【作者】 刘基巍；

【导师】 崔秀云；

【作者基本信息】 大连医科大学 ， 生物化学与分子生物学， 2004， 博士

【摘要】 人参皂甙Rg3[20(R)- Ginsenoside Rg3][1]是从红参中提取的微量中药单体,其分子式为C42H72O13,相对分子质量为784.30。人参皂甙Rg3的功效包括增效解毒、改善气虚症候、提高机体免疫力等[2,3]。另外,研究表明人参皂甙Rg3尚具有抑制肿瘤细胞的增殖、浸润和转移等作用[4]。肿瘤的发生和发展是一个极其复杂的过程,有多种转移相关的蛋白因子参与,并涉及多种转移途径和分子机制。人参皂甙Rg3抗肿瘤机理的研究为目前中药抗肿瘤研究热点之一。本研究工作主要包括:(1) 应用具有高淋巴道转移特性的肝细胞瘤株Hca-F25//6A3-F(F) 建立淋巴道转移模型,观察人参皂甙Rg3对肿瘤生长及淋巴道转移的抑制作用;采用免疫组化(IHC)方法分析人参皂甙Rg3对转移相关的蛋白因子,肿瘤细胞增殖核抗原[5,6](proliferating cell nuclear antigen , PCNA)、细胞周期蛋白依赖性激酶抑制蛋白[7,8](P16)和基质金属蛋白酶[9] (matrix metallo- proteinases, MMPs)表达的调节作用,探讨人参皂甙Rg3抗肿瘤作用的分子机制;并用电镜及流式细胞仪观察肿瘤细胞凋亡情况,探讨凋亡在人参皂甙Rg3抗淋巴道转移的作用。(2)建立核糖核酸酶抑制因子(Ribonuclease Inhibitor, RI)转基因B16黑色素瘤肺转移小鼠实验模型,观察人参皂甙Rg3对RI 转基因抗黑色素瘤细胞生长、浸润和转移的调控作用,探讨人参皂甙Rg3和/或RI 抗肿瘤新生血管作用及其相关性。(3)通过人参皂甙Rg3对体外培养的B16黑色素瘤细胞增殖的影响,观察肿瘤细胞凋亡情况,并通过RT-PCR和IHC方法检测凋亡的关键蛋白酶Caspase-3[10](胱氨酸-天冬氨酸蛋白激酶)基因及蛋白表达水平,进一步探讨人参皂甙Rg3诱导肿瘤细胞凋亡的机制。(4)临床应用人参皂甙Rg3辅助治疗乳腺癌化疗患者,观察其对乳腺癌患者化疗期间的生活质量和免疫功能的影响。 结果显示:(1)在肝细胞瘤株淋巴道转移的模型中,人参皂甙Rg3<WP=6>可抑制肿瘤的生长和转移,使PCNA及MMP-9在原发瘤肿瘤组织中的表达减少, P16的表达增加,与对照组相比差异显著(P<0.001);应用人参皂甙Rg3组在原发瘤及转移瘤中均观察到细胞凋亡的形态学变化,并检测到细胞凋亡峰。(2)在人参皂甙Rg3对RI 转基因B16黑色素瘤肺转移作用中,给药组(Rg3组)、RI 转基因组(RI 组)和联合组(Rg3+RI)荷瘤小鼠肺组织重量降低,降低的程度以联合组最明显,Rg3组和RI 组次之,与野生性对照组(W组)和空质粒转染组(B组)比较,有显著性差异(P(0.01);同时,肿瘤肺转移灶数目相应减少 (P(0.01);Rg3组、RI 组和联合组荷瘤小鼠的生存期延长, W组和B组动物全部死亡(至26天);肺内肿瘤组织的微血管密度经HE染色和第Ⅷ因子相关抗原的IHC分析显示, Rg3和RI使微血管密度降低, 降低程度与上述结果呈一致性。(3)体外培养的B16黑色素瘤细胞增殖实验显示人参皂甙Rg3抑制肿瘤细胞增殖呈量效关系;形态学观察、流式细胞学检测显示人参皂甙Rg3使S期细胞减少,肿瘤细胞的凋亡率增加; RT-PCR及IHC方法分析表明,人参皂甙Rg3上调Caspase-3基因及蛋白的表达。(4) 人参皂甙Rg3应用于乳腺癌化疗患者,使患者的CD4/ CD8 比值升高,明显高于对照组(P < 0. 001);Rg3组气虚证临床症状 (神疲乏力、呼吸气短、纳谷少馨、自汗等) 较对照组均有明显改善( P < 0. 05 或P < 0. 001)。本研究表明,人参皂甙Rg3具有抑制肿瘤生长和抗淋巴道转移的作用,与促进P16表达、降低PCNA和MMP-9表达及诱导肿瘤细胞凋亡有关。人参皂甙Rg3和RI转基因均使肿瘤肺转移灶内的微血管密度降低,肿瘤细胞浸润减少,说明人参皂甙Rg3和RI基因各自具有抗肿瘤生长和转移作用,其机理与抑制肿瘤新生血管生成相关,并且二者有协同作用。人参皂甙Rg3具有抑制B16黑色素瘤细胞增殖及促细胞凋亡作用,其机理与Rg3上调Caspase-3基因及蛋白的表达相关,其诱导肿瘤细胞凋亡作用为抗肿瘤机制的重要成因。乳腺癌患者化疗期间应用人参皂甙Rg3,可提高患者的免疫功能,改善气虚症状,并可提高生活质量。本文从基础研究及临床应用两个方面探讨人参皂甙Rg3抗肿瘤作用分子机制及在肿瘤辅助治疗中的价值,为临床合理应用人参皂甙Rg3提供依据,并为具有我国自主知识产权的抗癌I类新药人参皂甙Rg3走向国际市场奠定基础。更多还原

【Abstract】 Ginsenoside Rg3 is a kind of effective chemical component extracted tracely from the red Panix with molecular formula C42H72O13 and molecular weight 784.30 dalton. Many studies have shown that Ginsenoside Rg3 can decrease toxicity, improve symptoms of Qi –deficiency, increase immune function, and inhibit proliferation, infiltration and metastasis of the tumor cells. The etiology and development of tumor is very complicated, in which many related protein factors, some metastatic routes, as well as molecular mechanism are involved. The study of mechanism on anti-tumor effect of Ginsenoside Rg3 is now one of the attracting fields.The study includes: (1) By establishing the lymph-route metastatic model with high- potential metastatic tumor cell strain, Hca-F25//6A3-F(F), we observed the effect of Ginsenoside Rg3 on inhibiting proliferation and metastasis; analyzed the expression of metastasis related factors such as PNCA, P16 and MMPs by immunohistochemical assay (ICH) in order to study the molecular mechanism of Ginsenoside Rg3 in anti-lymphatic metastasis; moreover, the apoptosis analysis was undergone to evaluate its role of anti-tumor by light microscope, electronic microscope, and flow cytometry. (2) Using the lung metastasis mouse model implanted with RI-transgene B16 melanoma, we explored the anti-angiogenesis and the interaction of Ginsenoside Rg3 and RI by observing the inhibition of proliferation, invasion, and metastasis. (3) In vitro experiment of B16 melanoma cell line, we studied the gene and protein expression levels of Caspase-3 which is a key protease in apoptosis pathway by RT-PCR and IHC in order to explain the apoptosis mechanism induced by Ginsenoside Rg3. (4) We observed the quality of life and immune function of the breast cancer <WP=8>patients in chemotherapy while treated with Ginsenoside Rg3.The results showed that (1) in the lymph-route metastatic model experiment, Ginsenoside Rg3 could inhibit the growth and metastasis of tumor by increasing the expression level of p16, whereas decreasing those of PCNA and MMP-9 significantly compared with that of saline group in primary tumor tissue (P<0.001).The morphologic change showed that typical apoptic peak and more apoptosis micorobodies appeared in Ginsenoside Rg3 pretreatment and Ginsenoside Rg3 treatment groups compared with those of the other groups; (2) In the lung metastatic mouse model implanted with RI- transgene B16 melanoma, both Rg3 and RI could decrease the weight of lungs, with Rg3/RI group showing the greatest decrement among all other groups, the less extent for Rg3 and RI groups, which were significantly different from those of the W and B groups (P(0. 01).The lifespan of tumor- bearing mouse was shortened in W and B groups, with all animal dead at the 26th day of the experiment, while all mouse being alive during one and half month period of observation in Rg3/RI group. The microvessel density decreased among Rg3 and /or RI administrated groups, which showing consensus results with those of the inhibitory effect mentioned above by HE staining and Ⅷ-R Ag expression by IHC analysis; (3) The proliferation test of cultured B16 melanoma indicated that there was dose-dependent effect on inducing apoptosis by Ginsenoside Rg3. The morphological observation showed that characteristic change of apoptosis under light and fluorescent microscope. The results of RT-PCR and IHC showed that the expressions of Caspase-3 gene and protein were up-regulated; (4) In the clinical observation of the breast cancer patients in chemotherapy, the symptoms of Qi-deficiency in patients of Ginsenoside Rg3 group improved significantly, which was superior to the control group(P < 0. 05 or P < 0. 001). The level of T lymphocyte subtype ratio (CD4/ CD8) increased significantly in Ginsenoside Rg3 group compared with that of the control group ( P < 0. 001) .The study suggests that Ginsenoside Rg3 has anti-tumor effect, and the anti- lymphatic role , which is related to induce tumor apoptosis with up-regulation of p16 expression and down-regulation of PCNA an更多还原