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目的观察中药千金子对表达人G250基因小鼠renca细胞增殖的影响。方法利用DNA重组技术将成功扩增的人G250基因编码区序列定向插入到pRNAT-U6.1/Neo质粒真核表达载体中,得到重组质粒pRNAT-U6.1/Neo-G250,将该质粒转染renca小鼠细胞,经G418筛选获得抗性单克隆,扩大培养后经Western印迹鉴定人G250基因的表达。将该细胞在不同浓度含千金子二萜醇(111μg/ml、333μg/ml、1 000μg/ml、3 000μg/ml、9 000μg/ml)的培养基中培养,MTT法观察千金子二萜醇对表达人G250基因小鼠renca细胞体外增殖情况的影响。结果与空白对照组相比,24 h后实验组细胞增殖速度减慢,并随浓度增加增殖速度减慢越明显,空白组与3 000μg/ml及9 000μg/ml浓度组比较差异有明显统计学(P=0.006,P=0.000)。结论中药千金子使表达人G250基因小鼠renca细胞增殖能力降低,renca细胞体外生长被显著抑制。
Objective To observe the effect of Qianjinzi on the proliferation of renca cells expressing human G250 gene in mice. Methods The coding sequence of human G250 gene was successfully inserted into the eukaryotic expression vector pRNAT-U6.1 / Neo by DNA recombination technique to obtain the recombinant plasmid pRNAT-U6.1 / Neo-G250. The recombinant plasmid pRNAT-U6.1 / Renca mouse cells were stained with G418 and screened to obtain a monoclonal antibody. After expansion, the expression of human G250 gene was identified by Western blotting. The cells were cultured in different concentrations of diterpene alcohol (111μg / ml, 333μg / ml, 1000μg / ml, 3000μg / ml, 9000μg / ml) Effects of in vitro proliferation of renca cells expressing human G250 gene in mice. Results Compared with the blank control group, the proliferation rate of the experimental group was slowed down after 24 h, and the more obvious the slowed down proliferation rate was. The difference between the blank group and the 3 000 μg / ml and 9 000 μg / ml group was statistically significant (P = 0.006, P = 0.000). Conclusion Qianjinzi can decrease the proliferation of renca cells expressing human G250 and the growth of renca cells in vitro.