人IL-24基因对CD133阳性喉癌细胞作用的实验研究
发布时间：2019-01-19 10:57
【摘要】：目的：克隆人IL-24基因，并构建其真核表达载体；分离Hep-2喉癌细胞株中CD133阳性细胞（CD133+Hep-2），通过研究人IL-24基因在CD133+Hep-2细胞中的表达，探讨IL-24基因对CD133+Hep-2细胞的作用。 方法：分离正常人外周血单个核细胞，应用Trizol法提取单个核细胞的总RNA，将总RNA应用RT-PCR法反转录为cDNA，设计引物P1、P2，扩增人IL-24基因，琼脂糖凝胶电泳及基因测序正确后将其TA克隆入pMD19-T simple vector载体；通过NheI、XhoI双酶切人IL-24基因和真核表达载体pIRES2-ZsGreen1，构建人IL-24基因的真核表达载体pIRES2-ZsGreen1-hIL-24并应用酶切及基因测序验证；流式细胞仪（FCM）从Hep-2细胞中分选CD133+Hep-2细胞，将验证正确的pIRES2-ZsGreen1-hIL-24在脂质体2000介导下转入CD133+Hep-2细胞，并验证其表达情况；MTT、FCM检测人IL-24基因对CD133+Hep-2细胞生长的影响；裸鼠移植瘤实验检测转染人IL-24基因的CD133+Hep-2细胞的成瘤能力。 结果：在人外周血单个核细胞中成功克隆了621bp的人IL-24基因，基因序列测定结果与GenBank中报道一致；pMD19-T-hIL-24、pIRES2-ZsGreen1-hIL-24重组表达质粒酶切、基因测序验证正确；用脂质体介导法将重组质粒pIRES2-ZsGreen1-hIL-24成功转染入CD133+Hep-2细胞，经检验人IL-24基因整合入转染细胞并稳定表达；MTT法检测结果显示转入IL-24基因实验组的细胞生长与未转染及空载体对照组相比明显受到抑制（P0.05); FCM检测细胞凋亡的结果显示实验组与未转染及空载体对照组相比凋亡率明显增加（P0.05);裸鼠移植瘤实验证实转入hIL-24基因的实验组CD133+Hep-2细胞裸鼠移植瘤的体积、重量明显低于未转染及空载体对照组移植瘤（P0.05)。 结论：人IL-24基因的表达能够抑制CD133+Hep-2细胞的增殖，促进其凋亡，，抑制CD133+Hep-2细胞裸鼠移植瘤的生长。
[Abstract]:Objective: to clone human IL-24 gene and construct its eukaryotic expression vector. CD133 positive cells (CD133 Hep-2) were isolated from Hep-2 laryngeal carcinoma cell lines. The effect of IL-24 gene on CD133 Hep-2 cells was studied by studying the expression of human IL-24 gene in CD133 Hep-2 cells. Methods: normal human peripheral blood mononuclear cells (PBMC) were isolated. Total RNA, of mononuclear cells was extracted by Trizol method. Total RNA was reverse transcribed into cDNA, designed primer P1CP2to amplify human IL-24 gene. The TA was cloned into pMD19-T simple vector after correct agarose gel electrophoresis and gene sequencing. The eukaryotic expression vector pIRES2-ZsGreen1-hIL-24 of human IL-24 gene was constructed by NheI,XhoI double enzyme digestion of human IL-24 gene and eukaryotic expression vector pIRES2-ZsGreen1, and verified by enzyme digestion and gene sequencing. Flow cytometry (FCM) (FCM) was used to separate CD133 Hep-2 cells from Hep-2 cells. The correct pIRES2-ZsGreen1-hIL-24 was transferred into CD133 Hep-2 cells mediated by liposome 2000 and its expression was confirmed. The effect of human IL-24 gene on the growth of CD133 Hep-2 cells was detected by MTT,FCM, and the tumorigenic ability of CD133 Hep-2 cells transfected with human IL-24 gene was detected by tumor transplantation assay in nude mice. Results: the human IL-24 gene of 621bp was cloned successfully in human peripheral blood mononuclear cells, and the results of gene sequencing were consistent with those reported in GenBank. The recombinant plasmid pIRES2-ZsGreen1-hIL-24 was successfully transfected into CD133 Hep-2 cells by liposome-mediated transfection, and the human IL-24 gene was integrated into the transfected cells and expressed stably. The results of MTT assay showed that the growth of the cells transferred into the IL-24 gene group was significantly inhibited compared with the untransfected and empty vector control group (P0.05). The results of FCM showed that the apoptotic rate of the experimental group was significantly higher than that of the untransfected and empty vector control group (P0.05). Tumor transplantation in nude mice confirmed that the volume and weight of transplanted tumor of CD133 Hep-2 cells transferred to hIL-24 gene in nude mice were significantly lower than those in untransfected and empty vector control group (P0.05). Conclusion: the expression of human IL-24 gene can inhibit the proliferation of CD133 Hep-2 cells, promote their apoptosis and inhibit the growth of xenografts of CD133 Hep-2 cells in nude mice.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.65
[Abstract]:Objective: to clone human IL-24 gene and construct its eukaryotic expression vector. CD133 positive cells (CD133 Hep-2) were isolated from Hep-2 laryngeal carcinoma cell lines. The effect of IL-24 gene on CD133 Hep-2 cells was studied by studying the expression of human IL-24 gene in CD133 Hep-2 cells. Methods: normal human peripheral blood mononuclear cells (PBMC) were isolated. Total RNA, of mononuclear cells was extracted by Trizol method. Total RNA was reverse transcribed into cDNA, designed primer P1CP2to amplify human IL-24 gene. The TA was cloned into pMD19-T simple vector after correct agarose gel electrophoresis and gene sequencing. The eukaryotic expression vector pIRES2-ZsGreen1-hIL-24 of human IL-24 gene was constructed by NheI,XhoI double enzyme digestion of human IL-24 gene and eukaryotic expression vector pIRES2-ZsGreen1, and verified by enzyme digestion and gene sequencing. Flow cytometry (FCM) (FCM) was used to separate CD133 Hep-2 cells from Hep-2 cells. The correct pIRES2-ZsGreen1-hIL-24 was transferred into CD133 Hep-2 cells mediated by liposome 2000 and its expression was confirmed. The effect of human IL-24 gene on the growth of CD133 Hep-2 cells was detected by MTT,FCM, and the tumorigenic ability of CD133 Hep-2 cells transfected with human IL-24 gene was detected by tumor transplantation assay in nude mice. Results: the human IL-24 gene of 621bp was cloned successfully in human peripheral blood mononuclear cells, and the results of gene sequencing were consistent with those reported in GenBank. The recombinant plasmid pIRES2-ZsGreen1-hIL-24 was successfully transfected into CD133 Hep-2 cells by liposome-mediated transfection, and the human IL-24 gene was integrated into the transfected cells and expressed stably. The results of MTT assay showed that the growth of the cells transferred into the IL-24 gene group was significantly inhibited compared with the untransfected and empty vector control group (P0.05). The results of FCM showed that the apoptotic rate of the experimental group was significantly higher than that of the untransfected and empty vector control group (P0.05). Tumor transplantation in nude mice confirmed that the volume and weight of transplanted tumor of CD133 Hep-2 cells transferred to hIL-24 gene in nude mice were significantly lower than those in untransfected and empty vector control group (P0.05). Conclusion: the expression of human IL-24 gene can inhibit the proliferation of CD133 Hep-2 cells, promote their apoptosis and inhibit the growth of xenografts of CD133 Hep-2 cells in nude mice.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.65
【共引文献】
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