The Improvement Of Placental Perfusion In Preeclampsia Model Rat By Transplantation Of Angiogenic T Cells

Part Ⅰ Extraction and culture of angiogenic T lymphocytes from healthy pregnant womenObjective:To explore the method of isolation and culture of human umbilical cord blood derived angiogenic T lymphocytes,so as to lay the foundation for further study on the improvement of placental perfusion in preeclampsia rats by angiogenic cells.Methods:The umbilical cord blood of healthy pregnant women was collected with sterile blood bag.Isolation of cord blood mononuclear cells was performed by density gradient centrifugation,followed by magnetic activated cell sorting(MACS)and flow cytometry for the isolation and identification of CD3/CD31 double positive cells(angiogenic T lymphocytes).VEGF cells were cultured in M-199 medium supplemented with 20% fetal bovine serum.Results:The CD31 positive cells were sorted by MACS magnetic beads,and then the selected CD31 positive cells were sorted by flow cytometry for CD3 positive sorting.The obtained CD3/CD31 double positive cells were angiogenic T lymphocytes.After culture,the rate of CD3/CD31 double positive cells was identified to reach 81.3%.Conclusion: Mononuclear cells isolated from umbilical cord blood of healthy pregnant women by density gradient centrifugation were sorted with MACS magnetic beads sorting and flow cytometry.The study confirms that cultured cells are in line with the characteristics of angiogenic T lymphocytes after the observation of the growth and proliferation of the cultured cells and identification using flow cytometry after culture.This method applied in the present study is proved to be useful to obtain angiogenic T lymphocytes from the cord blood of healthy pregnant women.Part Ⅱ Transplantation of angiogenic T lymphocytes for the improvement of placental perfusion in preeclampsia ratsObjective: To investigate the effect of the transplantation of angiogenic T lymphocytes for improvement of placental perfusion in preeclampsia rats.Methods: A rat model of preeclampsia was established by intraperitoneal injection of NG-nitro-L-arginine methyl ester(L-NAME).Density gradient centrifugation was used to isolate umbilical cord blood mononuclear cells from healthy pregnant women.Then,angiogenic T lymphocytes were isolated and obtained combining MACS magnetic bead sorting and flow cytometry.Following in vitro culture and the preparation of cell suspension,angiogenic T lymphocytes were transplanted into pregnant rats with preeclampsia by tail intravenous injection.Fluorescence microscopy was used to observe the situation of placental neovascularization.Placenta and the newborn rats’ weight were examined by monitoring the changes of blood pressure and urine protein in pregnant mice to understand the pregnancy outcome,and to further understand the effect of angiogenic T lymphocytes transplantation on the improvement of placental perfusion in preeclampsia rats.Results: Semi-quantitative observation under fluorescence microscopy showed that compared with the control group,the density of placental neovascularization was significantly higher in the tail vein transplanted Tang cells.Meanwhile,blood pressure and urine protein values were significantly lower than those in the control group.Conclusion: Transplantation of angiogenic T lymphocytes by tail intravenous injection can improve placental perfusion and clinical symptoms in pregnant rats with preeclampsia.