【作者】 吴志贤；

【导师】 谭建明；

【作者基本信息】 第二军医大学 ， 外科学， 2011， 博士

【摘要】 研究背景英国前瞻性糖尿病研究(UKPDS)表明2型糖尿病(T2DM)的治疗效果随着时间的延长越来越差,糖化血红蛋白(HbA1c)水平越来越高,导致糖尿病并发症难以控制,患者最终难以避免致死和致残的威胁。其中一个重要原因是患者的胰岛β细胞功能随着时间的延长逐渐下降。在胰高血糖素样肽-1(GLP-1)类似物出现之前,T2DM的药物治疗不能改善β细胞功能,仅为对症治疗。虽然研究提示GLP-1类似物可改善β细胞功能,但是需要长期注射应用存在不方便,且具有抑制胃肠道蠕动等不良反应。新的保护和恢复胰岛功能的手段一直是医学界期待和研究的方向。近年来,干细胞以其促进再生等一系列功能成为研究的热点。动物实验把干细胞应用到糖尿病大鼠中得到了令人欣慰的结果,干细胞可以恢复糖尿病大鼠的胰岛功能,纠正高血糖,其机制可能与促进自体胰腺干细胞再分化有关。因此人们热切的期待干细胞治疗糖尿病特别是T2DM这一具有庞大人群疾病的临床结果。本试验通过自体骨髓干细胞及高压氧(HOT)联合治疗,观察治疗糖尿病胰岛功能改善及血糖控制好转情况,从临床角度研究干细胞对T2DM胰岛损伤的恢复能力,改善血糖控制的效果及可能的不良反应。目的探索从采集的人骨髓中分离、培养间充质干细胞(MSC)的方法,建立人骨髓间充质干细胞(bone marrow mesenchymal stem cells, BM-MSC)的体外培养体系,为后续的干细胞研究提供稳定的细胞模型。将体外培养的BM-MSC和体外分离的骨髓单个核细胞(bone marrow mononuclear cells, BM-MNC)同时移植入糖尿病患者体内,研究骨髓干细胞联合HOT治疗T2DM的有效性与安全性方法(l)采用经典的贴壁培养法培养BM-MSC,在含10%胎牛血清的DMEM-LG培养基中进行培养,获得人BM-MSC。吖啶橙-碘丙啶(AO-PI)染色鉴定其活性。通过流式细胞仪分析其表面标记(CD29,CD73,CD90,CD105,CD34,CD45)以及成骨、成脂肪等多向诱导分化等鉴定MSC特征。通过染色体分析MSC P5代遗传学改变。接种于裸鼠背部皮下,术后3天及1、3个月肉眼观察成瘤情况。取三代到五代(P3-P5)的BM-MSC用于后续实验。临床应用前进行细菌培养、真菌培养、支原体检测、内毒素检测等检查。(2)T2DM患者80例,男48例,女性32例,进入研究前均接受胰岛素治疗,随机分为骨髓干细胞(BM-SC)联合HOT治疗组(BM-SC+HOT组),骨髓干细胞治疗组(BM-SC组),HOT治疗组和常规治疗组(对照组)。BM-SC+HOT组和BM-SC组接受首次自体骨髓采集以培养自体BM-MSC,BM-MSC制备成功后拟进行输注,输注当日接受二次骨髓采集以分离BM-MNC。BM-SC+HOT组接受BM-MSC及BM-MNC胰腺动脉内输注治疗和输注前后共20次HOT治疗,BM-SC组接受BM-MSC及BM-MNC治疗,HOT组接受20次HOT治疗,以上3组均在胰岛素治疗基础上接受干预,对照组只接受胰岛素治疗。胰腺动脉采用术前CT血管造影(CTA)及术中数字减影血管造影(DSA)分析。4组患者随访1年,每3个月随访1次,观察患者体重、血压、体重指数(BMI)、腹围及胰岛素用量变化,评估血糖控制情况、生活质量评分,复查HbA1c、空腹血糖、空腹C肽及其它实验室指标。实验获得医院伦理委员会批准。所有患者均签署临床试验知情同意书。结果(1)经贴壁培养48h后可见散在细胞,形态呈成纤维样,72h后通过全量换液去除各种残留血细胞,MSC呈分散存在或几个形成细胞团,7-10天后,贴壁细胞数量明显增多,分布及生长均匀,排列整齐,呈梭形或长多边形,形态不均一。原代培养10-14天细胞可达80-90%融合,0.25%胰蛋白酶常规消化,按1:2的比例传代,传代后的BM-MSC形态与原代基本相似,经过3代后BM-MSC基本达到形态均一。AO-PI染色鉴定活性>95%。流式细胞术细胞表型的鉴定分析显示BM-MSC CD29、CD73、CD90、CD105高表达(99.65±0.44%,98.5±0.84%,98.7±0.78%,98.8±0.37%),而CD34、CD45低表达(0.77%±0.17%,1.86±0.21%)。MSC培养至第5代,未发现染色体核型异常改变。实验裸鼠在观察期内均未见结节形成或可疑病灶产生。诱导分化实验表明BM-MSC在体外具有向骨、脂肪细胞分化的能力。临床应用前细菌培养、真菌培养、支原体检测、内毒素检测等均为阴性。(2)BM-SC+HOT组和BM-SC组在随访结束时与治疗前相比HbA1c(%)降低(7.02±0.78 vs. 7.99±0.70, p<0.01; 6.98±0.80 vs. 8.48±0.69, p<0.01),胰岛素用量(IU)降低(25.25±12.57 vs. 35.05±13.34, p<0.01; 26.95±13.77 vs. 37.10±13.23 , p<0.01),空腹血糖(mmol·L-1)降低(6.47±1.17 vs. 7.78±2.20 , p<0.05; 7.25±1.72 vs. 8.13±2.13 , p<0.05),空腹C肽(ng·mL-1)升高(1.96±0.78 vs. 0.99±0.70, p<0.01; 1.81±0.80 vs. 1.02±0.69, p<0.01),生活质量评分升高(86.3±16.2 vs. 67.4±19.6, p<0.01, 83.3±16.0 vs. 62.8±14.2, p<0.01)。BM-SC+HOT组和BM-SC组在随访结束时与对照组相比较HbA1c降低(7.02±0.78 vs. 8.11±0.55, p<0.01; 6.98±0.80 vs. 8.11±0.55, p<0.01),胰岛素用量降低(25.25±12.57 vs. 38.60±8.20, p<0.01; 26.95±13.77 vs. 38.60±8.20, p<0.01),空腹血糖降低(6.47±1.17 vs. 8.37±2.35, p<0.01; 7.25±1.72 vs. 8.37±2.35, p<0.05),空腹C肽升高(1.96±0.78 vs. 1.11±0.53, p<0.01; 1.81±0.80 vs. 1.11±0.53, p<0.01),生活质量评分升高(86.3±16.2 vs. 68.2±18.1, p<0.01, 83.3±16.0 vs. 68.2±18.1, p<0.01)。HOT与对照组在随访结束时与治疗前相比HbA1c、胰岛素用量、空腹血糖、空腹C肽及生活质量评分差异无统计学意义。HOT组在随访结束时与对照组相比HbA1c、胰岛素用量、空腹血糖、空腹C肽及生活质量评分差异无统计学意义。干细胞治疗相关不良事件包括一过性发热(n=2),一过性腹痛(n=5),穿刺性出血(n=3)等。CTA及DSA发现胰体尾部优势动脉主要为胰背动脉(50%),其次为胰大动脉(15.7%)和胰横动脉(10.8%)。胰背动脉和胰大动脉共同作为优势动脉占21.6%。结论(l)利用贴壁培养法分离培养,可获得足量的人BM-MSC。培养的BM-MSC活性高,表型均一,并具有多向分化潜能,无致瘤性,临床安全性高,可用于进一步的组织工程、细胞和分子生物学研究。(2)骨髓干细胞胰腺动脉内输注治疗可以恢复T2DM患者部分胰岛功能,减少胰岛素用量,从而显著改善血糖控制,提高患者生活质量。干细胞治疗副作用较小,与HOT联合治疗未能进一步提高治疗效果。HOT单独治疗T2DM对病情无改善效果。(3)胰体尾优势供血动脉变异大,来源复杂,分析其变异状况有利于提高干细胞治疗的成功率。更多还原

【Abstract】 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.更多还原