CAJ | 학술논문

目的探讨高压氧预适应对体外血脑屏障内皮细胞紧密连接的影响及机制.方法通过hCMEC/D3细胞培养建立体外血脑屏障模型.将培养的hCMEC/D3细胞按数字表法随机分为3组:空白对照组(A组)、缺氧组(B组)及高压氧预处理组(C组).采用Millicell-ERS系统测定血脑屏障的跨内皮阻抗(TEER).采用细胞免疫荧光检测内皮细胞紧密连接蛋白oOccludin(≥)及ZO-1的表达变化.结果正常培养的hCMEC/D3细胞形态呈梭形,细胞之间紧密结合,TEER值达80 ～120 Q·cm2,提示成功建立体外血脑屏障.相对于A组[(107.17±10.41)Q·cm2],B、C两组TEER值[(50.02±6.87)Ω·cm2、(83.81±8.22)Ω·cm2]均有所下降,但B组下降更明显.免疫荧光结果显示:相对于A组,B组Occludin的表达水平明显降低,而C组无明显变化.相对于A组,B组ZO-1在细胞膜表达明显降低,而胞浆表达增多,而C组ZO-1的表达无明显变化.结论高压氧预适应可以缓解缺氧时紧密连接蛋白Occludin和ZO-1表达水平及方式的改变.
목적 탐토고압양예괄응대체외혈뇌병장내피세포긴밀련접적영향급궤제.방법 통과hCMEC/D3세포배양건입체외혈뇌병장모형.장배양적hCMEC/D3세포안수자표법수궤분위3조:공백대조조(A조)、결양조(B조)급고압양예처리조(C조).채용Millicell-ERS계통측정혈뇌병장적과내피조항(TEER).채용세포면역형광검측내피세포긴밀련접단백oOccludin(≥)급ZO-1적표체변화.결과 정상배양적hCMEC/D3세포형태정사형,세포지간긴밀결합,TEER치체80 ～120 Q·cm2,제시성공건입체외혈뇌병장.상대우A조[(107.17±10.41)Q·cm2],B、C량조TEER치[(50.02±6.87)Ω·cm2、(83.81±8.22)Ω·cm2]균유소하강,단B조하강경명현.면역형광결과현시:상대우A조,B조Occludin적표체수평명현강저,이C조무명현변화.상대우A조,B조ZO-1재세포막표체명현강저,이포장표체증다,이C조ZO-1적표체무명현변화.결론 고압양예괄응가이완해결양시긴밀련접단백Occludin화ZO-1표체수평급방식적개변.
Objective To explore the effects of hyperbaric oxygen preconditioning (HBOP) on close connection of endothelial cells in in-vitro blood-brain barrier and possible mechanism involved.Methods The blood-brain barrier model was developed by using hCMEC/D3 cell line.Then, the cultured cells were randomly divided into 3 groups: the blank control group (Group A), the hypoxia group (Group B) and the HBOP group (Group C).Millicel1-ERS was used to test TEER, while immunofluorescence was performed to detect changes in the expressions of occludin and ZO-1.Results The normal cultured hCMEC/D3 displayed a spindle shape,with the cells closely connected and the value of TEER as high as 80-120 Ω · cm2 , indicating that the in vitro blood-brain barrier model was successfully established.Relative to the TEER value of group A [107.17 ±l0.41)Ω·cm2], theTEER values of group B and group C [(50.02±6.87)Ω · cm2, (83.81 ±8.22) Ω · cm2] all decreased quite considerably, with the TEER value of group B decreasing more significantly.Immunofluorenscence detection results indicated that as compared with group A, the expression level of occludin in group B decreased significantly, while no significant expression level of occludin was noticed in group C.As compared with group A, ZO-1 expression level in the cell membrane of group B decreased significantly, while the expression level of cytoplasm increased, and there were no significant changes in ZO-1 expression level in group C.Conclusions HBO preconditioning could decrease changes in the expression levels of occludin and ZO-1 induced by hypoxia.