携带VEGF和Ang1基因的BMSCs对缺血缺氧诱导新生鼠PVL模型的干预研究

发布时间：2019-06-27 19:14

【摘要】：目的:探讨VEGF和Ang1双基因联合BMSCs干预对PVL模型小鼠NVU的保护作用。方法:本研究分两部分。第一部分:缺血缺氧诱导新生鼠PVL模型的建立及脑组织VEGF和Ang1水平变化选取7日龄昆明小鼠50只,随机分为模型组和假手术组,模型组30只,假手术组20只,模型组通过结扎单侧颈总动脉并在低氧环境下(6%O2+94%N2)饲养6小时的方法构建PVL新生小鼠模型,假手术组行同侧颈总动脉分离术后缝合手术切口,于造模后24小时、48小时、72小时和7天分别处死两组小鼠取脑组织,4%多聚甲醛固定后行HE染色、免疫组化染色等鉴定模型是否成功,同时通过RT-PCR检测脑组织VEGF和Ang1的水平变化,了解其与脑组织病理改变之间的关系。第二部分:携带VEGF和Ang1基因的BMSCs对缺血缺氧诱导PVL小鼠模型的干预研究将100只造模成功后的PVL新生小鼠随机分为BMSCs-VEGF、BMSCsAng1、BMSCs-A+V、BMSCs、模型盐水对照5组,每组20只,另外选取同日龄新生小鼠20只进行颈总动脉分离后设为空气对照组。每组小鼠于造模后24小时内在立体定位仪下侧脑室内注入相应干预因子,分别于干预后24小时、48小时、72小时和7天处死两组小鼠取脑组织,4%多聚甲醛固定后行HE染色、免疫组化染色等观察脑组织病理改变、VEGF和Ang1表达情况,同时做MBP免疫组化了解髓鞘化水平,同时通过RT-PCR检测脑组织VEGF和Ang1 m RNA的水平变化。结果:第一部分:应用单侧颈总动脉结扎结合低氧诱导的方法制作新生小鼠PVL模型,模型组动物体重增长缓慢,毛发粗糙凌乱无光泽,脑组织大体标本见软化灶形成,逐渐液化坏死形成囊腔,HE染色提示模型组神经细胞大小不等,核固缩、碎裂,证实PVL模型动物存在脑组织病理学的改变。RT-PCR显示术后模型组小鼠手术复苏后脑组织VEGF和Ang1表达逐渐增加,48小时后VEGF和Ang1 mRNA水平均高于假手术组。免疫组化提示两组小鼠VEGF和Ang1表达均随时间增加,但模型组平均光密度值明显高于假手术组(P0.05)。第二部分:1、BMSCs-V+A组及BMSCs-Ang1组小鼠在麻醉复苏后体重增长较理想,病理学检查证实模型鼠脑损伤较其他组减轻明显,BMSCs和BMSCsVEGF组体重增长较慢,与模型组相比无统计学差异(P0.05),脑组织病理学改善不明显,与模型对照组比较无统计学差异。2、免疫组化:BMSCs-V+A组和BMSCs-VEGF组VEGF平均光密度值增加,但BMSCs-VEGF组MBP平均光密度值与模型对照组无差异(P0.05)。BMSCs-V+A组、BMSCs-Ang1组及BMSCs组Ang1和MBP平均光密度值均增加,与模型对照组相比有显著差异(P0.05),BMSCs-V+A组MBP表达与空气组接近,无统计学差异(P0.05)。3、RT-PCR:BMSCs-VEGF组和BMSCs-V+A组VEGF mRNA水平迅速升高,与模型对照组差异显著(P0.05),BMSCs-V+A组和BMSCs-Ang1组Ang1mRNA水平显著升高,与模型对照组差异显著(P0.05)。结论:1、采用缺血缺氧诱导的方法可以建造理想的新生小鼠PVL模型,模型组小鼠脑组织VEGF和Ang1水平在损伤早期均升高。2、联合应用携带VEGF和Ang1基因的BMSCs可以有效的修复受损的NVU,保护神经细胞,促进神经纤维髓鞘化,减轻PVL脑损伤,从而为PVL的有效干预提供了新的思路。
[Abstract]:Objective: To study the protective effect of VEGF and Ang1 double gene (BMSCs) on the NU in the PVL model mice. Method: This study is divided into two parts. The first part: the establishment of the PVL model and the change of the level of VEGF and Ang1 in the brain tissue of the neonatal rats were randomly divided into two groups: model group and sham-operation group,30 in the model group and 20 in the sham operation group. In the model group, PVL new mouse model was constructed by ligation of one-sided common carotid artery and in a low-oxygen environment (6% O2 + 94% N2) for 6 hours. After 24 hours,48 hours,72 hours and 7 days after the establishment of the model, the two groups of mice were sacrificed to take brain tissue,4% paraformaldehyde was fixed, HE staining and immunohistochemical staining were performed to determine whether the model was successful, and the level of VEGF and Ang1 in brain tissue was detected by RT-PCR. To understand the relationship between the pathological changes of the brain and the brain. The second part: BMSCs carrying the VEGF and the Ang1 gene were randomly divided into BMSCs-VEGF, BMSC sang1, BMSCs-A + V, BMSCs and model saline control group 5 with the intervention of the BMSCs carrying the VEGF and the Ang1 gene in the ischemia-hypoxia-induced PVL mouse model. In addition,20 newborn mice of the same day were selected to be divided into the air control group only after the common carotid artery was separated. in each group, the corresponding intervention factors were injected into the lateral ventricle of the lateral ventricle of the three-dimensional positioning device within 24 hours after the model, respectively, and the two groups of mice were sacrificed at 24 hours,48 hours,72 hours and 7 days after the intervention, and the two groups of mice were sacrificed to obtain the brain tissue, and the HE staining was performed after the fixation of 4% paraformaldehyde, The pathological changes of brain tissue, the expression of VEGF and Ang1 were observed by immunohistochemical staining and the expression of VEGF and Ang1 was studied by immunohistochemistry, and the level of VEGF and Ang1 mRNA was detected by RT-PCR. Results: The first part: The PVL model of the new mouse was made by the method of single-sided common carotid artery ligation combined with the hypoxia-induced method, the body weight of the model group was slow, the hair was rough and unsmooth, the general specimen of the brain tissue was found in the softening range to form the capsule cavity, The pathological changes of the brain tissue of the PVL model animals were confirmed by HE staining. RT-PCR showed that the expression of VEGF and Ang1 in the brain after the post-operation of the model group was gradually increased, and the level of VEGF and Ang1 mRNA was higher in the 48-hour group than in the sham-operated group. The expression of VEGF and Ang1 in both groups increased with time, but the mean optical density of the model group was significantly higher than that of the sham-operated group (P0.05). The second part:1, BMSCs-V + A group and BMSCs-Ang1 group mice body weight increased after the anesthesia recovery, the pathological examination confirmed that the model rat brain injury was less than that of the other groups, the body weight of the BMSCs and the BMSC sVEGF group is slower, the body weight of the BMSCs and the BMSC sVEGF group is slower, the body weight of the BMSCs and the BMSC sVEGF group is not statistically different (P0.05), and the pathological improvement of the brain tissue is not obvious, The mean optical density of the BMSCs-V + A group and the BMSCs-VEGF group increased, but the average optical density of the BMSCs-V + A group, the BMSCs-Ang1 group and the BMSCs group Ang1 and MBP increased, Compared with the model control group, the expression of VEGF mRNA in BMSCs-V + A group and BMSCs-V + A group was significantly higher than that of the control group (P0.05). The expression of VEGF mRNA in BMSCs-V + A group and BMSCs-V + A group was significantly higher than that of the control group (P0.05), and the Ang1 mRNA levels in BMSCs-V + A and BMSCs-Ang1 group were significantly increased. The difference between the control group and the control group was significant (P0.05). Conclusion:1. The expression of VEGF and Ang1 in the brain tissue of the model group can be increased in the early stage of the injury by using the method of ischemia-hypoxia induction. It is a new way for the effective intervention of PVL to promote the myelopathy of the nerve fibers and to reduce the brain damage of the PVL.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R742

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