脂联素在L-02细胞中对肝糖代谢信号转导机制的研究

发布时间：2018-04-15 14:13

本文选题：人脂联素 + 糖尿病　；参考：《天津医科大学》2008年硕士论文

【摘要】：目的：在人肝细胞株L-02中获取具有生物学活性的脂联素重组蛋白的分泌性表达,并在L-02细胞中探讨脂联素抑制糖异生的信号转导机制。方法：①从人脂肪组织中提取总RNA,利用RT-PCR方法得到人脂联素基因cDNA序列,自行设计引物一对,经PCR扩增人脂联素基因完全编码序列,构建pMD18-T/ADPN重组克隆质粒,目的序列测序鉴定；PCR扩增脂联素目的片段,连至真核表达载体pCDNA3.1/CT-GFP-TOPO,构建脂联素真核表达质粒pCDNA3.1/CT-GFP-TOPO-ADPN。②将pCDNA3.1/CT-GFP-TOPO-ADPN转化入TOP10感受态细胞,筛选可能含有阳性克隆的白色菌落进行鉴定,提取阳性克隆中的重组质粒,命名为pCDNA3.1-ADPN。③将pCDNA3.1—ADPN经脂质体介导、G418筛选后转染至人肝细胞株L-02,免疫荧光法观察转染率,收获的细胞裂解液及细胞培养上清经SDS-PAGE、western blot、细胞免疫组织化学法检测脂联素表达情况。④用含0 mmol/L、5 mmol/L、15 mmol/L和30 mmol/L葡萄糖的培基分别模拟无糖、低糖、中糖、高糖环境。将转染pCDNA3.1—ADPN的L-02细胞和对照组分别在上述环境中处理24 h,酶学法测定培养基血糖,观察重组脂联素蛋白的活性。⑤以western blot测定正常L-02组、转染组、30 mmol/L glucose处理组和“饥饿”组(72 h未换液)中phospho-AMPK、及其下游环腺苷酸反应元件结合蛋白(CREB)、phospho-CREB蛋白表达情况,免疫组化法观察四组细胞中环腺苷酸反应元件结合蛋白辅激活物2(TORC2)的定位情况,葡萄糖脱氢酶偶联法测定葡萄糖-6-磷酸酶(G6P)活性。结果：①成功构建脂联素克隆质粒pMD18-T/ADPN和真核表达质粒pCDNA3.1/CT-GFP-TOPO-ADPN(简称pCDNA3.1-ADPN)。②转染pCDNA3.1-ADPN的L-02细胞在免疫荧光显微镜下可见绿色荧光,表明转染成功,转染率在60%以上。③在细胞培养上清和沉淀中均检测到表达产物,表明重组蛋白利用自身信号肽获得分泌性表达。细胞裂解液和细胞培养上清中表达的重组蛋白分子量约为57 kD,具有人脂联素抗原性。④转染pCDNA3.1—ADPN的L-02细胞高糖(30mmol/L)处理24 h后其培基血糖低于对照组(p0.05),但在无糖、低糖和中糖环境中无明显差异(p0.05)。⑤与其它组比较,转染组：phospho-AMPK蛋白表达升高(p0.05), TORC2主要在定位在细胞质中,G6P酶的活性显著降低(p0.05),但CREB、phospho-CREB蛋白表达无明显差异(p0.05)。结论：我们成功构建了人脂联素真核表达质粒,并在人肝细胞株L-02中得到可溶性、分泌性表达,获得的重组蛋白具有生物学活性。脂联素抑制糖异生的机制可能与活化phospho-AMPK,抑制TORC2进入核内,从而抑制糖异生酶G6P的表达有关,其信号转导通路可能为Adiponectin-AMPK-CREB-TORC2-糖异生关键酶。这为进一步研究脂联素的信号通路,并探寻新的信号分子奠定了坚实的基础。
[Abstract]:Aim: to obtain the secretory expression of adiponectin recombinant protein with biological activity in L-02 cell line and to explore the signal transduction mechanism of adiponectin in L-02 cells.Methods the cDNA sequence of human adiponectin gene was obtained from human adipose tissue by RT-PCR. A pair of primers were designed and amplified by PCR to construct the recombinant plasmid of pMD18-T/ADPN.Objective to sequence and amplify the adiponectin target fragment by PCR, and connect it to the eukaryotic expression vector pCDNA3.1 / CT-GFP-TOPO. construct the adiponectin eukaryotic expression plasmid pCDNA3.1/CT-GFP-TOPO-ADPN.2 to transform pCDNA3.1/CT-GFP-TOPO-ADPN into TOP10 receptive cells and screen the white colony which may contain positive clones for identification.The recombinant plasmid was extracted from the positive clone and named as pCDNA3.1-ADPN.3. The pCDNA3.1-ADPN was screened by liposome and transfected into human hepatocyte strain L-02. The transfection rate was observed by immunofluorescence.The expression of adiponectin in the cell lysate and cell culture supernatant was detected by SDS-PAGEG western blot.4 the glucose free, low, medium and high glucose environments were simulated by a culture containing 0 mmol / L 5 mmol 路L ~ (-1) L ~ (15) mmol/L and 30 mmol/L glucose, respectively.The L-02 cells transfected with pCDNA3.1-ADPN and the control group were treated in the above environment for 24 hours respectively. The blood glucose of the culture medium was measured by enzymatic method. The activity of recombinant adiponectin protein was observed. The activity of recombinant adiponectin protein was measured by western blot in normal L-02 group.Phospho-AMPK and its downstream cyclic adenylate response element binding protein (CREBEB-CREB) were expressed in 30 mmol/L glucose treatment group and "hunger" group for 72 h.The localization of cyclic adenylate response element binding protein coactivator (TORC2) was observed by immunohistochemical method. The activity of glucose-6-phosphatase (G6P) was determined by glucose dehydrogenase coupling method.Results pMD18-T/ADPN and eukaryotic expression plasmid pCDNA3.1 / CT-GFP-TOPO-ADPN (pCDNA3.1-ADPN).2 transfected L-02 cell line) were successfully constructed by using 1: 1 adiponectin clone plasmid. Green fluorescence was observed in L-02 cells transfected with pCDNA3.1-ADPN under immunofluorescence microscope, indicating that the transfection was successful.The expression products were detected in the supernatant and precipitate of cell culture with transfection efficiency of more than 60%, indicating that the recombinant protein was secreted by using its own signal peptide.The molecular weight of the recombinant protein expressed in the cell lysate and cell culture supernatant was about 57 kD.The L-02 cell line with human adiponectin antigenicity was treated with high glucose 30 mmol / L for 24 h.There was no significant difference between low and medium sugar environment. Compared with other groups, the expression of TORC2 protein in the transfected group was higher than that in the other groups. The activity of G6P enzyme was significantly decreased in the cytoplasm of the transfected group, but there was no significant difference in the expression of phospho-CREB protein in the cytoplasm of the transfected group.Conclusion: we successfully constructed the eukaryotic expression plasmid of human adiponectin and obtained soluble and secretory expression in L-02 cell line. The recombinant protein has biological activity.The mechanism of adiponectin inhibiting glycosylation may be related to the activation of phospho-AMPK and the inhibition of TORC2 into the nucleus, thus inhibiting the expression of G6P. The signal transduction pathway of adiponectin-AMPK-CREB-TORC2-glycosylated key enzyme may be related to adiponectin-AMPK-CREB-TORC2-glycosylation.This provides a solid basis for the further study of adiponectin signaling pathway and the exploration of new signaling molecules.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

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