CAJ | 학술논문

目的分析人不同发育阶段表皮干细胞基因表达变化特征,探讨其可能的生物学意义.方法收集胎龄28～32周胎儿、4～12岁少儿、35～55岁成人3组正常皮肤组织标本,每组10例.采用胰蛋白酶和乙二胺四乙酸联合消化法分离表皮,Ⅳ型胶原快速黏附法分离、纯化表皮干细胞,免疫细胞化学染色法行整合素β1、角蛋白19单克降抗体检测鉴定.Trizol一步法分别提取各组细胞总RNA,琼脂糖甲醛变性凝胶电泳质检.制备探针并与表达谱芯片进行杂交,扫描芯片荧光信号图像.对芯片图像进行分析,以2倍差异表达值筛选差异表达基因.选择明显上调或下凋的基因,用实时RT-PGR技术进一步验证相关基因.结果与少儿组比较,成人组差异表达基因1808个,其中上调的基因1089个、下调的基因719个,已知基因1462个、未知基因346个.少儿组样本与胎儿组比较,差异表达基因4534个,其中上调的基因1783个、下调的基因2751个,已知基因3577个、未知基因957个.根据基因功能分类,成人组与少儿组差异表达基因可分为128类,少儿组与胎儿组差异表达基因可分为216类.1104个基因在胎儿组、少儿组与成人组样本中呈持续差异表达,根据基因功能分为32类.实验检测到持续差异表达基因中,有94个差异表达基因呈持续上调状,75个差异表达基因呈持续下调趋势.实时RT-PCR验证结果与芯片筛选结果一致.结论体外培养的胎儿、少儿与成人表皮干细胞基因表达谱有明显不同,其差异可能与不同发育阶段人表皮干细胞的增殖分化能力及皮肤创伤修复能力不同密切相关.
목적 분석인불동발육계단표피간세포기인표체변화특정,탐토기가능적생물학의의.방법 수집태령28～32주태인、4～12세소인、35～55세성인3조정상피부조직표본,매조10례.채용이단백매화을이알사을산연합소화법분리표피,Ⅳ형효원쾌속점부법분리、순화표피간세포,면역세포화학염색법행정합소β1、각단백19단극강항체검측감정.Trizol일보법분별제취각조세포총RNA,경지당갑철변성응효전영질검.제비탐침병여표체보심편진행잡교,소묘심편형광신호도상.대심편도상진행분석,이2배차이표체치사선차이표체기인.선택명현상조혹하조적기인,용실시RT-PGR기술진일보험증상관기인.결과 여소인조비교,성인조차이표체기인1808개,기중상조적기인1089개、하조적기인719개,이지기인1462개、미지기인346개.소인조양본여태인조비교,차이표체기인4534개,기중상조적기인1783개、하조적기인2751개,이지기인3577개、미지기인957개.근거기인공능분류,성인조여소인조차이표체기인가분위128류,소인조여태인조차이표체기인가분위216류.1104개기인재태인조、소인조여성인조양본중정지속차이표체,근거기인공능분위32류.실험검측도지속차이표체기인중,유94개차이표체기인정지속상조상,75개차이표체기인정지속하조추세.실시RT-PCR험증결과여심편사선결과일치.결론 체외배양적태인、소인여성인표피간세포기인표체보유명현불동,기차이가능여불동발육계단인표피간세포적증식분화능력급피부창상수복능력불동밀절상관.
Objective To analyze expression characteristics of human skin epidermal stem cell at different developmental stages, and to explore its biological significance. Methods Health skin samples from 28-32 w fetuses(F group) , 4-12 y children(C group) , and 35-55 y adult(A group) were harvested,with 10 cases in each group. Epidermis were separated using trypsin digestion and EDTA, and human epidermal stem cells were isolated and purified with type Ⅳ collagen attachment method. The monoclonal antibody of integrin β1 and keratin 19 were used for detection and identification of epidermal stem cells by immunohistochemical staining. Total RNA was extracted from above cells by Trizol one-step method, and were detected by formaldehyde denaturing agarose gel electrophoresis. Probes were prepared and hybridized into cDNA microarray for scanning fluorescent signals and analysis of images, with two-fold differential expression value for screening. Significantly up/down-regulated genes were selected for verification by real time RT-PCR. Results By comparing expression profile between A and C groups, a total of 1808 genes with differential expression were detected, including 1089 up-regulated genes and 719 down-regulated genes, and they were classified into 128 categories. Among them, 1462 genes were known (found in GeneBank), 346 genes were unknown. A total of 4534 genes with differential expression were detected between C and F groups, in which 1783 genes were up-regulated and 2751 genes were down-regulated, and they were classified into 216 categories. Among them, 3577 genes were known (found in GeneBank), and 957 genes were unknown. There were 1104 genes with differential expression consistently detected in F, C and A groups,which were classified into 32 categories according to gene function. Among them, 94 genes were consistently up-regulated and 75 genes consistently down-regulated. Test results of real time RT-PCR were in accordance with above-mentioned results. Conclusions Gene expression profiles of epidermal stem cells cultured in vitro, harvested from fetuses, children, and adult, exhibit obvious difference. This may be closely related to different stages of proliferation and differentiation of human epidermal stem cell and self-repair ability of wound at different developmental stages.