| Objective:At present mesenchymal stem cells(MSCs) become the hot spot.In the cardiovascular domain they are used mainly to treat coronary heart disease whearas rarely to heart failure caused by dilated cardiomyopathy,and the research result about MSCs to heart failure caused by cardiomyopathy are disagreed.Because the MSCs' survival rate is very low in vivo,are dissatisfied.Our experimental study is to detect the function of recombinant human growth hormone(rhGH) to MSCs in vivo and in vitro,to observe the therapeutic effects of combining rhGH and MSCs transplantation to heart failure caused by adriamycin-induced cardiomyopathy, and to analysis its mechanism.In order to search a drug which not only can improve MSCs' survival rate,but also have no disadvantage contribution to elevate the therapeutic effects of MSCs transplantation,and provide theoretical evidence for clinical treatment.Methods:1.Making the model of heart failure caused by adriamycin-induced cardiomyopathy.We used adriamycin intraperitoneal injection(2.5 mg/kg every time,3 times every week for a week,2 weeks interval,injection for a week again,amount to 6 times,total dose 15 mg/kg) to make the model of heart failure caused by adriamycin -induced cardiomyopathy.We measured left ventricular end-diastolic inner diameter(LVDD),left ventricular end-systolic inner diameter(LVSD),left ventricular eject fraction(LVEF),left ventricular fractional shortening (LVFS) by color Doppler ultrasound,left ventricular systolic pressure(LVSP),positive rate of rise of left ventricular pressure(dp/dtmax),left ventricular end-diastolic pressure(LVEDP) and negative rate of rise of left ventricular pressure(—dp/dtmax) by hemodynamic measurement.The ratio of whole cardiac weight/body weight(HW/BW) and left ventricular weight/body weight (LW/BW) were obtained.Pathological test were done.All the above demonstrated the model of heart failure caused by adriamycin -induced cardiomyopathy are successful.2.MSCs' abstraction in vitro,cultivation,identification,derivation,differentiation,signature and ultrastructural change. We chose Wistar rats of seven-week aged whose body weight are about 160-170g,collected their bone marrow both of thigh bone and tibial bone and used whole bone marrow adherence method to separate and cultivate MSCs;observing their growth-up characteristics.When MSCs went down to the 3rd generation,we used flow cytometer(FCM) to measure cell surface antigen CD45,CD44 and CD71 to identify whether the cells cultured in vitro were MSCs;of the 3rd generation were added into 5-azacytidine(5-Aza) which was 10μmol/l for 24h,at the 2 weeks, 3 weeks and 4 weeks we detected the expression ofα-sarcomeric actin and myosin heavy chain(MHC) by immunohistochemical method and observed the transformation efficiency of MSCs to myocyte by FCM;at the same time we collected the MSCs which were not induced by 5-Aza and induced by 5-Aza for 2 weeks,3 weeks and 4 weeks to inspect their ultramicrostructure;before transplantation we used BrdU of 10mg/l concentration to sign MSCs for tracking their direction in vivo.3.The research of rhGH to MSCs in vitro and its possible mechanismWe chose the 2nd generation MSCs to measure the growth rate or inhibition ratio of different concentration(1ng/ml,10 ng/ml,50 ng/ml,100 ng/ml,200 ng/ml and 500 ng/ml) rhGH in different time(24h,48h and 72h).Then chose the best concentration(200ng/ml) of contributed to MSCs for the best time(72h) and measured the growth rate or inhibition ratio;and determined the expression of insulin-like growth factor 1(IGF-1) mRNA in different time(24h,48h and 72h of rhGH directive function and the 4th,9th,10th day and the 2nd,3rd,4th week after removing rhGH directive function) by RT-PCR to certify initially whether rhGH enhances the growth of MSCs by IGF-1 mediation.4.The effect of rhGH,MSCs transplantation and combined rhGH and MSCs transplantation to cardiac function of heart failure caused by adriamycin-induced cardiomyopathy.35 Wistar rats were divided into normal group(n=6),heart failure group(HF group,n=9), rhGH group(GH group,n=6),MSCs transplantation group(MSCs group,n=7) and rhGH combining MSCs transplantation group(G+M group,n=7).Rats of MSCs group and G+M group were accepted MSCs transplantation by punctiform injection into myocardium(the number of cells is 5×106);those of GH group and G+M group were accepted rhGH(2mg/kg,qod) by subcutaneous injection at three days before MSCs transplantation;those of normal group and HF group were injected the same voluminal saline,for a month.We measured the growth hormone (GH) level and brain natriuretic peptide(BNP) level in serum before and after all the therapy;a month later,all the haemodynamic index were obtained including systolic pressure(LVSP), positive rate of rise of left ventricular pressure(dp/dtmax),left ventricular end-diastolic pressure(LVEDP) and negative rate of rise of" left ventricular pressure(-dp/dtmax);whole heart quality index(HW/BW) and left ventricular quality index(LW/BW) were gained;left ventricular samples were accepted pathoscopy and ultramicro- pathoscopy.5.Mechanism research of combining rhGH and MSCs transplantation improving cardiac function of heart failure caused by adriamycin-induced cardiomyopathy.We used immunohistochemical method to measure the MSCs which were signed by BrdU before transplantation in order to detecting their survival and distribution in vivo; homeochronous detection of BrdU(in cellular nucleus) and MHC(in cytoplasm) in a cell can trail the myocytes which were transferred by MSCs;we referenced Mercadier JJ's method to extract MHC of myocardium,then to separated its isozyme:V1- type MHC and V3- type MHC, and observe their changes respectively and their reciprocal transformation information;using RT-PCR method to measure the expression of IGF-1 mRNA;by all the above observation we get the analysis about the mechanism of combining rhGH and MSCs transplantation improving cardiac function of heart failure caused by adriamycin-induced cardiomyopathy.Results:1.Making the model of heart failure caused by adriamycin-induced cardiomyopathy.Compared to normal group,LVDD and LVSD of model group were increased (5.71±0.27mm vs 4.16±0.20mm,3.17±0.31mm vs 1.07±0.11mm,P＜0.01),EF and FS decreased (76.79±8.56%vs 94.51±2.61%,44.52±3.51%vs 74.27±1.80%,P＜0.01);LVSP,dp/dtmax and -dp/dtmax were all lower than normal group(14.27±0.98 Kpa vs 15.73±0.25 Kpa,600.67±83.30 Kpa/s vs 731.67±34.96 Kpa/s和469.00±54.11 Kpa/s vs 583.00±32.19 Kpa/s,P＜0.05),LVEDP higher than normal group(0.85±0.29 Kpa vs -0.07±0.23 Kpa,P＜0.05);BW lessened than normal group(262.00±7.87g vs 303.33±6.11g,P＜0.01),HW/BW and LW/BW increased (3.74±0.29 g/kg vs 2.86±0.10 g/kg and 2.91±0.25 g/kg vs2.25±0.15 g/kg,P＜0.01);chambers heart of model group were larger than that of normal group,ventricle wall became thin and stiff; HE dyeing results showed cadiocyte were dropsy and balloon-like,cytoplasm dyeing of were uneven,and showed inflammatory cell infiltrate.2.MSCs' abstraction in vitro,cultivation,identification,derivation,differentiation,signature and ultrastructural change.MSCs in vitro presented adherence-like growth,most MSCs were fibroblast-like spindle cell;cell surface antigen CD45 expression were negative,CD44 and CD71 were positive;at the 2nd,3rd,4th week after 5—Aza induceα—sarcomeric actin and MHC were expressed,and with the time extend their expression were added;the expression of MHC mRNA were increased gradually at the 0,2nd,3rd,4th week(0.5497±0.0099,0.8376±0.0202,0.9525±0.0044 and 1.0352±0.3484,P＜0.05);Transformation efficiency of MSCs to myocyte at the 2nd,3rd,4th week via 5-Aza induce were higher than the non-induced group;ultramicroresults showed bigger cell nucleus,double amphinucleoli,less organelles,nevertheless MSCs via 5-Aza induce have myofilament structure,more organelles including Golgi's body,mitochondrium,rough endoplasmic reticulum,et al.With the time extend,the expression of myofilament and organelles increased.3.The research ofrhGH to MSCs in vitro and its mechanismAbove the 10ng/ml concentration,rhGH can enhance the MSCs growth.200ng/ml was the best concentration,the;at the 72nd hour the growth rate of 200ng/ml rhGH was 144.10%;the expression of IGF-1 mRNA increased gradually at Oh,24h,48h and 72h(0.4391±0.0058, 0.6749±0.0084,0.7781±0.0068 and 0.8230±0.0060,P＜0.05);when removing rhGH action,the expression of IGF-1 mRNA were reduced compared to rhGH direct-acting,but still increased with the time extended,at the 2nd week its expression got to summit,then decreased.The IGF-1 mRNA expression of no rhGH acting,the 4th day,the 9th day,the 10th day,the 2nd week,the 3rd week,the 4th week after removing rhGH direct-acting were 0.4391±0.0058,0.5287±0.0077, 0.5747±0.0050,0.6068±0.0056,0.7071±0.0089,0.5791±0.0057 and 0.5781±0.0081,comparing between every two groups all have statistic difference except the 9th day,the 3rd week,the 4th week.4.The effect of rhGH,MSCs transplantation and combined rhGH and MSCs transplantation to cardiac function of heart failure caused by adriamycin-induced cardiomyopathy.4.1 haemodynamics results LVSP and dp/dtmax of GH group rats were all higher than those of HF group(16.03±1.33 Kpa vs 12.28±2.35 Kpa,687.00±40.95Kpa/s vs 496.17±79.82Kpa/s,P＜0.05);all index of MSCs group had no statistic difference compared to HF group;LVSP,dp/dpmax and -dp/dpmax of G+M group were all higher than those of HF group(19.28±0.95 Kpa vs12.28±2.35 Kpa,824.25±42.79 Kpa/s vs 496.17±79.82Kpa/s, 681.75±30.98 Kpa/s vs 388.00±45.63 Kpa/s,P＜0.05 ),but LVEDP no statistic difference.4.2 HW/BW and LW/BW results HW/BW and LW/BW of GH group,MSCs group and G+ M group were all lower than HF group,but P＞0.05.4.3 Serum GH and BNP results4.3.1 serum GH results Before treatment,serum GH levels of every group were obviously lower than normal group(1.16±0.58 ng/ml,1.37±0.09 ng/ml,1.10±0.50 ng/ml,1.25±0.42 ng/ml vs 2.64±1.03 ng/ml,P＜0.05);after treatment,serum GH levels of every group had no statistic difference.After treatment,only GH levels of GH group and G+M group were higher than before treatment(2.50±0.68 ng/ml vs 1.37±0.09 ng/ml,2.48±0.90 ng/ml vs 1.25±0.42 ng/ml, P＜0.05);but had no statistic difference comparing to normal group.4.3.2 serum BNP results Before treatment,serum BNP levels of every group were obviously higher than normal group(1464.14±151.00 pg/ml,1462.44±242.87 pg/ml,1475.29±281.33 pg/ml,1451.78±180.93 pg/ml vs 1125.02±271.28 pg/ml,P＜0.05);after treatment,serum BNP levels of GH group,MSCs group and G+M group were significantly lower than HF group (1270.72±203.72 pg/ml,1385.00+250.13 pg/ml,1219.31±126.71 pg/ml vs 1684.46±196.20 pg/ml,P＜0.05 ).After treatment,only BNP levels of GH group and G+M group were obviously lower than before treatment(1270.72±203.72 pg/ml vs 1462.44±242.87 pg/ml,P＜0.05; 1219.31±126.71 pg/ml vs 1451.78±180.93 pg/ml,P=0.001).4.4 Pathological results4.4.1 HE stainCadiocytes of HF group ranked chaotic and showed different degree degeneration,necrosis and inflammatory cell infiltrate.After the treatments of rhGH,MSCs transplantation,and rhGH combined MSCs transplantation,the numbers of cadiocytes became more than those of HF group,and the degree of cadiocyte degeneration was lessen.Compared between the three treatment groups,G+M group was the best.4.4.2 VG stainA small quantity of interstitial substance were seen in cardiac muscle of normal group; interstitial fibrosis were serious in cardiac muscle of HF group;after the treatments of rhGH, MSCs transplantation,and rhGH combined MSCs transplantation,the degree of interstitial fibrosis lessened.4.5 Electron microscope resultsIn normal group cardiac muscle fibers lined up in order and mitochondrium had no swelling; the cardiac muscle fibers of HF group ranked chaotic,and mitochondrium swelled seriously; after the treatments of rhGH,MSCs transplantation,and rhGH combined MSCs transplantation, the numbers of mitochondrium became more,the swelling degree were lessened,cardiac muscle fibers ranked well-arranged than those of HF group.5.Mechanism research of combining rhGH and MSCs transplantation improving cardiac function of heart failure caused by adriamycin-induced cardiomyopathy. 5.1 The number of MSCs marked by BrdU in the myocardium of G+M group rats were obviously increased than those of MSCs group rats;and the number of MSCs which were transdifferentiated into myocyte in the myocardium of G+M group rats were increased than those of MSCs group.5.2 IGF-1 mRNA results:IGF-1 mRNA relative amount of HF group was lower than that of normal group(0.5397±0.0111 vs 0.7789±0.0057,P＜0.05);compared to HF group,IGF-1 mRNA relative amount of GH group,MSCs group and G+M group were all increased(1.0227±0.0106 vs 0.5397±0.0111,0.6518±0.0102 vs 0.5397±0.0111 and 1.0242±0.0166 vs 0.5397±0.0111, P＜0.05);that of MSCs group was lower than that of GH group(0.6518±0.0102 vs 1.0227±0.0106,P＜0.05).5.3 MHC results:Comparing to HF group,the contents of V1-type MHC,V3-type MHC and the ratio of V1/V3 of GH group had no statistic difference;V3-type MHC of MSCs group were increased(10.33±0.33 vs 7.43±2.08,P＜0.05);V1-type MHC and V3-type MHC of G+M group were all obviously increased(86.22±1.73 vs 77.47±2.02 and 12.44±0.31 vs 7.43±2.08,P＜0.05), whereas V1/V3 no statistic difference.Among the three therapy group,V3-type MHC of MSCs group and G+M group were all increased compared to GH group(10.33±0.33 vs 7.42±0.72 and 12.44±0.31 vs 7.42±0.72,P＜0.05);V1-type MHC of G+M group were higher than that of MSCs group and GH group(86.22±1.73 vs 81.66±1.79,86.22±1.73 vs 79.79±3.23, P＜0.05).the ratio of V1/V3 were no statistic difference among every group.Conclusion:1.The models of heart failure caused by adriamycin-induced cardiomyopathy were made successfully,and were considered as the models of dilated cardiomyopathy.2.The cells cultured in vitro are MSCs,which can be induced to myocyte by 5—Aza,but their transformation efficiency are lower.3.rhGH can enhance MSCs growth in vitro.Its function of growth promotion are by IGF-1 mediation.4.rhGH therapy can improve cardiac systolic function of rats which suffered from heart failure caused by adriamycin-induced cardiomyopathy;whereas MSCs transplantation has no significant curative effect.Combining recombinant human growth hormone and mesenchymal stem cells transplantation can improve cardiac systolic and diastolic function significantly.5.The mechanism of improving cardiac function by combining recombinant human growth hormone and mesenchymal stem cells transplantation therapy are:rhGH enhanced the MSCs growth in vivo;enlarged cadiocyte number and promotive protein synthetic action of rhGH can cause the contents of MHC added,which can improve cardiac function,rhGH and MSCs can cause the expression of IGF-1 in local cadiocyte which can enhance the cells proliferation and differentiation,enhance new vessels formation,restrain RAS,inhibit apoptosis.Above factors cause to cardiac function improvement. |
