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目的:研究益气活血软坚解毒方(YHRJ)含药血清对人肝癌细胞系Bel-7402细胞凋亡调控基因Fas,FasL,Bcl-2,Bax,P53,NF-kB表达影响.方法:将细胞分为对照组(NS)组、NS+DDP(顺氯氨铂)组、YHRJ等剂量组(YHRJD)、YHRJD+DDP组、YHRJ高剂量组(YHRJG)、YHRJG+DDP组,应用流式细胞术、免疫组化、原位杂交、RT-PCR等方法对用药24,48h的Bel-7402细胞凋亡的主要调控基因Fas,FasL,Bcl-2,Bax,P53,NF-kBmRNA与蛋白表达进行检测.结果:流式细胞术检测显示:与NS组相比,NS+DDP、YHRJD、YHRJD+DDP及YHRJG+DDP组Fas蛋白表达均明显提高(30.12%±22.94%,10.50%±8.41%,30.35%±22.98%,32.61%±26.87%vs8.77%±6.93%,P<0.01),YHRJG组效果不明显(P>0.05);NS+DDP、YHRJD+DDP组FasL蛋白表达也升高(16.40%±7.168%,8.41%±6.74%vs4.12%±2.60%,P<0.01),而YHRJG组FasL蛋白表达降低(3.05%±2.53%vs4.12%±2.60%,P<0.01).免疫组化结果显示:除YHRJG+DDP组外,其余各组突变型P53蛋白表达明显降低(30.2%,14.6%,19.8%,17.3%vs60.0%,P<0.05);各加药组Bax蛋白表达明显增高(40.7%,40.4%,72.1%,68.9%,42.2%vs30.0%,P<0.05);NS+DDP组与YHRJG组Bcl-2蛋白表达明显降低(26.3%,24.4%vs30.5%,P<0.05),而YHRJD、YHRJG+DDP、YHRJG+DDP组Bcl-2表达明显升高(41.8%,39.3%,45.6%vs30.5%,P<0.05);各加药组NF-kB蛋白表达明显降低(15.9%,13.3%,14.1%,7.8%,14.6%vs24.2%,P<0.05).原位杂交结果显示:各加药组NF-kBmRNA表达明显降低(30.5%,13.3%,21.4%,17.4%,53.2%vs58%,P<0.05).RT-PCR结果显示:YHRJ等效剂组凋亡调控基因Bcl-2mRNA表达明显降低(0.717±0.198vs1.327±0.097,P<0.001);DDP、YHRJD、YHRJG组凋亡调控基因BaxmRNA表达明显增高(46.22±6.22,56.19±7.36,62.32±11.06vs35.22±4.38,P<0.05).结论:YHRJ含药血清诱导人肝癌细胞系Bel-7402细胞凋亡可能的基因调控机制在于通过抑制凋亡信号转导基因FasL基因蛋白表达,促进凋亡调控基因Bax基因蛋白表达,抑制NF-kB基因mRNA及蛋白表达来实现的.
Objective: To study the effects of Yiqi Huoxue Ruanjian Jiedu Recipe (YHRJ) containing serum on the expression of Fas, FasL, Bcl-2, Bax, P53 and NF-kB in human hepatocellular carcinoma cell line Bel-7402.Methods: The rats were divided into NS group, NS + DDP group, YHRJ group, YHRJD + DDP group, YHRJ high dose group and YHRJG + DDP group. Flow cytometry The expressions of Fas, FasL, Bcl-2, Bax, P53, NF-kB mRNA and protein in Bel-7402 cells treated with drugs for 24,48 hours were detected by immunohistochemistry, in situ hybridization and RT-PCR The results of flow cytometry showed that the expression of Fas protein in NS + DDP, YHRJD, YHRJD + DDP and YHRJG + DDP groups were significantly increased compared with NS group (30.12% ± 22.94%, 10.50% ± 8.41% DDP and YHRJD + DDP groups (P <0.05). The levels of FasL protein in NSDH, DDP and YHRJD + DDP group were also significantly higher than those in YHRJG group (30.35% ± 22.98%, 32.61% ± 26.87% vs 8.77% ± 6.93%, P < 16.40% ± 7.168%, 8.41% ± 6.74% vs4.12% ± 2.60%, P <0.01), while the expression of FasL in YHRJG group was decreased (3.05% ± 2.53% vs4.12% ± 2.60%, P <0.01). The results of immunohistochemistry showed that except YHRJG + DDP group, the expression of mutant P53 protein in the other groups were significantly decreased (30.2%, 14.6%, 19.8%, 1 7.3% vs60.0%, P <0.05). The expressions of Bax protein in each group were significantly increased (40.7%, 40.4%, 72.1%, 68.9%, 42.2% vs30.0%, P < Compared with YHRJG group, the expression of Bcl-2 protein was significantly decreased (26.3%, 24.4% vs 30.5%, P <0.05), while the expression of Bcl-2 was significantly increased in YHRJD, YHRJG + DDP and YHRJG + DDP groups (41.8%, 39.3% , 45.6% vs30.5%, P <0.05). The protein expression of NF-kB in each group was significantly decreased (15.9%, 13.3%, 14.1%, 7.8%, 14.6% vs24.2%, P < Results of hybridization showed that the expression of NF-κB mRNA in each group was significantly decreased (30.5%, 13.3%, 21.4%, 17.4%, 53.2% vs58%, P <0.05) Bcl-2mRNA expression was significantly decreased (0.717 ± 0.198vs1.327 ± 0.097, P <0.001) in DDP, YHRJD and YHRJG groups (46.22 ± 6.22,56.19 ± 7.36,62.32 ± 11.06 vs35.22 ± 4.38, P <0.05) .Conclusion: The possible gene regulation mechanism of apoptosis induced by YHRJ-containing serum on Bel-7402 cells is that apoptosis is promoted by inhibiting the expression of FasL gene signal transduction gene Regulate gene Bax gene protein expression, inhibit NF-kB gene mRNA and protein expression to achieve.