重组腺病毒介导恒河猴MyD88基因沉默siRNA在恒河猴未成熟树突状细胞中的表达与鉴定
发布时间：2019-04-25 16:17
【摘要】：目的：鉴定恒河猴MyD88基因沉默siRNA的重组腺病毒载体在恒河猴未成熟树突状细胞(immature dendritic cell, imDC)中的表达情况及探讨恒河猴MyD88基因沉默siRNA腺病毒载体感染恒河猴imDC对T细胞免疫应答的影响。 方法：在静脉麻醉的情况下,抽取恒河猴骨髓血,使用猴淋巴细胞分离液将采取的骨髓血中的单个核细胞分离出来,利用免疫磁珠试剂盒和磁极正性分选CD34+阳性细胞,并对其进行纯化。在得到的细胞中添加培养基及细胞因子GM-CSF、IL-4进行培养。将上述CD34+阳性细胞培养并诱导至第7天,细胞计数后以每孔2.5×105个细胞接种于24孔板中,按MOI为100、200、300、400、500、600分别加入病毒,48小时后观察细胞绿色荧光色素蛋白的表达情况。将第一部分得到的CD34+阳性细胞培养并诱导至第7天,按MOI为500～600用重组腺病毒感染48小时提取蛋白,然后通过Western Blot检测技术检测蛋白表达情况。混合淋巴细胞反应,按照不同的方法处理刺激细胞将刺激细胞分为：DC组、imDC组、基因沉默imDC组、LPS+imDC组、LPS+基因沉默imDC组。以T细胞作为反应细胞,流式细胞术检测T细胞。根据刺激细胞：反应细胞比例为1：5,1：10,1：20,1：40加入反应细胞。通过统计学分析各组对T细胞的增殖情况。 结果：利用免疫磁珠法获得的DC纯度较高,使用细胞因子培养第四天后可诱导imDC成熟。重组腺病毒载体在恒河猴未成熟树突状细胞中的稳定感染的MOI为500-600。 Western Blot检测可见33kD处出现特异性条带,在43kD处可见β-actin内参成功表达,可推断目的蛋白在imDC中成功表达,且重组腺病毒载体在imDC中的蛋白表达明显低于空白组及阴性对照组。通过分析MyD88干扰组蛋白表达较空白对照组表达水平下调73.5%,MyD88干扰组蛋白表达较阴性对照组表达水平下调80.6%。尼龙毛柱法分离出的T细胞纯度大于80%,有实验意义,可作为反应细胞进行实验。混合淋巴细胞培养,由统计结果得出结论：imDC组刺激T细胞增殖的能力显著低于mDC组；重组腺病毒感染的imDC组刺激T细胞增殖的能力显著低于其他组。 结论：免疫磁珠法可纯化DC,细胞因子可诱导DC向imDC分化;当MOI为500～600时,重组腺病毒载体在恒河猴imDC中可获得最佳感染效率；恒河猴MyD88基因沉默siRNA在恒河猴imDC中的蛋白表达受到明显抑制；重组腺病毒感染的imDC可抑制T细胞的增殖。 展望：利用重组腺病毒感染未成熟树突状细胞,在进行体内实验时通过门静脉将感染后的imDC注入受体体内,希望可以诱导免疫耐受的产生,为移植领域开辟新道路。
[Abstract]:Objective: to identify the recombinant adenovirus vector of rhesus monkey MyD88 gene silencing siRNA in rhesus monkey immature dendritic cells (immature dendritic cell,). The expression of imDC in rhesus monkeys and the effect of rhesus monkey MyD88 gene silencing siRNA adenovirus vector on T cell immune response in rhesus monkeys infected with imDC were investigated. Methods: bone marrow blood was extracted from rhesus monkeys under intravenous anesthesia. Mononuclear cells from bone marrow blood were isolated from rhesus monkeys by using monkey lymphocyte separation solution. CD34 positive cells were sorted by immunomagnetic beads kit and magnetic polarization. And purify it. The culture medium and cytokine GM-CSF,IL-4 were added to the obtained cells. The CD34 positive cells were cultured and induced to the 7th day. After the cells were counted, 2.5 脳 105 cells per hole were inoculated in 24-well plate, and the virus was added to the cells according to the MOI value of 100200300400500600. 48 hours later, the expression of green fluorescent pigment protein (GFP) was observed. The CD34 positive cells obtained from the first part were cultured and induced to the 7th day. The protein was extracted from the cells infected with recombinant adenovirus for 48 hours according to the MOI value of 500? *. Mixed lymphocyte reaction, the stimulated cells were divided into three groups: DC group, imDC group with gene silencing, LPS imDC group with gene silencing, and imDC group with LPS gene silencing according to different methods. Using T cells as reactive cells, T cells were detected by flow cytometry. According to the stimulating cells: the proportion of reactive cells was 1? 5, 1? 10, 1? 20, and 1? 40 were added to the reactive cells. The proliferation of T cells in each group was analyzed statistically. Results: the purity of DC obtained by immunomagnetic beads was high, and the maturation of imDC could be induced by cytokine culture for 4 days. The stable infection MOI of recombinant adenoviral vector in rhesus monkey immature dendritic cells was 500x600. The specific band at 33kD was detected by Western Blot, and 尾-actin was successfully expressed in 43kD. It can be inferred that the target protein was successfully expressed in imDC, and the protein expression of recombinant adenovirus vector in imDC was significantly lower than that in blank group and negative control group. The expression of MyD88 interference histone protein was down-regulated by 73.5% compared with the blank control group, and MyD88 interference group was down-regulated by 80.6% compared with the negative control group. The purity of T cells isolated by nylon wool column method is more than 80%, which has experimental significance and can be used as a reaction cell. In mixed lymphocyte culture, the results showed that the ability of stimulating T cell proliferation in imDC group was significantly lower than that in mDC group, and the ability to stimulate T cell proliferation in recombinant adenovirus-infected imDC group was significantly lower than that in other groups. Conclusion: DC, cytokines can be purified by immunomagnetic beads to induce the differentiation of DC into imDC, and the recombinant adenovirus vector can obtain the best infection efficiency in rhesus monkey imDC when MOI is 500? The expression of MyD88 gene silencing siRNA protein in rhesus monkey imDC was significantly inhibited, and recombinant adenovirus-infected imDC could inhibit the proliferation of T cells. Prospect: the recombinant adenovirus was used to infect immature dendritic cells, and the infected imDC was injected into the recipient by portal vein in vivo, hoping to induce the production of immune tolerance and open up a new way for transplantation.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R657.3
本文编号：2465279
[Abstract]:Objective: to identify the recombinant adenovirus vector of rhesus monkey MyD88 gene silencing siRNA in rhesus monkey immature dendritic cells (immature dendritic cell,). The expression of imDC in rhesus monkeys and the effect of rhesus monkey MyD88 gene silencing siRNA adenovirus vector on T cell immune response in rhesus monkeys infected with imDC were investigated. Methods: bone marrow blood was extracted from rhesus monkeys under intravenous anesthesia. Mononuclear cells from bone marrow blood were isolated from rhesus monkeys by using monkey lymphocyte separation solution. CD34 positive cells were sorted by immunomagnetic beads kit and magnetic polarization. And purify it. The culture medium and cytokine GM-CSF,IL-4 were added to the obtained cells. The CD34 positive cells were cultured and induced to the 7th day. After the cells were counted, 2.5 脳 105 cells per hole were inoculated in 24-well plate, and the virus was added to the cells according to the MOI value of 100200300400500600. 48 hours later, the expression of green fluorescent pigment protein (GFP) was observed. The CD34 positive cells obtained from the first part were cultured and induced to the 7th day. The protein was extracted from the cells infected with recombinant adenovirus for 48 hours according to the MOI value of 500? *. Mixed lymphocyte reaction, the stimulated cells were divided into three groups: DC group, imDC group with gene silencing, LPS imDC group with gene silencing, and imDC group with LPS gene silencing according to different methods. Using T cells as reactive cells, T cells were detected by flow cytometry. According to the stimulating cells: the proportion of reactive cells was 1? 5, 1? 10, 1? 20, and 1? 40 were added to the reactive cells. The proliferation of T cells in each group was analyzed statistically. Results: the purity of DC obtained by immunomagnetic beads was high, and the maturation of imDC could be induced by cytokine culture for 4 days. The stable infection MOI of recombinant adenoviral vector in rhesus monkey immature dendritic cells was 500x600. The specific band at 33kD was detected by Western Blot, and 尾-actin was successfully expressed in 43kD. It can be inferred that the target protein was successfully expressed in imDC, and the protein expression of recombinant adenovirus vector in imDC was significantly lower than that in blank group and negative control group. The expression of MyD88 interference histone protein was down-regulated by 73.5% compared with the blank control group, and MyD88 interference group was down-regulated by 80.6% compared with the negative control group. The purity of T cells isolated by nylon wool column method is more than 80%, which has experimental significance and can be used as a reaction cell. In mixed lymphocyte culture, the results showed that the ability of stimulating T cell proliferation in imDC group was significantly lower than that in mDC group, and the ability to stimulate T cell proliferation in recombinant adenovirus-infected imDC group was significantly lower than that in other groups. Conclusion: DC, cytokines can be purified by immunomagnetic beads to induce the differentiation of DC into imDC, and the recombinant adenovirus vector can obtain the best infection efficiency in rhesus monkey imDC when MOI is 500? The expression of MyD88 gene silencing siRNA protein in rhesus monkey imDC was significantly inhibited, and recombinant adenovirus-infected imDC could inhibit the proliferation of T cells. Prospect: the recombinant adenovirus was used to infect immature dendritic cells, and the infected imDC was injected into the recipient by portal vein in vivo, hoping to induce the production of immune tolerance and open up a new way for transplantation.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R657.3
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