北京大学学报（医学版）
北京大學學報（醫學版）
북경대학학보（의학판）
JOURNAL OF BEIJING MEDICAL UNIVERSITY(HEALTH SCIENCES)
2015年
2期
305-311
,共7页
任玉兰%战园%路璐%李盛林%傅歆%俞光岩%曹彤%刘鹤
任玉蘭%戰園%路璐%李盛林%傅歆%俞光巖%曹彤%劉鶴
임옥란%전완%로로%리성림%부흠%유광암%조동%류학
胚胎干细胞%角蛋白细胞%核型分析%上皮细胞
胚胎榦細胞%角蛋白細胞%覈型分析%上皮細胞
배태간세포%각단백세포%핵형분석%상피세포
Embryonic stem cells%Keratinocytes%Karyotyping%Epithelial cells
目的：诱导人胚胎干细胞（ human embryonic stem cells，hESCs）定向分化为角质形成细胞K-hESCs（ kerati-nocyte derived from human embryonic stem cells），并分析诱导过程中K-hESCs不同标志物的表达特点。方法：运用含骨形成蛋白4、维甲酸和N2添加剂的上皮分化培养基直接诱导hESCs分化为K-hESCs，通过染色体核型分析检测K-hESCs核型，通过实时定量PCR检测K-hESCs在分化的不同时期基因的表达及其与原代人牙龈上皮细胞（ hu-man gingival epithelial cells，HGECs）、人永生化口腔上皮细胞（ human immortalized oral epithelial cells，HIOECs）、人永生化皮肤角质形成细胞HaCaT的基因表达差异；利用免疫组织化学检测不同标志物在K-hESCs的蛋白表达。结果：运用上皮分化培养基成功地诱导hESCs分化为上皮样细胞K-hESCs；K-hESCs核型具有正常46条染色体，无结构异常；K-hESCs中角质形成细胞标志物基因p63的表达明显低于HaCaT细胞（P＜0．05），而与HGECs和HIOECs中的表达差异无统计学意义（ P＞0．05）。结论：运用上皮分化培养基可成功诱导hESCs分化为具有正常核型的K-hESCs；标志物的表达特点提示诱导hESCs分化为K-hESCs的过程是从hESCs向单层上皮干细胞分化继而转向复层上皮终末分化发展的趋势，最终得到的K-hESCs类似于单层鳞状上皮干细胞向终末分化初始阶段的角质形成细胞，该阶段的细胞由上皮干细胞静止状态被激活，处于分化初始高增殖活力阶段。
目的：誘導人胚胎榦細胞（ human embryonic stem cells，hESCs）定嚮分化為角質形成細胞K-hESCs（ kerati-nocyte derived from human embryonic stem cells），併分析誘導過程中K-hESCs不同標誌物的錶達特點。方法：運用含骨形成蛋白4、維甲痠和N2添加劑的上皮分化培養基直接誘導hESCs分化為K-hESCs，通過染色體覈型分析檢測K-hESCs覈型，通過實時定量PCR檢測K-hESCs在分化的不同時期基因的錶達及其與原代人牙齦上皮細胞（ hu-man gingival epithelial cells，HGECs）、人永生化口腔上皮細胞（ human immortalized oral epithelial cells，HIOECs）、人永生化皮膚角質形成細胞HaCaT的基因錶達差異；利用免疫組織化學檢測不同標誌物在K-hESCs的蛋白錶達。結果：運用上皮分化培養基成功地誘導hESCs分化為上皮樣細胞K-hESCs；K-hESCs覈型具有正常46條染色體，無結構異常；K-hESCs中角質形成細胞標誌物基因p63的錶達明顯低于HaCaT細胞（P＜0．05），而與HGECs和HIOECs中的錶達差異無統計學意義（ P＞0．05）。結論：運用上皮分化培養基可成功誘導hESCs分化為具有正常覈型的K-hESCs；標誌物的錶達特點提示誘導hESCs分化為K-hESCs的過程是從hESCs嚮單層上皮榦細胞分化繼而轉嚮複層上皮終末分化髮展的趨勢，最終得到的K-hESCs類似于單層鱗狀上皮榦細胞嚮終末分化初始階段的角質形成細胞，該階段的細胞由上皮榦細胞靜止狀態被激活，處于分化初始高增殖活力階段。
목적：유도인배태간세포（ human embryonic stem cells，hESCs）정향분화위각질형성세포K-hESCs（ kerati-nocyte derived from human embryonic stem cells），병분석유도과정중K-hESCs불동표지물적표체특점。방법：운용함골형성단백4、유갑산화N2첨가제적상피분화배양기직접유도hESCs분화위K-hESCs，통과염색체핵형분석검측K-hESCs핵형，통과실시정량PCR검측K-hESCs재분화적불동시기기인적표체급기여원대인아간상피세포（ hu-man gingival epithelial cells，HGECs）、인영생화구강상피세포（ human immortalized oral epithelial cells，HIOECs）、인영생화피부각질형성세포HaCaT적기인표체차이；이용면역조직화학검측불동표지물재K-hESCs적단백표체。결과：운용상피분화배양기성공지유도hESCs분화위상피양세포K-hESCs；K-hESCs핵형구유정상46조염색체，무결구이상；K-hESCs중각질형성세포표지물기인p63적표체명현저우HaCaT세포（P＜0．05），이여HGECs화HIOECs중적표체차이무통계학의의（ P＞0．05）。결론：운용상피분화배양기가성공유도hESCs분화위구유정상핵형적K-hESCs；표지물적표체특점제시유도hESCs분화위K-hESCs적과정시종hESCs향단층상피간세포분화계이전향복층상피종말분화발전적추세，최종득도적K-hESCs유사우단층린상상피간세포향종말분화초시계단적각질형성세포，해계단적세포유상피간세포정지상태피격활，처우분화초시고증식활력계단。
Objective:To differentiate human embryonic stem cells ( hESCs ) into keratinocytes ( K-hESCs) and analyse the expression characteristics of biomarkers of K-hESCs.Methods: The hESCs of line H9 were seeded on matrigel in mTeSR1 medium.The hESCs were directly differentiated into kerati-nocytes in epithelial differentiation medium with bone morphogenetic protein 4, retinoic acid and N2 sup-plement.The karyotype of K-hESCs was analyzed, comparing the gene expression differences of K-hESCs with human gingival epithelial cells (HGECs), human immortalized oral epithelial cells (HIOECs) and HaCaT by Real-time PCR.Molecular characteristics of the cell differentiation were observed throughout the process by immunocytochemical techniques.Results:H9-hESCs were successfully differentiated into the cells that exhibited characteristics of keratinocytes in epithelial differentiation medium.The karyotype of K-hESCs was 46, XX; and the keratinocyte gene p63 expression in K-hESCs was significantly lower than that in HaCaT ( P<0.05) , but there was no significant difference of p63 expression in K-hESCs, comparing with that in HGECs and HIOECs ( P >0.05 ) .Conclusion: H9-hESCs could be directly differentiated into K-hESCs.The gene expression of K-hESCs was similar to that of epithelial cells in the early stage of monolayer cells differentiation with high proliferative activity.
