人垂体腺瘤细胞原代培养的纯化及其激素的表达与分泌

Primary Culture and Purification of Pituitary Adenoma Cells and Characteristics of Hormone Expression and Secretion

目的探讨原代培养垂体腺瘤的细胞纯化方法及其激素表达与分泌的特点。方法采用三种原代培养方法对垂体腺瘤细胞进行培养,方法Ⅰ为目前常用的垂体瘤细胞培养方法,方法Ⅱ结合使用右旋颉氨酸(D-valineDMEMD-valine)培养液,方法Ⅲ采用反复贴壁法结合DMEMD-valine培养液培养。观察其细胞形态变化和生长特征,放射免疫法测定不同培养时间培养液中相应激素的水平。免疫组织化学染色法检测培养细胞生长激素(growthhormone,GH)、泌乳素(prolactin,PRL)的表达。结果3种方法均能成功培养出垂体腺瘤细胞,呈圆形或椭圆形,其纯度随培养时间的延长逐渐下降,培养第20天其平均纯度分别为40%,46%,96%。方法Ⅲ明显高于方法Ⅰ和方法Ⅱ,差异具有显著性意义(P<0.01);培养液中均含有相应激素,且3种方法的激素分泌量没有统计学差异(P>0.05);腺瘤细胞分别呈GH,PRL阳性表达,成纤维细胞表达I型胶原。结论方法Ⅲ可得到纯度达95%以上的人垂体腺瘤细胞,纯化后的细胞可分别呈GH和PRL阳性表达并具有良好的激素分泌功能,为进一步研究人垂体腺瘤的发病机理、药物治疗和颅外移植等方面奠定了实验基础。

[Objective ] To explore the purification method and the characteristics of hormone expression and secretion of the primary culture of the pituitary adenoma cells.[Methods] Three different methods, Ⅰ the usual means, Ⅱ： the usual means with DMEM D-valine, Ⅲ： double adherence before DMEM D-valine, were used to culture the pituitary adenoma cells. To observe the changes in cell morphology and cell growth, and to detect the alteration of basic hormone secretion along the days of culture with radio-immunity method. Immunohistoehemical technique was used to detect the expression of GH and PRL of the cultured cells. [Results] All the pituitary adenoma were successfully cultivated. The morphology of the pituitary adenoma cells was round or elliptic. The purity of the adenoma cells gradually declined along the days of culture. The percentage of the adenoma cells of the three mothods were 40%, 46%, and 96%, respectively, 20 d after culture. The purity of Ⅲ was significantly highest among the three methods （P〈 0.01）. The GH and PRL secretion in the media have no significant difference among the three methods （P 〉0.05）. The positive expression of GH or PRL in the pituitary adenoma ceils was in accordance with the clinical classification of the pituitary adenoma. The expression of collagen I was positive in the fibroblast.[Conclusions] The purity of pituitary adenoma cell was over 95% by method Ⅲ. The expression of GH or PRL was positive in the purified pituitary adenoma ceils and these ceils have satisfactory hormone secretion function, which provide experiment foundation for studying the pathogenesis, chemical therapy, and extracranial cell transplantation, and so on.