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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:构建含有人内皮抑素基因的重组腺病毒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.DH5α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3d后,培养液中内皮抑素含量可达226μg/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。
BACKGROUND: Burned wounds are often accompanied with scar hyperplasia after wound healing. Endothelial cells play an important role in the proliferation of scar. Inhibition of the growth of endothelial cells can reduce scar hyperplasia to a certain extent. OBJECTIVE: To construct recombinant adenovirus Ad / hEnd containing human endostatin gene and observe the changes of endothelial cell proliferation characteristics when co-cultured with keratinocytes infected with the virus. DESIGN, TIME AND SETTING: Observational control experiments were performed at the State Key Laboratory of Burn Research, Southwest Hospital, Third Military Medical University, China from September 2006 to May 2007. Materials: pAdTrack-CMV and pAdEasy-1 were purchased from Stratagene Company, USA; 293 cells and E. coli Ecoli DH5α were stored in our laboratory. Methods: Endostatin gene sequence was obtained by reverse transcription - polymerase chain reaction and polymerase chain reaction using human fetal liver mRNA as a template. The recombinant plasmid pAdTrack-ES was inserted into the adenovirus shuttle plasmid pAdTrack-CMV. After identification, the positive recombinant was transformed into pAdeasy1 receptor and the positive clones were screened. Lipofectamine was transfected into 293 cells to obtain Ad / hEnd. After being identified by polymerase chain reaction and endostatin in the supernatant, keratinocytes were infected and co-cultured with endothelial cells using a cuff method. And compared with untransfected keratinocytes. MAIN OUTCOME MEASURES: The homologous recombination and identification of pAd / hEnd, the production and identification of Ad / hEnd, the expression of endostatin after transfection of 293 cells, the purification and titer of Ad / hEnd, the concentration of endostatin Content, percentage of endothelial cell apoptosis and inhibition of endothelial cells. Results: ① Ad / hEnd was successfully obtained and the virus titer reached 1.65 × 1012 PFU / L. ② The keratinocytes infected with Ad / hEnd could effectively express and secrete endostatin. After three days of continuous culture, the content of endostatin in the culture fluid reached 226μg / L. (3) The percentage of endothelial cell apoptosis and cell inhibition co-cultured with transgenic keratinocytes were significantly higher than that of the control group (P <0.05). CONCLUSION: When co-cultured with endothelial cells, transdifferentiation of Ad / hEnd keratinocytes can promote endothelial cell apoptosis and inhibit its proliferation by secreting endostatin.