SD大鼠腺垂体细胞体外分离方法的比较  

Comparison of in vitro isolation of adenohypophyseal cells from SD rats

作　　者：蒋小飞[1] 周惠芳[1] 周伯如[1] 刘贝[1] 袁艺祯 单进军[2] 徐建亚[2] 谢彤 

机构地区：[1]南京中医药大学第一临床医学院,南京医学博士研究生210029 [2]南京中医药大学儿科研究所,南京210029

出　　处：《医学研究生学报》2017年第12期1272-1276,共5页Journal of Medical Postgraduates

基　　金：国家自然科学基金(81473713);中国和欧洲的医疗保健业发展合作研究计划(FP7/2007-2013/PIRSES-GA-2013-612589);江苏省自然基金(BK20141463);江苏省中医药局重大项目(ZD201702)

摘　　要：目的体外分离腺垂体制备主要包括酶消化及机械法等方法,但何种方法效果更好尚无相关报道。现比较大鼠腺垂体细胞体外分散制备的几种主流方法并对其进行促性腺激素分泌细胞鉴定,明确何种制备方法更为便捷高效。方法将48只成年雌性SD大鼠按随机数字表法分为胰酶组(0.25%胰酶EDTA液消化)、Ⅳ型胶原蛋白酶组(Ⅳ型胶原蛋白酶消化)、机械分离组(200目细胞筛研磨)及胰酶+机械分离组(0.25%胰酶EDTA液消化+200目细胞筛研磨),每组12只。评价各种方法制备细胞的效果。培养各组原代细胞,动态观察各组细胞在体外生长的情况,并利用免疫细胞化学染色技术进行鉴定。结果与机械分离组细胞活率[(90.25±0.96)%]比较,胰酶组、胰酶+机械分离组[(94.11±1.71)%、(94.92±1.92)%]均升高;与Ⅳ型胶原蛋白酶细胞活率[(93.32±1.77)%]比较,胰酶组、胰酶+机械分离组亦明显升高(P<0.05)。形态学观察:各种方法制备的腺垂体细胞均呈圆形,折光性强,边缘完整清楚。培养16~24 h候开始部分贴壁,至48~72 h绝大部分细胞贴壁,早期腺细胞仍为圆形,体积较前减小,可见少量大体积的细胞散落分布其间。培养7 d后,细胞变为多角形或梭形,并连接成片,至10 d左右,纤维化逐渐明显;免疫细胞化学染色表明FSH阳性细胞体积较大,数量少,散在分布;其阳性产物位于细胞质内,呈蓝色;酶联免疫吸附试验测定细胞上清液证实离体细胞仍有激素分泌功能。结论对比单一制备细胞方法,胰酶消化+机械分离法具有一定优势;体外培养的垂体前叶细胞生长良好,分泌功能存在,可胜任相关科学实验。Objective In vitro isolation of adenohypophyseal mainly includes enzymatic digestion and mechanical methods.But there is no relevant report about which method is better. In this paper, several mainstream methods of the vitro isolation of adenohypophysis cells in rats are compared and the identification of gonadotropin secretory cells is carried out to figure out which preparation method is more convenient and efficient. Methods 48 mature female SD rats were randomly divided into 4 groups: trypsin group( 0.25% trypsin EDTA digestion),Ⅳ-type collagen enzyme group( IV type collagen enzyme digestion),mechanical separation group( 200 mesh cell sieves grinding) and trypsin digestion plus mechanical separation group( 0.25% trypsin EDTA digestion plus 200 mesh cell sieves grinding). 12 rats in each group. The effect of these 4 methods was evaluated. Secondly,the primary cells of each group were cultured. We dynamically observed the vitro growth of cells in each group.Finally,the cells were identified by using the immunocytochemical staining technique Results Compared with the mechanical separation group and-IV type collagen enzyme group [( 90. 2 ±0.96) %,( 93. 32 ± 1. 77) %],Cell viability of trypsin group and trypsin digestion plus mechanical separation group was elevated[( 94.11±1.71) %,( 94.92±1.92) %]( P<0.05). Morphological observation: The pituitary cells prepared by each methods were all round with strong refraction and clear edges and began to partly adhere to the wall after cultured for 16-24 hours. Then most of the cells adhered to wall after 48-72 hours,while the early glandular cells were still round. But the volume was smaller than before. Also,a small amount of large cells were scattered among them. 7 days later,the cells began to become polygonal or spindle shaped and were connected to pieces. Till around 10 days,the fibrosis gradually became obvious; Immunocytochemical staining: It is indicated that FSH positive cells were larger in volume while less in number and scattered in the cytoplasm. And the

关 键 词：SD大鼠 垂体前叶细胞 体外培养 免疫化学染色 

分 类 号：R36[医药卫生—病理学]