SiRNA沉默XIAP基因对骨肉瘤MG-63细胞生物学特性的影响
发布时间：2018-09-04 11:52
【摘要】：目的:骨肉瘤是一种临床常见的好发于青少年的成骨性恶性肿瘤,它恶性程度高,预后比较差。随着化学疗法、手术技术、骨重建等方法的进展,5年总生存率明显提高。阿霉素(adriamycin,ADM)、顺铂(diamminedichloroplantinum,DDP)是抗骨肉瘤的最有效药物,但肿瘤细胞对化疗药物产生耐药性是影响化疗疗效的主要原因。许多化疗药物通过诱导凋亡抗肿瘤生长。XIAP是凋亡调节因子家族中对凋亡调节作用最强的分子之一,同时是唯一的caspases抑制剂。研究显示它在多种恶性肿瘤中上调表达,并与肿瘤化疗耐药及预后差紧密相关。RNAi是近年来发展起来的新技术,si RNA已经成为继反义核酸、核酶之后基因治疗的又一新武器。为此,我们合成特异性干扰XIAP基因的si RNA,并在脂质体介导下转染骨肉瘤MG-63细胞,观察其对骨肉瘤细胞生长抑制以及细胞侵袭性的影响,以及对骨肉瘤MG-63细胞ADM药物敏感性及耐药性的影响,从而为新的骨肉瘤的治疗方法提供实验依据。方法:1根据XIAP基因已知序列化学合成1条含21-23个碱基的si RNA(XIAP-si RNA)及阴性对照(si RNA-neg);用化学合成的XIAP-si RNA转染骨肉瘤MG-63细胞,通过RT-PCR方法和流式细胞术检测转染前后MG-63细胞XIAP-m RNA和蛋白水平的表达情况,进一步观察转染前后骨肉瘤MG-63细胞增殖抑制的改变,流式细胞术和MTT法进一步检测转染前后MG-63细胞的凋亡及ADM对转染前后骨肉瘤MG-63细胞的半数抑制浓度(IC50),观察化学合成的特异性XIAP-si RNA对骨肉瘤细胞增殖抑制的影响以及对骨肉瘤MG-63细胞ADM药物敏感性的影响。2统计学方法采用SPSS 13.0进行分析处理,采用t检验和单因素方差分析,如多组间两两比较采用q检验,以P0.05认为差异有统计学意义。1 XIAP-si RNA对骨肉瘤MG-63细胞增殖有抑制作用,使细胞周期阻滞发生在细胞周期的G0/1期,并能诱导凋亡。2 XIAP-si RNA降低了XIAP m RNA和蛋白的表达水平。3 XIAP-si RNA联合ADM作用的细胞抑制率达(63.1±2.1)%,流式细胞仪分析显示XIAP-si RNA组细胞的凋亡率明显高于转染非特异性si RNA组、空载组及未转染组。结论:1特异性的化学合成XIAP-si RNA能够下调骨肉瘤MG-63细胞XIAP基因和蛋白表达,阻滞细胞周期于G0/1期和诱导凋亡。2特异性的化学合成XIAP-si RNA能增强骨肉瘤MG-63细胞对ADM的敏感性。RNAi技术有望成为肿瘤基因靶向治疗的一种新的途径。结果:
[Abstract]:Objective: osteosarcoma is a common osteogenic malignant tumor in adolescents, which has a high degree of malignancy and poor prognosis. With the progress of chemotherapy, surgical techniques and bone reconstruction, the 5-year overall survival rate improved significantly. Adriamycin (adriamycin,ADM) and cisplatin (diamminedichloroplantinum,DDP) are the most effective drugs against osteosarcoma. Many chemotherapeutic drugs are one of the most powerful apoptosis-regulating molecules in the family of apoptosis-regulating factors, and are the only caspases inhibitors. It has been shown that it is up-regulated in various malignant tumors and closely related to chemotherapy resistance and poor prognosis. RNAi, a new technique developed in recent years, has become a new weapon in gene therapy after antisense nucleic acid and ribozyme. Therefore, we synthesized si RNA, that specifically interfered with XIAP gene and transfected MG-63 cells into osteosarcoma MG-63 cells mediated by liposome, and observed its effects on the growth inhibition and cell invasion of osteosarcoma cells. The drug sensitivity and drug resistance of ADM in osteosarcoma MG-63 cells were also studied, so as to provide experimental evidence for the treatment of osteosarcoma. Methods one si RNA (XIAP-si RNA containing 21-23 bases and one negative control (si RNA-neg) were chemically synthesized according to the known sequence of XIAP gene, and then transfected into MG-63 cells of osteosarcoma by chemically synthesized XIAP-si RNA. The expression of XIAP-m RNA and protein in MG-63 cells before and after transfection was detected by RT-PCR and flow cytometry. The inhibition of proliferation of MG-63 cells in osteosarcoma was further observed before and after transfection. Flow cytometry and MTT were used to detect the apoptosis of MG-63 cells before and after transfection and the half inhibitory concentration (IC50) of ADM on osteosarcoma MG-63 cells before and after transfection. The effects of chemically synthesized specific XIAP-si RNA on the proliferation inhibition of osteosarcoma cells were observed. And the effect on the drug sensitivity of ADM in osteosarcoma MG-63 cells. 2 the SPSS 13.0 method was used to analyze the drug sensitivity of osteosarcoma cells. T test and single factor analysis of variance were used, such as Q test for comparison of multiple groups. The results showed that the difference was statistically significant (P0.05). 1 XIAP-si RNA could inhibit the proliferation of MG-63 cells of osteosarcoma, resulting in cell cycle arrest in G0 / 1 phase of cell cycle. Apoptosis induced by 2. 2 XIAP-si RNA decreased the expression level of XIAP m RNA and protein. 3. 3 XIAP-si RNA combined with ADM could inhibit the apoptosis of the cells (63.1 卤2. 1). Flow cytometry analysis showed that the apoptosis rate of XIAP-si RNA group was significantly higher than that of non specific si RNA transfected group. No load group and no transfection group. Conclusion the specific chemical synthesis of XIAP-si RNA can down-regulate the expression of XIAP gene and protein in osteosarcoma MG-63 cells. Blocking cell cycle at G0 / 1 phase and inducing apoptosis. 2 specific chemical synthesis of XIAP-si RNA can enhance the sensitivity of osteosarcoma MG-63 cells to ADM. RNAi technique may be a new approach to target tumor gene therapy. Results:
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R738.1
本文编号：2222024
[Abstract]:Objective: osteosarcoma is a common osteogenic malignant tumor in adolescents, which has a high degree of malignancy and poor prognosis. With the progress of chemotherapy, surgical techniques and bone reconstruction, the 5-year overall survival rate improved significantly. Adriamycin (adriamycin,ADM) and cisplatin (diamminedichloroplantinum,DDP) are the most effective drugs against osteosarcoma. Many chemotherapeutic drugs are one of the most powerful apoptosis-regulating molecules in the family of apoptosis-regulating factors, and are the only caspases inhibitors. It has been shown that it is up-regulated in various malignant tumors and closely related to chemotherapy resistance and poor prognosis. RNAi, a new technique developed in recent years, has become a new weapon in gene therapy after antisense nucleic acid and ribozyme. Therefore, we synthesized si RNA, that specifically interfered with XIAP gene and transfected MG-63 cells into osteosarcoma MG-63 cells mediated by liposome, and observed its effects on the growth inhibition and cell invasion of osteosarcoma cells. The drug sensitivity and drug resistance of ADM in osteosarcoma MG-63 cells were also studied, so as to provide experimental evidence for the treatment of osteosarcoma. Methods one si RNA (XIAP-si RNA containing 21-23 bases and one negative control (si RNA-neg) were chemically synthesized according to the known sequence of XIAP gene, and then transfected into MG-63 cells of osteosarcoma by chemically synthesized XIAP-si RNA. The expression of XIAP-m RNA and protein in MG-63 cells before and after transfection was detected by RT-PCR and flow cytometry. The inhibition of proliferation of MG-63 cells in osteosarcoma was further observed before and after transfection. Flow cytometry and MTT were used to detect the apoptosis of MG-63 cells before and after transfection and the half inhibitory concentration (IC50) of ADM on osteosarcoma MG-63 cells before and after transfection. The effects of chemically synthesized specific XIAP-si RNA on the proliferation inhibition of osteosarcoma cells were observed. And the effect on the drug sensitivity of ADM in osteosarcoma MG-63 cells. 2 the SPSS 13.0 method was used to analyze the drug sensitivity of osteosarcoma cells. T test and single factor analysis of variance were used, such as Q test for comparison of multiple groups. The results showed that the difference was statistically significant (P0.05). 1 XIAP-si RNA could inhibit the proliferation of MG-63 cells of osteosarcoma, resulting in cell cycle arrest in G0 / 1 phase of cell cycle. Apoptosis induced by 2. 2 XIAP-si RNA decreased the expression level of XIAP m RNA and protein. 3. 3 XIAP-si RNA combined with ADM could inhibit the apoptosis of the cells (63.1 卤2. 1). Flow cytometry analysis showed that the apoptosis rate of XIAP-si RNA group was significantly higher than that of non specific si RNA transfected group. No load group and no transfection group. Conclusion the specific chemical synthesis of XIAP-si RNA can down-regulate the expression of XIAP gene and protein in osteosarcoma MG-63 cells. Blocking cell cycle at G0 / 1 phase and inducing apoptosis. 2 specific chemical synthesis of XIAP-si RNA can enhance the sensitivity of osteosarcoma MG-63 cells to ADM. RNAi technique may be a new approach to target tumor gene therapy. Results:
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R738.1
【参考文献】
相关期刊论文 前2条
1 任淑华,王井伟,曲平,刘义,张伟,张林;THE EFFECT OF RNAI-MEDIATED GENE SILENCING ON HER-2/NEU GENE EXPRESSION IN LUNG ADENOCARCINOMA CELLS[J];Chinese Journal of Cancer Research;2005年02期
2 王井伟;曹磊;王宇;付军;田海梅;刘义;张伟;;EFFECT OF SURVIVIN-siRNA-MEDIATED GENE SILENCING ON SURVIVIN EXPRESSION IN OSTEOSARCOMA CELL LINE MG-63[J];Chinese Journal of Cancer Research;2006年04期
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