【作者】 廖晓星；

【导师】 孙庭；

【作者基本信息】 江西医学院 ， 泌尿外科， 2003， 硕士

【摘要】 目的：①研究肾小管上皮细胞体外培养及鉴定方法②研究过氧化氢诱导的肾小管上皮细胞凋亡，为模拟肾缺血再灌注损伤时凋亡的研究建立良好的模型。方法：①采用机械分离结合酶消化法获取肾小管，原代培养出肾小管上皮细胞。②利用动态观察、细胞化学染色法及肾小管上皮细胞的透射电镜观察进行肾小管上皮细胞的鉴定。③用含不同浓度过氧化氢（H2O2）的培养基对肾小管上皮细胞进行氧化应激培养。④利用凋亡细胞的形态变化及生物素原位末端标记法(TUNEL)观察氧化应激后肾小管上皮细胞的凋亡情况。结果：1、肾小管段不同换液时间24h,48h,72h,96h,120h贴壁率分别为：0.1378±2.048E-02，0.3300±7.969E-02，0.5411±3.919E-02，0.4389±3.018E-02，经多组间两两比较，除72h与96h两组间两两比较差别无意义外（p>0.05）,其余各组间比较差别均有显著意义（p<0.05）。肾小管上皮细胞成功的原代培养：培养的动态观察，从2-3天开始有呈卵圆形的肾小管上皮细胞从贴壁的肾小管周围长出，并于第4-7天处于对数生长期。2、原代培养的细胞鉴定为肾小管上皮细胞：（1）、培养细胞经碱性磷酸酶染色均呈阳性。（2）、透射电镜观察见细胞面向液层的刷状缘有大量微绒毛形成。3、肾小管上皮细胞经不同浓度0mmol/L,0.6mmol/L,1.0mmol/L,1.4mmol/L H2O2应激后的细胞贴壁率分别为1.0000±0.000，0.9400±2.366, 0.9217±1.941, 0.8467±4.676。其中除0.6mmol/L与1.0mmol/L间差别无显著意义外(p>0.05),其余各组差别均有显著意义（p<0.05）。4、肾小管上皮细胞经H2O2氧化应激后出现不同程度的凋亡变化，生物素原位末端标记法（TUNEL）检测，过氧化氢应激浓度为0mmol/L,0.6mmol/L,1.0mmol/L,1.4mmol/L时，细胞的凋亡指数分别是3.000E-02±8.944E-03,0.1450±2.510E-02,0.2317±2.858E-02,0.2500±2.366E-02。其中除1.0mmol/L与1.4mmol/L两组间差别无显著意义（p>0.05）,其余各组间差别均有显著意义（p<0.05）。结论：采用机械结合酶消化法可分离到较纯的肾小管，肾小管段培养72h首次换液细胞贴壁最佳,肾小管上皮细胞用常规方法可以成功培养及鉴定；肾小<WP=4>管上皮细胞经H2O2氧化应激可以产生凋亡；在保持肾小管上皮细胞贴壁率较高的条件下,1.0mmol/L H2O2应激可作为体外研究肾缺血性损伤凋亡的良好模型。更多还原

【Abstract】 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 .<WP=6>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 .更多还原