Effect Of Total Saponins Of Panaxginseng On Expression Of Erythropoietin Receptor On CD34~+ Cells

PART ONE:EFFECT OF TOTAL SAPONINS OF PANAX GINSENG (TSPG) ON EXPRESSION OF ERYTHROPOIETIN RECEPTOR ON CD34+ CELLSObjective:Since 1980s, umbilical cord blood (UCB), as hematopoietic stem/ progenitor cells source to cure the blood desease patients induced extensive attention, UCB rich in hematopoietic stem / progenitor cells with original, strong self-proliferation and the ability to differentiate, which is another source to get hematopoietic stem / progenitor cells. Erythropoietin (Epo) is an important hematopoietic growth factor that maintain erythroid progenitor cell survival, proliferation, differentiation and maturation.Panax Ginseng is known as"invigorating qi"in traditional Chinese medicine and Total saponins of Panaxginseng (TSPG) is one of the main effective ingredient extracted from Panax Ginseng which plays a vital role in"invigorating qi"or promoting haematopoietic function.Our investigation indicated TSPG in combine with Epo may induce CD34+ cells differentiate into erythrocytic cells. The most significant finding is that TSPG would not play the part in it without exogenous growth factor, which indicated that TSPG promote hematopoiesis in cell factor-dependent manner. Therefore, we postulate TSPG probably induce blood cells to proliferate through regulating EpoR on the surface of hematopoietic cells. Our purpose is to clarify the effect of TSPG on expression of EpoR on CD34+ cells.Methods :1.The purified CD34+ cells were induced by 50mg/L TSPG with combination of 100ng/ml stem cell factor for 24 hours, compared with the control group containing 100ng/ml stem cell factor alone.2. By means of flow cytometry, observe the positive cell percentage of EpoR expression on CD34+ cells membrane by 50mg/L TSPG for 24 hours.3.By means of semiquatitative RT-PCR, detect the EpoR expression CD34+ cells by 50mg/L TSPG for 24 hours.4.Under laser scanning confocal microscope (LSCM), observe the EpoR expression on CD34+ cells by 50mg/L TSPG for 24 hours.5.By methods of spectrophotometric,detect effect of erythrocytic differentiation of CD34+ cells induced by 50mg/L TSPG.Results:1. Flow cytometry assay showed that the positive cell percentage of EpoR expression on CD34+ cells induced by 50mg/L TSPG increased, P=0.035.2. Semiquatitative RT-PCR indicated that the ratio of EpoR/β-actin are upregulated after induced by 50mg/L TSPG , P=0.016.3. Under LSCM, the intensity of fluorescence on CD34+ cells membrane became increased after induced by 50mg/L TSPG for 24 hours.4. The results of spectrophotometric methods indicated that 50mg/L TSPG promoted the expression of hemoglobin of CD34+ cells, P=0.037.Conclusion:1. 50mg/L TSPG may enhance the EpoR expression on CD34+ cell membrane.2. 50mg/L TSPG may increase the EpoR mRNA expression of CD34+ cells.3. 50mg/L TSPG can promoted the expression of hemoglobin of CD34+ cells .PART TWO:EFFECT OF TOTAL SAPONINS OFPANAX GINSENGON EXPRESSION OF ERYTHROPOIETIN RECEPTOR ON HUMAN ERYTHROLEUKEMIA CELL LINE (K562) Objective:Leukemia is a malignant tumor of the human race, is the result of the abnormal proliferation and differentiation of hematopoietic stem cells. So far the treatment of leukemia is still mainly traditional high-dose combination chemotherapy, but the side effects of this therapy, high recurrence rate cause injury to normal hematopoietic and immune system. Total saponins of panax ginseng (TSPG) is one of the main effective ingredient extracted from Panax Ginseng which plays a vital role in"invigorating qi''or promoting haematopoietic. Our previous study showed that TSPG may inhibit the proliferation of leukemia cells. However, its molecular biological mechanism is still unclear. EpoR plays an important role in the leukemia proliferation and differentiation, therefore we assumed that TSPG probably induced K562 cells to apoptosis by the way of decreasing the expression of EpoR. To test this idea,we used cell culture technique in vitro to observe the effect of TSPG on expression of EpoR on the K562 cells, in order to clarify the mechanism by which TSPG control the leukemia growth, and to privide the theoretical basis and the experimental evidence for its clinic application.Methods:1. The leukemia cell line (K562) was cultured in vitro. Add 200mg/L TSPG into the K562 culture system after 24, 48, 72 hours.2. By means of flow cytometry, observe the positive cell percentage of EpoR expression in K562 cells by 200mg/L TSPG for 24, 48, 72 hours. 3. By means of semiquatitative RT-PCR, detect the EpoR mRNA expression on K562 cells by 200mg/L TSPG for 24, 48, 72 hours.Results:1. Flow cytometry assay showed that the positive cell percentage of EpoR expression in K562 cells induced by 200mg/L TSPG decreased in time-dependent manner, P<0.05.2. Semiquatitative RT-PCR assay indicated that the ratios of EpoR/β-actin are downregulated after induced by 200mg/L TSPG in time-dependent manner, P<0.01.Conclusion:1. 200mg/L TSPG may reduce the positive cell percentage of EpoR expression in K562 cells in time-dependent manner.2. 200mg/L TSPG may reduce the EpoR mRNA expression of K562 cell in time-dependent manner.