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

【摘要】：細菌性腦膜炎被認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的神經系統(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)病中起關鍵作用,發(fā)病機制未明,治療較困難。因此闡明細菌性腦膜炎時BBB通透性改變及調控機制有非常重要的實用意義。 研究表明腦微血管內皮細胞(brain micro vascular endothelial cells, BMECs)及緊密連接(tight junction, TJ)是BBB結構和功能的主要基礎。LPS為革蘭氏陰性細菌細胞壁的組成成分,釋放入血后被稱為內毒素,對BBB屏障功能破壞有重要作用。我們團隊的前期實驗證實中樞神經系統(tǒng)感染性時LPS表達顯著升高,且可引起B(yǎng)EMC緊密連接蛋白Occludin和ZO-1表達下調,但其具體的調控方式不明。目前研究顯示PKC、Rho、PI3K和酪氨酸激酶以及核轉錄因子NF-κB均可能參與調控緊密連接的組裝和分解,維持內皮細胞低通透性。但對于上述信號分子是否參與細菌性腦膜炎或感染性腦損傷時LPS致BBB通透性升高的調控過程,他們之間的調控關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細胞株具有原代鼠腦微血管內皮細胞屏障特性 目的：評價永生化小鼠腦微血管內皮細胞株Bend.3是否具有原代培養(yǎng)的鼠腦微血管內皮細胞的屏障及生理特性。 方法：將小鼠腦微血管內皮細胞株Bend.3和原代培養(yǎng)的鼠腦微血管內皮細胞接種于細胞培養(yǎng)插內,跨內皮細胞電阻抗(transendothelial electrical resistance,TEER)和辣根過氧化物酶(horseradish peroxidase,HRP)通透性實驗檢測其屏障功能。Westernblot法和直接熒光染色法觀察其緊密連接相關蛋白Occludin、ZO-1的表達及細胞骨架蛋白F-actin的分布。 結果：Bend.3細胞的TEER隨培養(yǎng)時間延長逐漸升高,10d達82.33±6.03Ω·cm2,與培養(yǎng)3d時相比,差異有統(tǒng)計學意義(P0.05),與同期原代培養(yǎng)的鼠腦微血管內皮細胞的TEER相比,無明顯差異(P0.05)。Bend.3細胞培養(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)計學意義(P0.05),與同期原代培養(yǎng)的鼠腦微血管內皮細胞的TEER相比,無明顯差異(P0.05)。培養(yǎng)10d時Bend.3細胞和原代培養(yǎng)的鼠腦微血管內皮細胞均表達高濃度的緊密連接蛋白Occludin、ZO-1,且F-actin主要分布在細胞周邊,線條完整連續(xù),未見明顯縫隙形成。 結論：小鼠腦微血管內皮細胞株Bend.3具有原代培養(yǎng)的腦微血管內皮細胞的屏障特性,且其屏障功能在接種10d后可達到最理想的狀態(tài)。 第二章初步篩選脂多糖致腦微血管內皮細胞通透性改變的信號分子 目的：證實脂多糖通過引起Actin重組、緊密連接表達和分布變化導致腦微血管內皮細胞通透性增高,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皮細胞通透性改變的調控。 方法：運用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皮細胞通透性,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)預處理Bend.3細胞,再通過TEER檢測LPS對各組細胞屏障功能的影響,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控過程。 結果：LPS可致Bend.3細胞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表達下調,Claudin-5蛋白分布改變,以及細胞骨架F-actin重組。PKC、Rho抑制劑可改善LPS引起的Bend.3細胞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。 結論：脂多糖引起腦微血管內皮細胞Actin重組、緊密連接表達和分布變化而導致其通透性增高；PKC和Rho,而非PI3K和酪氨酸激酶,參與了此過程調控。 第三章PKC和RhoA信號相互作用調控脂多糖致腦微血管內皮細胞通透性升高過程 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皮細胞通透性升高調控過程,他們與RhoA之間的調控關系如何。 方法：1.pull-down法和免疫共沉淀結合體外酶學實驗法分別檢測LPS作用不同時間Bend.3細胞的RhoA和PKC各亞基(α、β和ζ)活化狀態(tài)。2.分別利用脂質體2000將PcDN A3.1hygro-n19RhoA、 PcDNA3.1hygro-vector(空載對照質粒)導入Bend.3細胞,利用潮霉素B篩選出穩(wěn)定表達株。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導入Bend.3細胞,利用嘌呤霉素B篩選出穩(wěn)定表達株。Western blot分別鑒定PKC-α PKC-β和PKCζ蛋白的表達抑制情況。并根據導入質粒不同分5組,運用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轉移酶抑制RhoA活性后,體外酶學法檢測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調控關系；為進一步確認在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N19RhoA和vector-1質粒的兩組Bend.3細胞在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控關系,抑制穩(wěn)定表達PKCζ-ShRNA和vector-2的Bend.3細胞的RhoA活性,比較其在LPS作用前后的TEER值的改變。 結果：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PKC-ακ、PKC-β、PKC-ζ及N19RhoA的Bend.3細胞株。分別抑制RhoA及PKC各亞型(α、β和ζ)均可改善LPS對Bend.3細胞TJ的破壞作用。3.PKC-ζ活性受RhoA調控,RhoA活性受PKC-α調控,且在LPS致Ben.3細胞通透性上升調控過程中,PKC-ζ PKC-α分別是RhoA的下游及上游調控信號。 結論：PKC各亞基(α,β,ζ)和RhoA活化均促使BBB-TJ開放而導致BBB通透性上升,其中PKC-a、PKC-ζ分別為RhoA的上游調控分子和下游調控事件。 第四章RhoA/NF-κB/MLCK信號參與調控脂多糖致腦微血管內皮細胞通透性升高過程 目的：探討RhoA/NF-κB/MLCK信號是否參與LPS致BBB通透性升高的調控。 方法：利用脂質體2000將DNMu-IκBa質粒導入Bend.3細胞,潮霉素B篩選出穩(wěn)定表達株。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細胞的RhoA和NF-κB的活化作用。同時比較LPS對穩(wěn)定表達N19RhoA和DNMu-IκBa質粒的Bend.3細胞的通透性、F-actin分布以及緊密連接蛋白表達分布等指標的影響。為明確上述過程中NF-κB是否由RhoA活化而激活,進一步比較了LPS作用不同時間,Bend.3細胞、穩(wěn)定表達Vector-1和N19RhoA質粒的Bend.3細胞的NF-κB活性變化,同時比較了LPS對Bend.3細胞、穩(wěn)定表達Vector-1和DNMu-IκBα質粒的Bend.3細胞的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轉錄,導致肌球蛋白輕鏈(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轉錄水平。 結果：穩(wěn)定表達DNMu-IκBα和N19RhoA質粒均可改善LPS致Bend.3細胞緊密連接破壞、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 .
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R725.1

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