本文選題：肝細胞生長因子 + 腦動脈內皮細胞��； 參考：《新鄉(xiāng)醫(yī)學院》2015年碩士論文

【摘要】：背景血腦屏障主要是由腦動脈內皮細胞(Cerebral artery endothelial cell,CAEC)構成,后者常見于機體的生理活動中,如細胞或組織的相關合成及代謝功能,并分泌某種細胞因子及生物活性物質,是參與各種生理、病理活動的相關作用細胞。越來越多的研究顯示肝細胞生長因子(Hepatocyte growth factor, HGF)具有相關神經(jīng)營養(yǎng)以及促神經(jīng)生長,促進微小血管及軸突的發(fā)生、并能減少瘢痕的形成以及作用于腦室相關管膜下區(qū)(Subventricular zone,SVZ)的神經(jīng)干細胞,并使其增殖、分化的作用。TGF-β1是一種確保血腦屏障完整的重要因子,肝細胞生長因子與其的作用效應根據(jù)細胞的外環(huán)境以及細胞內環(huán)境的相關活性因子的不同而不同,尤其是對神經(jīng)膠質化的調控HGF-TGF-β1平衡具有重要作用。Ad-HGF感染RMMC培養(yǎng)上清液是通過基因工程技術構建的Ad-HGF轉染RMMC得到的細胞培養(yǎng)上清液。根據(jù)前期Ad-HGF轉染大鼠腦膜間皮細胞(rat meningeal mesothelial cells,RMMCs),我們得到了細胞培養(yǎng)上清液,并進行相關細胞活性的研究,期望為明確Ad-HGF感染RMMC培養(yǎng)上清液對大鼠的CAEC是否能促進相關的微小血管再生和相關的神經(jīng)保護提供進一步的實驗基礎,從而為下一步臨床應用Ad-HGF基因治療老年性缺血性腦血管病提供相關實驗基礎。目的采用Ad-HGF轉染原代培養(yǎng)的SD大鼠相關腦膜間皮細胞,并取得相關條件細胞培養(yǎng)上清液,使其作用于大鼠的CAEC,觀察細胞增殖和遷移能力及細胞中的TGF-β1蛋白表達情況的影響。方法通過基因工程技術構建的Ad-HGF轉染大鼠腦膜間皮細胞(rat meningeal mesothelial cells,RMMCs)得到的細胞培養(yǎng)上清液作用于體外原代培養(yǎng)SD大鼠腦動脈內皮細胞(Cerebral artery endothelial cell,CAEC),觀察上清液作用后CAEC增殖和遷移能力的影響,利用分子生物學技術,通過細胞研究和整體研究,正向-反向設計,檢測TGF-β1在CAEC中的表達,分析HGF-TGF-β1及相關活性因子在CAEC新生、神經(jīng)再生中的作用,嘗試揭示TGF-p1因子信號在促腦血管新生中的分子調控及神經(jīng)保護機制作用。結果1.通過倒置相差顯微鏡觀察大鼠腦動脈內皮細胞,常規(guī)培養(yǎng)96h,規(guī)范換液后,顯微鏡下可以看到少許細胞從腦動脈血管內側段游出,呈不規(guī)則多角形。繼續(xù)培養(yǎng)8-10天之后,可見細胞鋪滿,呈“鋪路石”樣,大小一致,形狀均一,融合度高達90%。2.原代細胞培養(yǎng)8-10 d后,細胞鋪滿瓶底行消化傳代,經(jīng)傳代的第3代細胞用于鑒定,HE染色,細胞呈短梭形或多角形,大小不等,細胞漿豐富,薄染為淡紅色,著色均勻。3.進一步熒光顯微鏡下進行觀察,可見傳代細胞的胞核及周圍胞漿內均有黃綠色熒光出現(xiàn),即相關第Ⅷ因子抗原陽性,陽性率90%,其胞質與內部胞核間界限較明顯,對照組細胞不染色(呈陰性),證明細胞為大鼠腦動脈內皮細胞。4.細胞遷移實驗中,PVPE膜上的大量CAEC被結晶紫染色；隨機選取100倍視野下細胞遷移的數(shù)量進行比較,實驗組與對照組相比,差異有顯著性。5.MTT實驗檢測Ad-HGF感染LMC培養(yǎng)上清液不同時間長度作用于CAEC酶聯(lián)免疫檢測儀OD570nm處測量各孔的吸光值,實驗組與對照組相比,差異有顯著性。6.同一時間點,隨著Ad-HGF感染RMMC培養(yǎng)上清液及外源性HGF濃度增加,CAEC中TGF-p1平均光密度值逐漸降低,TGF-p1平均光密度值呈一定量相關性,差異有顯著性。結論1.采用組織塊培養(yǎng)法及胰酶消化法可以成功獲取高純度大鼠腦動脈內皮細胞。2. Ad-HGF感染RMMC培養(yǎng)上清液能促進大鼠腦動脈內皮細胞的增殖和遷移。3. Ad-HGF感染RMMC培養(yǎng)上清液及HGF具有抑制CAEC中TGF-p1蛋白表達的作用。
[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.
【學位授予單位】：新鄉(xiāng)醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R743.3

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