Establishment Of The Method For Isolation And Culture Of Early Pregnant Rabbit Endometrial Cells

Endometrium is main tissue of ovarian hormones, which is important in the studying on procreating. Endometrial cells take morphological and biochemistrical change by estrogen and progesterone, which behives not only the epithelial cells and the stromal cells transform, but endometrial proliferation and secretion change. During the pregnancy, endometrium turns secretive membrane. Through culturing endometrinm in vitro, we can learn the optimal condition of the isolation and the incubation of endometrial cells of early pregnant rabbit in vitro, of which were also dealt with the morphological modality, growth and proliferation characteristic. So it is the base of separating and culturing the stromal cells and epithelial cells. Further we can focuse on the endometrial action in the endometrium partial immunity. The experiment used different digestion and isolation methods to prepare the cells. Growth character of early pregnant rabbit endometrial cells in vitro were studied through primary culture, passage culture, the identification. At the same time, we learn growth and augmentation of being stimulated EC by hormones. The results as follows:1. The endometrium was digested by type I collagenase or trypsin can provide enough dispersed cells for primary culture. But the number was little and its viability was not strong using the trypsin, while using 1 g/Lcollagenase to digest the cells, the number of cells was 5×106 and the viability ratio was 90%. After implanting 24 h, the cells mainly stick the wall and grow, which could form incomplete monolayer in four to five days. The stromal cells and epithelial cells were able to be separated through 74 μm filter, the stromal cells can through the filter, while the epithelial cells ware the shape of pipe, it can not through the filter. The cultured cells are observed with microscopy, the epithelial cells were similar the shape of "tadpole" or "polygon", mostly appearing whirlpool, assembling together and growth, the nucleus was round, while the stromal cells stretch out, prolong appear wide in the middle, tip in the two ends. Cells like spindle, the nucleus are egg-shape, Contact inhibition in existing among cells. After four to five days, cells assemble and are motionless relatively.DMEM/F12 (1:1) is more appropriate as culture medium to culture EC compared with M199 and RPMI-1640 medium.10 μg/mL Insulin can evidently accelerate cells growth and proliferation. The identification was done by immunohostochemical experiments, If the cytoplasm is yellow by rat antagonize rabbit keratin, then keratin is positive, If the cytoplasm is yellow by rat antagonize rabbit vinemetin, then vinemetin is positive. We use the EC as positive compare and substitute PBS for the first antigen as negative compare. The EC are in a lag phase in 18 h to 24 h after passage, then they enter logarithmic growth phase and can last two to four days. From the seventh day, they enter plateau, then begin to fall and die from the eighth day.The early pregnant endometrial cells are easily cultured than endometrial cells in other phases, growth and proliferating rapidly, and the number of received cells is more.