CAJ | 학술논문

背景: 烧创伤后创面愈合多伴有瘢痕增生,内皮细胞在瘢痕增生中具有重要作用,抑制内皮细胞的生长可以在一定程度上减轻瘢痕增生.目的: 构建含有人内皮抑索基因的重组腺病毒Ad/hEnd,并观察与感染该病毒的角朊细胞共培养时内皮细胞增殖特性的改变.设计、时间及地点: 观察对照实验,于2006-09/2007-05在解放军第三军医大学西南医院烧伤研究所国家重点实验室完成.材料: pAdTrack-CMV及pAdEasy-1购自美国Stratagene公司:293细胞及大肠杆菌Ecoli.DH5 α由本室保存.方法: 以人胎肝组织mRNA为模板,通过反转录-聚合酶链反应及聚合酶链反应获得内皮抑素基因序列,插入到腺病毒穿梭质粒pAdTrack-CMV中,获得重组质粒pAdTrack-ES.经鉴定后将阳性重组子转化至pAdeasy1受体菌,筛选阳性克隆.脂质体介导转染293细胞,获取Ad/hEnd,经聚合酶链反应鉴定及上清中内皮抑素含量检测后,感染角朊细胞,采用套皿法与内皮细胞共培养.并与未转染的角朊细胞进行对照观察.主要观察指标: pAd/hEnd的同源重组及其鉴定,Ad/hEnd的产生与鉴定,转染293细胞后内皮抑素的表达,Ad/hEnd的纯化与滴度测定,培养液中内皮抑素含量,内皮细胞凋亡百分数及内皮细胞抑制率测定.结果: ①成功获取了Ad/hEnd,病毒滴度可达1.65×1012PFU/L.②感染Ad/hEnd的角朊细胞可有效表达并分泌内皮抑素,连续培养3 d后,培养液中内皮抑素含量可达226 μg/L.③与转基因角朊细胞共培养的内皮细胞凋亡百分数与细胞抑制率均显著高于对照组(P<0.05).结论: 与内皮细胞共培养时.转Ad/hEnd角朊细胞可通过分泌内皮抑素促进内皮细胞凋亡,并抑制其增殖.
배경: 소창상후창면유합다반유반흔증생,내피세포재반흔증생중구유중요작용,억제내피세포적생장가이재일정정도상감경반흔증생.목적: 구건함유인내피억색기인적중조선병독Ad/hEnd,병관찰여감염해병독적각원세포공배양시내피세포증식특성적개변.설계、시간급지점: 관찰대조실험,우2006-09/2007-05재해방군제삼군의대학서남의원소상연구소국가중점실험실완성.재료: pAdTrack-CMV급pAdEasy-1구자미국Stratagene공사:293세포급대장간균Ecoli.DH5 α유본실보존.방법: 이인태간조직mRNA위모판,통과반전록-취합매련반응급취합매련반응획득내피억소기인서렬,삽입도선병독천사질립pAdTrack-CMV중,획득중조질립pAdTrack-ES.경감정후장양성중조자전화지pAdeasy1수체균,사선양성극륭.지질체개도전염293세포,획취Ad/hEnd,경취합매련반응감정급상청중내피억소함량검측후,감염각원세포,채용투명법여내피세포공배양.병여미전염적각원세포진행대조관찰.주요관찰지표: pAd/hEnd적동원중조급기감정,Ad/hEnd적산생여감정,전염293세포후내피억소적표체,Ad/hEnd적순화여적도측정,배양액중내피억소함량,내피세포조망백분수급내피세포억제솔측정.결과: ①성공획취료Ad/hEnd,병독적도가체1.65×1012PFU/L.②감염Ad/hEnd적각원세포가유효표체병분비내피억소,련속배양3 d후,배양액중내피억소함량가체226 μg/L.③여전기인각원세포공배양적내피세포조망백분수여세포억제솔균현저고우대조조(P<0.05).결론: 여내피세포공배양시.전Ad/hEnd각원세포가통과분비내피억소촉진내피세포조망,병억제기증식.
BACKGROUND: Scar hypertrophy is always followed by the wound healing in burn and trauma. Endothelial cells play a key role in scar hypertrophy, so inhibitory growth of endothelial cells can relieve scar hypertrophy to a certain degree. OBJECTIVE: To construct a recombinant adenovirus vector expressing human endostatin (Ad/hEnd), and to investigate the cooperative effect of Ad/hEnd and keratinocyte on endothelial cell proliferation. DESIGN, TIME AND SETTING: Observational study, which was performed in the State Key Laboratory of Trauma, Burn and Combined Injury, Institute of Burn Research, Southwest Hospital of the Third Military Medical University of Chinese PLA between September 2006 and May 2007. MATERIALS: pAdTrack-CMV and pAdEasy-1 were obtained from Stratagene Company, USA; 293 cell and Ecoli.DH5α were stored in our laboratory. METHODS: The endostatin gene sequence was obtained by reverse transcription-polymerase chain reaction (RT-PCR) and polymerase chain reaction (PCR) based on mRNA of human fetal hepatic tissue and inserted into the adenovims shuttle plasmid pAdTrack-CMV to obtain recombinant plasmid pAdTrack-ES. After identification, positive recon was transformed into pAdeasy 1 recipient virus to screen positive clones. The adenovirus Ad/hEnd was generated from 293 cells and identified by PCR and fluorescence microscope. Then the keratinocytes were infected with Ad/hEnd, and co-cultured with endothelial cells by nest dish culture method. The content of endostatin was detected, and the non-transfection keratinocytes were used as the controls. MAIN OUTCOME MEASURES: Homologous recombination and identification of pAd/hEnd; generation and identification of Ad/hEnd; endostatin expression after 293 cell transfection; purification and titer measurement of Ad/hEnd; content of endostatin in culture solution; apoptotic percentage of endothelial cells; inhibitory ratio of endothelial cells. RESULTS: Ad/hEnd was constructed and the virus titer was generally up to 1.65×1012 PFU/L. Ad/hEnd-infected keratinocytes could effectively express and secrete endostatin of which the content reached 226 μg/L after 3 days of co-culture. The apoptotic percentage and inhibitory ratio of the endothelial cells co-cultured with Ad/hEnd-infected keratinocytes were significantly higher than those in control group (P<0.05). CONCLUSION: Ad/hEnd-infected keratinocytes co-cultured with endothelial cells can promote apoptosis and inhibit proliferation of endothelial cells through excretion of endostatin.