常规培养条件下成年大鼠颈动脉体原代细胞培养

Primary cell culture of adult rat carotid body under conventional culture conditions

目的 探讨成年大鼠离体颈动脉体的细胞培养方法.方法 选取8~9周龄健康成年雄性SD大鼠,体重250~270 g,游离双侧颈总动脉分叉,体视显微镜下分离出完整颈动脉体,置于100μl胶原酶缓冲液中,采用酶加机械切割方法消化细胞,离心后通过常规培养基重悬细胞后种于预先包被多聚-D-赖氨酸的玻片上.借助颈动脉体Ⅰ型细胞标志物酪氨酸羟化酶和Ⅱ型细胞标志物胶质纤维酸性蛋白鉴定所培养细胞,并通过巢蛋白染色鉴定有无干细胞特性.结果 常规培养基可以培养出有活性的成年8周龄大鼠的颈动脉体原代细胞,Ⅰ型细胞可以保持类似于体内生长状态的簇状,Ⅱ型细胞散在分布.在这些原代培养细胞中含有巢蛋白阳性干细胞.结论 常规培养基能够培养出活性较好且符合体内生长特性的成年大鼠颈动脉体细胞,满足后续颈动脉体分子机制的研究.

Objective To explore the primary cell culture method of the carotid body from adult SD rat.Methods After the carotid bifurcations were dissected from adult SD rats,the carotid bodies were isolated from the surrounding tissues and placed in the enzyme solutions.Then the carotid body cells were dissociated by incubation in 37℃-5%CO2 incubator for 8 min followed by the cut for 3 min,and repeated these steps.The carotid body growth medium was added to stop enzyme activity after gently triturated the cells,then centrifuged for 5 min at 1000 r/min.And the suspended carotid body cells were placed on glass coverslips coated with poly-D-lysine,and incubated at 37℃.The cultured cells were identified by typeⅠand typeⅡcell markers,respectively.Results The carotid body cells of 8-week-old rats could be cultured in conventional medium.And the nestin positive stem cells could be seen in these primary cells.Conclusions The primary culture established in the research retains activity and growth characteristics of carotid body in vivo from adult rat and therefore might improve the research that involves the mechanisms of carotid body.