| Object: To investigate the variance of expression of Paxillin (Pax) in human renal tubular epithelial cells (HKCs) during undergoing tubular epithelial to mesenchymal transition (TEMT) induceded by transforming growth factor β1 (TGF-β1) and the result of intervention of protein tyrosine kinase (PTK) inhibitor genistein (Gen).Methods: The HKC cells were cultured in vitro with Dulbecco's modified Eagle's medium/F12 medium supplanted with 10% fetal bovine serum (FBS), and seeded on twenty-four-well culture plates to approximatedly 60% to 70% confluence, and then shifted to serum-free medium for 24 hours. These cells were divided into four groups: ?Control(C) group: The HKC cells were treated with free serum medium (FSM) for 48 hours. (2)T group: The HKC cells were co-incubated FSM containing TGF-β1 including dose-effect TA group and phrase-effect TB group. TA group is that the HKC cells were co-incubated different concentration TGF-Pi (5 ng/ml, 10 ng/ml, 20 ng/ml) with (FSM) for 48 hours and were divided into T5,T10,T20 for three subsections; TB group is that the HKC cells were co-incubated TGF-β1(10 ng/ml) with FSM for 24,48,72 hours and were divided into T24,T48,T72 for three subsections. (3)T+G group: The HKC cells were co-incubated TGF-β1 (10ng/ml) and Gen (50μmol/L) with FSM for 48 hours. The morphological changes in cells were observed through the invert phase-contrast microscope. At the same time, The mRNA expression of E-cadherin, alpha-smooth mucle actin (α-SMA) and Pax was detected by reverse transcription polymerase chain reaction(RT-PCR), the protein expression of E-cadherin,α-SMA and Pax was proved by immuno-histochemical staining, also the protein expression of E-cadherin and a-SMA was proved by indirect immunofluorescence, and the protein of Pax was detected by Western blot.Result: 1. Cells shape observation (1)C group: The shape of HKC cells showed classical cobblestone morphology and grew in a compact mode. (2)TA group: no visible...
|
PDF Full Text Request