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利用抗肿瘤药靶—酪氨酸激酶从广西特色中草药中筛选和研究选择性抗肿瘤药
Screening and Research Selective Anti-Hepatoma Drugs from the Traditional Chuang and Yao Medicine in Guangxi by Tyrosine Kinase, Angiogenesis as Antitumor Drug Targets
【作者】 王金妮；
【导师】 梁钢；
【作者基本信息】 广西医科大学 ， 药理学， 2012， 硕士
【摘要】 目的：利用抗肿瘤药靶-酪氨酸激酶从18种广西常用中草药(瑶药、壮药)中筛选和研究选择性抗肿瘤药物方法：醇提取十八种广西特色中草药(其中瑶药8种,壮药10种),采用MTT法测定其体外对人肝癌SMMC-7721细胞的抑制作用；选取体外抑瘤作用较好的药物,根据血清药理学方法制备含药血清,观察其体外对人肝癌细胞SMMC-7721的抑制作用；通过细胞裂解液提取不同作用时间不同作用剂量下的人肝癌细胞SMMC-7721总蛋白,采用ELISA法测定人肝癌细胞SMMC-7721总蛋白中酪氨酸激酶(TPK)含量,观察不同药物提取物对其含量影响；通过建立鸡胚绒毛尿囊膜模型测定不同药物提取物抗血管生成的抑制作用；建立人肝癌细胞SMMC-7721裸鼠移植瘤模型,观察不同药物治疗组对裸鼠移植瘤的影响,HE染色观察肿瘤生长及其血管生长情况,免疫组化法检测瘤组织中VEGF.CD34的表达。结果：在筛选的特色药物中,瑶药2号、瑶药5号体外抑制人肝癌SMMC-7721的作用较强,IC50分别为O.080mg/ml,0.281mg/ml;血清药理学实验结果显示瑶药2号、瑶药5号、索拉菲尼的含药血清均显示出不同程度的体外抗肿瘤作用,其中10%瑶药2号含药血清组抑瘤作用与10%索拉菲尼含药血清组相比,抑制率为11.15%,抑制作用变强(P<0.05)；瑶药2号、瑶药5号可不同程度的降低酪氨酸激酶的含量(P<0.01)：12.5mg/L索拉菲尼、50mg/mL瑶药2号、12.5mg/mL～50mg/mL瑶药5号均有不同程度的抑制鸡胚尿囊血管生长作用,与空白对照组相比,索拉菲尼对各级血管均有明显抑制作用(P<0.01),瑶药2号高剂量组对二、三级血管有抑制作用(P<0.01),瑶药5号对三级血管有抑制作用,高剂量时可同时抑制一、二级血管(P<0.01)；索拉菲尼组,瑶药2号高剂量组、瑶药2号中剂量组肿瘤瘤重、相对体积明显低于阴性对照组(P<0.01),其抑瘤率分别为51.75%,47.71%,21.27%；HE染色显示索拉菲尼组、瑶药2号高剂量组肿瘤坏死区域明显大于阴性对照组；索拉菲尼组、瑶药2号高剂量组MVD明显低于阴性对照组(P<0.01),瑶药2号中剂量组与阴性对照组相比也有差异(P<0.05)；索拉菲尼组、瑶药2号高剂量组可明显降低肿瘤组织内VEGF的表达(P<0.01)。结论：瑶药2号、瑶药5号均可不同程度的抑制血管生长,可降低肿瘤细胞内酪氨酸激酶的含量。此外,瑶药2号还可降低肿瘤内微血管密度以及VEGF的表达。
【Abstract】 Objective:To screen and research targeting anti-hepatoma drugs from18kinds of the traditional Chinese herbal medicines from Yao medicin,Chuang medicine in Guangxi by antitumor drug target as tyrosine kinase.Methods:18Ethanol extracts of Yao medicines,Chuang medicines in Guangxi were obtained by cold-extraction alcohol,then the inhibitory activity of human hepatoma cell line SMMC-7721with MTT were determined in vitro.The medicated serums with obviously anti-tumor effect of enthanol extracts were prepared according to the serum pharmacology method and further observed its inhibiting effect in same tumor cell line by MTT.The total protein of human hepatoma cell line SMMC-7721at different time or concentration of extracts was extracted by cell lysis buffer,respectively.The content of tyrosine kinase in total protein of tumor cells was detected by Enzyme-linked immunosorbent assay (ELISA). The model of angiogenesis in allantocheriionof chick embroyo was established so as to observe antiangiogenesis effect of extracts by CAM assay in vivo.The models of xenografted tumor in nude mice with human hepatoma cell line SMMC-7721were established,and then observated the tumor growth by HE staining and expression of VEGF,CD34in tumor tissues of each group by immunohistochemistry method.Results:Yao medicine2and Yao medicine5had stronger anti-tumor ability to human hepatoma cell line SMMC-7721among the screening drugs,the IC50was0.080mg/ml,0.281mg/ml,respectively.Experimental results of serum pharmacology showed medicated serum inhibited the growth of human hepatoma cell line SMMC-7721in varying degrees.10%serum of Yao medicine2group had an obvious inhibition effect on tumor compared with10%serum of sorafenib group, the inhibitory rate was11.15%.There was significant differences between two groups (P<0.05).Yao medicine2and Yao medicine5reduced the level of tyrosine kinase (p<0.01).12.5mg/L sorafenib,50mg/mL Yao medicine2and12.5mg/mL-50mg/mL Yao medicine5inhibited the angiogenesis of CAM in varying degrees.Compared with control group,sorafenib had a significant inhibition on different levels of blood vessels(p<0.01).High dose of Yao medicine2had inhibition on two or three-level blood vessels (p<0.01).Yao medicine5had inhibition on three-level blood vessels,while inhibiting one or two-level blood vessels under high dosage (p<0.01).The tumor weight and tumor relative volume in the following groups,such as sorafenib group,high-dose Yao medicine2group and medium-dose Yao medicine2group,was significantly lower than control group(P<0.01). The result of HE staining showed that tumor necrotic area in sorafenib group and high-dosage Yao medicine2group was significantly greater than control group.MVD were lower of sorafenib group and high-dosage Yao medicine2group than that of control group (P<0.01).There were significant differences between medium-dose Yao medicine2group and control group (P<0.05). Sorafenib group and high-dose Yao medicine2group reduced significantly the expression of VEGF in tumor tissues (P<0.01).Conclusion:Yao medicine2and Yao medicine5not only can inhibit the growth of blood vessels in varying degrees,but also can reduces the content of tyrosine kinases in tumor cells.In addition,MVD and the expression of VEGF may be reduced because of Yao medicine2.