Mouse Blastocyst Invasion Tumor Cells In Vitro

1. Mouse Blastocyst Invade Tumor Cells in vitro During blastocyst implantation, apoptosis is observed morphologically at the implantation site of endometrium. The objective of this study was to demonstrate biochemical evidence of apoptosis in tumor and normal cells using a mouse implantation model--co-culture model. Blastocyst from day 4 pregnant mice were cultured on tumor cell lines of human (human lung cancer cells line--A549,human ovary cancer cells line--3AO,HO-8910,SKOV3, Henrietta Lacks strain of cancer cells--Hela ,human bladder cancer cells line--T24,human osteoblastoma cells line--MG-63 , human kidney cancer cells line--GRC-1), mouse melanoma cells line--B16,human umbilicus vein endothelial cells line -- EUV-304,mouse embryo fibroblast--PA317,Chinese hamster ovary --CHO cells line and primary rabbit embryo osteoblast--RF10,primary newborn mouse lung fibroblast--MF1 for 96 hours. Attachment was observed after 12 hours period of culture and almost all hatched blastocysts attached by 48 hours period. Blastocyst outgrowth was initiated after 24-36 hours of co-culture and almost all attached blastocysts demonstrated outgrowth after 60 hours of incubation. Trophoblastic cells between co-cultured cells-trophoblast units dislodged co-cultured cells.2. Effect of Tumor and Normal Cells on Attachment and Outgrowth of Mouse Blastocyst in vitro Some types of co-cultured cell monolayers were significantly efficient at promoting advanced stages of blastocyst in vitro, such as attachment and outgrowth. The mouse embryo fibroblast--PA317 were selected as control for testing. When co-cultured 48 hours to 72 hours later all kinds of cells can affected the ability of blastocyst attachment, significantly different between normal cells and tumor cells (P<0.001), but there were no significantly difference between normal cells (P>0.05). At first stage when co-cultured 36 hours later there were nosignificantly difference from all kinds of cells (P>0.05). But after that times some tumor cells can affect the ability of blastocyst outgrowth. There were significantly differences between these cells. And then we took normal and tumor cells as the whole to analysis, co-cultured with tumor cells and normal cells did not affect the ability of development of mouse blastocyst in vitro (P>0.05). 3．Interactions Between Mouse Blastocyst and Tumor Cell Lines in vitroSome co-cultured cells dislodged by trophoblastic cells in tumor-trophoblast unit demonstrated morphological features of apoptosis by TUNEL and Annexin V--EGFP staining when co-cultured 48 hours days later such as A549 cell. But on the other way PCNA were observed in the detached tumor cells (A549,3AO )around the blastocyst, or in tumor epithelial cells at the tumor-fetal interface when co-cultured 96 hours in vitro.These results suggested there were autocrine/paracrine regulation of the biological of tumor and normal cells at mouse blastocyst implantation, that blastocyst may produce specific factors that can induce apoptosis, in both tumor cells and normal cells,then can stimulate tumor cells proliferate.4. A Three-dimensional Model of Mouse Blastocysts Implantation in vitroThe cultured mouse blastocyst obtained on the 4th day of pregnancy on human fibrin gel and observed the invasive behavior of blastocyst to develop a new embryo invasion three-dimensional model. This study showed that: When mouse blasocyst was cultured on it, human fibrin gel did not have influenced behaviors of mouse blastocyst. The whole blastocyst retained three-dimensional structure. Trophoblastic cells did not flatten but retained a polarized removal and invasion like did in vivo. It suggested human fibrin gel could be used as substratum of three-dimensional model for investigating blastocyst invasive behavior.