本文選題：原代培養(yǎng) + 腦微血管內(nèi)皮細(xì)胞��； 參考：《昆明醫(yī)科大學(xué)》2014年碩士論文

【摘要】：[目的]卒中的發(fā)病率逐年上升,病后的高致殘率讓其備受關(guān)注。在卒中的進(jìn)展過程中,血腦屏障(blood brain barrier,BBB)的破壞發(fā)揮著重要作用。BBB是介于血液和腦組織之間的一個(gè)對物質(zhì)有選擇性通過作用的動(dòng)態(tài)界面,起著維持中樞神經(jīng)系統(tǒng)(central nervous system,CNS)內(nèi)環(huán)境穩(wěn)定的重要作用。臨床診療中我們發(fā)現(xiàn)卒中病人常伴有發(fā)熱,而發(fā)熱會(huì)加重BBB的破壞,進(jìn)而加重腦損傷,影響預(yù)后。然而發(fā)熱加重BBB破壞的具體機(jī)制還未能完全闡明。已有研究發(fā)現(xiàn)基質(zhì)金屬蛋白酶(matrix metalloproteinases, MMPs)特別是MMP-9,可能通過破壞細(xì)胞間的緊密連接(tight junction,TJ)導(dǎo)致BBB破壞。我們前期動(dòng)物實(shí)驗(yàn)也發(fā)現(xiàn),發(fā)熱通過增高M(jìn)MP-9活性,降解TJ的重要成分claudin-1蛋白,從而導(dǎo)致BBB通透性升高。但是在常溫情況下,MMP-9是否具有破壞BBB結(jié)構(gòu)claudin-1蛋白的作用,以及發(fā)熱是否加劇MMP-9對claudin-1蛋白的破壞,目前仍不完全清楚。為此,本實(shí)驗(yàn)采用原代培養(yǎng)的SD大鼠腦微血管內(nèi)皮細(xì)胞和星形膠質(zhì)細(xì)胞通過Transwell小室共培養(yǎng),構(gòu)建體外BBB模型,予以外源性添加MMP-9及發(fā)熱處置,并觀察BBB模型緊密連接蛋白claudin-1的變化,從而探討MMP-9及發(fā)熱對體外BBB模型緊密連接蛋白claudin-1的影響。 [方法]首先用酶消化法進(jìn)行SD大鼠腦微血管內(nèi)皮細(xì)胞和星形膠質(zhì)細(xì)胞原代及傳代培養(yǎng),并通過HE染色、免疫熒光法鑒定所培養(yǎng)的細(xì)胞為所需要的腦微血管內(nèi)皮細(xì)胞和星形膠質(zhì)細(xì)胞；再將2種經(jīng)過鑒定傳至3代的細(xì)胞通過Transwell小室進(jìn)行共培養(yǎng),建立體外BBB模型；并通過滲漏實(shí)驗(yàn)和Lucifer yellow (LY)滲透性測定,以確定成功建立了體外BBB模型；將模型隨機(jī)分為37℃常溫、39℃高溫、37℃常溫+MMP-9、39℃高溫+MMP-9四組,并在30min、60min、90min進(jìn)行LY滲透性測定,觀察各組體外BBB模型通透性的變化；進(jìn)而采用免疫組織化學(xué)法和蛋白印跡法觀察各組BBB緊密連接蛋白claudin-1的變化。 [結(jié)果]1、經(jīng)HE染色、免疫熒光法鑒定所培養(yǎng)的細(xì)胞為SD大鼠腦微血管內(nèi)皮細(xì)胞和星形膠質(zhì)細(xì)胞。通過Transwell小室建立的非接觸BBB模型。滲漏試驗(yàn)結(jié)果成陽性。模型具有低通透系數(shù)(permeability coefficient,Pe,1.08±0.16x10"3cm/min)。 2、不同時(shí)間點(diǎn)組內(nèi)比較,模型通透性實(shí)驗(yàn)顯示,37℃常溫組和37℃常溫+MMP-9組,體外BBB模型的通透性無明顯改變,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。39℃高溫組和39℃+MMP-9發(fā)熱組,體外BBB模型的通透性隨時(shí)間延長而逐漸增高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 3、相同時(shí)間點(diǎn)組間比較,模型通透性實(shí)驗(yàn)顯示,37℃常溫+MMP-9組與37℃常溫組比較,體外BBB模型的通透性無明顯改變,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。而39℃高溫組與37℃常溫組比較、39℃高溫+MMP-9組與39℃高溫組、39℃高.溫+MMP-9組與37℃常溫+MMP-9組比較,體外BBB模型的通透性均明顯升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 4、不同時(shí)間點(diǎn)組內(nèi)比較,免疫組織化學(xué)法顯示,四組的claudin-1陽性表達(dá)細(xì)胞數(shù)差異均無統(tǒng)計(jì)學(xué)意義(P0.05)。而western blot法則顯示,37℃常溫組與37℃常溫+MMP-9組的組內(nèi)claudin-1陽性表達(dá)細(xì)胞數(shù)差異也無統(tǒng)計(jì)學(xué)意義(P0.05),但是39℃高溫組和39℃高溫+MMP-9組,claudin-1陽性表達(dá)細(xì)胞數(shù)隨時(shí)間延長而逐漸下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 5、相同時(shí)間點(diǎn)組間比較,免疫組織化學(xué)法及western blot法都顯示,37℃常溫+MMP-9組與37℃常溫組比較,緊密連接蛋白claudin-1的表達(dá)均無明顯改變,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。而39℃高溫組與37℃常溫組比較、39℃高溫+MMP-9組與37℃常溫+MMP-9組比較,緊密連接蛋白claudin-1的表達(dá)均明顯下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 6、相同時(shí)間點(diǎn)組間比較,免疫組織化學(xué)法還顯示,39℃高溫+MMP-9組與39℃高溫組相比,僅在90min時(shí),claudin-1陽性表達(dá)細(xì)胞數(shù)進(jìn)一步下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 7、相同時(shí)間點(diǎn)組間比較,Western Blot法還顯示,39℃高溫+MMP-9組與39℃高溫組相比,claudin-1的表達(dá)均進(jìn)一步下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 [結(jié)論]1.原代培養(yǎng)出了SD大鼠腦微血管內(nèi)皮細(xì)胞和星形膠質(zhì)細(xì)胞,并成功建立了體外BBB模型。 2、本實(shí)驗(yàn)條件下,單純外源性添加MMP-9不能改變體外BBB模型的通透性,也不能破壞緊密連接蛋白claudin-1。 3、39℃高溫能明顯增加體外BBB模型的通透性,外源性添加MMP-9能進(jìn)一步增加發(fā)熱對體外BBB模型完整性的破壞。 4、39℃高溫會(huì)導(dǎo)致體外BBB模型緊密連接蛋白claudin-1的表達(dá)下降；外源性添加MMP-9能進(jìn)一步加重發(fā)熱對體外BBB模型緊密連接蛋白claudin-1的破壞。
[Abstract]:[Objective] the incidence of stroke is increasing year by year, and the high disability rate after the disease has attracted much attention. In the process of stroke, the destruction of the blood brain barrier (BBB) plays an important role in.BBB, a dynamic interface between the blood and brain tissue, which has a selective interaction between the substance and the brain tissue, to maintain the central nervous system. (central nervous system, CNS) the important role of environmental stability in the clinical diagnosis and treatment of stroke patients often accompanied by fever, and fever will aggravate the destruction of BBB, aggravate brain damage and affect the prognosis. However, the specific mechanism of the exacerbation of BBB has not been fully elucidated. The study of matrix metalloproteinases (matrix metallop) has been found. Roteinases, MMPs), especially MMP-9, may cause BBB destruction by destroying the close connections between cells (tight junction, TJ). In our previous animal experiments, we also found that fever can degrade claudin-1 protein, an important component of TJ, by increasing the activity of MMP-9, which leads to the increase of BBB permeability. The effect of structure of claudin-1 protein and whether fever aggravates the destruction of MMP-9 to claudin-1 protein is still not completely clear. To this end, the primary cultured SD rat brain microvascular endothelial cells and astrocytes were co cultured in the Transwell chamber, and the BBB model was constructed in vitro, and the exogenous MMP-9 and fever treatment were added. The changes of tight junction protein Claudin-1 in BBB model were observed, and the effects of MMP-9 and fever on tight junction protein Claudin-1 in BBB model were explored.
[Methods] the original and passages of SD rat brain microvascular endothelial cells and astrocytes were cultured with enzyme digestion method, and HE staining was used to identify the cultured cells as required brain microvascular endothelial cells and astrocytes by immunofluorescence, and then 2 kinds of cells which were identified and passed to 3 generations were carried out through the Transwell chamber. Co culture, established in vitro BBB model, and through the leakage test and Lucifer yellow (LY) permeability determination to determine the successful establishment of the BBB model in vitro. The model was randomly divided into 37 C room temperature, 39 C high temperature, 37 C at normal temperature +MMP-9,39 temperature +MMP-9 four groups, and 30min, 60min, 90min LY permeability determination, observe each group of BBB model in vitro Immunohistochemical staining and Western blotting were used to observe the changes of BBB tight junction protein claudin-1.
[results]1, HE staining, immunofluorescence assay showed that the cultured cells were SD rat brain microvascular endothelial cells and astrocytes. The non contact BBB model established by the Transwell chamber. The results of the leakage test were positive. The model had low permeability coefficient (permeability coefficient, Pe, 1.08 + 0.16x10 "3cm/min).
2, compared with the time point group, the model permeability test showed that the permeability of BBB model in the normal temperature group and +MMP-9 group at 37 C was not significantly changed, the difference was not statistically significant (P0.05).39 C high temperature group and 39 C +MMP-9 fever group, the permeability of BBB model in vitro increased gradually with time, the difference was statistically significant (P0.05).
3, compared with the same time point group, the model permeability test showed that the permeability of BBB model in +MMP-9 group at 37 C and 37 C at normal temperature had no significant change, and the difference was not statistically significant (P0.05). The 39 C high temperature group was compared with the normal temperature group at 37 degrees C, the high temperature +MMP-9 group and 39 C high temperature group at 39 C, the high temperature group, and the temperature +MMP-9 group and 37 temperature +MMP-9 at normal temperature. Compared with the control group, the permeability of BBB model in vitro increased significantly, and the difference was statistically significant (P0.05).
4, compared with the different time point group, the immuno histochemical method showed that there was no significant difference in the number of claudin-1 positive cells in the four groups (P0.05), but the Western blot rule showed that there was no significant difference in the number of claudin-1 positive cells in the group of 37 C and 37 C at normal temperature +MMP-9 group, but there was no statistical significance (P0.05), but at 39 centigrade and 39 C In high temperature group +MMP-9, the number of claudin-1 positive cells decreased gradually with time, and the difference was statistically significant (P0.05).
5, compared with the same time point group, immunohistochemistry and Western blot method showed that there was no significant change in the expression of close connexin claudin-1 at normal temperature at 37 C and 37 C at normal temperature group, and the difference was not statistically significant (P0.05). The 39 C high temperature group was compared with the normal temperature group at 37, and the ratio of the high temperature +MMP-9 group to the 37 C at normal temperature +MMP-9 group was compared with that of the normal temperature group at 37. The expression of tight junction protein claudin-1 was significantly lower than that of the control group (P0.05).
6, compared with the same time point group, immuno histochemical method also showed that the number of claudin-1 positive expression cells in the high temperature +MMP-9 group at 39 C was further lower than that of the high temperature group at the temperature of 39 (P0.05), and the difference was statistically significant (P0.05).
7, compared with the same time point group, the Western Blot method also showed that the expression of Claudin-1 in the high temperature +MMP-9 group at 39 C was further decreased compared with the 39 C high temperature group, and the difference was statistically significant (P0.05).
[conclusion]1. primarily cultured SD rat brain microvascular endothelial cells and astrocytes, and successfully established BBB model in vitro.
2, under the experimental conditions, simply exogenous MMP-9 addition can not change the permeability of BBB model in vitro, nor destroy the tight junction protein claudin-1..
3,39 high temperature significantly increased the permeability of BBB model in vitro. Exogenous addition of MMP-9 could further increase the damage of fever to BBB model integrity in vitro.
High temperature at 4,39 C can lead to the decrease of the expression of tightly connexin Claudin-1 in the BBB model in vitro, and exogenous MMP-9 can further aggravate the destruction of the close connexin claudin-1 of the BBB model in vitro by the fever.

【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R743.3

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 趙康峰;王，

本文編號(hào)：1819398