Effect Of Non-platelet RNA Particles In Peripheral Blood On Mesenchymal Stem Cells

Objective Non-platelet RNA particles(NPRPs)in human circulation can participate in tissue regeneration,repair damage and other processes,and have repair and regeneration characteristics similar to mesenchymal stem cells(MSCs)and extracellular vesicles(EVs).NPRPs or its combined application with MSCs is expected to improve the efficacy of MSCs cell replacement therapy.This experiment aims to determine whether NPRPs have an effect on the proliferation,cell cycle,and expression of surface markers of MSCs by co-culturing NPRPs in human peripheral blood with MSCs,and provide a new theoretical basis for improving or improving the efficacy of MSCs.Method In a sterile environment,NPRPs were obtained from human peripheral blood by high-speed centrifugation and cultured,and primary MSCs were subcultured.Co-cultivate NPRPs with different concentrations or amounts(0ul,20 ul,40ul,60 ul,80ul,100ul)and P4 generation MSCs in three 96-well plates.The group added with 0ul NPRPs was recorded as the control group,and the group added with 20 ul,40ul,60 ul,80ul,and 100 ul NPRPs was recorded as the experimental group I,II,III,IV,and V,and each group was set with 6 parallel auxiliary holes,and the three well plates were tested for the absorbance(OD)of mesenchymal stem cells in each group by MTT method after co-culture for 24,48,and 72 hours,record the data,and calculate the relative proliferation rate of each group of MSCs by the formula;NPRPs with different concentrations or amounts(0ul,200 ul,400ul,600 ul,800ul,1000ul)and P4 generation MSCs in 6 wells Co-culture in the plate,add 0ul as the control group,and add 200 ul,400ul,600 ul,800ul,and1000 ul as the experimental group I,II,III,IV,and V,respectively.After 48 hours,the cell cycle and surface marker expression of MSCs were detected by flow cytometry,and the MSCs proliferation index was calculated according to the cell cycle results by formula.SPSS20.0 statistical software was used to analyze the data and draw conclusions.Results 1.After co-cultivation for 24 hours,the difference of pairwise comparison of the relative proliferation rates of the MSCs in the experimental I-V groups and the control group had no statistical significance(P>0.05);After co-cultivation for 48 hours,the difference of relative proliferation rates of MSCs in the experimental IV and V groups were statistically significantcompared with the relative proliferation rates of the MSCs in the control group(P <0.05),and there were no statistically significant differences between the other groups(P> 0.05);After co-culture for 72 hours,the difference of relative proliferation rate of MSCs in the experimental III group was statistically significant compared with the relative proliferation rate of the MSCs in the control group(P <0.05),the difference of relative proliferation rates of MSCs in the experimental IV and V groups were significantly higher than those of the control group(P<0.01),and there were no statistically significant differences between the other groups(P> 0.05);and the correlation between the concentration(amount)of NPRPs and the relative proliferation rate of MSCs at different time in co-culture was statistically significant(P<0.01).2.After 48 hours of co-cultivation,the difference of pairwise comparison between each experimental groups and the control group G0 / G1 phase of MSCs was not statistically significant(P>0.05);the difference of pairwise comparison between each experimental group and control group of G2 / M phase was not statistical significance(P>0.05),the difference of pairwise comparison between each experimental group and control group of S phase was not statistical significance(P>0.05);the difference of pairwise comparison between each experimental group and control group of MSC proliferation index was not statistical significance(P>0.05).3.After 48 hours of co-cultivation,the difference of pairwise comparison between each experimental group and control group of the expression levels of each MSCs surface markers(Ig G1,CD44,CD73,CD90,CD105,CD29,CD34,CD45,HLA-DR)was not statistical significance(P>0.05).Conclusion NPRPs can promote the proliferation of MSCs,and the concentration of NPRPs has a significant correlation with the proliferation rate of MSCs.NPRPs have no significant effect on the cell cycle and surface marker expression of MSCs.