本文選題：CD59　切入點(diǎn)：Cbp　出處：《青島大學(xué)》2013年碩士論文

【摘要】：目的研究GPI錨固蛋白CD59與Cbp在細(xì)胞上的定位及在T細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)通路中的相互作用,以探討CD59向T細(xì)胞內(nèi)傳遞信號(hào)的相關(guān)機(jī)制。 方法應(yīng)用細(xì)胞免疫熒光技術(shù),通過(guò)激光共聚焦熒光顯微鏡系統(tǒng)對(duì)CD59、Cbp的定位進(jìn)行了分析。利用RNA干擾技術(shù),設(shè)計(jì)針對(duì)Cbp基因的寡核營(yíng)酸序列,克隆至線性化的pSUPER載體中構(gòu)建pSUPER-siCbp重組質(zhì)粒并進(jìn)行鑒定。采用電轉(zhuǎn)染方法轉(zhuǎn)染Jurkat細(xì)胞,熒光顯微鏡觀察GFP基因表達(dá),并行RT-PCR和western blot檢測(cè)轉(zhuǎn)染細(xì)胞中Cbp的表達(dá)。將(?)DSUPER-siCbp、pEGFP-N3-Cbp、突變型pEGFP-N3-Cbp三種質(zhì)粒進(jìn)行擴(kuò)增并酶切鑒定,同樣轉(zhuǎn)染Jurkat細(xì)胞,檢測(cè)各轉(zhuǎn)染組Cbp蛋白的表達(dá)。CD59mAb交聯(lián)刺激細(xì)胞后,western blot檢測(cè)各組Csk、p-ZAP-70和p-Fyn表達(dá)量；MTT檢測(cè)細(xì)胞增殖；ELISA檢測(cè)細(xì)胞上清中自介素2(IL-2)的水平。 結(jié)果激光共聚焦結(jié)果顯示：細(xì)胞靜止時(shí),Cbp分子點(diǎn)狀聚集于膜上,CD59分子廣泛分布；隨著細(xì)胞活化,Cbp少量的散在分布,而CD59表達(dá)增多并點(diǎn)狀聚集。pSUPER-siCbp重組質(zhì)粒經(jīng)菌液PCR、 DNA PCR、限制性?xún)?nèi)切酶雙酶切分析和DNA測(cè)序鑒定正確。各轉(zhuǎn)染組可表達(dá)綠色熒光蛋自,轉(zhuǎn)染J2-pSUPER-siCbp重組質(zhì)粒的Jurkat細(xì)胞中Cbp基因表達(dá)量明顯低于其他各組。選取J2組質(zhì)粒進(jìn)行后續(xù)檢測(cè)結(jié)果：與未轉(zhuǎn)染組比較,在基因與蛋白水平,干擾組Cbp表達(dá)減少,而高表達(dá)組和突變組Cbp表達(dá)增加。CD59mAb交聯(lián)刺激細(xì)胞后,與未轉(zhuǎn)染組比較,干擾組和突變組細(xì)胞增殖快,IL-2分泌增多,Csk、p-ZAP-70和p-Fyn增多,突變組變化更明顯；高表達(dá)組細(xì)胞則與之相反。 結(jié)論CD59可借助棕櫚酰基團(tuán)使Cbp分子磷酸化,向細(xì)胞內(nèi)傳遞抑制信號(hào),引起細(xì)胞內(nèi)相關(guān)分子的一系列變化,為探討CD59與Cbp在T細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)通路中相互作用及CD59GP(?)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)機(jī)制奠定基礎(chǔ)。
[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.

【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R392.11

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