Experimental Study Of The Role Of Protein Phosphatase 2A To Migration Of Bone Marrow Mesenchymal Stem Cells

Background: Coronary circulation changes broken the balance between coronary blood flow and myocardial demand which cause myocardial damage is ischemic heart disease(IHD). Its pathological change is necrosis of cardiomyocytes due to ischemia hypoxia ischemia area. Infarction area was gradually replaced by fibrous scars. Residual myocardial compensatory hypertrophy and ventricular remodeling. Ventricular remodeling is a main factor of refractory heart failure and death of patients with myocardial infarction.Although drugs and intervention treatment developed nowedays, it is still unable to fundamentally reverse the ventricular remodeling and subsequent heart failure. Bone marrow mesenchymal stem cells(BMSCs) induce myocardial cell division and regeneration,and repair pathological myocardial,which maintain the steady state of the heart and improve heart function.BMSCs transplantation has become a treatment of heart failure. In recent years a number of studies show that: migration of BMSCs exists in myocadial tissue repair process. How of reactive BMSCs migrate to the pathological myocardial becomes a research hotspot. Numbers of studies have shown that chemokines and protein phosphokinase participate in the migration of BMSCs, but the studies of protein phosphatase remains to be elucidated.Objective: 1) Study the regulation of protein phosphatase 2A(PP2A) to the basal level and SDF-1/CXCR4-mediated migration of bone marrow mesenchymal stem cells(BMSCs); 2)Explore functioning regulatory subunits of PP2 A in SDF-1/CXCR4-mediated migration of BMSCs.Methods:To the experiment we use vitro culture bone marrow mesenchymal stem cells(BMSCs) of SD rat as the research model, and we use the specificity of PP2 A inhibitors,and combining the liposome transfection technique import si RNA, adjust PP2 A activity from different angles. We adopt the scratches and Transwell chamber as the standards to evaluate migration of BMSCs which is regulated under PP2 A.(1) Scratch experiment(wound-ranging) : Culturing different treatment groups of cells with 12-wells plate, eachwell inoculated 1.0 x 105/ml cells, Observing the wound-ranging process with a microscope, comparing the horizontal-motion ability of BMSCs under differant-treated in each group.(2) In Vitro experiment in Transwell chamber: Take BMSCs in good condition and culture after 48 h, digest and harvest cells, using DMEM medium wash three times,respectively inoculated 2.0 x 105 cells in each Transwell chamber which has been treated by OA or SDF-1(membrane pore diameter 8 microns), Cuting filter membrane after 4h,dying with DAPI fluorescent,Observing in fluorescence microscope with 20 times model,each well count 5 horizons and calculate average number, comparing the motion ability of BMSCs under differant-treated in each group. Take BMSCs which are in good condition for liposome transfection with si NC and si PPP2 Ca, the former as the control group and the latter silencing PP2 A catalytic subunit PPP2 Ca, using Transwell to compared with the migration of BMSCs in control group and the si PPP2 Ca group. Exploring PP2 A functional regulatory subunits which may play a role in the process of SDF-1/CXCR4 mediated migration of BMSCs by real-time quantitative PCR.Results:(1) In vitro experiment with Transwell chamber indicated that migration of BMSCs in OA 10 n M,OA 25 n M and OA 50 n M group were significantly reduced,(p <0.05); Migration of BMSCs is negatively correlated to the concentration of OA. Migrated numbers between OA 10 nm group and 25 n M OA group has no statistically significant difference(p > 0.05), but the number OA 50 n M group and OA 10 nm or 25 nm OA group have difference(p < 0.05). Stimulation with both OA and SDF-1, the migration of BMSCs were reduced compared with single stimulus SDF-1 group and it is significantly(p < 0.05).(2) scratch experiment, the bottom level of BMSCs migration distance comparison, OA under the action of BMSCs scratches distance is much smaller than the control group, the healing time of 12 h point OA group and control group no statistical difference(p > 0.05),24 h, 48 h time points are difference than the control group(p < 0.05). Verify the SDF-1/CXCR4 axis chemotaxis, introducing the SDF-1 as migration after chemical inducer, SDF-1 and OA stimulation group in 12 h, 24 h, 48 h scratches healing from the SDF-1 group were significantly reduced, statistically significant(p < 0.05).(3) si PPP2 Ca transfection greatly reduced expression of PPP2 Ca to 8.88 ± 1.52%(p < 0.05). Knockdown of si PPP2 Ca significantly decreased migration ability of BMSCs both at basal level and SDF-1-mediated level(p < 0.05);(4) SDF-1 increased expression of XX(209.37 ± 41.03%)and decreased expressiono of XX(39.58 ± 10.77).Conclusion: 1) Inhibition of phosphatase activity of PP2 A with chemical inhibitors OA suppresseed basal and SDF-1-mediated migration of BMSCs. 2) Knockdown of the main catalytic subunit of PP2 A with si RNA suppressed basal and SDF-1-mediated migration of BMSCs. 3) Regulatory subunits of PP2 A, which named PPP2R2 B in B subgroup and STRN3 in B "’ subgroup, whose expression is regulated by SDF-1. SDF-1 induces high expression of PPP2R2 B and inhibits the expression of STRN3 in BMSCs.