Primary Study Of Differentiation Of Bone Marrow Stroma Cells Into Insulin-producing Cells

[Background] Diabetes is a chronic disease that make people weaking and expend costly, it results in severe secondary complications for poor glycaemic control, such as renal, retinal, and heart diseases. Diabetes in all its forms currently afflicts at least 200 million people in the world and this number is expected to double by the year 2025. Diabetes has become a burden on both patients and society, and increasinglyserious, especially in the developing countries. Pancreas or islet transplantation has been impressive in treating some type 1 diabetes (Insulin Dependent Diabetes Mellitus, IDDM) and part of type 2 diabetes(Non-Insulin Dependent Diabetes Mellitus, NIDDM), but this requires possibly toxic immunosuppression and there will never be enough donor islets to satisfy demand. In recent years, followed by the presentation of the concepts of Adult Stem Cells and Multipotent Stem Cells, stem cell replacing treatment for IDDM has become a new hotspot in worldwide research fields. The bone marrow stroma cells are un-hemopoietic cells with multi-differentiating potential, they have much greater advantages than others because they are easy to obtain, that they have no immunosuppression, have moral support and steady genetic background, they have been regarded as good cells for cell engineering and genetic engineering. Researches have showed that MSCs have a pluripotent ability to differentiate a variety of cells and tissues in vitro. [Objectives] The objectives of this research are to culture MSCs and pancreaticstroma cells. To explore a simple way of differentiating the MSCs into insulin-producing cells(IPCs) in vitro.[Methods] 1. The culture of SD rat MSCs: Adult Sprague-Dawley rat was put to death by breakdown the cervical cord and soaked in 75% of alcohol for 5 min. Isolating the thighbones and the shinbones in sterile condition, cutting epiphyseals, washing the inner corical with D-hanks solution, density gradient centrifugation and erythrocyte dissolving with ammonium chloride method were used in marrow nucleated cell isolation; DMEM/F12 (1:1) culture medium (containing 10% of fetal bovine serum and 2.438g/L of sodium bicarbonate) was used as the culture system; MSCs were purified with adherence method.2. The culture of rabbit MSCs: We chose the tibial tubercle of rabbit's posterior limb as the puncture site; the marrow was punctured and aspirated with a 20ml single-use syringe; heparin was used for anticoagulation; density gradient centrifugation and erythrocyte dissolving with ammonium chloride method were used in marrow nucleated cell isolation; DMEM/F 12 (1:1) culture medium (containing 10% of fetal bovine serum and 2.438g/L of sodium bicarbonate) was used as the culture system; MSCs were purified with adherence method.3. The culture of SD rat pancreas stroma cells: Newborn Sprague-Dawley rat (without sucking the breast) was put to death by breakdown the cervical cord, opening the abdomen under sterile operation, eviscerating the pancreas and isolating cells by mechanical dispersion and enzymatic digestion, washing 2 times with D-Hanks solution, the cells were suspended with DMEM/F 12 (1:1) complete medium and cultured in 37 , 5%CO2 incubator. For comparing, Stroma cells of pancreas from adult rat were cultured. Take the medium of the P2-P5 cells to differentiate experiment.4. Inducing culture of MSCs: Pancreas stroma cells were cultured in DMEM/F12(1:1) complete medium. 3 days later, the medium was sucked out and purified by centrifugation. The top liquid was mixed with DMEM/F 12 (1:1) complete medium in the proportion of 1 to 3. MSCs of passage 3 were cultured with the mixture for 5-10 days (The medium was changed every other day). The cells were harvested and dyedwith DTZ and insulin immunocytochemistry (ICC). Insulin gene RT-PCR was also done to identify IPCs[Results] l.The marrow nucleated cells isolated by density gradient centrifugation grew rapidly, The cells showed fibroblast-like morphology, subculture steadily, proliferate rapidly. The adherence method can discard the miscellaneous cells. But the erythrocyte dissolving with ammonium chloride method may impact the proliferative ability of the MSCs. The density gradient centrifugation and the adherence method can isolate and purify MSCs perfectly, this method was obviously better than erythrocyte dissolving method with ammonium chloride method in marrow nucleated cells isolation. The effect of DMEM/F12 (1:1) culture system (containing 10% of fetal bovine serum and 2.438g/L of sodium bicarbonate) in culture MSCs was confirmed.2.It was difficult to isolate and culture the pancreas stroma cells of the adult rat, and there are too many cell mass. There are few cells adhered and no proliferation. In 72 hours, these cells died one after the other. It was esy to isolate and culture the pancreas stroma cells of the newborn rat. The cells grew rapidly, and the cell morphology were fusiform-shaped and stelliform mostly, minority cells were large rounded or stelliform. When subculture to passage 2, the pancreas stroma cells adhered rapidly, and we can see that the cells grow to the circumference in radiative or spiring shape, part area in focal shape. On the mass, there are positive cells dyeing with dithizone (DTZ) and insulin immunocytochemistry (ICC). 3. After being cultured with mixed medium, MSCs sticked to the wall and proliferated quickly in the mixed medium. The main appearances of the cells were shuttle and polygon. As time goes on, round and spindle cells with thick grains inside appeared and increased. All distributed on a centralized island appearance. At the 6th day, some round or spindle cells with thick grains inside and island like cell mass showed positive on DTZ and insulin ICC coloration. Moreover, expression of insulin gene in the cells was detected by RT-PCR. It showed that some of MSCs could be induced into IPCs in the mixed medium.[Conclusions] 1. MSCs can be isolated by density gradient centrifugation andadherence method successfully. The pancreas stroma cells of newborn rat can be isolated by mechanical dispersion and enzymatic digestion successfully. We established a convenient method for isolating and culturing pancreas stroma cells. Discovered that there were pancreas stem cells in the pancreas stroma cells, and they have the abilities of differentiation to IPCs by pancreas stroma cells external inducing.2.The medium of rat's pancreas stroma cells can induce rat's MSCs into IPCs. Induceing-cells can be positive cells dyeing with dithizone (DTZ) and insulin immunocytochemistry (ICC).3.Rabbit's MSCs can be induced into IPCs by the medium of rat's pancreas stroma cells. This conclusion offers a new choice for inducing MSCs into other cells in different species. This confirms the feasibility of inducing MSCs into IPCs.