IL-24基因治疗小鼠黑色素瘤的实验研究

发布时间：2018-05-29 21:44

本文选题：重组质粒 + IL-24　；参考：《延边大学》2012年硕士论文

【摘要】：目的：研究重组质粒pEGFP-N1-IL-24基因在小鼠黑色素瘤移植瘤体内的表达及其对黑色素瘤的抑制作用。方法： 1.B16细胞培养从液氮罐里取出并复苏B16细胞,接种于含10%小牛血清的RPMI-1640培养液的培养瓶中进行培养,传代。 2.黑色素瘤B16细胞小鼠皮下移植瘤模型的建立待观察C57BL/6小鼠一周后,一般状况良好的情况下,收集对数生长期的B16细胞,在无菌条件下每只以5×10.cells/ml接种于小鼠背部皮下,观察小鼠移植瘤的生长情况。 3.IL-24基因治疗黑色素瘤B16细胞小鼠移植瘤的影响将荷瘤鼠随机分为3组,每组6只,在瘤体内注射脂质体包裹的重组质粒pEGFP-N1-IL-24(10μg)为实验组,注射脂质体包裹的空载质粒pEGFP-N1(10μg)、注射PBS(0.2m1)为阴性对照组。每3天注射一次,共注射4次。每次注射药物前先用游标卡尺测量肿瘤的长径(L)和短径(W),计算肿瘤体积,并绘制肿瘤的生长曲线。 4.在末次注射药物后7天断颈处死小鼠,剥离肿瘤组织。小部分组织冰冻用于RT-PCR鉴定,剩余部分组织冰冻用于Western蛋白印迹法检测Bax蛋白的表达。 5. RT-PCR方法检测移植瘤组织中IL-24mRNA的表达从各组移植瘤组织中提取总RNA,逆转录合成cDNA,与IL-24上下游引物,进行PCR反应,扩增产物进行琼脂糖凝胶电泳,并查看目的条带 6. Western蛋白印迹法检测移植瘤组织中的Bax蛋白的表达提取各组移植瘤组织中的蛋白,进行聚丙烯酰胺凝胶电泳,转膜,封闭,加入抗体与目的蛋白产生反应,显影、定影后,观察目的蛋白的表达水平。结果： 1.成功建立了黑色素瘤移植瘤小鼠模型,接种细胞后第12天小鼠皮下肿瘤直径均达5mm,肿瘤移植率达100%； 2.肿瘤生长曲线图表明重组质粒pEGFP-N1-IL-24组移植瘤的体积明显小于空载质粒pEGFP-N1组和PBS组,其差异有统计学意义(P0.05),而空载质粒组和PBS组移植瘤体积则无明显差异(P0.05)。与空载质粒pEGFP-N1组和PBS组相比,重组质粒pEGFP-Nl-IL-24组移植瘤生长缓慢,而空载质粒pEGFP-N1组和PBS组移植瘤生长曲线几乎平行； 3.各组的肿瘤组织中提取了RNA,进行RT-PCR检测,扩增产物经琼脂糖凝胶电泳显示重组质粒pEGFP-N1-IL-24组在611bp处有一阳性条带而空载质粒pEGFP-N1组和PBS组未出现此预期的条带； 4.各组移植瘤组织中提取蛋白进行Western蛋白印记法,结果在重组质粒pEGFP-N1-IL-24组上出现了Bax蛋白的表达,而空载质粒pEGFP-N1组和PBS组未出现Bax蛋白的表达。结论： 1. IL-24基因在黑色素瘤组织中稳定表达了其蛋白。 2. IL-24具有抑制黑色素瘤B16细胞小鼠移植瘤生长的作用。 3. IL-24对黑色素瘤组织有促进Bax蛋白分泌的作用。
[Abstract]:Aim: to investigate the expression of recombinant plasmid pEGFP-N1-IL-24 gene in murine melanoma xenografts and its inhibitory effect on melanoma. Methods: 1.B16 cells were extracted from liquid nitrogen tank and resuscitated. The B16 cells were cultured in RPMI-1640 culture flask containing 10% calf serum. 2. Establishment of subcutaneous tumor transplantation model of melanoma B16 cells in mice to be observed after one week after C57BL/6 mice were in good condition, B16 cells in logarithmic growth phase were collected and inoculated subcutaneously with 5 脳 10.cells/ml on the back of mice under aseptic condition. The growth of transplanted tumor in mice was observed. The effect of 3.IL-24 gene therapy on transplanted melanoma B16 cells mice were randomly divided into 3 groups, 6 mice in each group, and the recombinant plasmid pEGFP-N1-IL-24(10 渭 g encapsulated with liposome was injected into the tumor as experimental group. The blank plasmid pEGFP-N1(10 渭 g was injected with liposome, and the control group was injected with PBSX 0.2 m ~ (1). Once every 3 days, a total of 4 times. The tumor volume was calculated and the tumor growth curve was drawn by using Vernier caliper to measure the long diameter L) and the short diameter of the tumor before each injection. 4. The mice were killed 7 days after the last injection and the tumor tissue was removed. A small number of tissues were frozen for RT-PCR identification, while the rest were used for Western Western blot assay to detect the expression of Bax protein. 5. RT-PCR method was used to detect the expression of IL-24mRNA in transplanted tumor tissues. Total RNAs were extracted from each group of transplanted tumor tissues, then synthesized by reverse transcription, then reacted with IL-24 primers, reacted with PCR, amplified products were analyzed by agarose gel electrophoresis, and the target bands were examined. 6. Western Western blotting was used to detect the expression of Bax protein in the transplanted tumor tissue. The protein was extracted from the transplanted tumor tissue by polyacrylamide gel electrophoresis, transmembrane, blocking, reaction with the antibody and the target protein. The expression level of target protein was observed. Results: 1. The mouse model of melanoma transplanted tumor was established successfully. The diameter of subcutaneous tumor was 5 mm and the tumor transplant rate reached 100 mm on the 12th day after inoculation. 2. The tumor growth curve showed that the volume of transplanted tumor in the recombinant plasmid pEGFP-N1-IL-24 group was significantly smaller than that in the blank plasmid pEGFP-N1 group and the PBS group, and the difference was statistically significant (P 0.05), but there was no significant difference in the volume of the transplanted tumor between the no-load plasmid group and the PBS group. Compared with the no-load plasmid pEGFP-N1 group and PBS group, the growth of transplanted tumor was slower in the recombinant plasmid pEGFP-Nl-IL-24 group, but the growth curve was almost parallel between the no-load plasmid pEGFP-N1 group and the PBS group. 3. RNAs were extracted from tumor tissues of each group and detected by RT-PCR. Agarose gel electrophoresis showed that there was a positive band at 611bp in the recombinant plasmid pEGFP-N1-IL-24 group, but there was no such expected band in the pEGFP-N1 group and PBS group. 4. The expression of Bax protein was found in the recombinant plasmid pEGFP-N1-IL-24 group, but no Bax protein expression was found in the blank plasmid pEGFP-N1 group and PBS group. Conclusion: 1. IL-24 gene expressed its protein stably in melanoma tissue. 2. IL-24 can inhibit the growth of transplanted melanoma B 16 cells in mice. 3. IL-24 can promote the secretion of Bax protein in melanoma tissues.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.5

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