| Objective: To investigate the expression and effect of connective tissue growth factor (CTGF) during the process of the human tubular epithelial-myofibriblast transdifferentiation (TEMT) induced by transforming growth factor β1(TGFβ1).Methods: Human tubular epithelial cell (HKCs) line were cultured in vitro with DMEM/F12 1:1 medium. Based on the feasibility of CTGF antisense oligonucleotide(AS) conjugated with 5'-fluorescent isothiocyanate (5'FITC) encapsulated with cationic liposome DOTAP transfecting into HKCs was proved by MTT method, fluorescence microscopy and fluorescence spectrophotometer, HKCs were divided into four groups: ①Control(C) group:HKCs were treated with free serum medium (FSM). ②TGFβ1(T) groups: HKCs were treated with FSM contained diverse concentration TGFβ1 (Ta:20ng/ml, Tb:40ng/ml). ③Tb+AS group: HKCs were treated with FSM contained TGFβ140ng/ml and AS(encapsulated with DOTAP). ④Tb+SC group: HKCs were treated with FSM contained TGFβ140ng/ml and SC (CTGF scrambled oligonucleotide, encapsulated with DOTAP). After cells had been cultured for 96 hours, the morphological change were observed by microscope, the expression of cell keratin, alpha-smooth muscle actin(α-SMA) and CTGF were analyzed by immunohistochemistry, the expression of α-SMAmRNA and CTGFmRNA were investigated by RT-PCR. The correlation between the relative expression of CTGFmRNA and α-SMAmRNA was surveyed by statistical analysis.Results: MTT method.:1. Cell shape observation: ①C group: The shape of HKCs showed a classic cobblestone morphology. ②T group: Some HKCs became elongated in shape and disassociated from neighboring cells. The percentage of elongated cells of Tb group was larger than that of Ta group, suggesting that TGFβ1 can induce HKCs morphology transform in shape. ③Tb+AS group: The percentage of elongated cells was less than that of Tb group(P<0.01), suggesting that AS can inhibit HKCs morphology transform in shape induced by TGFβ1. ④Tb+SC group: The percentage of elongated cells was similar to that of Tb group(P>0.05). 2. Immunohistochemistry assays: ①C group: CTGF and α-SMA expression were negative,but cell keratin (CK) expression was strong positive. ②T group: Compared with C group,in Ta group and Tb group, the expression ofCTGF and α-SMA were positive but the positive expression of CK decreased. average optical density(AOD)CTGF and AODα-SMA increased significantly (P<0.01) but AODCK decreased significantly(P<0.01). In Tb group, AODCTGF and AODα-SMA increased significantly(P<0.01) but AODCK decrease significantly(P<0.01), compared with Ta group. It suggested TGFβ1 can induce the expression of CTGF and α-SMA at the same time inhibit the expression of CK. ③Tb+AS group: Compared with Tb group, AODCTGF and AODα-SMA decreased significantly (P<0.01) but AODCk increased significantly(P<0.01). It suggested AS restrain the expression of CTGF and α-SMA and the expression inhibiting of CK induced by TGFβ1. ④Tb+SC group: Compared with Tb group, AODCTGF,AODα-SMA,AODCK were similar(P>0.05). 3. RT-PCR assays: ①C group: The expression of CTGFmRNA and α-SMAmRNA were negative. ②T group: The expression of CTGFmRNA and α-SMAmRNA were positive. Compared with C group, the relative expression quantity of CTGFmRNA (C/G) and the relative expression quantity of α-SMAmRNA(α/G) increased significantly(P<0.01). In Tb group, C/G andα/G increased significantly (P<0.01), compared with Ta group. It suggested TGFβ1 induce the expression of CTGFmRNA and α-SMAmRNA. ③Tb+AS group: Compared with Tb group, C/G and α/G decreased significantly(P<0.01). suggesting that AS inhibit the expression of CTGFmRNA and α-SMAmRNA induced by TGFβ1 ④Tb+SC group: C/G andα/G were similar in Tb+SC group and Tb group(P>0.05). Statistical analysis showed that there was a positive correlation between the expression of CTGFmRNA and α-SMAmRNA (R=0.96,P<0.01).Conclusions: ①TGFβ1 induce TEMT and CTGF expression in HKCs. ② CTGF is a key factor in TGFβ1 inducing TEMT. Blocking the expression and/or... |
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