Experimental Study On The Low Limb Ischemia Diseases Treated With Combined Application Of Bone Marrow Mesenchymal Stem Cells Transplantation And Erythropoietin

BackgroundThe lower limb ischemic disease is very common in clinical practice.It mainly includes arteriosclerosis obliterans,diabetic arteriosclerosis and thromboangitis obliterans,etc.These diseases can cause arterial stenosis or obliteration which could lead to intermitten claudication,pain and limb ischemic necrosis.Vascular prosthesis or autogenous blood vessel bypass grafting and interventional therapy are usual treatments.However,when it comes to the critical chronic lower limb ischemia induced distal out-flow tract defective limbs,surgeries mentioned above are no longer the answer.Singular medicine treatment is not so effective either.Many patients can not escape amputation in the end which is a painful disaster.Hopefully,therapeutic angiogenesis brings new hope to these patients.Therapeutic angiogenesis promotes the formation of both new blood vessels and collateral circulation by means of transferring heterogeneous angiogenesis inducing factors into ischemic tissue.It mainly contains two strategies.One is using cytokines or cytokine gene carriers,the other is transplanting multi-directional differentiation potent stem cells to ischemic tissue,where they would differentiate into vascular endothelium and produce vascular growth factors to achieve angiogenesis.Cells that could be used to treat limb ischemic diseases can be embryonic stem cells(ES), endothelial progenit cells(EPCs),bone marrow mononuclear cells,(BMMNCs), peripheral blood stem cells(PBSCs),and mesenchymal stem cells(MSCs) and so on.Using cell growth factors and gene therapy treating ischemic disease has gained certain improvement in both fundamental research and clinical practice,but still these treatments have some problems.First,aside from angiogenesis,these growth factors have some side effects,for example they could cause transient hypotension,tissue edema;Second,they may accelerate coronary damage,and even lead to embryonic cardiovascular malformation or embryo death,etc.Third,in gene therapy,naked DNA plasmid and non-virus vector have low transfection rate and short time duration, whereas virus vectors like adenovirus have relatively high transfection efficiency, nonetheless hold the hidden danger of immune rejection.Forth,both the effectiveness and safety of long-term use of growth factor proteins or gene therapy to treat lower limb ischemic disease is yet to be confirmed by extended clinical researches.Fifth,for patients with aging,diabetic mellitus,hypercholesteremia and hyperhomocysteinemia, etc,their vascular regenerative ability is defective,in addition to decreased endogenous vascular growth factor synthesis,the dysfunction of vascular endothelium makes declined reactivity to cytokines,therefore vascular growth factors alone could not efficiently stimulate vascular regeneration.As to embryonic stem cell transplantation,it may be oncogenic,and allograft antigenicity is another unavoidable problem,even autografting may not escape immune rejection because of genetic variation.What's more,there is a high incidence rate of aneuploid during the development and differentiation process,although embryonic stem cell can differentiated into various cell types,it's non-orientational.Additionally,before transplantation,embryonic stem cell donors should be checked for severe genetic disease.These disadvantageous factors as well as ethical issues confined the extensive application of embryonic stem cells.Adult stem cells transplantation is another strategy for therapeutic angiogenesis. The cells used for the transplantation maily included EPCs,BM-MNCs,PBSCs, MSCs.At present,Autologous transplantation of BM-MNCs is most common in clinical practice.BM-MNCs contain EPCs which can transformed into vascular endothelia cells to promote vasculogenesis.BM-MNCs could also secrete pro-angiogenesis cytokines to promote vascular proliferation.Accordingly,local blood supply is improved due to increased tissue microvascular number and the establishment of collateral circulation.The therapeutic effects of BM-MNCs transplantation have gained both experimental and clinical confirmation.But still BM-MNCs transplantation has the following flaws:both lab and clinical research show that the pro-angiogenesis effects are related to the number of stem cells transplanted,however,the number of EPCs contained in bone marrow and peripheral blood is extremely limited,and continually declined with the growing age;As to patients with senior age,unsound physical fitness complicated with critical diabetic mellitus,it's increasingly hard to collect enough high quality BM-MNCs to achieve optimal therapeutic effects due to the decreased number and declined quality of EPCs. Compared with the cells above,MSCs is a better source for inducing therapeutic angiogenesis,because it could secrete multiple angiogenic cytokines and differentiate into ECs or SMCs,and it could be amplified in vitro to get considerable high quality cells for transplantation without immunologic rejection.However,preliminary studies revealed that ischemia and mechanical stress could induce apoptosis of transplanted MSCs which were injected immediately after femoral ligation resulting in failed improvement of flow recovery.Kinnaird found that the vasculogenesis effect depend on the number of the transplanted MSCs,but the vasculogenesis effect could not increase unlimitedly along with the increase of the MSCs.So combining MSC transplantation and other treatment is very important for a better curative effect. EPO has been recently demonstrated to be able to inhibit apoptosis,enhance the activity of MSCs,amplify the MSCs to secrete VEGF,induce angiogenesis by mobilizing EPCs from bone marrow to home to the ischemia area.Combined treatment with MSC transplantation and EPO may achieve a more effective angiogenesis.This study including three parts investigates those questions:(1) To observe the clinical efficacy and analyze the influcing factors of autologous transplantation of bone marrow mononuclear cells(BM-MNCs) in the treatment of lower extremity ischemic disorders.(2) To research the separation,in vitro culture and expanding method of mesenchymal stem cells(MSCs) and identifying the MSCs cultured by this mothod.Study the biological characteristics of MSCs and result of 4',6-diamidino-2-phenylindole(DAPI) labeling MSCs.(3) To investigate the effect of EPO on the therapeutic potency of MSCs transplantation in a rat model of limb ischemia.Part 1.The clinical efficacy and influcing factors of autologous transplantation of bone marrow mononuclear cells in the treatment of lower extremity ischemic disordersObjective:To observe the clinical efficacy and analyze the influcing factors of autologous transplantation of bone marrow mononuclear cells(BM-MNCs) in the treatment of lower extremity ischemic disorders.Methods:Totally 26 cases were treated by autologous transplantation of BM-MNCs.After aspiration of bone marrow blood from the iliums,the BM-MNCs were separated and then injected into the skeletal muscles of the ischemic legs.The pain,cool feeling and Intermittent claudication were observed as well as the clinical subjective score,ankle-brachial index(ABI) and the blood peak speed of the ischemic legs were monitored 3 months after the surgery.All the clinical and laboratory findings were compared with that before the surgery in order to estimated the clinical efficacy of autologous transplantation of BM-MNCs.Then the cases were divided into the high concentration group(＞1×10⁷/ml) and the low concentration group (＜1×10⁷/ml) by the concentration of the BM-MNCs injected into the muscles.The improvement degree of the two groups were compared.The efficacy of the BM-MSCs concentration on the therapy were evaluated.Results:3 months after the BM-MNCs transplantation,The lower limbs pain alleviating rate,cool feeling alleviating rate and the Intermittent Claudication alleviating rate were respectively 92%,84%and 88%.The clinical subjective score after the transplantation were lower than that before the transplantation(P＜0.01),as well as the ABI and the blood peak velocity of the lower limbs signicantly increased than that before the transplantation(0.33±0.22 vs 0.24±0.17),(26.65±7.31 vs 18.32±5.74)(P＜0.05).The difference value of the pain,cool feeling,intermittent claudication and the blood peak velocity in the high concentration group before and after the transplantation is sigenificantly higher than that of the low concentration group(P＜0.05).However,the difference value of ABI in the high concentration group is higher than that of the low concentration group with no statistical significance (P＞0.05).All the patients had no obvious complications during the follow up period.Conclusions:Autologous transplantation of the BM-MNCs could be a safe and effective method for treating patients with lower extremity ischemic disorder.The quantity of the mononuclear cells,the extent of ischemia,especially the concentration of the mononuclear cells injected into the muscles significantly influent the result of the treatment.Part 2.In vitro culture and biological characteristics observe of mesenchymal stem cells Objective:To research the separation,in vitro culture and expanding method of mesenchymal stem cells(MSCs) and identifying the MSCs cultured by this mothod. Study the biological characteristics of MSCs and result of 4',6-diamidino-2-phenylindole(DAPI) labeling MSCs.Method:MSCs were isolated and purified from rats using density centrifugation and anchoring culture,then cultured in low-glucose DMEM supplement with 10% fetal bovine serum(FBS) for expanding.Observed the morphous and growth velocity of MSCs,identify the phenotype of MSCs,draw the growth curve of MSCs and determine the mitotic index and cloning efficiency of MSCs.Observed the survival rate and growth state of MSCs underwent freeze thawing.Also observed the efficiency of DAPI labeling MSCs and impact of DAPI on MSCs.Result:The cultured MSCs were typical spindle-shaped,grew in whirlpool anchoring the culture plates,arranged gyrately.Through detection by flow cytometry, the expressions of CD44 and CD29 were positive,the exprssions of CD11b and CD45 were negative,which was in accordance with the phenotype of MSCs.MSCs got together at 9^th～12^th day.The passage MSCs proliferated fast.MSCs were purified at 3^rd generation.As the passage increases,the cloning efficiency and proliferative ability of MSCs decrease.The survival rates of MSCs thawed after frozen 1 month and 6 months were 90%and 85%respectively.The efficiency of DAPI labeling MSCs was 100%.The fluor colouration didn't weaken.DAPI had no influence on the morphous and growth of MSCs.Conclusion:MSCs can be harvested using density centrifugation and anchoring culture.In vitro expanding of MSCs is feasible.Freeze thawing has no obvious influence on MSCs.DAPI can label the nucleus of MSCs effectively and has no obvious influence on the morphous and growth of MSCs.Part 3.Erythropoietin augments the efficacy of therapeutic angiogenesis induced by allogenic bone marrow stromal cells in a rat model of limb ischemiaObjective:Transplantation of mesenchymal stem cells(MSCs) has been developed as a new method of treating severe ischemia diseases by therapeutic angiogenesis.Erythropoietin(EPO) is capable of inducing angiogenesis and inhibiting MSCs apoptosis.This study was to investigate the effect of EPO on the therapeutic potency of MSCs transplantation in a rat model of limb ischemia. Methods:The included rats(n=32) underwent right femoral artery ligation to create a model of hind limb ischemia and were randomly divided into four groups (n=8,each).24h after the operation,rats in the MSC+EPO group were injected intramuscularly with 0.4ml DMEM loaded with MSCs(1×10⁷cells) and EPO (1000U/kg body weight) into eight points of the ischemic area(0.05 ml each).In like manner,the MSC group was injected with the same dose of DMEM loaded with only MSCs,whereas the control group and the EPO group were injected with the same dose of DMEM only.EPO(1000U/kg body weight in 0.4ml saline solution) was injected once daily during the first 3 days each week for the three-week studies period to the EPO+MSC group and the EPO group.Other rats were treated in like manner with the same dose of saline solution.Laser Doppler Perfusion Imaging(LDPI), Angiography,immunohistochemistry and RT-PCR were performed to examine the results of treatments at 22 days after ligation.DAPI labeled MSCs were transplanted into another five rats and detected by Immunofluorescence double staining.Results:The blood perfusion was more affluent in the EPO group and the MSC group than those in the control group and it was significantly higher in the MSC+EPO group than in the other groups(P＜0.01).Angiography demonstrated that more collateral establishment were seen in MSC group,EPO group and in MSC+EPO group,in which there were the most affluent collateral branches ever,than in the control group.The quantitative analysis of angiographic score(AS) showed that AS was better in MSC group,EPO group and MSC+EPO group than that in the control group(P＜0.05),and the MSC+EPO group got the highest score(P＜0.05). Immunohistochemistry exhibited that the concentration of vessel was significantly increased in MSC group,EPO group and MSC+EPO group than in the control group (P＜0.05),especially in MSC+EPO group(P＜0.05).Most of the new vessels were capillaries without vascular smooth muscle,whose areas were not significantly different among these groups(P＞0.05),whereas less of them were capillaries with smooth muscle,whose quantities were not significantly different among these groups (P＞0.05).Moreover,when it comes to the area of the new vessels with smooth muscle, MSC group is more significant than the control group(P＜0.05) and less significant than MSC+EPO group(P＞0.05),as well as there was no significant difference between EPO group and the control group(P＞0.05).Immunofluorescence double staining demonstrated that MSC could survive in vivo and differentiated into vascular endothelial cells(ECs) and smooth muscle cells(SMCs).Immunohistochemistry and computer imaging analysis found that VEGF and Bcl-2 were up regulated and Bax was down regulated in the MSC+EPO group compared with the other three groups (P＜0.05).RT-PCR found that VEGF mRNA was highest in the MSC+EPO group, fllowed by the MSC group and the EPO group,lowest in the control group(P＜0.05).Conclusion:MSC transplantation or EPO injection alone can accelerate angiogenesis,development of the collateral vessels and tissue blood flow recovery. The combined treatment with MSC transplantation and EPO infusion is superior to MSC transplantation alone in the treatment of limb ischemia.