本文選題：緊密連接 + 蛋白激酶C��； 參考：《中南大學(xué)》2012年博士論文

【摘要】：細(xì)菌性腦膜炎被認(rèn)為是全球前十大致死性感染性疾病之一,致死、致殘率高,目前仍約30-50%的患者留有不可逆轉(zhuǎn)的神經(jīng)系統(tǒng)后遺癥。血腦屏障(Blood-brain barrier, BBB)通透性增加致血管源性腦水腫在其發(fā)病中起關(guān)鍵作用,發(fā)病機(jī)制未明,治療較困難。因此闡明細(xì)菌性腦膜炎時BBB通透性改變及調(diào)控機(jī)制有非常重要的實(shí)用意義。 研究表明腦微血管內(nèi)皮細(xì)胞(brain micro vascular endothelial cells, BMECs)及緊密連接(tight junction, TJ)是BBB結(jié)構(gòu)和功能的主要基礎(chǔ)。LPS為革蘭氏陰性細(xì)菌細(xì)胞壁的組成成分,釋放入血后被稱為內(nèi)毒素,對BBB屏障功能破壞有重要作用。我們團(tuán)隊(duì)的前期實(shí)驗(yàn)證實(shí)中樞神經(jīng)系統(tǒng)感染性時LPS表達(dá)顯著升高,且可引起B(yǎng)EMC緊密連接蛋白Occludin和ZO-1表達(dá)下調(diào),但其具體的調(diào)控方式不明。目前研究顯示PKC、Rho、PI3K和酪氨酸激酶以及核轉(zhuǎn)錄因子NF-κB均可能參與調(diào)控緊密連接的組裝和分解,維持內(nèi)皮細(xì)胞低通透性。但對于上述信號分子是否參與細(xì)菌性腦膜炎或感染性腦損傷時LPS致BBB通透性升高的調(diào)控過程,他們之間的調(diào)控關(guān)系如何等方面的研究均少見報道。對上述問題的深入研究,有助于進(jìn)一步闡明感染性腦損傷時BBB通透性增高的病理過程和發(fā)病機(jī)制,為其臨床防治提供新的思路。 本研究分為四部分： 第一章永生化Bend.3細(xì)胞株具有原代鼠腦微血管內(nèi)皮細(xì)胞屏障特性 目的：評價永生化小鼠腦微血管內(nèi)皮細(xì)胞株Bend.3是否具有原代培養(yǎng)的鼠腦微血管內(nèi)皮細(xì)胞的屏障及生理特性。 方法：將小鼠腦微血管內(nèi)皮細(xì)胞株Bend.3和原代培養(yǎng)的鼠腦微血管內(nèi)皮細(xì)胞接種于細(xì)胞培養(yǎng)插內(nèi),跨內(nèi)皮細(xì)胞電阻抗(transendothelial electrical resistance,TEER)和辣根過氧化物酶(horseradish peroxidase,HRP)通透性實(shí)驗(yàn)檢測其屏障功能。Westernblot法和直接熒光染色法觀察其緊密連接相關(guān)蛋白Occludin、ZO-1的表達(dá)及細(xì)胞骨架蛋白F-actin的分布。 結(jié)果：Bend.3細(xì)胞的TEER隨培養(yǎng)時間延長逐漸升高,10d達(dá)82.33±6.03Ω·cm2,與培養(yǎng)3d時相比,差異有統(tǒng)計(jì)學(xué)意義(P0.05),與同期原代培養(yǎng)的鼠腦微血管內(nèi)皮細(xì)胞的TEER相比,無明顯差異(P0.05)。Bend.3細(xì)胞培養(yǎng)10d和3d的平均HRP通透率在120min分別為(2.±20.05)%和(4.3±0.20)%,差異有統(tǒng)計(jì)學(xué)意義(P0.05),與同期原代培養(yǎng)的鼠腦微血管內(nèi)皮細(xì)胞的TEER相比,無明顯差異(P0.05)。培養(yǎng)10d時Bend.3細(xì)胞和原代培養(yǎng)的鼠腦微血管內(nèi)皮細(xì)胞均表達(dá)高濃度的緊密連接蛋白Occludin、ZO-1,且F-actin主要分布在細(xì)胞周邊,線條完整連續(xù),未見明顯縫隙形成。 結(jié)論：小鼠腦微血管內(nèi)皮細(xì)胞株Bend.3具有原代培養(yǎng)的腦微血管內(nèi)皮細(xì)胞的屏障特性,且其屏障功能在接種10d后可達(dá)到最理想的狀態(tài)。 第二章初步篩選脂多糖致腦微血管內(nèi)皮細(xì)胞通透性改變的信號分子 目的：證實(shí)脂多糖通過引起Actin重組、緊密連接表達(dá)和分布變化導(dǎo)致腦微血管內(nèi)皮細(xì)胞通透性增高,初步探討PKC、Rho、PI3K和酪氨酸激酶等信號是否參與脂多糖致腦微血管內(nèi)皮細(xì)胞通透性改變的調(diào)控。 方法：運(yùn)用TEER測定法、F-actin染色法、western blot和免疫熒光法分別檢測了LPS作用不同時間下Bend.3細(xì)胞的通透性,F-actin分布以及緊密連接蛋白cluaudin-5,Occludin和ZO-1的狀態(tài),以動態(tài)觀察LPS是否通過破壞緊密連接蛋白增加腦血管內(nèi)皮細(xì)胞通透性,然后分別利用calphostin C (PKC抑制劑)、C3transferase (Rho抑制劑)、wortmannin (PI3K抑制劑)、PP2(酪氨酸激酶抑制劑)預(yù)處理Bend.3細(xì)胞,再通過TEER檢測LPS對各組細(xì)胞屏障功能的影響,了解PKC、酪氨酸激酶、PI3K、Rho信號是否參與了LPS致血腦屏障通透性升高的調(diào)控過程。 結(jié)果：LPS可致Bend.3細(xì)胞TEER以時間依賴性的方式下降,同時伴有緊密連接蛋白ZO-1、Occludin和Claudin-5表達(dá)下調(diào),Claudin-5蛋白分布改變,以及細(xì)胞骨架F-actin重組。PKC、Rho抑制劑可改善LPS引起的Bend.3細(xì)胞TEER下降；而PI3K、酪氨酸激酶抑制劑不能阻斷LPS對Bend.3屏障功能的破壞。 結(jié)論：脂多糖引起腦微血管內(nèi)皮細(xì)胞Actin重組、緊密連接表達(dá)和分布變化而導(dǎo)致其通透性增高；PKC和Rho,而非PI3K和酪氨酸激酶,參與了此過程調(diào)控。 第三章PKC和RhoA信號相互作用調(diào)控脂多糖致腦微血管內(nèi)皮細(xì)胞通透性升高過程 目的：探討PKC各亞型(α、β和ζ)如何參與脂多糖致腦微血管內(nèi)皮細(xì)胞通透性升高調(diào)控過程,他們與RhoA之間的調(diào)控關(guān)系如何。 方法：1.pull-down法和免疫共沉淀結(jié)合體外酶學(xué)實(shí)驗(yàn)法分別檢測LPS作用不同時間Bend.3細(xì)胞的RhoA和PKC各亞基(α、β和ζ)活化狀態(tài)。2.分別利用脂質(zhì)體2000將PcDN A3.1hygro-n19RhoA、 PcDNA3.1hygro-vector(空載對照質(zhì)粒)導(dǎo)入Bend.3細(xì)胞,利用潮霉素B篩選出穩(wěn)定表達(dá)株。Pull Down法鑒定RhoA活性的抑制情況。并將PLKO.1-puro-PKCα-shRNA、PLKO.1-puro-PKCβ-shRNA PLKO.1-puro-PKCζ-shRNA和empty PLKO.1-puro vector導(dǎo)入Bend.3細(xì)胞,利用嘌呤霉素B篩選出穩(wěn)定表達(dá)株。Western blot分別鑒定PKC-α PKC-β和PKCζ蛋白的表達(dá)抑制情況。并根據(jù)導(dǎo)入質(zhì)粒不同分5組,運(yùn)用F-actin染色法、western blot和免疫熒光法分別檢測了LPS作用不同時間下各組Bend.3細(xì)胞F-actin分布以及緊密連接蛋白cluaudin-5,Occludin和ZO-1的狀態(tài),以了解抑制RhoA及PKC亞基(α、β和ζ)后LPS致緊密連接破壞作用的改變。3.利用N19RhoA和C3轉(zhuǎn)移酶抑制RhoA活性后,體外酶學(xué)法檢測LPS對PKC各亞基活化作用；利用shRNA分別抑制PKC-α、PKC-β和PKC-ζ活性后Pull Down法分別檢測各組LPS對RhoA活化作用,以初步探討PKC各亞型與RhoA調(diào)控關(guān)系；為進(jìn)一步確認(rèn)在LPS致BBB屏障功能破壞過程中PKC-α是否為RhoA上游分子信號,抑制PKC-a活性后,比較穩(wěn)定表達(dá)N19RhoA和vector-1質(zhì)粒的兩組Bend.3細(xì)胞在LPS作用前后的TEER值變化；為明確PKC-ζ和RhoA在LPS致BBB屏障功能破壞過程中的調(diào)控關(guān)系,抑制穩(wěn)定表達(dá)PKCζ-ShRNA和vector-2的Bend.3細(xì)胞的RhoA活性,比較其在LPS作用前后的TEER值的改變。 結(jié)果：1.LPS作用5min RhoA及PKC各亞型(α、β和ζ)均開始活化。2.成功建立穩(wěn)定表達(dá)PKC-ακ、PKC-β、PKC-ζ及N19RhoA的Bend.3細(xì)胞株。分別抑制RhoA及PKC各亞型(α、β和ζ)均可改善LPS對Bend.3細(xì)胞TJ的破壞作用。3.PKC-ζ活性受RhoA調(diào)控,RhoA活性受PKC-α調(diào)控,且在LPS致Ben.3細(xì)胞通透性上升調(diào)控過程中,PKC-ζ PKC-α分別是RhoA的下游及上游調(diào)控信號。 結(jié)論：PKC各亞基(α,β,ζ)和RhoA活化均促使BBB-TJ開放而導(dǎo)致BBB通透性上升,其中PKC-a、PKC-ζ分別為RhoA的上游調(diào)控分子和下游調(diào)控事件。 第四章RhoA/NF-κB/MLCK信號參與調(diào)控脂多糖致腦微血管內(nèi)皮細(xì)胞通透性升高過程 目的：探討RhoA/NF-κB/MLCK信號是否參與LPS致BBB通透性升高的調(diào)控。 方法：利用脂質(zhì)體2000將DNMu-IκBa質(zhì)粒導(dǎo)入Bend.3細(xì)胞,潮霉素B篩選出穩(wěn)定表達(dá)株。報告基因法鑒定NF-κB活性的抑制情況。首先為了解RhoA和NF-κB是否參與LPS致BBB通透性升高的調(diào)控：利用pull down法和熒光素酶報告基因法分別測定LPS對Bend.3細(xì)胞的RhoA和NF-κB的活化作用。同時比較LPS對穩(wěn)定表達(dá)N19RhoA和DNMu-IκBa質(zhì)粒的Bend.3細(xì)胞的通透性、F-actin分布以及緊密連接蛋白表達(dá)分布等指標(biāo)的影響。為明確上述過程中NF-κB是否由RhoA活化而激活,進(jìn)一步比較了LPS作用不同時間,Bend.3細(xì)胞、穩(wěn)定表達(dá)Vector-1和N19RhoA質(zhì)粒的Bend.3細(xì)胞的NF-κB活性變化,同時比較了LPS對Bend.3細(xì)胞、穩(wěn)定表達(dá)Vector-1和DNMu-IκBα質(zhì)粒的Bend.3細(xì)胞的RhoA活性的影響。最后,為闡明上述過程中NF-κB是否通過增加MLCK轉(zhuǎn)錄,導(dǎo)致肌球蛋白輕鏈(Myosin light Chain, MLC)磷酸化,本研究利用western blot及RT-PCR分別檢測了MLC磷酸化和MLCK轉(zhuǎn)錄水平。 結(jié)果：穩(wěn)定表達(dá)DNMu-IκBα和N19RhoA質(zhì)粒均可改善LPS致Bend.3細(xì)胞緊密連接破壞、通透性升高的作用。LPS作用5min RhoA活化,作用30min NF-κB活化；抑制RhoA活化,LPS致NF-κB活化的作用也被明顯抑制,但抑制NF-κB活化對RhoA活性水平無影響。LPS作用0.5h, MLCK轉(zhuǎn)錄水平上升,3h MLC磷酸化水平明顯增高,阻斷NF-κB活化后上述表現(xiàn)被抑制。 結(jié)論：RhoA/NF-KB/MLCK信號通過磷酸化MLC,參與了LPS致BBB-TJ破壞、通透性升高過程的調(diào)控。
[Abstract]:Bacterial meningitis is considered one of the top ten fatal infectious diseases in the world . It is fatal and has a high disability rate . At present , there are still some 30 - 50 % of patients with irreversible nervous system sequelae . Blood - brain barrier ( BBB ) permeability increases vascular - derived brain edema plays a key role in the pathogenesis . The pathogenesis is not clear , and the treatment is difficult . Therefore , it is very important to clarify the change of BBB permeability and control mechanism in bacterial meningitis .

The study shows that brain micro vascular endothelial cells ( BMECs ) and tight junction ( TJ ) are the main bases of BBB structure and function . LPS is the component of the cell wall of Gram - negative bacteria , which is called endotoxin after release of blood , and it has important effect on the function of BBB barrier .

This study is divided into four parts :

In the first chapter , the cell line has the barrier properties of the rat brain microvascular endothelial cells .

Objective : To evaluate the protective barrier and physiological characteristics of cultured mouse brain microvascular endothelial cells ( Bendothelial cells ) in primary cultured rat brain microvascular endothelial cells .

Methods : The rat brain microvascular endothelial cells were inoculated into the cell culture , and the barrier function was measured by the permeability test of transendothelial electrical resistance ( TEER ) and horseradish peroxidase ( HRP ) . Western blot and direct fluorescence staining were used to observe the expression of the related proteins in human brain microvascular endothelial cells , the expression of ZO - 1 and the distribution of F - actin in the cells .

Results : The TEER of Bend . 3 cells increased gradually with culture time , 10d reached 82.33 鹵 6.03 惟 路 cm ~ 2 , and the difference was statistically significant ( P0.05 ) . Compared with TEER of rat brain microvascular endothelial cells cultured in the same period , there was no significant difference ( P0.05 ) .

Conclusion : The mouse brain microvascular endothelial cell strain Bend . 3 has the barrier property of primary cultured brain microvascular endothelial cells , and its barrier function can reach the ideal state after 10d .

In chapter 2 , the signal molecules of lipopolysaccharide - induced permeability change in brain microvascular endothelial cells were preliminarily selected .

Objective : To demonstrate the increase of permeability of microvascular endothelial cells induced by lipopolysaccharide ( Actin ) recombination , closely linked expression and distribution changes .

Methods : The permeability , F - actin distribution and tight connexin cluaudin - 5 , F - actin distribution and the state of tight connexin cluaudin - 5 were detected by TEER assay , F - actin staining , western blot and immunofluorescence staining . The effects of LPS on the cell barrier function of each group were observed by using calphostin C ( PKC inhibitor ) , C 3 transferase ( inhibitor of tyrosine kinase ) , wortmannin ( inhibitors of tyrosine kinase ) , and the effects of LPS on the barrier function of each group were investigated .

Results : The TEER decreased in time - dependent manner , and the expression of Claudin - 5 protein was down - regulated . The expression of Claudin - 5 protein was changed , and the F - actin of cytoskeletal framework was reduced . PKC and rho inhibitor could improve the decrease of TEER in Bend . 3 cells induced by LPS .
however , that inhibitor of tyrosine kinase can not block the damage of LPS to the function of the Bend . 3 barrier .

Conclusion : The expression and distribution of Actin in brain microvascular endothelial cells induced by lipopolysaccharide ( LPS ) resulted in increased permeability .
PKC and rho are involved in the regulation of this process , while non - PI3 and tyrosine kinases are involved .

In chapter 3 , PKC and RhoA signaling interact to regulate the permeability of microvascular endothelial cells induced by lipopolysaccharide .

Objective : To investigate how PKC isoforms ( 偽 , 尾 and zeta ) participate in the regulation of permeability of microvascular endothelial cells induced by lipopolysaccharide ( LPS ) , and their relationship with RhoA .

Methods : The expression of PKC - 偽 - PKC - 尾 and PKC - 尾 - shRNA was determined by the method of pull - down and immune co - precipitation combined with in vitro enzymatic experiment . The expression of PKC - 偽 PKC - 尾 and PKC - zeta protein was determined by using liposome 2000 . The expression of PKC - 偽 PKC - 尾 and PKC - zeta protein were determined by Western blot . The activation of PKC - 偽 PKC - 尾 and PKC - 偽 - shRNA was investigated by Western blot .
The effects of PKC - 偽 , PKC - 尾 and PKC - zeta on the activation of RhoA were respectively detected by using shRNA , and the relationship between PKC - 偽 , PKC - 尾 and PKC - zeta activation was studied .
In order to further confirm whether PKC - 偽 was the upstream molecular signal of RhoA and inhibited PKC - a activity after LPS - induced BBB function destruction , two groups of Bend . 3 cells stably expressing N19RhoA and vector - 1 plasmid changed TEER values before and after LPS .
In order to clarify the regulatory relationships of PKC - zeta and RhoA in the process of LPS - induced BBB dysfunction , the activity of RhoA in Bend . 3 cells stably expressing PKC - zeta - ShRNA and vector - 2 was inhibited , and the changes of TEER value before and after LPS action were compared .

Results : 1 . All of the subtypes of RhoA and PKC were activated by LPS for 5 min . PKC - 偽 - 魏B , PKC - 尾 , PKC - zeta and N19RhoA were successfully established .

Conclusion : PKC - a , PKC - zeta , PKC - a , PKC - zeta , PKC - a , PKC - zeta , PKC - a , PKC - zeta , PKC - a , PKC - zeta , PKC - a , PKC - zeta , PKC - a and PKC - zeta , respectively , increased BBB - TJ opening in the presence of PKC - a and PKC - zeta , respectively .

In chapter 4 , RhoA / NF - 魏B / MLCK signal is involved in regulating the permeability of microvascular endothelial cells induced by lipopolysaccharide .

Objective : To investigate whether the signal of RhoA / NF - 魏B / MLCK is involved in the regulation of BBB permeability in LPS - induced BBB .

Methods : The effects of LPS on the activity of RhoA and NF - 魏B in Bend . 3 cells of Bend . 3 cells were determined by using Lipofectamine 2000 . The effects of LPS on the activity of RhoA and NF - 魏B in Bend . 3 cells were determined by using the method of pull down and luciferase reporter gene .

Results : The stable expression of DNMu - I魏B 偽 and N19RhoA plasmid could improve the adhesion of LPS - induced Bend . 3 cells and increase the permeability . LPS acts 5 min for activation of RhoA and activates NF - 魏B in 30min .
LPS could inhibit the activation of RhoA and LPS - induced NF - 魏B activation , but the inhibition of NF - 魏B activation did not affect the activity of RhoA . The level of NF - 魏B activation increased , the level of MLCK transcription increased , the phosphorylation level of 3h MLC was obviously increased , and the above expression was inhibited after blocking NF - 魏B activation .

Conclusion : RhoA / NF - KB / MLCK signal is involved in the regulation of BBB - TJ damage and increase of permeability in LPS - induced BBB - TJ .
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R725.1

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