OBJECTIVE: To determine the effect of transforming growth factor-β1 (TGF-β1) on the expression of telomerase in hepatic stellate cells (HSCs) in rats and the role of TGF-β1 in the development of liver fibrosis. METHODS: Primary HSCs were isolated from normal rats by density gradient separation and divided into 2 groups for culturing. The morphology of HSCs was identified by the inverted fluorescence microscope. The purity of HSCs was identified by immunohistological expression and fluorescence analysis. One group of HSCs was treated with different concentrations (0, 0.1, 1, and 10 ng/mL) of TGF-β1 for 24 h, while the other group was treated with 1 ng/mL TGF-β1 and cultured for 3, 6, and 9 days. The mRNA expression of telomerase reverse transcriptase (TERT) was assessed and compared by polymerase chain reaction. RESULTS: Cell morphology showed that TGF-β1 triggered the differentiation of HSCs from a quiescent phenotype into highly activated myofibroblasts. TERT mRNA expression in the primary HSCs showed slight increase with the culture time, though with no statistical difference between the results at various time points (P>0.05). TGF-β1 at 0.1 ng/mL did not significantly affect the TERT mRNA level compared with the 0 ng/mL group, while 1 ng/mL and 10 ng/mL TGF-β1 significantly decreased the level of TERT mRNA (P<0.05). TGF-β1 at 1 ng/mL had only weak effect on TERT mRNA expression after the 3 day treatment compared with the 0 ng/mL group (P>0.05). TGF-β1 at 1 ng/mL significantly inhibited TERT mRNA expression 6 days after the treatment (P<0.05). TGF-β1 inhibited the expression of TERT mRNA level in the HSCs in both dose- and time-dependent manner. CONCLUSION TGF-β1 may contribute to the transdifferentiation of HSCs by reducing TERT levels to develop hepatic fibrosis.
Animals ; Cell Transdifferentiation ; Cells, Cultured ; Hepatic Stellate Cells ; drug effects ; metabolism ; RNA, Messenger ; Rats ; Telomerase ; metabolism ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo study effects of Yishen Kangxian Compound (YKC) and benazepril containing serums on HK-2 cells (human renal proximal tubule epithelial cells) in the process of renal tubular epithelial cells to mesenchymal myofibroblasts transdifferentiation (TEMT) by gene chip.

METHODSYKC and benazepril containing serums were prepared. Their inhibitory effects on HK-2 cells in the transforming growth factor-beta1 (TGF-beta1)-induced TEMT process were observed. HK-2 cells were randomly divided into four groups, i.e., the blank control group, the model group, the benazepril group, and the YKC group. The gross RNAs were extracted and purified by taking advantage of the HumanHT-12 v4 of IlluminaBeadChip. Differentially expressed genes were obtained after they were reversely transcribed to cDNA, incorporating biotin labeling probe, hybridized with GeneChip, picture signals of fluorescence in gene array scanned and compared with differential genes by computer analysis.

RESULTSDifferentially expressed genes were successfully identified by gene chip. Compared with the model group, there were 227 differentially expressed genes in the benazepril group, including 118 up-regulated genes and 109 downregulated genes. Compared with the model group, there were 97 differentially expressed genes in the YKC group, including 69 up-regulated genes and 28 down-regulated genes. The Gene Ontology (GO) analysis indicated that YKC was more actively involved in the regulatory process than benazepril in terms of cell damage, apoptosis, growth, NF-KB, protein kinase, neuron, and blood vessel growth.

CONCLUSIONSYKC and benazepril could inhibit the TEMT process of HK-2 cells. But YKC also had taken part in cell damage, apoptosis, growth,and more pathways of early stage TEMT.

Cell Line ; Cell Transdifferentiation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Epithelial Cells ; cytology ; drug effects ; Genomics ; Humans ; Kidney Tubules, Proximal ; cytology ; pathology

OBJECTIVETo observe the antagonist effect of Curcuma Aromatica (CA) on renal tubular epithelial-myofibroblast transdifferentiation (EMT) induced by transforming growth factor-beta1 (TGF-beta1).

METHODSNormal renal tubular epithelial NRK-52E cells in vitro cultured were randomly divided into 6 groups, i.e., the normal control group (Group C), the TGF-beta1 induced model group (Group T), the low dose CA treated group (Group E1), the moderate dose CA treated group (Group E2), the high dose CA group (Group E3), and the Benazepril Hydrochloride Tablet treated group (Group Y). Except Group C, corresponding medication (with an action of 48 h) was administered to cells in the rest groups after they were induced by TGF-beta1 for 24 h. The morphological changes were observed by inverted phase contrast microscope. The distribution of beta-actin protein was detected by immunohistochemical assay. The mRNA expressions of alpha-smooth muscle actin (alpha-SMA) and E-cadherin (E-cad) were detected by real-time PCR. The concentration of fibronectin (FN) was detected by ELISA.

RESULTSAfter induced by TGF-beta1 for three days, hypertrophy and elongated cells in fusiform-shape occurred,with increased expressions of beta-actin protein in the cytoskeletal structure (P < 0.05), bundle fibrous structure occurred inside cytoplasm with significantly up-regulated intracellular alpha-SMA mRNA expressions (P < 0.05), E-cad mRNA expression decreased (P < 0.05), the FN content in the supernate increased (P < 0.05) in Group T. Compared with Group T, partial cells in Group E1, E2, and E3 showed fibrous changes, accompanied with decreased expression of beta-actin protein and FN concentration (P < 0.05). The expression of alpha-SMA mRNA increased and the expression E-cad mRNA decreased in Group E2 and E3 (both P < 0.05). But there was no statistical difference in the expression levels of E-cad and alpha-SMA mRNA (P > 0.05). Compared with Group E1, the expression of beta-actin protein and FN concentration decreased in Group E2 and E3 (P < 0.05). The expressions of alpha-SMA mRNA decreased and E-cad mRNA increased in Group E3 (P < 0.05). Compared with Group Y, the expression of beta-actin mRNA and FN concentration increased in Group E1 (P < 0.05); the expression of beta-actin mRNA increased in Group E3 (P < 0.05); the expression of E-cad mRNA decreased in Group E3 (P < 0.05). There was no statistical difference in the expression of alpha-SMA mRNA among Group E1, E2, and E3 (P > 0.05).

CONCLUSIONCA could inhibit the occurrence of TGF-beta1 induced EMT, which could be used as an effective drug for treating chronic renal insufficiency.

Animals ; Cell Transdifferentiation ; drug effects ; Cells, Cultured ; Curcuma ; chemistry ; Drugs, Chinese Herbal ; pharmacology ; Epithelial Cells ; drug effects ; Kidney Tubules ; cytology ; Male ; Myofibroblasts ; drug effects ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo explore the effect of resveratrol on transforming growth factor-beta1 (TGF-beta1) induced transdifferentiation of podocytes.

METHODSMouse podocytes in vitro cultured under differentiating conditions for 10 days were divided into the normal group, the model group, the high dose resveratrol group, and the low dose resveratrol group. The podocytes in the high and low dose resveratrol groups were intervened with 5 micromol/L and 2 micromol/L resveratrol respectively for 30 min. Those in the model group and the two resveratrol treated groups were continually incubated with 5 ng/mL TGF-beta1 for 72 h. Those in the normal group were routinely cultured. The protein expression of podocyte phenotypic protein molecules such as E-cadherin, P-cadherin, zonula occludens-1 (ZO-1), NEPH1, and alpha-smooth muscle-actin (alpha-SMA) were detected by immunocytochemistry, flow cytometry (FCM), and Western blot. A simple albumin influx assay was used to evaluate the filtration barrier function of podocyte monolayer.

RESULTSCompared with the normal control group, E-cadherin (+) percentage rate, the protein expression of P-cadherin, ZO-1, and NEPH1 significantly decreased in the model group (P < 0.05), but the expression of alpha-SMA and albumin permeability across podocyte monolayers increased significantly (P < 0.05). Compared with the model group, E-cadherin (+) percentage rate significantly increased (P < 0.05) and albumin permeability across podocyte monolayers decreased significantly (P < 0.05) in the high and low dose resveratrol groups. In the low dose resveratrol group, the expression of P-cadherin and NEPH1 significantly increased (P < 0.05). In the high dose resveratrol group, the expression of P-cadherin, ZO-1, and NEPH1 increased significantly, and the expression of alpha-SMA decreased significantly (P < 0.05). The correlations between resveratrol concentrations and the expression of E-cadherin (+), P-cadherin, and NEPH1 were significantly positive (r(E-cadherin (+)) = 0.772, r(P-cadherin) = 0.756, r(NEPH1) = 0.809, P < 0.05).

CONCLUSIONThe role of resveratrol in inhibiting TGF-beta1 induced phenotype abnormality might be an important mechanism for preserving the integrality of glomerular filtration barrier and decreasing proteinuria.

Animals ; Cell Transdifferentiation ; drug effects ; Cells, Cultured ; Kidney Tubules ; cytology ; drug effects ; Mice ; Podocytes ; cytology ; drug effects ; Stilbenes ; pharmacology ; Transforming Growth Factor beta1 ; metabolism

Human embryonic stem cells (hESCs) are pluripotent cells that have the ability of unlimited self-renewal and can be differentiated into different cell lineages, including neural stem (NS) cells. Diverse regulatory signaling pathways of neural stem cells differentiation have been discovered, and this will be of great benefit to uncover the mechanisms of neuronal differentiation in vivo and in vitro. However, the limitations of hESCs resource along with the religious and ethical concerns impede the progress of ESCs application. Therefore, the induced pluripotent stem cells (iPSCs) via somatic cell reprogramming have opened up another new territory for regenerative medicine. iPSCs now can be derived from a number of lineages of cells, and are able to differentiate into certain cell types, including neurons. Patient-specifi c iPSCs are being used in human neurodegenerative disease modeling and drug screening. Furthermore, with the development of somatic direct reprogramming or lineage reprogramming technique, a more effective approach for regenerative medicine could become a complement for iPSCs.
Cell Differentiation ; Cell Lineage ; Cell Transdifferentiation ; Cellular Reprogramming ; drug effects ; Embryonic Stem Cells ; cytology ; Humans ; Induced Pluripotent Stem Cells ; cytology ; transplantation ; Neural Stem Cells ; cytology ; transplantation ; Neurodegenerative Diseases ; therapy ; Regenerative Medicine ; Transcription Factors ; genetics ; metabolism

Cancer stem cells (CSCs) are resistant to chemo- and radio-therapy, and can survive to regenerate new tumors. This is an important reason why various anti-cancer therapies often fail to completely control tumors, although they kill and eliminate the bulk of cancer cells. In this study, we determined whether or not adenine nucleotide translocator-2 (ANT2) suppression could also be effective in inducing cell death of breast cancer stem-like cells. A sub-population (SP; CD44+/CD24-) of breast cancer cells has been reported to have stem/progenitor cell properties. We utilized the adeno-ANT2 shRNA virus to inhibit ANT2 expression and then observed the treatment effect in a SP of breast cancer cell line. In this study, MCF7, MDA-MB-231 cells, and breast epithelial cells (MCF10A) mesenchymally-transdifferentiated through E-cadherin knockdown were used. ANT2 expression was high in both stem-like cells and non-stem-like cells of MCF7 and MDA-MB-231 cells, and was induced and up-regulated by mesenchymal transdifferentiation in MCF10A cells (MCF10AEMT). Knockdown of ANT2 by adeno-shRNA virus efficiently induced apoptotic cell death in the stem-like cells of MCF7 and MDA-MB-231 cells, and MCF10AEMT. Stem-like cells of MCF7 and MDA-MB-231, and MCF10AEMT cells exhibited increased drug (doxorubicin) resistance, and expressed a multi-drug resistant related molecule, ABCG2, at a high level. Adeno-ANT2 shRNA virus markedly sensitized the stem-like cells of MCF7 and MDA-MB-231, and the MCF10AEMT cells to doxorubicin, which was accompanied by down-regulation of ABCG2. Our results suggest that ANT2 suppression by adeno-shRNA virus is an effective strategy to induce cell death and increase the chemosensitivity of stem-like cells in breast cancer.
ATP-Binding Cassette Transporters/*genetics/metabolism ; Adenine Nucleotide Translocator 2/antagonists & inhibitors/genetics ; Adenoviridae/*genetics ; Antineoplastic Agents/pharmacology ; Apoptosis/drug effects/genetics ; Breast Neoplasms ; Cadherins/antagonists & inhibitors/genetics ; Cell Line, Tumor ; Cell Survival/drug effects/genetics ; Cell Transdifferentiation/drug effects ; Doxorubicin/pharmacology ; Drug Resistance, Neoplasm/drug effects/*genetics ; Epithelial-Mesenchymal Transition/drug effects ; Female ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Knockdown Techniques ; Humans ; Neoplasm Proteins/*genetics/metabolism ; Neoplastic Stem Cells/drug effects/*metabolism/pathology ; RNA, Small Interfering/*genetics ; Signal Transduction/drug effects

OBJECTIVETo observe the expressions of Wnt/beta-catenin and the effects of tanshinone IIA (TII A) on Wnt/beta-catenin signaling pathway in high glucose induced renal tubular epithelial cell transdifferentiation.

METHODSHuman kidney proximal tubular epithelial cells (HK-2) were divided into three groups, i. e., the normal glucose group, the high glucose group, and the high glucose plus tanshinone IIA group. The expression of beta-catenin was observed using immunocytochemical staining. The protein expression of beta-catenin, E-cadherin, and alpha-smooth muscle actin (alpha-SMA) were detected by Western blot. The mRNA levels of beta-catenin and E-cadherin were detected by RT-PCR.

RESULTSCompared with the normal glucose group, both the protein and the mRNA expressions of beta-catenin were significantly enhanced (P < 0.01), the expression of E-cadherin significantly decreased (P < 0.01), the expression of beta-catenin increased in the cytoplasm and nucleus in the high glucose group. TIIA at the final concentration of 100 micromol/L significantly reduced the ectopic expression of beta-catenin. At that concentration, the protein and mRNA expressions of beta-catenin in the nucleus significantly decreased, while the protein and mRNA expressions of E-cadherin were up-regulated. Meanwhile, the expression of alpha-SMA obviously decreased.

CONCLUSIONSWnt/beta-catenin signaling pathway participated in the high glucose induced renal tubular epithelial cell transdifferentiation. TIIA inhibited the transdifferentiation process possibly through down-regulating the activities of Wnt/beta-catenin signaling pathway, thus further playing a role in renal protection.

Cadherins ; metabolism ; Cell Line ; Cell Transdifferentiation ; drug effects ; Diterpenes, Abietane ; pharmacology ; Epithelial Cells ; cytology ; drug effects ; metabolism ; Glucose ; adverse effects ; Humans ; Kidney Tubules, Proximal ; cytology ; drug effects ; metabolism ; Wnt Signaling Pathway ; drug effects ; beta Catenin ; metabolism

We aimed to examine the effect of pioglitazone on transdifferentiation of preosteoblasts from rat bone marrow mesenchymal stem cells (BMSCs) into adipocytes and investigate its effect on bone metabolism. BMSCs were harvested from the femurs and tibias of a rat, then separated, purified, proliferated for 3 generations and differentiated into preosteoblasts for 5 days and 14 days respectively in the presence of osteogenic medium. Thereafter, the preosteoblasts were cultured for 21 days in the presence of adipogenic medium with and without pioglitazone (1 μg/mL). Partially-differentiated osteoblasts were identified by mineralized nodules with Alizarin red S staining. Transdifferentiated adipocytes were identified by Oil Red O staining. Reverse transcription PCR (RT-PCR) was performed to assay the expression levels of osteogenic markers Runx2 and ALP, and an adipogenic marker PPARγ. Those cells cultured for 5 days did not show mineralized nodules as detected by staining of Alizarin red S, while those cultured for 14 days showed dispersed mineralized centers in the form of brown spots, although without obvious red mineralized nodules. After adipogenic transdifferentiation for 21 days, adipose-drops were found in cells of 5CG and 5EG earlier than those of 14CG and 14EG, and the former showed much more adipocytes separately as detected by Oil Red O staining. Whatever the time was 5 days or 14 days of BMSCs osteogenic differentiation, the cells cultured with pioglitazone showed much more adipocytes than those without pioglitazone. Our experiment showed that the less time it took for BMSCs osteogenic differentiation, a stronger ability remained for BMSCs to transdifferentiate into adipocytes. The mRNA expression levels of Runx2 and ALP were decreased by 1.79 and 1.90 times respectively in 5EG (P< 0.05) as compared with 5CG, and that of PPARγ was increased by 1.31 times in 5EG (P<0.05) as compared with 5CG. The mRNA expression levels of Runx2 and ALP were decreased by 1.45 and 1.54 times respectively in 14EG (P<0.05) as compared with 14CG, and that of PPARγ was increased by 1.39 times in 14EG (P<0.05) as compared with 14CG. It was concluded that pioglitazone stimulated the transdifferentiation of BMSCs into adipocytes. These observations provided a potential mechanism of imbalance in thiazolidinedione induced bone metabolism.
Adipocytes ; drug effects ; Animals ; Cell Transdifferentiation ; drug effects ; Female ; Male ; Mesenchymal Stromal Cells ; drug effects ; Osteoblasts ; drug effects ; Osteogenesis ; drug effects ; Rats ; Rats, Sprague-Dawley ; Thiazolidinediones ; pharmacology

OBJECTIVETo explore the DNA methylation levels of genome in cFb transdifferentiation induced by SiO2 in rats.

METHODSThe primary macrophages and fibrocytes of SD rats were co-cultured directly and indirectly, which were exposed to SiO2 at the doses of 25, 50 and 100 g/ml. The transdifferentiation of cFb was identified with immunohistochemical assay. The genomic DNA methylation levels of cFb were detected with HPLC.

RESULTSUnder the condition of indirect co-culture, as compared with control group, the genomic DNA methylation levels of cFb exposed to SiO2 at the doses of 25, 50 and 100 g/ml reduced by 19.9%, 26.9% and 30.3%, respectively (P < 0.05); as compared with cFb exposed to 100 g/ml SiO2, the genomic DNA methylation levels of cFb exposed to 5-aza-dC decreased by 22.0% (P < 0.05). Under the condition of ThinCert(TM) direct co-culture, as compared with control group, the genomic DNA methylation levels of cFb exposed to SiO2 at the doses of 25, 50 and 100 g/ml reduced by 22.2%, 30.2% and 36.7%, respectively (P < 0.05); as compared with cFb exposed to 100 g/ml SiO2, the genomic DNA methylation levels of cFb exposed to 5-aza-dC decreased by 20.6% (P < 0.05).

CONCLUSIONUnder the co-culture condition in vitro, SiO2 could reduce the genomic DNA methylation levels of cFb. The ThinCert(TM) direct co-culture can be used to study the silicosis fibrosis.

Animals ; Cell Transdifferentiation ; drug effects ; Cells, Cultured ; Coculture Techniques ; DNA Methylation ; Fibroblasts ; cytology ; drug effects ; Genome ; drug effects ; Lung ; cytology ; drug effects ; Male ; Rats ; Rats, Sprague-Dawley ; Silicon Dioxide ; adverse effects

PURPOSE: To investigate the role of focal adhesion kinase (FAK) in transforming growth factor (TGF)-beta-induced myofibroblast transdifferentiation of human Tenon's fibroblasts. METHODS: Primary cultured human Tenon's fibroblasts were exposed to TGF-beta1 for up to 48 hours. The mRNA levels of FAK, alpha smooth muscle actin (alphaSMA), and beta-actin were determined by quantitative real time reverse transcription polymerase chain reaction. The protein levels of collagen type I, FAK, phospho-FAK, alphaSMA, and beta-actin were determined by Western immunoblots. After the small interfering RNA targeting FAK (siRNA(FAK)) molecules were delivered into the cells, the expressions of alphaSMA proteins were determined by Western immunoblots. RESULTS: In human Tenon's fibroblasts, TGF-beta1 significantly increased the mRNA and protein expressions of alphaSMA. However, when the action of FAK was inhibited using siRNAFAK, the TGF-beta1-induced expression of alphaSMA was attenuated. CONCLUSIONS: Our data suggest that FAK may be associated with the TGF-beta1-induced transdifferentiation of human Tenon's fibroblasts to myofibroblasts, which is the essential step of subconjunctival fibrosis.
Actins/metabolism ; Analysis of Variance ; Blotting, Western ; Cell Transdifferentiation/drug effects ; Cells, Cultured ; Collagen/metabolism ; Fibroblasts/cytology/drug effects/metabolism ; Focal Adhesion Protein-Tyrosine Kinases/*metabolism ; Humans ; Myofibroblasts ; RNA, Messenger/metabolism ; RNA, Small Interfering/metabolism ; Real-Time Polymerase Chain Reaction ; Transforming Growth Factor beta/*pharmacology