本文選題：SH2B1β + 克隆��； 參考：《南方醫(yī)科大學》2009年碩士論文

【摘要】： 目前,肥胖迅速向全球蔓延,已經波及到人們概念中該問題并不嚴重的東南亞地區(qū)。因此,肥胖已成為當今全球醫(yī)學上越來越嚴重的問題。當人們的體重大大超過推薦的指標就可以確診為肥胖癥,隨著肥胖患病率的增高,與肥胖相關的其他慢性非傳染性疾病,如糖尿病、代謝綜合征、高血壓、心腦血管疾病、睡眠障礙、哮喘、腫瘤等的患病率也呈明顯上升的趨勢。根據(jù)流行病學統(tǒng)計,肥胖發(fā)生率的增加與成年人群死亡率的增加呈高度相關。近年來的研究表明,肥胖是由特定的生化因子引起的一系列進食調控和能量代謝紊亂而導致的疾病,與遺傳、環(huán)境、基因、膳食結構有關,而基因是主要的決定因素。從宏觀的水平上看,食物攝入增加、體力活動減少和生熱機制的改變導致了脂肪的過多堆積;但從微觀水平上分析,成熟脂肪細胞的增多是脂質發(fā)生累積的關鍵因素。肥胖已經不僅僅是影響人體形象美觀的問題,還是威脅人類健康的問題。被列為世界上第六位的影響全人類疾病負擔的危險因素。 1950年,Ingalls等發(fā)現(xiàn)一個基因的陰性突變可以導致肥胖,他將該基因稱為肥胖基因(obese gene,ob gene),第一次出現(xiàn)了ob基因的概念。Coleman等用交叉灌流實驗證明,某種血源性因子能調節(jié)動物攝食、代謝、參與肥胖形成,且這種因子的產生與ob基因有關。1991年Friedman描述了五種可以使小鼠形成肥胖的單基因突變,分別是obese(ob)、diabetes(db)、fat(fa)、tubby(tub)和obese yellow(A~y),為研究肥胖的分子機制奠定了基礎。1994年Zhang等利用突變基因的定位克隆技術克隆了小鼠和人的ob基因,同時發(fā)現(xiàn)ob基因的突變可以導致肥胖,使肥胖研究真正進入分子時代。 瘦素蛋白(Leptin)是ob基因的產物,是由白色脂肪細胞分泌的一種有167個氨基酸殘基組成的蛋白質。它除了通過作用于下丘腦特異性受體發(fā)揮抑制飲食、減少能量攝入、減輕體重和增加能量消耗的作用外,同時還通過其它組織器官上的受體(胰島、肝臟、性腺、腎上腺、甲狀腺等)在體內共同形成一個復雜的網絡作用系統(tǒng),影響著機體許多生理系統(tǒng)和代謝通路。瘦素蛋白的外周作用還包括調節(jié)糖代謝的平衡、促進脂肪分解、抑制脂肪合成、參與免疫調節(jié)功能和促進生長發(fā)育等。經過長期的研究,人們對瘦素蛋白的生物學作用已經有更進一步的認識:瘦素蛋白在能量代謝調節(jié)中并不是一個孤立的激素,而是與許多神經肽、神經遞質、激素及其它因子協(xié)同發(fā)揮作用;在人群中存在有不可忽視的“瘦素抵抗”現(xiàn)象等。這些都是致力把瘦素蛋白單獨用作為治療肥胖癥藥物的研究者今后需要解決的問題。 隨著研究的深入,研究者發(fā)現(xiàn)SH2B1β蛋白能與瘦素激活的細胞信號蛋白JAK2(非受體酪氨酸激酶)相互作用,調節(jié)能量代謝和體重。SH2B1β是SH2B信號接頭蛋白家族中的一員,其結構上有SH2結構域,可以結合磷酸化的酪氨酸殘基,進而連接不同的信號蛋白,發(fā)揮其銜接信號及強化信號通路的作用。SH2B1β廣泛分布于下丘腦、肝臟、胰臟、脂肪組織等組織中,參與胰島素敏感性和血糖平衡的調節(jié)以及增強神經生長因子誘導的神經分化。由于SH2B1β是Leptin/JAK2信號通路活化所必需的,如果敲除小鼠的SH2B1β基因,將使小鼠的體重是同窩正常組小鼠的兩倍,SH2B1β基因缺失導致嚴重的瘦素抵抗,食欲過盛和肥胖。SH2B1β通過自身結合JAK2同時募集胰島素底物蛋白(IRS)結合JAK2復合體,以JAK2/STAT3和IRS2/PI3K兩條路徑來增強瘦素依賴的信號轉導,因而SH2B1β是一種內源的瘦素敏感性增強劑,同時,SH2B1β的過表達還可以抵消蛋白酪氨酸磷酸酶1B(PTP1B)介導的瘦素抑制信號的作用。 目前有最新的文獻報道指出,脂肪組織與神經組織中的SH2B1β在脂肪細胞的生長分化過程中產生兩種相反的效應。雖然神經組織SH2B1β的負調節(jié)效應占優(yōu),但是脂肪組織SH2B1β是以一種細胞自主的方式來調節(jié)脂肪細胞的分化,并在3T3-L1前脂肪細胞的分化過程中呈現(xiàn)表達量進行性增加,同時促進細胞中脂滴的累積以及脂肪分化轉錄因子PPARγ、C/EBPα的表達。 脂肪細胞數(shù)量的增多和體積的增大能導致肥胖,而前脂肪細胞分化的增加則是導致成熟脂肪細胞數(shù)量增多的主要原因,近年來脂肪細胞的分化調控已成為肥胖及其相關疾病的研究熱點。人或動物的脂肪組織成份包括微血管、神經組織、成纖維細胞、脂肪細胞以及前脂肪細胞,脂肪細胞系的發(fā)育過程為多能干細胞—間充質干細胞—前脂肪細胞—成熟脂肪細胞,所以有效的調節(jié)前脂肪細胞的分化能控制脂肪組織增生、肥大。脂肪細胞分化與胰島素和胰島素樣生長因子相關,胰島素和胰島素樣生長因子通過結合其受體激活PI3K和MAPK兩條信號轉導通路,促進脂肪細胞的分化。SH2B1β作為一種胰島素受體(IR)和胰島素樣生長因子受體(IGF-IR)活性的正調節(jié)蛋白,參與調控脂肪細胞分化的信號通路。 目前有關脂肪組織SH2B1β的研究很少,而且研究主要是針對鼠源蛋白。根據(jù)文獻報道SH2B1β鼠源基因與人源基因的相似性達到85%-87%,本課題著眼于使用人源的SH2B1β基因在體外原核表達重組蛋白后,制備抗SH2B1β的單克隆抗體,通過定性及定量的方法初步探討該人源蛋白在人源前脂肪細胞分化中的調控作用,為治療與前脂肪細胞分化關系密切的肥胖及其并發(fā)癥提供新的藥物靶點。 由于脂肪干細胞是臨界于間充質干細胞與前脂肪細胞時期之間的一種細胞狀態(tài),所以本研究通過體外原代培養(yǎng)脂肪干細胞(ADSC)并誘導其成脂分化,收集分化成熟的脂肪細胞后,用TRIZOL一步法提取細胞總RNA。運用RT-PCR方法成功擴增并克隆出SH2B1β基因片段,將所得的目的基因克隆至pET28a(+)表達載體,并在大腸桿菌中進行了大量可溶性表達。用飽和硫酸銨鹽析法和DEAE陰離子交換法純化重組蛋白,重組蛋白通過抗His-tag單抗鑒定后免疫小鼠制備單克隆抗體,分別用ELISA、Western-Blot法檢測及鑒定單克隆抗體的特異性。 利用RT-PCR技術成功克隆了SH2B1β基因,其大小約為2000bp。經測序并與GenBank中序列進行比對,結果顯示插入片段與已報道的SH2B1β基因序列配對一致,無移碼突變,大小為2016bp。將編碼基因克隆到pET-28a(+)原核表達質粒中,經PCR、限制性酶切分析等鑒定,證實成功構建了SH2B1β-pET28a(+)重組原核表達質粒。 經IPTG誘導后,重組質粒SH2B1β-pET28a(+)在大腸桿菌E.coli DE3中表達出His-tag融合重組蛋白,分子量約為80kDa,與理論值基本符合。表達產物主要以可溶性形式存在。經過純化后,重組蛋白純度可達80%以上。用抗His-tag單抗對表達產物進行Western-blot分析,可見特異性區(qū)帶出現(xiàn),表明該蛋白確實是所要表達的His-tag融合蛋白。進一步用純化的表達產物免疫小鼠制備免疫血清,ELISA分析表明重組蛋白能與該免疫血清起反應,而與正常小鼠血清不發(fā)生交叉反應,說明重組蛋白具有抗原活性。 將免疫小鼠脾細胞與小鼠骨髓瘤細胞NS-1融合后篩選到2株單抗分泌細胞。以純化重組蛋白為抗原,用ELISA法檢測單抗細胞株培養(yǎng)上清的效價,OD值分別為1.553～2.210。Western-blot結果顯示:抗SH2B1β的2株單抗與人源脂肪組織內提取的天然蛋白發(fā)生特異性反應,而陰性對照組則無明顯條帶出現(xiàn),說明制備的單抗均為特異性單抗。 以上結果表明,我們已經成功地構建了SH2B1β-pET28a(+)原核重組表達質粒,目前國內尚未見關于SH2B1β重組并表達的相關報道。而且在大腸桿菌中大量表達出具有免疫活性的可溶性重組蛋白SH2B1β;篩選并建立了2株分泌抗SH2B1β單克隆抗體的雜交瘤細胞株,為進一步研究該蛋白的生物學功能、在脂肪細胞發(fā)育分化過程中所起的作用、對肥胖癥潛在治療價值的驗證奠定基礎。
[Abstract]:Obesity has spread rapidly around the world and has spread to Southeast Asia, which is not a serious problem in people's concept. Obesity has become a growing problem in global medicine today. The prevalence of chronic non communicable diseases, such as diabetes, metabolic syndrome, hypertension, cardiovascular and cerebrovascular diseases, sleep disorders, asthma, and cancer, has also increased significantly. According to epidemiological statistics, the increase in the incidence of obesity is highly related to the increase in the mortality of adults. Recent studies have shown that obesity is specific. The disease caused by a series of eating regulation and energy metabolism disorder caused by biochemical factors, related to heredity, environment, gene, and dietary structure, and genes are the main determinants. From the macro level, the increase in food intake, the decrease of physical activity and the change of the mechanism of heat generation have resulted in excessive accumulation of fat; but from the micro level. The increase in mature adipocytes is the key factor in the accumulation of lipid. Obesity is not only a problem that affects the beauty of the human body, but also a threat to human health. It is listed as the sixth risk factor affecting the burden of disease in the world.
In 1950, Ingalls and others found that a negative mutation of a gene could lead to obesity. He called the gene obese gene (OB gene), and the first occurrence of the concept.Coleman of the ob gene showed that a certain blood source factor could regulate animal feeding, metabolism, and participate in the formation of obesity, and the production of such factors and ob based factors Five kinds of single gene mutations that could make the mice obese were described in.1991. They were obese (OB), diabetes (DB), fat (FA), tubby (tub) and obese yellow, which laid the foundation for the study of the molecular mechanism of obesity. Now mutations in the ob gene can lead to obesity, making obesity research truly enter the molecular age.
Leptin is a product of the ob gene, a protein composed of 167 amino acid residues secreted by white adipocytes. It is not only able to inhibit diet, reduce energy intake, reduce weight and increase energy consumption by acting on the hypothalamic specific receptors, but also through other tissues and organs. The body (islets, livers, gonads, adrenal glands, thyroid glands, etc.) forms a complex network of networks in the body that affects many physiological and metabolic pathways in the body. The peripheral action of leptin proteins also includes regulating the balance of sugar metabolism, promoting fat decomposition, inhibiting fat synthesis, participating in immunoregulation and promoting growth and development. After a long period of study, there is a further understanding of the biological effects of leptin protein: leptin protein is not an isolated hormone in the regulation of energy metabolism, but a synergistic effect with many neuropeptides, neurotransmitters, hormones and other factors; there is a phenomenon of "leptin resistance" that can not be ignored in the population. All these are the problems to be solved by researchers who are trying to use leptin alone as a treatment for obesity drugs.
With the further research, the researchers found that SH2B1 beta protein can interact with the cell signaling protein JAK2 (non receptor tyrosine kinase) activated by leptin. The regulation of energy metabolism and body weight.SH2B1 beta is a member of the SH2B signal junction protein family. The structure has a SH2 domain, which can be combined with the phosphorylated tyrosine residues and then connect to different types. The role of signal protein, the function of its cohesive signal and intensification signal pathway,.SH2B1 beta is widely distributed in the hypothalamus, liver, pancreas, adipose tissue and other tissues, and participates in the regulation of insulin sensitivity and blood glucose balance and the enhancement of neural differentiation induced by nerve growth factor. Since SH2B1 beta is essential for the activation of Leptin/JAK2 signaling pathway, If the SH2B1 beta gene was knocked out of the mice, the weight of the mice would be two times that of the normal group, SH2B1 beta gene deletion led to severe leptin resistance, excessive appetite and obesity.SH2B1 beta through its own combination of JAK2 and the insulin substrate protein (IRS) combined with the JAK2 complex, with the JAK2/STAT3 and IRS2/PI3K two paths to enhance leptin. Dependent signal transduction, SH2B1 beta is an endogenous leptin sensitifier, and the overexpression of SH2B1 beta can also counteract the role of the protein tyrosine phosphatase 1B (PTP1B) mediated leptin suppression signal.
The latest literature reports that the SH2B1 beta in adipose tissue and nerve tissue produces two opposite effects during the growth and differentiation of adipocytes. Although the negative regulatory effect of SH2B1 beta is dominant, the adipose tissue SH2B1 beta regulates the differentiation of adipocyte by a cellular autonomic formula and before 3T3-L1 In the process of adipocyte differentiation, the expression of the expression is increased, and the accumulation of lipid droplets in the cells and the expression of the fat differentiation transcription factor PPAR gamma, C/EBP a are also promoted.
The increase in the number of adipocytes and the increase in volume can lead to obesity, but the increase in the differentiation of pre adipocytes is the main cause of the increase in the number of mature adipocytes. In recent years, the regulation of adipocyte differentiation has become a hot spot in obesity and related diseases. The composition of human or animal fat tissue includes microvessels and nerve tissue. Fibroblasts, adipocytes, and preadipocytes, the development of the adipocyte system is pluripotent stem cells - mesenchymal stem cells - preadipocytes - mature adipocytes. Therefore, effective regulation of pre adipocyte differentiation can control adipose tissue hyperplasia and hypertrophy. Adipocyte differentiation is associated with insulin and insulin-like growth factors. Insulin and insulin-like growth factors (insulin-like growth factors) promote the differentiation of adipocyte differentiated.SH2B1 beta as a positive regulator of insulin receptor (IR) and insulin like growth factor receptor (IGF-IR) activity by combining its receptor to activate the two signal transduction pathways of PI3K and MAPK, and participate in the regulation of the signal transduction pathway of adipocyte differentiation.
At present, there are few studies on SH2B1 beta in adipose tissue, and the research is mainly aimed at the mouse source protein. According to the literature reports, the similarity of SH2B1 beta mouse source gene and human source gene has reached 85%-87%. This topic focuses on the preparation of monoclonal antibodies against SH2B1 beta by using human SH2B1 beta gene in the expression of recombinant protein of SH2B1 beta in vitro. Quantitative method is a preliminary study of the regulation of the human source protein in the differentiation of prehuman adipocytes, which provides new drug targets for the treatment of obesity and its complications which are closely related to the differentiation of preadipocyte.
Since adipose stem cells are critical to a cell state between mesenchymal stem cells and preadipocytes, this study was used to extract and differentiate mature adipocytes by primary culture of fat stem cells (ADSC) in vitro and to induce adipocyte differentiation. The TRIZOL one step method for extracting total RNA. was successfully amplified by RT-PCR method. The pET28a (+) expression vector was cloned from the SH2B1 beta gene fragment, and a large amount of soluble expression was carried out in Escherichia coli. The recombinant protein was purified by saturated ammonium sulfate salting out and DEAE anion exchange method. The recombinant protein was immunized by anti His-tag monoclonal antibody to prepare monoclonal antibodies, and ELISA and Wes were used respectively. Tern-Blot method was used to detect and identify the specificity of monoclonal antibodies.
The SH2B1 beta gene was cloned successfully by RT-PCR technology. The size of 2000bp. was sequenced and compared with the sequence in GenBank. The results showed that the inserted fragment was matched with the reported SH2B1 beta gene sequence, without the code mutation, the size was 2016bp., and the encoding gene was cloned into the pET-28a (+) prokaryotic expression plasmid, and the restriction enzyme was cut through PCR. It was confirmed that Recombinant Prokaryotic Expression Plasmid of SH2B1 beta -pET28a (+) was successfully constructed.
After IPTG induction, the recombinant plasmid SH2B1 beta -pET28a (+) expressed a His-tag fusion recombinant protein in Escherichia coli E.coli DE3. The molecular weight of the recombinant protein was approximately 80kDa, which was basically in accordance with the theoretical value. The expression product was mainly in the form of soluble form. After purification, the purity of the recombinant protein could reach more than 80%. The expression product was expressed as Western-blo by anti His-tag monoclonal antibody. T analysis showed that the specific zone appeared, indicating that the protein was indeed a His-tag fusion protein to be expressed. Further, the purified expression product was used to immunize mice to prepare the immune sera. ELISA analysis showed that the recombinant protein could react with the immune sera and did not cross reaction with the normal mice serum, indicating that the recombinant protein had antigen activity. Sex.
2 mAb secretory cells were screened by fusion of immunized mouse splenocytes and murine myeloma cell NS-1. The recombinant protein was purified as antigen and ELISA method was used to detect the titer of McAb culture supernatant. The OD value was 1.553 to 2.210.Western-blot, respectively: 2 monoclonal antibodies against SH2B1 beta and natural protein extracted from human adipose tissue Specific reaction was observed, while no obvious bands appeared in the negative control group, indicating that the mAbs prepared were specific mAbs.
The above results show that we have successfully constructed the recombinant expression plasmid of SH2B1 beta -pET28a (+). There is no report on the recombinant and expression of SH2B1 beta, and a large number of soluble recombinant protein SH2B1 beta with immune activity is expressed in Escherichia coli, and 2 monoclonal antibodies secreting anti SH2B1 beta monoclonal antibodies have been screened and established. In order to further study the biological function of the protein, the hybridoma cell line plays a role in the development and differentiation of adipocyte, and lays the foundation for the verification of the potential therapeutic value of obesity.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R589.2;R346

【參考文獻】

相關期刊論文 前10條

1 亓玉民,尹鴻軫,陳遠耀;處理雜交瘤細胞污染的有效方法[J];白求恩醫(yī)科大學學報;1989年01期

2 羅盛康,席菁樂;成人前脂肪細胞的原代培養(yǎng)[J];第一軍醫(yī)大學學報;2005年03期

3 劉江,姜述德;細胞污染支原體的檢測及清除[J];國外醫(yī)學.預防.診斷.治療用生物制品分冊;1995年02期

4 ;槲皮素和甘黃芩甙元對氧化修飾低密度脂蛋白的抑制作用[J];國外醫(yī)學(中醫(yī)中藥分冊);1999年03期

5 鮑榮琦;調脂湯治療高脂血癥32例[J];南京中醫(yī)藥大學學報(自然科學版);2002年06期

6 李斌,趙紅藝,馬曉冰,王曉軍,錢文江;新法吸脂整形術──高頻電場的應用,附百例臨床報告[J];實用美容整形外科雜志;1999年03期

7 解乃昌;肥胖模型及病態(tài)肥胖外科減肥治療機理[J];陜西醫(yī)學雜志;1996年03期

8 梁柳娟,黃朝，

本文編號：1968213