【作者】 王阳；

【导师】 黄巨恩；

【作者基本信息】 广西医科大学 ， 组织学与胚胎学， 2010， 硕士

【摘要】 目的：通过体外培养SD大鼠肾小管上皮细胞的方法,探讨改构型酸性成纤维细胞生长因子(MaFGF)是否对庆大霉素(GM)引起的肾小管上皮细胞损伤有预防作用。方法：在无菌条件下分离SD大鼠原代肾小管上皮细胞,然后接种于50ml培养瓶、6孔板或96孔板内：(1)培养24小时后于细胞处于对数生长期时在96孔板内向实验组加入不同浓度的MaFGF,12小时后再加入一系列浓度的GM,再继续培养24小时后用MTT法检测每组细胞的光密度(OD)值并计算细胞生长抑制率、相应每组的GM的IC50。(2)将处于对数生长期的细胞分为三组：对照组、GM组、MaFGF+GM组。首先给予MaFGF+GM组一定量的MaFGF作用12小时,然后GM组和MaFGF+GM组同时给予相同剂量的GM继续培养24小时,从形态学上观察MaFGF对GM诱导的肾小管上皮细胞损伤的预防作用；通过检测对照组、GM组、MaFGF+GM组相应的生化指标(MDA、NO)和酶学指标(SOD、GSH-Px),观察MaFGF对GM诱导的肾小管上皮细胞损伤的预防作用。结果：(1)MaFGF预处理12h能使GM对肾小管上皮细胞的半数抑制浓度(IC50)升高。(2)加入2.0g／L以上浓度的GM作用24h后可见细胞由原有的多边鹅卵石样变圆皱缩、形态异常,细胞边界较为模糊甚至消失,细胞胞浆稀薄,立体感较差,细胞富集度降低,多数细胞胞浆内出现较多的黑色颗粒和空泡呈明显的细胞老化现象,有些甚至脱落悬浮于培养基中；MaFGF预处理12h后再加GM继续培养24 h,可见细胞变圆皱缩程度减轻,细胞轮廓较清晰,细胞边界较为清晰且折光性尚好,细胞胞浆较丰富,立体感较强,细胞富集度提高,胞浆内少见黑色颗粒和空泡。(3)GM组与对照组比较,MDA、NO的含量升高而SOD、GSH-Px的活性降低,差异均有统计学意义(P<0.01); MaFGF+GM组与GM组比较,MDA、NO的含量有所下降而SOD、GSH-Px的活性有所升高,差异均有统计学意义(P<0.01);而MaFGF+GM组与对照组比较,各生化和酶学指标含量或活性均未回落至正常水平,差异仍有统计学意义(P＜0.01)。结论：MaFGF对庆大霉素诱导的肾小管上皮细胞损伤具有一定的预防作用。MaFGF对庆大霉素肾毒性的预防作用与其直接或间接拮抗自由基的产生,加强抗氧化酶对自由基的清除和保护抗氧化酶的活性有关。更多还原

【Abstract】 Objective:To explore the preventive role of MaFGF in the injury of gentamicin on renal tubular epithelial cells of SD rats.Method:SD rat’primary renal tubular epithelial cells were sterilely colleccted, and then cultured in 6-well plates,96-well plates and culture bottles: (1) After the cells grow in logarithmic growth phase in 96-well plates, the cells were divided into five groups randomly. Control group were incubated for 36h normally; GM group were incubated with DMEM culture medium containing GM respectively and the final concentrations were followed by 1.0g/L,2.0 g/L,3.0 g/L,4.0 g/L and 5.0g/L at the latter 24 hours; MaFGF groups(Ⅰ,Ⅱ,Ⅲ) were incubated with DMEM culture medium containing MaFGF respectively and the final concentrations were followed by 5.20μg/L,6.24μg/L and 7.28μg/L at the former 12 hours, and then added GM as the above serious of drug concentrations at the latter 24 hours. Assay the activity of each group cells by MTT methord and calculate the inhibition rate and the corresponding IC50 values of GM. (2) The cells were divided into three groups randomly when they grow in logarithmic growth phase:Control group, GM group and MaFGF+GM group. Control group were incubated for 36h normally; GM group were incubated with DMEM culture medium containing 3.0g/L GM at the latter 24 hours; MaFGF+GM group were incubated with DMEM culture medium containing 7.28μg/L MaFGF at the former 12 hours, and then added 3.0 g/L GM at the latter 24 hours. And the preventive role of MaFGF in the injured renal epithelial cells was observed by using inverted phase contrast microscope and some biochemical and enzymological indexes.Result:(1) MaFGF pretreatment renal tubular epithelial cells,which can increase IC50 value of GM. (2) Added 2.0g/L or higher concentrations of GM at the latter 24 hours, renal tubular epithelial cells became abnormal shape, which lost the original multilateral cobblestone-like shape and became rounded shrinkage. Cell borders became fuzzier or even disappear.Cytoplasm became thin, and three dimensional sense became worse, and cell concentration was low. More black particles and vacuoles existed in cytoplasm. Some cells even were sheded and suspended in culture medium. But the cells which were pretreated whith MaFGF at the former 12 hours and added 3.0g/L GM at the latter 24 hours also showed that the extent of rounded shrinkage was remission, and the cell shapes became clearer, and the refraction of cell boundary was better. Cytoplasm became abundant, and three dimensional sense became better, and cell concentration was higher. A litter of black particles and vacuoles existed in cytoplasm. (3) Compared GM group and Control group, the content of MDA and NO was increased while the activity of SOD and GSH-Px was decreased, the differences were significant statistically (P<0.01); Compared MaFGF+GM group and GM group, the content of MDA and NO was decreased while the activity of SOD and GSH-Px was increased, the differences were significant statistically (P<0.01); Compared MaFGF+GM group and Control group, the content of the four indexes were not restored to the normal levels, the differences were still significant difference statistically (P<0.01).Conclusion:MaFGF could prevent the injury of renal tubular epithelial cells in some degree. The preventive effect of MaFGF on GM nephrotoxicity was related to antagonize free radicals directly or indirectly, enhance the activity of antioxidant enzymes which could scaven free radicals, and protect antioxidant enzymes.更多还原