中华创伤杂志
中華創傷雜誌
중화창상잡지
Chinese Journal of Traumatology
2014年
5期
394-399
,共6页
表皮%干细胞%基因表达谱
錶皮%榦細胞%基因錶達譜
표피%간세포%기인표체보
Epidermis%Stem cells%Gene expression profiling
目的 探讨人表皮中具有未分化特性的表皮干细胞和角质形成细胞之间微小RNA(micro-RNA,miRNA)表达谱的差异. 方法 (1)采用酶消化法和Ⅳ型胶原快速贴壁相结合的方法获得人原代表皮干细胞及角质形成细胞.倒置显微镜下观察培养细胞的生长状况,免疫细胞化学染色法行β1整合素、角蛋白1(keratin 1,CK1)、CK10、CK19单克隆抗体检测鉴定.(2)Trizol 一步法分别提取表皮干细胞和角质形成细胞总RNA,甲醛变性胶电泳质检.mirVanaTM miRNA分离试剂盒对其进行纯化,使用miRNA标记和杂交试剂盒进行荧光标记及芯片杂交,利用FeatureExtraction(V 10.7)软件对杂交图片进行分析,GeneSpring(GX 10.0)软件进行数据归一化及差异分析,同时应用RT-PCR法验证miRNA芯片结果的可靠性.(3)预测差异表达的miRNA的靶基因. 结果 (1)快速黏附于Ⅳ型胶原的细胞群培养3d能形成明显克隆,免疫细胞化学染色显示β1整合素及CK19呈阳性表达,为表皮干细胞;不能快速黏附于Ⅳ型胶原的细胞群培养3d无明显克隆形成,CK1及CK10呈阳性表达,为已分化角质形成细胞.(2)筛选出表皮干细胞中表达上调的miRNA 31个,表达下调的miRNA 153个.其中显著上调的miRNA有hsa-miRNA-125b-3p、hsa-miRNA-197-5p、hsa-miRNA-376a-3p等;显著下调的miRNA有hsa-miRNA-203、hsa-miRNA-29b-3p、hsa-miRNA-34a-3p等.其中上调的hsa-miRNA-197-5p和下调的hsa-miRNA-29b-3p的RT-PCR验证结果与芯片检测结果具有较好的一致性.(3)部分miRNA靶基因预测提示miRNA与细胞增殖分化、凋亡衰老等生物学特性有关. 结论 人表皮干细胞与角质形成细胞的miRNA表达存在明显差异,可能与两者不同的增殖分化能力等生物学特性有关.
目的 探討人錶皮中具有未分化特性的錶皮榦細胞和角質形成細胞之間微小RNA(micro-RNA,miRNA)錶達譜的差異. 方法 (1)採用酶消化法和Ⅳ型膠原快速貼壁相結閤的方法穫得人原代錶皮榦細胞及角質形成細胞.倒置顯微鏡下觀察培養細胞的生長狀況,免疫細胞化學染色法行β1整閤素、角蛋白1(keratin 1,CK1)、CK10、CK19單剋隆抗體檢測鑒定.(2)Trizol 一步法分彆提取錶皮榦細胞和角質形成細胞總RNA,甲醛變性膠電泳質檢.mirVanaTM miRNA分離試劑盒對其進行純化,使用miRNA標記和雜交試劑盒進行熒光標記及芯片雜交,利用FeatureExtraction(V 10.7)軟件對雜交圖片進行分析,GeneSpring(GX 10.0)軟件進行數據歸一化及差異分析,同時應用RT-PCR法驗證miRNA芯片結果的可靠性.(3)預測差異錶達的miRNA的靶基因. 結果 (1)快速黏附于Ⅳ型膠原的細胞群培養3d能形成明顯剋隆,免疫細胞化學染色顯示β1整閤素及CK19呈暘性錶達,為錶皮榦細胞;不能快速黏附于Ⅳ型膠原的細胞群培養3d無明顯剋隆形成,CK1及CK10呈暘性錶達,為已分化角質形成細胞.(2)篩選齣錶皮榦細胞中錶達上調的miRNA 31箇,錶達下調的miRNA 153箇.其中顯著上調的miRNA有hsa-miRNA-125b-3p、hsa-miRNA-197-5p、hsa-miRNA-376a-3p等;顯著下調的miRNA有hsa-miRNA-203、hsa-miRNA-29b-3p、hsa-miRNA-34a-3p等.其中上調的hsa-miRNA-197-5p和下調的hsa-miRNA-29b-3p的RT-PCR驗證結果與芯片檢測結果具有較好的一緻性.(3)部分miRNA靶基因預測提示miRNA與細胞增殖分化、凋亡衰老等生物學特性有關. 結論 人錶皮榦細胞與角質形成細胞的miRNA錶達存在明顯差異,可能與兩者不同的增殖分化能力等生物學特性有關.
목적 탐토인표피중구유미분화특성적표피간세포화각질형성세포지간미소RNA(micro-RNA,miRNA)표체보적차이. 방법 (1)채용매소화법화Ⅳ형효원쾌속첩벽상결합적방법획득인원대표피간세포급각질형성세포.도치현미경하관찰배양세포적생장상황,면역세포화학염색법행β1정합소、각단백1(keratin 1,CK1)、CK10、CK19단극륭항체검측감정.(2)Trizol 일보법분별제취표피간세포화각질형성세포총RNA,갑철변성효전영질검.mirVanaTM miRNA분리시제합대기진행순화,사용miRNA표기화잡교시제합진행형광표기급심편잡교,이용FeatureExtraction(V 10.7)연건대잡교도편진행분석,GeneSpring(GX 10.0)연건진행수거귀일화급차이분석,동시응용RT-PCR법험증miRNA심편결과적가고성.(3)예측차이표체적miRNA적파기인. 결과 (1)쾌속점부우Ⅳ형효원적세포군배양3d능형성명현극륭,면역세포화학염색현시β1정합소급CK19정양성표체,위표피간세포;불능쾌속점부우Ⅳ형효원적세포군배양3d무명현극륭형성,CK1급CK10정양성표체,위이분화각질형성세포.(2)사선출표피간세포중표체상조적miRNA 31개,표체하조적miRNA 153개.기중현저상조적miRNA유hsa-miRNA-125b-3p、hsa-miRNA-197-5p、hsa-miRNA-376a-3p등;현저하조적miRNA유hsa-miRNA-203、hsa-miRNA-29b-3p、hsa-miRNA-34a-3p등.기중상조적hsa-miRNA-197-5p화하조적hsa-miRNA-29b-3p적RT-PCR험증결과여심편검측결과구유교호적일치성.(3)부분miRNA파기인예측제시miRNA여세포증식분화、조망쇠로등생물학특성유관. 결론 인표피간세포여각질형성세포적miRNA표체존재명현차이,가능여량자불동적증식분화능력등생물학특성유관.
Objective To investigate the difference in expression profiles of micro-RNA (miR-NA) between human epidermal stem cells and epidermal keratinocytes.Methods (1) Human primary epidermal stem cells and keratinocytes were obtained with enzyme digestion method and type Ⅳ collagen coated chosen method.Growth of cells cultured in vitro was observed by inverted microscope.Monoclonal antibody of integrinβ1,keratin 1 (CK1),CK10,and CK19 were detected by immunocytochemical staining.(2)Total RNA was respectively isolated from epidermal stem cells and epidermal keratinocytes by Trizol-based single-step procedure,detected by formaldehyde denaturing gel electrophoresis,purified by mirVanaTM miRNA isolation kit,and then labeled and hybridized by miRNA labeling and hybridization kit.Images of hybridization were analyzed using the Feature Extraction (Version 10.7).Data normalization and difference analysis were performed with GeneSpring (GX 10.0).Moreover,miRNA microarray results were confirmed by RT-PCR.(3) Target genes of differently expressed miRNA were predicted.Results Epidermal stem cells exhibited rapid adherence to type Ⅳ collagen and formed distinct clones after 3 days of culture; expressions of integrinβ1 and CK19 were positive.Keratinocytes could not adhere rapidly to type Ⅳ collagen and formed few clones after 3 days of culture ; expressions of CK1 and CK10 were positive.(2) Of the epidermal stem cells,31 miRNAs were up-regulated and 153 down-regulated.Besides,significantly up-regulated miRNAs were hsa-miRNA-125b-3p,hsa-miRNA-197-5p,and hsa-miRNA-376a-3p,while significantly down-regulated miRNAs were hsa-miRNA-203,hsa-miRNA-29b-3p,and hsa-miRNA-34a-3p.Findings of RT-PCR on hsa-miRNA-197-5p and hsa-miR-29b-3p revealed high concordance with the microarray results.(3) Some miRNAs target genes indicated that miRNA related to cell proliferation,differentiation,apoptosis,aging and other biological characteristics.Conclusion Distinct differences in miRNA expression profiles are detected between human epidermal stem cells and keratinocytes and this may correlate closely with their different biological characteristics such as proliferation and differentiation ability.
