| Objective:Bone marrow-derived cells with the self-renewal ability and multipotency of differentiation have shown the capacity of regenerating damaged myocardial cells and vascular endodermis cells. At present, In the dilated cardiomyophy, mesenchymal stem cells (MSCs) intravenous transplantation and bone marrow mobilization are rarely used to treat heart failure caused by cardiomyopathy and the research results about MSCs and bone marrow mobilization to heart failure caused by cardiomyopathy lack theoretical evidence. This experiment includes two parts:The first part is experimental study of combining mesenchymal stem cells intravenous transplantation with recombinant human growth hormone to treat heart failure caused by adriamycin-induced cardiomyopathy, the second part is experimental study of combining bone marrow mobilization with recombinant human growth hormone to treat heart failure caused by adriamycin-induced cardiomyopathy.First, we need confirm that MSCs and CD34+MNC can attract and retain injured myocardial. second,we analyse whether recombinant human growth hormone enhance MSCs and CD34+MNC' survival and differentiation. Our experimental study is to observe the cardiac function and myocardial matrix remodeling between combining rhGH with MSCs intravenous transplantation and bone marrow mobilization to heart failure caused by adriamycin-induced cardiomyopathy and to analysis their mechanism.All of these will provide us to select better transplantation way and get theoretical evidence for clinical treatment.Methods:1.selecting the model of heart failure caused by adriamycin-induced cardiomyopathy.We made the model of heart failure caused by adriamycin-induced cardiomyopathy through adriamycin intraperitoneal injection It included two kinds of models; model one(2.5 mg/kg every time,3 times every week, amount to 6 times, total dose 15 mg/kg) and model two (2.5 mg/kg every time,1times every week, amount to 6 times, total dose 15 mg/kg).We evaluated cardiac function by color Doppler ultrasound and hemodynamic measurement. cardiac weight, body weight,left Ventricular weight are measured. Heart,kidney,liver s pathological test were done. All the above are to select the better model of heart failure caused by adriamycin-induced cardiomyopathy.2.MSCs' abstraction in vitro, cultivation, identification, signature and MSCs'retaining heart.Five-week Wistar rats whose body weight were about 100g were selected.we collected their bone marrow from thigh bone and tibial bone which were separated and cultivated MSCs by adherence method and observed their shape change. When MSCs were the 3rd generation, we measured cell surface antigen CD45, CD44 and CD34 by flow cytometer (FCM) and identified whether these stem cells were MSCs. Before MSCs intravenous transplantation, we signed MSCs by Brdu and observed the distribution of MSCs in heart,kidney and live by mmunohistochemical method.3.The effect of MSCs intravenous transplantation and combined MSCs intravenous transplantation with rhGH to cardiac treatment of heart failure caused by adriamycin-induced cardiomyopathy.90 Wistar rats were divided into heart failure group (HF group, n=30), MSCs intravenous transplantation group(MSCs group, n=30) and combining MSCs intravenous transplantation group with rhGH (GH+MSCs group, n=30). Rats of MSCs group and GH+MSCs group were accepted MSCs transplantation by tail vein injection(1×109个/ml); GH+MSCs were accepted rhGH (2mg/kg, qd,10d) by subcutaneous injection; HF group did not do any treatment. We measured serum ET-1 level and brain natriuretic peptide (BNP) level before the therapy and after the therapy 4 weeks.At the same times,We measured cardiac function by color Doppler ultrasound and hemodynamic measurement. left ventricular samples were accepted pathological test.We detected MSCs' survival in damaged myocadial tissue by Brdu immunohistochemical method and used Brdu and MHC or Actin immunohistochemical two dye method to confirm differentiation into cardiomyocytes.we also used Brdu immunohistochemical method to find vascular formation and observed the number of vascular by HE dye. using RT-PCR method to measure Myocardial expression of IGF-1 mRNA in all groups.In order to observe ventricular remodeling,we tested VG dye and TUNEL experiment.4. The effect of bone marrow mobilization and combined bone marrow mobilization with rhGH to cardiac treatment of heart failure caused by adriamycin-induced cardiomyopathy.70 Wistar rats were divided into heart failure group (HF group, n=20), bone marrow mobilization (MNC group, n=30) and combining bone marrow mobilization with rhGH group(GH+MNC group, n=20). Rats of MNC group and GH+MNC group were accepted rhG-GSF(50ug/Kg, qd,5d) by subcutaneous injection; GH+MSCs were accepted rhGH (2mg/kg, qd,10d) by subcutaneous injection;HF group were given the same voluminal saline. bromodeoxyuridine (Brdu,50mg/kg,qd,4w) were given to all rats of MNC group,HFgroup, GH+MNC group before they sacrificed. After G-CSF stimulating 5 day, Complete blood counts and leukocytes differentiation were obtained and the mononuclear cells(MNCs) were separated by Ficoll gradient centrifugation and labeled with anti-CD34for FACS analysis. After G-CSF stimulating 5 day, hearts of 10 rat in MNC group were left and tested cardiac CD34. We measured serum ET-1 level and brain natriuretic peptide (BNP) level before the therapy and after all the therapy 4 weeks.At the same times,We measured cardiac function by color Doppler ultrasound and hemodynamic measurement. left ventricular samples were accepted pathological test.We used Brdu immunohistochemical method to detect MNC' survival distribution. we also used Brdu and MHC or Actin immunohistochemical two dye method to confirm differentiation into cardiomyocytes. we also used Brdu immunohistochemical method to find vascular formation and observed the number of vascular by HE dye.using RT-PCR method to measure Myocardial expression of IGF-1 mRNA in all groups. In order to observe ventricular remodeling,we tested VG dye and TUNEL experiment.Results:1. Selecting the model of heart failure caused by adriamycin-induced cardiomyopathyLVIDd and LVIDs of model one group were higher than normal group (4.11±0.26 mm vs 3.91±0.23mm, P>0.05; 2.47±0.31 mm vs 0.97±0.21mm,P<0.05); LVIDd and LVIDs of model two Group were increased (5.55±0.36mmvs3.91±0.23mm;3.67±0.44mmvs0.97±0.21m m,P<0.05).EF and FS of model one group and model two group decreased (76.79±8.56%, 70.74±7.86% vs 98.51±3.61%;44.52±3.51%,35.11±3.67% vs 76.77±1.90%,P<0.05); Compa-red to normal group,LVSP,+dp/dtmax and-dp/dtmax were decreased (13.25±0.88Kp a,12.55±0.78 Kpa vs 16.21±0.24K pa;590.67±73.40Kpa/s,489.12±53.4Kpa/svs 861.67±39.96 pa/;534.00±59.22 Kpa/s,499±45.22 Kpa/s vs 632.00±45.19 Kpa/s, P＜0.05), LVEDP higher than normal group(0.97±0.13 Kpa,2.20±0.40 vs-0.08±0.17 Kpa, P＜0.05); B W were lower than normal group (200.33±8.43g,218.11±8.64g vs 240.33±7.11g,P<0.05),HW/BW and LW/BW raised (3.68±0.29g/kg,3.70±0.22 g/kgvs2.73±0.44 g/kg;2.84±0.18 g/kg,2.92±0.22 g/kg vs2.15±0.10 g/kg, P<0.05); Compared to normal group, Ventricular cavity of model group were larger and ventricle wall were thinner; HE dyeing results showed that cardiomyocytes cell existed degeneration and model two showed necrosis; model one in kidneyand liver'injury is bigger than model two. models two of heart failure caused by adriamycin-induced cardiomyopathy could be provided the use of our study by experiment. 2.MSCs' abstraction in vitro, cultivation, identification, signature and MSCs' retaining heartMSCs growed in vitro by adherenc, MSCs presented Spindle-shaped.MSCs did not express surface antigen CD34,CD45and expressed surface antigen CD44.we could see one kind of cell which liked mononuclear Cell in cardiac by HE dye, we detected their survival and distribution in cardiac injuring position by brdu immunohistochemical method and saw brown cellular nucleus in cardiac injuring position whearas rarely saw brown cellular nucleus in kidney,liver.we confirmed that the cells cultured in vitro were MSCs and MSCs could retain and settle cardiac injured position.3.The effect of MSCs intravenous transplantation and combined MSCs intravenous Transpa-ntation with rhGH to cardiac treatment of heart failure caused by adriamycin-induced cardiomyopathy3.1 cardiac echocardiography resultsCompared with HF group, LVIDd and LVIDs of MSCs group and GH+MSCs group rats were decreased (3.41±0.72 mm,2.50±0.44 mm vs 5.50±0.34 mm;1.51±0.32 mm,1.06±0.33 mm vs3.61±0.56, P＜0.001); EF and FS increased(89.11±7.58%,93.12±6.44%vs 71.13±8.48%;54.32±2.89%,61.77±3.12% vs35.44±1.91,P<0.001).MSCs and MSC+GH therapy could improve cardiac function of rats which suffered from heart failure caused by adriamycin-induced cardiomyopathy by color Doppler ultrasound.3.2 Haemodynamics resultsLVSP,+dp/dpmax and-dp/dpmax of MSCs group and GH+MSCs group rats were all higher than those of HF group(14.88±0.78Kpa,15.66±0.21Kpa vs 12.55±0.88Kpa;790.67±73.88 Kpa/s,827.67±33.96 Kpa/svs 489.12±53.40 Kpa/s;600.33±45.11 Kpa/s,611.00±43.19 Kpa/s vs 499±45.22 Kpa/s,P<0.05); LVEDP were all lower than those of HF group (-0.01±0.11 Kpa,-0.03±0.13 Kpa vs 2.20±0.40 Kpa/s, P＜0.05).MSCs and MSC+GH therapy could improve cardiac function of rats which suffered from heart failure caused by adriamycin-induced cardiomyopathy by hemodynamic measurement.3.3 Serum BNP and ET-1 resultsAfter treatment, BNP levels of MSCs group and GH+MSCs group were obviously lower than before treatment (210.12±14.44pg/ml vs390.62±11.98 pg/ml;177.34±21.33pg/ml vs 401.33±22.12 pg/ml P＜0.001);BNP levels of HF group did not have stastics meaning between before treatment and after treatment (408.25±31.45pg/ml vs388.12±9.52pg/ml, p>0.05). After treatment, ET-1 levels of MSCs group and GH+MSCs group were obviously lower than before treatment (2.56±0.34 ng/ml vs4.92±0.59 ng/ml;1.88±0.21 ng/ml vs 5.01±0.77 ng/ml,P<0.001);ET-1 levels of HF group did not have stastics meaning between before treatment and after treatment (5.22±0.30 ng/ml vs5.12±0.36 ng/ml, p>0.05).MSCs and MSC+GH therapy could improve cardiac function of rats which suffered from heart failure caused by adriamycin-induced cardiomyopathy by serum ET-1 level and serum brain natriuretic peptide (BNP) level.3.4 cardiomyocytes HE dye,immunohistochemica and Myocardial expression of IGF-1 mRNAwe could observ brown cellular nucleus in vascular endodermis cell between MSCs group and GH+MSCs group and also found that the vascular number in MSCs group and GH+MSCs group increased(16.33±1.41/0.2mm,18.44±1.17/0.2mmvs9.90±1.6/0.2mm,P<0.001), if we added GH,we could increase this kind cell and vascular number.We could see red cellular nucleus and brown cytoplasm between MSCs group and GH+MSCs group by immunohistochemical two dye method, we also saw cell membrane and lateral stripe. if we added GH,we could increase this kind cell number.IGF-1 mRNA relative amount of MSCs group and GH+MSCs group were higher than that of HF group (0.7118±0.0106,1.0361±0.0157 vs 0.5017±0.0121,P<0.05); compared to MSCs group,IGF-1 mRNA relative amount of GH+MSCs group was increased (1.0361±0.0157 vs0.7118±0.0106,P<0.05).we found that MSCs could differentiate into cardiomyocytes and vascular cells in cardiac injured position.and GH could enhance this cell number in cardiac injured position; MSCs and MSC+GH therapy could increase the expression of IGF-1 in Myocardial which could enhance these cells'proliferation and differentiation.3.5 VG dye and the number of apoptosisAfter treatment,we found that collagen volume fraction (CVF) levels of MSCs group and GH+MSCs group were obviously lower than HF group(15.20±5.40%,14.33±5.11% VS19.88±2.78%, P<0.05); After treatment, we found that the levels of cardiomyocytes apoptosis of MSCs group and GH+MSCs group were obviously lower than HF group (17.44±6.78%,12.34±2.98% vs27.33±7.98 P＜0.05).we foud that MSCs and MSC+GH therapy could decrease the number of collagen and apoptosis and improve ventricular remodeling.4. The effect of bone marrow mobilization and combined bone marrow mobilization with rhGH to cardiac treatment of heart failure caused by adriamycin-induced cardiomyopathy.4.1 the number of MNC and CD34 marker G-CSF mobilization increased MNC numbers,we found that MNC numbers of MNC group and GH+MNC group after treatment were obviously higher than before treatment (15.79 X 109/L vs 5.36 X 109/L;15.99 X 109 vs6.30 X 109/L,P<0.05); MNC numbers of HF group did not have stastics meaning between before treatment and after treatment (4.92 X 109/L vs 5.39 X 109/L P>0.05); The number of circulating CD34+MNCs in peripheral Flow cytometric analys is showed CD34 surface marker between MNC group and GH+MNCgroup.cardiac immunofluore-scence showed CD34 surface marker between MNC group and GH+MNC group. We found that G-CSF mobilization increased CD34+MNC numbers and CD34+MNC could retain and settle cardiac injured position;GH could enhance CD34+MNC number in cardiac injured position.4.2 cardiac echocardiography resultsCompared with HF group, LVIDd and LVIDs of MNC group and GH+MNC group rats were decreased (2.91±0.66mm,2.77±0.77 mm vs 5.50±0.34 mm;1.51±0.55 mm,1.45±0.26mm vs3.61±0.56 mm, P＜0.001); EF and FS increased(84.55±7.48%,90.24±5.43% vs 71.13±8.48%;46.32±2.89%,54.42±5.12 vs35.44±1.91,P<0.001).MNC and GH+MNC therapy could improve cardiac function of rats which suffered from heart failure caused by adriamycin-induced cardiomyopathy by color Doppler ultrasound.4.3 Haemodynamics resultsLVSP,+dp/dpmax and-dp/dpmax of MNC group and GH+MNC rats were all higher than those of HF group(14.33±0.44Kpa,15.22±0.33Kpa vs 12.55±0.88Kpa; 767.44±71.47Kpa/s, 803.67±56.63 Kpa/s vs489.12±53.40 Kpa/s;601.33±42.33Kpa/s,603.00±46.29 Kpa/svs 499±45.22Kpa/s, P＜0.05); LVEDP were all lower than those of HF group (-0.08±0.11 Kpa,-0.11±0.17 Kpa vs 2.20±0.40 Kpa/s,P<0.05). MNC and GH+MNC therapy could improve cardiac function of rats which suffered from heart failure caused by adriamycin-induced cardiomyopathy by hemodynamic measurement.4.4 Serum BNP and ET-1 resultsAfter treatment, BNP levels of MNC group and GH+MNC group were obviously lower than before treatment (207.55±18.56pg/ml vs377.12±21.38 pg/ml;189.14±27.63pg/ml vs389.43±24.16 pg/ml, P＜0.001);BNP levels of HF group did not have stastics meaning between before treatment and after treatment (399.77±21.75pg/ml vs393.12±19.62pg/ml,p>0.05). After treatment, ET-1 levels of MNC group and GH+MNC group were obviously lower than before treatment (2.11±0.44 ng/ml vs4.99±0.67 ng/ml;1.89±0.17ng/ml vs 5.01±0.87ng/ml P＜0.001);ET-1 levels of HF group did not have stastics meaning between before treatment and after treatment (5.67±0.43ng/ml vs5.70±0.56 ng/ml p>0.05). MNC and GH+MNC therapy could improve cardiac function of rats which suffered from heart failure caused by adriamycin-induced cardiomyopathy by serum ET-1 level and serum brain natriuretic peptide (BNP) level.4.5 cardiomyocytes HE dye,immunohistochemica and myocardial expression of IGF-1 mRNAWe could observed brown cellular nucleus in vascular endodermis cell between MNC group and GH+MNC group and also found that the vascular number in MNC group and GH+MNCgroup increased(14.67±1.33/0.2mm,15.65±1.21/0.2mmVS9.47±1.21/0.2mm P＜0.00 1), if we added GH,we could increase this kind cell and vascular number. We could see red cellular nucleus and brown cytoplasm between MNC group and GH+MNC group by mmunohistochemic-ial two dye method, we also saw cell membrane and lateral stripe,if we added GH,we could increase this kind cell number.IGF-1 mRNA relative amount of MNC group and GH+MNC group were higher than that of HF group (0.7034±0.0113,0978±0.013 vs 0.497±0.0141 P＜0.05); compared to MNC group,IGF-1 mRNA relative amount of GH+MNC group was increased (0978±0.013 vs0.7034±0.0113 P＜0.05). We found that MNC could differentiate into cardiomyocytes and vascular cells in cardiac injured position.and GH could enhance these cells number in cardiac injured position; MNC and MNC+GH therapy could increasethe expression of IGF-1 in Myocardial which could enhance the cells'proliferation and differentiation.4.6 VG dye and the number of apoptosisAfter treatment, we found collagen volume fraction (CVF) levels of MNC group and GH+MN group were obviously lower than HF group(13.20±4.40%,12.89±4.65% vs21.21±4.33 %P＜0.05); After treatment, we find the levels of cardiomyocytes apoptosis of MNC group and GH+MNC group were obviously lower than HF group (8.44±6.34%,6..34±5.98% VS27.33±7.33 P＜0.05). We foud that MNC and MNC+GH therapy could decrease the number of collagen and apoptosis and improve ventricular remodeling.Conclusion:1.model 2 of heart failure caused by adriamycin-induced cardiomyopathy were made successfully and could be provided the use of our study.2.The cells cultured in vitro were MSCs; MSCs could retain and settle cardiac injured position,GH could enhance MSCs number in cardiac injured position.3. MSCs could differentiate into cardiomyocytes and vascular cells in cardiac injured position., GH could enhance this cell number in. cardiac injured position.4. MSCs and MSCs+GH therapy could increase the expression of IGF-1 in Myocardial which could enhance MSCs'proliferation and differentiation,5. MSCs and MSCs+GH therapy could improve cardiac function of rats which suffered from heart failure caused by adriamycin-induced cardiomyopathy6. MSCs and MSCs+GH therapy could decrease the number of collagen and apoptosis and improve ventricular remodeling.7. G-CSF mobilization increased CD34+MNC numbers and retained and settled cardiac injured position.,GH could enhance CD34+MNC number in cardiac injured position.8. CD34+MNC could differentiate into cardiomyocytes and vascular cells in cardiac injured position,GH could enhance this cell number in cardiac injured position.9. MNC and MNC+GH therapy could increase the expression of IGF-1 in Myocardial which could enhance MNC' proliferation and differentiation,10. MNC and GH+MNC therapy could improve cardiac function of rats which suffered from heart failure caused by adriamycin-induced cardiomyopathy11. MNC and GH+MNC therapy could decrease the number of collagen and apoptosis and improve ventricular remodeling.
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