中华围产医学杂志
中華圍產醫學雜誌
중화위산의학잡지
CHINESE JOURNAL OF PERINATAL MEDICINE
2009年
6期
425-429
,共5页
唐洁%胡娅莉%刁振宇%孙海翔%颜桂军%陈林君%张艳青
唐潔%鬍婭莉%刁振宇%孫海翔%顏桂軍%陳林君%張豔青
당길%호아리%조진우%손해상%안계군%진림군%장염청
先兆子(癎)%细胞内信号肽和蛋白质类%血管内皮生长因子受体1%重组蛋白质类%腺病毒科
先兆子(癎)%細胞內信號肽和蛋白質類%血管內皮生長因子受體1%重組蛋白質類%腺病毒科
선조자(간)%세포내신호태화단백질류%혈관내피생장인자수체1%중조단백질류%선병독과
Pre-eclampsia%Intraeellular signaling peptides and proteins%Vascular endothelial growth factor receptor-1%Recombinant proteins%Adenoviridae vector
目的 构建腺病毒介导的小鼠可溶性endoglin(sEng)和可溶性血管内皮生长因子受体-1(sFlt-1)重组蛋白,观察其对内皮细胞管化及滋养细胞迁移的影响.方法 提取小鼠胎盘组织总RNA,通过RT-PCR获得sEng和sFlt-1 cDNA,插入到穿梭载体pAdTrack-CMV中,获得pATC-sEng和pATC-sFlt-1质粒,转化其至pAdeasy-1受体菌,选阳性克隆转染293A细胞,获得Ad/sEng和Ad/sFlt-1,制备高滴度病毒液.用病毒感染COS7细胞,检测sEng和sFlt-1蛋白表达,并通过成管和划痕实验观察两种蛋白对人脐静脉血管内皮细胞(HUVEC)管化和BeWo细胞迁移的影响.结果 成功获得sEng和sFlt-1重组腺病毒,其滴度分别为2.2×1011pfu/ml和2.0×1011pfu/ml.感染COS7细胞后,sEng和sFlt-1蛋白均较高表达,并能抑制HUVEC管化及BeWo细胞迁移,两者有协同作用.结论 构建的腺病毒介导的小鼠sEng和sFlt-1重组蛋白对血管生成和滋养细胞迁移有明显抑制作用.
目的 構建腺病毒介導的小鼠可溶性endoglin(sEng)和可溶性血管內皮生長因子受體-1(sFlt-1)重組蛋白,觀察其對內皮細胞管化及滋養細胞遷移的影響.方法 提取小鼠胎盤組織總RNA,通過RT-PCR穫得sEng和sFlt-1 cDNA,插入到穿梭載體pAdTrack-CMV中,穫得pATC-sEng和pATC-sFlt-1質粒,轉化其至pAdeasy-1受體菌,選暘性剋隆轉染293A細胞,穫得Ad/sEng和Ad/sFlt-1,製備高滴度病毒液.用病毒感染COS7細胞,檢測sEng和sFlt-1蛋白錶達,併通過成管和劃痕實驗觀察兩種蛋白對人臍靜脈血管內皮細胞(HUVEC)管化和BeWo細胞遷移的影響.結果 成功穫得sEng和sFlt-1重組腺病毒,其滴度分彆為2.2×1011pfu/ml和2.0×1011pfu/ml.感染COS7細胞後,sEng和sFlt-1蛋白均較高錶達,併能抑製HUVEC管化及BeWo細胞遷移,兩者有協同作用.結論 構建的腺病毒介導的小鼠sEng和sFlt-1重組蛋白對血管生成和滋養細胞遷移有明顯抑製作用.
목적 구건선병독개도적소서가용성endoglin(sEng)화가용성혈관내피생장인자수체-1(sFlt-1)중조단백,관찰기대내피세포관화급자양세포천이적영향.방법 제취소서태반조직총RNA,통과RT-PCR획득sEng화sFlt-1 cDNA,삽입도천사재체pAdTrack-CMV중,획득pATC-sEng화pATC-sFlt-1질립,전화기지pAdeasy-1수체균,선양성극륭전염293A세포,획득Ad/sEng화Ad/sFlt-1,제비고적도병독액.용병독감염COS7세포,검측sEng화sFlt-1단백표체,병통과성관화화흔실험관찰량충단백대인제정맥혈관내피세포(HUVEC)관화화BeWo세포천이적영향.결과 성공획득sEng화sFlt-1중조선병독,기적도분별위2.2×1011pfu/ml화2.0×1011pfu/ml.감염COS7세포후,sEng화sFlt-1단백균교고표체,병능억제HUVEC관화급BeWo세포천이,량자유협동작용.결론 구건적선병독개도적소서sEng화sFlt-1중조단백대혈관생성화자양세포천이유명현억제작용.
Objective To construct the recombinant adenovirus Ad/sEng and Ad/sFlt-1, and investigate their effects on angiogenesis and the migration of human umbilieal vein endothelial cells (HUVEC) and trophoblast. Methods The sEng and sFlt-1 cDNA were obtained from mouse placenta by RT-PCR, which were inserted into the shuttle plasmid pAdTrack-CMV. After confirmation by endonuclease and sequencing, we transferred them into Escherichia coli. BJ5183-AD-1 cells were applied to obtain the homologous reeombinants, which were transfected into 293A cells to obtain Ad/sEng and Ad/sFlt-1, and COS7 cells were infected with them. The expressions of sFlt-1 and sEng were detected by Western blot. The effects of both proteins on HUVEC tube formation and BeWo migration were investigated. Results High titers of Ad/sEng and Ad/sFlt-1 were obtained (2.2×1011pfu /ml and 2. 0 ×1011 pfu/ml, respeetively) and both sFlt1 and sEng proteins were efficiently expressed, whieh inhibited the HUVEC tube formation and BeWo cell migration. Conclusions Adenovirus mediated mouse sFlt-1 and sEng has been constructed and expressed successfully in vitro with inhibitory effects on angiogenesis and the migration of HUVEC and trophoblast.
