SFRP5和SLURP1相互作用及对胰岛素抵抗作用的研究

发布时间：2018-04-11 05:38

本文选题：SFRP5 + 免疫共沉淀　；参考：《重庆医科大学》2015年硕士论文

【摘要】：第一部分免疫共沉淀筛选SFRP5相互作用蛋白目的：免疫共沉淀筛选SFRP5相互作用蛋白。方法：免疫共沉淀分离SFRP5相互作用蛋白,聚丙烯酰胺凝胶电泳分离蛋白后银染,通过与对照组的比较切割差异蛋白条带,质谱鉴定、生物信息学分析,选取一个可能与SFRP5相互作用的蛋白质。结果：成功分离SFRP5相互作用的蛋白质,质谱检测差异银染条带筛选出7个差异蛋白,选取SLURP1为目标蛋白。结论：SLURP1可能是SFRP5的相互作用蛋白。第二部分验证SFRP5和SLURP1间的相互作用目的：SFRP5和SLURP1蛋白之间相互作用的验证。方法：提取脂肪组织T-RNA, PCR获的SFRP5和SLURP1基因片段,分别构建真核表达载体pCMV-HA-SFRP5和pCMV-Myc-SLURP1,利用PCR产物大小和DNA测序结果鉴定重组质粒是否成功,Western-blot检测蛋白表达情况。真核表达载体pCMV-HA-SFRP5和pCMV-Myc-SLURP1共转染HepG-2细胞,免疫共沉淀技术验证SFRP5和SLURP 1蛋白间相互作用。结果：真核表达载体pCMV-HA-SFRP5和pCMV-Myc-SLURP1成功构建,转染HepG-2细胞,HA蛋白抗体沉淀,Myc蛋白抗体检测,可见SLURP 1的表达；反之,Myc蛋白抗体沉淀,HA蛋白抗体检测,可见SFRP5蛋白表达。结论：成功构建了真核表达载体pCMV-HA-SFRP5和pCMV-My-c-SLURP1,成功验证SFRP5和SLURP1间的相互作用。第三部分SFRP5和SLURP1基因之间的影响和对胰岛素抵抗的作用目的：研究SFRP5和SLURP1基因间的影响和对胰岛素抵抗作用。方法：Q-PCR检测SFRP5 (SLURP1)基因在小鼠组织中的分布；SFRP5和SLURP 1过表达质粒分别转染hepG-2细胞,将细胞分为正常转染组和高浓度饱和脂肪酸处理组,Q-PCR方法检测SFRP5(SLURP1)表达上调对SLURP1 (SFRP5)的影响；用构建的三个不同目的片段的pGenesil-SFRP5和pGenesil-SLURP 1抑制质粒转染hepG-2细胞,Q-PCR方法筛选最佳抑制质粒；然后用pGenesil-SFRP5和pGenesil-SLURP1的最佳抑制质粒转染hepG-2细胞,检测TG、TC含量变化；同样用pCMV-HA-SFRP5和pCMV-Myc-SLURP1过表达质粒转染hepG-2细胞,检测TG、TC含量和葡萄糖摄取率(GUR)的变化；检测SLURP1基因在3T3-L1细胞诱导分化过程中表达变化。结果：SFRP5和SLURP1基因shRNA的最佳抑制率分别为68%和51.7%；不同小鼠组织均可见SFRP5基因分布,mRNA相对表达量由高到低分别为：脂肪、肌肉、脑、心脏、肝脏等；SLURP1基因mRNA相对表达量由高到低分别为：胃、皮肤、动脉、肺、心脏、睾丸、肌肉、脂肪等；无处理因素情况下SLURP1和SFRP5过表达质粒分别转染HepG-2, SFRP5或SLURP1表达无变化；SLURP 1和SFRP5分别转染过表达,然后用棕榈酸(PA)诱导,可见SLURP1变化对SFRP5影响显著,SFRP5变化对SLURP1影响效果不明显。脂肪诱导过程中SLURP1基因表达升高,第4天达到峰值；HepG-2细胞SLURP1基因过表达,检测SREBP-1C、ACC、FAS、SCD-1、HMGR mRNA, SREBP-1C、ACC、FAS表达降低,HMGR、SCD-1无变化。SLURP1基因过表达时TG、TC表达下降(P0.05)、糖摄取增加(P0.05)。SLURP1基因抑制,TG升高(P0.05)、TC无变化。SFRP5基因过表达,TG降低(P0.05)、TC无变化、糖摄取增加(P0.05)。SFRP5基因抑制,TG升高(P0.05)、TC无变化。结论：SLURP1基因过表达时可以抑制TG、TC,增加糖摄取。SFRP5基因过表达时可以仅可抑制TG。 SLURP1基因抑制时TG合成增加。SFRP5基因抑制时TG合成同样增加。
[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.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.6

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