慢病毒介导狨猴B2m基因沉默的初步研究
发布时间：2018-09-14 17:11
【摘要】：[背景]免疫缺陷动物是指由于先天性遗传缺陷或用人工方法造成免疫系统一种或多种成分缺陷的动物,在研究人类许多疾病机理、研发药物、器官移植等工作中有重要的作用。构建这类疾病相关的基因修饰动物模型是探究人类各种免疫缺陷疾病分子机理和治疗靶点的一种有效手段。近年来许多科研团队尝试利用新兴的基因编辑技术构建各种灵长类动物疾病模型并取得一定进展。慢病毒载体因其具有能介导外源基因在非分裂细胞中稳定高效的表达、感染率高、免疫反应小等优点,现已成为构建转基因动物和基因治疗研究的重要手段。将慢病毒载体特性与RNAi特异性抑制同源基因表达的作用相结合,能在多种细胞中实现针对目标基因的沉默,从而特异性降低其表达,并产生持续而稳定的诱导靶基因沉默的效果。β 2-微球蛋白是一个高度保守的低分子量蛋白质,是主要组织相容性复合物MHCI类分子的轻链部分,功能是维持MHCI类分子的结构稳定和辅助其向细胞表面表达。已有研究证实B2m基因缺陷会导致小鼠体内缺乏CD4CD8+T细胞,造成免疫缺陷现象;现探究在灵长类动物体内下调B2m基因的表达后,是否会与啮齿类动物发生相似的分子机制。狨猴是小型非人灵长类动物,凭借其体型小、与人类的亲缘关系接近等优点,适合建立成为肿瘤动物模型,以便研究肿瘤发病机理及治疗方案的研究。[目的]选择与人类已验证过的B2m基因shRNA靶点完全同源的狨猴B2m基因序列,在细胞水平筛选狨猴B2m基因的有效沉默靶点,并进行验证。利用RNA干扰技术沉默狨猴B2m基因,分析狨猴B2m基因沉默后其外周血CD4和CD8细胞数量上的变化,以期为构建免疫缺陷狨猴模型奠定一定基础。[方法](1)在细胞水平筛选狨猴B2m基因的有效沉默靶点,然后与狨猴B2m基因进行序列比对,筛选到2条序列完全匹配,将它们合成shRNA干扰序列,并分别插入在介导RNA干扰的慢病毒载体上。将构建的两个慢病毒表达质粒在聚乙烯亚胺(polyethylenimine,PEI)介导下转染293T细胞,转染后48h,用实时荧光定量法检测转染细胞中B2m基因mRNA的水平。(2)分析狨猴CD抗原(CD4+和CD8+)的一般表达水平:现有的动物资源中共有20只未进行过实验的狨猴,每只抽取300ml后肢静脉血用于流式统计狨猴CD4和CD8细胞的一般水平,为后续的动物实验提供实验依据。(3)慢病毒介导狨猴B2m基因沉默:选择干扰效率大于70%的靶点包装成介导RNAi的慢病毒,将介导B2m基因沉默的慢病毒和空载对照病毒通过狨猴后肢静脉分别注射到实验组和对照组动物体内,每周取500ml后肢静脉血进行流式分析和血常规检测,分析狨猴CD4和CD8细胞和CD4/CD8的比值变化。[结果](1)通过与人源B2m基因shRNA序列进行比对,筛选出2个与狨猴完全同源的B2m沉默靶位点,分别位于狨猴B2mmRNA的405bp～425bp,780bp～800bp。结果为两个沉默靶点在转录水平的沉默效率分别是(46.54±7.91)%(p0.05)和(83.22 ±4.37)%(p0.0001),均有统计学意义,其中第二个靶点沉默效率大于70%,适合进行后续的动物水平实验。(2)将抽取的20份狨猴静脉血样进行流式分析,得到的结果进行统计学分析,以x ± s表示:CD4%的值为(32.9±7.3)%;CD8%的值为(24.9±6.6)%。CD4/CD8 的值为(1.47±0.45)。(3)将每周抽取的血样进行流式分析,结果显示与对照组狨猴相比,实验组狨猴血样中,CD4、CD8细胞数量以及CD4/CD8比值明显下降,表明慢病毒介导的对狨猴B2m基因进行RNAi后,对狨猴免疫水平有影响。[结论]本实验筛选得到有效的狨猴B2m RNAi慢病毒表达载体,完成了细胞水平的基因沉默效率验证。统计了本单位现有狨猴的CD4和CD8细胞的一般平均水平;包装的高滴度慢病毒能安全有效的感染狨猴,使狨猴CD4、CD8细胞及CD4/CD8比值均有降低趋势。
[Abstract]:[BACKGROUND] Immunodeficient animals refer to animals with one or more components of the immune system defect caused by congenital genetic defects or artificial methods. They play an important role in the study of many human diseases, drug research and development, organ transplantation and other work. In recent years, many scientific research teams have attempted to construct a variety of primate disease models by using new gene editing techniques and have made some progress. Lentiviral vectors, because of their ability to mediate the stable and efficient expression of foreign genes in non-divisive cells, have a high infection rate and are immune to infection. The combination of lentiviral vector characteristics with the specific inhibition of homologous gene expression by RNAi can silence target genes in a variety of cells, thus specifically reducing their expression and producing a sustained and stable induction target. Beta-2-microglobulin is a highly conserved low molecular weight protein and a light chain part of the major histocompatibility complex MHCI molecules. Its function is to maintain the structural stability of MHCI molecules and assist their expression on the cell surface. This study aims to explore whether the down-regulation of B2m gene expression in primates may have a similar molecular mechanism to that in rodents. Marmosets are small non-human primates, which are suitable for establishing tumor animal models because of their small size and close relationship with humans. [Objective] To select the B2m gene sequence which is completely homologous to the shRNA target of human B2m gene and screen the effective silencing target of B2m gene in marmosets at the cellular level. [Methods] (1) Screening the effective silencing targets of the B2m gene at the cellular level, then aligning with the B2m gene of the marmoset, the two sequences were completely matched, and the shRNA interference sequences were synthesized and inserted into the slow disease mediated by RNA interference respectively. Two lentiviral expression plasmids were transfected into 293T cells mediated by polyethylenimine (PEI). The level of B2m gene mRNA in the transfected cells was detected by real-time fluorescence quantitative method 48 hours after transfection. (2) Analysis of the general expression level of CD antigen (CD4 + and CD8 +) in marmosets: 20 of the existing animal resources were not improved. The venous blood from each hind limb of the experimental marmosets was collected for flow cytometric analysis of the general level of CD4 and CD8 cells, providing experimental basis for subsequent animal experiments. (3) Lentivirus-mediated B2m gene silencing in marmosets: selected lentiviruses with interference efficiency greater than 70% were packaged into lentiviruses that mediated RNAi, and lentiviruses and CD8 cells that mediated B2m gene silencing were used for flow cytometric analysis. No-load control virus was injected into the hind limb vein of the experimental group and the control group respectively, and 500 ml hind limb venous blood was taken weekly for flow cytometry analysis and blood routine test to analyze the ratio of CD4 and CD8 cells to CD4/CD8 changes. [Results] (1) By comparing the shRNA sequence of human B2m gene, two of them were screened completely with the marmosets. The homologous B2m silencing target sites were located in 405-425 BP and 780-800 BP of B2mmRNA of marmosets, respectively. The results showed that the silencing efficiency of the two silencing targets at transcriptional level was (46.54 (7.91)% (p0.05) and (83.22 (4.37)% (p0.0001), respectively. The silencing efficiency of the second target was higher than 70%, which was suitable for subsequent animal level. Experiments. (2) 20 venous blood samples of marmosets were analyzed by flow cytometry and the results were statistically analyzed. The values of CD4% and CD8% were (32.9 + 7.3)% and (24.9 + 6.6)%. The values of CD4 / CD8 were (1.47 + 0.45). (3) Weekly blood samples were analyzed by flow cytometry. The results showed that compared with the control group, the values of CD4% and CD8% in the experimental group were (32.9 + 7.3)%. The number of CD4, CD8 cells and the ratio of CD4 to CD8 were significantly decreased in blood samples, indicating that lentivirus-mediated RNAi of B2m gene in marmosets had an effect on the immune level of marmosets. [Conclusion] The valid expression vectors of B2m RNAi lentivirus in marmosets were screened and the cell-level gene silencing efficiency was verified. The average level of CD4 and CD8 cells in monkeys, and the high titer lentiviruses packaged in the package could infect marmosets safely and effectively, which made the ratio of CD4, CD8 cells and CD4/CD8 decrease.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;R-332
[Abstract]:[BACKGROUND] Immunodeficient animals refer to animals with one or more components of the immune system defect caused by congenital genetic defects or artificial methods. They play an important role in the study of many human diseases, drug research and development, organ transplantation and other work. In recent years, many scientific research teams have attempted to construct a variety of primate disease models by using new gene editing techniques and have made some progress. Lentiviral vectors, because of their ability to mediate the stable and efficient expression of foreign genes in non-divisive cells, have a high infection rate and are immune to infection. The combination of lentiviral vector characteristics with the specific inhibition of homologous gene expression by RNAi can silence target genes in a variety of cells, thus specifically reducing their expression and producing a sustained and stable induction target. Beta-2-microglobulin is a highly conserved low molecular weight protein and a light chain part of the major histocompatibility complex MHCI molecules. Its function is to maintain the structural stability of MHCI molecules and assist their expression on the cell surface. This study aims to explore whether the down-regulation of B2m gene expression in primates may have a similar molecular mechanism to that in rodents. Marmosets are small non-human primates, which are suitable for establishing tumor animal models because of their small size and close relationship with humans. [Objective] To select the B2m gene sequence which is completely homologous to the shRNA target of human B2m gene and screen the effective silencing target of B2m gene in marmosets at the cellular level. [Methods] (1) Screening the effective silencing targets of the B2m gene at the cellular level, then aligning with the B2m gene of the marmoset, the two sequences were completely matched, and the shRNA interference sequences were synthesized and inserted into the slow disease mediated by RNA interference respectively. Two lentiviral expression plasmids were transfected into 293T cells mediated by polyethylenimine (PEI). The level of B2m gene mRNA in the transfected cells was detected by real-time fluorescence quantitative method 48 hours after transfection. (2) Analysis of the general expression level of CD antigen (CD4 + and CD8 +) in marmosets: 20 of the existing animal resources were not improved. The venous blood from each hind limb of the experimental marmosets was collected for flow cytometric analysis of the general level of CD4 and CD8 cells, providing experimental basis for subsequent animal experiments. (3) Lentivirus-mediated B2m gene silencing in marmosets: selected lentiviruses with interference efficiency greater than 70% were packaged into lentiviruses that mediated RNAi, and lentiviruses and CD8 cells that mediated B2m gene silencing were used for flow cytometric analysis. No-load control virus was injected into the hind limb vein of the experimental group and the control group respectively, and 500 ml hind limb venous blood was taken weekly for flow cytometry analysis and blood routine test to analyze the ratio of CD4 and CD8 cells to CD4/CD8 changes. [Results] (1) By comparing the shRNA sequence of human B2m gene, two of them were screened completely with the marmosets. The homologous B2m silencing target sites were located in 405-425 BP and 780-800 BP of B2mmRNA of marmosets, respectively. The results showed that the silencing efficiency of the two silencing targets at transcriptional level was (46.54 (7.91)% (p0.05) and (83.22 (4.37)% (p0.0001), respectively. The silencing efficiency of the second target was higher than 70%, which was suitable for subsequent animal level. Experiments. (2) 20 venous blood samples of marmosets were analyzed by flow cytometry and the results were statistically analyzed. The values of CD4% and CD8% were (32.9 + 7.3)% and (24.9 + 6.6)%. The values of CD4 / CD8 were (1.47 + 0.45). (3) Weekly blood samples were analyzed by flow cytometry. The results showed that compared with the control group, the values of CD4% and CD8% in the experimental group were (32.9 + 7.3)%. The number of CD4, CD8 cells and the ratio of CD4 to CD8 were significantly decreased in blood samples, indicating that lentivirus-mediated RNAi of B2m gene in marmosets had an effect on the immune level of marmosets. [Conclusion] The valid expression vectors of B2m RNAi lentivirus in marmosets were screened and the cell-level gene silencing efficiency was verified. The average level of CD4 and CD8 cells in monkeys, and the high titer lentiviruses packaged in the package could infect marmosets safely and effectively, which made the ratio of CD4, CD8 cells and CD4/CD8 decrease.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;R-332
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