siRNA干扰Cx37对鼠胃癌皮下移植瘤的影响

发布时间：2018-12-15 16:15

【摘要】：第一部分筛选Cx37基因的有效si RNA干扰序列目的:筛选有效Cx37-si RNA干扰序列。方法:根据Cx37基因序列设计si RNA干扰片段,与Cx37基因过表达载体共转染HEK293细胞,检测Cx37基因蛋白表达水平,确定有效Cx37-si RNA干扰序列。结果:通过Western-blot分析,从三种不同的Cx37-si RNA中确定了2条可有效抑制Cx37基因蛋白表达的si RNA序列。选择Cx37-si RNA-3片段,进行下游si RNA干扰序列慢病毒表达载体及慢病毒颗粒的构建。结论:成功筛选Cx37基因的有效si RNA干扰序列。第二部分构建携带Cx37-si RNA有效干扰序列载体的慢病毒颗粒及MFC胃癌细胞小鼠皮下移植瘤模型目的:构建MFC胃癌细胞小鼠皮下移植瘤模型,向瘤体注射携带Cx37-si RNA有效干扰序列载体的慢病毒颗粒,并确定Cx37基因表达的抑制情况。方法:利用RPMI 1640完全培养基培养MFC小鼠胃癌细胞系。在30只雌性BALB/c-nu/nu小鼠前肢皮下接种0.2 ml MFC小鼠胃癌细胞悬液(2.5×107cells/ml),建立皮下移植瘤模型。将小鼠随机分成3组,分别向实验鼠瘤体内注入携带Cx37-si RNA的慢病毒颗粒、携带阴性对照-si RNA的慢病毒颗粒和生理盐水。实验终结时,测量3组小鼠体重及移植瘤。用10%中性福尔马林溶液固定移植瘤,石蜡包埋后切片,进行苏木精-伊红染色,并用荧光显微镜观察Cx37-si RNA载体中GFP基因蛋白表达及分布情况。提取小鼠移植瘤总RNA和总蛋白,通过RT-PCR和Western-blot法检测Cx37 m RNA和蛋白表达水平。结果:利用荧光显微镜可见瘤体内存在GFP蛋白的大量表达,提示携带Cx37-si RNA的慢病毒转染成功。各组小鼠体重无明显差异。Cx37-si RNA组移植瘤中Cx37基因m RNA的表达水平明显低于mock-si RNA组和生理盐水组(P0.05),Cx37基因m RNA表达水平在mock-si RNA组和生理盐水组之间无明显差异。Western-blot分析显示,Cx37-RNAi组移植瘤中Cx37蛋白表达水平明显低于mock-si RNA组和生理盐水组(P0.05)。结论:向MFC胃癌细胞小鼠皮下移植瘤中注射携带Cx37-si RNA有效干扰序列载体的慢病毒颗粒有效抑制了Cx37基因表达。第三部分干扰MFC胃癌细胞小鼠皮下移植瘤中Cx37基因表达对瘤体细胞增殖和凋亡的影响目的:明确注射Cx37-si RNA慢病毒对MFC胃癌细胞小鼠皮下移植瘤瘤体细胞增殖和凋亡的影响。方法:按照第二部分的实验方法,利用40只雌性BALB/c-nu/nu小鼠构建MFC胃癌细胞小鼠皮下移植瘤模型,随机分成4组:未注射组、Cx37-si RNA组、mocksi RNA组和生理盐水组。分别向后三组实验鼠瘤体内注入携带Cx37-si RNA的慢病毒颗粒、携带阴性对照-si RNA的慢病毒颗粒和生理盐水。8周后,利用CCK8方法检测各组移植瘤的细胞增殖能力,利用Annexin V-FITC和PI染色方法、Hoechst33342染色方法,以及Bcl-2和Bax m RNA及蛋白表达水平的检测,明确瘤体内细胞凋亡情况。结果:CCK8方法检测各组移植瘤的细胞增殖能力结果显示,Cx37-si RNA组移植瘤细胞增殖能力明显低于其他三个实验组(P0.01)。Annexin V-FITC和PI染色方法检测各组移植瘤细胞凋亡情况结果发现,Cx37-si RNA组移植瘤中凋亡细胞数明显高于mock-si RNA组和生理盐水组(P0.05)。Hoechst33342染色实验结果发现,Cx37-si RNA组移植瘤中细胞核浓缩的阳性细胞数量明显高于mock-si RNA组和生理盐水组(P0.01)。RT-PCR及Western-blot实验结果显示,Cx37-si RNA组移植瘤中Bcl-2和Bax的m RNA和蛋白表达比值均明显低于mock-si RNA组和生理盐水组(P0.01),Mock-si RNA组与生理盐水组相比Bcl-2/Bax没有明显差异。结论:干扰Cx37基因表达水平可有效抑制MFC胃癌细胞小鼠皮下移植瘤细胞增殖能力,并可促进移植瘤细胞凋亡。
[Abstract]:The first part screens the effective si RNA interference sequence of the Cx37 gene: screening an effective Cx37-si RNA interference sequence. Methods: The expression level of Cx37 gene was detected and the effective Cx37-si RNA interference sequence was determined by co-transfecting the HEK293 cells with the Cx37 gene overexpression vector according to the Cx37 gene sequence. Results: The si-RNA sequence of the expression of Cx37 gene can be effectively inhibited from three different Cx37-si RNA by Western-blot analysis. The Cx37-si RNA-3 fragment was selected for the construction of the downstream si RNA interference sequence lentiviral expression vector and lentiviral particles. Conclusion: The effective si RNA interference sequence of Cx37 gene was successfully screened. the second part constructs the slow virus particles carrying the Cx37-si RNA effective interference sequence carrier and the model of the subcutaneous transplantation of the MFC gastric cancer cell mouse: constructing a subcutaneous transplantation tumor model of the MFC gastric cancer cell mouse, and injecting the slow virus particles carrying the Cx37-si RNA into the tumor body to effectively interfere with the sequence carrier, and the expression of Cx37 gene was determined. Methods: The mouse gastric cancer cell line was cultured with RPMI 1640 medium. In 30 female BALB/ c-nu/ nu mice, 0.2 ml of MFC mouse gastric cancer cell suspension (2.5-107cells/ ml) was subcutaneously inoculated with 0.2 ml of MFC mouse gastric cancer cell suspension (2.5-107cells/ ml). The mice were randomly divided into three groups, and the lentiviral particles carrying Cx37-si RNA were injected into the mouse tumor and the lentiviral particles and physiological saline of negative control-si RNA were carried. At the end of the experiment, the body weight and the transplanted tumor of group 3 mice were measured. The expression and distribution of GFP gene in Cx37-si RNA vector were observed by means of a 10% neutral buffered formalin, paraffin-embedded section, and hematoxylin-eosin staining. The total RNA and total protein of the mice were extracted, and the expression level of Cx37 mRNA and protein was detected by RT-PCR and Western-blot. Results: The expression of GFP in the tumor was detected by fluorescence microscope, and the transfection of the lentivirus with Cx37-si RNA was successful. There was no significant difference in body weight of each group. The expression of Cx37 mRNA in the Cx37-si RNA group was significantly lower than that of the Mock-si RNA group and the normal saline group (P0.05). The expression level of Cx37 gene was not significantly different between the Mock-si RNA group and the normal saline group. Western-blot analysis showed that the expression of Cx37 in Cx37-RNAi group was significantly lower than that of the Mock-si RNA group and the normal saline group (P0.05). Conclusion: The expression of Cx37 gene was effectively inhibited by the injection of the lentiviral particles with the effective interfering sequence vector of Cx37-si RNA into the subcutaneous transplanted tumor of the MFC. The third part of the study on the effect of Cx37 gene expression on the proliferation and apoptosis of the tumor cells in the mice with MFC gastric cancer cells: the effect of the injection of Cx37-si RNA lentivirus on the proliferation and apoptosis of the tumor cells of the mice with MFC gastric cancer cells was well-defined. Methods: According to the experimental method of the second part, 40 female BALB/ c-nu/ nu mice were used to construct the subcutaneous transplantation tumor model of MFC gastric cancer cells, and the mice were randomly divided into 4 groups: the non-injection group, the Cx37-si RNA group, the mcksi RNA group and the normal saline group. The lentiviral particles carrying Cx37-si RNA and the lentiviral particles and physiological saline with negative control-si RNA were injected into the mice of the three experimental mice respectively. After 8 weeks, the cell proliferation ability of each group of transplanted tumors was detected by the CCK8 method, and the Hoechst33342 staining method was used by the method of Annexin V-FITC and PI staining. and the expression level of the Bcl-2 and Bax mRNA and the protein expression level are detected, and the cell apoptosis in the tumor is determined. Results: The results showed that the proliferation ability of Cx37-si RNA group was significantly lower than that of the other three experimental groups (P0.01). The number of apoptotic cells in the Cx37-si RNA group was significantly higher than that of the Mock-si RNA group and the normal saline group (P0.05). The results of the Hoechst33342 staining showed that the number of positive cells in the nucleus of the Cx37-si RNA group was significantly higher than that of the Mock-si RNA group and the normal saline group (P0.01). The results of RT-PCR and Western-blot analysis showed that the number of cells in the Cx37-si RNA group was significantly higher than that of the Mock-si RNA group and the normal saline group (P0.01). The expression of Bcl-2 and Bax in the Cx37-si RNA group was significantly lower than that of the Mock-si RNA group and the normal saline group (P0.01). The Bcl-2/ Bax in the Mock-si RNA group was not significantly different from that of the normal saline group. Conclusion: The interference of Cx37 gene expression level can effectively inhibit the proliferation of the transplanted tumor cells in the mice with MFC, and can promote the apoptosis of the transplanted tumor cells.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.2

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