核心蛋白聚糖蛋白对人结肠癌HCT-8细胞的生物学作用

Biological Action of Decorin on HCT-8 Cells of Human Colonic Carcinoma

为探讨核心蛋白聚糖（decorin，DCN）蛋白对人结肠癌HCT8细胞的生物学作用，本实验将重组载体PcDNA3．1DCN／His（MDCN）稳定转染入HCT一8细胞，并将携带有目的基因的细胞进行扩增培养，然后应用RT—PCR及WesternBlot法对M—DCN在细胞mRNA及蛋白水平的表达分别进行鉴定，应用MagExtractor方法提取携带有M—DCN基因组HCT-8细胞（HCT-8-DCN）中的DCN蛋白，应用Bradford法测定提取蛋白的含量；同时，按DCN蛋白30μg／ml对HCT-8细胞进行培养，应用MTT法检测细胞增殖活力，细胞培养96h后用流式细胞术分析细胞周期。结果显示，RT—PCR及WesternBlot实验均可见到目的条带，证实MDCN转染HCT8细胞成功并实现稳定表达。实现了针对6个组氨酸标签使用MagExtractor方法对DCN蛋白的提取。加入DCN蛋白培养HCT-8细胞组细胞生长曲线呈现为生长抑制；流式细胞术结果提示G1期细胞显著增多。结果表明，DCN蛋白具有抑制HCT8细胞增殖的作用。

The objective of this experiment was to explore the biological action of decorin （DCN） on HCT- 8 cells of human colonic carcinoma,in this experiment recombinant vector PcDNA 3.1-DCN/His（M-DCN） was stably transfected into HCT-8 cells, and carried-objective gene ceils were amplificated and cultured, then by using RT-PCR and Western Blot to detect the expression of M-DCN at the levels of cellular mRNA and protein,respectively,and extracted carried M-DCN gene group HCT-8 cells（HCT-8-DCN） DCN pro- tein by using Mag Extractor method, and by using Bradford method to detect the content of extracted pro- tein;at same time,with 30μg/ml DCN HCT-8 cells were cultured,by using MTT method to detect the pro- liferation ability of the cells,finally,96h after cell culture the cell cycle was analysed by using flow cytome- try. As results, RT-PCR and Western Blot all measured the objective bands which demonstrates M-DCN successfully transfecting HCT 8 ceils and stably expressed;for six labeled histidine the extraction of DCN protein was complete by using MagExtractor method;adding DCN protein to culture HCT-8 cells obtained the growth curve showing growth-inhibited;flow cytometry data suggested G1 cells showed significant in- creased. Results show that DCN protein has the role of inhibiting proliferation of HCT-8 cells.