本文選題：SFRP5 + 免疫共沉淀　； 參考：《重慶醫(yī)科大學(xué)》2015年碩士論文

【摘要】：第一部分免疫共沉淀篩選SFRP5相互作用蛋白目的：免疫共沉淀篩選SFRP5相互作用蛋白。方法：免疫共沉淀分離SFRP5相互作用蛋白,聚丙烯酰胺凝膠電泳分離蛋白后銀染,通過(guò)與對(duì)照組的比較切割差異蛋白條帶,質(zhì)譜鑒定、生物信息學(xué)分析,選取一個(gè)可能與SFRP5相互作用的蛋白質(zhì)。結(jié)果：成功分離SFRP5相互作用的蛋白質(zhì),質(zhì)譜檢測(cè)差異銀染條帶篩選出7個(gè)差異蛋白,選取SLURP1為目標(biāo)蛋白。結(jié)論：SLURP1可能是SFRP5的相互作用蛋白。第二部分驗(yàn)證SFRP5和SLURP1間的相互作用目的：SFRP5和SLURP1蛋白之間相互作用的驗(yàn)證。方法：提取脂肪組織T-RNA, PCR獲的SFRP5和SLURP1基因片段,分別構(gòu)建真核表達(dá)載體pCMV-HA-SFRP5和pCMV-Myc-SLURP1,利用PCR產(chǎn)物大小和DNA測(cè)序結(jié)果鑒定重組質(zhì)粒是否成功,Western-blot檢測(cè)蛋白表達(dá)情況。真核表達(dá)載體pCMV-HA-SFRP5和pCMV-Myc-SLURP1共轉(zhuǎn)染HepG-2細(xì)胞,免疫共沉淀技術(shù)驗(yàn)證SFRP5和SLURP 1蛋白間相互作用。結(jié)果：真核表達(dá)載體pCMV-HA-SFRP5和pCMV-Myc-SLURP1成功構(gòu)建,轉(zhuǎn)染HepG-2細(xì)胞,HA蛋白抗體沉淀,Myc蛋白抗體檢測(cè),可見(jiàn)SLURP 1的表達(dá)；反之,Myc蛋白抗體沉淀,HA蛋白抗體檢測(cè),可見(jiàn)SFRP5蛋白表達(dá)。結(jié)論：成功構(gòu)建了真核表達(dá)載體pCMV-HA-SFRP5和pCMV-My-c-SLURP1,成功驗(yàn)證SFRP5和SLURP1間的相互作用。第三部分SFRP5和SLURP1基因之間的影響和對(duì)胰島素抵抗的作用目的：研究SFRP5和SLURP1基因間的影響和對(duì)胰島素抵抗作用。方法：Q-PCR檢測(cè)SFRP5 (SLURP1)基因在小鼠組織中的分布；SFRP5和SLURP 1過(guò)表達(dá)質(zhì)粒分別轉(zhuǎn)染hepG-2細(xì)胞,將細(xì)胞分為正常轉(zhuǎn)染組和高濃度飽和脂肪酸處理組,Q-PCR方法檢測(cè)SFRP5(SLURP1)表達(dá)上調(diào)對(duì)SLURP1 (SFRP5)的影響；用構(gòu)建的三個(gè)不同目的片段的pGenesil-SFRP5和pGenesil-SLURP 1抑制質(zhì)粒轉(zhuǎn)染hepG-2細(xì)胞,Q-PCR方法篩選最佳抑制質(zhì)粒；然后用pGenesil-SFRP5和pGenesil-SLURP1的最佳抑制質(zhì)粒轉(zhuǎn)染hepG-2細(xì)胞,檢測(cè)TG、TC含量變化；同樣用pCMV-HA-SFRP5和pCMV-Myc-SLURP1過(guò)表達(dá)質(zhì)粒轉(zhuǎn)染hepG-2細(xì)胞,檢測(cè)TG、TC含量和葡萄糖攝取率(GUR)的變化；檢測(cè)SLURP1基因在3T3-L1細(xì)胞誘導(dǎo)分化過(guò)程中表達(dá)變化。結(jié)果：SFRP5和SLURP1基因shRNA的最佳抑制率分別為68%和51.7%；不同小鼠組織均可見(jiàn)SFRP5基因分布,mRNA相對(duì)表達(dá)量由高到低分別為：脂肪、肌肉、腦、心臟、肝臟等；SLURP1基因mRNA相對(duì)表達(dá)量由高到低分別為：胃、皮膚、動(dòng)脈、肺、心臟、睪丸、肌肉、脂肪等；無(wú)處理因素情況下SLURP1和SFRP5過(guò)表達(dá)質(zhì)粒分別轉(zhuǎn)染HepG-2, SFRP5或SLURP1表達(dá)無(wú)變化；SLURP 1和SFRP5分別轉(zhuǎn)染過(guò)表達(dá),然后用棕櫚酸(PA)誘導(dǎo),可見(jiàn)SLURP1變化對(duì)SFRP5影響顯著,SFRP5變化對(duì)SLURP1影響效果不明顯。脂肪誘導(dǎo)過(guò)程中SLURP1基因表達(dá)升高,第4天達(dá)到峰值；HepG-2細(xì)胞SLURP1基因過(guò)表達(dá),檢測(cè)SREBP-1C、ACC、FAS、SCD-1、HMGR mRNA, SREBP-1C、ACC、FAS表達(dá)降低,HMGR、SCD-1無(wú)變化。SLURP1基因過(guò)表達(dá)時(shí)TG、TC表達(dá)下降(P0.05)、糖攝取增加(P0.05)。SLURP1基因抑制,TG升高(P0.05)、TC無(wú)變化。SFRP5基因過(guò)表達(dá),TG降低(P0.05)、TC無(wú)變化、糖攝取增加(P0.05)。SFRP5基因抑制,TG升高(P0.05)、TC無(wú)變化。結(jié)論：SLURP1基因過(guò)表達(dá)時(shí)可以抑制TG、TC,增加糖攝取。SFRP5基因過(guò)表達(dá)時(shí)可以?xún)H可抑制TG。 SLURP1基因抑制時(shí)TG合成增加。SFRP5基因抑制時(shí)TG合成同樣增加。
[Abstract]:The first part immunoprecipitation screening interaction protein of SFRP5 Objective: immunoprecipitation screening interaction protein of SFRP5. Methods: isolated SFRP5 interacting protein immunoprecipitation, polyacrylamide gel electrophoresis separation of proteins after silver staining, compared with the control group by cutting the difference of protein bands, mass spectrometry, bioinformatics analysis, selection a possible interaction between SFRP5 and protein. Results: the interaction of SFRP5 protein separation and mass spectrometric detection between silver staining bands screened 7 proteins, SLURP1 is selected as the target protein. Conclusion: SLURP1 interacts with SFRP5. The second part verify the interactions between SLURP1 and SFRP5 to verify each other the interaction of SFRP5 and SLURP1 protein. Methods: adipose tissue was T-RNA, PCR was SFRP5 and SLURP1 gene fragments were constructed eukaryotic expression vector pCMV- HA-SFRP5 and pCMV-Myc-SLURP1, using the PCR product size and DNA sequencing. The recombinant plasmid is successful, detect the expression of Western-blot protein. The eukaryotic expression vector pCMV-HA-SFRP5 and pCMV-Myc-SLURP1 were transfected into HepG-2 cells, immunoprecipitation verification of SFRP5 and SLURP 1 protein interactions. Results: the eukaryotic expression vector pCMV-HA-SFRP5 and transfected into HepG-2 pCMV-Myc-SLURP1 was successfully established. Cells, HA protein antibody precipitation, detection of Myc antibody, expression of SLURP 1 can be seen; on the other hand, Myc protein antibody precipitation, detection of HA antibody, SFRP5 protein expression was observed. Conclusion: the successful construction of the eukaryotic expression vector of pCMV-HA-SFRP5 and pCMV-My-c-SLURP1, SFRP5 and SLURP1 successfully verified the interaction between SFRP5 and SLURP1. The influence between parts third gene on insulin resistance and the role of objective: To study the effect of SFRP5 and SLURP1 between genes and On insulin resistance. Methods: the detection of Q-PCR SFRP5 (SLURP1) gene distribution in mouse tissues; SFRP5 and SLURP 1 expression plasmids were transfected into hepG-2 cells, the cells were divided into normal group and high concentration of saturated fatty acid treatment group, Q-PCR method for detection of SFRP5 (SLURP1) expression of SLURP1 (SFRP5). Effect; using three different fragments to construct the pGenesil-SFRP5 and pGenesil-SLURP 1 inhibited hepG-2 cells were transfected with Q-PCR method, screening the best inhibiting plasmid; and then use the pGenesil-SFRP5 and pGenesil-SLURP1 the best suppression plasmid was transfected into hepG-2 cells, detect TG, TC content changes; using the same pCMV-HA-SFRP5 and pCMV-Myc-SLURP1 expression plasmids were transfected into hepG-2 cells and detection of TG, TC the content and glucose uptake rate (GUR) changes; detection of SLURP1 gene expression induced by changes in differentiation in 3T3-L1 cells. Results: SFRP5 and The best inhibition of SLURP1 shRNA gene were 68% and 51.7%; different mouse tissues showed SFRP5 gene distribution, the relative expression of mRNA from high to low: fat, muscle, brain, heart and liver; SLURP1 gene relative expression of mRNA from high to low: stomach, skin, lung artery., heart, testis, muscle, fat and other factors under the condition of no treatment; SLURP1 and SFRP5 expression plasmids were transfected into HepG-2, SFRP5 or SLURP1 expression had no change; over expression of SLURP 1 and SFRP5 were transfected, then palmitic acid (PA) induced visible effects of SLURP1 changes on SFRP5 significant effects of SFRP5 change on SLURP1 fat is not obvious. In the process of induction of SLURP1 gene expression increased, reached the peak at the fourth day; the over expression of SLURP1 gene in HepG-2 cells, ACC, detection of SREBP-1C, FAS, SCD-1, HMGR, mRNA, SREBP-1C, ACC, HMGR, FAS expression decreased, no changes in SCD-1.SLURP1 gene Overexpression of TG, decrease the expression of TC (P0.05), sugar uptake (P0.05) inhibition of.SLURP1 gene, TG (P0.05, TC) increased over expression of.SFRP5, TG decreased (P0.05), no change in TC, increased glucose uptake (P0.05) inhibition of.SFRP5 gene, TG (P0.05), TC. Change. Conclusion: the overexpression of SLURP1 gene can inhibit TG, TC,.SFRP5 gene expression increased glucose uptake can only inhibit TG. gene SLURP1 inhibited TG synthesis increased.SFRP5 gene inhibited TG synthesis also increased.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R446.6

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