wtp53协同中药白花蛇舌草对原发性肝癌作用机制的实验研究
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摘要
目的：探讨wtp53基因协同中药白花蛇草对原发性肝癌的作用机制，寻找一条基因治疗与中医中药综合治疗的新方法。方法：制备小鼠原发性肝癌的动物模型后，随机分为4组，肝癌组；基因治疗组：肿瘤局部注射外源wtp53腺病毒液0.2ml／次，4天1次，连续12天；基因+中药组：白花蛇草煎剂(1mg／ml)灌胃1.0ml／只／天，同时肿瘤局部注射外源wtp53腺病毒液0.2ml／次，连续12天；及中药组：白花蛇草煎剂灌胃1.0ml／只／天，共12天。应用光镜、TRAP-PCR-ELISA法、TUNEL法、免疫组化及免疫学方法对小鼠端粒酶活性、细胞凋亡、相关基因bcl-2\PCNA、免疫功能进行了检测。结果：经wtp53治疗的小鼠肝癌组织明显缩小，协同白花蛇草效果更明显。基因组和基因+中药组端粒酶活性明显降低，可见较多的凋亡细胞，凋亡相关基因调控蛋白bcl-2\PCNA表达增高，阳性细胞与凋亡细胞分布一致。免疫功能检测发现，服用白花蛇草的小鼠T淋巴细胞对抗原刺激的反应明显增强，T淋巴细胞受体(TCR)受体数量增多、血清细胞因子IL-2、GM-CSF分泌增强，RT-PCR检测发现，基因组、基因+中药组和单纯中药组均可见肝癌组织穿孔素mRNA表达。结论：Wtp53能够抑制肝癌组织生长，协同中药白花蛇草能提高对肿瘤细胞的抑制作用。wtp53通过抑制肝癌组织中端粒酶活性，下调bcl-2蛋白表达，抑制PCNA蛋白高表达而促进肿瘤细胞凋亡，发挥抗肿瘤作用。白花蛇草通过增强T淋巴细胞的转化功能、TCR受体的表达、IL-2、GM-CSF分泌增强及CTL细胞对靶细胞的杀伤介质穿孔素的高表达而提高机体的免疫反应。本实验结果表明，wtp53协同白花蛇草既可有效的抑制肝癌细胞的生长，又可提高机体对肿瘤的免疫反应，显示出协同效应。
Objective:To explore the mediated effects of tumor suppressor gene wild-type p53 and Chinese traditional medicine oldenlandia on primary carcinoma of liver and research the new method against tumor of combining tumor suppressor gene and Chinese traditional medicine. Methods:Animal mode of mice with primary carcinoma of liver was estabalished and the animal were divided into four groups randomly.hepotacarcinoma group(group I ) ;Gene therapy group(group II) : wild-type p53 adenovirus liquor(0.2ml) was injected to the tumor area once a day for 12 days;Group III:Gene and oldenlandia group,each animal in this group was perfused with oldenlandia decoction (lmg/ml)1.0ml/day and 0.2ml wild-type p53 adenovirus liquor injection at the tumor simultaneously for 12day and Oldenlandia group(groupIV),in which mice was perfused oldenlandia decoction(1.0ml/day) for 12days.Then the telomerase activity of tumor,cell apoptosis,bcl-2\PCNA expression and immune funciton markers were detected by corresponding methods. Results:The
tumor tissue is notably diminished in size with the treatment of wild-type p53 and minished more significantly at the Gene and oldenlandia group.The telomerase activity is deregulated obviously at the Gene group and Gene oldenlandia group,and many apoptotic cells can be observed.The tumors treated with wild-type p53 or wild-type p53 plus oldenlandia showed that presence and expression of apoptosis-mediated protein bcl-2\PCNA were demonstrated using irnmunohistochemistry.that bcl-2\PCNA positive cells showed the accordant distribution with the apoptotic cells.The T-lymphatic cells in mice perfused with oldenlandia decoction display the salient reaction to antigen stimulation and the amount of T cell antigen receptor is increased concomitant with the prominent excretion of serum cytokine IL-2 and GM-CSF.The perform mRNA expression
was affirmed in hepatocarcinoma tissues in Gene group and Gene plus oldenlandia group assessed by RT-PCR,whereas nontreated cells didn't show these changes. Conclusions: Wild-type p53 can significantly inhibit the growth of hepatocarcinoma, and the inhibiting function was enhanced with the use of oldenlandia;Wild-type p53 repressed hepatocarcinoma growth through restraining telomerase activity, deregulating bcl-2 and PCNA expression and inducing cancerous cell apoptosis and oldenlandia can reinforce the immunologic reaction through enhancing the T-lymphocytic conversion function,expression of TCR receptor,IL-2/GM-CSF excretion and elevating the expression of perforin mRNA.The study suggested that wild-type p53 and oldenlandia had synergistic effect in both inhibiting hepatocarcinoma cell growth and sensitizing the immunologic reaction in vivo to cancerous cells.
Objective:To explore the mediated effects of tumor suppressor gene wild-type p53 and Chinese traditional medicine oldenlandia on primary carcinoma of liver and research the new method against tumor of combining tumor suppressor gene and Chinese traditional medicine. Methods:Animal mode of mice with primary carcinoma of liver was estabalished and the animal were divided into four groups randomly.hepotacarcinoma group(group I ) ;Gene therapy group(group II) : wild-type p53 adenovirus liquor(0.2ml) was injected to the tumor area once a day for 12 days;Group III:Gene and oldenlandia group,each animal in this group was perfused with oldenlandia decoction (lmg/ml)1.0ml/day and 0.2ml wild-type p53 adenovirus liquor injection at the tumor simultaneously for 12day and Oldenlandia group(groupIV),in which mice was perfused oldenlandia decoction(1.0ml/day) for 12days.Then the telomerase activity of tumor,cell apoptosis,bcl-2\PCNA expression and immune funciton markers were detected by corresponding methods. Results:The
tumor tissue is notably diminished in size with the treatment of wild-type p53 and minished more significantly at the Gene and oldenlandia group.The telomerase activity is deregulated obviously at the Gene group and Gene oldenlandia group,and many apoptotic cells can be observed.The tumors treated with wild-type p53 or wild-type p53 plus oldenlandia showed that presence and expression of apoptosis-mediated protein bcl-2\PCNA were demonstrated using irnmunohistochemistry.that bcl-2\PCNA positive cells showed the accordant distribution with the apoptotic cells.The T-lymphatic cells in mice perfused with oldenlandia decoction display the salient reaction to antigen stimulation and the amount of T cell antigen receptor is increased concomitant with the prominent excretion of serum cytokine IL-2 and GM-CSF.The perform mRNA expression
was affirmed in hepatocarcinoma tissues in Gene group and Gene plus oldenlandia group assessed by RT-PCR,whereas nontreated cells didn't show these changes. Conclusions: Wild-type p53 can significantly inhibit the growth of hepatocarcinoma, and the inhibiting function was enhanced with the use of oldenlandia;Wild-type p53 repressed hepatocarcinoma growth through restraining telomerase activity, deregulating bcl-2 and PCNA expression and inducing cancerous cell apoptosis and oldenlandia can reinforce the immunologic reaction through enhancing the T-lymphocytic conversion function,expression of TCR receptor,IL-2/GM-CSF excretion and elevating the expression of perforin mRNA.The study suggested that wild-type p53 and oldenlandia had synergistic effect in both inhibiting hepatocarcinoma cell growth and sensitizing the immunologic reaction in vivo to cancerous cells.
引文
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