1.Effect of pioglitazone on transdifferentiation of preosteoblasts from rat bone mesenchymal stem cells into adipocytes.
Linfang WANG ; Lihua LI ; Haibo GAO ; Yuming LI
Journal of Huazhong University of Science and Technology (Medical Sciences) 2012;32(4):530-533
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
2.Study of resveratrol suppressing TGF-beta1 induced transdifferentiation of podocytes.
Ru-Chun YANG ; Xiao-Ling ZHU ; Hua-Qin ZHANG ; Wei-Dong LI
Chinese Journal of Integrated Traditional and Western Medicine 2013;33(12):1677-1682
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
3.Genomics research on roles of yishen kangxian compound in the TEMT process of HK-2 cells.
Shi-zhu SUN ; Wei SUN ; Xue-mei QIN ; Jun BAO ; Yan CHEN ; Juan-juan JIANG ; Zhi-xiang SHEN ; Xue-yan GAO
Chinese Journal of Integrated Traditional and Western Medicine 2013;33(8):1129-1134
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
4.Effects of Panax notoginseng on the transdifferentiation of fibroblasts in human hypertrophic scar in vitro.
Heng YAO ; Shi-Rong LI ; Jian-Yi LIU ; Zhe LI ; Jun WU
Chinese Journal of Burns 2007;23(3):188-190
OBJECTIVETo observe the effects of Panax notoginseng on the transdifferentiation of the cultured human fibroblasts from hypertrophic scar in vitro, and explore its anti-fibrosis mechanism.
METHODSThe fibroblasts from human hypertrophic scar were cultured in vitro. Different amount of Panax notoginseng was added into the medium, respectively (400 microg/ml and 800 microg/ml). A culture without addition of the drug served as control. The fibroblast-populated collagen lattice method was used to detect the gel contraction, and contraction ratio was calculated. The immunocytochemistry staining method was used to detect the expression of alpha-smooth muscle actin. The flow cytometry method was used to detect the positive rate of alpha-smooth muscle actin.
RESULTSThe contraction degree of the fibroblasts after PNS administration was ameliorated at each time-point, with contraction index lower than that of controls (P < 0.05 or P < 0.01). Scattered distribution of alpha-SMA positive granules were observed in the cytoplasma, and the positive rate of alpha-SMA expression in 400 microg/ml (31.52%) and 800 microg/ml (24.28%) PNS groups were obviously lower than that in control group (45.74%, P < 0.05). The staining intensity of positive cells in 400 microg/ml and 800 microg/ml PNS groups was also obviously lower than that in control group (P < 0.05 or P < 0.01).
CONCLUSIONPanax notoginseng can inhibit the transdifferentiation of the cultured human fibroblasts from hypertrophic scar, and it exhibits an anti-fibrosis effect on hypertrophic scar in vitro.
Cell Division ; drug effects ; Cell Transdifferentiation ; Cells, Cultured ; Cicatrix, Hypertrophic ; pathology ; Fibroblasts ; cytology ; drug effects ; Ginsenosides ; pharmacology ; Humans ; Panax notoginseng ; chemistry ; Wound Healing
5.Effect of bone morphogenetic protein-7 on aristolochic acid induced renal tubular epithelial cells transdifferentiation.
Yong-zhi XU ; Hao HUANG ; Hua-feng LIU
Chinese Journal of Integrated Traditional and Western Medicine 2010;30(2):157-160
OBJECTIVETo observe the effect of bone morphogenetic protein-7 (BMP-7) on aristolchic acid induced renal tubular epithelial cell trans-differentiation to look for new therapeutic approach for aristolchic acid nephropathy (AAN).
METHODSIn vitro cultured human proximal renal tubular epithelial cell line HK-2 cells were treated with different concentrations of BMP-7 (75 ng/mL, 150 ng/mL and 300 ng/mL) after trans-differentiation of the cells was induced by AA (10 microg/mL). Levels of alpha-SMA mRNA and protein expressions were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively.
RESULTSBMP-7 reversed the AA inducing alpha-SMA expressions in HK-2 cells in a dose-dependent manner.
CONCLUSIONBMP-7 can inhibit the trans-differentiation of human renal tubular epithelial cell induced by AA, thereby might be a new potential drug for AAN prevention and treatment.
Actins ; metabolism ; Aristolochic Acids ; adverse effects ; Bone Morphogenetic Protein 7 ; pharmacology ; Cell Line ; Cell Transdifferentiation ; drug effects ; Epithelial Cells ; cytology ; Humans ; Kidney Tubules, Proximal ; cytology
6.Effects of tanshinone IIA on Wnt/beta-catenin signaling pathway of high glucose induced renal tubular epithelial cell transdifferentiation.
Bao-Ying HUANG ; Luo-Yuan CAO ; Xian-Guo FU
Chinese Journal of Integrated Traditional and Western Medicine 2012;32(7):965-969
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
7.Ginsenoside R(g1) inhibit transdifferentiation in rat renal tubular epethelial cells induced by TGF-beta1.
Xi-sheng XIE ; Heng-chuan LIU ; Hui-juan LI ; Jun-ming FAN
China Journal of Chinese Materia Medica 2008;33(17):2136-2141
OBJECTIVETo investigate the effects of ginsenoside R(g1) on the transdifferentiation of rat renal tubular epethelial cells induced by transforming growth factor-beta1, (TGF-beta1).
METHODCultured normal rat renal tubular epethelial cells (NRK-52E) were divided into control group, TGF-beta1-induced group and treated with ginsenoside R(g1) at different concentration (10, 20, 40 mg x L(-1)) group. The morphology of tubular epithelial-myofibroblast transdifferentiation induced by TGF-beta1 was observed through light microscope. alpha-SMA and E-cadherin protein expression were assessed by immunohistochemistry and western blot analyses. alpha-SMA, collagen I and and fibronectin gene expression were assessed by real-time quantitative chain reaction. Enzyme-linked immunosorbent assay was used to quantitatively detect collagen I and fibronectin in the supernatant.
RESULT10 mg x L(-1) TGF-beta1 could induce the transdifferentiation of tubular epithelial myofibroblast, showing fibroblast-like in morphology, with significantly enhanced expression of alpha-SMA, depressed expression of E-cadherin and increased secretion of fibronectin and collagen I (P < 0.05). Compared to TGF-beta1-induced group, ginsenoside R(g1) partly abrogated the alpha-SMA expression and E-cadherin depression triggered by TGF-beta1 in tubular epithelial cells in a dose-dependent manner (P < 0.05). Meanhile, ginsenoside R(g1) blocked morphologic transformation of tubular epithelial cells and decreased levels of collagen I and fibronectin (P < 0.05).
CONCLUSIONGinsenoside R(g1) could inhibit TGF-beta1 induced the tubular epithelial-myofibroblast transdifferentiation and decreased levels of collagen I and fibronectin in NRK52E.
Animals ; Cadherins ; genetics ; metabolism ; Cell Line ; Cell Transdifferentiation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Epithelial Cells ; cytology ; drug effects ; Gene Expression ; drug effects ; Ginsenosides ; pharmacology ; Kidney Tubules ; cytology ; drug effects ; Panax ; chemistry ; Rats ; Transforming Growth Factor beta1 ; pharmacology
8.Expression of fragile histidine triad (FHIT) protein and Ki-67 in transformed epithelial cells induced by Yunnan tin mine dust.
Li BIAN ; Yong-Wen HE ; Hong-Mei FU ; Yong-Hua RUAN ; Qian GAO ; Chun-Yan WANG ; Ke-Wei JIN
Chinese Journal of Industrial Hygiene and Occupational Diseases 2010;28(12):895-899
OBJECTIVETo study the expression and significance of fragile histidine triad (FHIT) and Ki-67 in transformed epithelial cells induced by Yunnan tin mine dust.
METHODSEvery second generation of immortalized human bronchial epithelial cells (BEAS-2B) and human embryo lung fibroblasts (WI-38) were exposed to 100 µg/ml Yunnan tin mine dust for 72 h, until the ninth generation. The cells were subsequently co-cultured from the 11th generation. Experimental setup: B group, B (W) group, B (W 100) group, B100 group, B100 (W) group, B100 (W100) group. The expressions of FHIT and Ki-67 in epithelial cells were determined by the method of immunocytochemistry at the 16th, 26th and 36th generation. The percentage of Ki-67 positive cells was calculated as proliferation index.
RESULTSThe expression of FHIT was observed in BEAS-2B cells. The expression levels of FHIT among B group, B (W) group and B (W 100) group had not instinctive difference. At the 16th generation, the expression of FHIT in the B100 group was decreased compared with that in the B group and the expression of FHIT between B100 (W) group and B100 (W100) group was lower than that in the B100 group. At the 26th generation, the expression of FHIT was decreased compared with that at the 16th generation in the B100, B100 (W) and B100 (W100) groups. However, At the 36th generation, positive expression were observed again in the B100, B100 (W) and B100 (W100) groups and the expression levels were in incremental order. At the 16th, 26th and 36th generation, the proliferation indexes of B group, B (W) group and B (W 100) group were all < 3%. The proliferation indexes of B100, B100 (W) and B100 (W100) were increased step by step with the generation elongation.
CONCLUSIONSFHIT could be a target at which Yunnan tin mine dust induces transformation of BEAS-2B cells. The proliferation activation of BEAS-2B cells can be improved by Yunnan tin mine dust.
Acid Anhydride Hydrolases ; metabolism ; Cell Line ; Cell Transdifferentiation ; China ; Dust ; Epithelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Ki-67 Antigen ; metabolism ; Lung ; cytology ; Neoplasm Proteins ; metabolism ; Tin ; toxicity
9.DNA methylation levels of genome in cFb transdifferentiation induced by SiO2 in rats.
Yong-xing WANG ; Wu YAO ; Bin GAO ; Yong-cheng YAO ; Yi-ming WU ; Chang-fu HAO
Chinese Journal of Industrial Hygiene and Occupational Diseases 2012;30(5):326-330
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
10.Effects of lentinan on interleukin-1β-induced transdifferentiation of human embryonic lung fibroblasts to myofibroblasts.
Long-Teng JIN ; He ZHANG ; Chang-Chong LI ; Wei-Xi ZHANG ; Li LIN
Chinese Journal of Contemporary Pediatrics 2011;13(4):331-335
OBJECTIVETo study the effects of interleukin-1β (IL-1β) on transdifferentiation of human embryonic lung fibroblasts to myofibroblasts and the effects of lentinan on the transdifferentiation.
METHODSThe human embryonic lung fibroblasts were cultured in vitro, and fibroblasts were treated with different concentrations of IL-1β and lentinan. The proliferation activity of the human embryonic lung fibroblasts was evaluated by the Cell Counting Kit-8 (CCK-8). The expression of α-smooth muscle actin (α-SMA) protein was measured by immunocytochemistry. The levels of fibronectin (FN), typeⅠcollagen (ColⅠ) and α-SMA mRNA were detected by RT-PCR.
RESULTSCompared with the untreated control group, the absorbance value of cell proliferation, α-SMA protein levels, FN, ColⅠand α-SMA mRNA expression were significantly up-regulated after different concentrations of IL-1β (0.1, 1, 10 ng/mL) treatment for 48 hrs (P<0.01). Lentinan treatment inhibited up-regulation of the cell proliferation activity, α-SMA protein levels, FN, ColⅠand α-SMA mRNA expression induced by IL-1β in a dose-independent manner (P<0.01).
CONCLUSIONSLentinan can suppress human embryonic lung fibroblast proliferation, fibroblast-myofibroblast transdifferentiation and extra cellular matrix synthesis induced by IL-1β.
Actins ; analysis ; genetics ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cell Transdifferentiation ; Cells, Cultured ; Fibroblasts ; cytology ; drug effects ; Fibronectins ; analysis ; genetics ; Humans ; Interleukin-1beta ; pharmacology ; Lentinan ; pharmacology ; Myofibroblasts ; cytology