【摘要】：目的：探討擬缺血性損傷對(duì)大鼠腦微血管內(nèi)皮細(xì)胞（Rat Brain MicrovascularEndothelial Cells，rBMECs）上P-糖蛋白（P-glycoprotein，P-gp）的影響及其相關(guān)機(jī)制。 方法：原代培養(yǎng)rBMECs、神經(jīng)元和神經(jīng)膠質(zhì)細(xì)胞并借助Transwell建立共培養(yǎng)體系；在共培養(yǎng)體系的基礎(chǔ)上用Na2S2O4（終濃度為2mM）建立損傷模型,損傷時(shí)間為4h。在共培養(yǎng)體系內(nèi)室、外室加入腫瘤壞死因子（Tumor necrosis factor-α，TNF-α）、一氧化氮合酶（Nitric-oxide synthase，NOS）、蛋白激酶C（Protein kinase C，PKC）的抑制劑H398、L-NMMA(NG-Monomethyl-L-arginine.monoacetate）、BIM（Bisindolylmaleimide)測(cè)定Rh123的表觀滲透系數(shù)（Apparent Permeability Coefficient，Papp）以反映擬缺血性損傷對(duì)rBMECs上P-gp功能的變化；測(cè)定外室熒光素鈉（Fluorescein Sodium Injection，NaF）的Papp以確保rBMECs緊密連結(jié)的完整性；采用Western Blot法確定可能引起P-gp表達(dá)和功能變化的因子，然后加入相應(yīng)的抑制劑測(cè)定各因子和P-gp表達(dá)變化的情況，研究擬缺血性損傷對(duì)rBMECs上P-gp的影響的機(jī)制。 結(jié)果：1.通過(guò)Transwell成功建立了rBMECs、神經(jīng)元和神經(jīng)膠質(zhì)細(xì)胞的共培養(yǎng)體系。2.以Na2S2O4（終濃度為2mM）誘導(dǎo)rBMECs損傷4h，與空白組比較，損傷組Rh123由內(nèi)室到外室的Papp值顯著降低，，由外室到內(nèi)室的Papp值顯著增強(qiáng)，說(shuō)明P-gp的功能是增強(qiáng)的；NaF的Papp值無(wú)顯著變化表明rBMECs緊密連結(jié)沒(méi)有受到影響，確保了實(shí)驗(yàn)結(jié)果的準(zhǔn)確性。3.Western Blot結(jié)果顯示，Na2S2O4（終濃度為2mM）誘導(dǎo)rBMECs損傷4h，TNF-α、NOS和PKC表達(dá)均明顯增強(qiáng)；H398對(duì)TNF-α表達(dá)無(wú)明顯影響但可下調(diào)NOS、PKC、P-gp的表達(dá)；L-NMMA對(duì)TNF-α表達(dá)無(wú)明顯影響但可下調(diào)、PKC、P-gp的表達(dá)；BIM對(duì)TNF-α、NOS的表達(dá)無(wú)明顯影響但可下調(diào)P-gp的表達(dá)。 結(jié)論：擬缺血性損傷時(shí)，rBMECs受到刺激合成并釋放促炎癥細(xì)胞因子TNF-α，TNF-α繼而激活NOS、PKC對(duì)rBMECs上P-gp起調(diào)控作用且上調(diào)其表達(dá)，為腦中風(fēng)藥物治療的研究提供了新的特異性靶點(diǎn)。
[Abstract]:Aim: to investigate the effect of ischemic injury on P-glycoprotein (P-glycoprotein P GP) in rat brain microvascular endothelial cells (Rat Brain MicrovascularEndothelial Cells,rBMECs) and its related mechanism. Methods: rBMECs, neurons and glial cells were cultured in primary culture and co-culture system was established by means of Transwell. On the basis of co-culture system, Na2S2O4 (final concentration was 2mM) was used to establish the injury model for 4 h. Tumor necrosis factor (Tumor necrosis factor- 偽 (TNF- 偽), nitric oxide synthase (Nitric-oxide synthase,NOS), protein kinase C (Protein kinase (C (Protein kinase) inhibitor H398 were added into the external chamber of co-culture system. L-NMMA (apparent osmotic coefficient (Apparent Permeability Coefficient,Papp) of Rh123 was determined by NG-Monomethyl-L-arginine.monoacetate), BIM (Bisindolylmaleimide) to reflect the changes of P-gp function on rBMECs induced by pseudo ischemic injury. The Papp of sodium fluorescein (Fluorescein Sodium Injection,NaF) was determined to ensure the integrity of rBMECs. Western Blot method was used to determine the factors that might cause the changes of P-gp expression and function, and then the corresponding inhibitors were added to determine the changes of factors and P-gp expression, to study the mechanism of the effect of pseudo ischemic injury on P-gp on rBMECs. Results: 1. The co-culture system of rBMECs, neurons and glial cells was successfully established by Transwell. 2. RBMECs injury induced by Na2S2O4 (final concentration 2mM) was induced for 4 h. Compared with the blank group, the Papp value of Rh123 in the injured group decreased significantly from the inner chamber to the outer chamber, and the Papp value from the outer chamber to the inner chamber was significantly enhanced, which indicated that the function of P-gp was enhanced. There was no significant change in the Papp value of NaF, which indicated that the close connection of rBMECs was not affected, and the accuracy of the experimental results was ensured. 3.Western Blot results showed that the expression of TNF- 偽, NOS and PKC in rBMECs injury induced by Na2S2O4 (final concentration was 2mM) was significantly increased. H398 had no obvious effect on the expression of TNF- 偽 but could down-regulate the expression of NOS,PKC,P-gp, L-NMMA had no obvious effect on the expression of TNF- 偽, but could down-regulate the expression of PKC,P-gp. BIM had no significant effect on the expression of TNF- 偽 and NOS, but could down-regulate the expression of P-gp. Conclusion: during ischemic injury, rBMECs is stimulated to synthesize and release pro-inflammatory cytokines TNF- 偽, TNF- 偽 and then activate NOS,PKC to regulate and up-regulate the expression of P-gp on rBMECs. It provides a new specific target for the drug therapy of cerebral apoplexy.
【學(xué)位授予單位】：河南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R743

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 劉曉東,劉國(guó)卿;P-糖蛋白調(diào)節(jié)血腦屏障上的谷氨酸轉(zhuǎn)運(yùn)(英文)[J];Acta Pharmacologica Sinica;2001年02期

2 ;Neuroprotective effects of scutellarin on rat neuronal damage induced by cerebral ischemia/reperfusion[J];Acta Pharmacologica Sinica;2005年12期

3 賈繼明;王宗權(quán);吳立軍;吳以嶺;;人參皂苷Rb_1的藥理活性研究進(jìn)展[J];中國(guó)中藥雜志;2008年12期

4 韓金安,胡威夷,孫增會(huì);顱腦損傷后鈣、鈣調(diào)蛋白的變化及三七總皂甙的治療作用[J];中國(guó)中西醫(yī)結(jié)合雜志;1999年04期

5 胡海燕;崔志慧;陳翔;王文花;張曉艷;;清心開(kāi)竅方含藥腦脊液對(duì)連二亞硫酸鈉所致PC12細(xì)胞損傷的保護(hù)機(jī)制研究[J];中國(guó)中藥雜志;2013年09期

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