The Study Of Rabbit Bone Marrow Mesenchymal Stem Cells Culture, Cryopreservation And Differentiation Into Pancreatic β Cells In Vitro

Diabetes is a serious metabolic disease for harming health of the growingpopulation. Islet transplantation can treat the diabetes, however was restricted with thedonors’ lack and immune injection. And the feasibility of cell replacement was significant.Many researches showed that stem cells,as embryonic stem cells(ESCs), inducedpluripotent stem cells (iPS)and adult stem cells(AS) differentiated into insulin-producingcells.The differentiated cells have been shown to partially/totally reverse the diabeticphenotypes and improve glucose control for the ability to cure the diabetes with celltransplantation. Bone Marrow Mesenchymal Stem Cells(BM-MSCs) is another type ofadult stem cells within intraosseous other than hematopoietic stem cell without the ethicalissues and technical problems that involved ESCs and iPS. However,bone marrowmesenchymal stem cells differentiate into pancreatic islet βcells in steady and efficientremains solved eagerly. On the basis of rabbit BM-MSCs culturation in vitro, we proposethis project to idtentify the proliferation and biological property after cryopreservation,constitute a method or technique system optimized in stable that directed differentiationinto pancreatic islet βcells in vitro.1. Rabbit bone marrow mesenchymal stem cells isolation with red blood cell lysis andamplification as its adherent capacity. To explore their growth properties bymorphological observation, cell surface marker identification and cell growth curve.Rabbit bone marrow mesenchymal stem cells separeated by red cell lysis are pure, alsowith stable properties and good proliferation activity, which above is simple and easy tocopy.2. The thawed cells after15d,30d, and90d frozen storage were detected with trypanblue to evaluate the cell recovery rate, and evaluated the influence to the recovery of eachgeneration cells (passage1,3and9).The cryopreserved cells proliferation ability and adipogenic differentiation of thawed BM-MSCs testified the characteristics ofdifferentiation.The result illustrated that cryopreserved rabbit BM-MSCs were culturedand passaged with limited numbers,and still have the pluripotency to differentiate intoadipocyte.So based on the above research, the cryopreserved rabbit BM-MSCs can alsobe used in tissue engineering,cell transplantation,and gene therapy. With rabbitBM-MSCs cryopreservation can solve the problems for reserves, materials or timesaving.3. We attempt to treat with DNA methyltransferase inhibitor—5-Aza-dC to explore thedecreased DNA methylation could or not strengthen expression of islet specific genePDX-1, then promote islet cells differentiation and β cells mature. The amplification ofdimethylsulfoxide (DMSO) on rabbit BM-MSCs was study simply, determined by MTTto select suitable concentration for differentiation and growth with smaller damage. Basedon previous foundation of our laboratory that5-Aza-dc appropriate concentration to effectrabbit BM-MSCs was1μmol/L，and DMSO along with high glucose can induce rabbitBM-MSCs into insulin producing cells. DMSO joint with nicotinamide, compared withDNA methylation inhibitors5-Aza-dc separately in different stages are similarmorphology. Dithizone staining showed that differentiated cells can produce insulin.However, the loss of islet specific gene PDX-1fails to to reach the experimental goal.The differentiation proposal still remains to further optimization.4. The experiment aimed at optimization of methods to induce rabbit BM-MSCs intopancreatic islet β cells for diabetes with cell replacement therapy. BM-MSCs wereinduced by growth factors, as bFGF, EGF, B27, HGF, β-cellulin and Activin A with highglucose in vitro. Islet clusters in terminal were proved by morphological changes andDTZ staining with inverted microscope. The protein like Insulin and PDX-1in derivedcells was investigated by immunofluorescence and the expression of genes(PDX-1，Insulin) related to islets in derived islet clusters was assessed by RT-PCR. Terminaldifferentiated cell clusters identified by glucose with different concentrations, whichrelease insulin at difference. The result showed that growth factors combined with highsugar could induce rabbit bone marrow mesenchymal stem cells into insulin-producingcells. More than before, the differentiated cells are relative mature.