat c-fos gene was 1 477 bp in length, in which the coding sequence was 1 143 bp, encoding a protein of 380 amino acids. Protein structure analysis showed that goat FOS protein has a basic leucine zipper structure, and subcellular localization prediction suggested that it was mainly distributed in the nucleus. The relative expression level of c-fos was higher in the subcutaneous adipose tissue of goats (P < 0.05), and the expression level of c-fos was significantly increased upon induced differentiation of subcutaneous preadipocyte for 48 h (P < 0.01). Overexpression of c-fos significantly inhibited the lipid droplets formation in goat subcutaneous adipocytes, significantly decreased the relative expression levels of the AP2 and C/EBPβ lipogenic marker genes (P < 0.01). Moreover, AP2 and C/EBPβ promoter are predicted to have multiple binding sites. In conclusion, the results indicated that c-fos gene was a negative regulatory factor of subcutaneous adipocyte differentiation in goats, and it might regulate the expression of AP2 and C/EBPβ gene expression.


Subject(s)
Animals , Goats/genetics , Cell Differentiation/genetics , Adipogenesis/genetics , Gene Expression Regulation , Proteins/genetics , Cloning, Molecular
13.
West China Journal of Stomatology ; (6): 175-184, 2023.
Article in English | WPRIM | ID: wpr-981109

ABSTRACT

OBJECTIVES@#This study aimed to investigate how naringenin (Nar) affected the anti-inflammatory, vascula-rization, and osteogenesis differentiation of human periodontal ligament stem cells (hPDLSCs) stimulated by lipopolysaccharide (LPS) and to preliminarily explore the underlying mechanism.@*METHODS@#Cell-counting kit-8 (CCK8), cell scratch test, and Transwell assay were used to investigate the proliferation and migratory capabilities of hPDLSCs. Alkaline phosphatase (ALP) staining, alizarin red staining, lumen-formation assay, enzyme-linked immunosorbent assay, quantitative timed polymerase chain reaction, and Western blot were used to measure the expression of osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), vascular endothlial growth factor (VEGF), basic fibroblast growth factor (bFGF), von Willebrand factor (vWF), tumor necrosis factor-α (TNF-α), and interleukin (IL)-6.@*RESULTS@#We observed that 10 μmol/L Nar could attenuate the inflammatory response of hPDLSCs stimulated by 10 μg/mL LPS and promoted their proliferation, migration, and vascularization differentiation. Furthermore, 0.1 μmol/L Nar could effectively restore the osteogenic differentiation of inflammatory hPDLSCs. The effects of Nar's anti-inflammatory and promotion of osteogenic differentiation significantly decreased and inflammatory vascularization differentiation increased after adding AMD3100 (a specific CXCR4 inhibitor).@*CONCLUSIONS@#Nar demonstrated the ability to promote the anti-inflammatory, vascularization, and osteogenic effects of hPDLSCs stimulated by LPS, and the ability was associated with the stromal cell-derived factor/C-X-C motif chemokine receptor 4 signaling axis.


Subject(s)
Humans , Anti-Inflammatory Agents/pharmacology , Cell Differentiation , Cell Proliferation , Cells, Cultured , Chemokine CXCL12 , Lipopolysaccharides/pharmacology , Osteogenesis , Periodontal Ligament/metabolism , Receptors, Chemokine/metabolism , Stem Cells , Interleukin-8/metabolism
14.
West China Journal of Stomatology ; (6): 165-174, 2023.
Article in English | WPRIM | ID: wpr-981108

ABSTRACT

OBJECTIVES@#This study aimed to investigate the effect of new biomimetic micro/nano surfaces on the osteoclastic differentiation of RAW264.7 macrophages by simulating natural osteons for the design of concentric circular structures and modifying graphene oxide (GO).@*METHODS@#The groups were divided into smooth titanium surface group (SS), concentric microgrooved titanium surface group (CMS), and microgroove modified with GO group (GO-CMS). The physicochemical properties of the material surfaces were studied using scanning electron microscopy (SEM), contact-angle measurement, atomic force microscopy, X-ray photoelectron spectroscopy analysis, and Raman spectroscopy. The effect of the modified material surface on the cell biological behavior of RAW264.7 was investigated by cell-activity assay, SEM, and laser confocal microscopy. The effect on the osteoclastic differentiation of macrophages was investiga-ted by tartrate-resistant acid phosphatase (TRAP) immunofluorescence staining and quantitative real-time polymerase chain reaction (qRT-PCR) experiments.@*RESULTS@#Macrophages were arranged in concentric circles along the microgrooves, and after modification with GO, the oxygen-containing groups on the surface of the material increased and hydrophilicity increased. Osteoclasts in the GO-CMS group were small in size and number and had the lowest TRAP expression. Although it promoted the proliferation of macrophages in the GO-CMS group, the expression of osteoclastic differentiation-related genes was lower than that in the SS group, and the difference was statistically significant (P<0.05).@*CONCLUSIONS@#Concentric circular microgrooves restricted the fusion of osteoclasts and the formation of sealing zones. Osteomimetic concentric microgrooves modified with GO inhibited the osteoclastic differentiation of RAW 264.7 macrophages.


Subject(s)
Graphite/pharmacology , Titanium/pharmacology , Haversian System , Macrophages , Cell Differentiation , Oxides/pharmacology , Surface Properties
15.
West China Journal of Stomatology ; (6): 140-148, 2023.
Article in English | WPRIM | ID: wpr-981105

ABSTRACT

OBJECTIVES@#To investigate the effect of recombinant human fibroblast growth factor 21 (rhFGF21) on the proliferation and mineralization of cementoblasts and its mechanism.@*METHODS@#Hematoxylin eosin, immunohistochemical staining, and immunofluorescence were used to detect the expression and distribution of fibroblast growth factor 21 (FGF21) in rat periodontal tissues and cementoblasts (OCCM-30), separately. Cell Counting Kit-8 was used to detect the proliferation of OCCM-30 under treatment with rhFGF21. Alkaline phosphatase staining and Alizarin Red staining were used to detect the mineralization state of OCCM-30 after 3 and 7 days of mineralization induction. The transcription and protein expression of the osteogenic-related genes Runx2 and Osterix were detected by real-time quantitative polymerase chain reaction (PCR) and Western blot analysis. The expression levels of genes of transforming growth factor β (TGFβ)/bone morphogenetic protein (BMP) signaling pathway in OCCM-30 were detected through PCR array analysis.@*RESULTS@#FGF21 was expressed in rat periodontal tissues and OCCM-30. Although rhFGF21 had no significant effect on the proliferation of OCCM-30, treatment with 50 ng/mL rhFGF21 could promote the mineralization of OCCM-30 cells after 7 days of mineralization induction. The transcriptional levels of Runx2 and Osterix increased significantly at 3 days of mineralization induction and decreased at 5 days of mineralization induction. Western blot analysis showed that the protein expression levels of Runx2 and Osterix increased during mineralization induction. rhFGF21 up-regulated Bmpr1b protein expression in cells.@*CONCLUSIONS@#rhFGF21 can promote the mineralization ability of OCCM-30. This effect is related to the activation of the TGFβ/BMP signaling pathway.


Subject(s)
Humans , Rats , Animals , Dental Cementum , Core Binding Factor Alpha 1 Subunit/metabolism , Cell Differentiation , Bone Morphogenetic Proteins/metabolism , Transforming Growth Factor beta/pharmacology
16.
Acta Physiologica Sinica ; (6): 429-438, 2023.
Article in Chinese | WPRIM | ID: wpr-981018

ABSTRACT

It has been well documented that exercise can improve bone metabolism, promote bone growth and development, and alleviate bone loss. MicroRNAs (miRNAs) are widely involved in the proliferation and differentiation of bone marrow mesenchymal stem cells, osteoblasts, osteoclasts and other bone tissue cells, and regulation of balance between bone formation and bone resorption by targeting osteogenic factors or bone resorption factors. Thus miRNAs play an important role in the regulation of bone metabolism. Recently, regulation of miRNAs are shown to be one of the ways by which exercise or mechanical stress promotes the positive balance of bone metabolism. Exercise induces changes of miRNAs expression in bone tissue and regulates the expression of related osteogenic factors or bone resorption factors, to further strengthen the osteogenic effect of exercise. This review summarizes relevant studies on the mechanism whereby exercise regulates bone metabolism via miRNAs, providing a theoretical basis for osteoporosis prevention and treatment with exercise.


Subject(s)
Humans , MicroRNAs/metabolism , Osteogenesis/genetics , Cell Differentiation , Osteoblasts , Bone Resorption/metabolism
17.
Acta Physiologica Sinica ; (6): 269-278, 2023.
Article in Chinese | WPRIM | ID: wpr-981004

ABSTRACT

DMRT, a gene family related to sexual determination, encodes a large group of transcription factors (DMRTs) with the double-sex and mab-3 (DM) domain (except for DMRT8), which is able to bind to and regulate DNAs. Current studies have shown that the DMRT gene family plays a critical role in the development of sexual organs (such as gender differentiation, gonadal development, germ cell development, etc.) as well as extrasexual organs (such as musculocartilage development, nervous system development, etc.). Additionally, it has been suggested that DMRTs may be involved in the cancer development and progression (such as prostate cancer, breast cancer, lung cancer, etc.). This review summarizes the research progress about the mammalian DMRTs' structure, function and its critical role in cancer development, progression and therapy (mainly in human and mice), which suggests that DMRT gene could be a candidate gene in the study of tumor formation and therapeutic strategy.


Subject(s)
Male , Animals , Humans , Mice , Transcription Factors/genetics , Mammals/metabolism , Cell Differentiation , Neoplasms/genetics
18.
Acta Physiologica Sinica ; (6): 205-215, 2023.
Article in Chinese | WPRIM | ID: wpr-980998

ABSTRACT

Vascular wall-resident stem cells (VW-SCs) play a critical role in maintaining normal vascular function and regulating vascular repair. Understanding the basic functional characteristics of the VW-SCs will facilitate the study of their regulation and potential therapeutic applications. The aim of this study was to establish a stable method for the isolation, culture, and validation of the CD34+ VW-SCs from mice, and to provide abundant and reliable cell sources for further study of the mechanisms involved in proliferation, migration and differentiation of the VW-SCs under various physiological and pathological conditions. The vascular wall cells of mouse aortic adventitia and mesenteric artery were obtained by the method of tissue block attachment and purified by magnetic microbead sorting and flow cytometry to obtain the CD34+ VW-SCs. Cell immunofluorescence staining was performed to detect the stem cell markers (CD34, Flk-1, c-kit, Sca-1), smooth muscle markers (SM22, SM MHC), endothelial marker (CD31), and intranuclear division proliferation-related protein (Ki-67). To verify the multipotency of the isolated CD34+ VW-SCs, endothelial differentiation medium EBM-2 and fibroblast differentiation medium FM-2 were used. After culture for 7 days and 3 days respectively, endothelial cell markers and fibroblast markers of the differentiated cells were evaluated by immunofluorescence staining and q-PCR. Furthermore, the intracellular Ca2+ release and extracellular Ca2+ entry signaling were evaluated by TILLvisION system in Fura-2/AM loaded cells. The results showed that: (1) High purity (more than 90%) CD34+ VW-SCs from aortic adventitia and mesenteric artery of mice were harvested by means of tissue block attachment method and magnetic microbead sorting; (2) CD34+ VW-SCs were able to differentiate into endothelial cells and fibroblasts in vitro; (3) Caffeine and ATP significantly activated intracellular Ca2+ release from endoplasmic reticulum of CD34+ VW-SCs. Store-operated Ca2+ entry (SOCE) was activated by using thapsigargin (TG) applied in Ca2+-free/Ca2+ reintroduction protocol. This study successfully established a stable and efficient method for isolation, culture and validation of the CD34+ VW-SCs from mice, which provides an ideal VW-SCs sources for the further study of cardiovascular diseases.


Subject(s)
Mice , Animals , Endothelial Cells , Cell Differentiation/physiology , Stem Cells , Adventitia , Fibroblasts , Cells, Cultured , Antigens, CD34/metabolism
19.
Braz. j. biol ; 83: e248024, 2023. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1355855

ABSTRACT

Abstract By applying the in-silico method, resveratrol was docked on those proteins which are responsible for bone loss. The Molecular docking data between the resveratrol and Receptor activator of nuclear factor-kappa-Β ligand [RANKL] receptors proved that resveratrol binds tightly to the receptors, showed the highest binding affinities of −6.9, −7.6, −7.1, −6.9, −6.7, and −7.1 kcal/mol. According to in-vitro data, Resveratrol reduced the osteoclasts after treating Marrow-Derived Macrophages [BMM] with Macrophage colony-stimulating factor [MCSF] 20ng / ml and RANKL 50ng / ml, with different concentrations of resveratrol (2.5, 10 μg / ml) For 7 days, the cells were treated with MCSF (20 ng / ml) and RANKL (40 ng / ml) together with concentrated trimethyl ether and resveratrol (2.5, 10 μg / ml) within 12 hours. Which, not affect cell survival. After fixing osteoclast cells with formaldehyde fixative on glass coverslip followed by incubation with 0.1% Triton X-100 in PBS for 5 min and after that stain with rhodamine phalloidin staining for actin and Hoechst for nuclei. Fluorescence microscopy was performed to see the distribution of filaments actin [F.actin]. Finally, resveratrol reduced the actin ring formation. Resveratrol is the best bioactive compound for drug preparation against bone loss.


Resumo Com a aplicação do método in-silico, o resveratrol foi ancorado nas proteínas responsáveis ​​pela perda óssea. Os dados de docking molecular entre o resveratrol e o ligante do receptor ativador do fator nuclear kappa-Β [Receptor Activator of Nuclear Factor kappa-B Ligant (RANKL)] provaram que o resveratrol se liga fortemente aos receptores, mostraram as afinidades de ligação mais altas de −6,9, −7,6, −7,1, −6,9, - 6,7 e -7,1 kcal / mol. De acordo com dados in-vitro, o resveratrol reduziu os osteoclastos após o tratamento de macrófagos derivados da medula óssea [Bone Marrow-derived Macrophage (BMM)] com fator estimulador de colônias de macrófagos [Macrophage Colony-Stimulating Factor (MCSF)] 20ng / ml e RANKL 50ng / ml, com diferentes concentrações de resveratrol (2,5, 10 μg / ml). Durante sete dias, as células foram tratadas com MCSF (20 ng / ml) e RANKL (40 ng / ml) juntamente com éter trimetílico concentrado e resveratrol (2,5, 10 μg / ml) em 12 horas, processo que não afeta a sobrevivência celular. Após a fixação de células de osteoclastos com fixador de formaldeído em lamela de vidro seguido de incubação com 0,1% Triton X-100 em PBS por 5 min, foi realizado posteriormente o procedimento para corar com rodamina faloidina a actina e Hoechst os núcleos. A microscopia de fluorescência foi realizada para ver a distribuição dos filamentos de actina [F.actina]. Finalmente, o resveratrol reduziu a formação do anel de actina. O resveratrol é o melhor composto bioativo para o preparo de medicamentos contra a perda óssea.


Subject(s)
Osteoclasts , RANK Ligand , Cell Differentiation , Molecular Docking Simulation , Resveratrol/pharmacology
20.
International Journal of Oral Science ; (4): 13-13, 2023.
Article in English | WPRIM | ID: wpr-971601

ABSTRACT

X-linked hypophosphatemia (XLH) represents the most common form of familial hypophosphatemia. Although significant advances have been made in the treatment of bone pathology, patients undergoing therapy continue to experience significantly decreased oral health-related quality of life. The following study addresses this persistent oral disease by further investigating the effect of DMP1 expression on the differentiation of XLH dental pulp cells. Dental pulp cells were isolated from the third molars of XLH and healthy controls and stable transduction of full-length human DMP1 were achieved. RNA sequencing was performed to evaluate the genetic changes following the induction of odontogenic differentiation. RNAseq data shows the upregulation of inhibitors of the canonical Wnt pathway in XLH cells, while constitutive expression of full-length DMP1 in XLH cells reversed this effect during odontogenic differentiation. These results imply that inhibition of the canonical Wnt pathway may contribute to the pathophysiology of XLH and suggest a new therapeutic strategy for the management of oral disease.


Subject(s)
Humans , Familial Hypophosphatemic Rickets , Wnt Signaling Pathway , Dental Pulp , Quality of Life , Cell Differentiation
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