【作者】 杜震生；

【导师】 吴万垠；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2012， 博士

【摘要】 背景原发性肝癌是我国常见恶性肿瘤之一,其治疗以手术、放化疗、介入性治疗、分子靶向药物治疗和中医药治疗为主。近些年,随着新健脾理气方(简称XJPLQF)治疗肝癌在临床上广泛应用并取得良好效果,许多实验对新健脾理气方治疗肝癌的机理进行了多方探讨,研究结果显示新健脾理气方抑制肿瘤的有多重作用。许多研究表明了EGFR-STAT3信号传导通路参与肿瘤的形成。本研究从EGFR-STAT3信号通路的角度初步探讨了新健脾理气方治疗肝癌的机理,为临床应用提供了依据。目的本试验目的在于从动物实验入手,研究新健脾理气方对肝癌EGFR-STAT3言号通路的作用及新健脾理气方治疗肝癌的机理。方法建立皮下实体瘤动物模型,然后将造模后的小鼠随即分成5组：荷瘤模型组(模型组)、CTX阳性对照组(CTX组)、新健脾理气方低剂量组(XJPLQF氏剂量组)、新健脾理气方中剂量组(XJPLQF中剂量组)和新健脾理气方高剂量组(XJPLQF高剂量组),另外设正常空白对照组(正常组),共6组,每组10只昆明种小鼠。空白对照组和荷瘤模型组用生理盐水灌胃,CTX组用环磷氮芥腹腔注射,各中药组以低、中、高剂量的新健脾理气方中药灌胃。停药后24小时取材。观察新健脾理气方对H22肝癌小鼠肿瘤细胞形态学的影响；应用流式细胞术检测新健脾理气方对H22肝癌小鼠外周血T淋巴细胞亚群CD4+、CD8+以及CD4+/CD8+的影响；应用免疫组化法检测H22肝癌小鼠肿瘤细胞FasL表达来判断新健脾理气方对肝癌小鼠肿瘤细胞免疫逃逸机制的影响,应用免疫组化法检测Bax、 Bcl-2表达判断新健脾理气方对H22肝癌小鼠肿瘤细胞凋亡相关基因蛋白表达的影响；应用免疫组化法检测EGFR、 TGF-α、 STAT3蛋白表达以及用原位杂交法检测H22肝癌小鼠肿瘤细胞的EGFR mRNA和STAT3mRNA表达,以判断新健脾理气方对EGFR-STAT3信号通路的影响。结果1.给药前各组小鼠体重无统计学差异(P>0.05)。给药第14天荷瘤模型组与各用药组及正常对照组的体重差异有统计学差异(P<0.05)。模型组体重大于其它用药组及正常对照组,提示模型组肿瘤不受抑制的生长,造成小鼠体重迅速增加的假象。抑瘤率比较发现,新健脾理气方各剂量组均有不同程度抑瘤作用,其中高剂量组效果较好。2.新健脾理气方各组的CD4+细胞值明显高于模型组(P<0.05),各组之间CD8+细胞值无统计学差异(P>0.05)；XJPLQF高剂量组的CD4+/CD8+比值明显高于模型组,差异有统计学意义(P<0.05)。3.各组小鼠肿瘤组织FasL均有阳性表达。组间比较,XJPLQF中、高剂量组小鼠肿瘤组织FasL的表达明显低于模型组(P<0.05)。而其他各组之间的FasL表达无统计学差异(P>0.05)。4.免疫组化法结果表明,各实验用药组和模型组在bax基因蛋白表达方面存在差异,XJPLQF高剂量组和CTX组bax基因蛋白表达均高于模型组,差异有统计学意义(P<0.05)。各实验用药组和模型组在bcl-2基因蛋白表达方面存在差异。XJPLQF高剂量组和CTX组bcl-2基因蛋白表达均低于模型组,差异有统计学意义(P<0.05)。5. EGFR-STAT3通路5.1免疫组化法：XJPLQF高剂量组及CTX组EGFR的染色阳性表达均低于模型组(P<0.05)。其余组之间EGFR染色阳性表达均无统计学差异(P>0.05)。XJPLQF高剂量组及CTX组TGF-α的染色阳性表达均低于模型组(P<0.05)。其余组之间TGF-α染色阳性表达均无统计学差异(P>0.05)。 XJPLQF高剂量组及CTX组STAT3的染色阳性表达均低于模型组(P<0.05)。其余组之间STAT3染色阳性表达均无统计学差异(P>0.05)。5.2原位杂交法：XJPLQF高剂量组及CTX组EGFR mRNA的阳性表达均低于模型组(P<0.05)。其余组之间EGFR mRNA阳性表达均无统计学差异(P>0.05)。XJPLQF高剂量组及CTX组STAT3mRNA的阳性表达均低于模型组(P<0.05)。其余组之间STAT3mRNA阳性表达均无统计学差异(P>0.05)。Spearman’s rho相关分析表明,EGFR mRNA与STAT3mRNA在H22肝癌小鼠肿瘤组织中的表现呈显著正相关(相关系数=0.530,P=-0.000)。结论1.新健脾理气方可能对H22肝癌小鼠肿瘤的生长的具有抑制作用。2.新健脾理气方可能具有改善机体的免疫功能状态,干预免疫逃逸的作用。3.新健脾理气方可能具有诱导H22肝癌小鼠肿瘤细胞凋亡的作用。4.新健脾理气方可能具有降低EGFR、 TGF-α(?)PSTAT3的表达,从而阻断EGFR-STAT3信号通路传导的作用。更多还原

【Abstract】 ObjectivesPrimary hepatoma is one of the common malignant tumors in China. Several familiar therapeutic means for patients with liver cancers are surgery radiotherapy and chemotherapy, interventional therapy, molecular targeted agents and TCM treatment. With the use of new jianpliqi formula (XJPLQF) the effect of XJPLQF on the formation and development of live cancer has been regarded as the key to the science. More and more researchs have shown that XJPLQF could increase immune status and upgrade survival rate, and the method of XJPLQF has been widely applied. XJPLQF is a kind of effective anti-tumor agent.PurposeDiscussion in role of XJPLQF on the hepatoma and EGFR-STAT3pathway can provide experimental proof for the treatment of hepatoma.MethodsH22hepato mouse model was made, and hepatocarcinoma cell line H22was chosen for tumor animal model. After tumors was built, animals were randomly divided into cancer bearing group, three dosages of XJPLQF groups, cyclophosphanmide group and normal group.Animals were feed with sodium, XJPLQF, and inject cyclophosphamide for14days. Then the effect of XJPLQF on hepatomal cells in mouse H22was observed by light microscope. Determination of lymphocyte subtypes CD4~+,CD8~+and CD4~+/CD8~+was performed by FACS. Determination in the expression of Bcl-2、 bax and FasL from tumor tissues were examed by immumohistochemistry. EGFR, TGF-α, STAT3in carcinogenesis were detected by immunohistochemistry too. In situ hybridization was applied to investigate the expression of STAT3mRNA and EGFR mRNA. Then numbers were statistically evaluated. Results1. Tumor quality in model group was bigger than in every treatment group after the tests (P<0.05).2. Through FACS, CD4~+lymphocytes raised in XJPLQF groups compared with model group (P<0.05), and there is no significant difference bewteen XJPLQF groups and cyclophosphamide group in CD8~+but there is significant difference in CD4~-/CD8~+bewteen XJPLQF high dosage group and model group(P<0.05).3. Through testing Bax and Bcl-2, increased apotosis can be deduced in treatment groups(P<0.05), especially in XJPLQF groups.There is no significant difference between XJPLQF groups and cyclophosphamide group.4. Through immumohistochemistry, compared with the cancer bearing group, the expression of FasL decreased in XJPLQF groups (P<0.05), and there is no statistical difference among indivial group.5. EGFR, TGF-α, STAT3were highly expressed at the model group, the expression of EGFR, TGF-α and STAT3has been found in all groups and the levels of EGFR and TGF-a in model group were statistically higher than that in treatment groups, similarlly the EGFR mRNA level in the model group was much higher than in XJPLQF high dosage group and cyclophosphamide group(P<0.05). The expression of STAT3mRNA was statistically different between XJPLQF high dosage group and model group (P<0.05).Conelution1. XJPLQF can inhibit the proliferation of hepatoma cells in mouse H22.2. XJPLQF can decrease the activity of hepatoma cells, and regulates lymphocytes subtypes CD4~+and CD4~+/CD8~+in peipheral blood, hence improves immune status in mouse H22. XJPLQF can reduce the activity of FasL, and hence promotes apoptosis in mouse H22.3. XJPLQF can degrade the expression of Bcl-2in mouse H22, and raised the expression of bax at the same time.4. XJPLQF can degrade the expression of EGFR, TGF-α and STAT3. In hepatoma, there was positive correlation between XJPLQF and EGFR-STAT3signal pathway expression.更多还原