Preparation And Function Detection Of Human Platelet Growth Factors

ObjectiveThe objective of this study is preparing functional platelet growth fac-tors of activity from human platelets applying for cell therapy and regener-ative medicine.MethodsHealthy human donors donated 400ml whole blood by national standards after test qualification,separating buffy coat and preparing platelet concentrates(PC)in a sterile environment,pooling PC of six bags and concentrating to adjust the concentration of platelets to about 1000 × 109/L.Selecting different methods to activate PC,experiment was divided into four groups:(A)S/D(1%TnBP and 1%TritonX-45)treated group;(B)2%TnBP treated group;(C)CaCl2(23mmol/ml)and S/D treated group;(D)CaCl2(23mmol/ml),thrombin(100u/ml)and S/D treated group.Detecting the content of PDGF-BB,TGF-? 1 and VEGF in PC and four groups by ELISA.Choosing an organic solvent/surfactant(S/D)for treatment of PC,S/D reagents TnBP and TritonX-45 to achieve a final concentration of 1 percent,in 310 C water bath,was stirred for 1 hour,and then added 7.5 percent castor oil,oil extraction,and then purified by column chromatography to remove TnBP and TritonX-45.Detecting the content of PDGF-BB,TGF-?1,and VEGF in the process of S/D treatment,oil extraction and column chromatography by ELIS-A.Assaying rudimental TritonX-45 after S/D treatment by high performance liquid chromatography.Selecting human keratinocytes(HaCaT)and human dermal fibroblasts(hDFbs)for cell experiment to detect the functional activity of growth factors.(1)To detect the effect of HPGF(human platelet growth factors)activated by differ-ent methods on the proliferation of HaCaT and hDFbs,the detection time was respectively 24h,48h and 72h,experiment was divided into five groups:control group,repeated freezing and thawing group,CaCl2 activated group,S/D treated group,lyophilized S/D treated group;(2)To detect the effect of S/D treated HPGF in different concentrations on the proliferation of HaCaT and hDFbs,the detection time was respectively 24h,48h and 72h,experiment was divided into six groups:control group,5%HPGF group,10%HPGF group,15%HPGF group,20%HPGF group,25%HPGF group;(3)Analyzing the effect of HPGF in different concentrations on the migration of HaCaT,the detection time was Oh,24h,48h and 72h,experiment was divided into four groups:control group,10%HPGF group,15%HPGF group,20%HPGF group,to observe the migration of HaCaT by microscopic and taking a photograph;(4)Analyzing the effect of HPGF on cell cycle of HaCaT and hDFbs by flow cytometer,the detection time was 6h,12h and 24h,experiment was divided into three groups:control group,7.5%HPGF group,15%HPGF group.ResultsThe content of three growth factors in PC was low,the content of growth factors was decreased in varying degrees after activated,the content of growth factors in S/D treatment group was the highest,there was significant differe-nce(P<0.01)in S/D treatment with control group and other experimental groups.During the process of S/D treatment,twice oil extraction and column chromatography of PC,the content of three growth factors was gradually decreased.In three experiment groups of removing the remaining S/D reagents,the effect of column chromatography and twice oil extraction was the best,the remaining S/D reagents after oil extraction three times was higher than column chromatography and twice oil extraction treated group,the effect of twice oil extraction treated group united with adsorbent centrifugal treated group was the worst.The final choice was the group of twice oil extraction and column chromatography,remaining content of TritonX-45 reduced from 10000ppm to 10-100ppm by HPLC.To detect the effect of HPGF activated by different methods on the proliferation of HaCaT and hDFbs by CCK-8,there was statistically significant differences(P<0.01)in experimental groups and control group,CaCl2 treated group was the best,S/D treated group promoted cell proliferation less effect-ively than CaCl2 treated group,but S/D treated group was superior to control group and other experimental groups(repeated freeze-thawing group and freeze-dried group).To detect the effect of S/D treated HPGF in different concentrations on the proliferation of HaCaT and hDFbs by CCK-8,in 24h,48h and 72h,the effect of promoting cell proliferation was significantly between control group and experimental groups(P<0.05),15%HPGF group was better than other experimental groups.The groups with different concentrations of HPGF,such as 5%,10%,15%,as the concentration increased,the better promoting cell proliferation,when the concentration exceed 20%,would gradually appear to the trend of inhibiting cell growth.To analyze the effect of HPGF in different concentrations on the migration of HaCaT by image analysis software,which showed that there was significant difference between experimental groups and control group(P<0.05),but the extent of cell migration with different concentrations of HPGF between experimental groups was no significant difference(P>0.05).Analyzing the effect of HPGF on cell cycle of HaCaT and hDFbs by flow cytometer,15%HPGF group,7.5%HPGF group with control group had significant difference(P<0.05).ConclusionThe method of S/D was used for conventional viral inactivation,which can lysis platelet effectively,to remove the organic solvent,the lysate was extracted twice by oil,once by column chromatography,the amount of residual organic solvent met clinical requirement.Function experiments showed that platelet growth factors prepared by S/D were able to promote cell proliferation of HaCaT and hDFbs.This laid the foundation for us to further separation and purification of platelet growth factors.