發(fā)布時(shí)間：2018-09-14 17:11

【摘要】：[背景]免疫缺陷動(dòng)物是指由于先天性遺傳缺陷或用人工方法造成免疫系統(tǒng)一種或多種成分缺陷的動(dòng)物,在研究人類許多疾病機(jī)理、研發(fā)藥物、器官移植等工作中有重要的作用。構(gòu)建這類疾病相關(guān)的基因修飾動(dòng)物模型是探究人類各種免疫缺陷疾病分子機(jī)理和治療靶點(diǎn)的一種有效手段。近年來(lái)許多科研團(tuán)隊(duì)嘗試?yán)眯屡d的基因編輯技術(shù)構(gòu)建各種靈長(zhǎng)類動(dòng)物疾病模型并取得一定進(jìn)展。慢病毒載體因其具有能介導(dǎo)外源基因在非分裂細(xì)胞中穩(wěn)定高效的表達(dá)、感染率高、免疫反應(yīng)小等優(yōu)點(diǎn),現(xiàn)已成為構(gòu)建轉(zhuǎn)基因動(dòng)物和基因治療研究的重要手段。將慢病毒載體特性與RNAi特異性抑制同源基因表達(dá)的作用相結(jié)合,能在多種細(xì)胞中實(shí)現(xiàn)針對(duì)目標(biāo)基因的沉默,從而特異性降低其表達(dá),并產(chǎn)生持續(xù)而穩(wěn)定的誘導(dǎo)靶基因沉默的效果。β 2-微球蛋白是一個(gè)高度保守的低分子量蛋白質(zhì),是主要組織相容性復(fù)合物MHCI類分子的輕鏈部分,功能是維持MHCI類分子的結(jié)構(gòu)穩(wěn)定和輔助其向細(xì)胞表面表達(dá)。已有研究證實(shí)B2m基因缺陷會(huì)導(dǎo)致小鼠體內(nèi)缺乏CD4CD8+T細(xì)胞,造成免疫缺陷現(xiàn)象;現(xiàn)探究在靈長(zhǎng)類動(dòng)物體內(nèi)下調(diào)B2m基因的表達(dá)后,是否會(huì)與嚙齒類動(dòng)物發(fā)生相似的分子機(jī)制。狨猴是小型非人靈長(zhǎng)類動(dòng)物,憑借其體型小、與人類的親緣關(guān)系接近等優(yōu)點(diǎn),適合建立成為腫瘤動(dòng)物模型,以便研究腫瘤發(fā)病機(jī)理及治療方案的研究。[目的]選擇與人類已驗(yàn)證過(guò)的B2m基因shRNA靶點(diǎn)完全同源的狨猴B2m基因序列,在細(xì)胞水平篩選狨猴B2m基因的有效沉默靶點(diǎn),并進(jìn)行驗(yàn)證。利用RNA干擾技術(shù)沉默狨猴B2m基因,分析狨猴B2m基因沉默后其外周血CD4和CD8細(xì)胞數(shù)量上的變化,以期為構(gòu)建免疫缺陷狨猴模型奠定一定基礎(chǔ)。[方法](1)在細(xì)胞水平篩選狨猴B2m基因的有效沉默靶點(diǎn),然后與狨猴B2m基因進(jìn)行序列比對(duì),篩選到2條序列完全匹配,將它們合成shRNA干擾序列,并分別插入在介導(dǎo)RNA干擾的慢病毒載體上。將構(gòu)建的兩個(gè)慢病毒表達(dá)質(zhì)粒在聚乙烯亞胺(polyethylenimine,PEI)介導(dǎo)下轉(zhuǎn)染293T細(xì)胞,轉(zhuǎn)染后48h,用實(shí)時(shí)熒光定量法檢測(cè)轉(zhuǎn)染細(xì)胞中B2m基因mRNA的水平。(2)分析狨猴CD抗原(CD4+和CD8+)的一般表達(dá)水平:現(xiàn)有的動(dòng)物資源中共有20只未進(jìn)行過(guò)實(shí)驗(yàn)的狨猴,每只抽取300ml后肢靜脈血用于流式統(tǒng)計(jì)狨猴CD4和CD8細(xì)胞的一般水平,為后續(xù)的動(dòng)物實(shí)驗(yàn)提供實(shí)驗(yàn)依據(jù)。(3)慢病毒介導(dǎo)狨猴B2m基因沉默:選擇干擾效率大于70%的靶點(diǎn)包裝成介導(dǎo)RNAi的慢病毒,將介導(dǎo)B2m基因沉默的慢病毒和空載對(duì)照病毒通過(guò)狨猴后肢靜脈分別注射到實(shí)驗(yàn)組和對(duì)照組動(dòng)物體內(nèi),每周取500ml后肢靜脈血進(jìn)行流式分析和血常規(guī)檢測(cè),分析狨猴CD4和CD8細(xì)胞和CD4/CD8的比值變化。[結(jié)果](1)通過(guò)與人源B2m基因shRNA序列進(jìn)行比對(duì),篩選出2個(gè)與狨猴完全同源的B2m沉默靶位點(diǎn),分別位于狨猴B2mmRNA的405bp～425bp,780bp～800bp。結(jié)果為兩個(gè)沉默靶點(diǎn)在轉(zhuǎn)錄水平的沉默效率分別是(46.54±7.91)%(p0.05)和(83.22 ±4.37)%(p0.0001),均有統(tǒng)計(jì)學(xué)意義,其中第二個(gè)靶點(diǎn)沉默效率大于70%,適合進(jìn)行后續(xù)的動(dòng)物水平實(shí)驗(yàn)。(2)將抽取的20份狨猴靜脈血樣進(jìn)行流式分析,得到的結(jié)果進(jìn)行統(tǒng)計(jì)學(xué)分析,以x ± s表示:CD4%的值為(32.9±7.3)%;CD8%的值為(24.9±6.6)%。CD4/CD8 的值為(1.47±0.45)。(3)將每周抽取的血樣進(jìn)行流式分析,結(jié)果顯示與對(duì)照組狨猴相比,實(shí)驗(yàn)組狨猴血樣中,CD4、CD8細(xì)胞數(shù)量以及CD4/CD8比值明顯下降,表明慢病毒介導(dǎo)的對(duì)狨猴B2m基因進(jìn)行RNAi后,對(duì)狨猴免疫水平有影響。[結(jié)論]本實(shí)驗(yàn)篩選得到有效的狨猴B2m RNAi慢病毒表達(dá)載體,完成了細(xì)胞水平的基因沉默效率驗(yàn)證。統(tǒng)計(jì)了本單位現(xiàn)有狨猴的CD4和CD8細(xì)胞的一般平均水平;包裝的高滴度慢病毒能安全有效的感染狨猴,使狨猴CD4、CD8細(xì)胞及CD4/CD8比值均有降低趨勢(shì)。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q78;R-332

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本文編號(hào)：2243340