人孤雌胚胎干细胞无饲养层培养体系的建立-论文.pdf

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1、中国修复重建外科杂志2013年5月第27卷第5期·559·人孤雌胚胎干细胞无饲养层培养体系的建立梁锐王志强陈天星朱静朱妹李英杨龙朱宝生【摘要】目的建立一种安全、有效、经济且适于人孤雌胚胎干细胞(humanparthenogeneticembryonicstemcells，hPESCs)体外培养的无饲养层培养体系。方法将常规体外培养的hPESCs分别以mTeSRr”1培养基(对照组)和人包皮成纤维细胞的条件培养基(humanforeskinfibrobtasts．conditionalmedium，hFFs．CM)(实验组)扩增培养，倒置显微镜下观察

2、两组无饲养层培养体系下hPESCs的生长状态；采用ALP检测和核型分析研究hPESCs生物学特性；采用RT-PCR检测hPESCs全能性标记物Oct．4的表达情况；通过体外和体内分化实验观察hPESCs向3个胚层分化的潜能。结果两组hPESCs形态规则、不易分化，在形态、扩增速度等方面无明显差异；已成功在体外培养l5代，两组均能保持正常女性的二倍体核型46，XX和全能性；RT-PCR检测示两组Oct．4mRNA均呈阳性表达；体外分化均可形成拟胚体；在裸鼠体内均可形成含有3个胚层组织成分的畸胎瘤。结论hFFs．CM无饲养层培养体系可长期支持hPESC

3、s的生长并维持其未分化状态，成功建立了一种不仅能维持hPESCs的有效扩增、减少动物源性污染、降低培养成本，还可满足l临床大规模应用的hPESCs无饲养层培养体系。【关键词】人孤雌胚胎干细胞无饲养层条件培养基人包皮成纤维细胞ESTABLISHMENTOFFEEDER．FREECUL．TURESYSTEM0FHUMANPARTHENOGENETICEMBRYONICSTEMCELLS／LIANGRui，WANGZhiqiange,CHENTianxingd，ZHUJing~，ZHUShu，LIYing~，YANGLo，ZHUBaoshen~．Depa

4、rtmentofPathology,GeneticDiagnosisCente~theFirstPeople’sHospitalofYunnanProvince,KunmingYunnan，650032，P．R．China；DepartmentofOncology,砌BFirstAfiliatedHospitalofKunmingMedicalUniversity．Correspondingauthor：WANGZhiqiang,E—mail：wzqcy2000@126．com【Abstract】ObjectiveToestablishasafe，

5、effective，andeconomicfeeder—freeculturesystemwhichissuitableforthecultureofhumanparthenogeneticembryonicstemcells(hPESCs)nvitro．MethodshPESCswereculturedwithmTeSR1medium(controlgroup)andhumanforeskinfibroblasts—conditionalmedium(hFFs—CM)(experimentalgroup)．ThegrowthstatusofhPE

6、SCsinbothfeeder—freeculturesystemswereobservedwithinvertedmicroscope．Alkalinephosphatase(ALP)analysisandkaryotypeanalysiswereusedtostudythebiologicalcharacteristicsofhPESCs．TheexpressionofhPESCspluripotentmarkerOct一4wasanalyzedbyRT—PCR．Differentiationexperimentinvivoandinvitro

7、wasappliedtoobservethedifferentiationpotentialofhPESCsintothreegermlayers．ResultshPESCshadregularmorphologywithdificultyindifferentiationinbothculturesystems．NoobviousdifferencewasobservedinmorphologyandexpansionspeedofhPESCsbetween2groups．Aftersubculturedfor15passagesinvitro，

8、hPESCsin2groupscouldmaintainnormalfemalediploidkaryotype46，XX