经外源性血管内皮生长因子处理的大鼠表皮干细胞对深Ⅱ度烧伤大鼠创面愈合的影响及机制

Effects and mechanism of rat epidermal stem cells treated with exogenous vascular endothelial growth factor on healing of deep partial-thickness burn wounds in rats

目的探讨经外源性血管内皮生长因子(VEGF)处理的大鼠表皮干细胞(ESC)对深Ⅱ度烧伤大鼠创面愈合的影响及机制。方法从1只3个月龄雌性SD大鼠躯干皮肤分离获得ESC,取第3代对数生长期细胞进行实验(1)^(3)。(1)取细胞,采用角质形成细胞专用无血清培养基(K-SFM)常规培养(常规培养条件下同),于倒置光学显微镜下观察培养3、5 d细胞形态。(2)取细胞常规培养24 h,流式细胞仪检测细胞表面标志物CD44、CD45、CD11b和CD11c的表达,样本数为3。(3)取4个批次细胞,各批次细胞均采用随机数字表法分为空白对照组和VEGF组。空白对照组细胞常规培养,VEGF组细胞用含终质量浓度为10 ng/mL VEGF的K-SFM培养。1个批次细胞培养10 d,采用蛋白质印迹法检测细胞角蛋白19(CK19)、CK10蛋白表达;1个批次细胞培养0(即刻)、2、4、6、8、10 d,分别取细胞采用实时荧光定量反转录PCR法检测Nanog mRNA表达;1个批次细胞培养2、4、6、8、10 d,分别取细胞采用细胞计数试剂盒8测定吸光度值;1个批次细胞培养10 d,光学显微镜下观察计数>50个细胞的克隆数,计算细胞克隆形成率。各批次细胞各时间点样本数均为3。(4)选取36只3个月龄雌雄不限SD大鼠,用100℃铁质圆片在每只大鼠背部两侧压迫10 s制作2个直径为10 mm的深Ⅱ度烫伤(下称烧伤)创面。按随机数字表法将致伤大鼠分为VEGF+ESC组、单纯ESC组及空白对照组,每组12只大鼠、24个创面。伤后0(即刻)~2 d,3组大鼠每个创面均每天一次性注射20μL磷酸盐缓冲液(PBS),其中VEGF+ESC组PBS中含0.8×10^6个/mL经10 ng/mL VEGF处理10 d的ESC、单纯ESC组PBS中含0.8×10^6个/mL未经任何处理的ESC、空白对照组PBS中不含任何其他物质。于首次注射后(下称注射)0(即刻)、3、7、14 d,先按随机数字表法于每组分别取3只大鼠观察并记录创面愈合情况,计算注射3、7、14 d创面愈合率;随后处死各时间点大鼠切取创面组织行苏木精-伊红染色,行组织学观察。对数据行独立样本t检验、析因设计方差分析、LSD检验、Bonferroni校正。结果(1)培养3 d细胞呈集群分布,细胞集群生长缓慢;培养5 d集群大而圆,集群内细胞主要为细胞核大而圆、胞质少的细胞,符合ESC形态特征。(2)细胞表面CD44阳性表达率为(94.3±1.2)%,CD45、CD11b、CD11c呈阴性表达。细胞鉴定为ESC。(3)培养10 d,与空白对照组比较,VEGF组细胞CK19蛋白表达水平明显升高(t=3.756,P<0.05),CK10蛋白表达水平显著降低(t=3.149,P<0.05)。与空白对照组比较,VEGF组细胞培养0、2 d Nanog mRNA表达量和培养2、4 d吸光度值无明显变化(t=0.58、0.77,0.53、3.02,P>0.05),培养4、6、8、10 d Nanog mRNA表达量和培养6、8、10 d吸光度值均明显升高(t=6.34、5.00、5.58、4.61,5.65、10.78、15.51,P<0.01)。培养10 d,VEGF组细胞克隆形成率为(56.4±1.3)%,显著高于空白对照组的(31.5±1.3)%(t=13.96,P<0.01)。(4)注射0 d,3组大鼠创面均可见深度达真皮浅层的烧伤创面。注射3 d,VEGF+ESC组大鼠创缘正常皮肤组织轻微收缩,早于其余2组;注射7 d,VEGF+ESC组大鼠新生表皮已覆盖大部分创面,单纯ESC组大鼠创周可见部分上皮爬行,空白对照组大鼠创面未见明显上皮化现象;注射14 d,VEGF+ESC组大鼠创面基本愈合,单纯ESC组大鼠尚余少部分创面未愈合,空白对照组大鼠尚余大部分创面未愈合。3组大鼠注射3 d创面愈合率相近(P>0.05);单纯ESC组大鼠注射7、14 d创面愈合率分别为(26.0±2.0)%、(64.4±4.7)%,明显高于空白对照组的(12.4±1.1)%、(29.1±3.3)%(P<0.01),2组均明显低于VEGF+ESC组的(41.0±2.4)%、(91.3±3.5)%(P<0.01)。注射3 d,VEGF+ESC组大鼠创面可见大量炎性细胞浸润,早于其余2组;注射7 d,VEGF+ESC组大鼠创面可见大量内皮细胞,单纯ESC组大鼠创面可见部分内皮细胞增殖,空白对照组大鼠创面可见大量炎性细胞浸润;注射14 d,VEGF+ESC组大鼠创面新生表皮细胞基本覆盖创面,单纯ESC组大鼠创面新生表皮细胞覆盖大部分创面,空白对照组大鼠创面基底可见大量内皮细胞且新生表皮细胞覆盖部分创面。结论经外源性VEGF处理的大鼠ESC可促进深Ⅱ度烧伤大鼠创面愈合,这可能与VEGF促进ESC增殖并降低其分化水平从而维持细胞干性有关。

Objective To explore the effects and mechanism of rat epidermal stem cells(ESCs)that were treated with exogenous vascular endothelial growth factor(VEGF)on the healing of deep partial-thickness burn wounds in rats.Methods ESCs were isolated and cultured from the trunk skin of a 3-month-old female Sprague-Dawley(SD)rat.The third passage of cultured cells in the logarithmic growth phase was used in experiments(1)-(3).(1)The cells were routinely cultured in keratinocytes-specified serum-free medium(K-SFM)(the same routine culture condition below).The morphology of cells cultured for 3 and 5 days was observed under the inverted optical microscope.(2)After 24 hours in routine culture,the expression of cell surface markers CD44,CD45,CD11b,and CD11c was detected by flow cytometer,with triplicate samples.(3)Four batches of cells were collected,and each batch was divided into VEGF group or blank control group according to the random number table.The cells in blank control group were routinely cultured,while the cells in VEGF group were cultured in K-SFM containing VEGF in the final mass concentration of 10 ng/mL.The protein expressions of cytokeratin 19(CK19)and CK10 in cells cultured for 10 days were detected by Western blotting.The Nanog mRNA expression in cells cultured for 0(immediately),2,4,6,8,and 10 day(s)was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction.The absorbance value was detected with cell counting kit-8 in cells cultured for 2,4,6,8,and 10 days.The clone number of more than 50 cells was observed and counted under the optical microscope in cells cultured for 10 days,and the cell colony formation rate was calculated.Three samples at each time point was analysed.(4)Thirty-six 3-month-old SD rats(either male or female)were used for the study,and two deep partial-thickness burn wounds(10 mm in diameter)were created in each rat by pressing a 100℃ electric iron plate on symmetric dorsal side.According to the random number table,the injured rats were divided into VEGF+ESCs group,ESCs alone group,and blank control group,with 12 rats and 24 wounds in each group.From 0(immediately)to 2 day(s)after injury,20μL phosphate buffer solution(PBS)was injected into each wound in the three groups in one time,once a day,with the solution in VEGF+ESCs group containing 0.8×10^6 cells/mL of ESCs treated by 10 ng/mL VEGF for 10 days,the solution in ESCs alone group containing 0.8×10^6 cells/mL of ESCs without any treatment,and the solution in blank control group being PBS only.On post first injection day(PFID)0(immediately),3,7,and 14,three rats from each group were taken respectively according to the random number table for wound healing assessment,and the wound healing rates on PFID 3,7,and 14 were calculated.The mice at each time point were sacrificed with wound tissue harvested for histology,and the skin structure was observed by hematoxylin-eosin staining.Data were statistically analyzed with independent sample t test,analysis of variance for factorial design,least significant difference test,and Bonferroni correction.Results(1)By day 3 in culture,cells distributed in slowly-growing clusters.By day 5,the clusters were large and round,in which the cells mainly with large and round nuclei and little cytoplasm were observed.The above results were consistent with the morphological characteristics of ESCs.(2)The positive expression rate of CD44 was(94.3±1.2)%,and the expressions of CD45,CD11b,and CD11c were negative.The cells were confirmed as ESCs.(3)Compared with those of blank control group,the protein expression of CK19 in the cells of VEGF group was significantly increased after 10 days in culture(t=3.756,P<0.05),while the protein expression of CK10 was significantly decreased(t=3.149,P<0.05).Compared with those of blank control group,the Nanog mRNA expression in the cells cultured for 0 and 2 day(s)and absorbance values of the cells cultured for 2 and 4 day(s)were not significantly changed in VEGF group(t=0.58,0.77,0.53,3.02,P>0.05),while the Nanog mRNA expression in the cells cultured for 4,6,8,and 10 days and absorbance values of the cells cultured for 6,8,and 10 days were significantly increased in VEGF group(t=6.34,5.00,5.58,4.61,5.65,10.78,15.51,P<0.01).After 10 days in culture,the cell colony-forming rate in VEGF group was(56.4±1.3)%,significantly higher than(31.5±1.3)%of blank control group(t=13.96,P<0.01).(4)The burn wounds of rats in the three groups were confined to the superficial dermis of the skin on PFID 0.On PFID 3,normal skin tissue at wound margins slightly contracted in the rats of VEGF+ESCs group,which was earlier than that in the other two groups.On PFID 7,the newly generated epidermis covered most parts of the rat wounds in VEGF+ESCs group,and some of the epithelium crawled around the rat wounds in ESCs alone group,but no obvious epithelialization was observed in the rat wounds in blank control group.On PFID 14,the rat wounds in VEGF+ESCs group were basically healed,while some parts of the rat wounds were unhealed in ESCs alone group,and most parts of the rat wounds were unhealed in blank control group.On PFID 3,the wound healing rates of rats in the three groups were similar(P>0.05).On PFID 7 and 14,the wound healing rates of rats in ESCs alone group,respectively(26.0±2.0)% and(64.4±4.7)%,were obviously higher than(12.4±1.1)%and(29.1±3.3)% of blank control group(P<0.01),all of which were obviously lower than(41.0±2.4)%and(91.3±3.5)% of VEGF+ESCs group(P<0.01).On PFID 3,infiltration of a large number of inflammatory cells were observed in the rat wounds in VEGF+ESCs group,which was earlier than those in the other two groups.On PFID 7,a large number of endothelial cells were observed in the rat wounds in VEGF+ESCs group,while proliferation of a few endothelial cells were observed in the rat wounds in ESCs alone group,and a large number of inflammatory cells infiltrated the rat wounds in blank control group.On PFID 14,the newly generated epidermal cells covered nearly all the rat wounds in VEGF+ESCs group and most parts of the rat wounds in ESCs alone group,while a large number of endothelial cells were observed and the newly generated epidermal cells covered some parts of the rat wounds in blank control group.Conclusions ESCs of rats treated with exogenous VEGF can promote the healing of deep partial-thickness burn wounds in rats,which may be related to VEGF's roles in promoting the proliferation of ESCs and reducing its differentiation level,so as to maintain the potency of stem cells.