应用RNA干扰技术敲除催乳素受体研究自分泌催乳素对激活的Jurkat细胞的免疫调控作用

发布时间：2018-01-14 18:33

本文关键词：应用RNA干扰技术敲除催乳素受体研究自分泌催乳素对激活的Jurkat细胞的免疫调控作用　出处：《福建医科大学》2008年博士论文　论文类型：学位论文

【摘要】： 目的:大量的文献报道已经证实了内分泌激素——催乳素(prolactin,PRL)在机体免疫应答中的调控作用,但是T淋巴细胞自分泌的催乳素的免疫调控作用至今不清楚。为了研究这种自分泌催乳素在被激活的T淋巴细胞中的作用,我们构建了慢病毒介导的RNA干扰技术敲除了催乳素受体(prolactin receptor,PRLR)的T淋巴细胞模型并以PHA刺激活化后检测其免疫效应的变化。本研究有三个目的:1.设计、构建慢病毒介导的RNA干扰技术敲除了PRLR的Jurkat细胞模型;2.利用上述细胞模型,探讨沉默PRLR基因的表达而阻断自分泌PRL的作用后对T淋巴细胞增殖的影响及其分子机制;3.进一步探讨自分泌PRL对Th1/Th2细胞因子平衡偏离和调节性细胞因子IL-10的影响。方法:1.按照小分子RNA干扰的设计原则和表达载体的要求,设计构建PRLR特异性的慢病毒RNA干扰重组体,并进行酶切鉴定及基因测序;包装慢病毒并测定其滴度;2.体外培养人淋巴白血病T细胞株Jurkat细胞,进行不同滴度的重组慢病毒感染,通过荧光显微镜观察重组慢病毒感染Jurkat细胞的情况;应用western blot检测PRLR基因沉默效果,确认慢病毒介导的RNA干扰技术敲除了催乳素受体的Jurkat细胞模型构建成功;3.用Nb2淋巴细胞生物测定法检测催乳素受体敲除的Jurkat细胞自分泌催乳素的水平;4. MTT比色法联合细胞计数检测PHA刺激Jurkat细胞模型后的增殖情况,应用western blot检测与PRL刺激Jurat细胞增殖相关的抗凋亡蛋白Bcl-2的表达情况;5.流式细胞仪检测PHA刺激Jurkat细胞模型后协同刺激分子CD28、CD154和CD137的表达变化;6.应用双抗体夹心ELISA检测PHA刺激Jurkat细胞模型后上清液中细胞因子IL-2、IL-4、IFN-γ和IL-10的变化。结果:1.构建的PRLR特异性的慢病毒RNA干扰重组体经酶切鉴定、基因序列测序,结果与设计序列完全相同,慢病毒重组体构建成功;2.慢病毒包装并测定浓度后感染Jurkat细胞,感染168h后收集细胞进行western blot检测,结果显示靶蛋白被明显抑制,催乳素受体敲除的Jurkat细胞模型构建成功;3.Nb2淋巴细胞生物检测法测定自分泌的催乳素水平有较高的敏感性和特异性,用这种方法检测出催乳素受体敲除的Jurkat细胞自分泌的催乳素水平比对照组和空载体组高;4.催乳素受体敲除的Jurkat细胞受PHA刺激后增殖反应明显下降,其分子机制是抗凋亡蛋白Bcl-2的下调;5.以PHA刺激敲除了催乳素受体的Jurkat细胞模型后,与对照组比较,协同刺激分子CD154、CD137的表达下调(P0.05),而CD28的表达不受影响;6.与对照组比较,细胞因子IL-2、IL-4的表达下调(P0.05),而IFN-γ和IL-10的表达不受影响。结论:1.构建的PRLR特异性慢病毒介导的RNA干扰能够在Jurkat细胞中保留较长时间的RNA干扰,阻断了自分泌催乳素对Jurkat细胞的作用;构建的这种细胞模型是研究自分泌催乳素作用于T淋巴细胞的一种很好的工具; 2.Jurkat细胞的催乳素受体敲除后自分泌的催乳素水平上调,表明PRLR对自分泌的PRL存在负反馈调节作用;3.自分泌PRL在维持淋巴细胞增殖起共辅助因子的作用,这种作用的机制是它可以通过PRL/PRLR信号转导途径调节抗凋亡蛋白Bcl-2的表达; 4.自分泌PRL通过调节协同刺激分子和细胞因子的表达来影响T淋巴细胞的免疫应答;5.自分泌PRL调节Th1和Th2淋巴细胞的平衡,对调节性细胞因子IL-10的表达没有影响;6.T淋巴细胞周围微环境中自分泌的催乳素对T淋巴细胞的增殖和对PHA刺激的免疫应答起重要的调控作用。
[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.

【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

【引证文献】