移植的人骨髓基质细胞在体内分布与安全性评价的实验研究

发布时间：2018-12-27 18:20

【摘要】： 目的在建立人骨髓基质细胞培养体系基础上,观察人骨髓基质细胞在裸鼠体内的分布情况和对脏器的影响,为人骨髓基质细胞作为干细胞移植的前期研究和安全性提供实验依据。方法采用密度梯度离心结合差速贴壁法分离培养人骨髓基质细胞,通过细胞形态学、表型特征对成人骨髓来源的基质细胞进行鉴定;采用细胞生长曲线分析和成骨诱导分化分析其体外增殖特性和分化潜能。而后建立人骨髓基质细胞静脉移植的裸鼠模型,观察受体裸鼠的一般情况,通过组织学分析、RT-PCR检测人Alu基因和CD45基因等技术手段,研究移植的骨髓基质细胞体内的分布情况和对脏器的影响。此外,还进行安全性检测,包括长期传代培养的观察和成瘤性实验检测。结果体外培养的人骨髓基质细胞为成纤维样细胞,细胞紧密排列呈漩涡状生长;流式细胞术检测CD34、CD45为阴性表达;细胞生长曲线提示第1至第5代人骨髓基质细胞增殖速度无显著性差异(P＞0.05);成骨诱导18天后,茜素红染色可见橙红色钙结节。在裸鼠移植模型中,输注第4代体外培养的人骨髓基质细胞后,受体裸鼠一般状况良好,解剖大体观、HE染色、瑞氏染色观察受体组织结构无异常。RT-PCR检测人Alu基因、CD45基因在不同移植时间相应脏器均有表达。此外,细胞长期传代后出现衰老征象,而无明显的成瘤特性。结论采用密度梯度离心结合差速贴壁法分离、DMEM-LG-10%FBS培养液可成功分离培养含有间充质干细胞的人骨髓基质细胞群。经静脉输注后,在不同移植时间检测,发现受体内各脏器均有分布,但进一步作安全检测发现移植的骨髓基质细胞尚无明显的恶变趋势和现象发生。
[Abstract]:Objective To establish a human bone marrow stromal cell culture system, observe the distribution of human bone marrow stromal cells in nude mice and the effect on the organs, and provide experimental basis for the early research and safety of human bone marrow stromal cells as stem cell transplantation. Methods The human bone marrow stromal cells were isolated and cultured by density gradient centrifugation and differential adhesion method. The matrix cells of adult bone marrow were identified by cell morphology and phenotypic characteristics. The proliferation and differentiation potential of human bone marrow stromal cells were analyzed by cell growth curve and bone-induced differentiation. Then, a nude mouse model of human bone marrow stromal cell vein transplantation was established, and the general situation of the receptor naked mice was observed. By means of histological analysis, RT-PCR, human Alu gene and CD45 gene were used to study the distribution of bone marrow stromal cells and the effect on the organs. In addition, safety detection is carried out, including the observation of long-term subculture and the test of tumorigenicity. Results The human bone marrow stromal cells cultured in vitro were fibroblast-like cells, and the cells were closely arranged in a vortex-like growth. The expression of CD34 and CD45 was detected by flow cytometry. The growth curve of the cells showed no significant difference in the proliferation of bone marrow stromal cells from the 1st to 5th generation (P> 0.05). After 18 days of bone-induced bone induction, the red-red calcium nodules were seen in the red-red staining. In the nude mouse transplantation model, after the fourth generation of the human bone marrow stromal cells cultured in vitro, the receptor naked mice were generally in good condition, the general view of the anatomy, the HE staining, and the Rechsler's staining showed no abnormality in the structure of the receptor. The expression of the human Alu gene and the CD45 gene in the corresponding organs of the human Alu gene was detected by RT-PCR. In addition, the cells showed signs of aging after long-term passage without obvious tumor characteristics. Conclusion The cell population of human bone marrow stromal cells containing mesenchymal stem cells can be isolated and cultured in DMEM-LG-10% FBS culture medium by means of density gradient centrifugation and differential adhesion. After intravenous infusion, the internal organs of the transplanted bone marrow were found to be distributed in different time, but there was no obvious tendency and phenomenon of malignant transformation in the bone marrow stromal cells of the transplanted bone marrow.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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