胰岛素抵抗状态下Betatrophin基因的表达变化及对相关信号通路的影响

发布时间：2018-01-07 03:05

本文关键词：胰岛素抵抗状态下Betatrophin基因的表达变化及对相关信号通路的影响　出处：《重庆医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：第一部分小鼠Betatrophin基因表达载体构建及鉴定目的:构建小鼠Betatrophin基因重组质粒(p IRES2-EGFP-Betatrophin),并用于C57 BL/6J小鼠的原代肝细胞转染。方法:提取小鼠肝脏组织Total RNA,经逆转录(RT-PCR)获得c DNA。后续PCR扩增得到Betatrophin CDS序列全长。经双酶切,连接到真核表达载体p IRES2-EGFP上,构建成p IRES2-EGFP-Betatrophin,后续通过双酶切+凝胶电泳、DNA测序、Blast对重组质粒进行鉴定。结果:将重组质粒(p IRES2-EGFP-Betatrophin)和经过双酶切的重组质粒进行琼脂糖凝胶电泳,凝胶成像后前者(p IRES2-EGFP-Betatrophin重组质粒)出现在对应Marker 5897bp的位置;后者出现两条线性化条带,分别在对应Marker 597bp(Betatrophin CDS全长序列)位置和5.3kb(p IRES2-EGFP线性条带)位置,初步确定重组质粒构建成功。测序结果同Genbank中Betatrophin m RNA所编码的CDS序列全长在NCBI中进行DNA序列比对,比对结果完全一致,证明所克隆的基因就是小鼠Betatrophin c DNA。结论:成功构建真核表达载体p IRES2-EGFP-Betatrophin。第二部分Betatrophin在小鼠组织中的表达目的:探索普食、高脂喂养的C57BL/6J小鼠组织中,Betatrophin m RNA和蛋白表达差异。方法:取高脂饲料喂养(HFD)28周的C57BL/6J小鼠(5只)、普食喂养(SD)的同龄C57BL/6J小鼠(5只)处死取肝脏、脂肪组织于液氮中保存,后续提取各对应组织RNA并逆转录为c DNA,再通过实时定量荧光PCR测定Betatrophin的m RNA表达差异;提取各对应组织总蛋白,通过Western blot方法测定Betatrophin的蛋白表达差异。结果:Betatrophin mRNA和蛋白表达量在HFD小鼠的肝脏和脂肪组织中明显高于(p0.05或p0.01)SD小鼠对应组织;结论:提示,Betatrophin的表达可能与小鼠机体的胰岛素抵抗相关。第三部分Betatrophin在肝细胞中的表达及其对胰岛素信号通路的影响目的:探索胰岛素抵抗状态的小鼠肝原代细胞Betatrophin基因表达情况,以及Betatrophin过表达对增加小鼠肝细胞胰岛素抵抗的机制。方法:分离C57BL/6J小鼠原代肝细胞,分组培养。使用浓度递增的胰岛素处理;经一定浓度胰岛素处理24h的原代肝细胞再通过罗格列酮或者二甲双胍作用,通过定量PCR及Western blot检测Betatrophin表达。原代肝细胞和巨噬细胞RAW264.7共培养;另外组使用罗格列酮或者二甲双胍处理共培养体系细胞,通过定量PCR及Western blot检测Betatrophin表达情况;另外各组原代肝细胞分别加2%(在培养基中所占体积比)的PBS(空白对照)、正常人血清(normal)、PCOS患者未治疗者血清(Before metformin)、PCOS患者二甲双胍治疗3个月血清(3months after metformin)、PCOS患者二甲双胍治疗6个月的血清(6months after metformin)培养24小时,再通过定量PCR及Western blot检测Betatrophin、P-Ins R以及P-AKT表达;最后用过表达质粒以及干扰RNA转染小鼠肝原代细胞48h,定量PCR以及Western blot检测Betatrophin、P-Ins R以及P-AKT表达情况。结果:随着insulin浓度梯度的增加,insulin处理的肝原代Betatrophin表达逐渐增高(p0.05或p0.01),同时经一定浓度insulin作用24h的肝原代细胞再用不同浓度罗格列酮或者二甲双胍处理后Betatrophin表达显著下降(p0.05或p0.01);肝原代细胞、巨噬细胞264.7共培养体系在经罗格列酮或者二甲双胍作用后Betatrophin表达显著降低(p0.05或p0.01);Betatrophin过表达组的P-Ins R、P-AKT表达同对照组比明显降低(p0.01),Betatrophin干扰组的P-Ins R、P-AKT表达相对于对照组显著升高(p0.01)。结论:Betatrophin表达与胰岛素抵抗相关,Betatrophin基因过表达可能抑制了PI3K/Akt信号通路,降低胰岛素敏感性,从而增强肝细胞胰岛素抵抗。
[Abstract]:The first part of the expression of Betatrophin gene in construction and identification of recombinant vector Objective: to construct the recombinant plasmid of mouse Betatrophin gene (P IRES2-EGFP-Betatrophin), and for primary liver cells transfected with C57 BL/6J mice. Methods: the extraction of liver tissue of mice Total RNA by reverse transcription (RT-PCR) C DNA. PCR further amplified the full-length CDS sequence of Betatrophin. After double enzyme cut, connected to the eukaryotic expression vector p IRES2-EGFP, construct P IRES2-EGFP-Betatrophin, following by double enzyme digestion and gel electrophoresis, DNA Blast sequencing, the recombinant plasmids were identified. Results: the recombinant plasmids (P and IRES2-EGFP-Betatrophin) after double digestion of recombinant plasmid was analyzed by agarose gel electrophoresis, gel imaging (after the former p the recombinant plasmid of IRES2-EGFP-Betatrophin Marker 5897bp) appears in the corresponding position; two linear bands which appeared respectively in the corresponding Marker 597bp (Betat The full-length sequence of rophin CDS 5.3kb (P) position and IRES2-EGFP linear belt) position, preliminary determination of the recombinant plasmid was constructed successfully. The sequencing results with CDS Betatrophin m RNA sequence is Genbank encoding in NCBI DNA sequences alignment result is completely consistent, which indicated that the cloned gene is mouse Betatrophin C DNA. conclusion: really the expression vector p IRES2-EGFP-Betatrophin. second Betatrophin in mice Objective: To explore the successful construction of normal diet, high-fat diet C57BL/6J mice, Betatrophin m RNA and protein expression differences. Methods: high fat diet (HFD) for 28 weeks C57BL/6J mice (5 rats), normal chow (SD) age C57BL/6J mice (n = 5) were sacrificed and the liver, adipose tissue stored in liquid nitrogen, the subsequent extraction of corresponding tissue RNA and reverse transcription of C DNA, then Betatrophin PCR real time fluorescence quantitative determination The m RNA expression; protein extraction of the corresponding tissue, expression determination of Betatrophin protein by Western blot method. Results: the expression of Betatrophin and mRNA protein in HFD mouse liver and adipose tissue was significantly higher than that of (P0.05 or P0.01) corresponding to SD mice; conclusion: the results suggest that, relative expression of Betatrophin may be related to mice insulin resistance in the body. The third part of the Betatrophin expression in liver cells and its effect on the insulin signaling pathway Objective: To explore the insulin resistance state of mouse liver primary cells the expression of Betatrophin gene, and Betatrophin overexpression on insulin resistance increase mechanism of liver cells in mice. Methods: primary cultured hepatic cells of C57BL/6J mouse. The use of insulin treatment. The increasing of the concentration of the primary hepatocytes; 24h treatment of insulin by rosiglitazone or a two Double guanidine, PCR and Western blot by quantitative detection of Betatrophin expression in primary hepatocytes and macrophages. RAW264.7 co cultured with rosiglitazone or metformin group; another treatment co culture system of cells, the expression of PCR and Western by quantitative detection of blot Betatrophin in each group; primary hepatocytes were 2% (in volume ratio based training in) PBS (blank control), normal human serum (normal), PCOS in patients with untreated serum (Before metformin), metformin in patients treated with PCOS for 3 months (3months after metformin) in serum, serum of metformin in patients treated with PCOS for 6 months (6months after metformin) for 24 hours, and then through the quantitative PCR and Western blot detection of Betatrophin, expression of P-Ins R and P-AKT; finally used the expression plasmid and interference RNA transfection of mouse primary hepatocytes 48h, PCR and Western blot quantitative detection of Betatroph In, P-Ins R and P-AKT expression. Results: with the increase of insulin concentration gradient, insulin treatment of primary liver Betatrophin expression gradually increased (P0.05 or P0.01), and the primary function of a certain concentration of insulin 24h in hepatic cells with different concentrations of rosiglitazone or metformin two Betatrophin after treatment was significantly decreased (P0.05 or P0.01); primary liver cell, macrophage co culture system in 264.7 by rosiglitazone or metformin significantly decreased expression of Betatrophin (P0.05 or P0.01); Betatrophin group of P-Ins R expression, P-AKT expression decreased significantly compared with the control group (P0.01), Betatrophin P-Ins R P-AKT interference group, the expression was significantly increased compared with the control group (P0.01). Conclusion: the expression of Betatrophin is associated with insulin resistance, overexpression of Betatrophin could inhibit the PI3K/Akt signaling pathway, reduced insulin sensitivity, thereby enhancing the Insulin resistance in liver cells.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R587.1

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