【作者】 马向华；

【导师】 马超英；

【作者基本信息】 西南交通大学 ， 中药学， 2013， 硕士

【摘要】 目的：探讨峨参提取物对于HEPG2、MG-63、B16、HELA细胞体外的抗肿瘤活性和促凋亡的作用研究；探讨峨参内酯的急性毒性以及峨参内酯对H22荷瘤小鼠的体内抗肿瘤活性及其分子机制。方法：应用MTT比色法检测不同浓度峨参提取物(0、2.5、5、10、20、40、80μg/m1)对HEPG2、MG-63、B16、HELA细胞增殖抑制作用,分别计算不同药物对肿瘤细胞生长的抑制率与半数抑制浓度IC5o；采用DAPI染色法、琼脂糖凝胶电泳法(DNA ladder法)检测峨参内酯对HEPG2、MG-63、B16、HELA细胞凋亡形态学观察；计算峨参内酯对昆明小鼠的半数致死量LDs0采用腹水型肝癌细胞株建立小鼠移植肿瘤模型,待造模成功后随机将小鼠分为7组,分别以峨参内酯高剂量、中剂量、低剂量组、峨参多糖组以及替加氟组作为治疗组,模型组及空白组给予0.5%CMC-Na作为对照。治疗结束后,观察小鼠的一般状态,胸腺指数、脾脏指数、药物的抑瘤率及不同药物组对小鼠的生命延长率,通过免疫组化技术初步检测给予不同剂量的肿瘤组织中bax、 bcl-2、P53的表达情况,探讨峨参抗肿瘤的分子机制。结果：1、峨参石油醚提取物对HEPG2、HELA细胞的抑制作用十分明显,经计算得出,峨参石油醚提取物对HEPG2、HELA细胞的IC50值分别为36.53μg/ml和18.25μg/ml,对于MG-63和B16细胞抑制作用较小；峨参氯仿提取物对HEPG2、HELA两种肿瘤细胞的IC50值分别为40.62μg/ml和45.66μg/ml,对于MG-63和B16细胞抑制作用较小；峨参乙酸乙酯提取物和峨参多糖几乎无体外抗肿瘤活性；用峨参内酯作用于四种肿瘤细胞48h后,对四种肿瘤细胞均具有显著的抑制作用,经计算得出,峨参内酯对HEPG2、MG-63、B16、HELA四种肿瘤细胞的IC50值分别为12.24μg/m1、14.37μg/ml、43.25μg/ml和18.72μg/ml,且随着药物浓度的增长,药物对肿瘤细胞的抑制率增长也十分显著,在药物浓度为到80gg/ml时,抑制率均达到了70%以上；而异峨参内酯仅对于B16细胞和MG-63细胞有较显著的抑制作用,对于Hela细胞和HEPG2细胞几乎无抗肿瘤作用,经统计学软件计算得出异峨参内酯对MG-63细胞和B16细胞的IC50值分别为34.68μg/ml和65.72μg/ml;2、DAPI染色后,荧光显微镜观察到,四种细胞的生长明显受到抑制,有明显的细胞核断裂和固缩,且随着峨参内酯浓度的增加,细胞凋亡现象更加明显,而对照组(0μg/ml)细胞完整,未见任何细胞凋亡迹象；DNA ladder检测细胞凋亡实验中,用不同浓度峨参内酯处理细胞后,药物组均产生不同程度的细胞凋亡特征性DNA梯带；3、通过对昆明小鼠给予峨参内酯的急性毒性实验实验表明,峨参内酯虽然具有较高的抗肿瘤活性,但是其急性毒性也较强,计算得出峨参内酯的LDso为151.52mg/kg,其95%可信区间为93.28mg/kg-252.94mg/kg;4、峨参内酯高剂量、中剂量、低剂量组以及替加氟组对H22小鼠肝癌实体瘤的抑制率分别为54.39%、63.74%、56.14%、53.80%；通过免疫组化技术初步检测给予不同药物剂量的肿瘤组织中bax、bcl-2、P53的表达情况,初步研究表明,与模型组相比,峨参内酯能显著提高小鼠的生命延长率,峨参内酯能够上调bax的表达,降低bcl-2/bax比值,从而起到诱导肝癌细胞H22凋亡的作用,而各剂量组p53虽然均有一定的表达,但是各实验组之间无显著差异。结论：峨参内酯对于HEPG2、MG-63、B16、HELA四种肿瘤细胞具有显著的体内外抗肿瘤活性,可以延长H22肝癌小鼠的生存期,其促进凋亡的途径之一可能为上调bax的表达,降低bcl-2/bax比值,从而起到诱导肿瘤细胞凋亡的作用。更多还原

【Abstract】 Objective To investigate the effect of the extracts of Anthriscus sylvestris (L.)Hoffm on inhibiting proliferation and inducing apoptosis HEPG2, MG-63, B16, HELA cells in vitro; To explore the acute toxicity of the Anthriscus lactone and the anti-tumor activity and molecular mechanism in H22tumor-bearing mice in vivo.Methods To detect the inhibitive effect of different concentration of the extracts of Anthriscus sylvestris (L.)Hoffm on proliferation of HEPG2, MG-63, B16, HELA cells in vitro after48h by MTT Assay method. And to calculate the IC50value of the petrol extract of the extracts of Anthriscus sylvestris (L.)Hoffm. To detect the cell apoptosis character of HEPG2, MG-63, B16, HELA cells by treating with different doses of the petrol extract of Anthriscus sylvestris (L.)Hoffm by the DAPI method and DNA ladder method. And to calculated the IC50value of the Anthriscus lactone; To establish the anmimal model by inoculating the H22cell line in the left oxter asepticly, and then devided the mice into7groups randomly. took the Anthriscus lactone high-dose, medium-dose and low-dose group, Anthriscus polysaccharide group and Tegafur group as the treatment group, model group and blank group were treated with0.5%CMC-Na as the control, then gave the mice drugs daily and weighed once a day respectively, after12days,7mice in each group were killed. Then to take out the tumor, thymus and spleen, and weighed them to calculate the mortality, inhibition rate, thymus index, spleen index, and the rate elongation of life. To detect the necrosis of tumor and expression of bax、bcl-2、P53by HE and immunohistochemistry method.Results1、The extracts of chloroform, petroleum of Anthriscus sylvestris (L.)Hoffm all had the better inhibitive effect on the proliferation of HEPG2、HELA cells, the IC50value were36.53μg/ml and18.25μg/ml on HEPG2、HELA cells by The extracts of petroleum. The IC50value were40.62μg/ml and45.66μg/ml on HEPG2、HELA cells by The extracts of chloroform. Anthriscus ethyl acetate extract and Anthriscus polysaccharide almost have no anti-tumor activity in vitro. The Anthriscus lactone had better inhibitive effect on the proliferation of the four tumor cells. The IC50value were12.24μg/ml、14.37μg/ml、43.25μg/ml and18.72μg/ml for HEPG2、MG-63、B16、HELA cells respectively. The rate of inhibitive effect was related with the concentration, and the rate of inhibitiv effect reached70%at the80μg/ml.The isoan-thricin had significantly inhibitive effect or the proliferation of B16cells and MG-63cells, and the IC50value were34.68μg/ml and65.72μg/ml for B16cells and MG-63cells respectively.2、By DAPI stained, then to observe the impact of different concentrations of Anthriscu lactone on HEPG2, MG-63, B16, HELA cells by gel electrophoresis method. These fou tumor cells’growth was suppressed, including the cell collapse, fragmentation of cel nucleus, pyknosis and so on. And higher concentrations, more obvious. The cells in the control group were intact. At different concentrations treated, The DNA apopsis electrophoresis lines were observed by DNA ladder method.3、The acute toxicity test of Anthriscus lactone used Kunming mice. The test showed the Anthriscus lactone althougth had higher anti-tumor activity, but also had higher acute toxicity. the LD50value was151.52mg/kg, the95%confidence interval was93.28mg/kg-252.94mg/kg.4、The inhibitive rate of high-dose group, medium-dose group, low-dose group, and the Tegafur group were54.39%、63.74%、56.14%、53.80%respectively. To detect the bax,bcl-2and P53expression in different group’s tumor tissues by immunohistochemical method. The preliminary study showed Anthriscus lactone can significantly improve the lives of mice and can upregulate the expression of bax and reduce the ratio of bcl-2and bax So Anthriscus lactone had the effect of inducing the H22cells apoptosis. Although each group’s p53had certain expression, but there was no significant differences.Conclusions The Anthriscus lactone had significant anti-tumor activity on HEPG2、 MG-63、B16、HELA cells in vivo. And can significantly improve the lives of mice which have H22tumor cells. Its one of the ways to promote apoptosis may be related with bax expression raised and the ratio of bcl-2and bax reduced.更多还原