| IntroductionAs indicated by UKPDS, type 2 diabetes mellitus is more resistant to traditional therapy as natural history develops. HbA1c levels are increasing, leading to uncontrollable complications, resulting in ultimately unavoidable morbidity and mortality. The reason is the patients'pancreaticβcell function gradually decreased along with time. T2DM medication was only symptomatic treatment rather than improvement ofβcell function before the advent of glucagon-like peptide -1 (GLP-1) analogues. Although GLP-1 analogues are supposed to improveβ-cell function, long-term injection requirement is inconvenient, and gastrointestinal motility is inhibited. Novel approach to preserve and rehabilitate the islet has long been pursued by researchers. Recently, stem cells were highlighted due to their multipotent potentials. Encouraging results were observed in diabetic animal models infused with stem cells, of which islet function and glucose control were improved. The probable mechanism was related with induction of re-differentiation of autologous pancreatic stem cells. Therefore, clinical outcomes of stem cells infusion in diabetes especially T2DM which possesses large population are earnestly expected.In this study, after autologous bone marrow stem cells infusion and hyperbaric oxygen (HOT) therapy in T2DM patients, islet function and glucose control were observed to prove the capability of stem cells rehabilitating impaired islets and promoting diabetic control with mild adverse effects in clinical practice. ObjectivesTo explore the procedure of mesenchymal stem cell (MSC) isolation and cultivation and establish the BM-MSC expansion model, supplying stable cell lines for subsequent research To investigate the efficacy and safety for diabetes patients of combined BM-MSC infusion and hyperbaric oxygen therapy (HOT)MethodsFirst, the classical bone marrow adhering method was used for the culture of MSC. The BM-MSC were cultured with DMEM-LG containing 10% fetal calf serum at 37℃in a humidified atmosphere containing 5% CO2. Viability was examined by AO-PI staining. The phenotype of MSC (CD29, CD73, CD105, CD34, CD45, and CD90) was identified by flowcytometry. The multipotent potentials of MSC were identified by induction into osteoblasts and lipoblasts. Genetic variation of MSC passage 5 was examined by chromosome analysis. The nude mice carcinogenic test was performed to test the tumorigenicity of cells. MSC of passage 3-5 were utilized in the subsequent practice. Before clinical application, bacterial cultures, fungal culture, mycoplasma, endotoxin tests were performed.Second, eighty type 2 diabetic patients (48 males) treated with exogenous insulin were randomized into 4 groups: stem cell infusion and hyperbaric oxygen therapy group (BM-SC+HOT), stem cell infusion group (BM-SC), hyperbaric oxygen therapy group (HOT), and conventional therapy group (CONTROL). The patients in BM-SC+HOT and BM-SC group underwent bone marrow aspiration to have MSC separated and cultured. After MSC product prepared, a second bone marrow aspiration was performed to separate BM-MNC. The patients in BM-SC+HOT underwent BM-MNC and BM-MSC infusion by pancreatic arterial interventional therapy and 20 sessions of peri-infusion HOT. BMSC group underwent BM-MNC and BM-MSC infusion. HOT group underwent 20 sessions of peri-infusion HOT. The above 3 groups underwent therapy on the base of conventional treatment. CONTROL group continued conventional therapy. Predominant artery of pancreatic body and tail was identified by CTA and DSA. All patients were followed up for 1 year, during which they were visited every 3 months. The endpoint included weight, exogenous insulin, HbA1c, fasting blood glucose, fasting c-peptide, quality of life and other laboratory parameters were measured. Consents were obtained from all.Results 1) After 48 hours,several adherent cells could be found. And after 72 hours,the nonadherent cells were removed and only the adherent ones were cultured following 3-7 days of culture. Fresh complete medium was replaced twice a week. At about 7-10 days,the isolated cells were developed to visible systematic colonies of adherent fibroblast-like cells. They arranged regularly with clear boundary and showed spindle-like or polygon morphology. Primary cultures were maintained for 10-14 days. Upon reaching near 80-90% confluence, cells were detached with a solution of 0.25% trypsin 3-5 minutes at 37℃. After centrifugation, cells were resuspended with DMEM-LG, replated at a ratio of l: 2 and referred to as first-passage cultures. And the morphology of MSC was about uniformat passage3. Viability >95% was observed with AO-PI staining. Flowcytometric analysis of the MSC at passage 3 showed that these cells were negative for CD34, CD45, and they expressed high levels of CD29, CD73, CD105, and CD90. No abnormal changes in the chromosome karyotype were detected in MSC culture up to the fifth passage. No abnormal changes such as skin nodules or doubtful focus in the nude mice were observed. In various induction differentiation conditions, BM-MSC could differentiate into the osteoblasts, adipocytes. Negative test results were found in bacterial cultures, fungal culture, mycoplasma and endotoxin tests.2) HbA1c in BMSC+HOT group and BMSC group was significantly lower at 1 year compared with that pre-infusion (7.02±0.78 vs. 7.99±0.70, p<0.01; 6.98±0.80 vs. 8.48±0.69, p<0.01). Exogenous insulin (25.25±12.57 vs. 35.05±13.34 , p<0.01; 26.95±13.77 vs. 37.10±13.23 , p<0.01) and FBG (6.47±1.17 vs. 7.78±2.20 , p<0.05; 7.25±1.72 vs. 8.13±2.13 , p<0.05) were also lower, while fasting c-peptide (1.96±0.78 vs. 0.99±0.70, p<0.01; 1.81±0.80 vs. 1.02±0.69, p<0.01) and quality of life (86.3±16.2 vs. 67.4±19.6, p<0.01, 83.3±16.0 vs. 62.8±14.2, p<0.01) was higher. HbA1c in BMSC+HOT group and BMSC group was significantly lower at 1 year compared with that of CONTORL (7.02±0.78 vs. 8.11±0.55, p<0.01; 6.98±0.80 vs. 8.11±0.55, p<0.01). Exogenous insulin (25.25±12.57 vs. 38.60±8.20, p<0.01; 26.95±13.77 vs. 38.60±8.20, p<0.01) and FBG (6.47±1.17 vs. 8.37±2.35, p<0.01; 7.25±1.72 vs. 8.37±2.35, p<0.05) were also lower compared with those of CONTORL, while fasting c-peptide (1.96±0.78 vs. 1.11±0.53, p<0.01; 1.81±0.80 vs. 1.11±0.53, p<0.01) and quality of life (86.3±16.2 vs. 68.2±18.1, p<0.01, 83.3±16.0 vs. 68.2±18.1, p<0.01) was higher. HbA1c, exogenous insulin, FBG, fasting c-peptide or quality of life at 1 year in HOT group was comparable to that pre-infusion, and to that at 1 year in CONTROL group. Adverse events included reversible fever (n=2), abdominal pain (n=5) and bleeding at puncture point (n=3). Dorsal pancreatic artery (50%), great pancreatic artery (15.7%) and transverse pancreatic artery accounts for predominant artery of pancreatic body and tail showed by CTA and DSA.Conclusions1) The isolation and cultivation of MSC from bone marrow were performed with the bone marrow adhering method. The cultured MSC have high viability, uniform phenotype, multipotent potentials, low tumorigenicity and qualified clinical safety. The cell lineage can be used for further research.2) Stem cells infusion intra-arterially improves diabetic control in type 2 diabetes patients, enhances islet function, decreases exogenous insulin and improves quality of life with mild adverse events, which have no additional effect when combined with HOT. HOT alone have no improvements for diabetes.3) Complicated variation and origins of predominant artery of pancreatic body and tail should be comprehensively studied before MSC infusion.
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