Ad-HGF感染RMMC培养上清液对大鼠CAEC的作用研究

发布时间：2018-05-20 06:33

本文选题：肝细胞生长因子 + 脑动脉内皮细胞　；参考：《新乡医学院》2015年硕士论文

【摘要】：背景血脑屏障主要是由脑动脉内皮细胞(Cerebral artery endothelial cell,CAEC)构成,后者常见于机体的生理活动中,如细胞或组织的相关合成及代谢功能,并分泌某种细胞因子及生物活性物质,是参与各种生理、病理活动的相关作用细胞。越来越多的研究显示肝细胞生长因子(Hepatocyte growth factor, HGF)具有相关神经营养以及促神经生长,促进微小血管及轴突的发生、并能减少瘢痕的形成以及作用于脑室相关管膜下区(Subventricular zone,SVZ)的神经干细胞,并使其增殖、分化的作用。TGF-β1是一种确保血脑屏障完整的重要因子,肝细胞生长因子与其的作用效应根据细胞的外环境以及细胞内环境的相关活性因子的不同而不同,尤其是对神经胶质化的调控HGF-TGF-β1平衡具有重要作用。Ad-HGF感染RMMC培养上清液是通过基因工程技术构建的Ad-HGF转染RMMC得到的细胞培养上清液。根据前期Ad-HGF转染大鼠脑膜间皮细胞(rat meningeal mesothelial cells,RMMCs),我们得到了细胞培养上清液,并进行相关细胞活性的研究,期望为明确Ad-HGF感染RMMC培养上清液对大鼠的CAEC是否能促进相关的微小血管再生和相关的神经保护提供进一步的实验基础,从而为下一步临床应用Ad-HGF基因治疗老年性缺血性脑血管病提供相关实验基础。目的采用Ad-HGF转染原代培养的SD大鼠相关脑膜间皮细胞,并取得相关条件细胞培养上清液,使其作用于大鼠的CAEC,观察细胞增殖和迁移能力及细胞中的TGF-β1蛋白表达情况的影响。方法通过基因工程技术构建的Ad-HGF转染大鼠脑膜间皮细胞(rat meningeal mesothelial cells,RMMCs)得到的细胞培养上清液作用于体外原代培养SD大鼠脑动脉内皮细胞(Cerebral artery endothelial cell,CAEC),观察上清液作用后CAEC增殖和迁移能力的影响,利用分子生物学技术,通过细胞研究和整体研究,正向-反向设计,检测TGF-β1在CAEC中的表达,分析HGF-TGF-β1及相关活性因子在CAEC新生、神经再生中的作用,尝试揭示TGF-p1因子信号在促脑血管新生中的分子调控及神经保护机制作用。结果1.通过倒置相差显微镜观察大鼠脑动脉内皮细胞,常规培养96h,规范换液后,显微镜下可以看到少许细胞从脑动脉血管内侧段游出,呈不规则多角形。继续培养8-10天之后,可见细胞铺满,呈“铺路石”样,大小一致,形状均一,融合度高达90%。2.原代细胞培养8-10 d后,细胞铺满瓶底行消化传代,经传代的第3代细胞用于鉴定,HE染色,细胞呈短梭形或多角形,大小不等,细胞浆丰富,薄染为淡红色,着色均匀。3.进一步荧光显微镜下进行观察,可见传代细胞的胞核及周围胞浆内均有黄绿色荧光出现,即相关第Ⅷ因子抗原阳性,阳性率90%,其胞质与内部胞核间界限较明显,对照组细胞不染色(呈阴性),证明细胞为大鼠脑动脉内皮细胞。4.细胞迁移实验中,PVPE膜上的大量CAEC被结晶紫染色；随机选取100倍视野下细胞迁移的数量进行比较,实验组与对照组相比,差异有显著性。5.MTT实验检测Ad-HGF感染LMC培养上清液不同时间长度作用于CAEC酶联免疫检测仪OD570nm处测量各孔的吸光值,实验组与对照组相比,差异有显著性。6.同一时间点,随着Ad-HGF感染RMMC培养上清液及外源性HGF浓度增加,CAEC中TGF-p1平均光密度值逐渐降低,TGF-p1平均光密度值呈一定量相关性,差异有显著性。结论1.采用组织块培养法及胰酶消化法可以成功获取高纯度大鼠脑动脉内皮细胞。2. Ad-HGF感染RMMC培养上清液能促进大鼠脑动脉内皮细胞的增殖和迁移。3. Ad-HGF感染RMMC培养上清液及HGF具有抑制CAEC中TGF-p1蛋白表达的作用。
[Abstract]:The background blood brain barrier is mainly composed of Cerebral artery endothelial cell (CAEC). The latter is common in the physiological activities of the body, such as the related synthesis and metabolic function of cells or tissues, and secreting some cytokines and bioactive substances, which are related to various physiological and pathological activities. The more studies show that Hepatocyte growth factor (HGF) has related neurotrophic and nerve growth promoting the occurrence of small blood vessels and axons, and can reduce the formation of cicatrix and the neural stem cells that act on the Subventricular zone (SVZ) in the ventricles of the submembrane of the brain (Subventricular zone, SVZ), and make them proliferate and differentiate into.T. GF- beta 1 is an important factor to ensure the integrity of the blood brain barrier. The action effect of hepatocyte growth factor is different according to the external environment of the cell and the related active factors in the cell environment, especially for the regulation of the HGF-TGF- beta 1 balance of neurogliosis, which is important for the use of.Ad-HGF infection RMMC culture supernatant. The cell culture supernatant was obtained by transfection of Ad-HGF into RMMC by engineering technology. According to Ad-HGF transfection of rat meningeal mesothelial cells (rat meningeal mesothelial cells, RMMCs), we obtained cell culture supernatant and related cell activity. It is expected that the CAEC of Ad-HGF infection RMMC culture supernatant to rat CAEC is to clear the Ad-HGF infection RMMC. Whether it can promote the related microvascular regeneration and related neuroprotection provides a further experimental basis for the next clinical application of Ad-HGF gene therapy for the treatment of Senile Ischemic cerebrovascular disease. Objective to transfect SD rat related mesothelial cells in primary cultured SD rats with Ad-HGF and to obtain related conditioned cell culture. The effect of the supernatant on the proliferation and migration ability of the cell and the expression of TGF- beta 1 protein in the cell was observed. Methods the cell culture supernatant obtained by Ad-HGF transfection of rat meningeal mesothelial cells (RMMCs) through gene engineering technology was used in the primary culture of the culture supernatant in vitro. Rat cerebral arterial endothelial cells (Cerebral artery endothelial cell, CAEC), observe the effect of CAEC proliferation and migration after the action of supernatant. By molecular biology technology, through cell research and overall study, positive and reverse design is used to detect the expression of TGF- beta 1 in CAEC, and to analyze HGF-TGF- beta 1 and related active factors in CAEC newborn and nerve. The role of regeneration is to try to reveal the molecular regulation of TGF-p1 factor signal and the role of neuroprotective mechanism in promoting cerebral angiogenesis. Results 1. the rat cerebral artery endothelial cells were observed by inverted phase contrast microscope, and 96h was routinely cultured. After changing the liquid, a few cells could swim out of the medial segment of the cerebral arteries under the microscope. After 8-10 days of continuous culture, 8-10 days, the cells were covered with "pave stone", the size was uniform, the shape was uniform, the fusion degree was up to 8-10 D in the primary cell culture of 90%.2., and the cells were paving the bottom of the bottle at the bottom of the bottle. The cells were used for identification by the third generation cells of the passages. The cells were stained with HE, the small cells were short shuttle or polygon, the size was different, the cytoplasm was rich and thin dyeing. For the light red and uniform.3. fluorescence microscope, it was observed that the nucleus and surrounding cytoplasm of the cell were yellow green fluorescence, that is, the positive rate of factor VIII antigen was positive, the positive rate was 90%, the boundary between the cytoplasm and the inner nucleus was more obvious, the control group was not stained (negative), which proved that the cell was the brain artery of the rat. In the.4. cell migration experiment of endothelial cells, a large number of CAEC on the PVPE membrane were stained with crystal violet, and the number of cell migration under 100 times of visual field was randomly selected. Compared with the control group, the experimental group was significantly different from that of the control group. The difference in the length of the LMC culture supernatant of the Ad-HGF infection was determined by the different length of the LMC culture supernatant at the OD570nm part of the CAEC enzyme immunoassay instrument. Compared with the control group, the difference was significant.6. at the same time point. With the increase of RMMC culture supernatant and exogenous HGF concentration in the Ad-HGF infection, the average density of TGF-p1 in CAEC decreased gradually, and the mean light density of TGF-p1 showed a certain correlation, and the difference was significant. Conclusion 1. by tissue mass culture method and The method of trypsin digestion can successfully obtain the.2. Ad-HGF infection of the cerebral arterial endothelial cells of high purity rats, RMMC culture supernatant can promote the proliferation of rat cerebral artery endothelial cells and migration of.3. Ad-HGF infection RMMC culture supernatant and HGF can inhibit the expression of TGF-p1 protein in CAEC.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R743.3

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