| Diabetes is a life time metabolic disorder caused by multiple incentives and often results in chronic hyperglycemia.International Diabetes Federation(IDF)published in 2013 that the number of diabetic patients had reached 383 million globally,which made it become a severe problem of public health.Diabetes doesn't cause direct damage to human body,however sustained abnormally high glucose level may lead to gradual degeneration in large and micro blood vessel and in turn cardio,cranial,renal,neuron,optical and pedal diseases.Thus numerous scientists participate in the research about the pathogenesis and treatment of diabetes.Glucagon-like peptide-1(GLP-1)is a 31-AA peptide insulin stimulation hormone secreted by intestine L-cell of mammalian.Because GLP-1 can enhance the secretion of insulin under hyperglycemia,it makes GLP-1 become a prospective medicine in clinic treatment against diabetes type II.But GLP-1 is rapidly degraded in presence of DPP? which limited the T1/2 of GLP-1 to 2 minutes in longest and in turn the performance of its biological function.Resistance of GLP-1 against DPP? acquired via recombination and modification of AA residues or overexpression endogenous GLP-1 are expected to make GLP-1 operational in anti-diabetic clinical use.In this study,the expression of GLP-1 in vivo was enhanced by the construction of GLP-1 tandem repeats,so as to make it more resistant to the degradation of DPP?,and finally improve the physiological function of GLP-1 in vivo.Thus,we designed a series of vectors which contained a gradient copy number of 4-copy-multimer GLP-1 sequence,named pET-22b-GLP4,GLP8,GLP12 and GLP16.After that,we transformed them into Escherichia coli BL21(D3),and then induced the expression by IPTG.The results showed that the expression of GLP4 was the highest,followed by GLP8,while GLP12 and GLP16 were not expressed.In addition to the structure of the gene itself and the efficiency of the expression system,the induction temperature,induction time and the concentration of the inducer have influence on the expression of GLP-1.Through the experiment,we ascertained 26? as the suitable temperature and 8 hours as the optimal inducing time and 0.6mM IPTG as the best induced concentration for GLP-1 expression.Under the above optimized conditions,we finally determined that the most suitable copy number for the in vitro prokaryotic expression system was 4.At present,plenty of researches show that the small intestine also has the function of secreting insulin,and the small intestinal epithelium cells(IEC)and pancreatic islet endocrine cells have a common origin in development.These findings indicated that embryonic IEC may have the potential to differentiate into insulin secreting cells.To further investigate the relationship between islet and IEC,IEC obtained from rat embryonic were cultured.Morphological observation revealed that the cultured cells possessed the typical character of epithelial cells.Immunofluorescence staining showed that cultured cells expressed specific markers of small intestinal epithelial cells Keratin-8.Taken together,we confirmed the cultured cells were IEC.In order to facilitate the reform of small intestinal epithelial cells in the futrue,we further constructed the lentiviral vector of hrGFP by gateway technology and successfully infected rat intestinal epithelial cells.Conclusion: In this study,we successfully induced the expression of GLP-1 multimer in vitro,determined the optimal expression conditions of GLP-1 multimer and the optimal copy numbers,which laid a solid foundation for the study of GLP-1 in vivo.At the same time,the establishment of the culture system of rat intestinal epithelial cells in the embryonic stage provides the possibility for the study of the function and mechanism of the next stage. |
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