【作者】 杜永强；

【导师】 苏云明；

【作者基本信息】 黑龙江中医药大学 ， 中西医结合基础， 2009， 博士

【摘要】 目的：通过蝙蝠葛酚性碱(PAMD)抗肿瘤活性的体内实验研究，观察PAMD对胰腺癌(BXPC-3)荷瘤小鼠肿瘤的抑制作用及外周血TNF-α含量的影响；PAMD对胰腺癌(BXPC-3)荷瘤小鼠肿瘤组织中k-ras和DPC4基因及蛋白表达的影响；PAMD对胰腺癌(BXPC-3)细胞凋亡率的影响。方法：裸鼠40只，雌雄各半，采用右腋皮下接种胰腺癌(BXPC-3)肿瘤细胞，建立裸鼠胰腺癌动物模型，40只造模后小鼠随机分为五组，每组8只，分别为PAMD高剂量20mg／kg，PAMD中剂量10mg／kg，PAMD低剂量5mg／kg，阳性对照组给予注射用环磷酰胺1 5 6mg／kg，模型组给予同体积的生理盐水，另取8只作为空白组对照组(不接种瘤株，给予同体积的生理盐水)。观察PAMD对胰腺癌(BXPC-3)荷瘤小鼠移植瘤及免疫器官的影响，并按公式计算抑瘤率、脾脏指数；采用酶联免疫吸附试验(ELISA法)检测BXPC-3荷瘤小鼠外周血TNF-α含量；采用荧光定量PCR(Real-time PCR)技术、蛋白印迹技术(Western-blot)检测肿瘤k-ras和DPC4基因及蛋白表达的差异；采用流式细胞术(FCM)检测胰腺癌(BXPC-3)细胞凋亡率，从而判断PAMD对在体肿瘤的抑制作用及相关靶基因(k-ras、DPC4)表达的影响，探讨PAMD抗胰腺癌作用靶点。结果：PAMD高、中、低剂量对BXPC-3荷瘤小鼠均有明显抑制肿瘤生长的作用，抑瘤率分别为34．91％、52．83％、41．51％，与模型对照组比较具有统计学意义(P＜0．0 1)；同时PAMD中、低剂量组荷瘤小鼠的脾脏指数与模型对照组比较，差异无统计学意义(P＞0．05)，PAMD高剂量组荷瘤小鼠的脾脏指数与模型对照组比较，差异具有统计学意义(P＜0．05)；PAMD高、中、低剂量组、阳性对照组BXPC-3荷瘤小鼠外周血TNF-α含量较模型对照组明显升高，差异具有统计学意义(P＜0．01)；PAMD高、中、低剂量下调BXPC-3荷瘤小鼠瘤组织中K-ras mRNA及蛋白表达量，上调DPC4 mRNA及蛋白表达量，与模型对照组比较，差异具有统计学意义(P＜0．0 1)；PAMD高、中剂量组肿瘤细胞凋亡率明显升高，与模型对照组比较，差异具有统计学意义(P＜0．01)。结论：PAMD对BXPC-3荷瘤小鼠具有明显的抑瘤作用，其抑瘤作用可能通过调节机体免疫功能，充分调动细胞因子生物学活性，下调K-ras基因mRNA及蛋白表达，上调DPC4基因表达，影响TGF-β信号传导通路，诱导细胞凋亡和分化，从而抑制肿瘤生长。更多还原

【Abstract】 Objective:To observe the anti-tumor effect of PAMD(phenolicalkaloids of Menispermum dauricum)on tumor-bearingmice,so that we can study the content of TNF-αin peripheral bloodof tumor-bearing mice to observe the effect of on expression ofgene and protein content of k-ras and DPC4 in tumour tissues oftumor-bearing mice and to observe the effect on apoptosis rate ofBXPC-3.Methods:The 40 metastatic tumor model of tumor-bearingmice were established with BXPC-3 cell line by hypodermic ofright oxter.After 24h the mice divided into 5 groups at random,andeach group has eight mice.PAMD high-dose group was treated with20mg/kg、mid-dose group was treated with 10mg/kg、low-dosegroup was treated with 5mg/kg、control group was given bycyclophosphamidum 156mg/kg,was model group given byisopyknic physiological saline and blank group given isopyknicphysiological saline but no tumor established.The effect onimmune organ index was detected and the tumor control rate andthe index of spleen was calculate.ELISA was carried out aftertreatment with PAMD to detect the cotent of TNF-αin peripheralblood.Meanwhile,the effect on extression of gene and protein ofk-ras and DPC4 in tumour tissues of tumor-bearing mice wasdetected by Real-time Fluorescence Quantitative PCR andWestern-blot.Flow cytometry was used to detect the apoptosis rateof BXPC-3 cells line.So that the inhibition rate of tumorproliferation were detected and the effect of express on relatedtarget gene was decided.We can investigate the target of PAMD tocancer of pancreas.Results:PAMD could inhibite the tumor growth oftumor-bearing mice of BXPC-3 cell line,The inhibition rates were 34.91%,52.83%,41.51%,respectively,which has the statisticalsignificance(P＜0.01).Compared with the control group,the spleenindex of mid-dose group and low-dose grouphas statisticalsignificance(P＞0.05),while,the PAMD high-dose group could notshowed markedly effect on immune organ index (P＜0.05)Meanwhile,PAMD could decrease the content of TNF-αinperipheral blood of tumor-bearing mice,compared with the modelgroup with statistical significance (P＜0.01)PAMD coulddown-regulate the expression of gene of K-ras mRNA in tumortissues and increase the DPC4 mRNA and its protein expression,compared with the model group with statistical significance(P＜0.01)Meanwhile,PAMD could increase the apoptosis rate,showed markedly inhibiting action (P＜0.01)Conclusions:PAMD could inhibite the tumor growth intumor-bearing mice of BXPC-3 cell line.The effect of inhibitionwas related by regulating immunologic function,transferringbiological activity of cytokine,inhibiting gene product of K-ras,protein of P21,over-expression of K-ras mRNA,hoisting theexpression of gene DPC4,influenting TGF-βaccess of signaltransmit,induceing apoptosis and split apart,so that it caninhibiting the growth of tumor.更多还原