重組腺病毒介導(dǎo)恒河猴MyD88基因沉默siRNA在恒河猴未成熟樹突狀細(xì)胞中的表達(dá)與鑒定
發(fā)布時(shí)間：2019-04-25 16:17
【摘要】：目的：鑒定恒河猴MyD88基因沉默siRNA的重組腺病毒載體在恒河猴未成熟樹突狀細(xì)胞(immature dendritic cell, imDC)中的表達(dá)情況及探討恒河猴MyD88基因沉默siRNA腺病毒載體感染恒河猴imDC對(duì)T細(xì)胞免疫應(yīng)答的影響。 方法：在靜脈麻醉的情況下,抽取恒河猴骨髓血,使用猴淋巴細(xì)胞分離液將采取的骨髓血中的單個(gè)核細(xì)胞分離出來,利用免疫磁珠試劑盒和磁極正性分選CD34+陽性細(xì)胞,并對(duì)其進(jìn)行純化。在得到的細(xì)胞中添加培養(yǎng)基及細(xì)胞因子GM-CSF、IL-4進(jìn)行培養(yǎng)。將上述CD34+陽性細(xì)胞培養(yǎng)并誘導(dǎo)至第7天,細(xì)胞計(jì)數(shù)后以每孔2.5×105個(gè)細(xì)胞接種于24孔板中,按MOI為100、200、300、400、500、600分別加入病毒,48小時(shí)后觀察細(xì)胞綠色熒光色素蛋白的表達(dá)情況。將第一部分得到的CD34+陽性細(xì)胞培養(yǎng)并誘導(dǎo)至第7天,按MOI為500～600用重組腺病毒感染48小時(shí)提取蛋白,然后通過Western Blot檢測技術(shù)檢測蛋白表達(dá)情況。混合淋巴細(xì)胞反應(yīng),按照不同的方法處理刺激細(xì)胞將刺激細(xì)胞分為：DC組、imDC組、基因沉默imDC組、LPS+imDC組、LPS+基因沉默imDC組。以T細(xì)胞作為反應(yīng)細(xì)胞,流式細(xì)胞術(shù)檢測T細(xì)胞。根據(jù)刺激細(xì)胞：反應(yīng)細(xì)胞比例為1：5,1：10,1：20,1：40加入反應(yīng)細(xì)胞。通過統(tǒng)計(jì)學(xué)分析各組對(duì)T細(xì)胞的增殖情況。 結(jié)果：利用免疫磁珠法獲得的DC純度較高,使用細(xì)胞因子培養(yǎng)第四天后可誘導(dǎo)imDC成熟。重組腺病毒載體在恒河猴未成熟樹突狀細(xì)胞中的穩(wěn)定感染的MOI為500-600。 Western Blot檢測可見33kD處出現(xiàn)特異性條帶,在43kD處可見β-actin內(nèi)參成功表達(dá),可推斷目的蛋白在imDC中成功表達(dá),且重組腺病毒載體在imDC中的蛋白表達(dá)明顯低于空白組及陰性對(duì)照組。通過分析MyD88干擾組蛋白表達(dá)較空白對(duì)照組表達(dá)水平下調(diào)73.5%,MyD88干擾組蛋白表達(dá)較陰性對(duì)照組表達(dá)水平下調(diào)80.6%。尼龍毛柱法分離出的T細(xì)胞純度大于80%,有實(shí)驗(yàn)意義,可作為反應(yīng)細(xì)胞進(jìn)行實(shí)驗(yàn)。混合淋巴細(xì)胞培養(yǎng),由統(tǒng)計(jì)結(jié)果得出結(jié)論：imDC組刺激T細(xì)胞增殖的能力顯著低于mDC組；重組腺病毒感染的imDC組刺激T細(xì)胞增殖的能力顯著低于其他組。 結(jié)論：免疫磁珠法可純化DC,細(xì)胞因子可誘導(dǎo)DC向imDC分化;當(dāng)MOI為500～600時(shí),重組腺病毒載體在恒河猴imDC中可獲得最佳感染效率；恒河猴MyD88基因沉默siRNA在恒河猴imDC中的蛋白表達(dá)受到明顯抑制；重組腺病毒感染的imDC可抑制T細(xì)胞的增殖。 展望：利用重組腺病毒感染未成熟樹突狀細(xì)胞,在進(jìn)行體內(nèi)實(shí)驗(yàn)時(shí)通過門靜脈將感染后的imDC注入受體體內(nèi),希望可以誘導(dǎo)免疫耐受的產(chǎn)生,為移植領(lǐng)域開辟新道路。
[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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R657.3
本文編號(hào)：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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R657.3
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