同种异体EGFP-BMSCs在小鼠缺血肌袋存活和归巢能力的实验研究

发布时间：2018-07-08 13:17

本文选题：骨髓间充质干细胞 + 缺血肌袋　；参考：《昆明医学院》2011年硕士论文

【摘要】：目的：1、建立培养增强型绿色荧光蛋白(enhanced green fluorescent protein, EGFP)转基因C57BL/6雄性小鼠骨髓间充质干细胞(bone Mesenchymal stem cells, BMSCs)的分离培养方法。 2、研究同种异体骨髓间充质干细胞(bone Mesenchymal stem cells, BMSCs)在同种小鼠缺血损伤肌袋内定植存活及向缺血损伤肌袋归巢的能力。方法：1、EGFP-BMSCs勺分离培养和纯度鉴定：贴壁法培养扩增增强型绿色荧光蛋白(enhanced green fluorescent protein, EGFP)转基因C57BL/6雄性小鼠的EGFP-BMSCs,体外扩增传代至第5代,行流式细胞学鉴定EGFP-BMSCs纯度。 2、动物分组及模型建立：取C57BL/6小鼠90只,随机分为三组,每组30只：A组(股四头肌缺血肌袋模型局部注射组)：制作小鼠右侧股四头肌缺血肌袋模型(游离股四头肌,结扎其远近端,下同),注入同种异体浓度为2x106个/ml的EGFP-BMSCs悬液0.01ml(下同),左侧股四头肌肌袋注入0.01ml生理盐水做为对照；B组(正常股四头肌肌袋模型局部注射组)：暴露小鼠双侧股四头肌,取0.01mlEGFP-BMSCs注入小鼠右侧股四头肌肌袋内,取0.01ml生理盐水注入左侧股四头肌肌袋做为对照；C组(股四头肌缺血肌袋模型尾静脉注射组)：暴露小鼠双侧股四头肌,制作小鼠右侧股四头肌缺血肌袋,经尾静脉注射0.01mlEGFP-BMSCs。 3、术后一天,四天,一周,两周,四周随机处死A、B、C三组各6只受体小鼠,分别获取各组小鼠双侧肌袋组织,提取不同时间点不同的肌袋组织蛋白行Western-blot检测,计算其灰度比值,并做统计分析。原代培养,经过换液和传代,可以获得大量较纯的EGFP-BMSCs。 2、注入同种异体EGFP-BMSCs后,宿主C57BL/6小鼠均无明显异常反应。 3、术后一天、四天、一周、两周、四周时处死动物并提取A、B、C三组双侧股四头肌肌袋蛋白,Western-blot检测结果显示A组、B组和C组的右侧肌袋内不同时间段均可以检测到增强型绿色荧光蛋白(EGFP),以术后一天最多,但随时间延长而逐渐减少。 4、三组对照侧左侧不同时间段肌袋标本内均未检测到EGFP。 5、统计分析：用Quantity one软件分析EGFP在不同时间局部缺血注射组、局部未缺血注射组和全身注射组的Western blot条带,所得各数据经SPSS16.0统计学软件进行统计学分析。结论：1、全骨髓贴壁法可以从骨髓中成功分离培养出EGFP转基因C57BL/6小鼠的EGFP-BMSCs. 2、通过结扎股四头肌可成功建造小鼠缺血肌袋模型。 3、宿主对同种异体EGFP-BMSCs无明显免疫排斥反应。 4、局部注射同种异体EGFP-BMSCs可在宿主局部肌袋存活4周以上；局部注射EGFP-BMSCs有局部富集现象,可作为干细胞治疗的一条可行途径。 5、尾静脉注射(全身注射)EGFP-BMSCs能通过非血管途径归巢至宿主缺血损伤肌袋,并能定植存活4周以上,但随时间延长EGFP-BMSCs量逐渐减少。 6、归巢至小鼠缺血肌袋的EGFP-BMSCs可能参与损伤修复及血管的形成。
[Abstract]:Objective to establish a method for isolation and culture of bone mesenchymal stem cells (BMSCs) from C57BL / 6 male mice transgenic with enhanced green fluorescent protein (enhanced green fluorescent protein, EGFP). (2) to study allogeneic bone marrow mesenchymal stem cells (BMSCs). The ability of bone mesenchymal stem cells (BMSCs) to survive and homing to ischemic injury muscle pouch in allogeneic mice. Methods the EGFP-BMSCs were isolated, cultured and purified. EGFP-BMSCs of C57BL / 6 transgenic C57BL / 6 male mice were amplified by adherent method and then subcultured to the fifth generation in vitro. The purity of EGFP-BMSCs was determined by flow cytometry, and the animal groups and models were established. Ninety C57BL / 6 mice were randomly divided into three groups. 30 rats in each group (local injection group of quadriceps femoris ischemia muscle pouch model): the right quadriceps femoris ischemia muscle pouch model (free quadriceps femoris, ligation of its far and near end), In the same way, EGFP-BMSCs suspension (0.01ml) was injected with allogenic concentration of 2x106 / ml, and the left quadriceps muscle bag was injected with 0.01ml normal saline as control group B (normal quadriceps muscle pouch model local injection group): bilateral quadriceps muscle was exposed to mice. 0.01 ml EGFP-BMSCs were injected into the right quadriceps muscle pouch of mice, and 0.01ml saline was injected into the left quadriceps muscle bag as control group C (caudal vein injection group): bilateral quadriceps muscle was exposed. The right quadriceps femoris muscle ischemic muscle bag was made in mice, and 0.01ml EGFP-BMSCs.3 was injected into caudal vein. One day, four days, one week, two weeks, and four weeks after operation, the mice in each group were killed at random. The tissue proteins of muscle bags were extracted at different time points and detected by Western-blot. The grayscale ratio was calculated and analyzed statistically. After primary culture, a large number of pure EGFP-BMSCs 路2 could be obtained by medium exchange and passage. After injection of EGFP-BMSCs, host C57BL / 6 mice had no obvious abnormal response. 3, one day, four days, one week, two weeks after operation, The results of Western-blot analysis showed that enhanced green fluorescent protein (EGFP) could be detected in the right muscle pouch of group A and group C at different time points, the most of which was on the first day after operation, and the results of Western-blot analysis showed that the enhanced green fluorescent protein (EGFP) could be detected at different time points in the right muscle pocket of group A and C. However, with the prolongation of time, EGFP was decreased gradually. 4. EGFP was not detected in the muscle bag specimens of the left side of the three groups at different time points. Statistical analysis: the quantitative one software was used to analyze EGFP in the different time ischemic injection group. All the data were analyzed by SPSS 16.0 software. Conclusion the EGFP transgenic C57BL / 6 mouse EGFP-BMSCs.2 can be successfully isolated from bone marrow by whole bone marrow adherent method. By ligating quadriceps femoris, the ischemic muscle bag model of mice can be successfully constructed. 4. Local injection of EGFP-BMSCs could survive for more than 4 weeks. Local injection of EGFP-BMSCs can be a feasible way to treat stem cells. 5. Caudal vein injection (whole body injection) EGFP-BMSCs can homing to host ischemic injury muscle bag through non-vascular pathway, and can be colonized for more than 4 weeks. However, the number of EGFP-BMSCs gradually decreased with the prolongation of time. The EGFP-BMSCs homing to the ischemic muscle pouch of mice may participate in the repair of injury and the formation of blood vessels.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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