Co-transplantation of bone marrow mesenchymal stem cells and islet cells in the treatment of diabetes mellitus

doi:10.3969/j.issn.2095-4344.2013.31.021

Abstract

Abstract:

BACKGROUND: Drug treatment is the main method for the treatment of diabetes currently, but the development of the disease and occurrence of related complications are the challenges to the effect of drugs.
OBJECTIVE: To investigate the effect and feasibility of co-transplantation of bone marrow mesenchymal stem cells and islet cells for the treatment of diabetes.
METHODS: The rat bone marrow mesenchymal stem cells were separated and purified, and cultured in vitro to establish the diabetes models. The rat diabetes models were injected with bone marrow mesenchymal stem cells, co-cultured mixture of bone marrow mesenchymal stem cells and islet cells, and normal saline or phosphate buffer (control). The effect of transplantation was evaluated through observing the blood glucose levels, insulin secretion, and pathological changes of pancreatic tissue in the rat diabetes models.
RESUTLS AND CONCLUSION: In the diabetes rats treated with bone marrow mesenchymal stem cell transplantation, the C-peptide levels were significantly increased after transplantation, while the blood glucose levels were significantly decreased, but not lower than the normal level, and the blood glucose levels were increased again with the time prolonging. In the diabetes rats treated with co-transplantation of bone marrow mesenchymal stem cells and islet cells, the blood glucose levels were decreased significantly and lower than the normal level which was maintained in a certain time, and the decreasing degree was larger than that in the rats treated with simple bone marrow mesenchymal stem cell transplantation. Co-transplantation of bone marrow mesenchymal stem cells and islet cells is feasible for the treatment of diabetes with a certain effect.

Key words: organ transplantation, organ transplantation academic discussion, bone marrow mesenchymal stem cells, islet cells, diabetes, transplantation, pancreas, blood glucose, insulin, immunofluorescence, provincial grants-supported paper

CLC Number:

R318

Wu Li-ping, Li Meng, Li Rui-yu, Sun Yan-fu, Yan Jun-li. Co-transplantation of bone marrow mesenchymal stem cells and islet cells in the treatment of diabetes mellitus[J]. Chinese Journal of Tissue Engineering Research, doi: 10.3969/j.issn.2095-4344.2013.31.021.

Figures/Tables 1

2.1 骨髓间充质干细胞移植治疗糖尿病肖红珍等[11]对外源性骨髓间充质干细胞移植至糖尿病模型大鼠体内分化为胰岛细胞治疗糖尿病进行了研究。实验采用密度梯度离心结合贴壁培养的方法分离、纯化雄性大鼠骨髓间充质干细胞，并采用免疫组织荧光法进行鉴定。鉴定后将30只大鼠随机分为生理盐水组、骨髓间充质干细胞组以及糖尿病模型组，每组10只大鼠，骨髓间充质干细胞组给予大鼠尾静脉注射骨髓间充质干细胞悬液0.2 mL，生理盐水组给予大鼠尾静脉注射0.2 mL生理盐水，糖尿病模型组检测各大鼠血糖值及体质量，按一定剂量腹腔注射链脲佐菌素，检测各大鼠血糖值，当血糖≥ 16.7 mmol/L且稳定2 d后给予尾静脉注射骨髓间充质干细胞0.2 mL。应用原位杂交法检测各大鼠胰腺组织内Y染色体标志物及放射免疫法测定C-肽值。研究结果显示，大鼠腹腔注射链脲佐菌素后 2 d，血糖水平开始迅速升高，4 d后血糖值大于 16.7 mmol/L，且稳定在21.0 mmol/L左右，注射骨髓间充质干细胞后，血清中C-肽值明显升高，而生理盐水组未予注射骨髓间充质干细胞，C-肽值无明显变化，且生理盐水组中各大鼠胰腺组织中未见黄绿荧光Y染色体标记物存在，而骨髓间充质干细胞组和糖尿病模型组各大鼠胰腺组织中均可见黄绿荧光Y染色体标记物存在，且糖尿病模型组大鼠胰腺组织中黄绿荧光强度较大，Y染色体标记物的数目较多。刘弘光等[12]将体外培养的骨髓间充质干细胞移植至糖尿病模型大鼠体内，观察移植后各大鼠的血糖水平变化，结果发现，骨髓间充质干细胞移植3 d后，糖尿病模型大鼠的血糖为(21.84± 3.37) mmol/L，移植7 d后，糖尿病模型大鼠的血糖为(20.90±3.46) mmol/L，移植10 d后，糖尿病模型大鼠的血糖为(18.86±2.80) mmol/L，而未予以骨髓间充质干细胞移植糖尿病模型大鼠始终处于高血糖状态，并无下降趋势。此外，研究中还发现骨髓间充质干细胞移植3 d后，在大鼠胰腺血管周围可见大量BrdU阳性细胞。孟凡彪[13]在实验中也对骨髓间充质干细胞进行分离培养，并将培养后的骨髓间充质干细胞注射至糖尿病模型大鼠尾静脉中，监测空腹血糖的变化，利用胰岛素免疫荧光染色评估胰岛的大小。结果发现，骨髓间充质干细胞移植后11 d，糖尿病模型大鼠的空腹血糖水平明显降低，但此后又再次升高。胰岛免疫荧光染色面积增大，损伤的胰岛细胞获得再生。胰腺组织中发现带有标记物的骨髓间充质干细胞，移植的骨髓间充质干细胞移植入胰腺组织内部，获得一定的增殖，但骨髓间充质干细胞本身不表达胰岛素。与胰岛细胞共培养后，能够增加胰岛细胞的表达量。高斌等[14]对分离培养骨髓间充质干细胞并注射至糖尿病模型的大鼠进行对比观察，将注射骨髓间充质干细胞的糖尿病模型大鼠与注射生理盐水的糖尿病模型大鼠进行血糖监测比较，结果显示，移植2周后，骨髓间充质干细胞移植治疗糖尿病大鼠血糖为(23.63±6.42) mmol/L，胰岛素水平为(13.89± 7.89) mU/L，生理盐水对照组糖尿病大鼠血糖为(27.96±1.87) mmol/L，胰岛素水平为(11.57± 3.25) mU/L，而正常对照大鼠血糖水平为(7.97± 1.00) mmol/L，胰岛素水平为(33.68±6.21) mU/L。免疫组织化学染色观察可见正常对照大鼠胰岛中心部胰岛细胞的胰岛素为强阳性，细胞内可见深褐色粗大颗粒；生理盐水对照组大鼠可见胰岛及胰岛中心细胞数明显减少，胰岛素反应基本阴性，仅见个别胰岛细胞内出现少量微弱淡黄色细颗粒；而骨髓间充质干细胞移植糖尿病模型大鼠胰岛中心部细胞增多，大部分胰岛素反应阳性或强阳性呈黄色或褐色颗粒。高赟等[15]和董庆玉[16]同样对同种异体骨髓间充质干细胞移植治疗糖尿病大鼠分别进行了研究，研究均显示移植后大鼠的血糖有明显下降趋势，胰岛中增殖细胞明显增多。 2.2 骨髓间充质干细胞与胰岛细胞联合移植治疗糖尿病田烁[17]、叶永峰[18]和贾辰乐等[19]分别对骨髓间充质干细胞与胰岛细胞联合移植治疗糖尿病进行了实验研究。田烁[17]在实验中选取三四周龄的大鼠，采用密度梯度离心法结合贴壁培养法分离纯化骨髓间充质干细胞，并进行传代扩增，取第4代细胞与胰岛细胞共培养。通过腹腔注射链脲佐菌素破坏胰腺组织设立糖尿病模型大鼠，经大鼠尾静脉分别注射磷酸盐缓冲液、骨髓间充质干细胞、骨髓间充质干细胞和胰岛细胞设立对照组及实验组。研究结果显示，磷酸盐缓冲液治疗糖尿病模型大鼠血糖较治疗前无明显下降，骨髓间充质干细胞移植治疗糖尿病大鼠和骨髓间充质干细胞与胰岛细胞联合移植治疗糖尿病大鼠在移植后14 d血糖开始下降，且血糖水平与移植前相比较，差异有显著性意义(P < 0.01)，并且骨髓间充质干细胞与胰岛细胞联合移植治疗糖尿病大鼠在移植后血糖下降更明显。叶永峰[18]对骨髓间充质干细胞与胰岛细胞共培养进行了实验研究，分离大鼠骨髓间充质干细胞和胰岛，设立共培养组和单独培养组，并进行体外培养，结果显示，随体外培养时间的延长，胰岛与骨髓间充质干细胞混合培养组的存活率与胰岛单独培养组的存活率差异有显著性意义(P < 0.05)，随培养时间延长，胰岛对葡萄糖反应性逐渐减弱，刺激指数从第1天的3.1-3.3降至第7天的1-1.6。此外，研究结果还显示单独胰岛移植治疗(10.2±2.0) d内，血糖可控制在16.7 mmol/L以下，但随后血糖逐渐上升，半量胰岛联合骨髓间充质干细胞移植后(15.2±1.3) d内，血糖能够控制在16.7 mmol/L以下，胰岛联合骨髓间充质干细胞移植后(21.0±1.7) d内，血糖能够控制在16.7 mmol/L以下。胰岛联合骨髓间充质干细胞移植治疗的糖尿病模型大鼠，移植治疗前尾部皮肤存在溃疡病变，移植治疗15 d后，皮毛变光滑，动作敏捷，尾部病变好转。贾辰乐等[19]也对骨髓间充质干细胞与胰岛细胞共培养移植治疗糖尿病模型大鼠进行了研究，实验采用密度梯度离心法分离骨髓间充质干细胞，胶原酶消化法分离胰岛细胞，链脲佐菌素制备糖尿病模型大鼠，分别给予糖尿病模型大鼠单纯胰岛细胞移植和胰岛细胞联合骨髓间充质干细胞共移植，并设立生理盐水对照，比较血糖的变化。结果显示，生理盐水对照组糖尿病模型大鼠血糖水平一直较高，移植后1 d，血糖水平为(26.22±3.37) mmol/L，移植后5 d，血糖水平为(24.83±3.97) mmol/L，移植后 15 d，血糖水平为(27.47±2.65) mmol/L；单纯胰岛细胞移植治疗糖尿病模型大鼠，移植后1 d，血糖水平为(8.43±1.34) mmol/L，移植后5 d，血糖水平为(10.46±1.37) mmol/L，移植后15 d，血糖水平为(12.31±1.87) mmol/L；胰岛细胞联合骨髓间充质干细胞共移植治疗糖尿病模型大鼠，移植后1 d，血糖水平为(6.61±1.95) mmol/L，移植后5 d，血糖水平为(7.35±2.03) mmol/L，移植后15 d，血糖水平为(7.71±1.84) mmol/L。骨髓间充质干细胞与胰岛细胞联合移植和单纯胰岛细胞移植后的血糖水平相比较，差异有显著性差异意义。 2.3 国外学者对骨髓间充质干细胞移植以及胰岛细胞移植治疗糖尿病的研究文献举例见表1。"

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