| As a life-threatening metabolic disease, diabetes mellitus seriously influences people’s life and health. Diabetes mellitus is characterized with absolute and/or relative islet β-cell deficiency and insulin resistance. Islet transplantation is a promising therapy for diabetes. However, the lack of islet donors and the occurrence of immune rejection limit its application in clinical work. The transplantation of insulin-producing cells(IPCs) differentiated from non-pancreatic cells may solve this problem and become a new therapy for diabetic patients.Among multiple types of stem cells, BMSCs has been shown to be a safe and abundant stem cells. It has the ability to differentiate into multiple cells and elude detection by the host’s immune system. So, it has been selected as seed cells.The biological processes of pancreatic β-cells, such as differentiation,proliferation and apoptosis, are controlled by transcription factors. Among these transcription factors, Pdx-1 is a master regulator of pancreatic development and the maintenance of pancreatic β-cell function. Pdx-1 expressed in the embryonic gut epithelium and maintained in pancreatic precursor cells co-expressing several hormones. As shown in many studies, exogenous Pdx-1 expression induces the expression of various β-cell-specific genes, especially the insulin gene, innon-pancreatic cells. Pdx-1 expression initiates the expression of downstream transcription factors, especially neurogenin 3(Ngn3). During the development of budding ducts of the embryonic pancreas, Ngn3 activation initiates the differentiation of epithelial progenitors to pancreatic endocrine cells and plays a key role in pancreatic β-cell differentiation. V-maf muscu-loaponeurotic fibrosarcoma oncogene homolog A(Maf A) is another transcription factor that functions as an insulin gene trans-activator in the presence of Pdx-1 and maintains β-cell formation and function.During pancreatic β-cell development, Maf A expression is first detected at the beginning of the principal phase of insulin-producing cell production, while other important transcription factors, such as Pdx-1 and Ngn3, are expressed from the early stages of β-cell development. Recent studies discovered that together, Pdx-1, Ngn3 and Maf A could successfully reprogram BMSCs into IPCs in vitro and that transcription factors expression in IPCs plays an important role in maintaining the secretory function of the cells.The regeneration of pancreatic β-cells and the maintenance of insulin biosynthesis also require some gastrointestinal hormones such as gastrin and glucagon-like peptide-1(GLP-1). GLP-1 is an incretin hormone that induces pancreatic β-cell regeneration, protects against β-cell damage and stimulates insulin biosynthesis and glucose-dependent insulin secretion to regulate blood glucose levels.In addition, GLP-1 can differentiate non-pancreatic cells into IPCs in the presence of Pdx-1. Exenatide is responsible for pancreatic β-cell regeneration and proliferation by elevating endogenous antioxidant and gene levels in vitro. The presence of GLP-1R in h MSCs increases cell proliferation, reduces trans-differentiation into adipocytes,and prevents apoptosis. Gastrin increases the pancreatic β-cell mass in the ligated part of the exocrine ducts. Furthermore, combined Pdx-1 and gastrin double the β-cell mass, reduce hyperglycemia and induce pancreatic regeneration, and combined exendin-4 and gastrin are beneficial for treating diabetes and preserving the β-cell mass in diabetic mice. However, little information is available regarding the mechanism of GLP-1 and/or gastrin in BMSCs. Therefore, we hypothesized that GLP-1 and Gastrin can further promote differentiation of the IPCs, and reduce the blood glucose of diabetic rats after transplantation. Therefore, this study carried outthe following research.PartⅠ The cultivation of rat BMSCs and the construction of adenovirus vectorObjective The isolation, cultivation and identification of rat BMSCs. The Construction of p Ad-Pdx-1-Ngn3-Cherry and p Ad-Maf A-EGFP.Methods1. The isolation and culture of rat BMSCs: To obtain the rat BMSCs by adopting the method of whole bone marrow adherent. The identification of surface markers of CD34, CD44 and CD105 on rat BMSCs by flow cytometry. The growth feature were detected by cell counting kit 8(CCK 8).2. The construction of adenovirus vectors: p Ad- Cherry, p Ad- EGFP, p Ad- Pdx- 1- Ngn3- Cherry, and p Ad- Maf A- EGFP were packed and amplification in 293 t cells, which were buy from Shanghai Ji Kai gene chemical co.LTD. The virus drops degrees were the 10-9-10-10/ml. The adenovirus vectors were stored in- 80 ℃refrigerator.Results1. In the process of cultivate primary rat BMSCs, the appearance of cells gradually change from brightly round to long spindle shape. which conform to the morphological characteristics of rat BMSCs. The positive rate of CD34 expression was less than 5%, while the positive rate of CD44 and CD105 more than 95%. It conform to the surface characteristics of rat BMSCs. The growth curve showed that cell proliferation ability was well.2. The adenovirus vector sequence conform to gene sequences.Conclusion1. The rat BMSCs was obtained successfully.2. The adenovirus vector p Ad-Pdx-1- Ngn3-Cherry and p Ad-Maf A-EGFP were constructed successfully.PartⅡ Differentiation of BMSCs into IPCs in vitroObjective Differentiation of BMSCs into IPCs by infecting with Pdx-1、Ngn3 and Maf A,and to investigate the mechanism of GLP-1 and Gastrin induce IPCs.Methods1. The rat BMSCs were infected by adenovirus vector: The optimal multiplicity of infection(MOI) that p Ad- Cherry and p Ad- EGFP infected rat BMSCs respectively alone or in combination, which were detected by preliminary experiment.p Ad-Pdx-1-Ngn3-Cherry and p Ad-Maf A-EGFP infected rat BMSCs respectively alone or in combination on optimal MOI, and then rat BMSCs were cultivated in high glucose medium. The uninfected cells as control group.2. The identification and function test of infected cells(first phase) :The infected BMSCs were divided into3 groups. Pdx-1-Ngn3 infection group: BMSCs were infected with p Ad-Pdx-1-Ngn3-Cherry; Maf A infection group: BMSCs were infected with p Ad-Maf A-EGFP; combination group: BMSCs were infected with p Ad-Pdx-1-Ngn3-Cherry and p Ad-Maf A-EGFP. The uninfected BMSCs as uninfected group. The protein expression of Pdx-1,Ngn3 and Maf A in each group were detected by protein imprinting(Western blot); The insulin secrete feature was detected by Dithizone(DTZ) staining; The insulin secretion ability of each group was detected by enzyme-linked immunosorbent(ELISA) in different induce periods; The Pdx-1,Nestin,GK,Glucagon,insulin2 m RNA expression of induced cells in each group were detected by Real-time fluorescent quantitative PCR(RT-PCR).3. The functional analysis of GLP-1 and Gastrin induced co-infected cells(the second stage): The co-infected cells were cultured in high glucose medium for 7 days.Then, they were divided into 4 groups, respectively cultured in high glucose medium(co-infection group), high glucose medium containing glucagon like peptide-1(GLP-1 induced group) and Gastrin(Gastrin induced group), GLP-1 and Gastrin(co-induction group), To detect the insulin secretion of each group cells by enzyme linked immunosorbent assay(ELISA) in different periods; The m RNA expression of Pdx-1, nestin, GK, glucagon and insulin2 in each group were detected by real time fluorescence quantitative PCR(real time PCR) after induce 7 days.Results1. The optimal MOI of p Ad-Pdx-1-Ngn3-Cherry and p Ad-Maf A-EGFP alone and co-infection were 50 and 30, respectively.2. The first stage: Western blots showed that the infection group had the corresponding protein expression, which confirmed that p Ad-Maf A-EGFP and p Ad-Pdx-1-Ngn3-Cherry were successfully infected with BMSCs. After infection 5days, the infected cells began to gather, On the 7 day, they significantly poly integrated together, and can be dyed scarlet by DTZ, which conform to cells have the characteristics of IPCs. ELISA results showed that the infected cells could secrete insulin, and the insulin secretion ability of co-infection group was significantly better than the other group, insulin secretion of each group gradually increased on infection0 th, 3, 5, 7, 9 day, and co-infection group increased most significantly(P < 0.05);RT-PCR results showed compared with uninfected, Pdx-1, Insulin2, Glucagon and GK gene expression in infection groups was significantly increased(P < 0.05);compared with Maf A infection group, Pdx-1-Ngn3 infection group, the expression of Pdx-1, Pdx-1 and Insulin2 in co-infection group was up-regulation, the expression of Glucagon was down-regulation(P < 0.05); compared with Pdx-1-Ngn3 infection group, co-infection group Glucagon expression up-regulation(P < 0.05), the GK,Insulin2 m RNA expression in co-infection group is significantly better than other induction group.3. The second stage: The results of ELISA showed that each group cell could secrete insulin, especially in co-induction group the insulin secretion levels were significantly better than other groups, each group on induced 0, 3, 5, 7 day insulinsecretion increased gradually and co-induction group increased obviously(P < 0.05).RT-PCR results showed that compared with the co-infected group, GLP-1 induced group GK and insulin2 m RNA expression level was significantly increased(P < 0.05),PDX-1, GK, Insulin2 m RNA expression in co-induction group was significantly increased(P < 0.05).Conclusion1. Pdx-1-Ngn3 combined with Maf A successfully induced BMSCs into IPCs.2. GLP-1 and Gastrin promote the differentiation and function of IPCs by promoting the expression of GK, Pdx-1 and insulin2 genes.PartⅢ IPCs transplantation into DM ratsObjective To investigate the effects of IPCs on the changes of body weight and blood glucose of T1 DM rats and its mechanism.Methods1. Establishment of DM rat model: 8-week-old SPF male Sprague-Dawley(SD)rats(220 ± 10g) were adaptive feeding for 1 weeks. After fasting 12 h, rats were intraperitoneal injected with a single dose of 60mg/kg streptozotocin(STZ) to induced diabetes(DM). Random blood glucose(BG) were measured for three times to confirm the induction of diabetes(≥16.7mmol/l) 72 h after STZ injection. Rats that received an injection of diluents buffer alone were served as normal control group.2. DM rats were injected with a dose of 0.3ml/100 g 10% chloral hydrate anesthesia by intraperitoneal injection, IPCs were transplanted into DM rats renal capsule. Fixed in the prone position of rat limbs, prepped left renal region governor about 2 cm longitudinal incision, layer by layer to open the abdominal cavity, kidney singled out the abdominal cavity, small tweezers kidney film, micro syringe of prepared cell suspension was injected into the lower renal pole. 30 DM rats were randomly divided into five groups,sham operation group(left kidney was membranetransplantation of 10 ul PBS),transplant group A(left kidney was membrane transplantation combined with infection cell group 2 x 106),transplantation group B(left kidney was membrane transplantation of gastrin cells in the induced group 2 x106),transplantation group C(left kidney was membrane transplantation GLP-1 cells in the induced group 2 x 106),transplantation group D(left kidney was membrane transplantation combined with cells in the induced group 2 x 106).The other six normal rats left renal capsule membrane transplantation 10 ul PBS as control group.3. The change of rat body weight and fasting blood glucose were monitored after transplantation 7,14,21,28 days, the weight of the rats were weighed and recorded, the fasting blood glucose of rats were monitored and record after fasting 12 h.4. The intraperitoneal glucose tolerance test(IPGTT) were performed after transplantation 28 day. After fasting 12 h, rats were intraperitoneal injected with a dose of 0.2g per kilogram of weight 50% glucose. The blood glucose of rats were detected before and after injection 30 min, 60 min, 120 min.5. Renal immunohistochemistry: After intraperitoneal glucose tolerance test, the kidney tissue of rats were resected and fixed in 4% paraformaldehyde for immunohistochemical detection.Results1. After transplantation, the normal group rats weight increased 6-8g weekly,sham operated rats weight loss 3-5g weekly, transplantation rats weight not only did not appear significantly decreased but also increased, and the most significant increases in body weight of rats is transplantation group D. The blood glucose of rats in the sham operation group were sustain at high blood glucose level. Along with time,the blood glucose of the transplantation group decreased, especially in the transplantation group D.2. Intraperitoneal glucose tolerance tests showed, normal rats blood glucose reached a peak at 30 min after injection of glucose, 120 min down to normal levels.Sham operation group rats blood glucose have continued to rise after glucose injection; transplantation rats blood glucose change showing increased first and then decreased trend which similar to normal rats, but after injiection 120 min the bloodglucose has not declined to the level of fasting.3. Kidney immunofluorescence in transplantation group showed that the transplanted cells can survive and secrete insulin, and the transplantation group D cell survival number and insulin secretion were the most significant.Conclusion BMSCs successful differentiation into IPCs in vitro, which not only can survive and can secrete insulin after transplantation in vivo. GLP-1and Gastrin not only induced IPCs more perfect in vitro, but also the number of survived in vivo is the best after transplantaion. |
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