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研究资料表明,肝细胞提取物(hepatocyteextraction)在诱导BEL-7402细胞凋亡的同时能促进p53和Fas基因表达,而对bcl-2和C-myc表达具有一定的抑制作用.本研究采用免疫组化技术,进一步观察纯化的肝提取肽(S4)对4种肝癌和4种非肝癌细胞凋亡及凋亡相关基因的影响.1 材料与方法1.1 肝细胞提取物和S4(subfraction 4)的制备参见文献[2].1.2 细胞系及培养条件SMMC 7721、QGY-7703人肝癌细胞、HCT-8结肠癌细胞、GLC-82低分化肺腺癌细胞引自中国科学院细胞所.BEL-7402人肝癌细胞引自北京中日友好医院中西医结合研究所.Hepe小鼠肝癌细胞、CNE-2鼻咽癌细胞引自中山医科大学.SGC-7901胃腺癌细胞由第四军医大学生化室惠赠.实验时将对数生长期细胞用0.25%胰酶加0.025%EDTA消化,配成(2.5～5)×10~4/ml细胞悬液,加入内置盖玻片的24孔Costar培养板中,每孔0,5ml.37℃,5%CO_2条件下孵育24小时后,置4℃水浴1小时后立即升到37℃,将S4以不同浓度加入培养孔,每孔0.5ml,继续孵育.洗涤固定细胞,分别做DNA原位末端标记检测和癌基因免疫组化测定.根据预实验结果,选定S4为5μg/ml,作用48小时为统一条件,以利于结果的可比性及准确性.
Research data show that hepatocyte extract can induce the apoptosis of BEL-7402 cells and promote the expression of p53 and Fas genes, but inhibit the expression of bcl-2 and C-myc. In this study, the immune group was used. And further observe the effect of purified hepatic extract peptide (S4) on apoptosis and apoptosis-related genes in 4 hepatocellular carcinoma and 4 non-hepatoma cells.1 Materials and methods 1.1 Preparation of hepatocyte extracts and S4 (subfraction 4) See [2].1.2 Cell lines and culture conditions SMMC 7721, QGY-7703 human hepatoma cells, HCT-8 colon cancer cells, and GLC-82 poorly differentiated lung adenocarcinoma cells were introduced from the Institute of Cellular Sciences, Chinese Academy of Sciences. BEL-7402 human liver cancer Cells were drawn from Beijing Sino-Japanese Friendship Hospital Institute of Integrated Traditional Chinese and Western Medicine. Hepe mouse hepatoma cells, CNE-2 nasopharyngeal carcinoma cells were imported from Sun Yat-sen University of Medicine. SGC-7901 gastric adenocarcinoma cells were benefited from the fourth military college student room. The logarithmic growth phase cells were digested with 0.25% trypsin plus 0.025% EDTA, and (2.5 to 5) x 10~4/ml cell suspension was prepared and added to a 24-well Costar culture plate with built-in coverslips. After incubating for 24 hours at 5ml.37°C, 5% CO 2 , set it in a 4°C water bath for 1 hour. Rise to 37°C, add S4 to culture wells at different concentrations, 0.5ml per well, and continue to incubate. Wash and fix the cells and perform DNA end-of-end labeling detection and oncogene immunohistochemistry respectively. According to the results of the preliminary experiments, select S4 For 5μg/ml, 48 hours for the uniform conditions, in order to facilitate the comparability and accuracy of the results.