本文選題：血腦屏障模型 + 腦微血管內(nèi)皮細胞　； 參考：《昆明醫(yī)科大學(xué)》2015年碩士論文

【摘要】：[目的]卒中是全球人口死亡和殘疾的主要原因,其發(fā)病機制十分復(fù)雜。缺血性卒中是最常見的卒中類型,約占全部卒中的87%。血腦屏障(blood-brain barrier, BBB)是位于血液與腦組織之間的一個選擇性通透的屏障體系,嚴(yán)格控制血管兩側(cè)的物質(zhì)轉(zhuǎn)運,從而維持中樞神經(jīng)系統(tǒng)(central nervous system, CNS)內(nèi)環(huán)境的相對穩(wěn)定。卒中后BBB破壞直接影響CNS功能,導(dǎo)致血管源性腦水腫、出血性轉(zhuǎn)化和神經(jīng)元凋亡,加重神經(jīng)功能缺損。基質(zhì)金屬蛋白酶(matrix metalloproteinases,MMPs)的激活被認為是破壞BBB結(jié)構(gòu)和功能的重要分子機制。腦缺血時,基質(zhì)金屬蛋白酶-2(matrix metal loproteinase-2, MMP-2)可能通過降解內(nèi)皮細胞緊密連接蛋白和基底膜成分使血管壁完整性破壞。發(fā)熱是卒中患者早期常見并發(fā)癥,有研究發(fā)現(xiàn)發(fā)熱可加重卒中患者預(yù)后,而具體機制還未闡明。因此,研究發(fā)熱加重卒中后腦損傷的機制,以及發(fā)熱是否上調(diào)MMP-2表達而加劇對BBB緊密連接蛋白的損傷,就具有一定現(xiàn)實意義。為此,本課題通過原代培養(yǎng)SD大鼠乳鼠腦微血管內(nèi)皮細胞和星形膠質(zhì)細胞,建立簡便、易行的能夠模擬在體BBB的體外細胞模型。予以外源性添加MMP-2和發(fā)熱處置干預(yù),分析BBB模型緊密連接蛋白occludin的變化,從而探索MMP-2及發(fā)熱在腦缺血后BBB破壞方面可能的作用機制,為臨床缺血性卒中患者并發(fā)癥的防治提供理論依據(jù)。[方法]以SD大鼠乳鼠為實驗材料,采用酶消化法原代分離、純化、傳代培養(yǎng)獲得腦微血管內(nèi)皮細胞和星形膠質(zhì)細胞,倒置顯微鏡下觀察細胞的形態(tài)。經(jīng)過HE染色,免疫熒光鑒定細胞及其純度,使用培養(yǎng)3代的兩種細胞通過Transwell小室進行共培養(yǎng),構(gòu)建體外BBB接觸模型。并通過滲漏試驗和LY滲透性測定,鑒定模型屏障功能。將構(gòu)建成功的模型隨機分為37℃常溫、39℃發(fā)熱、37℃常溫+MMP-2、39℃發(fā)熱+MMP-2四組,進而采用免疫細胞化學(xué)法和western blot方法觀察各組模型中BBB緊密連接蛋白occludin的變化。同時用免疫細胞化學(xué)法檢測37℃常溫及39℃發(fā)熱條件下星形膠質(zhì)細胞MMP-2的表達。[結(jié)果]1.對分離培養(yǎng)的腦微血管內(nèi)皮細胞和星形膠質(zhì)細胞進行了形態(tài)學(xué)觀察,呈現(xiàn)出各自典型的生長特征。經(jīng)HE染色和免疫熒光法鑒定,細胞純度達95%以上,符合兩種細胞共培養(yǎng)的實驗要求。2.兩種細胞通過Transwell小室進行共培養(yǎng),成功建立與12孔板相匹配的BBB接觸模型。選擇鏡下細胞完整融合的模型進行4h滲漏試驗,結(jié)果成陽性。LY滲透性檢測模型具有低通透系數(shù)(permeability coefficient, Pe),其值為0.95 ±0.06×10-3cmin。3.免疫細胞化學(xué)法顯示,相同時間點39℃發(fā)熱組星形膠質(zhì)細胞MMP-2陽性表達細胞數(shù)較37℃常溫組增高,差異具有統(tǒng)計學(xué)意義(P0.05)。其中,39℃發(fā)熱組隨著處置時間的延長,星形膠質(zhì)細胞MMP-2陽性表達細胞數(shù)亦增加,差異具有統(tǒng)計學(xué)意義(P0.05)。4.免疫細胞化學(xué)法和Western blot顯示,相同時間點37℃常溫+MMP-2組與37℃常溫組比較,內(nèi)皮細胞occludin的表達在60min和90min時均下降,差異有統(tǒng)計學(xué)意義(P0.05)。且37℃常溫+MMP-2組隨著處置時間的延長,內(nèi)皮細胞occludin的表達有下降趨勢,差異具有統(tǒng)計學(xué)意義(P0.05)。5.免疫細胞化學(xué)法和Western blot顯示,相同時間點39℃發(fā)熱+MMP-2組與39℃發(fā)熱組比較,內(nèi)皮細胞occludin的表達在60min和90min時均下降,差異有統(tǒng)計學(xué)意義(P0.05)。且39℃發(fā)熱+MMP-2組隨著處置時間的延長,內(nèi)皮細胞occludin的表達有下降趨勢,差異具有統(tǒng)計學(xué)意義(P0.05)。6.免疫細胞化學(xué)法和Western blot顯示,相同時間點39℃發(fā)熱組內(nèi)皮細胞occludin的表達較37℃常溫組下降,差異有統(tǒng)計學(xué)意義(PO.05)。且39℃發(fā)熱組隨著處置時間的延長,內(nèi)皮細胞occludin的表達有下降趨勢,差異具有統(tǒng)計學(xué)意義(P0.05)。7.免疫細胞化學(xué)法和Western blot還顯示,相同時間點39℃發(fā)熱+MMP-2組內(nèi)皮細胞occludin的表達較37℃常溫+MMP-2組明顯下降,差異具有統(tǒng)計學(xué)意義(P0.05)。[結(jié)論]1.原代分離、培養(yǎng)出了SD大鼠乳鼠腦微血管內(nèi)皮細胞和星形膠質(zhì)細胞,并成功建立了符合實驗需要的體外BBB接觸模型。2.體外培養(yǎng)的星形膠質(zhì)細胞可表達MMP-2,發(fā)熱可上調(diào)星形膠質(zhì)細胞MMP-2的表達。3.37℃常溫下,外源性添加MMP-2可降低體外BBB模型內(nèi)皮細胞緊密連接蛋白occludin的表達,并隨時間的延長而加劇。4.39℃發(fā)熱條件下,體外BBB模型內(nèi)皮細胞緊密連接蛋白occludin的表達下調(diào),并隨時間的延長而加劇。5.發(fā)熱進一步加劇MMP-2對體外BBB模型內(nèi)皮細胞緊密連接蛋白occludin的下調(diào),并隨時間的延長而加劇。6.發(fā)熱可能通過上調(diào)MMP-2的表達從而加劇體外BBB模型的破壞。
[Abstract]:[Objective] stroke is a major cause of death and disability in the world, its pathogenesis is very complex. Ischemic stroke is the most common type of stroke, 87%. blood brain barrier total stroke (blood-brain barrier BBB) is a selective permeability between the blood and brain barrier system, strictly control the transport vessel on both sides of the material, so as to maintain the central nervous system (central nervous system, CNS) in a relatively stable environment. After stroke BBB damage directly affects the function of CNS, leading to vasogenic brain edema and hemorrhagic transformation and apoptosis of neurons, aggravated neurological deficits. Matrix metalloproteinases (matrix, metalloproteinases, MMPs) activation is considered an important molecular mechanism of damage of the structure and function of BBB. Cerebral ischemia, matrix metalloproteinase -2 (MMP-2 matrix metal loproteinase-2) by degradation of endothelial cells closely connected The protein and basement membrane components of the integrity of vessel wall damage. Fever is a common complication in patients with early stroke, studies have found that fever can worsen the prognosis of patients with stroke, but the specific mechanism is still unclear. Therefore, study on the mechanism of fever aggravate brain injury following stroke, and whether fever and increased expression of MMP-2 increased on tight junction proteins BBB damage and it has a certain practical significance. Therefore, this topic through the primary culture of neonatal rat SD rat microvascular endothelial cells and astrocytes, establish a simple, can simulate the in vitro cell model of BBB. And to add MMP-2 and exogenous fever management intervention, change BBB model analysis of tight junction protein occludin thus, to explore the mechanism of MMP-2 and fever after cerebral ischemia BBB damage may, for the prevention and treatment of complications in patients with ischemic stroke and provide a theoretical basis for the method to S. D rat as experimental material, using enzyme digestion of primary separation, purification, to obtain brain microvascular endothelial cells and astrocytes were cultured. Cell morphology was observed under inverted microscope. After HE staining, immunofluorescence and cell purity, the use of two kinds of cells were co cultured for 3 generations by Transwell by in vitro BBB contact model. And through the leakage test and LY permeability determination, identification model of barrier function. The constructed model is divided for the success of 37 degrees at room temperature, 39 degrees fever, 37 degrees of room temperature +MMP-2,39 C +MMP-2 four fever group, then observe the change of BBB of tight junction protein occludin in each group by immune cells chemical method and Western blot method. At the same time. The expression of MMP-2 of astrocytes was detected by immunocytochemistry at room temperature 37 degrees and 39 degrees fever]1. under the condition of isolated brain capillaries Skin cells and astrocytes were observed, showing their typical growth characteristics. After HE staining and immunofluorescence, cell purity was more than 95%, with total of two cell culture experiments.2. two cells were co cultured with Transwell cells to establish BBB contact model matching with 12 hole plate the choice model of cell fusion. Complete endoscopic 4H leakage test, the positive.LY detection model has low permeability permeability coefficient (permeability coefficient, Pe), and its value is 0.95 + 0.06 * 10-3cmin.3. immunocytochemical method showed that the same time point 39 degrees fever group of astrocytes MMP-2 positive cells with 37 degrees at room temperature was increased, the difference was statistically significant (P0.05). Among them, 39 degrees fever group with prolonged treatment time, astrocytes and MMP-2 positive cells also increased the number of poor. Wasstatisticallysignificant (P0.05).4. immunocytochemistry and Western blot showed that the same time point of 37 DEG C at room temperature and 37 DEG C temperature group +MMP-2 group, occludin expression of endothelial cells were decreased in 60min and 90min, the difference was statistically significant (P0.05). And 37 normal C + MMP-2 group along with the prolonging of treatment time the expression of occludin in endothelial cells was decreased, the difference was statistically significant (P0.05).5. immunocytochemistry and Western blot showed that the same time point of 39 DEG C and 39 degree fever fever +MMP-2 group group, occludin expression of endothelial cells were decreased in 60min and 90min, the difference was statistically significant (P0.05). And 39 degrees fever group +MMP-2 with prolonged treatment time, the expression of occludin in endothelial cells was decreased, the difference was statistically significant (P0.05).6. immunocytochemistry and Western blot showed that the same time point of 39 DEG C The expression of occludin in endothelial cells of the fever group compared to 37 DEG C at room temperature was decreased, the difference was statistically significant (PO.05). And 39 degrees fever group along with the prolonging of treatment time, the expression of occludin in endothelial cells was decreased, the difference was statistically significant (P0.05).7. immunocytochemistry and Western blot also showed that the expression of the same time point 39 degrees fever group +MMP-2 endothelial cell occludin than 37 C ambient temperature +MMP-2 group decreased significantly, the difference was statistically significant (P0.05). Conclusion the primary separation of]1., developed a rat brain SD rat microvascular endothelial cells and astrocytes, and successfully established in accordance with the experimental requirements of in vitro BBB contact model of.2. in vitro cultured astrocytes express MMP-2,.3.37 expression can be up-regulated at room temperature heating MMP-2 of astrocytes, exogenous addition of MMP-2 can reduce the BBB model in vitro endothelial cell tight junction protein OC The expression of cludin, and with the extension of time and increase the temperature of.4.39 DEG C heating conditions, decrease the expression of BBB in endothelial cell model in vitro of tight junction protein occludin, and with the extension of time and increase the.5. heating further down-regulation of MMP-2 on endothelial cells in vitro BBB model of tight junction protein occludin, and with the extension of time and aggravation of.6. fever by upregulating the expression of MMP-2 in vitro BBB model thereby increasing damage.

【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R743.3

【引證文獻】

相關(guān)會議論文 前1條

1 魏峰;田新華;王鵬;林曉寧;黃志純;豐偉;王玉華;孟晗;黃延林;陳陸馗;;不同培養(yǎng)方式體外血腦屏障模型的建立及性能分析[A];2011中華醫(yī)學(xué)會神經(jīng)外科學(xué)學(xué)術(shù)會議論文匯編[C];2011年

，

本文編號：1738515