| BackgroundDiabetes mellitus (DM) has been a major public health problem in the world,especially in China. More than92million Chinese men and women have diabetes mellitus,in which type2diabetes mellitus(T2DM) accounts for more than90%of DM. one of thebasic pathogenic mechanisms of T2DM is that β-cells fail to compensate the effect ofinsulin resistance. The another mechanism includes the reduction of β-cell number.Progressive exacerbation of β-cell number and functional impairment result in poorglycemic control and development of systemic complications of DM. It is necessary todevelop new agents or methods that can attenuate deterioration of β-cell function or/andincrease the number of insulin-producing cells. Stem cells are primitive cells capable ofself-renewing and differentiation into cells of other line-age. Bone marrow stem cells iseasy to get，it contains mutable cell types, especial mesenchymal stem cells(MSCs). MSCscan induce the regeneration of recipient-derived pancreatic insulin-secreting cells. Thereare a lot of experiments studies indicate that BM-MSCs have a good therapeutic effect onDM. Autologous bone marrow-derived mesenchymal stem cells (BM-MSCs) treatment inT2DM into the preclinical application research stage. Autologous BM-MSCs by infusing invivo, is expected to control and even cure diabetes mellitus.Based on the above understanding, we have embarked on this project. To establish theapproaches of isolation, culture, and identify the human BM-MSCs in vitro and observe thecellular growth status and morphology changes of the primary and passage MSCs withinverted microscope. BM-MSCs without any inducing treatment through the blood vesselsinvolved in directional migration to the pancreas. Serum levels of blood glucose, HbA1c,and islet function were measured before and after infusing BM-MSCs. Meanwhile, adverseevent was recorded. From the clinical point of view, to study the ability of bone marrow repair T2DM islet function and improve blood glucose control and observed possibleadverse reactions.The first part: Experimental Study on Isolation, Culture and Identify of HumanBone Marrow-derived Mesenchymal Stem Cells in VitroObjective: To establish the approaches of isolation, culture, and identify the humanBM-MSCs in vitro.Methods:200ml bone marrow(BM) blood from each patient was collected. MSCswere separated by human bone marrow stem cell purification kits in vitro. The BM waspurified by its adhesiveness. We observed the cellular growth status and morphologychanges of the primary and passage MSCs with inverted microscope. We draw cell growthcurves by cells counting. Cell surface antibody of CD34, CD44, CD45and CD90wasexamined by flow cytometry. Trypan blue staining identify their activity.Results: The BM-MSCs show spindle appearance. Between the cells arranged inparallel and swirling. The primary culture cells fused into monolayer after10-12days,while passage culture cells need7-10days. BM-MSCs were expressed positively CD44,CD90, but absence of CD34, CD45. Identification of Trypan blue staining activity is greaterthan95%.The second part: The clinical observational study of effect and safety ofautologous bone marrow mesenchymal stem cells on patients with type2diabetesmellitusObjective: To study the clinical effect of BM-MSCs on patients with T2DM viasplenic artery transplantation, to explore its safety and validity and try to explain themechanisms.Methods: A total of18patients with T2DM had been enrolled. Patients gave informedconsent and accept transplantation.①Bone marrow stem cells were mobilized withgranulocyte colony stimulating factor;②Patients received multipoint puncturation in thebilateral posterior superior iliac spine in a prone position following lumber anesthesia. Atotal of200ml bone marrow blood was collected. Stem cell was separated from bonemarrow blood;③BM-MSCs was infused into dorsal pancreatic artery or/and transversepancreatlc artery(TPA) through femoral artery intubation;④Serum levels of blood glucose,Glycosylated hemoglobin (HbA1c), C-peptide levels, area under curve for C-peptide(AUCC) were measured before and after infusing BM-MSCs. Meanwhile,adverse event was recorded, and to explore its safety and efficacy.Result:18patients (12males and6females),8cases of patients with follow-up in3month,5cases of patients with6month follow-up,5cases of patients with12monthfollow-up.①During autologous BM-MSCs transplantation, no adverse reaction wasdetected;②At3month,6month and12month after infusing BM-MSCs, mean levels ofHbA1c were lower than before treatment. Fasting C-peptide and Postprandial2h C-peptidelevels increased after infusing BM-MSCs. fasting C-peptide achieve statistical significance.AUCC was higher at3month,6month,12months compared with thatpre-infusion(5.93±5.85vs7.26±5.73,3.8±1.6vs5.8±2.38,4.73±5.32vs7.64±4.68). one casehave incracranial hypotension syndrome(IH) at the first postoperative day.Conclusion1.human BM-MSCs can be isolated and purified by density gradient centrifugation andadherent filtration. Derived cells can be cultured stably and expanded.2.BM-MSCs were expressed positively CD44, CD90, but absence of CD34, CD45.3.Autologous BM-MSCs transplantation for T2DM showed high safety and a certaineffect for islet function. |
