| Diabetes is one of serious diseases impact on human health. Type2diabetes is the most important type of diabetes, accounting for more than90%of the total number of diseases. The incidence of type2diabetes is closely related to the standard of living and diet. High-fat diet and obesity are the main factors to induce type2diabetes. In the past30years, with the economic development and the improvement of people’s living standard, the prevalence of type2diabetes has rapidly risen from less than1%to more than10%. The total number of patients has up to more than100million. Diabetes can cause cardiovascular disease, renal failure, peripheral neuropathy and retinopathy and other complications. Prevention and treatment of diabetes is most concerned about one of the hot spots in the field of medicine today.The outstanding performance of type2diabetes is insulin resistance, elevated blood sugar and decreased glucose tolerance. Existing antidiabetic drugs, although there are some hypoglycemic or improve the efficacy of insulin resistance, but are unable to restore islet beta cell function. No matter how medication, islet function is in constant decline. Therefore, it only can delay the progress of the disease, can not be completely repaired islet function, so that the disease has been effectively controlled.Glucagon-like peptide the-1(glucagon-like peptide1, GLP-1) is a peptide hormone secreted by the L cells of the intestinal epithelial belongs to Incretin. As some report has been shown that GLP-1stimulate pancreatic beta cell proliferation and insulin secretion, inhibition of beta cell apoptosis, increase muscle, liver cell sensitivity to insulin, also through the regulation of appetite, inhibit the energy absorption and fat mobilization role in lowering blood glucose and improve insulin resistance. GLP-1peptide-Liraglutide and Exenatide (Exenatide or Extendin-4, EX4) has become the new drugs for obesity and diabetes prevention. However, similar to GLP-1peptide by chemical synthesis methods of production, can not be separated from the separation and purification of the peptides, only injection route of administration but not oral, there is a complex production process, high cost and inconvenience of shortcomings. The need to improve the production and the mode of administration of the GLP-1analog peptide forced to reduce drug side effects.Bifidobacterium mammals the safest, most beneficial health probiotic group, is one of the first inhabitants of the human gut, infant gut flora. Bifidobacteria not only improve the intestinal microbial environment, inhibit the growth of pathogenic bacteria, promote nutrient absorption, but also has immune conditioning, decomposition of toxic metabolic intermediates and prevention of tumor. Bifidobacterium as food, medicines have been a few hundred years of history, its security has been fully validated. Bifidobacterium as the host strain to express functional polypeptide is the safest, easiest way, is conducive to long-term medication. Therefore, we take advantage of bifidobacteria to express similar peptide, and to explore its hypoglycemic and improve the efficacy of insulin resistance.To this end, while, its short half-life and the presence of renal toxicity side effects limit its application in real life. We constructed reconstructed GLP-1peptide GLP-1(8G) and similar peptide EX4Bifidobacterium expression vector on the basis of the Bifidobacterium expression system, and transformed and carry GLP-1(8G) and the EX4gene conversion Bifidobacterium. Analysis of the expression of bifidobacteria in vitro GLP-1(8G) and EX4expression, Expression and biological activity; vivo high-fat and high-sugar diet and streptozotocin (STZ)-induced mouse model of type2diabetes treatment experimental observation of its hypoglycemic effect, and to explore the molecular mechanism of action. The specific experimental design scheme is as follows:(1) Select the previous experiments have constructed E. coli-Bifidobacterium shuttle the plasmid vector pBBADs-EV and biosynthetic GLP-1altered peptide GLP-1(8G) and similar peptide EX4gene of fragment constructed and sequenced the identification shuttle plasmid pBBADs-GLP-1(8G) and pBBADs-EX4, then plasmid power transformed into Bifidobacterium longum (NCC2705), anaerobic culture48h, the positive clone colonies screened, which was inoculated into MRS medium containing ampicillin and0.2%L-arabinose (L-arabinose, L-Arb) inducing expression12h,24h,36h, respectively, with the ELISA and Western blot was used to verify the transgenic Bifidobacterium GLP-1(8G), EX4protein expression levels, and choose the best time of induction of expression.(2) Great growth status of INS-1cells (rat insulinoma cell) was added containing BL-GLP-1(8G) and BL-EX4induction of expression of the culture supernatant, cell cytotoxicity with CCK-8reagent cartridge toxicity testing.(3) BL-GLP-1(8G) and BL-EX4secreted proteins in vitro biological activity test: cultured on the transformed Bifidobacterium induced with0.2%L-arabinose (L-arabinose, L-Arb)Qing culture of INS-1cells for24hours, after48hours, the use of ELISA assay insulin levels in the culture fluid changes.(4) BL-GLP-1(8G) and BL-EX4secreted protein animal model of treatment of functional test:the use of high-fat and high-sugar fed C57BL/6J mice after intraperitoneal injection of streptozotocin (STZ) cause of type2diabetes model, the packet given to mice fed0.2%L-arabinose (L-arabinose, L-Arb)-induced expression of the BL-GLP-1(8G) for28consecutive days with the BL-EX4, BL-EV group as a positive control.①diabetic mice modeling and judgment:high sugar high fat diet feeding mice48days after intraperitoneal injection of the dose of10mg/kg streptozotocin (STZ)12h fasting, cut rat tail blood, blood glucose meter blood glucose, and reached diabetic mice forming standard non-fasting blood glucose (11.1mmol/L) was initially determined for type2diabetic mice, and continue feeding high fat and sugar;②Gavage BL-GLP-1(8G) and BL-EX4continuous treatment for28days after0.2%L-arabinose (L-arabinose, L-Arb) induced, eyeball blood,2000rpm low temperature and centrifuged for10min and the plasma stored at4℃refrigerator, alanine aminotransferase (ALT), blood urea nitrogen (BUN), creatinine (CR), triglycerides (TG), low density lipoprotein (LDL), very low density lipoprotein (VLDL) and other biochemical markers using automatic biochemical detector to detect;③Eyeball blood,3000rpm low temperature and centrifuged for10min to separate the plasma, the use of adiponectin enzyme reaction (ELISA) kit in accordance with its operation to detect the Adiponectin;④Deal with mice, each group fresh liver tissue quickly in liquid nitrogen. Liver tissue is removed from the liquid nitrogen, clipping about100mg of each group of organizations, grinding into the grinding dish, add Triol the RNA was extracted, reverse transcribed into DNA immediately, the use of real-time PCR was used to detect Adiponectin, adiponectin receptor and resistin in liver tissue expression levels of oil red O staining analysis of the degree of fatty liver disease;⑤From each part of the liver tissue,4%paraformaldehyde fixed, paraffin-embedded, HE staining their form of liver tissue for analysis;⑥Islet part of each group,4%paraformaldehyde, embedded in paraffin, cut into10μm, immunohistochemical analysis after treatment after the beta islet morphology changes and secretion.(5) Verify that the GLP-1receptor in mouse colon and INS-1cells in the presence of:①Clipping part of the colon of normal mice, sonication, centrifuged, the supernatant stored at-20℃spare, using Western blot identification;②Normal medium INS-1cells after48hours, sonication, centrifuged, the use of the Western blot method identified;③Clipping part of the colon of normal mice, fixed in4%paraformaldehyde, embedded in paraffin, immunohistochemistry processing identified;④Taken part of the colon of normal mice, embedded in paraffin, cut into10μm handled by organizational immunofluorescence method, after finalizing the use of the copolymer according to the microscope (confocal image)405/488nm wavelength camera identification;⑤In6-well plates, conventional medium to train in good condition, dealt with in accordance with the immunofluorescence method, the use of the the copolymer according microscope (confocal image) photo identification405/488nm wavelength;The results showed that:(1) In the Bifidobacterium longum (NCC2705) was successfully constructed, capable of secreting GLP-1altered peptide GLP-1(8G) and similar peptide EX4, BL-GLP-1(8G)and the BL-EX4transfer body; identification of selected BL-GLP-1(8G) and BL-EX4turn the gene bifidobacteria and no significant change in the biological and morphological. L-arabinose induction of in vitro, the secretion of the protein in transgenic Bifidobacteria supernatant and cell detected, the determination of the optimal induction of expression is24h;(2) BL-GLP-1(8G) of the culture supernatant after the induction of expression of the0.2%L-Ara with BL-EX4through the INS-1cell proliferation, and ELISA to detect the cell culture supernatant, prove its induction the culture supernatant of insulin levels were significantly increased;(3) Fasting blood glucose, oral glucose tolerance test, after gavage BL-GLP-1(8G) and BL-EX4, relative to BL-EV and type2diabetic mice had a significant effect (P<0.05);(4) For plasma biochemical detected, compared with the BL-EV Group, gavage BL-GLP-1(8G) of the mice treated VLDL, TG, LDL has a significant change;(5)Compared with the BL-EV Group, after gavage BL-GLP-1(8G) and BL-EX4treatment28days after the ELISA method to prove that the Adiponectin serum levels were significantly elevated;(6) The use of Western blot the copolymer according microscope (confocal) and immunohistochemical methods to prove the existence of INS-1cells and C57BL/6J mice colon the same GLP-1R receptor.In conclusion, we have successfully constructed carrying the GLP-1(8G) and EX4the Bifidobacterium expression vector, and screened to obtain the desired gene conversion Bifidobacterium; Gene into bacterial cells of Bifidobacterium and the culture supernatant can be detected to GLP-1(8G) and EX4expression; vitro experiments show that transformation of bifidobacteria GLP-1(8G) and EX4expression product can promote the proliferation of INS-1cells and stimulate insulin secretion; vivo experiments show that the treatment of type2diabetes model mice induced high-fat and high-sugar diet and STZ, confirmed that oral administration of GLP-1(8G) and EX4transformed Bifidobacterium and its hypoglycemic effect can be observed, and to explore the molecular mechanism.Production is simple with respect to the GLP-1analogs injection in the market of transgenic BL-GLP-1(8G) and BL-EX4possess the in vivo induction of expression is directly absorbed by the intestinal receptor, to reduce the incidence of renal toxicity; without any extraction and purification can be taken to reduce the production and purification costs, improve social benefits; simply by oral way, simple, non-toxic, painless, can significantly reduce pain and enhance their adaptability; induced in vivo expression, reducing its short half-life and play a role of uninterrupted, have a significant treatment effect. Efficacy results combined with animal experiments, expected future treatment of human type2diabetes, has a very large potential for development. Thus, BL-GLP-1(8G) and BL-EX4is expected to become a new dosage form of GLP-1analogs for the treatment of diabetes, and can be adjusted to the new generation of patients with intestinal microbial probiotics. |
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