CAJ | 학술논문

探讨人促红细胞生成素(rhEPO)对低氯损伤胶质细胞的影响及对金属蛋白酶-9(MMP-9)表达的影响.通过体外纯化培养第3代星形胶质细胞,将其分为正常组、低氧组、rhEPO预处理组以四唑蓝(MTT)比色法测定低氧培养12、24、36h细胞的存活率,倒置显微镜和透射电镜观察低氧对胶质细胞形态的影响;免疫荧光和逆转录一聚合酶链反应(RT-PCR)方法研究低氧对星形胶质细胞MMP-9表达的影响.结果显示:低氧组星形胶质细胞在低氧培养下出现细胞肿胀.且随时间的延长而加重,rhEPO预处理组在各时间点细胞肿胀明显轻于低氧组.rhEPO能减轻细胞超微结构的改变及降低MTT比值.RT-PCR及免疫荧光检测表明:低氧组MMP-9 mRNA及蛋白的表达在低氧各时间点均高于正常组.在24h达到最高,在36h开始降低(P＜0.05);rhEPO预处理组MMP-9mRNA及蛋白的表达变化在12、24、36h较低氧组低(P＜0.05).由此得出结论:rhEPO通过抑制MMP-9的表达促进低氯条件下星形胶质细胞的存活.
탐토인촉홍세포생성소(rhEPO)대저록손상효질세포적영향급대금속단백매-9(MMP-9)표체적영향.통과체외순화배양제3대성형효질세포,장기분위정상조、저양조、rhEPO예처리조이사서람(MTT)비색법측정저양배양12、24、36h세포적존활솔,도치현미경화투사전경관찰저양대효질세포형태적영향;면역형광화역전록일취합매련반응(RT-PCR)방법연구저양대성형효질세포MMP-9표체적영향.결과현시:저양조성형효질세포재저양배양하출현세포종창.차수시간적연장이가중,rhEPO예처리조재각시간점세포종창명현경우저양조.rhEPO능감경세포초미결구적개변급강저MTT비치.RT-PCR급면역형광검측표명:저양조MMP-9 mRNA급단백적표체재저양각시간점균고우정상조.재24h체도최고,재36h개시강저(P＜0.05);rhEPO예처리조MMP-9mRNA급단백적표체변화재12、24、36h교저양조저(P＜0.05).유차득출결론:rhEPO통과억제MMP-9적표체촉진저록조건하성형효질세포적존활.
To investigation the effect of recombinant human erythropoietin preconditioning on the injury of astrocytes and the expression of MMP-9 after hypoxia, cerebral cortical astrocytes, newborn SD rat within 2 days were collected with the pure culture and the astrocytes were divided into control group, hypoxia group and rhEPO preconditioning group. The survival rate of astrocytes of 12h, 24h and 36h were determined by MTI' assay. Inverted microscope and transmission electron microscope were used to observe the cell changes. The expression of MMP-9 mRNA and protein of astrocytes were detected by immunofluorescence and RT-PCR. It is shown that the astrocytes were swelled after hypoxia cultured 12h, with the maximal cell swelling observed at 36h. The rhEPO preconditioning can reduce the cell swelling remarkably. Ultrastruetural alterations of astrocytes such as mitochondrial swelling after hypoxia can also be alleviated remarkably by rhEPO preconditioning. The results of MTT assay show that the survival rate of rhEPO preconditioning group were obviously higher than that of the hypoxia group (P＜0.05). RT-PCR and immunocytochemistry analysis confirm that the expression of MMP-9 mRNA and protein was remarkably increased at 12h, peaked at 24h and dechned at 36h after hypoxia cultured. Compared with hypoxia group, the expression of MMP-9 mRNA and protein in the rhEPO preconditioning group was significantly lower at 12h, 24h and 36h (P＜0.05). The results show that rhEPO can improve the survival rate of astrocytes under hypoxia by inhibition of MMP-9.