本文選題：人脂聯(lián)素 + 糖尿病�。� 參考：《天津醫(yī)科大學》2008年碩士論文

【摘要】：目的：在人肝細胞株L-02中獲取具有生物學活性的脂聯(lián)素重組蛋白的分泌性表達,并在L-02細胞中探討脂聯(lián)素抑制糖異生的信號轉(zhuǎn)導機制。 方法：①從人脂肪組織中提取總RNA,利用RT-PCR方法得到人脂聯(lián)素基因cDNA序列,自行設計引物一對,經(jīng)PCR擴增人脂聯(lián)素基因完全編碼序列,構(gòu)建pMD18-T/ADPN重組克隆質(zhì)粒,目的序列測序鑒定；PCR擴增脂聯(lián)素目的片段,連至真核表達載體pCDNA3.1/CT-GFP-TOPO,構(gòu)建脂聯(lián)素真核表達質(zhì)粒pCDNA3.1/CT-GFP-TOPO-ADPN。②將pCDNA3.1/CT-GFP-TOPO-ADPN轉(zhuǎn)化入TOP10感受態(tài)細胞,篩選可能含有陽性克隆的白色菌落進行鑒定,提取陽性克隆中的重組質(zhì)粒,命名為pCDNA3.1-ADPN。③將pCDNA3.1—ADPN經(jīng)脂質(zhì)體介導、G418篩選后轉(zhuǎn)染至人肝細胞株L-02,免疫熒光法觀察轉(zhuǎn)染率,收獲的細胞裂解液及細胞培養(yǎng)上清經(jīng)SDS-PAGE、western blot、細胞免疫組織化學法檢測脂聯(lián)素表達情況。④用含0 mmol/L、5 mmol/L、15 mmol/L和30 mmol/L葡萄糖的培基分別模擬無糖、低糖、中糖、高糖環(huán)境。將轉(zhuǎn)染pCDNA3.1—ADPN的L-02細胞和對照組分別在上述環(huán)境中處理24 h,酶學法測定培養(yǎng)基血糖,觀察重組脂聯(lián)素蛋白的活性。⑤以western blot測定正常L-02組、轉(zhuǎn)染組、30 mmol/L glucose處理組和“饑餓”組(72 h未換液)中phospho-AMPK、及其下游環(huán)腺苷酸反應元件結(jié)合蛋白(CREB)、phospho-CREB蛋白表達情況,免疫組化法觀察四組細胞中環(huán)腺苷酸反應元件結(jié)合蛋白輔激活物2(TORC2)的定位情況,葡萄糖脫氫酶偶聯(lián)法測定葡萄糖-6-磷酸酶(G6P)活性。 結(jié)果：①成功構(gòu)建脂聯(lián)素克隆質(zhì)粒pMD18-T/ADPN和真核表達質(zhì)粒pCDNA3.1/CT-GFP-TOPO-ADPN(簡稱pCDNA3.1-ADPN)。②轉(zhuǎn)染pCDNA3.1-ADPN的L-02細胞在免疫熒光顯微鏡下可見綠色熒光,表明轉(zhuǎn)染成功,轉(zhuǎn)染率在60%以上。③在細胞培養(yǎng)上清和沉淀中均檢測到表達產(chǎn)物,表明重組蛋白利用自身信號肽獲得分泌性表達。細胞裂解液和細胞培養(yǎng)上清中表達的重組蛋白分子量約為57 kD,具有人脂聯(lián)素抗原性。④轉(zhuǎn)染pCDNA3.1—ADPN的L-02細胞高糖(30mmol/L)處理24 h后其培基血糖低于對照組(p0.05),但在無糖、低糖和中糖環(huán)境中無明顯差異(p0.05)。⑤與其它組比較,轉(zhuǎn)染組：phospho-AMPK蛋白表達升高(p0.05), TORC2主要在定位在細胞質(zhì)中,G6P酶的活性顯著降低(p0.05),但CREB、phospho-CREB蛋白表達無明顯差異(p0.05)。 結(jié)論：我們成功構(gòu)建了人脂聯(lián)素真核表達質(zhì)粒,并在人肝細胞株L-02中得到可溶性、分泌性表達,獲得的重組蛋白具有生物學活性。脂聯(lián)素抑制糖異生的機制可能與活化phospho-AMPK,抑制TORC2進入核內(nèi),從而抑制糖異生酶G6P的表達有關,其信號轉(zhuǎn)導通路可能為Adiponectin-AMPK-CREB-TORC2-糖異生關鍵酶。這為進一步研究脂聯(lián)素的信號通路,并探尋新的信號分子奠定了堅實的基礎。
[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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R363

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