siRNA干擾Cx37對鼠胃癌皮下移植瘤的影響
發(fā)布時間：2018-12-15 16:15
【摘要】：第一部分篩選Cx37基因的有效si RNA干擾序列目的:篩選有效Cx37-si RNA干擾序列。方法:根據(jù)Cx37基因序列設計si RNA干擾片段,與Cx37基因過表達載體共轉染HEK293細胞,檢測Cx37基因蛋白表達水平,確定有效Cx37-si RNA干擾序列。結果:通過Western-blot分析,從三種不同的Cx37-si RNA中確定了2條可有效抑制Cx37基因蛋白表達的si RNA序列。選擇Cx37-si RNA-3片段,進行下游si RNA干擾序列慢病毒表達載體及慢病毒顆粒的構建。結論:成功篩選Cx37基因的有效si RNA干擾序列。第二部分構建攜帶Cx37-si RNA有效干擾序列載體的慢病毒顆粒及MFC胃癌細胞小鼠皮下移植瘤模型目的:構建MFC胃癌細胞小鼠皮下移植瘤模型,向瘤體注射攜帶Cx37-si RNA有效干擾序列載體的慢病毒顆粒,并確定Cx37基因表達的抑制情況。方法:利用RPMI 1640完全培養(yǎng)基培養(yǎng)MFC小鼠胃癌細胞系。在30只雌性BALB/c-nu/nu小鼠前肢皮下接種0.2 ml MFC小鼠胃癌細胞懸液(2.5×107cells/ml),建立皮下移植瘤模型。將小鼠隨機分成3組,分別向實驗鼠瘤體內注入攜帶Cx37-si RNA的慢病毒顆粒、攜帶陰性對照-si RNA的慢病毒顆粒和生理鹽水。實驗終結時,測量3組小鼠體重及移植瘤。用10%中性福爾馬林溶液固定移植瘤,石蠟包埋后切片,進行蘇木精-伊紅染色,并用熒光顯微鏡觀察Cx37-si RNA載體中GFP基因蛋白表達及分布情況。提取小鼠移植瘤總RNA和總蛋白,通過RT-PCR和Western-blot法檢測Cx37 m RNA和蛋白表達水平。結果:利用熒光顯微鏡可見瘤體內存在GFP蛋白的大量表達,提示攜帶Cx37-si RNA的慢病毒轉染成功。各組小鼠體重無明顯差異。Cx37-si RNA組移植瘤中Cx37基因m RNA的表達水平明顯低于mock-si RNA組和生理鹽水組(P0.05),Cx37基因m RNA表達水平在mock-si RNA組和生理鹽水組之間無明顯差異。Western-blot分析顯示,Cx37-RNAi組移植瘤中Cx37蛋白表達水平明顯低于mock-si RNA組和生理鹽水組(P0.05)。結論:向MFC胃癌細胞小鼠皮下移植瘤中注射攜帶Cx37-si RNA有效干擾序列載體的慢病毒顆粒有效抑制了Cx37基因表達。第三部分干擾MFC胃癌細胞小鼠皮下移植瘤中Cx37基因表達對瘤體細胞增殖和凋亡的影響目的:明確注射Cx37-si RNA慢病毒對MFC胃癌細胞小鼠皮下移植瘤瘤體細胞增殖和凋亡的影響。方法:按照第二部分的實驗方法,利用40只雌性BALB/c-nu/nu小鼠構建MFC胃癌細胞小鼠皮下移植瘤模型,隨機分成4組:未注射組、Cx37-si RNA組、mocksi RNA組和生理鹽水組。分別向后三組實驗鼠瘤體內注入攜帶Cx37-si RNA的慢病毒顆粒、攜帶陰性對照-si RNA的慢病毒顆粒和生理鹽水。8周后,利用CCK8方法檢測各組移植瘤的細胞增殖能力,利用Annexin V-FITC和PI染色方法、Hoechst33342染色方法,以及Bcl-2和Bax m RNA及蛋白表達水平的檢測,明確瘤體內細胞凋亡情況。結果:CCK8方法檢測各組移植瘤的細胞增殖能力結果顯示,Cx37-si RNA組移植瘤細胞增殖能力明顯低于其他三個實驗組(P0.01)。Annexin V-FITC和PI染色方法檢測各組移植瘤細胞凋亡情況結果發(fā)現(xiàn),Cx37-si RNA組移植瘤中凋亡細胞數(shù)明顯高于mock-si RNA組和生理鹽水組(P0.05)。Hoechst33342染色實驗結果發(fā)現(xiàn),Cx37-si RNA組移植瘤中細胞核濃縮的陽性細胞數(shù)量明顯高于mock-si RNA組和生理鹽水組(P0.01)。RT-PCR及Western-blot實驗結果顯示,Cx37-si RNA組移植瘤中Bcl-2和Bax的m RNA和蛋白表達比值均明顯低于mock-si RNA組和生理鹽水組(P0.01),Mock-si RNA組與生理鹽水組相比Bcl-2/Bax沒有明顯差異。結論:干擾Cx37基因表達水平可有效抑制MFC胃癌細胞小鼠皮下移植瘤細胞增殖能力,并可促進移植瘤細胞凋亡。
[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.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.2
本文編號：2380945
[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.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.2
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