经血管内皮细胞生长因子刺激的骨髓间充质干细胞治疗急性放射性肠损伤的实验研究

发布时间：2019-06-29 14:40

【摘要】：第一部分SD大鼠骨髓间充质干细胞的分离、培养、纯化及其生物学特性鉴定 目的:SD大鼠BMSCs的体外分离、培养和纯化，以及生物学特性鉴定，为整个课题打下基础。 方法：联合采用密度梯度离心法和全骨髓贴壁培养法，分离培养和纯化BMSCs，同时显微镜下观察细胞生长状况。流式细胞仪鉴定细胞表面标记物，鉴定其向成骨、成脂分化潜能。 结果：分离培养的BMSCs呈梭形、大小均一，类似成纤维细胞样。流式结果显示BMSCs表面CD44、CD90阳性表达，而CD45阴性表达。P3代BMSCs可向成骨、成脂诱导分化。 结论：本部分实验成功分离培养了BMSCs，纯度高、形态均一，可用于之后的 相关实验研究。 第二部分VEGF刺激骨髓间充质干细胞后CXCR4表达的实验研究 目的：探讨BMSCs经VEGF刺激后对其归巢的影响 方法：将第一部分分离培养的BMSCs随机分成3组，A组：空白对照组，完全培养基培养；B组：在BMSCs的完全培养液中加入VEGF，使最终VEGF浓度为10ng/ml。C组：在BMCs的完全培养液中加入VEGF，使最终VEGF浓度为50ng/ml。流式细胞分别测量刺激48h后各组胞膜和胞膜内CXCR4的表达。 结果：各组BMSCs胞膜和胞膜内均有CXCR4表达，且胞膜内高于胞膜的表达，其中C组表达均最高，P0.05 结论：1、BMSCs和经VEGF刺激的BMSCs胞膜和胞膜内均有CXCR4表达；2、VEGF刺激BMSCs后，胞膜和胞膜内CXCR4表达增高；且50ng/ml浓度下表达最高； 第三部分SDF-1在放射性损伤的肠组织中的表达 目的：1、建立急性放射性肠损伤的模型；2、进一步探讨VEGF对BMSCs的归巢影响。 方法：90只雌性的SD大鼠随机分成3组（每组30只），即对照组（A组）、BMSCs治疗组（B组）和经VEGF刺激的BMSCs的治疗组（C组）。B组和C组在照射后4小时内通过尾静脉注射1×106/ml的雄性大鼠BMSCs悬液1ml及经50ng/ml的VEGF刺激48小时后的BMSCs悬液1ml；A组于照射后4小时内尾静脉输注生理盐水1ml。造模后第1、3、7、14、21天每组随机处死6只大鼠，留取全血与两份末端回肠标本。显微镜下观察肠道组织结构，测量绒毛高度及隐窝深度；ELISA法测量血清中瓜氨酸和VEGF的浓度。免疫组化SP法检测造模后3天肠组织SDF-1的表达。 结果：1、成功建立了放射性肠损伤模型；造模后第3、7、14天，C组大鼠精神状态，运动量，，饮食量，排泄状况均优于A组、B组，而造模后第1、21天三组大鼠之间无明显差异。HE染色结果显示，造模后第3、7、14天，C组肠道结构较A、B组完整，肠粘膜上皮细胞坏死少，伴有的炎性细胞较少，而腺体结构却较丰富。通过测量肠道绒毛高度和隐窝深度评估肠道功能，造模后第3、7、14天，同一时期绒毛高度和隐窝深度，CBA，P0.05有统计学差异。造模后第1、21天，同一时期绒毛高度和隐窝深度，各组无统计学差异，P0.05. 2、免疫组化SP法测量各组受损肠组织SDF-1的表达量，发现C组SDF-1的表达量明显高于A、B组，且B组表达量高于A组。 结论：1、直线加速器建立放射性肠损伤模型的剂量为12Gy； 2、放射性肠损伤可以自我修复，周期在2～3周左右； 3、经VEGF刺激的BMSCs能够促进急性放射性肠损伤的修复，能提高其修复的效率，但从远期效果来看无明显差异； 4、经VEGF刺激的BMSCs能通过提高BMSCs的归巢率促进损伤肠道修复，机制与SDF-1/CXCR4轴有关。
[Abstract]:Isolation, Culture, Purification and Biological Characterization of Bone Marrow Mesenchymal Stem Cells from SD Rat Objective: To study the isolation, culture and purification of BMSCs in SD rats, and to identify the biological characteristics and lay a foundation for the whole project. Methods: BMSCs were cultured and purified by density gradient centrifugation and full-bone marrow adherent culture, and the cells were observed under the microscope. Long condition. The cell surface marker was identified by flow cytometry to identify its osteogenic, lipid-forming score. Results: BMSCs isolated and cultured were in the form of a shed, and the size of BMSCs was similar to that of the cultured BMSCs. Fibroblast-like. The results showed that the expression of CD44 and CD90 on the surface of BMSCs was positive. 5 negative expression. The P3 generation BMSCs can be formed into an osteogenic, Conclusion: BMSCs were successfully isolated and cultured in this part. I. can be used after The second part of VEGF stimulates the bone marrow mesenchymal stem cells. The purpose of the study on the expression of CXCR4: to investigate the expression of BMSCs The effect of VEGF on the homing of BMSCs was divided into 3 groups and group A: empty. White control group, complete medium culture; Group B: VEGF was added to the complete culture solution of BMSCs to make the final VEGF concentration of 10 ng/ ml. Group C: VEGF was added to the complete culture solution of BMCs to make the most The final VEGF concentration was 50 ng/ ml. The results showed that the expression of CXCR4 in the cell membrane and the cell membrane of each group was higher than that of the cell membrane in the cell membrane. The expression of C group was the highest, and the expression of the C group was the highest, and the expression of CXCR4 was found in the cells of BMSCs and the membrane of BMSCs stimulated by VEGF. The expression of CXCR4 in the cell membrane and the membrane after Cs up-to-date; with the highest expression at 50 ng/ ml; and Expression of three-part SDF-1 in a radiation-induced intestinal tissue:1. Establishment of a model of acute radiation-induced intestinal injury 2. The effect of VEGF on the homing of BMSCs was further discussed. Methods:90 female SD rats were randomly divided into three groups (30 rats in each group), namely, the control group (group A) and the BMSCs treatment group (group A). Group B) and the treatment group (group C) of BMSCs stimulated with VEGF. The BMSCs suspension 1 ml and the BMSCs suspension 1 after 48 hours were stimulated with 1 ml of 1-106/ ml of BMSCs and 50 ng/ ml of VEGF in 4 hours after irradiation. ml; in group A,1 ml of normal saline was infused in the tail vein after 4 hours of irradiation. The first, third, 7th, 14th, and 21st days after the model were made In each group,6 rats were sacrificed at random, and whole blood and two terminal ileal specimens were taken. The structure of the intestinal tissue was observed under the microscope, and the height of the villi and the crypt were measured. The concentration of citrulline and VEGF in serum was measured by ELISA. The expression of SDF-1 in 3 days after the model was detected by SP method. Results:1. The model of intestinal injury was established successfully. The mental state, amount of exercise, amount of diet and excretion of group C rats were superior to those of group C in group 3,7,14 and C after the model. In group A and group B, there was no significant difference between group A and group B in group A and group B. The necrosis of the epithelial cells is small, with less inflammatory cells, and the glandular structure is rich. The intestinal function is evaluated by measuring the height of the intestinal villus and the depth of the crypt, and the third, the 7th and the 14th day of the model are the same. There was a statistical difference between the height of the villi and the depth of the crypt, the CBA and P05. The first and the 21st days after the model were the same. The expression of SDF-1 in the damaged intestinal tissue was measured by immunohistochemical SP method, and the expression of SDF-1 was measured by immunohistochemical SP method. The expression of SDF-1 in group B was significantly higher than that of group A and group B, and the expression of group B was higher than that of group A. Conclusion:1. The linear accelerator was used to establish the model of the radiation-induced intestinal injury. the amount is 12 Gy;2, the injury of the radioactive intestine can be self-repaired, the period is about 2-3 weeks; and 3, through the VE, GF-stimulated BMSCs can promote The repair of acute radioactive intestinal injury can improve the efficiency of the repair, but there is no significant difference from the long-term effect;4, the BMSCs stimulated by VEGF can pass through
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R818

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