【作者】 梁慧芳；

【导师】 李卓娅；

【作者基本信息】 华中科技大学 ， 分子免疫学， 2007， 博士

【摘要】 当今肥胖发病率越来越高,已经成为世界上普遍的社会问题之一。肥胖与胰岛素抵抗密切相关,是非胰岛素依耐型糖尿病发病的高危因素之一。胰岛素抵抗(insulin resistance , IR)是指机体对一定量胰岛素的生物学反应低于正常水平,即胰岛素敏感性下降,常伴随2型糖尿病、脂代谢异常、高血压及心血管疾病,将之统称为胰岛素抵抗综合征,又称X综合征。TNF-α是由激活的单核/巨噬细胞、淋巴细胞等分泌的细胞因子,介导炎症反应、免疫应答、抗肿瘤等多种生物学效应。近来发现脂肪组织也合成和分泌TNF-α,它在胰岛素抵抗的发生机制中起关键性作用。已知TNF-α可通过胰岛素受体底物-1(IRS-1)丝、苏氨酸磷酸化抑制其酪氨酸磷酸化,从而抑制胰岛素的信号转导,这个效应究竟是TNFRI或TNFRII介导的,现在尚无定论。本室前期工作从噬菌体12线肽库中筛选出TNFRI封闭肽(blocking peptide,BP),它可与分泌型TNF-α竞争性结合TNFRI,发挥拮抗TNF-α的作用,为了增加该肽的亲合力和延长其半衰期,我们将该肽的核苷酸序列插入到分泌性表达IgG1Fc段的真核表达载体pIG/3C,得到TNFRIBP和人IgG1Fc融合蛋白的表达载体pIG/3C-TNFRIBP-Fc。本课题用该载体与带neo筛选标记的pcDNA3.1共转染CHO-K1和BHK-21建立稳定细胞系,得到大量纯化的融合蛋白用于治疗肥胖相关的胰岛素抵抗,为开发治疗TNF-α相关疾病的肽类药物奠定基础。一、TNFRIBP-Fc融合蛋白的稳定表达与纯化1.建立稳定细胞系:分别将pIG/3C-TNFRIBP-Fc和pIG/3C-IgG1Fc载体和带有G418筛选标记的pcDNA3.1载体共转染CHO-k1和BHK-21细胞,经G418筛选,挑取高表达株,经三次有限稀释,得到七株稳定表达TNFRIBP-Fc融合蛋白的细胞系,三株稳定表达Fc蛋白的细胞系。2.融合蛋白和Fc蛋白的纯化:无血清扩大培养这些细胞株,收集上清,经protein A-Sepharose CL-4B亲和层析,得到纯化的融合蛋白和Fc蛋白,经双抗体夹心ELISA定量,证实TNFRIBP-Fc融合蛋白的平均表达效率约为500ng/ml,Fc蛋白的平均表达效率约为400ng/ml。3.SDS-PAGE鉴定融合蛋白和Fc蛋白的分子量:SDS-PAGE显示纯化后的融合蛋白的分子量为37kD左右,Fc蛋白的分子量大约是36kD。4.纯化的融合蛋白生物活性的检测:间接免疫荧光和MTT试验证实融合蛋白能特异性结合鼠细胞系L929细胞上的TNFRI,且160ng/ml的融合蛋白就可完全阻断TNFRI介导的100U/ml的分泌性TNF-α(sTNF-α)引起的细胞毒效应。二、TNFRIBP-Fc融合蛋白的体外试验1.体外诱导前脂肪细胞3T3-L1分化成脂肪细胞:前脂肪细胞系3T3L1先用0.5mM IBMX、1μM地塞米松和10μg/ml的胰岛素诱导三天,再用10μg/ml的胰岛素诱导两天,撤去胰岛素培养两天,诱导其分化成脂肪细胞的效率达90%以上。2.体外高糖诱导脂肪细胞胰岛素抵抗:用不同浓度的葡萄糖作用上述分化的脂肪细胞24小时,结果显示25mM的葡萄糖即可诱导分化的3T3-L1发生胰岛素抵抗,表现为在胰岛素刺激下对3H-2-脱氧葡萄糖的摄取降低150倍左右(p<0.05)。3.内源性两型TNF-α与高糖诱导脂肪细胞胰岛素抵抗的关系:用不同浓度的葡萄糖作用上述分化的脂肪细胞24小时,结果显示与15mM葡萄糖作用组相比,25mM葡萄糖诱导胰岛素抵抗时,脂肪细胞表面TM-TNF-α表达从81.4%降至3.56%,而培养上清中sTNF-α从63.73升高113.03 (pg/ml)。在诱导胰岛素抵抗的同时加入160ng/ml的融合蛋白或TACE抗体,明显改善高糖诱导的胰岛素抵抗,与25mM葡萄糖诱导抵抗组比,前者把细胞在胰岛素刺激下对糖摄取提高了20倍,后者提高了50倍。4.外源性两型TNF-α对脂肪细胞胰岛素应答的影响:分别用sTNF-α和TM-TNF-α作用分化的脂肪细胞24小时,观察细胞在胰岛素的刺激下对糖的摄取。结果显示与未刺激的对照组比,sTNF-α使脂肪细胞对胰岛素刺激的糖摄取降低了96%,而TM-TNF-α却使之增加了约2倍;同时加入融合蛋白或抗TNF抗体,可几乎完全逆转sTNFα诱导的胰岛素抵抗,同时也几乎完全阻断TM-TNFα提高脂肪细胞对胰岛素的敏感性的作用。5.两型TNFα对脂肪细胞胰岛素信号转导的影响:分别用sTNFα和TMTNF-α作用分化的脂肪细胞24小时,用Western检测IRS-1酪氨酸和丝氨酸磷酸化水平。结果显示sTNFα使IRS-1270位丝氨酸磷酸化水平升高而酪氨酸磷酸化水平下降,TM-TNF-α不影响正常胰岛素诱导的丝氨酸/酪氨酸磷酸化水平。三、TNFRIBP-Fc融合蛋白的体内试验1.高糖高脂饮食建立大鼠胰岛素抵抗的模型:选取体重在110～120g的雄性Wistar大鼠,给与普通饮食(ND)或者高糖高脂饮食(HFS)喂养16周,结果显示与普通饮食相比,高糖高脂饮食大鼠体重每月增加18%,血糖维持正常的同时表现出明显的高胰岛素血症,胰岛素抵抗指数HOMAIR升高了3倍。提示成功建立肥胖引起的胰岛素抵抗。2.治疗分组:第17周开始HFS组随机分成四组:融合蛋白治疗组(HFS+Fusion)、Fc蛋白治疗组(HFS+Fc)、TNF-α兔多抗治疗组(HFS+Ab)和单纯高糖高脂饮食对照组(HFS),每只大鼠尾静脉注射0.1ml生理盐水含500μg纯化的TNFRIBP-Fc融合蛋白或Fc蛋白或TNF-α兔多抗或0.1ml生理盐水,每周两次持续4周。普通饮食组(ND)保持不变。3.融合蛋白对体重、脂肪沉积和血清甘油三酯、总胆固醇的影响:HFS组体重增加值是ND组的2倍,附睾周围脂肪组织的重量是ND组1.8倍,脂肪细胞明显增大,血中甘油三酯水平是ND组的3.7倍。融合蛋白治疗明显改善了HFS诱导的脂肪沉积和高甘油三酯血症:体重增加值下降了26%,脂肪组织重量下降了30%,脂肪细胞体积也明显减小,甘油三酯水平下降了54%。血清胆固醇水平各组没有差异。抗体组的治疗效果最好,上述指标抗体组与普通饮食组之间没有显著差异。4.融合蛋白对糖代谢及胰岛素敏感性的影响:各组治疗前后空腹葡萄糖都在正常水平,但HFS组胰岛素水平、C肽和HOMAIR是ND组2倍左右,胰岛代偿性肥大,胰岛素和葡萄糖耐受实验说明胰岛素的敏感性相对ND组显著下降。融合蛋白治疗后,与HFS组比,胰岛素、C肽和HOMAIR都几乎下降了50%,胰岛代偿性肥大也得到缓解。胰岛素和葡萄糖耐受试验直接说明融合蛋白治疗后胰岛素敏感性显著增加,而Fc治疗则没有效果。抗体组治疗后敏感性与普通饮食组之间几乎没有显著差异。5.ELISA显示融合蛋白治疗对TNF-α蛋白水平的影响:用ELISA检测血清和组织蛋白提取液中TNF-α的水平。结果显示HFS组血清sTNF-α和肌肉、脂肪组织中TNF-α水平都明显增高,融合蛋白治疗使血清和脂肪组织中TNF-α的水平下降50%,使肌肉中TNF-α的水平下降了80%。肝脏组织各组之间没有差异。抗体组脂肪和肌肉组织TNF-α的水平仍比ND组高2.5和1.4倍。6.免疫组化显示融合蛋白对脂肪和胰腺组织TNFα蛋白水平的影响:用免疫组化比较脂肪和胰腺组织之间TNF-α表达水平。结果显示HFS组这两个组织TNF-α表达水平比ND组明显升高,融合蛋白和抗体治疗后TNFα水平显著下降。7.融合蛋白对IRS-1酪氨酸磷酸化水平的影响:用免疫共沉淀和Western比较各组IRS-1酪氨酸磷酸化水平。结果显示HFS组脂肪、肌肉组织和肝脏组织IRS-1酪氨酸磷酸化水平相对与ND组下降80%、68%和32%,融合蛋白治疗使脂肪和肌肉IRS-1酪氨酸磷酸化水平相对HFS组分别升高了54%和50%,肝脏组织IRS-1酪氨酸磷酸化水平各治疗组之间没有区别。抗体治疗使脂肪和肌肉IRS-1酪氨酸磷酸化水平相对HFS组分别升高了75%和59%。四、sTNFRⅡ的原核表达、活性鉴定及其多克隆抗体的制备1.sTNFRⅡ的原核表达:采用RT-PCR方法,从人外周血的单核细胞中扩增出人TNFRⅡ基因的胞外区,将其克隆入pET28a(+)高效表达载体,测序鉴定后得到正确的重组子,用IPTG诱导其表达后经镍柱纯化,得到sTNFRⅡ重组蛋白。经10%SDS-PAGE分析其分子量约为32kD,扫描分析,其表达量占菌体蛋白总量的30%左右。2.纯化后sTNFRⅡ的生物活性鉴定:sTNFRⅡ经western鉴定可以与TNFRⅡ的抗体结合;间接免疫荧光显示sTNFRⅡ可以抑制GFP-sTNF-α融合蛋白与细胞表面TNFR的结合;MTT实验显示20μg/ml sTNFRⅡ几乎可完全阻断sTNF-α引起的细胞毒效应。3.多克隆抗体的制备:用纯化的sTNFRⅡ与完全佛氏佐剂免疫两只家兔后得到抗TNFRⅡ多克隆抗体,效价分别为1:32000和1:16000。结论:(1)成功真核表达和纯化具有生物活性的融合蛋白TNFRIBP-Fc;(2)体外实验证实融合蛋白可以改善高糖和sTNF-α诱导脂肪细胞胰岛素抵抗,其作用机制主要是下调sTNF-α的表达和阻断sTNF-α与TNFRI结合介导的胰岛素抵抗;(3)两型TNF-α在胰岛素抵抗中发挥不同的作用:sTNF-α导致脂肪细胞胰岛素抵抗,TMTNF-α提高脂肪细胞对胰岛素的敏感性;(4)体内实验证实融合蛋白通过下调和抑制sTNF-α作用而改善高糖高脂饮食造成的肥胖和胰岛素抵抗。更多还原

【Abstract】 Obesity, with an increasing prevalence, has been become the most common metabolic disorder in the world. Obesity is highly associated with insulin resistance and is the biggest risk factor for non-insulin-dependent diabetes mellitus. Insulin resistance or the decrease in insulin sensitivity, defined as a smaller than expected biological response to a given dose of insulin, preceds overt diabetes and signifies a metabolic disorder called syndrome X (concurrence of type 2 diabetes, dyslipidemias, hypertension, and cardiovascular disease).Tumor necrosis factor (TNF-α), produced chiefly by activated monocytes/macrophages and T lymphocytes, is a pleiotropic cytokine with a wide range of biological effects, such as anti-infection, antitumor, immune regulation and inflammation. Recently TNF-αhas been found is synthesised and secreted by adipose tissue and plays a key role in insulin resistance. The general mechanism of TNF-α-induced insulin resistance involves inhibition of insulin receptor signaling as demonstrated in variety of cell types and obese animals. TNF-αinhibits insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1) at least in part by induction of serine phosphorylation of IRS-1. Although it has been previously shown that the lack of TNF-αfunction improve insulin sensitivity in obese animals, no definite conclusion about which TNFR mediates this action of TNF-α.TNFRI blocking peptide (TNFRIBP) was obtained by screening a phage displayed random 12 amino acid peptide library using soluble recombinant human TNFRI in our previous study. TNFRIBP was able to block TNF-αbioactivity by competitively binding to TNFRI. In order to enhance its affinity and prolong its half-life time, we inserted the oligonucleotide sequence of this peptide into the pIg/3C eukaryotic expression vector that contains an upsteam signal peptide sequence necessary for the secretion of chimeras and a downstream human IgG1-Fc fragment sequence, so we got the recombinant vector pIg/3C-TNFRIBP-Fc to secretively express TNFRIBP-Fc fusion protein. The aim of this study to establish stable cell lines to express TNFRIBP-Fc fusion protein by cotransfection CHO-K1 and BHK-21 cells with this recombinant vector and pcDNA3.1 which contains neo gene for selection, gain plenty of purified protein for treatment insulin resistance linked to obesity in vitro and in vivo. This study is expected to lay the foundaton for development of new peptide drug to treat TNF-αrelated diseases.1. Stable express and purify fusion protein TNFRIBP-Fc⑴Establish stable cell lines: CHO-K1 and BHK-21 cells were cotransfected with pIg/3C-TNFRIBP-Fc vector and pcDNA3.1 which contained neo gene for selection, then G418 resistant transformants were selected. After subcloning through three limited-dilution , seven stable cell lines which had high productivity of fusion protein and three stable cell lines which had high productivity of Fc protein were picked out, assessed by ELISA.⑵Purify fusion protein and Fc protein: We cultured these stable cell lines in quantity in the medium without serum and collected the culture supernatants. Fusion protein and Fc protein from supernatants were purified by chromatography on protein A-sepharose CL-4B. Approximately 50μg of fusion protein or 40μg of Fc protein was purified from 100ml of cultural supernatant, quantitied by a sandwich ELISA.⑶Identify molecular weight by SDS-PAGE: The molecule weight of the purified protein and Fc protein were about 37kD and 36kD respectively analyzed by SDS-PAGE.⑷Identify its biological activities: Indirect immunofluorescene showed that fusion protein was able to bind murine TNFRI. TNF-αbioassay suggested that fusion protein, but not Fc protein, suppressed TNF-α-induced cell death in a dose dependent manner. As low as 160ng/ml, the fusion protein completely inhibited the cytotoxicity induced by 100U/ml TNF-α.2. Effect of fusion protein on insulin resistance in vitro⑴Induce 3T3-L1 preadipocytes to differentiate into adipocytes: Briefly, the monolayer cell were maintained in medium A (DMEM with 10% fetal calf serum). Differentiation was induced by incubating the cells with medium A supplemented with 10μg/ml insulin, 1μM dexamethasone, 0.5mM IBMX for 3 days, followed by another 2 days incubation with medium A supplemented with 10μg/ml insulin. The cells were further incubated in medium A for an additional 2 days to complete the adipocyte conversion. Followed this procedure, great than 90% of the cells had the morphological and biochemical properties of adipocytes.⑵High glucose induce insulin resistance in 3T3-L1 adipocytes: 3T3-L1 adipocytes were treated with different concentration glucose for 24 hours, the insulin-stimulated 3H-2-deoxyglucose incorporation test were used to determin insulin sensitivity of 3T3-L1 cells. Results showed that 25mM glucose induced the adipocytes insulin resistance, for decreasing insulin-stimulated increase in glucose uptake by 150 times.⑶The effect of the two forms of endogenous TNF-αon insulin resistance induce by high glucose: 3T3-L1 adipocytes treated with different concentration glucose for 24 hours. Results showed when the cells were induced insulin resistance by 25mM glucose, the TM TNF-αprotein level was declined from 81.4% to 3.56% (p<0.01) and sTNF-αlevel was increased from 63.7 to 113.0 pg/ml (p<0.01), compared to group with 15mM glucose. Insulin resistance could be blocked in part by 160ng/ml anti-TACE antibody or 160ng/ml TNFR1BP-Fc fusion protein, compared to group induced insulin resistance by 25mM glucose, fusion protein and anti-TACE antibody increased insulin-stimulated glucose uptake by 20 and 50 times, respectively.⑷The effect of the two forms of exogenous TNF-αon reponse of 3T3-L1 adipocytes to insulin: 3T3-L1 adipocytes were treated with the two forms of exogenous TNF-αfor 24 hours and detected glucose uptake with insulin stimulation. Compared to the control group without any stimulation, sTNF-αdecreased insulin-stimulated glucose uptake by 96%, but TMTNF-αincreased two times. We alse found that fusion protein or anti-TNF-αantibody can completely block both of sTNF-αand TMTNF-αeffects when they were put together alone.⑸The effect of the two forms of exogenous TNF-αon insulin signaling: 3T3-L1 adipocytes were treated with the two forms of TNF-αfor 24 hours and after that we detected the level of IRS-1 tyrosine phospharylation and serine phopharylation on 270 site (Ser270) to find out its molecular mechanism. We found that sTNF-αstimulated serine phospharylation of IRS-1 and inhibited tyrosine phopharylation, TM TNF-αacted on the contrary: TM TNF-αinhibited IRS-1 Ser270 phospharylation without effect on tyrosine phopharylation.3. Effect of fusion protein on insulin resistance in vivo⑴Establish insulin resistance animal model: Four-week-old male wistar rats weighing between 110 and 130g were fed either with a normal diet (ND) or a high-fat and high-sucrose diet (HFS). Obesity associated insulin resistance was induced by HFS diet for 16 weeks, manifesting as increased body weight by 18%, significant hyperinsulinaemia but euglycemia maintenance and increased HOMAIR index by three times when compared with ND group.⑵Treat animals: After 16 weeks, besides the ND group, the rats with HFS were randomly assigned into four treated groups: fusion protein treatment group, Fc protein treatment group, rabbit anti-TNF-αpolyclonal antibody treatment group and the HFS control group. The rats were given ingtravenoursly with 500μg TNFR1BP-Fc fusion protein or Fc protein or rabbit anti-TNF-αpolyclonal antibody in 0.1 ml normal saline (NS) or just 0.1ml NS treatmeny respectively, twice a week for four weeks. ⑶Inhibitory effect of the fusion protein on body weight, adiposity, serum triglyceride and total chcholesteraemia: At the end of 20 weeks, the mean body weight gain, the weight of epididymal fat pads and the level of serum triglyceride of HFS group was 2 times,1.8 and 3.7 times than that of ND group. Besides that histological analysis of adipose tissue also showed that adipocytes were larger in HFS group than in ND group. The treatment with fusion protein, but not Fc protein for 4 weeks resulted in a significantly decrease weight gain by 26%, fat pads by 30% and the triglyceride level by 50%。Similarity, histological analysis of adipose tissue also shows that adipocytes were larger in HFS fed rats than in control rats. In contrast, the treatment of fusion protein, but not Fc protein, diminished their larger diameter. All these measurements of anti-TNF-αantibody (HFS+Ab) group showed no difference between the ND group. Total cholesterol levels were unchanged in the plasma among the four group.⑷The effect of the fusion protein on glucose metabolism and insulin sensitivity: The blood glucose leveles of all groups showed no difference among them and kept in normal limits, but the serum insulin, C-peptide and HOMAIR were about two folds of ND. The pancreatic islet was compensatory hypertrophy. The Insulin and glucose tolerance experiment explained that the sensitivity of insulin significantly impaired by HFS diet compared to ND group. After treatment with fusion protein, the parameter of insulin, C-peptide and HOMAIR nearly descended to 50%. The compensatory hypertrophy of pancreatic was released too. The Insulin and glucose tolerance experiment directly illustrated that the insulin sensitivity notably increased after post-treatment, but there was no effect of treatment with Fc. The insulin sensitivity of HFS+Ab group showed no difference between the ND group.⑸The effect of the fusion protein on TNFαprotein level by ELISA: We detected the TNFαin serum and in the extract of tissue protein. In HFS group, the content of TNFαin muscle and adipose tissue, obviously upgraded. In fusion protein group, the content of TNFαin serum and adipose tissue downgraded to 50%, however, in muscle downgraded to 80%. In the HFS+Ab group, the content of TNFαin serum and adipose tissue downgraded to 60%, however, in muscle downgraded to 80%.There was no difference in liver tissue of all groups. The levels of TNFαin adipose tissue and muscle tissue were 2.5 and 1.4 folds than that of ND group. ⑹The effect of fusion protein on TNFαprotein in adipose and pancreatic tissue by immunity histochemistry: We compared the expression level of TNFαin adipose and pancreatic tissue by immunity histochemistry. Expression level of TNFαobviously increased compared to ND group and decreased after post-treatment by fusion protein.⑺The effect of fusion protein on IRS-1 tyrosine phosphorylation: We tested tyrosine phosphrylation of IRS-1 by IP-Western blotting. The phosphorylation of IRS-1 was significantly inhibited in fat by 80%, muscle by 68% and liver by 32% for HFS-fed rats compared with that of ND-fed rats. Treatment with fusion protein led to a striking increase of IRS-1 phosphorylation by 54% in fat and by 50% in muscle. Treatment with antibody led to increase of IRS-1 phosphorylation by 75% in fat and by 59% in muscle.4. Cloning, expression and bioactivity appraisement of human sTNFRII and preparation its polyclonal antibody⑴Construct and express the recombiant of human soluble TNF receptorII(sTNFRII) by E.coli: The extracellular domain cDNA of human TNFRII was obtained by RT-PCR from human monocytes. The gene fragment was inserted into pET-28a(+) and positive recombinant was identified by sequencing without mutation. Protein expression was induced by IPTG in E.coli. The expression product was isolated and purified by Ni-NTA agrose. Results showed that the highest expression was achieved after induction for 6 h with IPTG.. SDS-PAGE showed an extra protein band which was around 32kD in size, which occuppied 30% of the total protein in E.coli.⑵Identify its biological activities: Western blotting showed that recombinant sTNFRII was able to bind anti-TNFRII antibody. Indirect immunofluorescene indicated that sTNFRⅡcould inhibit the binding of GFP-sTNF-αto TNFR on L929. TNF-αbioassay suggested that as low as 20μg/ml, sTNFRII completely inhibited the cytotoxicity induced by 100U/ml TNF-α.⑶Prepare sTNFRII polyclonal antibody: we used recombinant sTNFRII as antigen to immune rabbit and gained polyclonal antibody. The titres of the sTNFRII specific ployclonal antibody obtained were 1:32000 and 1:16000.Conclusion:⑴Eukaryotic stable expresses and purifies the fusion protein TNFRIBP-Fc which it is bioactive in murine system;⑵In vitro we identify Fusion protein ameliorates insuin resistant of 3T3-L1 adipocyte induced by high glucose and sTNF-αthrough downregulation sTNF-αexpression level and inhibit their interaction between sTNF-αand TNFRI;⑶TM TNF-αand sTNF-αplay different role in insulin resistance:TM TNF-αcan improve insulin sensitivity but sTNF-αinduced insulin resistance;⑷TNFRIBP-Fc fusion protein improves obesity and insulin resistance induced by high fat and high sucrose diet and protected form the obesity-related reduction in the insulin recetor signaling in muscle and fat tissues, mainly responsible for peripheral glucose uptake with stimulation by insulin.更多还原