CAJ | 학술논문

目的探讨分泌型凋亡相关蛋白1 (secreted apoptosis-related protein 1,SARP1)调控增生性瘢痕患者的成纤维细胞(fibroblast,FB)凋亡的作用.方法构建SARP1腺病毒载体(Ad-SARP1),感染瘢痕患者的皮肤FB,促进其表达SARP1蛋白,观察其蛋白表达后人FB增殖与凋亡的变化,明确SARP1蛋白对FB的调控作用,并通过转移酶介导的三磷酸脱氧鸟苷-生物素刻痕末端标记(TUNEL)方法、流式细胞仪(FACS)分析细胞增殖与凋亡动态变化.结果成功构建Ad-SARP1,并能够感染人FB,通过逆转录-聚合酶链式反应(RT-PCR)检测到SARP1的mRNA值明显增加,Western印迹方法检测SARP1蛋白表达明显增多(P＜0.05);其蛋白表达后四甲基偶氮噻唑蓝(MTT)法检测,与带荧光的腺病毒空载体(Ad-EGFP)及对照组相比,Ad-SARP1感染的FB增殖活力均显著增高;TUNEL检测Ad-SARP1感染FB前后细胞凋亡结果显示,细胞凋亡明显受到抑制,凋亡阳性细胞变少,接近瘢痕成纤维细胞(HSFB)凋亡;FACS分析表明,感染组FB的细胞凋亡指数与对照组相比明显下降(P＜0.01).结论 SARP1参与调控增生性瘢痕患者的FB功能,抑制细胞凋亡,促进细胞增殖加快.
목적 탐토분비형조망상관단백1 (secreted apoptosis-related protein 1,SARP1)조공증생성반흔환자적성섬유세포(fibroblast,FB)조망적작용.방법 구건SARP1선병독재체(Ad-SARP1),감염반흔환자적피부FB,촉진기표체SARP1단백,관찰기단백표체후인FB증식여조망적변화,명학SARP1단백대FB적조공작용,병통과전이매개도적삼린산탈양조감-생물소각흔말단표기(TUNEL)방법、류식세포의(FACS)분석세포증식여조망동태변화.결과 성공구건Ad-SARP1,병능구감염인FB,통과역전록-취합매련식반응(RT-PCR)검측도SARP1적mRNA치명현증가,Western인적방법검측SARP1단백표체명현증다(P＜0.05);기단백표체후사갑기우담새서람(MTT)법검측,여대형광적선병독공재체(Ad-EGFP)급대조조상비,Ad-SARP1감염적FB증식활력균현저증고;TUNEL검측Ad-SARP1감염FB전후세포조망결과현시,세포조망명현수도억제,조망양성세포변소,접근반흔성섬유세포(HSFB)조망;FACS분석표명,감염조FB적세포조망지수여대조조상비명현하강(P＜0.01).결론 SARP1삼여조공증생성반흔환자적FB공능,억제세포조망,촉진세포증식가쾌.
Objective To explore the effects of secreted apoptosis-related protein 1 (SARP1) on apoptosis of the hyperstrophic scar fibroblasts (HSFB) and its regulating mechnisms.Methods The recombinant vector was identified by enzyme digestion analysis.And the virus supernatant of the recombinant vector was extracted from packaged 293 cells,then it infected the skin fibroblasts from hypertrophic scar patients,which aimed to promote its expression of SARP1 protein.After adenovirus infection,the expression of SARP1 in the fibroblasts was confirmed by RT-PCR and Western blot.The effect of SARP1 on proliferation of HSFB was detected by MTT assay,and the effect of SARP1 on apoptosis of HSFB was detected and change of the cells functions were analyzed by FACS.Results Recombinants were confirmed.After adenovirus infection,both protein and mRNA of SARP1 were detected in HSFB.And the mRNA value of SARPlwas detected to increase significantly by RT-PCR and the protein expression was detected to increase significantly by Western blot (P＜0.05).The proliferation in the groups of the adenovirus infection and HSFB was positively regulated by SARP1 (P＜0.01) and the apoptosis of them was inhibited by the expression of SARP1 as compared to the control groups of HSFB and Ad-EGFP.It showed that the apoptosis index decreased as compared the group of infected fibroblasts to the control group by flow cytometry.Conclusions SARP1 could be highly expressed in HSFB by adenovirus infection,exhibiting the proliferation-enhancing and apoptosis-inhibiting effects on HSFB.