Establishment Of Rabbit Model Of Atherosclerosis And The Treatment By Transplantation Of Autologous Bonemarrow Stem Cells

Objective:This research is to establish the model of atherosclerosis through high fat diet in the japanese white rabbits.The lipid-lowering effect by traasplantation of autologous bone marrow mononuclear(BMMNCs)and bone marrow derived mesenchymal stem cells(MSCs)into the blood circulation through ear vein is observed.Whether the transplanted bone marrow-derived stem cells have the ability of homing to the tunica intima and the distribution of the stem cells in tunica intima are also observed. The possible mechanism of the transplanted autologous bone marrow-derived stem cells accelerating the repair of the intima injury is discussed.We also compare the therapeutic efficacy of BMMNCs and MSCs.Methods:1,Establishment of experimental atherosclerosis model in japanese white rabbits:AS rabbits model was established by high fat feed for 10 weeks.2,Culture of bone marrow-derived stem cells:The rabbits derived BMMNCs and MSCs were isolated and cultived by red blood cells disintegration and adherent culture.3,Characterization of the cultured cells:Cellular morphology was observed under the phase-contrast microscope,the phenotypes were analyzed by immunohistochemistry.3,labeling the BMMNCs and MSCs with BrdU:To definite the best concentration and time of BrdU labeled cells.4,Transplant BMMNCs and MSCs:We transplanted autologous BMMNCs and MSCs via the ear vein to study the effects on abdominal aorta function recovery following AS in rabbits.The measurement of blood lipid,pathological investigation and immunohistochemistry analysis of abdominal aorta tissue were carried out at different times of the experiment,including before(TO)and after BMMNCs and MSCs transplant on 3rd,7th,14th,21th,28th day(T3,T7,T14,T21,T28).Result:1,Culture and characterization of bone marrow-derived stem cells: After 3 days of culture,there are many rounded cells and some heterogeneous small spindle-shaped fibroblastoid cells growing in the culture flask.After 1 week of culture,MSCs grew in colonies that more heterogeneous small spindle-shaped fibroblastoid cells and less rounded cells.The amplification P3-MSCs were homogeneous,spindle and colony-shape.MSCs were positive for CD105,CD106＞98%and negative for CD34.2,Concentration and time of BrdU:The best concentration of Brdu for labeling BMMNCs and MSCs is 20μmol/L 30μmol/L,respectively.And the best time for labeling BMMNCs and MSCs is 72h.Under the circumstance,the amounts of labeled positive cells reach 97%.3,Blood biochemical indicator:Before built up of the model,there are no statistical significance(P＞0.05)on TG,TC,HDL-C,LDL-C in each group.5w after the model,TG,TC and LDL-C levels increased significantly to the highest level,HDL-C reach a minimum level in three model groups. 10w after the model,TG,TC,HDL-C,LDL-C levels in there model group have no obvious difference compared with 5th week.On the 7th day blood lipid level in the two transplantation groups were remarkably lower than the control group.There are statistically significant differentiation between the three groups(P＜0.05).On the 21th day,the TG,TC,HDL-C, LDL-C of the control group of being(4.65±0.95)mmol/L,(16.09±1.89)stool/L,(1.55±0.50)mmol/L,(9.37±1.26)mmol/L respectively,the BMMNCs transplantation group was(2.88±0.49)mmol/L,(13.70±3.52)mmol/L,(2.35±0.50)mmol/L,(3.11±1.16)mmol/L respectively,the MSCs transplantation group being(1.11±0.20)mmol/L,(1.37±0.38)mmol/L,(5.34±0.75)mmol/L,(0.61±0.33)mmol/L respectively, differences among the three groups was significant(P＞0.01).4,The result of pathological observation in abdominal aorta tissue: 10w after the model,lots of convex isabel spots and perthitic lipids on abdominal aorta are visible by naked eyes occurs.Under the light microscope,intima and intima-media thickened,edemaed,there were 3 to 4 layers of foam cells under tunica intima.With the time passing by, atherosclerosis gradually recovered,it was recovered better in the two transplantation groups than in the control group,differences among the two transplantation groups was not obvious.5,The result of immunohistochemistry of abdominal aorta tissues: Two transplantation groups BrdU staining were negative after the transplantation.Conclusion:1,The white rabbits could be made the model of atherosclerosis and hyperlipidemia through feeding with high fat diet for 10 weeks.2,MSCs and BMMNCs both have lipid-lowering capacity,can stop or regress atherosclerotic lesion.3,With regard to hyperlipidemia,the lipid-lowering effect of bone marrow derived mesenchymal stem cells(MSCs)are better than autologous bone marrow mononuclear(BMMNCs).4,The transplanted bone marrow-derived stem cells were not home to abdominal aorta.