Cbp分子在CD59 GPI介导的T细胞抑制信号转导中的作用研究

发布时间：2018-04-05 09:14

本文选题：CD59　切入点：Cbp　出处：《青岛大学》2013年硕士论文

【摘要】：目的研究GPI锚固蛋白CD59与Cbp在细胞上的定位及在T细胞信号转导通路中的相互作用,以探讨CD59向T细胞内传递信号的相关机制。方法应用细胞免疫荧光技术,通过激光共聚焦荧光显微镜系统对CD59、Cbp的定位进行了分析。利用RNA干扰技术,设计针对Cbp基因的寡核营酸序列,克隆至线性化的pSUPER载体中构建pSUPER-siCbp重组质粒并进行鉴定。采用电转染方法转染Jurkat细胞,荧光显微镜观察GFP基因表达,并行RT-PCR和western blot检测转染细胞中Cbp的表达。将(?)DSUPER-siCbp、pEGFP-N3-Cbp、突变型pEGFP-N3-Cbp三种质粒进行扩增并酶切鉴定,同样转染Jurkat细胞,检测各转染组Cbp蛋白的表达。CD59mAb交联刺激细胞后,western blot检测各组Csk、p-ZAP-70和p-Fyn表达量；MTT检测细胞增殖；ELISA检测细胞上清中自介素2(IL-2)的水平。结果激光共聚焦结果显示：细胞静止时,Cbp分子点状聚集于膜上,CD59分子广泛分布；随着细胞活化,Cbp少量的散在分布,而CD59表达增多并点状聚集。pSUPER-siCbp重组质粒经菌液PCR、 DNA PCR、限制性内切酶双酶切分析和DNA测序鉴定正确。各转染组可表达绿色荧光蛋自,转染J2-pSUPER-siCbp重组质粒的Jurkat细胞中Cbp基因表达量明显低于其他各组。选取J2组质粒进行后续检测结果：与未转染组比较,在基因与蛋白水平,干扰组Cbp表达减少,而高表达组和突变组Cbp表达增加。CD59mAb交联刺激细胞后,与未转染组比较,干扰组和突变组细胞增殖快,IL-2分泌增多,Csk、p-ZAP-70和p-Fyn增多,突变组变化更明显；高表达组细胞则与之相反。结论CD59可借助棕榈酰基团使Cbp分子磷酸化,向细胞内传递抑制信号,引起细胞内相关分子的一系列变化,为探讨CD59与Cbp在T细胞信号转导通路中相互作用及CD59GP(?)胞内信号转导机制奠定基础。
[Abstract]:Objective to study the location of GPI anchorage protein CD59 and Cbp on cells and the interaction in T cell signal transduction pathway, so as to explore the mechanism of CD59 signaling to T cells.
Methods by using cell immunofluorescence technique by laser confocal fluorescence microscopy system of CD59, the localization of Cbp is analyzed. Using RNA interference technology, designed according to Cbp gene oligonucleotide sequences, cloned into linearized pSUPER vector to construct pSUPER-siCbp recombinant plasmid and identification. Using electroporation transfected Jurkat cells. Fluorescence microscopy was used to observe the expression of GFP gene, the expression of the transfected cells parallel detection of RT-PCR and western in blot Cbp. (DSUPER-siCbp?), pEGFP-N3-Cbp, mutant pEGFP-N3-Cbp three plasmids were amplified and identified by enzyme digestion, also transfected into Jurkat cells, the expression of.CD59mAb Cbp protein crosslinking group transfected cells stimulated by western, blot were detected Csk, p-ZAP-70 expression and p-Fyn cell proliferation assay; MTT; detection of ELISA cell supernatant of interleukin 2 (IL-2) level.
The results of confocal laser scanning results showed that cell, Cbp molecular punctate aggregation on the membrane, CD59 molecules are widely distributed; with Cbp cell activation, a small amount of scattered, and the increased expression of CD59 and dot aggregation of.PSUPER-siCbp recombinant plasmids are detected by PCR, DNA, PCR, restriction enzyme digestion analysis and DNA sequencing. Right. Each transfection group the expression of green fluorescent protein, transfection of J2-pSUPER-siCbp recombinant plasmid in Jurkat cells and the expression of Cbp gene was significantly lower than other groups. Group J2 plasmid for subsequent detection results: compared with untransfected group, the gene and protein level, the expression of Cbp and reduce the interference group and high expression group and mutation group Cbp the increased expression of.CD59mAb cells stimulated by cross-linking, compared to untransfected groups, interference group and mutation group cell proliferation, IL-2 secretion, Csk, p-ZAP-70 and p-Fyn increased, mutation group change is more obvious; the high table The cells in the group were the opposite.
Conclusion CD59 can make Cbp molecules phosphorylated by palmityl group, transfer the inhibitory signals to cells, and cause a series of changes in the related molecules. It will lay a foundation for exploring the interaction between CD59 and Cbp in T cell signal transduction pathway and CD59GP (intracellular) signal transduction mechanism.

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R392.11

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