【摘要】：目的:探討VEGF和Ang1雙基因聯(lián)合BMSCs干預(yù)對(duì)PVL模型小鼠NVU的保護(hù)作用。方法:本研究分兩部分。第一部分:缺血缺氧誘導(dǎo)新生鼠PVL模型的建立及腦組織VEGF和Ang1水平變化選取7日齡昆明小鼠50只,隨機(jī)分為模型組和假手術(shù)組,模型組30只,假手術(shù)組20只,模型組通過結(jié)扎單側(cè)頸總動(dòng)脈并在低氧環(huán)境下(6%O2+94%N2)飼養(yǎng)6小時(shí)的方法構(gòu)建PVL新生小鼠模型,假手術(shù)組行同側(cè)頸總動(dòng)脈分離術(shù)后縫合手術(shù)切口,于造模后24小時(shí)、48小時(shí)、72小時(shí)和7天分別處死兩組小鼠取腦組織,4%多聚甲醛固定后行HE染色、免疫組化染色等鑒定模型是否成功,同時(shí)通過RT-PCR檢測腦組織VEGF和Ang1的水平變化,了解其與腦組織病理改變之間的關(guān)系。第二部分:攜帶VEGF和Ang1基因的BMSCs對(duì)缺血缺氧誘導(dǎo)PVL小鼠模型的干預(yù)研究將100只造模成功后的PVL新生小鼠隨機(jī)分為BMSCs-VEGF、BMSCsAng1、BMSCs-A+V、BMSCs、模型鹽水對(duì)照5組,每組20只,另外選取同日齡新生小鼠20只進(jìn)行頸總動(dòng)脈分離后設(shè)為空氣對(duì)照組。每組小鼠于造模后24小時(shí)內(nèi)在立體定位儀下側(cè)腦室內(nèi)注入相應(yīng)干預(yù)因子,分別于干預(yù)后24小時(shí)、48小時(shí)、72小時(shí)和7天處死兩組小鼠取腦組織,4%多聚甲醛固定后行HE染色、免疫組化染色等觀察腦組織病理改變、VEGF和Ang1表達(dá)情況,同時(shí)做MBP免疫組化了解髓鞘化水平,同時(shí)通過RT-PCR檢測腦組織VEGF和Ang1 m RNA的水平變化。結(jié)果:第一部分:應(yīng)用單側(cè)頸總動(dòng)脈結(jié)扎結(jié)合低氧誘導(dǎo)的方法制作新生小鼠PVL模型,模型組動(dòng)物體重增長緩慢,毛發(fā)粗糙凌亂無光澤,腦組織大體標(biāo)本見軟化灶形成,逐漸液化壞死形成囊腔,HE染色提示模型組神經(jīng)細(xì)胞大小不等,核固縮、碎裂,證實(shí)PVL模型動(dòng)物存在腦組織病理學(xué)的改變。RT-PCR顯示術(shù)后模型組小鼠手術(shù)復(fù)蘇后腦組織VEGF和Ang1表達(dá)逐漸增加,48小時(shí)后VEGF和Ang1 mRNA水平均高于假手術(shù)組。免疫組化提示兩組小鼠VEGF和Ang1表達(dá)均隨時(shí)間增加,但模型組平均光密度值明顯高于假手術(shù)組(P0.05)。第二部分:1、BMSCs-V+A組及BMSCs-Ang1組小鼠在麻醉復(fù)蘇后體重增長較理想,病理學(xué)檢查證實(shí)模型鼠腦損傷較其他組減輕明顯,BMSCs和BMSCsVEGF組體重增長較慢,與模型組相比無統(tǒng)計(jì)學(xué)差異(P0.05),腦組織病理學(xué)改善不明顯,與模型對(duì)照組比較無統(tǒng)計(jì)學(xué)差異。2、免疫組化:BMSCs-V+A組和BMSCs-VEGF組VEGF平均光密度值增加,但BMSCs-VEGF組MBP平均光密度值與模型對(duì)照組無差異(P0.05)。BMSCs-V+A組、BMSCs-Ang1組及BMSCs組Ang1和MBP平均光密度值均增加,與模型對(duì)照組相比有顯著差異(P0.05),BMSCs-V+A組MBP表達(dá)與空氣組接近,無統(tǒng)計(jì)學(xué)差異(P0.05)。3、RT-PCR:BMSCs-VEGF組和BMSCs-V+A組VEGF mRNA水平迅速升高,與模型對(duì)照組差異顯著(P0.05),BMSCs-V+A組和BMSCs-Ang1組Ang1mRNA水平顯著升高,與模型對(duì)照組差異顯著(P0.05)。結(jié)論:1、采用缺血缺氧誘導(dǎo)的方法可以建造理想的新生小鼠PVL模型,模型組小鼠腦組織VEGF和Ang1水平在損傷早期均升高。2、聯(lián)合應(yīng)用攜帶VEGF和Ang1基因的BMSCs可以有效的修復(fù)受損的NVU,保護(hù)神經(jīng)細(xì)胞,促進(jìn)神經(jīng)纖維髓鞘化,減輕PVL腦損傷,從而為PVL的有效干預(yù)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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R742

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