中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2008年
8期
1586-1590
,共5页
人胚胎干细胞%成纤维细胞%饲养层%混合
人胚胎榦細胞%成纖維細胞%飼養層%混閤
인배태간세포%성섬유세포%사양층%혼합
背景:人胚胎干细胞传代培养的关键是抑制其自发分化、保证细胞的全能性.以小鼠胚胎成纤维细胞或人包皮成纤维细胞作为饲养层尽管能够维持胚胎干细胞的未分化状态,但存在细胞克隆不饱满、平铺情况明显等问题.目的:制备小鼠胚胎成纤维细胞与人包皮成纤维细胞的混合饲养层,观察人胚胎干细胞在其上面的生长状态.设计:多样本观察比较.单位:海南医学院附属医院生殖医学中心.材料:实验于2006-04/2007-07在海南医学院附属医院生殖医学中心完成.包皮来自于行包皮环切的儿童,由海南医学院附属医院泌尿外科提供,儿童家属对治疗及实验均签署知情同意书,实验经医院医学伦理委员会批准.人胚胎干细胞系HN-1由本实验室从人类囊胚中分离培养并鉴定.清洁级孕12.5～14.5 d的胎鼠11只,实验过程中对动物的处置符合动物伦理学标准.方法:胎鼠麻醉后去除头、四肢和内脏,按常规胰蛋白酶反复消化法获得细胞悬液进行接种培养,待生长汇合后冻存部分原代细胞,用丝裂霉素C处理2.0～3.0 h后,按1×108 L-1密度接种于明胶包被的中心皿内,即小鼠胚胎成纤维细胞饲养层.人包皮成纤维细胞的分离培养与饲养层制备同上.上述两种成纤维细胞分别计数后,按1∶0,3∶1,1∶1,1∶3,0∶1比例混合,然后以1×108 L-1密度接种于明胶包被的中心皿内,即混合饲养层.观察体外传代培养的人胚胎干细胞在3种不同饲养层上的生长状态,并对生长在混合饲养层上的人胚胎干细胞进行碱性磷酸酶检测、OCT-4表达免疫组化检测、OCT-4及端粒酶mRNA表达RT-PCR检测.撤除饲养层,观察人胚胎干细胞体外分化情况.主要观察指标:①不同饲养层上人胚胎干细胞的生长状态比较.②人胚胎干细胞在不同比例混合饲养层上的生长状态比较.③混合饲养层上人胚胎干细胞未分化状态的检测.④体外分化实验.结果:①:生长在小鼠胚胎成纤维细胞和人包皮成纤维细胞上的人胚胎干细胞克隆扁平、不饱满,而生长在混合饲养层上的人胚胎干细胞克隆饱满、厚实,其克隆形态显著好于其他两种饲养层.②:小鼠胚胎成纤维细胞:人包皮成纤维细胞按1∶1混合时,人胚胎干细胞显著堆积生长,克隆边缘清晰、隆起明显且饱满,按1:3混合时无明显变化,优于其余3种混合比例.③:碱性磷酸酶染色及OCT-4抗原表达均呈强阳性,分别在200～300 bp和300～400 bp处可见OCT-4和端粒酶mRNA表达的特异性条带.④:能形成拟胚体,贴壁后人胚胎干细胞可分化为多种形态的细胞.?＃
结论:①与小鼠胚胎成纤维细胞或人包皮成纤维细胞常规饲养层相比,两者混合饲养层能够更好的支持人胚胎干细胞的体外传代培养,获得更佳的克隆形态.②小鼠胚胎成纤维细胞与人包皮成纤维细胞的混合比例为1∶1时效果较好.
揹景:人胚胎榦細胞傳代培養的關鍵是抑製其自髮分化、保證細胞的全能性.以小鼠胚胎成纖維細胞或人包皮成纖維細胞作為飼養層儘管能夠維持胚胎榦細胞的未分化狀態,但存在細胞剋隆不飽滿、平鋪情況明顯等問題.目的:製備小鼠胚胎成纖維細胞與人包皮成纖維細胞的混閤飼養層,觀察人胚胎榦細胞在其上麵的生長狀態.設計:多樣本觀察比較.單位:海南醫學院附屬醫院生殖醫學中心.材料:實驗于2006-04/2007-07在海南醫學院附屬醫院生殖醫學中心完成.包皮來自于行包皮環切的兒童,由海南醫學院附屬醫院泌尿外科提供,兒童傢屬對治療及實驗均籤署知情同意書,實驗經醫院醫學倫理委員會批準.人胚胎榦細胞繫HN-1由本實驗室從人類囊胚中分離培養併鑒定.清潔級孕12.5～14.5 d的胎鼠11隻,實驗過程中對動物的處置符閤動物倫理學標準.方法:胎鼠痳醉後去除頭、四肢和內髒,按常規胰蛋白酶反複消化法穫得細胞懸液進行接種培養,待生長彙閤後凍存部分原代細胞,用絲裂黴素C處理2.0～3.0 h後,按1×108 L-1密度接種于明膠包被的中心皿內,即小鼠胚胎成纖維細胞飼養層.人包皮成纖維細胞的分離培養與飼養層製備同上.上述兩種成纖維細胞分彆計數後,按1∶0,3∶1,1∶1,1∶3,0∶1比例混閤,然後以1×108 L-1密度接種于明膠包被的中心皿內,即混閤飼養層.觀察體外傳代培養的人胚胎榦細胞在3種不同飼養層上的生長狀態,併對生長在混閤飼養層上的人胚胎榦細胞進行堿性燐痠酶檢測、OCT-4錶達免疫組化檢測、OCT-4及耑粒酶mRNA錶達RT-PCR檢測.撤除飼養層,觀察人胚胎榦細胞體外分化情況.主要觀察指標:①不同飼養層上人胚胎榦細胞的生長狀態比較.②人胚胎榦細胞在不同比例混閤飼養層上的生長狀態比較.③混閤飼養層上人胚胎榦細胞未分化狀態的檢測.④體外分化實驗.結果:①:生長在小鼠胚胎成纖維細胞和人包皮成纖維細胞上的人胚胎榦細胞剋隆扁平、不飽滿,而生長在混閤飼養層上的人胚胎榦細胞剋隆飽滿、厚實,其剋隆形態顯著好于其他兩種飼養層.②:小鼠胚胎成纖維細胞:人包皮成纖維細胞按1∶1混閤時,人胚胎榦細胞顯著堆積生長,剋隆邊緣清晰、隆起明顯且飽滿,按1:3混閤時無明顯變化,優于其餘3種混閤比例.③:堿性燐痠酶染色及OCT-4抗原錶達均呈彊暘性,分彆在200～300 bp和300～400 bp處可見OCT-4和耑粒酶mRNA錶達的特異性條帶.④:能形成擬胚體,貼壁後人胚胎榦細胞可分化為多種形態的細胞.?＃
結論:①與小鼠胚胎成纖維細胞或人包皮成纖維細胞常規飼養層相比,兩者混閤飼養層能夠更好的支持人胚胎榦細胞的體外傳代培養,穫得更佳的剋隆形態.②小鼠胚胎成纖維細胞與人包皮成纖維細胞的混閤比例為1∶1時效果較好.
배경:인배태간세포전대배양적관건시억제기자발분화、보증세포적전능성.이소서배태성섬유세포혹인포피성섬유세포작위사양층진관능구유지배태간세포적미분화상태,단존재세포극륭불포만、평포정황명현등문제.목적:제비소서배태성섬유세포여인포피성섬유세포적혼합사양층,관찰인배태간세포재기상면적생장상태.설계:다양본관찰비교.단위:해남의학원부속의원생식의학중심.재료:실험우2006-04/2007-07재해남의학원부속의원생식의학중심완성.포피래자우행포피배절적인동,유해남의학원부속의원비뇨외과제공,인동가속대치료급실험균첨서지정동의서,실험경의원의학윤리위원회비준.인배태간세포계HN-1유본실험실종인류낭배중분리배양병감정.청길급잉12.5～14.5 d적태서11지,실험과정중대동물적처치부합동물윤리학표준.방법:태서마취후거제두、사지화내장,안상규이단백매반복소화법획득세포현액진행접충배양,대생장회합후동존부분원대세포,용사렬매소C처리2.0～3.0 h후,안1×108 L-1밀도접충우명효포피적중심명내,즉소서배태성섬유세포사양층.인포피성섬유세포적분리배양여사양층제비동상.상술량충성섬유세포분별계수후,안1∶0,3∶1,1∶1,1∶3,0∶1비례혼합,연후이1×108 L-1밀도접충우명효포피적중심명내,즉혼합사양층.관찰체외전대배양적인배태간세포재3충불동사양층상적생장상태,병대생장재혼합사양층상적인배태간세포진행감성린산매검측、OCT-4표체면역조화검측、OCT-4급단립매mRNA표체RT-PCR검측.철제사양층,관찰인배태간세포체외분화정황.주요관찰지표:①불동사양층상인배태간세포적생장상태비교.②인배태간세포재불동비례혼합사양층상적생장상태비교.③혼합사양층상인배태간세포미분화상태적검측.④체외분화실험.결과:①:생장재소서배태성섬유세포화인포피성섬유세포상적인배태간세포극륭편평、불포만,이생장재혼합사양층상적인배태간세포극륭포만、후실,기극륭형태현저호우기타량충사양층.②:소서배태성섬유세포:인포피성섬유세포안1∶1혼합시,인배태간세포현저퇴적생장,극륭변연청석、륭기명현차포만,안1:3혼합시무명현변화,우우기여3충혼합비례.③:감성린산매염색급OCT-4항원표체균정강양성,분별재200～300 bp화300～400 bp처가견OCT-4화단립매mRNA표체적특이성조대.④:능형성의배체,첩벽후인배태간세포가분화위다충형태적세포.?＃
결론:①여소서배태성섬유세포혹인포피성섬유세포상규사양층상비,량자혼합사양층능구경호적지지인배태간세포적체외전대배양,획득경가적극륭형태.②소서배태성섬유세포여인포피성섬유세포적혼합비례위1∶1시효과교호.
BACKGROUND: Key point for subculture of human embryonic stem cells (hESCs) is to inhibit spontaneous differentiation and make sure totipotency of cells. Although mouse embryonic fibroblasts (MEF) or human foreskin fibroblasts (HFF) used as the feeder layer can maintain undifferentiated state of embryonic stem cells, cell clone is still imperfect and parallel arranged. OBJECTIVE: To establish mixed feeder layer of mouse embryonic fibroblasts plus human foreskin fibroblasts and to observe the hESCs growth.DESIGN: Multi-sample observation and comparison.SETTING: Medical Center of Reproduction, the Affiliated Hospital of Hainan Medical College. MATERIALS: This study was performed at the Medical Center of Reproduction, the Affiliated Hospital of Hainan Medical College from April 2006 to July 2007. Foreskin was derived from the children who underwent circumcision and came from Urinary Surgery, the Affiliated Hospital of Haihan Medical College. The children's family members provided the informed consent, and the experiment received confirmed consent from the local ethic committee. hESCs line HN-1 was separated from human blastula. Eleven 12.5-14.5-day-old fetal mice of clean grade were selected in this study. The experimental animals were disposed according to ethical criteria. METHODS: Heads, four extremities, and viscera were removed from fetal mice under anesthesia. Subsequently, cell suspension was prepared using routine trypsinase digestion and inoculated. When cells were cultured in confluent monolayer, some primary cells were frozen, processed with mitocin-C for 2.0-3.0 hours, and inoculated based on the density of 1×108 L-1 in gelatin-coated dish, I.e., MEF feeder layer. The HFF separation and culture and the preparation of HFF feeder layer were the same as above-mentioned processing. In addition, the two fibroblasts were respectively counted and mixed together according to the ratios of 1∶0, 3∶1, 1∶1, 1∶3, and 0:1. And then, the mixture was inoculated based on the density of 1×108 L-1 in gelatin-coated dish, I.e., mixed feeder layer. The growth of subcultured hESCs in vitro was observed in three different feeder layers, and hESCs in the mixed feeder layer underwent alkaline phosphatase test, OCT-4 expression immunohistochemical measurement, and OCT-4 and telomerase mRNA expression RT-PCR detection. Finally, differentiation in vitro of hESCs was observed after removing the feeder layer.MAIN OUTCOME MEASURES: ① Growth of hESCs in the three different feeder layers; ② Growth of hESCs in the mixed feeder layer based on different mixed ratios; ③ undifferentiated state of hESCs in the mixed feeder layer; ④ differentiation in vitro.RESULTS: ① hESCs clone in the MEF and HFF feeder layers was thin and flat and imperfect, but hESCs clone in the mixed feeder layer was perfect and thick and solid. Apparently, the clone form in the mixed feeder layer was superior to MEF and HFF feeder layers. ② When MEF and HFF was mixed together according to the ratio of 1∶1, hESCs grew in apparent accumulation; clone border was clear; eminentia was apparent and perfect. However, there were no changes according to the ratio of 1∶3. The ratio of 1∶1 was superior to the ratios of 1∶0, 3∶1, and 0∶1. ③ Alkaline phosphatase staining and OCT-4 antigen expression were strongly positive. Specific straps of OCT-4 and telomerase mRNA expression were observed at 200-300 bp and 300-400 bp, respectively. ④ Embryoid bodies were formed. hESCs could differentiate into multi-morphological cells after attachment.CONCLUSION: ① The mixed feeder layer may well support in vitro subculture of hESCs to acquire excellent clone form compared to MEF or HFF feeder layer. ② The mixture of MEF and HFF has excellent effect according to the ratio of 1∶1.
