基于DNA微卫星不稳定探讨从痰论治胃癌机制

Microsatellite instability (MSI) has been identified as one of important mechanisms for development of gastric cancer, which is closely related with tumor cell proliferation. we used MKN-45 human gastric cancer orthotopic transplantation model in nude mice and found that this model can not only simulate the clinical progress of gastric cancer, but also the detection rate of microsatellite locis was 100% in the pre-experimental (70% MSI-H and 30% MSI-L). The mechanism of gastric cancer development including the healthy qi insufficient, phlegm, blood stasis,in traditional Chinese Medicine. While tumor "phlegm" for the proliferation of gastric cancer in particular need attention. Previous research had suggested that the inhibition rate of the tumor proliferation was up to 58% in the Xiaotan Sanjie recipe high-dose group. This project intends to further study the relationship between TCM "phlegm" and MSI in gastric cancer. The project includes human gastric cancer MKN-45 cell line culture in vitro, Xiaotan Sanjie recipe Chinese medicine serum intervention, then detect gastric cancer microsatellite locies and their regulation of apoptosis network IGFR Ⅱ, IGF, Bax gene expression; human gastric cancer MKN-45 nude mice orthotopic transplantation experiment in vivo including Xiaotan Sanjie recipe intervention, detection of microsatellite instability in tumor sites and IGFR Ⅱ, IGF, Bax apoptotic gene expression, to study the effects of Xiaotan Sanjie recipe intervention on the MSI. Explore the mechanism by the method of eliminate phlegm in traditional Chinese on the treatment of gastric cancer in order to seek for the evidence of clinical effective treatment.

微卫星不稳定已确定为胃癌发生发展的重要机制之一,与肿瘤细胞异常增殖关系密切。我们采用MKN-45人胃癌裸鼠原位移植模型研究发现：本模型不仅能模拟临床胃癌进展,同时预实验100%检测出微卫星位点（高度不稳定70%，低度不稳定30%）。中医认为胃癌发生发展的机制离不开正虚、痰、血瘀等，其中肿瘤"痰"对于胃癌增殖的影响需要尤其关注，前期研究表明高剂量组消痰散结方抑瘤率为58%，因此，本项目拟进一步深入研究"从痰论治"胃癌与微卫星不稳定之间的关系。本课题以MKN-45人胃癌细胞株体外培养，消痰散结中药血清干预，检测胃癌微卫星位点及其调控的凋亡网络IGFRⅡ、IGF、Bax基因表达； MKN-45人胃癌裸鼠原位移植体内实验，消痰散结方干预，检测肿瘤组织中微卫星不稳定位点及IGFRⅡ、IGF、Bax凋亡基因表达，研究消痰散结法对MSI的干预效果，探索消痰散结法对胃癌治疗作用机制以期寻找其临床治疗证据。

项目背景：胃癌发病率和死亡率呈增高趋势，实验研究证实DNA微卫星不稳定在胃癌的发生及预后中起着重要的作用。前期研究表明，消痰散结方对胃癌有明确的抑瘤作用，本课题通过MSI为作用靶点，探讨中药干预胃癌的作用机制。 .主要研究内容：通过体内和体外实验，体外实验运用消痰散结方中药血清干预MKN-45人胃癌细胞，检测其对胃癌细胞增殖、侵袭、迁移能力及凋亡的影响，通过其对人胃癌细胞微卫星不稳定性的作用，进一步检测胃癌细胞中与微卫星不稳定密切相关的错配修复蛋白hMLHl及二次突变相关蛋白转化生长因子-βII型受体（TGF-βRⅡ）、胰岛素样生长因子II型受体（IGFⅡR）和Bcl-2相关X蛋白（Bax）的表达水平；体内实验运用消痰散结方干预MKN-45人胃癌裸鼠原位移植荷瘤鼠，测量中药消痰散结方对MKN-45人胃癌裸鼠原位移植瘤生长的影响，检测消痰散结方对人胃癌裸鼠原位移植瘤微卫星不稳定性的作用，分析消痰散结方干预后对MKN-45人胃癌微卫星不稳定密切相关的错配修复蛋白hMLHl及二次突变相关蛋白TGF-βRⅡ、IGFⅡR和Bax的表达水平的影响。.重要结果：通过本课题实验研究，我们认为：.1、消痰散结方及其中药血清可以抑制MKN-45人胃癌细胞及MKN-45人胃癌裸鼠原位移植瘤的微卫星不稳定性，使其处于微卫星稳定状态。.2、消痰散结方抑制胃癌微卫星不稳定性的作用机制如下：.（1）消痰散结方及其中药血清可以上调MKN-45人胃癌细胞及MKN-45人胃癌裸鼠原位移植瘤中错配修复蛋白hMLHl的表达水平；.（2）消痰散结方中药血清可以上调MKN-45人胃癌细胞中二次突变相关蛋白TGF-βRⅡ、IGFⅡR和Bax的表达水平。.关键数据：体外和体内实验中结果趋势一致：在空白对照组中，五个微卫星位点中的D2S123、D5S346出现短串重复序列的某些延长和缩短，Bat-25、Bat-26、D17S250则无延长或缩短。而在消痰散结方组中D2S123、D5S34携带短串重复序列的基因的变异出现稳定趋势，提示消痰散结方干预后，可以使不稳定的短串重复序列趋于稳定。.科学意义：明确消痰散结方对胃癌微卫星不稳定的干预效果，探讨中药消痰散结方发挥此作用的可能机理，深入探讨消痰散结方从痰论治胃癌作用机制。