Differentiation Of Bone Marrow Mesenchymal Stem Cells Towards Insulin-producing Cells Using Mafa In Combination With GLP-1

Background and ObjectiveIn2013, Internatinal Diabetes Federation (IDF) announced that there were more than383milliondiabetic patients worldwide, diabetes mellitus has been recognized as the most serious public healthproblems.The current conventional treatment of diabetes can not control all the patients’ blood glucoseideally. Due to the lacking of donor sources and requiring of life-long immunosuppressive therapy, clinicalapplication of pancreas and islet transplantation is limited. In recent years, experts and scholars have paidmuch attention to the regeneration of insulin-producing cells at home and abroad.Mus musculus v-maf musculoaponeurotic fibrosarcoma oncogene family protein A (MafA) isthe only specific transcription factors present in the islet β cells, which not only regulates the expression ofinsulin relevant genes, but also regulates the proliferation and apoptosis of pancreatic β cells. Due toparticipating in the amplification and survival of islet β cells, MafA plays a key role in this vital function ofinsulin and growth of pancreatic β cells. Bone marrow mesenchymal stem cells (BMSCs) are present inadult marrow of high self-renewal and differentiation potential, which are also easy to transfect and havethe advantages of stable expression of foreign genes and avoiding immune rejection. So BMSCs are safegene therapy cellular carrier. Glucagon-like peptide-1(GLP-1) is a kind of peptide hormone, which issecreted by intestinal epithelium L endocrine cells, which has certain influence on protection of pancreaticcell, cell proliferation, and inhibition the apoptosis of islet β cells.We planed to construct eukaryotic expression vectors pcDNA3.1/Hygro(+)-MafA andpEGFP–MafA. By lipofection transfecting, we have transfected eukaryotic expression vector into BMSCs.Screening of the stably transfected cells line, detecting the expression of insulin relevent gene mRNA by using quantitative PCR and detecting the expression of insulin by using ELISA. In order to exploredifferentiation of bone marrow mesenchymal stem cells towards insulin-producing cells using MafA incombination with GLP-1.Methods1. Total cellular RNA was isolated with Trizol reagent, the first strand cDNA was synthesizedusing First-Strand Synthesis System. Target fragment was amplified by PCR. After the fragment of MafAwas inserted into the Hindlll-BamHI restriction sites of the eukaryotic expression vectors pEGFP-C1andpcDNA3.1/Hygro(+) by molecular cloning techniques respectively, The connection direction of the genesequences was confirmed with colony PCR identification. The recombinant eukaryotic vectors wereidentified by digestion. After DNA sequence analysis, The new plasmid was named aspcDNA3.1/Hygro(+)-MafA and pEGFP-MafA respectively.2. Based on whole bone marrow cell culture method, BMSCs were cultured. The pure BMSCswere used for flow cytometry analysis to detect the expression of CD34, CD44, CD105in BMSCs.3. Using lipofection transfecting, we transfected eukaryotic expression vector into BMSCs.Screening of the stably transfected cells line by hygromycin, we verified the stably transfected cells line byWestern blot. Grouping A: BMSCs group, B: MafA+BMSCs group, C: GLP-1+BMSCs group, D:MafA+GLP-1+BMSCs group. Quantitative PCR was performed to detect the expression of Nestin, Pdx-1,NKx6.1, Ngn3and Tle.Results1. Total RNA extracted from mouse pancreatic tumor cell line integrityand no degradation,amplify specific DNA bands, approximately1080bp, successfully constructed the eukaryotic expressionvector pcDNA3.1/Hygro (+)-MafA and pEGFP-MafA. 2. Isolation and primary culture rat BMSCs. Flow cytometry showing CD44, CD105positive,CD34negative expression, consistent with characteristics of BMSCs.3. The results of Western blot showed that the expression of the MafA gene was only detected inthe BMSCs transfected with pcDNA3.1/Hygro(+)-MafA, suggesting that MafA gene was successfullytransfected into BMSCs.4. The results of quantitative PCR: compared with BMSCs group，the mRNA levels of Pdx1,Ngn3, Nestin and Tle genes increased in MafA+BMSCs group, GLP-1+BMSCs group andMafA+GLP-1+BMSCs group. Compared with BMSCs group, the mRNA levels of Nkx6.1increased inGLP-1+BMSCs group and MafA+GLP-1+BMSCs group, while the expression of the mRNA levels ofNkx6.1in MafA+BMSCs group has no significant change. MafA+GLP-1+BMSCs group is furtherincreased the mRNA levels of Pdx1, Ngn3, Nestin, Nkx6.1and Tle genes. In contrast, no significantchanges were observed on the expression of these genes in MafA+BMSCs group and GLP-1+BMSCsgroup.5. The results of ELISA: there was almost no insulin release in BMSCs group, Compared withMafA+BMSCs group, the insulin secretion in GLP-1+BMSCs group and MafA+GLP-1+BMSCs groupboth has been increased(P<0.05), The increase of insulin secretion in MafA+GLP-1+BMSCs group wasmore than in GLP-1+BMSCs group(P<0.05).ConclusionThe introduced MafA gene played important roles in the differentiation process from BMSCsinto insulin-producing cells, which may shuttle to the nucleoplasm of BMSCs under high glucose, theninitiate the expression of native transcription factors, resulting in transactivation of the relevant genesincluding insulin and generation of β cell phenotype. GLP-1analogue can further enhance the differentiation process from BMSCs into insulin-producing cells.