| BackgroundWith the improvement of living standards,the incidence of Diabetes Mellitus(DM)showed a rising trend year by year.In recent years,basic researches have proved that adult stem cells show promising effect on the treatment of animal models of DM,they provide a new hope for clinical treatment of DM.Menstrual blood-derived Endometrial Stem Cells(MenSCs)have attracted increasing attention due to their abundant sources,low immunogenicity,higher multiple differentiation potential,and without tumorigenicity.However,the conventional MenSCs isolation is based on lymphocyte separation medium through using density gradient centrifugation,which is high-cost and time-consuming.Therefore,the aim of this study is to optimize the isolation of MenSCs by lysing the red cells directly,in order to save the isolation cost and time,meanwhile,improve the sample extraction efficiency.In addition,the pathogenesis of DM is not yet fully understood,then,the establishment of efficient and stable animal models is necessary for us to explore the pathogenesis of DM,and also provide supports for MenSCs based therapy for DM.Part I.Establishment and Evaluation of Rat Models of Type 2 Diabetes Mellitus Objective1.To examine the effect of building SD rat model of type 2 diabetes mellitus(T2DM),induced by injecting STZ in the way of a low dose and high frequency and eating high-fat diet for providing reference to T2 DM with high success rate and stability.2.To examine the similarity between the rat model of type 2 diabetes and clinical patients of diabetes by analyzing the biochemical indexes of clinical diabetic patients.Methods1.Male SD rats were fed with a special high-fat diet for 4 weeks,to induce insulin resistance,then STZ(30mg/kg)was injected intraperitoneally in d28,d35 and d42,in turn,continue to raise to d49,The experimental group was divided into the continuous high-fat diet feeding group and the normal feeding group.By testing the blood glucose,fasting blood glucose and glucose tolerance of each group,and insulin and blood fat,liver and kidney function related to the physiological and biochemical indicators,to evaluate the Establishment and evaluation of SD rat model of type 2 diabetes mellitus.2.Collection of clinical biochemical data of diabetes patients using SPSS 11.0 software for statistical analysis.Results1.After being i.p injected STZ for three times,all the rats reach to the T2 DM diagnostic criteria,and the symptoms of continuously high-fat diet feeding rats remained stable for whole experimental period,meanwhile,the liver and kidney function of these rats deteriorated gradually.In contrast,the rats feeded with regular chow at d49 had a recovery,and all the indexes of blood glucose,blood lipid,liver and kidney were reaching to normal.2.By analyzing the blood biochemical indexes of clinical diabetic patients,the biochemical indicators of the rat models of type 2 diabetes mellitu are basically consistent with this experiment.ConclusionRat models of type 2 diabetes mellitu(T2DM)induced by injecting STZ in the way of a low dose and high frequency and eating high-fat diet were successfully established,and which maintain of the stability needed to feed consecutively with high-fat diet.Combined with clinical data analysis,it was further proved that our experimental model approached to clinical patients,which provided a reliable source of animal models for exploring basic researches on the pathogenesis of DM.Part ?.Optimization of the separation of Menstrual blood-derived Endometrial Stem Cells ObjectiveTo establish an effecient and massive-sample method for extracting MenSCs,the Density Gradient Centrifugation and Red Blood Cell Lysis Method were performed respectively,to separate MenSCs from collected menstrual blood of healthy women and then the separation time and efficiency of these two methods were assessed.MethodsIn the first few days of the menstrual cycle,30 ml menstrual blood samples were collected from healthy female volunteers by using menstrual cup(Divacup),evenly divided them into A group and B group under the same condition(15ml per group)and then stored at 4 C.The primary cells were isolated and extracted in 48 h.In group A,6-10 times volume of red cell lysate to blood sample was added after centrifugation of A sample to smash red cells,and the percipitated cells were collected and seeded to culturing dishes.B group sample was treated with density gradient centrifugation,and cells in buffy coat were collected and then seeded to culture dishes after being washed by PBS.MTT colorimetric assay and flow cytometry were used to detect the proliferative activity of the third,ninth and 18 generation(P3,P9 and P18 generation)of MenSCs extracted by these two methods and the expression of adult stem cell surface markers.The difference between the two methods of separation time and separation efficiency was assessed.ResultsMenSCs extracted by these two methods showed positive signals of surface markers of adult stem cells such as CD29,CD44,CD73,CD90,CD105,and HLA-ABC,but without the detection of CD45 and CD34(hematopoietic stem cell surface markers)and HLADR.Statistical results showed no significant difference between these two different methods of isolation and culture of MenSCs by MTT method.But the separation time of Red Blood Cell Lysis Method is shorter than that of the traditional Density Gradient Centrifugation,and the separation efficiency is relatively higher.ConclusionTwo different methods of isolation and culture of MenSCs not only meet our criteria for identification of adult stem cells,but also have the same biological characteristics.It is worth noting that Red Blood Cell Lysis Method has several merits such as time-saving and low-cost for the basic and clinical study of MenSCs. |
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