骨髓间充质干细胞和体细胞的共微囊化研究

发布时间：2018-08-26 15:30

【摘要】：骨髓间充质干细胞(BMSCs)来源丰富、易分离纯化且具有多向分化潜能、可自体移植,是作为种子细胞移植的研究热点。微胶囊具有良好的免疫隔离性能和生物相容性,为干细胞大规模、高活性体外培养及长期保存提供了技术支持。共微囊化技术则利用干细胞与体细胞间的相互接触与支持作用为细胞移植疗法开辟了一种新途径。本文制备了载BMSCs细胞的海藻酸钙-几丁聚糖-海藻酸钙(ACA)微胶囊,考察了其理化力学性能及生物相容性。共微囊化BMSCs/肝细胞(Chang liver),考察细胞代谢性能。共微囊化BMSCs/胰岛β细胞(Beta-TC-6)用于治疗Ⅰ型糖尿病,并在分子水平探讨其机理。首先,体外培养BMSCs细胞,绘制其细胞生长曲线。然后采用免疫细胞组织化学染色方法鉴定其表面抗原CD29、CD44、CD117,检测结果为CD29呈阳性,CD44呈阳性,CD117呈阴性,成骨诱导分化实验结果也表明BMSCs在体外培养时具有分化的能力。其次,采用高压静电法制备载BMSCs细胞的ACA微胶囊,优化制备工艺为:两步法制备,海藻酸钠浓度1.75%(w/v),成膜时间10~ min,细胞密度1×10~6个/mL。制出的载BMSCs微囊球形度好、表面光滑,粒径主要分布在300~400μm间且7天内随时间变化不大,机械强度较好且囊内细胞分布均匀、活性高,破囊后重新培养的BMSCs细胞生长良好。再次,考察体外培养的微囊化BMSCs细胞的活性(CCK-8法),发现与平板培养和非液化核心的微囊化细胞相比,液化核心的微囊更适于细胞的长期生长。体外培养的第15天,细胞密度达到峰值为4.3×10~7个/mL。将载BMSCs细胞的微囊植入小鼠腹腔考察微胶囊的体内生物相容性,观察发现小鼠生理活动正常,未见毒性症状,体重增长平稳。28天内回收微囊,微囊表面光滑,球形度好,未出现纤维化或巨噬细胞聚集现象。然后,采用平板、微囊化、共培养及共微囊化四种培养方式来培养肝细胞(Chang liver),以白蛋白及尿素分泌量为检测指标考察比较肝细胞的代谢性能。结果表明,四组分泌白蛋白量及尿素的大小关系均为:共微囊化BMSCs/Chang liver组 BMSCs/Chang liver组微囊化Chang liver组 Chang liver细胞组。15天内,共微囊化BMSCs/Chang liver组白蛋白分泌量最高为3.08 mg/mL。11天内,尿素分泌量最高为3.75 mmol/L。最后,制备微囊化胰岛β细胞(Beta-TC-6)及共微囊化BMSCs/Beta-TC-6细胞,测定比较28天内胰岛素分泌量。第28天,共微囊化组分泌胰岛素含量为98.60 pg/mL。将两种微囊化细胞植入Ⅰ型糖尿病小鼠体内,监测比较其降血糖作用。结果表明,两者均能在28天内明显降低糖尿病小鼠的血糖,且共微囊化组具有更良好的效果,移植后的第28天,微囊化Beta-TC-6细胞组及共微囊化BMSCs/Beta-TC-6细胞组糖尿病小鼠血糖浓度分别为5.08 mmol/L和6.08 mmol/L。采用RT-PCR的方法检测细胞胰岛素mRNA表达水平,结果表明BMSCs与胰岛β细胞在微囊微环境中共培养后能在其诱导下在一定程度上分化为胰岛素分泌细胞。本研究证明了微胶囊可以为骨髓间充质干细胞诱导分化成成体细胞提供微环境,共微囊化骨髓间充质干细胞和体细胞用于移植治疗疾病具有潜在应用前景。
[Abstract]:Bone marrow mesenchymal stem cells (BMSCs) are abundant in origin, easy to isolate and purify, and have multi-directional differentiation potential. They can be transplanted by themselves. Microencapsulation has good immunoisolation and biocompatibility, which provides technical support for large-scale, high-activity in vitro culture and long-term preservation of stem cells. In this paper, calcium alginate-chitosan-calcium alginate (ACA) microcapsules loaded with BMSCs cells were prepared and their physical and chemical properties and biocompatibility were investigated. Metabolic properties of cells. Co-microencapsulated BMSCs/islet beta cells (Beta-TC-6) were used to treat type 1 diabetes mellitus and the mechanism was discussed at the molecular level.
Firstly, BMSCs cells were cultured in vitro and their growth curves were drawn. Then the surface antigens CD29, CD44 and CD117 were identified by immunocytochemical staining. The results showed that CD29, CD44 and CD117 were positive, CD44 were positive and CD117 were negative.
Secondly, the ACA microcapsules loaded with BMSCs cells were prepared by high-voltage electrostatic method. The optimized preparation process was as follows: sodium alginate concentration 1.75% (w/v), film forming time 10-min, cell density 1 x106 / mL. The prepared BMSCs-loaded microcapsules had good sphericity, smooth surface, mainly distributed between 300-400 microns and had little change with time within 7 days, and mechanical properties. BMSCs cells grew well after burbreaking.
Thirdly, the activity of microencapsulated BMSCs cells cultured in vitro (CCK-8 method) was investigated, and it was found that the microcapsules with liquefied core were more suitable for the long-term growth of cells than those with non-liquefied core and plate culture. The biocompatibility of the microcapsules in vivo was observed. The mice showed normal physiological activities, no toxic symptoms and stable weight gain. Within 28 days, the microcapsules were recovered. The surface of the microcapsules was smooth and spherical, and no fibrosis or macrophage aggregation was observed.
Then, Chang liver cells were cultured by plate, microencapsulation, co-culture and co-microencapsulation. The metabolic properties of hepatocytes were evaluated by the levels of albumin and urea. The results showed that the relationship between the amount of albumin and urea in the four groups was as follows: BMSCs/Changlive group BMSCs/Chang. The highest albumin secretion and urea secretion were 3.08 mg/mL and 3.75 mmol/L in the co-microencapsulated BMSCs/Changlive group within 15 days and 3.08 mg/mL respectively.
At last, we prepared microencapsulated islet beta cells (Beta-TC-6) and co-microencapsulated BMSCs/Beta-TC-6 cells, and measured and compared their insulin secretion in 28 days. On the 28th day, the content of insulin secreted by co-microencapsulated fraction was 98.60 pg/mL. Two kinds of microencapsulated cells were implanted into type I diabetic mice to monitor and compare their hypoglycemic effects. The blood glucose levels of diabetic mice in the microencapsulated Beta-TC-6 cell group and the co-microencapsulated BMSCs/Beta-TC-6 cell group were 5.08 mmol/L and 6.08 mmol/L respectively on the 28th day after transplantation. The results showed that BMSCs and islet beta cells could differentiate into insulin-secreting cells to some extent after co-culture in microcapsule microenvironment.
This study demonstrates that microcapsules can provide a microenvironment for the induction and differentiation of bone marrow mesenchymal stem cells into adult cells, and co-microencapsulated bone marrow mesenchymal stem cells and somatic cells have potential applications in transplantation therapy.
【学位授予单位】：华侨大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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