| Purpose: To investigate the effects of LIF on the proliferation and differentiationendothelial progenitor cells in vitro. To explore the possible way of efficientamplification on endothelial progenitor cells in vitro.Method: Under sterile conditions,80ml umbilical cord blood is taken forresearch from the Obstetrics, the affilicated Hospital of Luzhou Medical Collegewith informed consent form. The umbilical cord blood is isolated by densitygradient separation for mononuclear cells and the cell density is adjusted to3x106/ml. In vitro liquid culture system, The cells are divided randomly into twogroups: one is setted up as the control group for the EGM-2complete medium,,and the other is setted up as experimental group with LIF (10ng/ml) added inEGM-2complete medium. The endothelial progenitor cells are placed in twodifferent culture systems at37℃and put in5%C02incubator for culture.Thecell morphology and growth are observed under inverted microscope on1d,3d,7d,14d separately. The CD133expression of cells in two groups is tested byparallel flow cytometry detection. The cells on two groups are tested by MTT atthe72h,96h,120h,144h,168h to calculate the proliferation rate and draw thegrowth curve. The data are showed by the “average±standard deviation" meansand statistical analysized by SPSS17.0statistical software. The compare betweentwo groups is done by t test. There is significant difference when P <0.05. Results:①The mononuclear cells from umbilical cord blood presented the formwhich was a kind of circular, the smaller, diopter, evenly distributed. The CD133positive expression rate of mononuclear cells which tested by Flow cytometrydetection was low, only0.9%. The cells in the control group and the experimentalgroup began to stick wall for growth on3d. The cells gathered to form cluster andpresented the colony formation growth. The cell colony formation in experimentalgroup was more obvious. The cell size increased gradually, and changed graduallyfrom round to short spindle. On the seventh day cultured, the cells in two groups allhad rapid cell proliferation. Cell size increased obviously, cells gathered, density ofcells increased, the Number of cells increased and more colon formed. Colonycentral was given priority to round cell group, revolves around the round cell is aspindle radial arrangement of spindle cells. Compared with the control group, thecell colony and cell number in the experimental group were obvious advantage. Thecell volume in the experimental group was smaller than it in the control group. On14d, the cell fusion in control group reached to80%. The central cells were givenpriority to with polygonal cells, also spindle cells. The growth of cells was in pavingstone form. Cell size was noticeably bigger. The cell fusion in experimental groupreached to90%, with oval cells and spindle cells. Peripheral polygonal cells werenot obvious.②During the course of culture, the expression of CD133in controlgroup presented expression increasable at first and declined later. The expression ofCD133in cells was highest on7d. The expression of CD133in experimental group showed the similar change as it in control group, but it was higher than that incontrol group at the same time.③The results determined by MTT test showed thatthe difference of A490absorbance value between two groups increased along withthe extension of incubation time. the absorbance values of A490(nm) in the controlgroup was smaller than that in experimental group at the same time (P <0.05). Itindicated that the cell proliferation rate in experimental group was higher than that incontrol group.Conclusion: The specific culture system, in which a certain concentration ofleukemia inhibitory factor (10ng/ml) is added, can promote endothelial progenitorcells to proliferate and inhibit its differentiation. |
PDF Full Text Request