抗人脑微血管内皮细胞单域抗体P5的制备及对人和大鼠血脑屏障的影响  

Construction and Effect of Single Domain Antibody P5 against Human Brain Microvascular Endothelial Cells on the Blood-brain Barrier in Human and Rats

作　　者：王萍[1] 王义宝[2] 薛一雪[1] 

机构地区：[1]中国医科大学基础医学院神经生物学教研室 [2]中国医科大学第一附属医院神经外科,辽宁沈阳110001

出　　处：《解剖科学进展》2009年第1期29-31,共3页Progress of Anatomical Sciences

基　　金：国家自然科学基金(No.30670723);辽宁省自然科学基金(No.20052102);辽宁省教育厅科学技术计划项目(No.2008850);沈阳市科学技术计划基金(No.1072033-1-00;1081266-9-00)

摘　　要：目的制备抗人脑微血管内皮细胞单域抗体P5,研究P5对大鼠血脑屏障结构的影响。方法采用PCR技术将已获得的P5基因片段克隆至载体pET-30a-c并测序确证,重组载体经转化诱导后通过Western blot鉴定。采用免疫荧光和免疫组织化学方法观察P5在人及大鼠脑微血管内皮细胞中的结合水平,透射电镜观察大鼠血脑屏障超微结构的变化。结果Western blot结果显示表达产物的分子量为128kD,随着单域抗体P5(1μg/ml)作用时间的延长,P5与人及大鼠的脑微血管内皮细胞结合水平增加。透射电镜结果显示随着给予单域抗体P5浓度的增大(1μg/kg,10μg/kg),脑微血管内皮细胞中吞饮小泡的数量呈现一定程度的增加。结论成功获得抗人脑微血管内皮细胞单域抗体P5,P5能增加血脑屏障的胞吞转运。Objective To construct single domain antibody （sdAb） P5 against human brain microvascular endothelial cells （BMECs） and study the effect of P5 on the structure of blood-brain barrier （BBB） in rats. Methods PCR was used to clone the obtained P5 gene into pET-30a-c vector and sequenced. After transformation and induction, recombinant vector was judged by Western blot. Immunofluorescence and immunohistochemistry methods were used to observe the combination of P5 with BMEC of human and rats. Transmission electron microscope （TEM） was used to observe the ultrastrnctural changes of BBB in rats. Results Western blot results showed that the molecular weight of the protein was 128 kD. The uptake of sdAb P5 by BMECs of human and rats was increased with the prolonged treatment of sdAb P5（1μg/ml）. The number of pinocytotic vesicles also rose to a higher level with the increase of the concentration of sdAb PS（1 μg/kg, 10 μg/kg）. Conclusion The sdAb P5 against hBMEC was constructed successfully and increased the transcytosis of BBB.

关 键 词：抗人脑微血管内皮细胞单域抗体P5 血脑屏障 胞吞转运 人 大鼠 

分 类 号：Q463[生物学—生理学]