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1.
Adv Exp Med Biol ; 1164: 91-99, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31576542

RESUMO

Prostate cancer is the most common male cancer in the USA and the second leading cause of male cancer death in the USA. African American men have higher incidence and mortality rate from prostate cancer compared to Caucasian men in North America, indicating the prostate cancer is a major public health problem in this population. Studies of prostate cancer have been hampered by various factors including (1) restricted access to tissues, (2) difficulties in propagating premalignant lesions and primary prostate tumors in vitro, and (3) limited availability of prostate cell lines for in vitro studies. There is no commercially available pair of non-malignant and tumor cells derived from the same prostate cancer patient. Primary prostate epithelial cells grow for a finite life span and then senesce. Immortalization is defined by continuous growth of otherwise senescing cells and is believed to represent an early stage in tumor progression. To examine these early stages, we have developed in vitro models of prostate epithelial cell immortalization. Generation of human primary epithelial (HPE) cells has been achieved using the serum-free keratinocyte growth medium. Retrovirus containing human telomerase reverse transcriptase (hTERT) was also used for the generation of primary non-malignant and malignant tumor cells. In addition, we have established the first immortalized cell lines of a pair of non-malignant and malignant tumors derived from an African American prostate cancer patient. Interestingly, we have found that the Rock inhibitor and feeder cells induced the conditioned reprogramming (CR) of epithelial cells-normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro, without transduction of viral or cellular genes. More recently, using CR, we have established normal and tumor cultures respectively from a patient prostatectomy. These CR cells grow indefinitely in vitro and retain stable karyology. The tumor-derived CR cells produced tumors in SCID mice. The use of novel pair of non-malignant and malignant tumor cells derived from the same patient provides a unique in vitro model for studies of early prostate cancer and for testing preventive and therapeutic regimens.


Assuntos
Linhagem Celular , Células Epiteliais , Animais , Células Epiteliais/citologia , Humanos , Masculino , Camundongos , Camundongos SCID , Neoplasias da Próstata
2.
Adv Exp Med Biol ; 1164: 101-108, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31576543

RESUMO

Prostate cancer is the most frequently diagnosed solid malignancy in men. Epidemiological studies have shown African-American men to be at higher risk for developing prostate cancer and experience higher death as compared to other ethnic groups. Establishment of prostate cancer cell lines paired with normal cells derived from the same patient is a fundamental breakthrough in cell culture technology and provides a resource to improve our understanding of cancer development and pertinent molecular events. Previous studies have demonstrated that conditional reprogramming (CR) allows the establishment and propagation of patient-derived normal and tumor epithelial cell cultures from a variety of tissue types. Here, we report a new AA prostate cell model, paired normal and cancer epithelial cells from the same patient. "Tumor" cell culture AA-103A was derived from malignant prostate tissues, and "normal" cell culture AA-103B was derived from non-malignant prostate tissues from the prostatectomy specimen of an African-American male. These paired cell cultures have been propagated under CRC conditions to permit direct comparison of the molecular and genetic profiles of the normal epithelium and adenocarcinoma cells for comparison of biomarkers, enabling patient-specific pathological analysis, and molecular and cellular characterization. STR confirmed human origin albeit no karyotypic abnormalities in the two cell lines. Further quantitative PCR analyses demonstrated characteristic markers, including the high level of basal cell marker, the keratin 5 (KRT5) in normal cells and of luminal marker, the androgen receptor (AR) as well as the programmed death-ligand 1 (PD-L1) in tumor cells. Although 3-D sphere formation was observed, the AA-103A of tumor cells did not generate tumors in vivo. We report these paired primary epithelial cultures under CRC growth as a potentially useful tool for studies to understand molecular mechanisms underlying health disparities in prostate cancer.


Assuntos
Afro-Americanos , Linhagem Celular Tumoral , Disparidades nos Níveis de Saúde , Neoplasias da Próstata , Linhagem Celular , Células Epiteliais/citologia , Humanos , Masculino
3.
Adv Exp Med Biol ; 1164: 109-118, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31576544

RESUMO

Choosing an appropriate cell model(s) is the first decision to be made before starting a new project or programme of study. Here, we address the rationale that can be behind this decision and we summarize the current cell models that are used to study prostate cancer. Researchers face the challenge of choosing a model that recapitulates the complexity and heterogeneity of prostate cancer. The use of primary prostate epithelial cells cultured from patient tissue is discussed, and the necessity for close clinical-academic collaboration in order to do this is highlighted. Finally, a novel quantitative phase imaging technique is described, along with the potential for cell characterization to not only include gene expression and protein markers but also morphological features, cell behaviour and kinetic activity.


Assuntos
Linhagem Celular Tumoral , Células Epiteliais , Neoplasias da Próstata , Linhagem Celular , Células Epiteliais/citologia , Humanos , Masculino
4.
Phys Rev Lett ; 123(5): 058101, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31491312

RESUMO

We study the influence of cell-level mechanical heterogeneity in epithelial tissues using a vertex-based model. Heterogeneity is introduced into the cell shape index (p_{0}) that tunes the stiffness at a single-cell level. The addition of heterogeneity can always enhance the mechanical rigidity of the epithelial layer by increasing its shear modulus, hence making it more rigid. There is an excellent scaling collapse of our data as a function of a single scaling variable f_{r}, which accounts for the overall fraction of rigid cells. We identify a universal threshold f_{r}^{*} that demarcates fluid versus solid tissues. Furthermore, this rigidity onset is far below the contact percolation threshold of rigid cells. These results give rise to a separation of rigidity and contact percolation processes that leads to distinct types of solid states. We also investigate the influence of heterogeneity on tumor invasion dynamics. There is an overall impedance of invasion as the tissue becomes more rigid. Invasion can also occur in an intermediate heterogeneous solid state that is characterized by significant spatial-temporal intermittency.


Assuntos
Células Epiteliais/citologia , Modelos Biológicos , Fenômenos Biomecânicos , Forma Celular/fisiologia , Epitélio/fisiologia
5.
Pestic Biochem Physiol ; 159: 144-153, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400776

RESUMO

Ivermectin is a pesticide that has been used for over 30 years in livestock. Although there are a number of studies on the therapeutic potential of ivermectin, little is known about the effects of the drug during the early stage of pregnancy. In this study, we investigated the detrimental effects of ivermectin on porcine trophectoderm (pTr) and uterine luminal epithelial (pLE) cells. Ivermectin not only inhibited the proliferation of both cells via the regulation of cell cycle-associated genes, but also induced apoptosis in pTr and pLE cells. We also verified its effect on mitochondrial dysfunction as shown by loss of mitochondrial membrane potential, mitochondrial Ca2+ overload, and reactive oxygen species (ROS) generation in pTr and pLE cells. As a mechanistic approach, we evaluated ivermectin-mediated cell signaling interactions including PI3K, AKT and MAPK pathways. Overall, our results suggest that constant exposure to and accumulation of ivermectin may cause abnormal fetal morphogenesis and placentation during the early stages of pregnancy. Our results may further provide a comprehensive understanding of the detrimental effects of ivermectin during pregnancy and will contribute to the establishment of a complete safety profile for ivermectin and its association with environmental pollution and public health in humans and livestock.


Assuntos
Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Ivermectina/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Útero/citologia , Animais , Proliferação de Células/efeitos dos fármacos , Feminino , Suínos , Trofoblastos/citologia , Trofoblastos/efeitos dos fármacos
6.
Life Sci ; 234: 116735, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31394124

RESUMO

AIMS: The present study was to investigate the protective effects of Zn supplementation in OTA-induced apoptosis of Madin-Darby canine kidney (MDCK) epithelial cells and explore the potential mechanisms. Aiming to provides a new insight into the treatment strategy of OTA-induced nephrotoxicity by nutritional regulation. MAIN METHODS: Initially, through MTT and LDH assay revealed that Zn supplementation significantly suppressed OTA-induced cytotoxicity in MDCK cells. Then, the production of reactive oxygen species (ROS) was detected by using a DCFH-DA assay. Annexin V-FITC/PI, Hoechst 33258 staining and Flow cytometry were used to detect the apoptosis. The expressions of apoptosis-related molecules were determined by RT-PCR, Western blotting. Interestingly, OTA treatment slightly increased the levels of Metallothionein-1 (MT-1) and Metallothionein-2 (MT-2) by using RT-PCR, Western blotting assay; while Zn supplementation further improved the increase of MT-1 and MT-2 induced by OTA. However, the inhibitive effects of Zn supplementation were significantly blocked after double knockdown of MT-1 and MT-2 by using Small Interfering RNA (siRNA) Transfection method. KEY FINDINGS: Our study provides supportive data for the potential roles of Zn in reducing OTA-induced oxidative stress and apoptosis in MDCK cells. SIGNIFICANCE: Zn is one of the key structural components of many proteins, which plays an important role in several physiological processes such as cell survival and apoptosis. This metal is expected to contribute to the conservative and adjuvant treatment of kidney disease and should therefore be investigated further.


Assuntos
Apoptose/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Metalotioneína/genética , Ocratoxinas/toxicidade , Substâncias Protetoras/farmacologia , Zinco/farmacologia , Animais , Citoproteção/efeitos dos fármacos , Cães , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Madin Darby de Rim Canino , Estresse Oxidativo/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
7.
Vet Parasitol ; 273: 67-70, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31442896

RESUMO

Suckling calves are prone to Cryptosporidium infection. The variable degree of clinical disease is influenced by keeping conditions and immune status of the host, but diversity of isolate virulence may also contribute. The aim of the current study was to evaluate the cytopathogenic effects of 26 C. parvum field isolates by using a MTT assay in HCT-8 cell monolayers. Cell viability of monolayers inoculated with oocysts of the field isolates varied considerably with values of 17.7% (± 5.1%) to 99.5% (± 7.1%). A standard deviation of 18.6% was detected for cell viability of the in house reference strain, which were tested alongside in every assay. Field isolates were grouped in three categories of cytopathogenicity. Probably the length of storage has an effect on the level of the cell destruction category detected post infection in vitro. The applied tool may help to better understand the variable course of cryptosporidiosis in the field.


Assuntos
Doenças dos Bovinos/parasitologia , Criptosporidiose/parasitologia , Cryptosporidium parvum/patogenicidade , Animais , Bovinos , Linhagem Celular , Sobrevivência Celular , Células Epiteliais/citologia , Células Epiteliais/parasitologia , Interações Hospedeiro-Parasita/imunologia , Humanos , Virulência/imunologia
8.
Adv Exp Med Biol ; 1165: 101-116, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31399963

RESUMO

Kidney transplantation is a life-change measurement for the patients of end-stage renal disease (ESRD). However, the renal allograft cannot avoid initial acute kidney injury (AKI) and subsequent chronic allograft dysfunction (CAD), gradually develops fibrosis and eventually loses function. It is imperative to disclose the pathogenesis of AKI and CAD in order to facilitate interventions. We have studied the involvement of immunity, inflammation, and apoptosis in ischemia-reperfusion injury (IRI) and/or immunosuppressant induced AKI models, with associated chronic damage. Our research mainly focused on tubular epithelial cells (TECs) that are passive victims and also active participators in injury and mediate following repair or fibrosis. Targeting not only fibroblasts/myofibroblasts, but also TECs, might be a fundamental strategy to prevent and treat renal fibrosis. We have also evaluated the potential application of siRNA targeting caspase-3 and tissue protective erythropoietin derivatives, HBSP and CHBP, aiming to treat AKI and prevent CAD. Significant improvements have been obtained, but timely diagnosis and precise therapy of AKI and prevention of CAD progressing to ESRD are still very challenging. Modern technologies such as microarray and sequencing analysis have been used to identify biomarkers and potentially facilitate individual cell target treatment for transplant patients.


Assuntos
Lesão Renal Aguda , Transplante de Rim/efeitos adversos , Rim/patologia , Traumatismo por Reperfusão , Aloenxertos , Apoptose , Células Epiteliais/citologia , Fibrose , Humanos , Imunidade , Inflamação , Túbulos Renais/citologia
9.
Chem Commun (Camb) ; 55(57): 8329-8332, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31257378

RESUMO

Cell imaging heavily depends on fluorescent labels typically incompatible with Raman microscopy. The europium(iii) complex based on dipicolinic acid (DPA) presented here is an exception from this rule. Although its luminescence bands are very narrow, their intensity is comparable to the background Raman bands. This makes it complementary to less luminous compounds referred to as Raman tags. Through several examples we show that the complex provides a morphological context in otherwise unstained cells, thus acting as a spectral-counterstaining agent.


Assuntos
Complexos de Coordenação/química , Análise Espectral Raman , Candida albicans/metabolismo , Parede Celular/química , Células Epiteliais/química , Células Epiteliais/citologia , Európio/química , Células HeLa , Humanos , Microscopia de Fluorescência , Ácidos Picolínicos/química
10.
J Agric Food Chem ; 67(31): 8668-8676, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31271028

RESUMO

This study investigated the effect of Chlorella vulgaris (C. vulgaris) on genotoxicity, cytotoxicity, and apoptosis in Caco-2 and HT-29 cells. C. vulgaris significantly induced DNA damage in both cell lines at a concentration of 200 µg dry matter/mL (comet tail intensity CTI: 24.6 ± 4.7% for Caco-2, 16.6 ± 0.9% for HT-29). The application of processing (sonication, ball-milling) did not affect the genotoxicity negatively and lowered the lipid peroxidation in C. vulgaris preparations. C. vulgaris-induced intracellular formation of reactive oxygen species in human cell lines and might be responsible for the genotoxic effect. A solid fraction mainly triggered the observed DNA damage (CTI: 41.5 ± 1.9%), whereas a hydrophilic (CTI: 7.9 ± 1.7%) and lipophilic (CTI: 10.2 ± 2.1%) fraction revealed a significantly lower tail intensity. C. vulgaris significantly induced DNA damage in both cell lines possibly through intracellular formation of reactive oxygen species; however, it was repaired after a 2 h recovery time or was even avoided at lower concentrations. In addition, none of the preparations indicated an adverse effect on cell proliferation or revealed apoptotic activity.


Assuntos
Chlorella vulgaris/química , Dano ao DNA/efeitos dos fármacos , Células Epiteliais/citologia , Mutagênicos/toxicidade , Extratos Vegetais/toxicidade , Apoptose/efeitos dos fármacos , Processos Autotróficos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Chlorella vulgaris/crescimento & desenvolvimento , Chlorella vulgaris/efeitos da radiação , Ensaio Cometa , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Luz , Peroxidação de Lipídeos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
11.
Altern Lab Anim ; 47(2): 63-70, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31333044

RESUMO

Quantitative real-time polymerase chain reaction is a widely used technique that relies on reference genes for the normalisation of gene expression. These reference genes are constitutively expressed and must remain stable across all samples and treatments. Stability of housekeeping genes may vary and must be optimised for a specific tissue, sample or cell line. Here we present a study screening for possible reference gene candidates, eef1a1, rpl8, sub1.L, clta, H4 and odc1, in the Xenopus laevis (A6) kidney cell line. Quantification cycle results were analysed using geNorm to calculate the average expression stability and the coefficient of variation (CV) for each candidate reference gene. All of the tested genes met the guidelines for stable reference genes, namely an average expression stability of < 0.5 and a CV value of < 0.2, with eef1a1 > sub1.L > rpl8 > clta > odc1 > H4. By using pairwise variation analysis, the optimal number of reference targets was determined to be 2. As such, we report that the reference genes eef1a1 and sub1.L should be used to achieve optimal normalisation in A6 cells.


Assuntos
Anfíbios , Alternativas aos Testes com Animais , Células Epiteliais , Reação em Cadeia da Polimerase em Tempo Real , Alternativas aos Testes com Animais/métodos , Animais , Linhagem Celular , Células Epiteliais/citologia , Expressão Gênica
12.
Cell Physiol Biochem ; 53(1): 49-61, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31169991

RESUMO

BACKGROUND/AIMS: The most prevalent infectious disease, chronic periodontitis which leads to alveolar bone destruction and subsequent tooth loss, develops due to proinflammatory cytokine production induced by periodontopathic bacteria. Chronic obstructive pulmonary disease (COPD), a non-infectious disease, is the third leading cause of death globally. This condition exacerbates frequently, and which is attributable to proinflammatory cytokine production induced by infection by respiratory microorganisms such as Streptococcus pneumoniae. Although a positive association has recently been revealed between chronic periodontitis and COPD, how periodontitis contributes to the pathogenesis of COPD remains unclear. Therefore, we hypothesized that some periodontopathic bacteria are involved in the exacerbation of COPD through the induction of proinflammatory cytokine production by respiratory epithelial cells. In this connection, COPD develops in the airways; however, because most periodontopathic bacteria are anaerobic, they are unlikely to exhibit stable virulence in the lower respiratory organs in humans. Hence, we aimed to elucidate whether exposure to heat-inactivated periodontopathic bacteria induces proinflammatory cytokine production by several human respiratory epithelial cell lines and in the lower respiratory organs and serum in mice. METHODS: Real-time polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) were used to investigate in vitro induction by heat-inactivated periodontopathic bacteria and S. pneumoniae for mRNA expression and protein production of interleukin (IL)-8 and IL-6 by human respiratory epithelial cell lines. ELISA was also used to determine in vivo induction of cytokine production in the lower respiratory organs and serum of intratracheally heat-inactivated Fusobacterium nucleatum-inoculated mice. RESULTS: Some, but not all, periodontopathic bacteria, especially F. nucleatum, strongly induced IL-8 and IL-6 production by BEAS-2B bronchial epithelial cells. In addition, F. nucleatum induced IL-8 production by A549 alveolar epithelial cells as well as IL-8 and IL-6 production by Detroit 562 pharyngeal epithelial cells. Furthermore, F. nucleatum induced considerably higher cytokine production than S. pneumoniae. This was also observed in the entire lower respiratory organs and serum in mice. CONCLUSION: Exposure to increased number of F. nucleatum potentially induces proinflammatory cytokine production by human bronchial and pharyngeal epithelial cells, which may trigger exacerbation of COPD.


Assuntos
Fusobacterium nucleatum/patogenicidade , Interleucina-6/metabolismo , Sistema Respiratório/microbiologia , Animais , Brônquios/citologia , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Humanos , Interleucina-6/sangue , Interleucina-6/genética , Interleucina-8/sangue , Interleucina-8/genética , Interleucina-8/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sistema Respiratório/metabolismo , Streptococcus pneumoniae/patogenicidade
13.
Gene ; 710: 186-192, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31175924

RESUMO

EMT (Epithelial-Mesenchymal Transition) is one of the factors in the pathogenesis of adenomyosis. FMNL2 induced invasion of cancer cell through promoting EMT, but it is unclear the role of FMNL2 in the adenomyosis. By IHC staining, we found the expression level of FMNL2 was significantly higher in the ectopic endometrial stromal cells from women with adenomyosis when compared with normal endometrial stromal cells. Knockdown of FMNL2 inhibited the invasion and migration of ectopic endometrial stromal cells and promoted the protein levels of E-cadherin and Vimentin. Meanwhile, inhibition of FMNL2 could induce the cell membrane localization of E-cadherin. Our findings reveal that the aberrant activation of FMNL2 promotes the pathogenesis of adenomyosis through inducing the EMT process. On the contrary, inhibition of FMNL2 promotes the transition of ectopic endometrial stromal cells to epithelial cells in adenomyosis through a MET-like process.


Assuntos
Adenomiose/metabolismo , Células Epiteliais/metabolismo , Proteínas/metabolismo , Células Estromais/metabolismo , Regulação para Cima , Adenomiose/genética , Adulto , Antígenos CD/metabolismo , Caderinas/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Células Epiteliais/citologia , Transição Epitelial-Mesenquimal , Feminino , Técnicas de Silenciamento de Genes , Humanos , Pessoa de Meia-Idade , Proteínas/genética , Transdução de Sinais , Células Estromais/citologia , Vimentina/metabolismo
14.
J Biol Regul Homeost Agents ; 33(3): 675-685, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31189490

RESUMO

Endometrial cells undergo very specific changes associated with reproductive processes. Cells prepare for embryo development by increasing their volume. Then, if fertilization fails, endometrial cells are liable for apoptosis, preparing new cells that are ready for subsequent processes related to the possibility of embryo implantation and the development of pregnancy. PTX3 and TNFAIP6 are absent or reduced in cultured COCs, resulting in a functional change in COC in vitro. In this work, we want to check how PTX3, HAS2 and TNFAIP6 behave in luminal epithelium primary cell culture. Cells obtained during slaughter from porcine specimens were cultured primarily in vitro for 7 days. Their proliferation patterns were then analysed using RTCA, with the expression of genes of interest evaluated with the use of immunofluorescence and RT-qPCR. The results of these changes in the expression of the genes of interest were analysed on each of the seven days of the porcine luminal primary cell culture. Our study showed the increased level of PTX3, HAS2 and TN¬FAIP6 expression at the same hours of primary culture. Rt-qPCR showed a higher level of expression of the PTX3 gene in the first 72 h, at the end of the lag phase (in the phase of stasis in which the cells adapt to the new environment and often die). In contrast, TNFAIP6 expression increases about 96 hours when the cells are in the full log phase (logarithmic phase growth) and continue this trend in the plateau phase. We did not observe such drastic changes in the HAS2 expression pattern, which leads us to hypothesize that PTX3 and TNFAIP6 are designed to maintain a constant level of HAS2 in the cell throughout its lifetime. The obtained results could become a point of reference for further in vivo and clinical research.


Assuntos
Proteína C-Reativa/genética , Moléculas de Adesão Celular/genética , Endométrio/citologia , Células Epiteliais/citologia , Hialuronan Sintases/genética , Componente Amiloide P Sérico/genética , Animais , Proliferação de Células , Feminino , Cultura Primária de Células , Suínos
15.
Klin Lab Diagn ; 64(6): 348-350, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31200406

RESUMO

Analysis of the cellular composition of the eyeball conjunctival prints and evaluation of the informative of the cytomorphological method of research in the diagnosis of dry eye syndrome (DES). Cytological examination using impression cytology was performed in 72 patients: 60 patients with DES and 12 without eye disease (control group). The main component of cytological sample in patients with DES are (a) squamous epithelium (hyperkeratocytes); (b) groups and clusters of flattened epithelial cells; (c) a small amount (up to five in the preparation) of goblet cells. Impression cytology method improves the accuracy of diagnosis of DES.


Assuntos
Túnica Conjuntiva/citologia , Citodiagnóstico , Síndromes do Olho Seco/diagnóstico , Estudos de Casos e Controles , Células Epiteliais/citologia , Humanos
16.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(4): 313-319, 2019 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-31167690

RESUMO

Objective To explore the mechanism of transdifferentiation of human renal tubular epithelial cells induced by high glucose based on reactive oxygen species (ROS)/NF-κB signaling pathway. Methods HK-2 normal human proximal tubular epithelial cells were randomly divided into blank control group, osmotic pressure control group, high glucose group and pyrrolidine dithiocarbamate (PDTC) group. Cell morphology was observed by phase contrast microscope. Cell viability was measured by MTT assay. Flow cytometry was used to detect intracellular ROS content. Malondialdehyde (MDA) content and superoxide dismutase activity were tested by ELISA. Western blot analysis was used to examine the protein levels of NF-κBp65, phosphorylated IκBα (p-IκBα) and IKKα, monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1). The expressions of NF-κBp65, epithelial cadherin (E-cadherin) and alpha smooth muscle actin (α-SMA) were assessed by immunocytochemistry. Results In high glucose group, the cells became spindle-shaped or irregular, with radial edges, enlarged intercellular space, decreased refractive index and irregular arrangement. At the same time, the cell activity decreased with the prolongation of treatment time, and the cell activity in PDTC group was higher than that in high glucose group. Compared with the blank control group, the content of ROS and MDA increased and the activity of SOD decreased in the high glucose group, while the content of ROS and MDA decreased and the activity of SOD increased in the PDTC group compared with the high glucose group. Compared with the blank group, the protein levels of NF-κBp65, p-IκBα, IKKα, MCP-1 and ICAM-1 increased in the high glucose group, while the protein levels above decreased in the PDTC group compared with the high glucose group. Compared with the blank group, the proportion of positive cells of NF-κBp65 and α-SMA increased and the proportion of positive cells of E-cadherin decreased in the high glucose group. Compared with the high glucose group, the proportion of positive cells of NF-κBp65 and α-SMA decreased and the proportion of positive cells of E-cadherin increased in the PDTC group. Conclusion High glucose can induce epithelial-mesenchymal transition in HK-2 cells. Activation of NF-κB signaling pathway mediated by ROS participates in the above process, which can be blocked by PDTC.


Assuntos
Transdiferenciação Celular , Células Epiteliais/citologia , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Linhagem Celular , Meios de Cultura , Glucose , Humanos , Túbulos Renais/citologia , Pirrolidinas , Tiocarbamatos
17.
Eur J Histochem ; 63(2)2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31189296

RESUMO

The Kölliker's organ is a transient epithelial structure during cochlea development that gradually degenerates and disappears at postnatal 12-14 days (P12-14). While apoptosis has been shown to play an essential role in the degeneration of the Kölliker's organ, the role of another programmed cell death, autophagy, remains unclear. In our study, autophagy markers including microtubule associated protein light chain 3-II (LC3-II), sequestosome 1 (SQSTM1/p62) and Beclin1 were detected in the supporting cells of the Kölliker's organ through immunohistochemistry staining. In addition, Western blot and real-time PCR revealed a gradually decreased expression of LC3-II and an increased expression of p62 during early postnatal development. Compared to apoptosis markers that peaks between P7 and P10, autophagy flux peaked earlier at P1 and decreased from P1 to P14. By transmission electron microscopy, we observed representative autophagosome and autolysosome that packaged various organelles in the supporting cells of the Kölliker's organ. During the degeneration, these organelles were digested via autophagy well ahead of the cellular apoptosis. These results suggest that autophagy plays an important role in transition and degeneration of the Kölliker's organ prior to apoptosis during the early postnatal development.


Assuntos
Apoptose/fisiologia , Autofagia/fisiologia , Cóclea/embriologia , Cóclea/metabolismo , Animais , Anticorpos/imunologia , Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Proteína Beclina-1/metabolismo , Caspase 3/genética , Caspase 3/imunologia , Caspase 3/metabolismo , Cóclea/citologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Imuno-Histoquímica/métodos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/imunologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/imunologia , Proteína Sequestossoma-1/metabolismo , Fatores de Tempo
18.
Cell Biochem Funct ; 37(4): 290-300, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31211440

RESUMO

Vascular endothelial growth factor C (VEGF-C) is an angiogenic and lymphangiogenic growth factor. Recent research has revealed the role for VEGF-C in regulating autophagy by interacting with a nontyrosine kinase receptor, neuropilin-2 (NRP-2). However, whether VEGF-C participates in regulating cell survival and autophagy in renal proximal tubular cells is unknown. To address this question, we employed a cell modal of serum deprivation to verify the role of VEGF-C and its receptor NRP-2 in regulating cell survival and autophagy in NRK52E cell lines. The results show that VEGF-C rescued the loss of cell viability induced by serum deprivation in a concentration-dependent manner. Furthermore, endogenous VEGF-C was knocked down in NRK52E cells by using specific small-interfering RNAs (siRNA), cells were more sensitive to serum deprivation-induced cell death. A similar increase in cell death rate was observed following NRP-2 depletion in serum-starved NRK52E cells. Autophagy activity in serum-starved NRK52E cells was confirmed by western blot analysis of microtubule-associated protein-1 chain 3 (LC3), immunofluorescence staining of endogenous LC3, and the formation of autophagosomes by electron microscopy. VEGF-C or NRP-2 depletion further increased LC3 expression induced by serum deprivation, suggesting that VEGF-C and NRP-2 were involved in controlling autophagy in NRK52E cells. We further performed autophagic flux experiments to identify that VEGF-C promotes the activation of autophagy in serum-starved NRK52E cells. Together, these results suggest for the first time that VEGF-C/NRP-2 axis promotes survival and autophagy in NRK52E cells under serum deprivation condition. SIGNIFICANCE OF THE STUDY: More researchers had focused on the regulation of autophagy in kidney disease. The effect of VEGF-C on cell death and autophagy in renal epithelial cells has not been examined. We first identified the VEGF-C as a regulator of cell survival and autophagy in NRK52E cell lines. And VEGF-C/NRP-2 may mediate autophagy by regulating the phosphorylation of 4EBP1 and P70S6K. VEGF-C treatment may be identified as a therapeutic target in renal injury repair due to its capacity to promote tubular cell survival in the future.


Assuntos
Autofagia , Células Epiteliais/citologia , Túbulos Renais/citologia , Neuropilina-2/metabolismo , Soro , Fator C de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Ratos , Transdução de Sinais
19.
Phys Chem Chem Phys ; 21(24): 13099-13108, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31169276

RESUMO

With the aim of designing an efficient procedure for producing biocompatible drug delivery systems based on nanoparticle carriers for in situ controlled antibiotic release, we have defined a novel computational approach resorting to a reactive force field capable of realistically describing hybrid systems. The modeling procedure was focused on well-known components, namely gold nanoparticles, citrate, chitosan and gentamicin, and the experiments tuned on purpose. On the one hand, gold nanoparticles were synthesized, fuctionalized with chitosan, loaded with gentamicin and characterized by means of transmission electron microscopy (TEM), scanning electron microscopy (SEM), dynamic light scattering (DLS), UV-visible (UV-vis) spectroscopy, and Fourier transform infrared spectroscopy (FTIR). On the other hand, an effective model of a functionalized gold nanoparticle was created and its structure and dynamics were explored by classical reactive molecular dynamics simulations in solution based on the ReaxFF atomistic description. The structure, dynamics and drug release were reproduced realistically disclosing the motion of all the molecular components, their adsorption on the metal support, desorption, intermolecular interactions and self-assembly. The system size was very close to the experimental conditions and all the calculations could efficiently identify the most probable binding modes, the locations of the adsorbed molecules, the characteristic arrangements of the chains and the effects due to the surrounding environment. The role played by the substrate and water molecules in the releasing process was described in detail. In line with the literature it was found that the antibiotic activity was preserved and the drug release from the carrier could be tuned by changing the chitosan/getamicin weight ratio and the deposition pattern of the adsorbed layers.


Assuntos
Antibacterianos/química , Quitosana/química , Portadores de Fármacos/química , Gentamicinas/química , Ouro/química , Nanopartículas Metálicas/química , Simulação de Dinâmica Molecular , Antibacterianos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Liberação Controlada de Fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Gentamicinas/farmacologia , Humanos , Álcool de Polivinil/química , Staphylococcus aureus/efeitos dos fármacos
20.
Sheng Wu Gong Cheng Xue Bao ; 35(5): 892-900, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31223007

RESUMO

To investigate the effect of miR-331-3p on the proliferation of porcine renal epithelial cells (PK15) and its mechanism, the pcDNA 3.1(+) overexpression vector of miRNA-331-3p (pcDNA 3.1(+)-miR-331-3p) was constructed. PK15 cells were divided into four groups, including experimental group, experimental control group, inhibitor group and inhibitor control group. Experimental group and experimental control group were transfected with pcDNA 3.1(+)-miR-331-3p and pcDNA 3.1(+), respectively. Inhibitor group and inhibitor control group were transfected with miR-331-3p inhibitor and miR-331-3p negative control (miR-331-3p NC), respectively. Above all, CCK-8 reagent was used to plot the cell proliferation curve and Propidium (PI) staining was used to detect the proportion of cell stages. Secondly, its expression change were detected by quantitative real-time PCR that included the growth inhibitory protein family member 5 (ING5), cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 3 (CDK3), cyclin-dependent kinase 4 (CDK4), Cyclin B and cyclin-dependent kinase inhibitor 1A (CDKN1A). The results showed that the expression of miRNA-331-3p was significantly increased in the experimental group. The cell proliferation curve showed that the number of cells in experimental group was significantly higher than that in experimental control group or inhibitor control group at 48 h and 72 h (P<0.05). Simultaneously, Inhibitor group was significantly lower than experimental control group or inhibitor control group in the number of cells at 48 h and 72 h (P<0.05), but there was no significant difference between the experimental group and the control group. Compared with the experimental control group, the proportion of cells of experimental group in G0/G1 phase decreased, the proportion of S phase and G2/M phase increased, and the inhibitor control group showed the opposite trend. Simultaneously, the expression levels of CDK2, CDK3, CDK4 and Cyclin B genes in the experimental group were significantly increased, while ING5 and CDKN1A genes inhibiting proliferation showed a significant downward trend. These results demonstrate that the miR-331-3p overexpression vector was successfully constructed, and miR-331-3p has the ability to promote the proliferation of PK15 cells. The study lays a solid foundation for further research for its role in pig growth and development.


Assuntos
Proliferação de Células , Vetores Genéticos , MicroRNAs , Animais , Linhagem Celular , Proliferação de Células/genética , Células Epiteliais/citologia , MicroRNAs/genética , Suínos
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