| Obesity has reached epidemic proportions globally and becomes more and more serious.Obesity and overweight pose a major risk for serious diet-related chronic diseases,including type 2 diabete,cardiovascular disease,hypertension and stroke, and certain forms of cancer.The health consequences range from increased risk of premature death,to serious chronic conditions that reduce the overall quality of life. According to the epidemiological statistics,the increased risks of obesity were highly correlated with the increased adult group mortality.In recent years,studies have shown that obesity is the disease of biochemical regulation and energy metabolism in eating disorders,which is caused by a series of specific factors such as environmental, genetic,diet,and genes which may be the major determinant factor.From the macro level,the increasing in food intake,the decreasing in physical activity and the changing in the heat lead to the excessive accumulation of fat.However,from the micro-level,the number of mature fat cells is the key factor of the occurrence of lipid accumulation.Obesity has not only affected the figure of the body,but also been a threat to human health.In 1950,The gene mutation which led to obesity was found by Ingalls.It was called obesity gene(obese gene,ob gene).Coleman took cross-perfusion experiments to show that a certain blood-borne factors regulated the animal feeding,energy metabolism and was involved in the formation of fat,this emergence was related to the ob gene.1991,Friedman described five gene of mutation that caused mice obese, which respectively were obese(ob),diabetes(db),fat(fa),tubby(tub) and obese yellow(Ay),it was the foundation for studying the molecular mechanism of obesity. In 1994,Zhang used the positional cloning of mutant gene to clone ob gene of mice and human,and also found that the mutation of ob gene could lead to obesity.This meaned the obesity research went into the molecular time.Leptin protein(Leptin) is the expression product of ob gene,which is a protein of 167 amino acid residues and is secreted by white fat cells.In addition,by linking specific receptor in the hypothalamus,it can lead to suppress the appetite,reduce energy intake,lose weight and increase the consumption of energy.It also forms a complex network systems to affect many other body physiological systems and metabolic pathways by the receptor in other tissues and organs(islet,liver,gonad, adrenal gland,thyroid etc.).The peripheral roles of leptin protein also include regulation of balance in glucose metabolism,and promotion of lipolysis,inhibition of fat synthesis,involvement in immunomodulatory functions,promotion of the growth and development and so on.After a long-term researching,the biological role of leptin protein has been further understood:Leptin protein is not an isolated hormone in the regulation of energy metabolism,but the one which plays a role with a large number of neuropeptides,neurotransmitters,other hormones and factors.There is a phenomenon of "leptin resistance" in the population which can not be ignored.It is an issue that researchers who are committed to leptin protein need to address in the future.With the further studying,the researchers found that SH2B1βprotein could interact with cell signaling proteins JAK2(non-receptor tyrosine kinase).It was activated by leptin to regulate the energy metabolism and body weight.SH2B1βis a member of SH2B signal joint protein family,and its structure contains a SH2 domain which can be combined with the tyrosine phosphorylated residues to connect different signaling protein and exert its role of signal convergence and enhancement of signaling pathway.SH2B1βis widely distributed in the hypothalamus,liver,pancreas, adipose tissue and other tissues,which is involved in insulin sensitivity,the regulation of glucose balance and enhancement of nerve neural differentiation induced by growth factor.Because SH2B1βis necessary for the activation of Leptin/JAK2 signaling pathway,the knockout of SH2B1βgene will cause that the weight of these mice is twice more than that of the normal mice in the same nest.The deletion of SH2B1βgene can lead to severe leptin resistance,bulimia and obesity. SH2B1βcan combine with JAK2 and make insulin protein substrate(IRS) combine with JAK2 complex to enhance leptin-dependent signal transduction by the way of JAK2/STAT3 and IRS2/PI3K.Therefore,SH2B1βis supposed to be an endogenous enhancer of leptin sensitivity.Meanwhile,the over-expression of SH2B1βcan also offset inhibition of leptin-mediated signal caused by protein tyrosine phosphatase 1B (PTP1B).At present,It is reported that the SH2B1βin nerve tissue and adipose tissue brings two opposite effects in the growth and differentiation of fat cells.Although negative effect of regulation of SH2B1βin nerve tissue is dominant,SH2B1βin adipose tissue regulates differentiation of fat cells in a cell autonomous manner,and has a progressive increase in expression in differentiation process of 3T3-L1 preadipocyte,a promotion of lipid droplets accumulation in ceils and fat differentiation transcription factor PPARγ,C/EBPαexpression.The increase in number and size of fat ceils can lead to obesity,and the differentiation of pre-adipocyte is the main cause of increase in the number of mature fat cells.In recent years,the regulation of fat cell differentiation has become the research topic of obesity and its related diseases..The differentiation process of fat cell lines is multipotent stem cells-mesenchymal stem cells-pre-adipocyte-mature fat cells,the effective regulation in the differentiation of preadipocyte can control the hyperplasia and hypertrophy of adipose tissue.The adipocyte differentiation is related with insulin and insulin-like growth factor,which combine with their receptor to activate the two signal transduction pathway of PI3K and MAPK and promote differentiation of fat ceils.As an activity regulatory protein of insulin receptor(IR) and insulin-like growth factor receptor(IGF-IR),SH2B1βis involved in signaling pathway of regulation of adipocyte differentiation.There is little study about SH2B1βin adipose tissue and the research is mainly focus on the rat source protein.According to reports in the literature,human gene and murine gene of SH2B1βshare the similarity of 85%-87%.In this research we use human SH2B1βgene to have the prokaryotic expression of recombinant protein in vitro,and prepare anti-monoclonal antibody of SH2B1β,then explore effect of this protein in regulation of human pre-adipocyte differentiation through qualitative and quantitative methods,to provide some new drug targets for the treatment of obesity and its complications that are closely related to pre-adipocyte differentiation.Because adipose stem Cells(ADSC) are a form of cells which is between the periods of mesenchymal stem cells and pre-adipocyte cells,so we firstly cultured adipose stem cells and induced adipogenic differentiation in vitro by Dexameth and Insuline,collected matured fat cells,TRIZOL one-step was used to method to extract total RNA from cells.Using RT-PCR method,we successfully amplified and cloned gene fragments of SH2B1β,and combined gene fragments with expression vector of pET28a(+),then had a great number of soluble expression in E.coli.We used saturated ammonium sulfate method and DEAE anion exchange to purify recombinant protein which is identified by the His-tag monoclonal antibody,and then had recombinant protein immune mice to prepare monoclonal antibodies,the specificity of which was finally detected with ELISA and Western-Blot.The research results showed that we had successfully cloned SH2B1βgene by using RT-PCR.The result of sequencing and GenBank comparison of sequences showed that the sequence of inserted fragments accorded with the reported gene, without any frameshift mutation,the size of it is 2016bp.Encoding gene was cloned into prokaryotic expression plasmid of pET-28a(+).The result of identification by PCR,restriction enzyme analysis and sequencing result showed that we had successfully constructed recombinant prokaryotic expression plasmid of SH2B1β-pET28a(+).Recombinant plasmid of SH2B1β-pET28a(+) was expressed in E.coli as His-tag fusion protein,and expression products existed mainly in soluble form,the molecular weight of which was about 80kDa that in accordance with the theoretical value.The purity of recombinant protein was up to 80%after purification.The result that specific zone could be seen when we used anti-His-tag monoclonal antibody to carry out Western-blot analysis of expression products indicated that the protein is indeed to be the objective protein.ELISA analysis showed that the recombinant protein can react with the immune serum of immunized mice which was prepared by taking the purified expression products to immunize mice,but not with the normal mouse serum.All of these suggested that there was antigen in recombinant protein.We had prepared two monoclonal antibody-secreting cell lines with mice immunized with purified recombinant proteins.When we used the ELISA assay to detect the titer of supernatant secreted by monoclonal antibody-secreting cells,we found that OD values were 1.553～2.210.Results of western-blot showed that:Two anti-SH2B1βmonoclonal antibody and natural protein extracted from the human adipose tissue could react;while the negative control group had no obvious bands appeared.All of these suggested both of the monoclonal antibody were specific.These results suggested that we had successfully constructed the recombinant prokaryotic expression plasmid of SH2B1β-pET28a(+).Meanwhile,we have obtained substantial expression of a soluble recombinant protein SH2B1βwith immunological activity in E.coli,and then screened and established two hybridoma cell lines that can secrete anti-SH2B1βmonoclonal antibody.All of these can help the further research on biological function of SH2B1β,its roles in differentiation of fat cells,and its potential therapeutic value for obesity.
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