本文關(guān)鍵詞：應(yīng)用RNA干擾技術(shù)敲除催乳素受體研究自分泌催乳素對(duì)激活的Jurkat細(xì)胞的免疫調(diào)控作用　出處：《福建醫(yī)科大學(xué)》2008年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 催乳素 催乳素受體 慢病毒 RNA干擾 蛋白Bcl-2 CD28 CD154 CD137 IL-2 IL-4 IL-10 IFN-γ

【摘要】： 目的:大量的文獻(xiàn)報(bào)道已經(jīng)證實(shí)了內(nèi)分泌激素——催乳素(prolactin,PRL)在機(jī)體免疫應(yīng)答中的調(diào)控作用,但是T淋巴細(xì)胞自分泌的催乳素的免疫調(diào)控作用至今不清楚。為了研究這種自分泌催乳素在被激活的T淋巴細(xì)胞中的作用,我們構(gòu)建了慢病毒介導(dǎo)的RNA干擾技術(shù)敲除了催乳素受體(prolactin receptor,PRLR)的T淋巴細(xì)胞模型并以PHA刺激活化后檢測(cè)其免疫效應(yīng)的變化。本研究有三個(gè)目的:1.設(shè)計(jì)、構(gòu)建慢病毒介導(dǎo)的RNA干擾技術(shù)敲除了PRLR的Jurkat細(xì)胞模型;2.利用上述細(xì)胞模型,探討沉默PRLR基因的表達(dá)而阻斷自分泌PRL的作用后對(duì)T淋巴細(xì)胞增殖的影響及其分子機(jī)制;3.進(jìn)一步探討自分泌PRL對(duì)Th1/Th2細(xì)胞因子平衡偏離和調(diào)節(jié)性細(xì)胞因子IL-10的影響。 方法:1.按照小分子RNA干擾的設(shè)計(jì)原則和表達(dá)載體的要求,設(shè)計(jì)構(gòu)建PRLR特異性的慢病毒RNA干擾重組體,并進(jìn)行酶切鑒定及基因測(cè)序;包裝慢病毒并測(cè)定其滴度;2.體外培養(yǎng)人淋巴白血病T細(xì)胞株Jurkat細(xì)胞,進(jìn)行不同滴度的重組慢病毒感染,通過(guò)熒光顯微鏡觀察重組慢病毒感染Jurkat細(xì)胞的情況;應(yīng)用western blot檢測(cè)PRLR基因沉默效果,確認(rèn)慢病毒介導(dǎo)的RNA干擾技術(shù)敲除了催乳素受體的Jurkat細(xì)胞模型構(gòu)建成功;3.用Nb2淋巴細(xì)胞生物測(cè)定法檢測(cè)催乳素受體敲除的Jurkat細(xì)胞自分泌催乳素的水平;4. MTT比色法聯(lián)合細(xì)胞計(jì)數(shù)檢測(cè)PHA刺激Jurkat細(xì)胞模型后的增殖情況,應(yīng)用western blot檢測(cè)與PRL刺激Jurat細(xì)胞增殖相關(guān)的抗凋亡蛋白Bcl-2的表達(dá)情況;5.流式細(xì)胞儀檢測(cè)PHA刺激Jurkat細(xì)胞模型后協(xié)同刺激分子CD28、CD154和CD137的表達(dá)變化;6.應(yīng)用雙抗體夾心ELISA檢測(cè)PHA刺激Jurkat細(xì)胞模型后上清液中細(xì)胞因子IL-2、IL-4、IFN-γ和IL-10的變化。 結(jié)果:1.構(gòu)建的PRLR特異性的慢病毒RNA干擾重組體經(jīng)酶切鑒定、基因序列測(cè)序,結(jié)果與設(shè)計(jì)序列完全相同,慢病毒重組體構(gòu)建成功;2.慢病毒包裝并測(cè)定濃度后感染Jurkat細(xì)胞,感染168h后收集細(xì)胞進(jìn)行western blot檢測(cè),結(jié)果顯示靶蛋白被明顯抑制,催乳素受體敲除的Jurkat細(xì)胞模型構(gòu)建成功;3.Nb2淋巴細(xì)胞生物檢測(cè)法測(cè)定自分泌的催乳素水平有較高的敏感性和特異性,用這種方法檢測(cè)出催乳素受體敲除的Jurkat細(xì)胞自分泌的催乳素水平比對(duì)照組和空載體組高;4.催乳素受體敲除的Jurkat細(xì)胞受PHA刺激后增殖反應(yīng)明顯下降,其分子機(jī)制是抗凋亡蛋白Bcl-2的下調(diào);5.以PHA刺激敲除了催乳素受體的Jurkat細(xì)胞模型后,與對(duì)照組比較,協(xié)同刺激分子CD154、CD137的表達(dá)下調(diào)(P0.05),而CD28的表達(dá)不受影響;6.與對(duì)照組比較,細(xì)胞因子IL-2、IL-4的表達(dá)下調(diào)(P0.05),而IFN-γ和IL-10的表達(dá)不受影響。 結(jié)論:1.構(gòu)建的PRLR特異性慢病毒介導(dǎo)的RNA干擾能夠在Jurkat細(xì)胞中保留較長(zhǎng)時(shí)間的RNA干擾,阻斷了自分泌催乳素對(duì)Jurkat細(xì)胞的作用;構(gòu)建的這種細(xì)胞模型是研究自分泌催乳素作用于T淋巴細(xì)胞的一種很好的工具; 2.Jurkat細(xì)胞的催乳素受體敲除后自分泌的催乳素水平上調(diào),表明PRLR對(duì)自分泌的PRL存在負(fù)反饋調(diào)節(jié)作用;3.自分泌PRL在維持淋巴細(xì)胞增殖起共輔助因子的作用,這種作用的機(jī)制是它可以通過(guò)PRL/PRLR信號(hào)轉(zhuǎn)導(dǎo)途徑調(diào)節(jié)抗凋亡蛋白Bcl-2的表達(dá); 4.自分泌PRL通過(guò)調(diào)節(jié)協(xié)同刺激分子和細(xì)胞因子的表達(dá)來(lái)影響T淋巴細(xì)胞的免疫應(yīng)答;5.自分泌PRL調(diào)節(jié)Th1和Th2淋巴細(xì)胞的平衡,對(duì)調(diào)節(jié)性細(xì)胞因子IL-10的表達(dá)沒(méi)有影響;6.T淋巴細(xì)胞周?chē)h(huán)境中自分泌的催乳素對(duì)T淋巴細(xì)胞的增殖和對(duì)PHA刺激的免疫應(yīng)答起重要的調(diào)控作用。
[Abstract]:Objective: a large number of literatures have suggested that endocrine hormone prolactin (prolactin, PRL) in the regulation of immune response, but the immune regulation of T lymphocytes of autocrine prolactin remains unclear. In order to study the role of autocrine prolactin in activated T lymphocytes, we constructed lentiviral RNA interference technology mediated by prolactin receptor knockout (prolactin receptor, PRLR) T model and changes in the PHA lymphocyte activated to detect its immune effects. This study has three purposes: 1. design, construction of RNA interference lentivirus mediated knock out the Jurkat cell model of PRLR; 2. by using the cell model, explore the expression of PRLR gene silencing effects on T lymphocyte proliferation and blocking the autocrine role of PRL and its molecular mechanism; 3. to further explore the autocrine PRL of Th1/Th2 cells The effect of cytokine balance deviation and regulatory cytokine IL-10.
Methods: 1. according to the design principles and the expression of small interfering RNA vector, lentivirus RNA interference recombinant construct specific PRLR, and enzyme digestion and gene sequencing; packaging lentivirus and determine its titer; cell culture human T lymphocyte leukemia cell line Jurkat 2. in vitro, different titers of recombinant slow virus infection by fluorescence microscopy of recombinant lentivirus infected Jurkat cells; the application of Western blot detection of PRLR gene silencing effect confirmed RNA interference lentiviral mediated knock out the Jurkat cell model of prolactin receptor was successfully constructed; 3. Nb2 lymphocyte bioassay assay of prolactin receptor knockout Jurkat autocrine prolactin the 4. MTT; proliferation assay and cell count of Jurkat cells after stimulation of PHA model, the application of Western blot detection and PRL stimulated Jurat cells The expression of proliferation related anti apoptotic protein Bcl-2; 5. flow cytometry PHA stimulated Jurkat cell model of costimulatory molecule CD28, expression of CD154 and CD137; IL-4 6. application of double antibody sandwich ELISA PHA stimulated Jurkat cell model in the supernatant after cytokine IL-2, changes of IFN- gamma and IL-10.
Results: the identification of lentiviral RNA interference recombinant cut 1. to construct the PRLR specific enzyme, gene sequencing, and sequence design results are exactly the same, the recombinant was successfully constructed; Jurkat 2. cells infected with lentiviral packaging and determination of the concentration of 168h after infection were collected for Western blot detection showed that the targeted the protein was inhibited obviously, prolactin receptor knock Jurkat cell model in addition to the successful construction of 3.Nb2 lymphocyte; biological detection method for the determination of autocrine prolactin level has high sensitivity and specificity, using this method to detect the serum prolactin levels of prolactin receptor on Jurkat cells in autocrine than control group and empty vector group; 4. prolactin receptor knockout Jurkat cell proliferation stimulated by PHA was significantly decreased, the molecular mechanisms of down-regulation of the anti apoptotic protein Bcl-2; 5. were stimulated by PHA addition of prolactin receptor Jurkat Compared with the control group, the expression of CD154 and CD137 was down regulated (P0.05), but the expression of CD28 was not affected compared with the control group. 6., compared with the control group, the expression of cytokine IL-2 and IL-4 was down regulated (P0.05), while the expression of IFN- and IL-10 was not affected.
Conclusion: PRLR specific RNA interference lentiviral construct 1. by RNA interference can keep a long time in the Jurkat cells, blocking the role of autocrine prolactin on Jurkat cells; the cell model is the study of action of autocrine prolactin on T lymphocyte good tool 2.Jurkat cells; prolactin receptor knock prolactin levels are upregulated by autocrine after, showed that PRLR of autocrine PRL exist negative feedback effect; 3. PRL autocrine factors in maintaining lymphocyte proliferation, the mechanism of this kind of role is the expression of it can regulate the anti apoptotic protein Bcl-2 through PRL/PRLR signal transduction; 4. autocrine PRL by regulating the expression of costimulatory molecules and cell factor to influence the immune response of T lymphocytes; 5. PRL autocrine regulation of Th1 and Th2 lymphocyte balance on regulatory cells by The expression of IL-10 was not affected. Autocrine prolactin in the surrounding microenvironment of 6.T lymphocytes plays an important regulatory role in the proliferation of T lymphocytes and the immune response to PHA stimulation.

【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 王紫騫;奶山羊催乳素受體（PrlR）基因CDS區(qū)的克隆、序列分析與腺病毒干擾載體的構(gòu)建[D];西北農(nóng)林科技大學(xué);2011年

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本文編號(hào)：1424801