Primary Culture Of Renal Tubular Epithelial Cells And Establishment Of The Apoptosic Model Induced By Oxidative Stress

Objective:①To investigate the method of primary culture and identify renal tubular epithelial cells②To study the apoptosis of renal tubular epithelial cells induced by Hydrogen peroxide (H2O2 ) and establish the satisfactory simulated model of apoptosis of injury of renal tubular epithelial cells in renal ischemic reperfusion.Methods:①We harvested the renal tubular segments by machanical and chemical digestive method and primaryly cultured the renal tubular epithelial cells.②By dynamic observing ,cytochemistry staining ,and transmission electron microscope we identified the renal tubular epithelial cells.③Oxidizative stressed the renal tubular epithelial cells with medium containing different concentrations of H2O2.④We detected the apoptosis of renal tubular epithelial cells by the morphologic change and TUNEL(terminal-deoxynucleotidyl transferase mediated nick end labeling) after oxidative stress.Results:①,The anchor rates of renal tubular respectively were 0.1378±2.048E-02,0.3300±7.969E-02,0.5411±3.919E-02,0.4389±3.018E-02 after different time of replacing medium and the difference (p<0.05) between each two groups had statistical significance except the two groups (72h) and (96h)(p>0.05).The primary culture of the renal tubular epithelial cells succeed: by dynamic observing ,the oval-like renal tubular epithelial cells start to grow around the anchoring renal tubular segment after cultured 2 to 3 days.The cells grow in logarithm growing phase from 4 to 7days.②The primary cultured cells were identified successfully as the renal tubular epithelial cells:(a)The cultured cells were all positive by alkali-phosphatase staining. (b)Under transmission electron microscopy the brush border of the renal tubular epithelial cells have a great many of microvilli .③The renal tubular epithelial cells present different changes of apoptosic and the apoptosic cells confirmed by HE staining and DeadEndTM system detecting.The apoptosic indexes of the cells were 3.000E-02±8.944E-03,0.1450±2.510E-02,0.2317±2.858E-02,0.2500±2.366E-02 as the H2O2 stress concentrations were 0mmol/L, 0.6mmol/L, 1.0mmol/L, 1.4mmol/L respectively. The differences between each two groups had statistical significance (p<0.05) except the two groups 1.0mmol/L and 1.4mmol/L(p>0.05)。 ④The anchoring rates or renal tubular epithelial cells were 1.0000±0.000,0.9400±2.366, 0.9217±1.941, 0.8467±4.676 after stressed by H2O2 of different concentrations 0mmol/L, 0.6mmol/L, 1.0mmol/L,1.4mmol/L respectively. The differences between each two groups have statistical meaning (p<0.05) except the two groups (0.6mmol/L) and (1.0mmol/L)(p>0.05).Conclusion:The machanical and chemical digestive method that we modified can obtain fairy pure renal tubular segments.The best growth time is the third day after the first replacing medium.The renal tubular epithelial cells can be cultured successfully with our culture method. The apoptosis of renal tubular epithelial cells can be induced by oxidative stress ( H2O2 ).On the condition of keeping the high anchoring rate of the cells , it is a good model to research apoptosis of the renal ischemic reperfusion injury with 1.0mmol/L H2O2 stress in vitro .