CAJ | 학술논문

目的观察P311在小鼠浅Ⅱ度烧伤及体外细胞创伤模型中对表皮干细胞(ESC)迁移的作用并探讨其机制. 方法 (1)取18只C57BL/6小鼠,其中15只行背部浅Ⅱ度烧伤,于伤后6、12、24、48、72 h取创面及创周皮肤组织,各时相点3只；余下3只小鼠作为正常对照,同法取正常皮肤标本.采用生物素-链霉亲和素-过氧化物酶(SP)染色法观察P311在上述皮肤组织中的表达.(2)取6只新生的C57BL/6小鼠,连续3d腹腔注射50 μg/g溴脱氧尿苷(BrdU,2次/d)建立ESC示踪模型.7周后于其背部制作浅Ⅱ度烧伤创面.伤后72 h取创面皮肤组织制作连续切片,SP法免疫组织化学染色,观察P311阳性和BrdU阳性细胞空间共定位情况.(3)构建腺病毒空载体pAdEasy-增强型绿色荧光蛋白(EGFP)及P311腺病毒表达载体pAdEasy-EGFP-P311,并进行包装.应用Ⅳ型胶原快速黏附法分离培养人ESC,按照随机数字表法将其分为P311高表达组和EGFP对照组,每组3孔,分别以pAdEasy-EGFP-P311及pAdEasy-EGFP腺病毒感染ESC.2组ESC经丝裂霉素C处理2h后行划痕实验.划痕后0(即刻)、24、48、72 h测量固定范围内划痕剩余面积,计算划痕愈合面积百分比.对数据进行独立样本t检验. 结果 (1)浅Ⅱ度烧伤后不同时相点,P311在创面不同部位的表达量各不相同.伤后创面毛囊、新生表皮中P311表达量随时间延长逐渐上升；创周表皮及毛囊的P311表达量于伤后12 h达高峰,72 h回落至正常.(2)应用BrdU标记的小鼠浅Ⅱ度烧伤创面新生表皮层可见P311阳性细胞和BrdU阳性细胞共定位.(3)划痕后48、72 h,P311高表达组ESC划痕愈合面积百分比分别为(69±31)％、(89±26)％,明显高于EGFP对照组[(35 ±12)％、(46±31)％,t值分别为-2.336、-2.611,P值均小于0.05]. 结论 P311可明显促进小鼠浅Ⅱ度烧伤创面及体外细胞创伤模型中的ESC迁移,可能在创面修复中起重要作用.
목적 관찰P311재소서천Ⅱ도소상급체외세포창상모형중대표피간세포(ESC)천이적작용병탐토기궤제. 방법 (1)취18지C57BL/6소서,기중15지행배부천Ⅱ도소상,우상후6、12、24、48、72 h취창면급창주피부조직,각시상점3지；여하3지소서작위정상대조,동법취정상피부표본.채용생물소-련매친화소-과양화물매(SP)염색법관찰P311재상술피부조직중적표체.(2)취6지신생적C57BL/6소서,련속3d복강주사50 μg/g추탈양뇨감(BrdU,2차/d)건립ESC시종모형.7주후우기배부제작천Ⅱ도소상창면.상후72 h취창면피부조직제작련속절편,SP법면역조직화학염색,관찰P311양성화BrdU양성세포공간공정위정황.(3)구건선병독공재체pAdEasy-증강형록색형광단백(EGFP)급P311선병독표체재체pAdEasy-EGFP-P311,병진행포장.응용Ⅳ형효원쾌속점부법분리배양인ESC,안조수궤수자표법장기분위P311고표체조화EGFP대조조,매조3공,분별이pAdEasy-EGFP-P311급pAdEasy-EGFP선병독감염ESC.2조ESC경사렬매소C처리2h후행화흔실험.화흔후0(즉각)、24、48、72 h측량고정범위내화흔잉여면적,계산화흔유합면적백분비.대수거진행독립양본t검험. 결과 (1)천Ⅱ도소상후불동시상점,P311재창면불동부위적표체량각불상동.상후창면모낭、신생표피중P311표체량수시간연장축점상승；창주표피급모낭적P311표체량우상후12 h체고봉,72 h회락지정상.(2)응용BrdU표기적소서천Ⅱ도소상창면신생표피층가견P311양성세포화BrdU양성세포공정위.(3)화흔후48、72 h,P311고표체조ESC화흔유합면적백분비분별위(69±31)％、(89±26)％,명현고우EGFP대조조[(35 ±12)％、(46±31)％,t치분별위-2.336、-2.611,P치균소우0.05]. 결론 P311가명현촉진소서천Ⅱ도소상창면급체외세포창상모형중적ESC천이,가능재창면수복중기중요작용.
Objective To study effects of P311 on the migration of epidermal stem cells (ESCs) in mice with superficial partial-thickness burn and injured cell model in vitro and to explore the mechanism.Methods ( 1 ) Eighteen male C57 BL/6 mice were used.Fifteen of them were inflicted with superficial partial-thiekness burn on the back.In three injured mice wound tissue and skin of wound edge were obtained at post burn hour (PBH) 6,12,24,48,72 respectively.The rest three mice were used as normal control,and samples were harvested with the same method as above.The expressions of P311 in harvested samples were assessed with biotin-streptavidin-peroxidase (SP) staining.(2) Six newly born C57 BL/6 mice were intraperitoneally injected with 50 μg/g BrdU (two times a day) for three days for ESCs-labelling.Seven weeks later,the mice were inflicted with superficial partial-thickness burn on the back.Serial slices of burn wound tissue were prepared at PBH 72 and immunohistochemically stained with SP for observation of the co-localization of BrdU-positive ESCs and P31 1-positive cells.(3) The empty vector pAdEasy-enhanced green fluorescence protein (EGFP) and the adenovirus P311-expressing vector named pAdEasy-EGFP-P311 were constructed and packed.Human ESCs were isolated by the method of rapid adhesion to collagen Ⅳ.After being divided into P311 high-expressing group ( n =3) and EGFP control group ( n =3),the ESCs in two groups were respectively infected by pAdEasy-EGFP-P311 and pAdEasy-EGFP.Scratching assay was performed on ESCs in both groups after they were treated by mitomycin C for 2 hours.The remaining area within the fixed range was measured at post scratching hour (PSH) 0,24,48,and 72,and the wound-area healing rate was calculated.Data were processed with independent samples t test. Results ( 1 ) Expression amount of P311 was different in different parts of wound at different time points after burn.Expression amount of P311 in the newly formed epidermis and hair follicle of wound increased along with prolongation of time.Expression amount of P311 in the epidermis and hair follicle of wound edge peaked at PBH 12 and then decreased to normal levels at PBH 72.(2) Co-localization of BrdU-positive ESCs and P311-positive cells was observed in the new epidermal layer of wound tissue of mice,where ESCs were labeled by BrdU. (3) At PSH 48 and 72,wound-area healing rate was obviously higher in P311 high-expressing group [ (69 ±31)％,(89 ±26)％ ] than in EGFP control group [(35 ±12)％,(46 ±31)％,with t values respectively - 2.336,- 2.611,P values all below 0.05 ]. Conclusions P311 may promote the migration of ESCs both in rats with superficial partial-thickness burns and in injured cell model in vitro,and it may play an important role in wound healing.