【作者】 王俊华；

【导师】 何靖康；

【作者基本信息】 苏州大学 ， 心胸外科， 2011， 硕士

【摘要】 第一部分南美响尾蛇神经毒素对人肺鳞癌SK-MES-1细胞体外生长抑制作用及其相关机制目的:观察南美响尾蛇神经毒素(Crotoxin,CrTX)体外抗肿瘤作用,并初步探讨其相关作用机制。方法:以人肺鳞癌SK-MES-1细胞为实验对象,通过四氮唑盐还原法(MTT)、细胞集落平板克隆实验观察CrTX、吉非替尼(Iressa)及两药联合应用对SK-MES-1细胞的生长抑制作用;应用流式细胞术检测CrTX对SK-MES-1细胞凋亡率及细胞周期的影响;细胞间接免疫荧光染色法检测p-JNK蛋白表达情况;蛋白免疫印迹法(Western blot)测定CrTX作用后SK-MES-1细胞内p-JNK、BCL-2及Caspase-3活性亚单位P17蛋白表达水平,并以SP600125抑制JNK活性后观察CrTX对人肺鳞癌SK-MES-1细胞的影响。结果:CrTX对人肺鳞癌SK-MES-1细胞有生长抑制作用,与Iressa联用可增强Iressa抗肿瘤效果;CrTX对SK-MES-1细胞有诱导凋亡和G1期阻滞作用,并可引起人肺鳞癌SK-MES-1细胞p-JNK、Caspase-3活性亚单位P17蛋白表达上调及抑凋亡基因BCL-2下调;而用SP600125抑制JNK活性后,CrTX对人肺鳞癌SK-MES-1细胞诱导凋亡作用基本消失,p-JNK、Caspase-3活性亚单位P17及抑凋亡基因BCL-2蛋白表达水平无明显变化。结论:CrTX对人肺鳞癌SK-MES-1细胞体外生长有抑制作用,其作用与诱导凋亡和G1期阻滞有关,其中诱导凋亡作用系激活JNK信号通路、磷酸化BCL-2,进而活化Caspase-3所致。第二部分南美响尾蛇神经毒素对人肺鳞癌SK-MES-1细胞裸鼠移植瘤生长抑制作用研究目的:观察南美响尾蛇神经毒素(Crotoxin,CrTX)对人肺鳞癌SK-MES-1细胞裸鼠移植瘤生长抑制作用及其对移植瘤组织微血管生成的影响。方法:裸鼠皮下接种人肺鳞癌SK-MES-1细胞建立裸鼠移植瘤模型,分对照组,CrTX(10ug/kg生理盐水稀释后腹腔注射2次/周)组,吉非替尼(Iressa)(100 mg/kg/day)组,联合用药组(CrTX+Iressa);给药四周后比较移植瘤瘤重,计算抑瘤率;并用免疫组化法测定移植瘤微血管密度(microvessd density,MVD)及透射电镜观察移植瘤微血管内皮超微结构改变。结果:CrTX,Iressa均对人肺鳞癌SK-MES-1细胞移植瘤生长有抑制作用,二者联合应用可显著提高抑瘤作用,其抑瘤率分别是21.36%、22.88%及35.92%,差异有统计学意义(均P<0.05);免疫组化MVD计数示CrTX组、Iressa组及联合用药组均较对照组明显减少,差异有统计学意义(均P<0.05);电镜观察示:CrTX组、Iressa组及联合用药组移植瘤微血管数量较对照组明显减少,微血管内皮细胞结构有不同程度损害,其中联合用药组最为明显。结论: CrTX对人肺鳞癌SK-MES-1细胞移植瘤生长有抑制作用,并可减少移植瘤组织内微血管生成。更多还原

【Abstract】 Part 1 In vitro inhibition of Crotoxin on hunman lung squamous cell line SK-MES-1 and the related mechanismsObjective To observe the anti-tumor effect of Crotoxin in vitro and explore its related mechanisms.Methods SK-MES-1 cells, a cell line of human lung squamous, were used as the experimental materials. MTT colorimetry was used to test the growth inhibition ratio, and the flat colony experiment reflected the colony formation of cells. Both of the experiments were used to estimate the effects of Crotoxin and its combination with Iressa. The flow cytometry was used to detect the apoptosis rate and the cell cycle of SK-MES-1 cells treated with Cortoxin,Iressa and their combination. Changes in the expression of p-JNK were determined with immunofluorescence cytochemistry. Additionally, we also used Western Blot to detect the expression of some proteins, such as p-JNK, BCL-2, p17, the active subunit of Caspase-3. Futhermore,we used SP600125 to inhibit the activity of JNK to observe whether SK-MES-1 cells have some changes after treated by Crotoxin.Results Crotoxin can not only inhibit the growth of SK-MES-1 but also has additive effect when it is combined with Iressa. Cortoxin can induce SK-MES-1 cells to apoptosis and block them in G1 period, and it can also down-regulate the expression of BCL-2, and up-regulate the expression of p17, p-JNK. When using SP600125 to inhibit the activity of JNK, the apoptosis almost disappeared.Conclusion Crotoxin can inhibit the growth of SK-MES-1 cells and the anti-tumor effect was closely related to the induction of apoptosis and cell cycle blockage. The mechanism of Crotoxin to induce apoptosis was contributed to the activation of p-JNK and the down-regulate the expression of BCL-2, which further activate Caspase-3. Part 2 Growth-inhibiting effect of Crotoxin on SK-MES-1 cells xenografts in nude miceObjective To observe the inhibitory effects of Crotoxin on the SK-MES-1 cells in mude mice.Methods SK-MES-1 cells were incubated and were inoculated subcutaneously in athymic nude mice to establish xenograft models. The mice were divided into control group, Crotoxin group, Iressa group and the CrTX+Iressa group. The inhibitory rate was calculated according to the weights of xenografts. The intratumoral microvessel density(MVD) was evaluated by immunohistochemical staining, and the electron microscop was used to observe the morphological changes of microvess endothelium.Result The inhibitory rate of Crotoxin group, Iressa group and the CrTX+ Iressa group were respective21.36%, 22.88%and35.92%. Immunohistochemical results revealed that the expression of MVD in Crotoxin group, Iressa group and the Crotoxin+Iressa group were significantly lower than those in control group(P<0.05); The decrease of microvessels in the xenograft tumor and morphological changes of endothelium were observed under electron microscop.Conclusion Crotoxin can inhibit the growth of SK-MES-1cell xenografts in nude mice, which may be due to the inhibit tumor angiogenesis.更多还原