An integrative bioinformatics investigation and experimental validation of chromobox family in diffuse large B-cell lymphoma.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is one of the most aggressive malignant tumors. Chromobox (CBX) family plays the role of oncogenes in various malignancies. METHODS: The transcriptional and protein levels of CBX family were confirmed by GEPIA, Oncomine, CCLE, and HPA database. Screening of co-expressed genes and gene function enrichment analysis were performed by GeneMANIA and DAVID 6.8. The prognostic value, immune cell infiltration and drug sensitivity analysis of CBX family in DLBCL were performed by Genomicscape, TIMER2.0, and GSCALite database. Confirmatory Tests of CBX family protein expression in DLBCL were performed by immunohistochemistry. RESULTS: The mRNA and protein expressions of CBX1/2/3/5/6 were higher in DLBCL tissues than control groups. Enrichment analysis showed that the functions of CBX family were mainly related to chromatin remodeling, methylation-dependent protein binding, and VEGF signaling pathway. The high mRNA expressions of CBX2/3/5/6 were identified to be associated with short overall survival (OS) in DLBCL patients. Multivariate COX regression indicated that CBX3 was independent prognostic marker. Immune infiltration analysis revealed that the mRNA expressions of CBX family (especially CBX1, CBX5, and CBX6) in DLBCL were significantly correlated with the infiltration of most immune cells (including B cells, CD8 + T cells, CD4 + T cells, neutrophils, monocytes, macrophages, and Treg cells). Meanwhile, there was a strong correlation between the expression levels of CBX1/5/6 and surface markers of immune cells, such as the widely studied PVR-like protein receptor/ligand and PDL-1 immune checkpoint. Notably, our study found that DLBCL cells with CBX1 over-expression were resistant to the common anti-tumor drugs, but CBX2/5 had two polarities. Finally, we confirmed the higher expressions of CBX1/2/3/5/6 in DLBCL tissues compared with control groups by immunohistochemistry. CONCLUSION: We provided a detailed analysis of the relationship between the CBX family and the prognosis of DLBCL. Distinguished from other studies, We found that high mRNA expressions of CBX2/3/5/6 were associated with poor prognosis in DLBCL patients, and Multivariate COX regression indicated that CBX3 was independent prognostic marker. Besides, our study also found an association between the CBX family and anti-tumour drug resistance, and provided a relationship between CBX family expression and immune cell infiltration.

WEHI-3 cells inhibit adipocyte differentiation in 3T3-L1 cells.

By investigating the anti-adipogenic effects of WEHI-3 cells - a murine acute myelomonocytic leukemia cell line - we sought to improve the efficiency of hematopoietic stem cell transplantation (HSCT). Analysis of Oil Red O staining and the expression of adipogenic genes, including PPARγ, C/EBPα, FAS and LPL, indicated that WEHI-3 cells significantly inhibited 3T3-L1 mouse preadipocyte cells from differentiating into adipocytes. In vivo, fat vacuoles in mice injected with WEHI-3 cells were also remarkably reduced in the murine bone marrow pimelosis model. Moreover, the key gene in the Rho signaling pathway, ROCKII, and the key gene in the Wnt signaling pathway, ß-catenin, were both upregulated compared with the control group. siRNA-mediated knockdown of ROCKII and ß-catenin reversed these WEHI-3-mediated anti-adipogenic effects. Taken together, these data suggest that WEHI-3 cells exert anti-adipogenic effects and that both ROCKII and ß-catenin are involved in this process.

Effects of nicotine on proliferation and survival in human umbilical cord mesenchymal stem cells.

Cigarette smoking is known to have negative effects on tissue repair and healing. The aim of this study is to investigate the effects of nicotine in human umbilical cord mesenchymal stem cells (MSCs). After nicotine treatment, MSCs became pyknotic, vacuoles appeared in the cytoplasm and nucleus, and the nuclear boundary became fuzzy as observed using atomic force microscopy. Cell proliferation was inhibited in a dose-dependent manner (P < 0.05 for all concentrations). The proportion of apoptotic MSCs was significantly increased in a dose-dependent manner. The mitochondrial membrane potential was significantly decreased (P < 0.05). Nicotine-treated MSCs had a significantly higher G0/G1 ratio (P < 0.05). Peptide mass fingerprinting identified 27 proteins that were differentially expressed between MSCs with and without nicotine treatment. These nicotine exerted toxic effects on MSCs are likely related, at least in part, to the altered expression of multiple proteins that are essential to the health and proliferation of these cells.

Impaired activity of circulating EPCs and endothelial function are associated with increased Syntax score in patients with coronary artery disease.

It has previously been shown that the number of endothelial progenitor cells (EPCs) is negatively correlated with Syntax score in patients with coronary artery disease (CAD). However, the association between alterations in EPC function and Syntax score is still unknown. The present study evaluated the association between the activity of EPCs as well as endothelial function and Syntax score in patients with CAD and investigated the underlying mechanisms. A total of 60 patients with CAD were enrolled in 3 groups according to Syntax score, and 20 healthy subjects were recruited as the control group. The number and migratory, proliferative and adhesive activities of circulating EPCs were studied. The endothelial function was measured by flow­mediated dilatation (FMD) and the levels of nitric oxide (NO) in plasma or secreted by EPCs were detected. The number and activity of circulating EPCs were lower in patients with a high Syntax score, which was similar to the alteration in FMD. The level of NO in plasma or secreted by EPCs also decreased as Syntax score increased. There was a negative association between FMD or circulating EPCs and Syntax score. A similar association was observed between the levels of NO in plasma or secreted by EPCs and Syntax score. Patients with CAD who had a higher Syntax score exhibited lower EPC numbers or activity and weaker endothelial function, which may be associated with attenuated NO production. These findings provide novel surrogate parameters for evaluation of the severity and complexity of CAD.

Identification of six hub genes and analysis of their correlation with drug sensitivity in acute myeloid leukemia through bioinformatics.

BACKGROUND: Acute myeloid leukemia (AML) is a common hematopoietic malignancy and have unsatisfactory prognosis. Our study aimed to identify hub genes in AML and explore potential biomarkers through integrated bioinformatics. METHODS: Microarray datasets were analyzed to screen the differentially expressed genes (DEGs). Functional enrichment analysis was performed, and protein-protein interaction (PPI) network was generated by the STRING (11.0) database and Cytoscape (3.7.2) software. Hub genes were screened and verified through GEPIA2 and GEO microarray database. Sensitivity of AML cell lines with high expression of hub genes to the small-molecule drugs were identified using GSCA Lite. RESULTS: A total of 456 DEGs were identified and top 100 genes were screened out, of which six genes (FLT3, PF4, CD163, MRC1, CSF2RB, PPBP) were upregulated in AML and individually had a worse prognosis by the overall survival (OS) analysis. AML cell lines with FLT3-overexpression and CSF2RB-overexpression were sensitive to most small-molecule drugs, while, AML cells with CD163-overexpression were only sensitive to a few drugs. However, sensitivity to Erlotinib was correlated with high expression of PF4 and PPBP. CONCLUSIONS: In summary, FLT3, PF4, CD163, MRC1, CSF2RB, PPBP may be potential biomarkers and potential sensitive small-molecule drugs were correlated with overexpression of the biomarkers in AML.

Hypofunction of Circulating Endothelial Progenitor Cells and Aggravated Severity in Elderly Male Patients With Non-ST Segment Elevation Myocardial Infarction: Its Association With Systemic Inflammation.

Background: Aging patients easily suffer from non-ST segment elevation myocardial infarction (NSTEMI). Our previous studies revealed declined function of endothelial progenitor cells (EPCs) in the elderly. However, the impact of aging on EPC function and severity in male NSTEMI patients and its possible mechanism is unclear until now. Methods: We measured the circulating EPC function including migration, proliferation, and adhesion in aging or young male patients with NSTEMI. The GRACE and TIMI risk score were evaluated. Plasma levels of interleukin-6 (IL-6) and interleukin-17 (IL-17) were also detected in all patients. Results: Compared with the young group, the old male patients with NSTEMI had higher GRACE score and TIMI score and decreased function of circulating EPCs. EPC function was negatively correlated with GRACE score and TIMI score. IL-6 and IL-17 level were higher in the old group than those in the young group. There was a significant negative correlation between EPC function and IL-6 or IL-17. Moreover, IL-6 and IL-17 positively correlated with GRACE and TIMI score. Age was positively related with GRACE or TIMI score and plasma level of IL-6 or IL-17, but inversely correlated with EPC function. Conclusions: The current study firstly illustrates that the age-related decrement in EPC function is related to the severity of NSTEMI in male patients, which may be connected with systemic inflammation. These findings provide novel insights into the pathogenetic mechanism and intervention target of aging NSTEMI.

The detrimental qualitative and quantitative alterations of circulating endothelial progenitor cells in patients with bronchiectasis.

BACKGROUND: Bronchiectasis is an independent risk factor for cardiovascular disease（CVD）and cardiac dysfunction. Endothelial progenitor cells (EPCs) play a crucial role in maintaining endothelial function, and is inversely correlated with cardiovascular risk factors or cardiac dysfunction. However, the relationship between EPCs and bronchiectasis is unknown. METHODS: Twenty-nine patients with stable bronchiectasis and 15 healthy controls were recruited. Fasting venous blood were collected for determining circulating EPC number and activity as well as systemic inflammatory cytokines. RESULTS: The number and migratory or proliferative activity of circulating EPCs in bronchiectasis patients were significantly reduced (p < 0.001). In high E-FACED group, the number of circulating EPCs evaluated by cell culture assay and EPC proliferation were decreased (p < 0.05). Similarly, the number and function of circulating EPCs were both reduced in low forced expiratory volume in 1 s (FEV1) or high mMRC group (p < 0.05). There was a significant correlation between circulating EPCs and bronchiectasis disease severity, according to the E-FACED score (p < 0.05), particularly to FEV1 (p < 0.05) and mMRC dyspnea score (p < 0.05). The count and activity of EPCs inversely correlated with hsCRP levels and IL-6 levels (p < 0.01). CONCLUSIONS: Deficiencies in the number and function of circulating EPCs are present in patients with bronchiectasis. The changes are related to disease severity and may be partly attributed to systemic inflammation. The current findings may provide novel surrogate evaluation biomarkers and potential therapeutic target for bronchiectasis.

Reduced Number and Activity of Circulating Endothelial Progenitor Cells in Acute Aortic Dissection and Its Relationship With IL-6 and IL-17.

This study investigates the alteration in function and number of circulating endothelial progenitor cells (EPCs) in patients with aortic dissection (AD), compared with hypertensive patients, and its possible mechanism. Thirty-four patients with acute aortic dissection (AAD) and 20 patients with primary hypertension were involved. Flow cytometry analysis was performed to detect the number of CD34+/KDR+ cells, and acetylated low density lipoprotein (ac-LDL) and lectin fluorescent staining method was applied to test the number of cultured EPCs. In addition, EPC migration and proliferation were measured, and plasma interleukin 6 (IL-6) and interleukin 17 (IL-17) levels were investigated. The number of circulating EPCs in the AAD group was lower than that in the non-AD group, and the proliferation and migration of circulating EPCs in the AAD group were lower than that in the non-AD group. In addition, the number, proliferation, and migration of circulating EPCs were significantly inversely correlated with the aortic dissection detection risk score (ADD-RS). More importantly, increased plasma IL-6 and IL-17 level was found in the AAD group, and the two inflammatory factors were inversely associated with the function and number of circulating EPCs in the AAD group. We first demonstrated that the number and function of circulating EPCs are reduced in the AAD group, which may be partly related to upregulated plasma IL-6 and IL-17. Our study provides novel insight on the underlying mechanism and potential therapeutic target of AAD.

The miR-203a Regulatory Network Affects the Proliferation of Chronic Myeloid Leukemia K562 Cells.

To study the molecular mechanism by which miR-203a affects the development of CML, bioinformatics software was used to predict the upstream transcription factors and downstream target genes of miR-203a. A 5'-rapid amplification of cDNA ends assay was performed to detect gene transcription initiation sites. A chromatin immunoprecipitation assay was used to verify the binding of transcription factors and promoter regions. A double luciferase reporter gene vector was constructed to demonstrate the regulatory effect of miR-203a on target genes. Real-time PCR and western blotting were used to detect the relative expression levels of genes and proteins, respectively. The results showed that there was a binding site for the transcription factor EGR1 in the upstream promoter region of miR-203a. WT1, BMI1, and XIAP were identified as target genes regulated by miR-203a. EGR1 and miR-203a were downregulated in human peripheral blood mononuclear cells and the CML K562 cell line, while WT1, BMI1, and XIAP were upregulated. The transcription initiation site of miR-203a was identified in the upstream promoter region (G nucleotide at -339 bp), and the transcription factor EGR1 could bind to the promoter region (at -268 bp) of miR-203a and increase its expression. Over expression of miR-203a inhibited the proliferation of K562 cells. A rescue assay showed that overexpression of WT1, BMI1, and XIAP offset the antitumor effect of miR-203a. Conclusion, EGR1 positively regulated the expression of miR-203a, thus relieving the inhibition of miR-203a on the translation of its target genes (WT1, BMI1, and XIAP) and affecting the proliferation of K562 cells.

[Anti-aging effect of transplantation of mouse fetus-derived mesenchymal stem cells].

To determine the role of allogeneil graft of mesenchymal stem cells in mammalian longevity, mesenchymal stem cells were isolated from BALB/c mouse uterine-incision delivery fetus by two successive density gradient centrifugations, and then were purified and amplified by adherent culture. Identified P1 mesenchymal stem cells were injected (i.v.) through vena caudalis into the 15-month-old female BALB/c mice three times. The mice were evaluated with ultrasoundcardiogram, autopsy, score of cardiac, skin, lung, kidney, colon histopathology and serum total superoxide dismutase activity, maleic dialdehyde content, glutathione peroxidase activity. The results showed that after transplantation, the long-term surviving stem cells were found to be located in many organ tissues with in situ Y chromosomal hybridization dyeing. Median life span was increased in these animals after transplantation. Skin, cardiac, lung, kidney and colon pathology development were delayed. The retrogradation of heart function was attenuated, the increase of heart mass index (the mass of heart/the mass of the body), and serum maleic dialdehyde content, the decrease of spleen mass index (the mass of spleen/the mass of the body), serum total superoxide dismutase activity and glutathione peroxidase activity were reduced three months after transplantation (all P<0.05). These results support the idea that longevity can be enhanced by transplantation of mesenchymal stem cells and reinforce the hypothesis of mesenchymal stem cell as antiager.

[Research on anti-aging effect of mouse placenta cells transplantation].

To determine the role of placenta cells allogeneic graft in mammalian longevity, the 15-month-old female BALB/c mice (n = 50) were divided into Control group (A), Short-term transplanted group (B) and Long-term transplanted many times group (C). Their placentae (at 18 days of gestation) were taken out and ground with 50-eye cell grit, and the cells were intraperitoneally injected into the mice of B group and C group three times at intervals of 10 days; then the cells were transplanted into the mice of C group many times till the time of death. The mice were evaluated by use of ultrasound-cardiogram; autopsy; score of cardia, spleen, skin, lung, kidney; histopathology; serum total superoxide dismutase activity, serum maleic dialdehyde content, and serum glutathione peroxidase activity. The long-term surviving stem cells were found to be located in many organ tissues of B and C groups' mice with in situ Y chromosomal hybridization dyeing. Median life span of B group mice was 1.7 times that of A group's after transplantation, but there was no statistically significant difference between B group and C group. Three months after transplantation, in B group, the pathological developments of significant skin, cardia, lung, and kidney were delayed; the retrogradation of heart function was attenuated; the data on heart mass index (mass of heart/mass of body), left ventricular mass and serum Maleic Dialdehyde content, and on spleen mass index (mass of spleen/mass of body), left ventricular diastolic volume, serum Total Superoxide Dismutase activity and serum Glutathione Peroxidase activity, were all in a direction favourable to B group (P < 0.05). These results were in line with the hypothesis, i. e. longevity can be enhanced to some extent by transplantation of placenta cells.

Effect of Chinese Medicine Xinmaitong on Blood Pressure in Spontaneously Hypertensive Rats.

OBJECTIVE: To investigate the effect of traditional Chinese antihypertensive compound Xinmaitong on blood pressure and vasoactive factors of vasoconstrictor endothelin-1 (ET-1) and vasodilator calcitonin gene related peptide (CGRP) in spontaneously hypertensive rats (SHRs) with early stage hypertension. METHODS: Twenty male SHRs were randomly divided into two groups: 10 for hypertensive control group and 10 for hypertensive treatment group. In addition, 10 Wistar rats were used as the normal control group without any intervention. SHRs of hypertensive treatment group were orally treated with Xinmaitong, while the hypertensive control group was treated with the normal saline (NS) for a total of eight weeks. The blood pressure in SHRs was examined before and after the end of the eight-week study. After treatment, the rats were killed and the blood samples were collected to measure plasma levels of ET-1 and CGRP by ELISA method, respectively. Meanwhile, the aorta rings were isolated for measuring the mRNA expression of ET-1 and CGRP by PCR. Moreover, the protein levels of ET-1 and CGRP were studied by immunohistochemical. RESULTS: Daily oral administration of Xinmaitong resulted in significant fall in the SHRs' blood pressure, including systolic and diastolic blood pressures (SBP and DBP), mean blood pressure (MBP), and pulse pressure (PP). The plasma ET-1 levels were reduced and CGRP increased. In parallel, the mRNA and protein expression of ET-1 were decreased, whereas the mRNA and protein expression of CGRP were enhanced in SHRs treated with Xinmaitong. CONCLUSION: The present study demonstrated for the first time that Xinmaitong leads to the fall in blood pressure of SHRs and that this antihypertensive effect is, at least in part, due to improvement of arterial tone.

Elevated GTP Cyclohydrolase I Pathway in Endothelial Progenitor Cells of Overweight Premenopausal Women.

Background/Aims. Sexual differences exist in endothelial progenitor cells (EPCs), and various cardiovascular risk factors are associated with the preservation of endothelial function in premenopausal women. However, it is unclear whether differences in endothelial function and circulating EPCs exist between overweight premenopausal women and age-matched men. Methods. We compared EPC counting and functions in normal-weight and overweight premenopausal women and men, evaluated endothelial function in each group, and detected the expression of the guanosine triphosphate cyclohydrolase I (GTPCH I) pathway. Results. The number of EPCs was lower in the male group than in the female group, regardless of normal-weight or overweight status, and there was no significant difference between the different weight groups among females or males. Endothelial function and EPC migration and proliferation were preserved in overweight premenopausal women compared with overweight men as were nitric oxide (NO) levels in plasma and secreted by EPCs. Endothelial function, the circulating EPC population, and NO levels were not different between normal-weight and overweight premenopausal women. Flow-mediated dilatation was significantly correlated with EPC function, plasma NO levels, and EPC-secreted NO. Conclusions. This investigation provides the first evidence for sex-based differences in EPC activity and endothelial function in overweight middle-aged individuals; these differences are associated with alterations in NO production and may partly occur through downregulation of the GTPCH I pathway. The present results provide new insights into the mechanism underlying the preserved endothelial function in overweight premenopausal women and may uncover a potential therapeutic target for endothelial repair in overweight population.

[Effects of dihydroartemisinin on radiosensitivity of Raji cells].

OBJECTIVE: To study the effects of dihydroartemisinin (DHA) on radiation sensitivity of Raji cells, and explore its mechanisms. METHODS: CCK8 was used to determine the effect of DHA on cell viability of Raji cells; apoptosis, intracellular reactive oxygen speies(ROS) and mitochondrial membrane potential of Raji cells were detected by flow cytometry; and the protein expressions of protein kinase B(AKT), phospho-rylated-protein kinase B(p-AKT), Bcl-2 and Bax were determined by Western blot. RESULTS: The cells were randomly divided into four groups:control group, DHA(5µmol/L DHA), irradiation(IR, 4 Gy), IR+DHA group (4 Gy IR+5 µmol/L DHA). Compared with the other three groups, cells in DHA+IR group exhibited lower mitochondrial membrane potential (P<0.01). While the intracellular ROS content and apoptosis rate of Raji cells in DHA+IR group were increased significantly(P<0.01). In addition, compared with the other three groups, there was no significant difference in the expression of AKT, but the phosphorylation of AKT protein were significantly inhibited and the expression of Bcl-2 protein was markedly decreased. However, the expressions of Bax and Cleaved-Caspase-3 protein were markedly increased. CONCLUSIONS: DHA might activate the mitochondrial apoptotic signal via inhibiting phosphoinositide 3-kinase (PI3K/AKT) pathway and increase oxidative stress to enhance the radiosensitivity of Raji cells.

Inhibition of Adipogenesis Is Involved in the Protective Effects of 1,25-Dihydroxy Vitamin D3 on the Radiation-Injured Bone Marrow Microenvironment in Mice.

To explore the protective effects of 1,25-dihydroxy vitamin D3 (1,25-(OH)2D3) on the bone marrow microenvironment in mice after irradiation and the underlying molecular mechanisms, a total of 150 7-wk-old male BALB/c mice were randomly divided into a normal group, an irradiation (IR) group and an irradiation+1,25-(OH)2D3 (IR+VD3) group. The mice in the IR+VD3 group were treated with 6.0 Gy 60Coγ rays, and 1,25-(OH)2D3 (dissolved in DMSO, 2.5 µg/kg) was administered once per day from 2 d before to 8 d after irradiation. Mice in the IR group were treated with the same dose of γ rays and an equal volume of DMSO. Subsequently, the body weights and the numbers of peripheral white blood cells (WBCs) were measured. Histological analysis of femur bone marrow was conducted to determine the proportion of adipose area as well. Finally, the expression of peroxisome proliferator-activated receptor-gamma (PPARγ) in bone marrow was detected by immunohistochemistry. After irradiation, the percentage of adipose area in the bone marrow was significantly increased, and the WBC number and body weight were markedly reduced. Compared with irradiation alone, the co-administration of 1,25-(OH)2D3 with irradiation markedly attenuated radiation-induced adipogenesis in bone marrow, resulted in fewer bone marrow stromal cells expressing PPARγ and enhanced the recovery of body weight and WBCs. These results indicate that 1,25-(OH)2D3 could accelerate the recovery of body weight and WBCs in irradiated mice and protect the bone marrow by inhibiting radiation-induced adipogenesis via the down-regulation of PPARγ expression.

[Salinomycin Enhances the Apoptosis of T-cell Acute Lymphoblastic Leukemia Cell Line Jurkat Cells Induced by Vincristine].

OBJECTIVE: This study was aimed to investigate the effect of salinomycin combined with vincristine on the proliferation and apoptosis of Jurkat cells and its possible mechanisms. METHODS: The proliferation of Jurkat cells was examined by CKK-8 assay. Flow cytometry was used to assess cellular apoptosis. Levels of BCL-2, caspase-3, and caspase- 8 were measured by Western blot. RESULTS: The salinomycin or vincristine, either alone or in combination, inhibited the proliferation of Jurkat cells in a dose-dependent manner. Salinomycin combined with vincristine produced more obveous inhibition of cell proliferation than either compound used alone (P<0.05). Western blot analysis showed that the combined use of Sal and VCR reduced the expression of BCL-2 protein, and increased expression of caspase 3 and caspase 8 protein, more significantly. Furthermore, combination of Sal and VCR synergistally promoted apoptosis of the Jurkat cells (P<0.05). CONCLUSION: The combination of salinomycin and vincristine synergistically inhibits proliferation and promotes apoptosis of T-cell acute lymphoblastic leukemia Jurkat cells.

Promoting the recovery of injured liver with poly (3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) scaffolds loaded with umbilical cord-derived mesenchymal stem cells.

Cell-based therapies are major focus of current research for treatment of liver diseases. In this study, mesenchymal stem cells were isolated from human umbilical cord Wharton's jelly (WJ-MSCs). Results confirmed that WJ-MSCs isolated in this study could express the typical MSC-specific markers and be induced to differentiate into adipocytes, osteoblasts, and chondrocytes. They could also be induced to differentiate into hepatocyte-like cells. Poly (3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (PHBVHHx) is a new member of polyhydroxyalkanoate family and biodegradable polyester produced by bacteria. PHBVHHx scaffolds showed much higher cell attachment and viability than the other polymers tested. PHBVHHx scaffolds loaded with WJ-MSCs were transplanted into liver-injured mice. Liver morphology improved after 30 days of transplantation and looked similar to normal liver. Concentrations of serum alanine aminotransferase and total bilirubin were significantly lower, and albumin was significantly higher on days 14 and 30 in the WJ-MSCs+scaffold group than in the carbon tetrachloride (CCl4) group. Hematoxylin-eosin staining showed that liver had similar structure of normal liver lobules and similar size and shape of normal hepatic cells, and Masson staining demonstrated that liver had less blue staining for collagen after 30 days of transplantation. Real-time reverse transcription-polymerase chain reaction (RT-PCR) showed that the expression of the bile duct epithelial cell gene CK-19 in mouse liver is significantly lower on days 14 and 30 in the WJ-MSCs+scaffold group than in the CCl4 group. Real-time RT-PCR, immunocytochemistry, and periodic acid-Schiff staining showed that WJ-MSCs in scaffolds differentiated into hepatocyte-like cells on days 14 and 30 in the WJ-MSCs+scaffold group. Real-time RT-PCR also demonstrated that WJ-MSCs in scaffolds expressed endothelial cell genes Flk-1, vWF, and VE-cadherin on days 14 and 30 in the WJ-MSCs+scaffold group, indicating that WJ-MSCs also differentiated into endothelial-like cells. These results demonstrated that PHBVHHx scaffolds loaded with WJ-MSCs significantly promoted the recovery of injured liver and could be further studied for liver tissue engineering.

Endothelin-1, an important mitogen of smooth muscle cells of spontaneously hypertensive rats.

OBJECTIVE: To study the features of vascular smooth muscle cell (VSMC) proliferation induced by endothelin-1 (ET-1). METHODS: VSMCs of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats were cultured and treated with ET-1. Basic fibroblast growth factor (bFGF) gene expression was measured using both Northern blot and an enzyme-linked immunoassay. RESULTS: ET-1 resulted in an increase in bFGF transcripts at 8 - 24 h; bFGF levels were significantly higher in VSMCs treated with ET-1 than in those not treated. However, VSMCs growth responses in SHR and WKY were different. Smooth muscle cells of SHR were hyper-responsive to ET-1. Maximal bFGF mRNA levels were elevated 3.5-fold at 4 h of stimulation in WKY and 8-fold at 8h in SHR4. Moreover, the proliferation of VSMCs induced by ET-1 was inhibited by antisense phosphorothioate oligodeoxynucleotides (10 micromol/L AS-bFGF) but not sense bFGF oligomers at the same concentrations, being reduced by 80% in SHR and 40% in WKY vs control, respectively. Furthermore, the effect of AS-bFGF oligomers on SHR SMC proliferation is significantly greater than on WKY SMC proliferation. CONCLUSION: ET-1 may be required for exaggerated vascular growth responses in SHR and bFGF may be involved.

PHBVHHx scaffolds loaded with umbilical cord-derived mesenchymal stem cells or hepatocyte-like cells differentiated from these cells for liver tissue engineering.

More attention has recently been focused on the treatment of various kinds of hepatic diseases based on cell-based therapies. In this study, mesenchymal stem cells were isolated from umbilical cord (UC-MSCs). Results confirmed that UC-MSCs could differentiate into adipocytes, osteoblasts and hepatocytes. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (PHBVHHx), a new member of polyhydroxyalkanoate (PHA) family, was produced by bacteria. Liver-injured mouse model was established by CCl4 injection. PHBVHHx scaffolds were transplanted into the liver-injured mice. Liver morphology on day 28 post-transplantation of scaffolds loaded with UC-MSCs or hepatocyte-like cells differentiated from UC-MSCs significantly improved and looked similar to the normal liver. Concentrations of albumin (ALB) significantly increased, and total bilirubin (TB) and alanine axminotransferase (ALT) significantly decreased on days 14 and 28 post-transplantation of scaffolds loaded with UC-MSCs or differentiated UC-MSCs. HE staining showed that on day 28 post-transplantation of scaffolds loaded with UC-MSCs or differentiated UC-MSCs, livers had similar tissue structure of normal livers. Masson staining showed that on day 28 post-transplantation of scaffolds loaded with UC-MSCs or differentiated UC-MSCs, livers had less blue staining for collagen deposition compared with the others. These results demonstrated that PHBVHHx scaffolds loaded with UC-MSCs or differentiated UC-MSCs had the similar effect on injured livers and significantly promoted the recovery of injured livers.

Enhancement of skin wound healing with decellularized scaffolds loaded with hyaluronic acid and epidermal growth factor.

Current therapy for skin wound healing still relies on skin transplantation. Many studies were done to try to find out ways to replace skin transplantation, but there is still no effective alternative therapy. In this study, decellularized scaffolds were prepared from pig peritoneum by a series of physical and chemical treatments, and scaffolds loaded with hyaluronic acid (HA) and epidermal growth factor (EGF) were tested for their effect on wound healing. MTT assay showed that EGF increased NIH3T3 cell viability and confirmed that EGF used in this study was biologically active in vitro. Scanning electron microscope (SEM) showed that HA stably attached to scaffolds even after soaking in PBS for 48 h. ELISA assay showed that HA increased the adsorption of EGF to scaffolds and sustained the release of EGF from scaffolds. Animal study showed that the wounds covered with scaffolds containing HA and EGF recovered best among all 4 groups and had wound healing rates of 49.86%, 70.94% and 87.41% respectively for days 10, 15 and 20 post-surgery compared to scaffolds alone with wound healing rates of 29.26%, 42.80% and 70.14%. In addition, the wounds covered with scaffolds containing EGF alone were smaller than no EGF scaffolds on days 10, 15 and 20 post-surgery. Hematoxylin-Eosin (HE) staining confirmed these results by showing that on days 10, 15 and 20 post-surgery, the thicker epidermis and dermis layers were observed in the wounds covered with scaffolds containing HA and EGF than scaffolds alone. In addition, the thicker epidermis and dermis layers were also observed in the wounds covered with scaffolds containing EGF than scaffolds alone. Skin appendages were observed on day 20 only in the wound covered with scaffolds containing HA and EGF. These results demonstrate that the scaffolds containing HA and EGF can enhance wound healing.