Comparison of in vitro isolation of adenohypophyseal cells from SD rats / 医学研究生学报

ABSTRACT

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 immunocytochemi-cal staining technique Results Compared with the mechanical separation group and-ⅣV 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 ceils 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 ceils 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 positive products,showing blue,were located in the cytoplasm;The Enzyme linked immunosorbent assay (ELISA) was used to determine the supematant of cells and confirmed that the cells in vitro still had hormone secretion functions.Conclusion Compared with the single cell preparation method,the trypsin digestion plus mechanical separation method has certain advantages;The anterior pituitary cells vitro cultured are in good health conditions and still have secrete functions.They are tested to be competent for related scientific experiments.