【作者】 孙蓓；

【导师】 娄建石；

【作者基本信息】 天津医科大学 ， 药理学， 2016， 硕士

【摘要】 目的:研究丹参类化合物,主要是二氢丹参酮（dihydrotanshinone,DTS）及丹参酮Ⅰ（tanshinoneⅠ,TanⅠ）对肺癌细胞凋亡作用及调控蛋白表达的影响。通过实验,探讨二氢丹参酮（dihydrotanshinone,DTS）对人肺腺癌GLC-82细胞增殖的抑制作用,检测其对细胞周期和凋亡的影响,验证前期试验显著变化的蛋白表达并探讨其作用机制。建立动物模型,探讨丹参酮Ⅰ（tanshinoneⅠ,TanⅠ）联合照射对Lewis肺癌荷瘤小鼠的抑瘤作用及其机制。方法:体外培养人肺腺癌GLC-82细胞株,分为空白对照组和DTS给药组,MTT法检测不同浓度DTS(5,10,20,40,80,100μg·mL^-1)分别在24h和48h对GLC-82细胞增殖的抑制作用,确定其抑制作用是否具有时间和剂量依赖性,并计算抑制率;设置不同的给药时间梯度给予DTS,通过流式细胞术,PI单染发检测肺癌细胞的各个周期分布时相比例,通过AnnexinV/PI双染法检测DTS作用不同时间对GLC-82细胞凋亡的影响;运用蛋白印记法（Western blot）检测Bcl-2,Bax和Caspase-3蛋白的表达情况。通过接种Lewis肺癌细胞建立C57BL/6小鼠Lewis肺癌移植瘤模型,随机分为模型对照组、5-FU联合照射对照组、TanⅠ（10、20、40mg·kg-1）联合照射组。观察荷瘤鼠饮食状况,计算肿瘤相对体积、肿瘤生长延缓时间;剥瘤称重,计算抑瘤率;TUNEL法检测组织的凋亡情况。免疫蛋白印记法（Western blot）检测Bcl-2和Bax蛋白的表达情况。结果:MTT法检测表明,给予不同浓度的DTS后,对GLC-82细胞的生长有明显抑制作用且呈时间依赖性,随DTS给药浓度的增加,细胞增殖抑制率明显升高,与空白对照组相比有显著性差异（P<0.05或P<0.01）;DTS可诱导GLC-82细胞凋亡,并随给药时间的增加诱导凋亡的作用增强,给药组与空白对照组相比有显著性差异（P<0.05或P<0.01）;Western blot表明:随着DTS作用时间的增加,Bax蛋白表达无明显变化,Bcl-2蛋白表达逐渐减少,同时激活caspase家族蛋白,上调Caspase-3蛋白表达,下调Bcl-2的蛋白表达。不同剂量的TanⅠ联合照射后对Lewis肺癌移植瘤小鼠的研究表明:不同剂量TanⅠ给药联合照射后,对肿瘤的生长有一定的抑制作用,高剂量TanⅠ（40mg·kg-1）联合照射的抑瘤作用较强,且TanⅠ中剂量（20mg·kg-1）、高剂量（40mg·kg-1）组具有较强的放射增敏作用,并随剂量的增加,放射增敏作用逐渐增强。其抑瘤率分别为:27.14%、38.46%、59.78%,显著高于模型对照组（P<0.01）。高剂量TanⅠ组可见细胞皱缩,凋亡指数（apoptosis index AI）为51.3%,显著高于模型对照组（P<0.05）,同时能下调Bcl-2蛋白表达,并显著上调Bax蛋白表达,进而诱导细胞凋亡,且高剂量TanⅠ（40mg·kg-1）联合照射组与阳性对照5-FU联合照射组的放射增敏作用效果相近,均能诱导细胞凋亡。结论:DTS对肺腺癌GLC-82细胞有较强的细胞毒性,并诱导GLC-82细胞凋亡,其作用机制可能是通过下调Bcl-2的蛋白表达并激活Caspase-3蛋白的活性,从而起到诱导细胞凋亡的作用。TanⅠ能有效抑制Lewis肺癌移植瘤小鼠肿瘤的生长,高剂量TanⅠ联合照射组可见细胞皱缩的凋亡细胞,其机制可能是通过下调Bcl-2蛋白表达,上调Bax蛋白表达诱导细胞凋亡,进而发挥放射增效作用。更多还原

【Abstract】 Objective:This study aims to（1）determine the influence of dihydrotanshinone（DTS）and tanshinone I（Tan I）on the apoptosis of lung carcinoma cells and the mechanisms by which these compounds regulate protein expression;（2）determine,through experiments,the inhibitory effect of DTS on human lung adenocarcinoma GLC-82 cell proliferation and the influence of DTS on cell cycles and apoptosis;and（3）confirm the significant change of the protein expression in preliminary research and explore the mechanism of protein expression.An animal model was established to investigate the tumor suppression effect and mechanism of Tan I combined with radiotherapy on Lewis lung carcinoma-bearing mice.Methods:Human lung adenocarcinoma GLC-82 cells cultured in vitro were divided into blank control group and DTS group.The inhibitory effects of different concentrations(5,10,20,40,80,and 100μg·mL^-1)of DTS on the proliferation of GLC-82 cell were examined through MTT assay to confirm if the inhibitory effect of DTS was time-and dose-dependent,and the suppression ratio was calculated.Different time gradients of DTS administration were set.The proportion of different GLC-82 cell cycle phases was examined through flow cytometry and single propidium iodide（PI）staining.The effects of different action time of DTS on GLC-82 cell apoptosis were examined using annexin V/PI double staining.Expression of Bcl-2,Bax,and Caspase-3 proteins were detected through Western blot.A cancer xenograft model of Lewis lung in C57BL/6mice was established by inoculating Lewis lung cancer cells and randomly dividing into model control group,5-FU+radiotherapy control group（postive control group）,and Tan I(10,20,and40mg·kg^-1)+radiotherapy group.Dietary status of tumor-bearing mice was observed,and the relative volume of tumor and tumor growth delay time were calculated.The tumors were isolated and weighed to calculate the tumor inhibition rate.Cell apoptosis was analyzed through TUNEL.The expression of Bcl-2 and Bax proteins were analyzed through Western blot.Results:MTT assay showed a significant,time-dependent inhibitory effect of different concentrations of DTS on GLC-82 cell growth.With the increase of the DTS concentration,the inhibition rate of cell proliferation significantly increased compared with that of the blank control group（P<0.05 or P<0.01）.DTS could induce GLC-82 cell apoptosis,and the effect increased with increased administration time.A significant deference was observed between treatment groups and blank control group（P<0.05 or P<0.01）.Western blot indicated that increased DTS action time did not significantly change the expression level of Bax protein,whereas the expression of Bcl-2 protein decreased gradually.The caspase family proteins were activated,and the expression of caspase-3 protein was upregulated,whereas that of Bcl-2 protein was downregulated.Different doses of Tan I combined with radiotherapy had a certain inhibitory effect on the growth of Lewis lung cancer xenograftin mice.The inhibitory effect of high-dose(40 mg·kg^-1)Tan I combined with radiotherapy was stronger,whereas moderate-(20 mg·kg^-1)and high-dose(40 mg·kg^-1)Tan I alone had a relatively strong and positively dose-dependent radiosensitization effect.The inhibitory rates were 27.14%,38.46%,and 59.78%,respectively,which were significantly higher than that of the model control group（P<0.01）.In high-Tan-I-dose group,cell shrinkage was observed with an apoptosis index of 51.3%,which is significantly higher than that of the model control group（P<0.05）.Additionally,high-dose Tan I downregulated Bcl-2 protein expression but significantly upregulated Bax protein expression to induce cell apoptosis.Moreover,high-dose Tan I(40mg·kg^-1)+radiotherapy and 5-FU+radiotherapy presented a similar radiosensitization effect,and both could induce cell apoptosis.Conclusions:DTS has a potent cytotoxity against lung adenocarcinoma GLC-82cells and could induce cell apoptosis.The mechanism of action may be related to the downregulation of Bcl-2 protein expression and activation of Caspase-3 protein to induce apoptosis.Tan I can effectively suppress the growth of cancer xenograft of Lewis lung in mice and has a certain tumor inhibition effect.High Tan I dose causes cell shrinkage possibly by downregulating Bcl-2 protein expression and upregulating Bax protein expression to induce cell apoptosis and then exerting a radiosensitization effect.更多还原