| Objective Non-purified bone marrow stem cell suspension and the suspension of purified bone marrow stem cells were transplanted into the hindlimb ischemia of clean male Sprague-Dawley (SD) rats, which are inbred rats. The expression of vascular endothelial growth factor (VEGF) and the effects on angiogenesis and the function of ischemic hindlimb was observed after transplantation, then the value of autologous bone marrow stem cell transplantation in the treatment of Chronic lower extremity ischemic disease was explored, and the validity and invalidity of the two methods was compared, in order to provide theoretical basis on its clinic application.Methods:30 SD rats, randomly divided into 3 groups, experimental group 1 (n= 10): non-purified bone marrow cells suspension was injected directly into the muscles of hindlimb ischemia; experimental group 2 (n= 10):suspension of purified bone marrow stem cells for cell transplantation was injected into the muscles of hindlimb ischemia; the control group (n= 10):only injected with the same amount of PBS solution the same ways as group 1 and group 2;Successively puncture SD rats'femoral and shin bone marrow cavity both sides once, then get lml bone marrow iquid four times, which was made to filtration liquid 0.8 ml in group 1.In group 2, bone marrow cell suspension 0.8ml,added into PBS solution,was purified with density gradient centrifugation method, then added it into PBS solution again to made purified cells suspension; As a second step,ligated and resected SD rats'right femoral artery and its branches to establish experimental animal model of femoral artery occlusion with Yang Cheng jia reported method. Did the bone marrow cells transplantation as soon as the models were built successfully.4 weeks after transplantation, used RT-PCR to detect VEGF mRNA expression in thigh muscle homogenate made of femoral ischemic muscle,and used ELISA method to detect vascular endothelial growth factor (VEGF) levels in blood serum, then the blood flow of skin microcirculation was measured with laser Doppler flowmeter(LDF),finally, and the limb function score was made.Results (1) LDF detecting blood flow improvement in lower extremity 4 weeks after transplantation, the skin perfusion difference between both of the hind limbs treatment group 1 was 14.1±6.6, treatment group 2 was 19.9±7.2, control group was 31.4±9.6, both of treatment groups significantly decreased compared with the control group, The differences between treatment groups and control group were statistically significant (p<0.05); In contrast to treatment group 2, the result of treatment group 1 was better, (p<0.05).(2) the assessment of limb function 2 weeks after transplantation, compared with the control group, the hindlimb function scores of the treatment groups have no significant difference (P> 0.05), but after 3 weeks and 4 weeks are significantly different (P<0.05), and after 4 weeks, function score of the treatment group 1 is less than that of group 2, the difference is statistically significant (P<0.05).(3) Comparison of the blood serum level of VEGF and VEGF mRNA of each group after 4 weeks,both treatment groups'serum VEGF levels are significantly higher than the control group, and the serum content of VEGF of treatment group 1 is higher than treatment group 2, the difference is statistically significant (p<0.05); VEGF mRNA expression among hindlimb muscles increase relatively in the two treatment groups(P<0.05), the level of VEGF mRNA expression in treatment group 1 was higher than treatment 2 group (P<0.05).Conclusion (1) Both of non-purified bone marrow cells suspension transplantation and purified bone marrow stem cell suspension transplantation for treatment of the rat's ischemic hindlimb were to be effective, which possibly through increase secretion of VEGF in the ischemic hindlimb to promote the establishment of collateral circulation,and to improve the rats'limb function; VEGF expression level is positively correlated with the establishment of collateral circulation in hindlimb tschemia(2)Non-purified bone marrow cells transplantation has better therapeutic effect on collateral circulation formation in ischemic tissue than purified bone marrow stem cell transplantation. |
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