人肾间质成纤维细胞体外培养及染色体核型分析

In vitro culture and chromosome karyotype analysis of human renal interstitial fibroblasts

目的:体外培养人肾间质成纤维细胞并进行染色体核型分析。方法:实验于2004-06/2005-10在泸州医学院分子病理实验室和中心实验室(国家中医管理局二级实验室)完成。①实验材料:取慢性梗阻性肾病患者手术切除的部分肾脏标本,患者知情同意。研究方案获伦理委员会批准。②实验方法:通过组织块培养法体外分离、纯化并鉴定人肾间质成纤维细胞。③实验评估:观察培养过程中细胞的形态变化、免疫细胞化学方法鉴定波形蛋白、结蛋白和角蛋白的表达及体外培养第2～5代细胞做染色体核型分析。结果:①培养细胞形态学变化:第2～5代细胞形态较为均一,呈长梭形,显微镜下可见1～2个核仁;细胞之间界限清晰,呈鱼群状或栅栏状排列。②免疫细胞化学鉴定结果:免疫细胞化学检测第2代细胞表达波形蛋白强阳性,角蛋白散在灶性个别细胞弱阳性,结蛋白呈阴性表达;第3～5代细胞均表达波形蛋白强阳性,结蛋白和角蛋白呈阴性表达。③核型鉴定结果:第2～5代人肾间质成纤维细胞内含有46条染色体,呈正常人体细胞二倍体核型。结论:经免疫细胞化学鉴定及核型鉴定,体外培养第3～5代人肾间质成纤维细胞不仅达到纯化而且遗传性状稳定。

AIM： To culture human renal interstitial fibroblasts （hRIFs） in vitro, and analyze the chromosome karyotype. METHODS： The experiment was conducted in the Molecular Pathological Laboratory and Central Laboratory of Luzhou Medical College （Second Class Laboratory of State Administration Bureau of Traditional Chinese Medicine） from June 2004 to October 2005. （1）Partial kidney tissue was taken from human renal interstitial sample of patient who suffered from chronic obstructive nephropathy and agreed to the experiment. The study was permitted by Ethics Committee. （2） hRIFs were isolated, purified and identified by tissue culture method in vitro. Cell morphological changes were observed by inverted phase contrast microscope, and the expressions of vimentin, desmin and keratin and chromosome karyotype analysis of the 2^nd to 5^th passage cells were identified by immunocytochemistry （ICC）. RESULTS： （1）The cells of 2^nd to 5^th passage cultured in vitro were shuttle-shaped with one or two chromatospherite. The margin between cells was clear and arranged in fish or palisade. （2）In immunocytochemical study, the vimentin was found strong positive in the second passage cells, keratin was scattered and weak positive, and desmin was negatively expressed; The cells of 3^nd to 5^th passage did not express keratin and desmin but vimentin. （3）There was diploid karyotype in the cells of 2^nd to 5^th passage with 46 chromosomes. CONCLUSION： The hRIFs of the third to fifth passage cultured in vitro were successfully purified and with stable hereditary feature after identified by immunocytochemistry and karyotype analysis.