Experiment Study Of Placental Factor Purification And Its Activity On PC12 Cells

Part1 PF Purification and activity detectionObjective: To purify PF and select the most effective active component from purified products.Method: Anadesma was removed from fresh human placenta, washouted it by sotonic Na chloride and homogenated, freezed and thawed three times, then dialyzed by bag filter and collected dialysating extracellular fluid, freeze-dried. PF was purified by Sephadex G-25 gel filtration, Eluted, Collected each component. Through cultivating of lymphocytes, the product activity was detected by MTT test. The purity of product was analyzed by RP-HPLC. The molecular weight was measured by matrix-assisted laser desorption ionization time of flight serial mass spectrometry.Result: The fourth peak purified by Sephadex G-25 gel filtration from PF that has obvious activity to promote lymphocyte proliferation was the main active ingredient of placental factor. The fourth peak was polypeptide,the molecular weight of which was 6737.813Da. The purity of the fourth peak was 82%. Conclusion: During this experiment, we had got the fourth peak purified by Sephadex G-25 gel filtration from PF was the main active ingredient of placental factor. The purity of the fourth peak was 82%,Molecular weight was 6737.813Da.Part2 The effect of a placenta factor on the growth of PC12 cellsObjective To observe the effects of Placenta factor on the growth of PC12 cells.Method The effect of PF on the survival rate of PC12 cells in Serum-free medium and the proliferation of PC12 cells in the low serum medium were determined by MTT test. The morphology Changes of PC12 cells were observed by ordinary microscopic.Results At the concentrations of 50μg/ml,25μg/ml,12.5μg/ml,6.25μg/ml, PF could significantly increase the survival rate of PC12 cells in Serum-free medium by different concentrations of PF for PC12 cells 44 hours (P<0.05).At the concentrations of 12.5μg/ml,6.25μg/ml, PF could significantly increase the number of PC12 cells in the low serum medium by different concentrations of PF for PC12 cells 68 hours(P<0.01). During treating PC12 cells in the low serum medium with different concentrations of PF for 10 days, PC12 cells grew protuberance at the concentration of 3.13μg/ml and 1.56μg/ml of PF when PC12 cells had grown six days. Conclusion The study showed that PF could enhance the survival rate of PC12 cells in Serum-free medium, inhibit the proliferation of PC12 cells and induce the differentiation of PC12 cells.Part3 The effect of the fourth peak of placenta factor (PFIV) on the growth of PC12 cellsObjective To observe the effects of the fourth peak of Placenta factor (PFIV) on the growth of PC12 cells.Method The effect of PFIV on the survival rate of PC12 cells in Serum-free medium and the proliferation of PC12 cells in the low serum medium were determined by MTT test. The morphology Changes of PC12 cells were observed by ordinary microscopic.Results At the concentrations of 15μg/ml, 7.5μg/ml, 3.75μg/ml, 1.88μg/ml, PFIV could significantly increase the survival rate of PC12 cells in Serum-free medium by different concentrations of PFIV for PC12 cells 44 hours (P<0.05). At the concentrations of 0.47μg/ml,0.235μg/ml, PF could significantly increase the number of PC12 cells in the low serum medium by different concentrations of PFIV for PC12 cells 68 hours(P<0.01).During treating PC12 cells in the low serum medium with different concentrations of PFIV for 10 days, PC12 cells grew protuberance at the concentration of 0.118μg/ml and 0.059μg/ml of PFIV when PC12 cells had grown six days. Conclusion The study showed that PFIV could enhance the survival rate of PC12 cells in Serum-free medium, inhibit the proliferation of PC12 cells and induce the differentiation of PC12 cells.