| Backgrounds and Objective:Endothelial progenitor cells which was considered as the pre-endothelial progenitor cells,are not only involved in human embryonic angiogenesis, but in postnatal angiogenesis and endothelial repair process after injury. It plays an important role in the occurrence and development of endothelial dysfunction-related diseases, especially in coronary heart disease and idiopathic pulmonary hypertension. For the endothelial dysfunction-related diseases, transplantation of EPC with high proliferative activity may be a kind of treatment in the future. Assmus etc. used the progenitor cells from in vitro expansion for the treatment of acute myocardial infarction directly. It confirmed that transplantation treatment of EPC was safe and feasible. Chen Jun zhu etc. used the autologous EPC after in vitro expansion in clinical treatment of idiopathic pulmonary hypertension and this randomized controlled trial ended in 2006.The results showed that transplantation of EPC for the treatment of idiopathic pulmonary hypertension was safe, feasible and effective.However, EPC transplantation treatment performed on the clinic needs a large number of cells, and the cells source can,t be guaranteed. It is necessary to establishment cell bank for EPC. Because its immunogenic, allogeneic transplant of EPC will lead to immune rejection, which limits its clinical applications. EPC from human umbilical vein blood rarely has antigenicity. There were no immune rejection to the host, and the cell source are rich and have proliferation ability in vitro. Therefore it is suitable for the establishment of cell bank.The purpose of this study: Isolating and culturing human umbilical cord blood progenitor cells and the cells were preservated in the low temperature and then were recoveried. We get the EPC cells, and then compare cryopreservation of cell morphology and biological function. EPC will provide lots of seeds for studying the biology of human function,differentiation and endothelial dysfunction disease.We are aiming at establishing the human umbilical cord blood EPC bank and applying it in the treatment for coronary heart disease and idiopathic pulmonary hypertension.Methods:1.Collection of human umbilical cord blood: 38 cases were chosen from the first hospital of Hang zhou city from March 2009 to September 2009. The human umbilical vein blood was taken from the full-term pregnancy healthy women. After newborns parturited,we clamp the umbilical cord at a distance of about 5 ~ 10cm from umbilical round with the hemostatic forceps, and cut the umbilical cord between the two clamps, and then select the exposed thick umbilical vein near the maternal side of the umbilical cord.We puncture with needles of 50ml syringes toward the direction of placenta,then relax the clamp immediately after the success of puncture .The blood will flow into the 50ml syringes using the uterine intermittency contractions, and then clamp the umbilical cord immediately after blood collection.2.Isolation and culture of the mononuclear cells: the mononuclear cells were isolated from umbilical cord blood using density gradient centrifugation in the super-clean workplace.The cell suspension was inoculated into the 6-well plate pre-coated with fibronectin (FN) and cultured in M199 medium supplemented with 20% fetal bovine serum,and were maintained in a 5% CO2 atmosphere at 37℃. Change the medium every fifth day.The cells were stained with acLDL-Dil and FITC-UEA-I,then were identified with laser scanning confocal microscope.The cells were considered as the EPC if the cell were double-positive.3.Cryopreservation and recovery of cells: Each cord blood were divided into two groups after density gradient separation and in virtro culture.The low-temperature cryopreservation procedures was considered as the experimental group and in vitro cell which was not frozen was considered as the control group. Adjust the cell concentration and count the total cells number. Then move the cells to 1.5ml frozen pipes with transferpettor.Store the cells at low temperatures (-70℃) in the freezing box,in accordance with 4℃20min -- 0℃30min --- 20℃30min --- 70℃.Bath the cells in 37℃water rapidly after removing from frozen box and kept shaking it for the rapid melting within 1min. Then wash the cells twice with the culture medium mentioned above to remove the toxic effects of freezing protection solution. Collect the cells ,and count the total cells number ,and its survival rate for further experiments.4.Measurement of the proliferation activity for EPC: Digeste the adherent cells with 2.5% trypsin and 0.2% EDTA. Adjust the cell concentration with culture medium mentioned above with M199.Inoculate the cells into 96-well plates according with 200μl/hole. Then removed the culture medium after 48h and added 20μl MTT solution (5mg/ml) to each well for another 4h. Inoculate 200μl DMSO (purity 99.99%) to each well and measured the proliferation activity(OD) using Microplate Luminometer (λ=490nm) after full oscillation for 10min.5.Measurement of adhesion ability for the EPC: Digeste the adherent cells as described above and adjust the cell concentration, and then inoculate the cells into 6-well plate pre-coated by FN after incubation for 1h.Count the number of the cells under the microscope.Results:1.Dilute the human umbilical vein blood by adding human lymphocyte separation medium. Then the blood shows a clear stratification after centrifugation: the top layer is the plasma and platelets, and the mononuclear cell layer are in the middle,and the nest layer is the separated liquid layer, and the lowest layer is the red blood cells.2.Cell morphology:The mononuclear cells derived from density gradient centrifugation were inoculated into culture plates. The cells display round and uniform size. Remove the suspension cells and change the culture medium after five days. The adherent cells display slightly round and the number is little. There are more cell colony formation. At 15th day the cells number are more and cell are round or oval-shaped spindle, but less cell colony formation.3.The cells were stained with acLDL-Dil and FITC-UEA-I, then were identified with laser scanning confocal microscope. The cells were considered as the EPC which are double-positive.4.Cryopreservation and recovery of the cells: After cryopreservation and recovery, survival rate of EPC which was cultured in vitro was (84.17±4.02)% and there was no significant change in cell morphology.5.Changes of cell proliferative activity and adherence ability: Compared with non-cryopreserved cells,the cryopreserved cells showed a significant change in proliferative activity and adherence ability ( P <0.05).After incubation for 48h after recovery the cryopreserved cells,there was no significant difference (P>0.05) of proliferative activity and adherence ability between non-cryopreserved cells and cryopreserved cells.Conclusion:We detected the proliferation and adhesion capacity of EPC immediately which was recoveried after short-term cryopreservation. It shows that the capacity is decreased but there was no significant difference for them after recoveried and cultured 48h. |
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