Primary Study Of Treating Diabetes Mellitus With Bone Marrow Stem Cell Transplantation

[Background] According to a report of International Diabetes Federation in 2005, more than 194 million people have diabetes around the world and 5%~10% of them are type 1 diabetes (Insulin Dependent Diabetes Mellitus, IDDM). The number of people with diabetes is expected to increase alarmingly in the coming decades. In 1985 an estimated 30 million people worldwide had diabetes, and in 2000, little over a decade later, the figure had risen to over 150 million. This figure is expected to rise to almost 333 million by the year 2025. Usually, lifelong injections of insulin are main treatment for IDDM and some NIDDM (Non-Insulin Dependent Diabetes Mellitus) patients. But the effect of insulin is not satisfying. It can't hold back the complications of diabetes. To treat IDDM with pancreas or pancreas islets transplantation are attractive methods and amazing outcome had been reported. But the shortage of suitable donors and graft rejection are two restrictive problems. 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.[Objectives] The objectives of this research are to discuss the modes and feasibility of treating IDDM with bone marrow stem cells with diabetes animal models and clinical IDDM patient. Concretely, to study the following aspects: (1) To explore the curative effects of bone marrow stem cells mobilization with different projects onalloxan(ALX) induced rabbit diabetes animal models; (D To study the effects of autologous bone marrow stroma cells(MSCs) transfusion, which was cultured and amplified In vitro, to animal model diabetes; (3) To explore new methods of inducing MSCs to insulin producing cells(IPCs) in vitro; (4) To observe the distribution of IPCs, which was marked with 5-bromodeoxyuridine(BrdU) before transfusion, in diabetes rabbits induced with ALX and its curative effects; (5) From the point of view of IDDM patient, to explore the effects of bone marrow stem cell mobilization and autologous peripheral blood stem cell transplantation (APBSCT) on IDDM. [Methods] First, we explored the method of inducing diabetes animal models with ALX. 15 rabbits were divided into 3 groups according to the methods of medication: rabbits in group A were given ALX without abrosia, freshly prepared ALX 5%(in normal saline) was slowly injected intravenously at 150mg/kg; rabbits in group B were given ALX at twice, 50mg/kg of ALX 5% was intravenously injected without arosia firstly, 2 weeks later, lOOmg/kg of ALX 5% was given again followed by 12 hours' arosia; rabbits in group C were directly given ALX 5% at lOOmg/kg followed by 12 hours' arosia.Secondly, the feasibility of treating diabetes with different projects of bone marrow stem cell mobilization on diabetes animal models was studied. Model rabbits were distributed to 3 groups. Rabbits in group A were injected granulocyte colony-stimulating factor(G-CSF) only by hypodermic at lO^g/kg-d"1 (total 5~10d). Rabbits in group B were slowly injected Cyclophosphamide(CTX) intravenously first at 40mg/kg, 24hours later, G-CSF was injected as group A. Group C was control group. Rabbits in this group were injected normal saline 0.9% by hypodermic at O.lmL/d for 5 days. Indexes as fasting blood glucose, urine glucose, serum insulin and C peptide were detected for effect evaluation. Thirdly, we explored the methods of isolating and culturing rabbit's MSCs in vitro.We chose 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/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.Fourthly, we isolated and cultured stroma cells of pancreas. Newborn New Zealand Rabbit (without sucking the breast) was put to death by breakdown the cervical cord and soaked in 75% of alcohol for 5 min. Opening the abdomen under sterile operation, isolating the pancreas and simply cutting in a penicillin bottle, washing 2 times with D-Hanks solution, the cells were suspended with DMEM/F12 (1 '. 1) complete medium and cultured in 37°C, 5% CO2 incubator. For comparing, Stroma cells of pancreas from adult rabbit were cultured.Fifthly, 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^O ? 1) complete medium in the proportion of 1 to 4. MSCs of passage 3 were cultured with the mixture. The medium was changed every other day. From the 3rd day, the cells were harvested and dyed with DTZ and insulin ICC. Insulin gene RT-PCR was also done to identify IPCs. Sixthly, Observation of the distribution and curative effects of MSCs after being transfused back. We isolated and cultured MSCs as before. And induced them into IPCs, marked the cells with BrdU in vitro and transfused back intravenously. In the two weeks after that, the fasting blood glucose and insulin level were detected. At the end of the observation, BrdU and insulin immunohistochemistry on pancreas and liver section were studied.And for the clinical research, an IDDM patient was treated with bone marrow stem cell mobilization and autologous peripheral blood stem cell transplantation (APBSCT) successively. The indexes as general condition, fasting serum insulin and C-peptide levels, the daily dosage of insulin were observed.[Results] First, the method of inducing diabetes animal models with ALX was improved. The results showed that the twice administration method was the best with a highest model achievement ratio and lowest mortality.Secondly, the results of the marrow stem cell mobilization test showed that, no visible curative effect was found in group A. The high blood glucose level was also not controlled in rabbits of group B, but their fasting serum insulin level was raised to a certain extent.Thirdly, we isolated and cultured rabbit's MSCs in vitro successfully. ? We chose an optimal puncture site—tibial tubercle on rabbit. It is clear on anatomic structure, easy to be fixed, sterilized and punctured, the marrow capacity is big, too. (D The puncture method for bone marrow was improved. Instead of a myeloid puncture needle, a 20mL single-use syringe was used to puncture the bone. The method is more convenient and practicality. (3) Comparing with citrate sodium, heparin is more efficient for anticoagulation, should be the first choice for marrow aspiration. ? A contrast test showed that density gradient centrifugation was obviously better than erythrocyte dissolving method with ammonium chloride in marrow nucleated cell 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. Fourthly, pancreas stroma cells of newborn rabbit were cultured successfully in vitro. Positive cells dyeing with dithizone (DTZ) and insulin immunocytochemistry (ICC) were seen from the passage 2. These cells were IPCs. Fifthly, after being cultured with mixed medium, MSCs sticked to the wall andproliferated 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 6 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.Sixthly, in the term of observation, the fasting blood glucose and insulin level didn't improved significantly, but from the result of BrdU and insulin immunohisto-chemistry on pancreas section, we found that IPCs could homing to pancreas and maintained its insulin producing function.In the clinical research part, the patient's condition improved visibly and the daily dosage of insulin decreased significantly. Especially after APBSCT, the patient's fasting serum insulin and C-peptide level recovered normal. That was a similar short-term curative effect as islet transplantation. But the long-term effects need more observation. Combining with the stem cell mobilization experiment of diabetes animal models, the results was analyzed as follows: ? Obvious dissimilarity of pathogenesy lies between diabetes animal model and IDDM patient. The former is direct destruction of p cells by ALX, the latter is damage to p cells caused by autoimmunity. Both of bone marrow stem cell mobilization and APBSCT have remarkable effect in controlling autoimmune response. So, the curative effects on IDDM might be a result of controlling on autoimmune response. (2) Stem cells mobilized to the peripheral blood might homing to pancreas tissue. Induced in the microenvironment of pancreas, the stem cells differentiated to islet p cells and produced insulin. There were three bases supporting the conclusion. First of all, fasting serum C peptide level, which is a sign of the function of p cells, recoverednormal after APBSCT. Secondly, fasting serum insulin level was raised to a certain extent in the bone marrow stem cell mobilization experiment of animal models. Thirdly, the result of animal model test showed that MSCs amplified in vitro could homing to pancreas tissue after being transfused back. (3) Any organ has the ability of self-regeneration to a certain extent. Pancreas is not an exception. So pancreas stem cells might be self-induced to islet cells secondary to the controlling of autoimmune response to regenerate the endocrine secretion function. It might be the key meaning of APBSCT for IDDM that the former could promote the self-regeneration of islet cells.[Conclusions] In the animal model research part: (1) The method of inducing diabetes animal models with ALX was improved; (2) The treatment of stem cell mobilization with G-CSF only has no effects for DM of rabbit. But with the method of G-CSF+CTX, although the high level of blood glucose didn't get any significant improvements, the fasting serum insulin level was raised to a certain extent; (3) We optimized the methods of bone marrow aspirating and MSCs culturing; (4) We established a convenient method for culturing pancreas stroma cells; (5) Discovered firstly in the world that pancreas stroma cells have the abilities of self-inducing and external inducing. To a certain extent, this conclusion explains the regeneration and rehabilitation mechanism of |3 cells in islet, offers a new choice for cell replacement therapy for IDDM, and provides a new method for studies inducing MSCs into other cells. And further more, it proved that pancreas stroma cells not only have transdifferentiation function of stem cells, but also, as a composition of pancreas' microenvironment, have the ability of directional inducing for other stem cells; (6) The study manifested that IPCs induced from MSCs in vitro could homing to pancreas and maintained its insulin producing function. This confirms the feasibility of treating IDDM with autologous MSCs induced in vitro.In the clinical research part: The research received significant short-term curative effects. In view of the results of animal tests, the possible mechanisms for treating IDDM with APBSCT might be: promoting self-regeneration of islet cells and decreasing the destruction of circulating insulin secondary to the controlling of autoimmune response and/or mobilized MSCs homing to pancreas tissue and induced to islet cells under the influence of pancreas microenvironment.