中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2013年
5期
934-936
,共3页
项晓飞%程飚%唐建兵%彭艳%朱江婷
項曉飛%程飚%唐建兵%彭豔%硃江婷
항효비%정표%당건병%팽염%주강정
表皮干细胞%阿片受体%增殖%迁移
錶皮榦細胞%阿片受體%增殖%遷移
표피간세포%아편수체%증식%천이
Epidermal stem cells%Opioid receptor%Proliferation%Migration
目的 观察μ型阿片受体的活化对体外培养的人表皮干细胞(hESCs)增殖与迁移的影响.方法 分离培养原代hESCs,整合素β1、细胞角蛋白19 (CK19)染色鉴定.取第2代hESCs分别用含1 nmol/L β-内啡肽的K-SFM培养基(A组)、含10 nmol/L纳洛酮及1 nmol/L β-内啡肽的K-SFM培养基(B组)以及单纯的K-SFM培养基(C组)进行培养；连续5d,采用噻唑蓝(MTT)法检测hESCs分裂增殖活性.于培养后24 h刮擦生长融合成片的hESCs,制备宽度为100 μm的体外单层hESCs缺损模型,模型制备后24、48、72、96 h,倒置相差显微镜下观察各组表皮创面愈合,并拍照记录,计算模型制备后72 h各组创面愈合率.结果 原代培养的hESCs贴壁后呈鹅卵石样生长.免疫荧光染色法检测第2代hESCs的整合素β1、CK19均呈阳性表达.分组培养后,MTT法测得第2～4天中A组hESCs分裂增殖活性均高于C组,B组低于C组,各组间差异有统计学意义(P＜0.05).单层细胞缺损模型制备后24 h,各组均可见hESCs迁移越过创缘,其中A组越过创缘的细胞数多于C组,B组少于C组；模型制备72 h后,由Image-Pro Plus软件分析各组创面愈合率分别为A组(91.80±0.05)％、B组(79.40±0.06)％、C组(86.30 ±0.75)％,各组间差异均有统计学意义(P＜0.05).结论 μ型阿片受体活化后可促进hESCs分裂增殖和迁移活动,并可能藉此参与皮肤多种代谢调控过程.
目的 觀察μ型阿片受體的活化對體外培養的人錶皮榦細胞(hESCs)增殖與遷移的影響.方法 分離培養原代hESCs,整閤素β1、細胞角蛋白19 (CK19)染色鑒定.取第2代hESCs分彆用含1 nmol/L β-內啡肽的K-SFM培養基(A組)、含10 nmol/L納洛酮及1 nmol/L β-內啡肽的K-SFM培養基(B組)以及單純的K-SFM培養基(C組)進行培養；連續5d,採用噻唑藍(MTT)法檢測hESCs分裂增殖活性.于培養後24 h颳抆生長融閤成片的hESCs,製備寬度為100 μm的體外單層hESCs缺損模型,模型製備後24、48、72、96 h,倒置相差顯微鏡下觀察各組錶皮創麵愈閤,併拍照記錄,計算模型製備後72 h各組創麵愈閤率.結果 原代培養的hESCs貼壁後呈鵝卵石樣生長.免疫熒光染色法檢測第2代hESCs的整閤素β1、CK19均呈暘性錶達.分組培養後,MTT法測得第2～4天中A組hESCs分裂增殖活性均高于C組,B組低于C組,各組間差異有統計學意義(P＜0.05).單層細胞缺損模型製備後24 h,各組均可見hESCs遷移越過創緣,其中A組越過創緣的細胞數多于C組,B組少于C組；模型製備72 h後,由Image-Pro Plus軟件分析各組創麵愈閤率分彆為A組(91.80±0.05)％、B組(79.40±0.06)％、C組(86.30 ±0.75)％,各組間差異均有統計學意義(P＜0.05).結論 μ型阿片受體活化後可促進hESCs分裂增殖和遷移活動,併可能藉此參與皮膚多種代謝調控過程.
목적 관찰μ형아편수체적활화대체외배양적인표피간세포(hESCs)증식여천이적영향.방법 분리배양원대hESCs,정합소β1、세포각단백19 (CK19)염색감정.취제2대hESCs분별용함1 nmol/L β-내배태적K-SFM배양기(A조)、함10 nmol/L납락동급1 nmol/L β-내배태적K-SFM배양기(B조)이급단순적K-SFM배양기(C조)진행배양；련속5d,채용새서람(MTT)법검측hESCs분렬증식활성.우배양후24 h괄찰생장융합성편적hESCs,제비관도위100 μm적체외단층hESCs결손모형,모형제비후24、48、72、96 h,도치상차현미경하관찰각조표피창면유합,병박조기록,계산모형제비후72 h각조창면유합솔.결과 원대배양적hESCs첩벽후정아란석양생장.면역형광염색법검측제2대hESCs적정합소β1、CK19균정양성표체.분조배양후,MTT법측득제2～4천중A조hESCs분렬증식활성균고우C조,B조저우C조,각조간차이유통계학의의(P＜0.05).단층세포결손모형제비후24 h,각조균가견hESCs천이월과창연,기중A조월과창연적세포수다우C조,B조소우C조；모형제비72 h후,유Image-Pro Plus연건분석각조창면유합솔분별위A조(91.80±0.05)％、B조(79.40±0.06)％、C조(86.30 ±0.75)％,각조간차이균유통계학의의(P＜0.05).결론 μ형아편수체활화후가촉진hESCs분렬증식화천이활동,병가능자차삼여피부다충대사조공과정.
Objective To investigate the affects of μ-opioid receptor activation on proliferation and migration of epidermal stem cells (ESCs) in vitro.Methods Primary human ESCs (hESCs) were isolated and cultured,and marked by both integrin β31 and eytokeratin 19 (CK19) to be identified.The hESCs of the second generation were cultured with K-SFM supplemented with 1 nmol/L β-endorphins in group A,with K-SFM supplemented with 10 nmol/L naloxone and 1 nmol/L β-endorphins in group B,and with K-SFM supplemented with no drugs in group C.The proliferative ability of hESCs in each group was determined by using methyl thiazol tetrazolium (MTT) assay for 5 consecutive days.The 100 μm "scratch" wounds were created by scraping confluent hESCs plated on Petri-dishes with a sterile pipette tip in vitro.The pictures of migrating cells from the wound edge were taken at 24,48,72 and 96 h after incubarton with different media.The wound healing rate was calculated by Image-Pro Plus software at 72 h.Results Cultured primary hESCs could adhere to the wall and showed cobblestone-like shape.Immunofluorescence staining showed the positive results for integrin β1 and CK19.The proliferation rate of hESCs detected hy using MTT assay was significantly higher in group A than in groups C,visibly higher in group C than in group B during the day 2 to day 5,and the difference between each two groups was statistically significant (P ＜ 0.05).In group A,the number of cells migrating the wound edge was greater than in group C,and less in group B than in group C at 24 h.The wound healing rate in groups A,B and C at 72 h was (91.80 ± 0.05) ％,(79.40 ± 0.06) ％,and (86.30 ± 0.75) ％ respectively,showing significant differences among groups (P ＜ 0.05).Conclusion The activation of μ-opioid receptor can promote the proliferation and migration of hESCs in vitro.The μ-opioid receptor may be involved in some modutory processes of skin metabolisms.
