缺血损伤时骨髓间充质干细胞在种间归巢能力的实验研究
发布时间：2018-11-14 14:02
【摘要】：目的：研究缺血损伤时骨髓间充质干细胞(bone Mesenchymal stem cells, BMSCs)在种间的归巢能力。 方法：1.EGFP-BMSCs|的分离培养及纯度鉴定：贴壁培养法扩增增强型绿色荧光蛋白(enhanced green fluorescent protein, EGFP)转基因C57BL/6小鼠的EGFP-BMSCs,体外传代至第5代,并行流式细胞学鉴定EGFP-BMSCs的纯度。 2.缺血损伤模型的建立：受鼠麻醉后,双下肢大腿外侧剪毛消毒,右侧股部经前外侧切口切开,显露、分离股四头肌并用1号丝线结扎近、远端,止血关闭切口。左侧切开皮肤,股四头肌不分离不结扎,作为对照,止血后关闭切口。其他小鼠同法建立缺血模型,大鼠缺血模型分离股四头肌外侧头结扎。 3.消化相同供鼠(EGFP转基因小鼠)的BMSCs,计数并调整浓度为2×106个/ml,取0.1ml经尾静脉注入受鼠。分别于第1天、4天、7天、14天、28天处死受鼠,取右侧缺血肌肉模型及对侧肌肉组织,提取总蛋白,进行Western blotting检测EGFP,扫描并做灰度分析,统计分析。 结果：1.经Western blotting检测EGFP,缺血侧有EGFP表达,说明异体EGFP-BMSCs植入受鼠后,EGFP-BMSCs可归巢到缺血组织。 2.经统计分析各时间段种间缺血侧的灰度值无差异,P0.05,无统计学意义,表明种间EGFP-BMSCs归巢没有差异,异种EGFP-BMSCs具有低免疫原性。 结论：1BMSCs可以归巢到损伤组织。 2.利用EGFP转基因小鼠的BMSCs可以很好的示踪BMSCs的归巢。 3. BMSCs种间归巢没有差异,BMSCs具有低免疫原性。 4. BMSCs归巢是一种以主动运动为主的迁移。
[Abstract]:Aim: to study the homing ability of bone marrow mesenchymal stem cells (bone Mesenchymal stem cells, BMSCs) during ischemic injury. Methods: isolation, culture and purity identification of 1.EGFP-BMSCs: the EGFP-BMSCs, of (enhanced green fluorescent protein, EGFP) transgenic C57BL/6 mice with enhanced green fluorescent protein (GFP) was amplified by adherent culture method and was subcultured to the 5th generation in vitro. The purity of EGFP-BMSCs was identified by flow cytology. 2. The establishment of ischemic injury model: after anaesthesia, the lateral thigh shearing of both lower limbs was disinfected, the right thigh was cut and exposed through the anterolateral incision, the quadriceps femoris was separated and ligated, the distal side was ligated and the incision was closed by hemostasis. Left incision skin, quadriceps femoris not separated and not ligated, as a control, hemostasis closed incision. The ischemia model was established by the same method in other mice, and the lateral head of quadriceps femoris was isolated from the ischemic model of rats. 3. The BMSCs, count of the same donor mice (EGFP transgenic mice) was digested and the concentration of 0.1ml was adjusted to 2 脳 106 / ml,. 0.1ml was injected into recipient mice via tail vein. The recipient rats were killed on day 1, day 4, day 7, day 14 and day 28, respectively. The right ischemic muscle model and contralateral muscle tissue were taken, total protein was extracted, EGFP, scan was performed by Western blotting and gray scale analysis was performed. Results: 1. The expression of EGFP in ischemic side of EGFP, was detected by Western blotting, indicating that EGFP-BMSCs could homing to ischemic tissue after allogeneic EGFP-BMSCs implantation. 2. Statistical analysis showed that there was no difference in grayscale value of ischemic side between different species and there was no significant difference in homing of EGFP-BMSCs between species and in different species EGFP-BMSCs was low immunogenicity. Conclusion: 1BMSCs can homing to damaged tissue. 2. The BMSCs of EGFP transgenic mice can well trace the homing of BMSCs. 3. There was no difference in homing among BMSCs species, and BMSCs had low immunogenicity. 4. BMSCs homing is an active migration.
【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
本文编号：2331363
[Abstract]:Aim: to study the homing ability of bone marrow mesenchymal stem cells (bone Mesenchymal stem cells, BMSCs) during ischemic injury. Methods: isolation, culture and purity identification of 1.EGFP-BMSCs: the EGFP-BMSCs, of (enhanced green fluorescent protein, EGFP) transgenic C57BL/6 mice with enhanced green fluorescent protein (GFP) was amplified by adherent culture method and was subcultured to the 5th generation in vitro. The purity of EGFP-BMSCs was identified by flow cytology. 2. The establishment of ischemic injury model: after anaesthesia, the lateral thigh shearing of both lower limbs was disinfected, the right thigh was cut and exposed through the anterolateral incision, the quadriceps femoris was separated and ligated, the distal side was ligated and the incision was closed by hemostasis. Left incision skin, quadriceps femoris not separated and not ligated, as a control, hemostasis closed incision. The ischemia model was established by the same method in other mice, and the lateral head of quadriceps femoris was isolated from the ischemic model of rats. 3. The BMSCs, count of the same donor mice (EGFP transgenic mice) was digested and the concentration of 0.1ml was adjusted to 2 脳 106 / ml,. 0.1ml was injected into recipient mice via tail vein. The recipient rats were killed on day 1, day 4, day 7, day 14 and day 28, respectively. The right ischemic muscle model and contralateral muscle tissue were taken, total protein was extracted, EGFP, scan was performed by Western blotting and gray scale analysis was performed. Results: 1. The expression of EGFP in ischemic side of EGFP, was detected by Western blotting, indicating that EGFP-BMSCs could homing to ischemic tissue after allogeneic EGFP-BMSCs implantation. 2. Statistical analysis showed that there was no difference in grayscale value of ischemic side between different species and there was no significant difference in homing of EGFP-BMSCs between species and in different species EGFP-BMSCs was low immunogenicity. Conclusion: 1BMSCs can homing to damaged tissue. 2. The BMSCs of EGFP transgenic mice can well trace the homing of BMSCs. 3. There was no difference in homing among BMSCs species, and BMSCs had low immunogenicity. 4. BMSCs homing is an active migration.
【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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