梗阻性肾病成纤维细胞体外增殖规律的初步实验研究

目的]初步探讨梗阻性肾病人肾间质成纤维细胞体外培养的增殖规律。方法]取慢性梗阻性肾病(UUO)患者手术切除标本取材,体外培养1～7代细胞,在不同时间点,用相差倒置显微镜观察细胞形态变化;通过免疫细胞化学方法对体外培养细胞进行鉴定;用MTT比色法检测体外培养第3代成纤维细胞的吸光光度值。结果]原代培养5～7 d后,组织块周围开始长出数目不等的立方形或长梭形细胞;大约2～3周后,以占绝对优势的长梭形细胞可铺满瓶底。第2～5代细胞形态较为均一,多呈长梭形,胞浆丰富,核1～2个不等,偶见核仁,细胞呈栅栏状排列,周围有长短不一的轴状突起,并有无定性成分分布于4周。第6、7代细胞形态变化较大,细胞多数呈圆形或椭圆形,长梭形细胞数量减少,部分细胞形态不规则,胞内出现空泡和(或)颗粒状物质等细胞衰老、变性的形态改变。免疫细胞化学检测第2代细胞表达波形蛋白(vimentin)强阳性,角蛋白(keratin)散在灶性个别细胞弱阳性;第3～6代细胞均表达波形蛋白强阳性,结蛋白(desmin)和角蛋白阴性。传3代细胞MTT结果显示第3～5 d增殖速度最快,d 7细胞数量达到高峰,d 9后细胞生长相对稳定。结论](1)用组织块培养法在体外顺利培养出梗阻性肾病来源的hRIFs。(2)传3～5代hRIFs纯度高、活性佳,于接种培养后3～5 d进入对数增长期,体外实验研究选择该时期的hRIFs较好。

INITIAL RESEARCH ABOUT THE PROLIFEROUS RULE OF HUMAN RENAL INTERSTITIAL FIBROBLASTS FROM OBSTRUCTIVE NEPHROPATHY IN VITRO

Objective]To explore initially the proliferous rule of human renal interstitial fibroblasts from obstructive nephropathy patient in vitro.Methods]The tissue was taken from human renal interstitial sample of the patients who suffered from chronic ureteral obstruction and was cultured in vitro;the cells of 2ed passage were used to observe the change of figure and identify the cells by ICC;MTT test was used to exame the proliferous rule about the cells of 3rd passage in vitro.Results]After primary culture for 5-7 days,fusiform and cube cells began to erupt surrounding tissue clump,after about 2-3 weeks,the spindle cell which held absolute preponderance could spread out full bottle base.The cells cultured in vitro were shuttle-shapped and owned one or tow nuclear;cells did not express keratin and desmin but vimentin;cells proliferated quickly from the 3rd day to the 5th day;the number reached the pinnacle in the 7th day,and kept on proliferating steadyly during the time from the 9th day.Conclusion](1)We obtain the model of culture of human renal interstitial fibroblasts from fibrosis patient in vitro successfully.(2)The cells of the 3rd-5th passage is appropriated for the research about proliferation of fibroblasts in the 3rd-5th day after inoculated.