人孤雌胚胎干细胞在人包皮成纤维细胞饲养层上的生长状态

人孤雌胚胎干细胞(human parthenogenetic embryonic stem cells,hPESCs)体外培养常需饲养层的支持以保持干细胞特性.通过原代培养获得人包皮成纤维细胞(human foreskin fibroblasts,hFFs)并将其制备成饲养层,使hPESCs在hFFs上进行体外培养及传代.倒置显微镜下观察hPESCs的生长状态,采用碱性磷酸酶(alkalinephosphatase,AKP)检测、核型分析和体内分化实验研究hPESCs的生物学特性及分化潜能,以探索hFFs能否长期支持hPESCs的生长并维持其未分化状态.经原代培养成功获得了hFFs,通过形态学观察和免疫细胞化学染色鉴定符合成纤维细胞的生物学特性;在hFFs上生长的hPESCs克隆形态规则,不易分化;已成功在体外培养20余代,hPESCs仍能够保持基本生物学特性和正常核型,在裸鼠体内可形成含有3个胚层组织成分的畸胎瘤.作为人源性饲养层,hFFs可长期支持hPESCs的生长并维持其未分化状态.     

胰蛋白酶消化法和组织块法原代培养人包皮成纤维细胞的比较

小鼠胚胎成纤维细胞(MEFs)是目前国际上公认的人胚胎干细胞(hESCs)体外培养的饲养层细胞,但MEFs存在寿命短、动物源性污染等问题,需要探索适于hESCs体外培养且寿命长的人源性饲养层.采用胰蛋白酶消化法和组织块法分别原代培养人包皮成纤维细胞(hFFs),探索hFFs原代培养的最佳方法,为hFFs作为饲养层在hESCs研究中的应用提供可靠的科学依据;通过倒置显微镜下观察其生长状态和免疫细胞化学染色鉴定,结果显示两种原代培养方法获得的hFFs的形态及生物学特性均符合成纤维细胞;通过测定细胞生长曲线及MTT法检测,结果显示两种原代培养方法获得的不同代数的hFFs均具有较高的增殖活性,传代10余代仍能保持较好的细胞形态,可以制备成饲养层用于hESCs的研究.     

The Axenic Cultivation of Insect Forms of Trypanosoma (Duttonella) vivax and Development to the Infective Metacyclic Stage

A method for axenic cultivation of epimastigote and metacyclic forms of Trypanosoma (Duttonella) vivax at 27°C in vitro is described. Iscove's medium was supplemented with specific concentrations of foetal bovine serum, L-proline, L-glutamine, hypoxanthine, adenosine, pyruvate, and 2-mercaptoethanol. Bloodstream form parasites rapidly transformed into epimastigote forms that grew as surface-adherent colonies in plastic culture flasks. Transformation of epimastigotes to metacyclic forms was first observed 9–12 days after initiation of cultures. Percentages of metacyclics varied: East African T. vivax ranged up to 40% and West African T. vivax ranged up to 24%. Subcultures were made at two-week intervals and maintained for several months. Transformation of bloodstream forms to epimastigotes depended on initial attachment to the bottom of culture flasks and the presence of L-proline. The number and maturity of metacyclic forms was influenced by the concentrations of foetal bovine serum, L-proline, L-glutamine, and 2-mercaptoethanol. Trypanosomes from cultures were cryopreserved, revived, and used to re-establish fresh axenic cultures. These results represent a significant advance in cultivation of T. vivax insect forms that should enable studies to be accomplished on metabolism, differentiation, and pharmacology of this parasitic protozoan, free from the influence of extraneous cells.