重组人生长激素和骨髓干细胞移植联合治疗阿霉素性心肌病心力衰竭的实验研究
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摘要
目的：
骨髓干细胞是具有自我复制和多向分化潜能的多能干细胞,可修复损伤的心肌细胞和血管内皮细胞。目前骨髓间充质干细胞(mesenchymal stem cells, MSCs)和骨髓动员出CD34+单个核细胞(CD34+mononuclear Cell CD34+MNC)在扩张型心肌病(DCM)中的研究仍处于起步阶段,尚缺乏理论证据。本研究分为两部分,为重组人生长激素(rhGH)联合MSCs静脉移植对阿霉素性心肌病心力衰竭大鼠的治疗研究和rhGH联合骨髓动员对阿霉素性心肌病心力衰竭大鼠的治疗研究。首先确定MSCs和CD34+MNC是否归巢到心脏受损部位,探讨重组人生长激素(recombinant human growth hormone, rhGH)对MSCs和CD34+MNC的存活和分化的影响,从心功能和心室重塑两方面观察其疗效,并推测其可能的机制,试图寻找一种干细胞移植更为简便有效的方法,为临床治疗扩张性心肌病所致心力衰竭提供理论依据。
方法：
1.阿霉素性心肌病心力衰竭大鼠模型的选择
通过腹腔注射阿霉素的方法制作阿霉素性心肌病心力衰竭大鼠模型。模型1组采用2.5 mg/kg、3次／周、用2周、共计6次,总剂量15 mg/kg。末次注射完成后观察2周。模型2组采用2.5 mg/kg、1次／周、用6周、共计6次、总剂量15 mg/kg。末次注射完成后观察2周。通过心脏彩色多普勒超声和血液动力学检测评价其心功能指标。留取心脏标本,测定体重、心脏重量和左心室重量,并观察心腔大小。留取左心室和肝、肾标本用HE和电镜观察病理变化,确定最佳制作模型。
2.间充质干细胞(MSCs)的体外分离、培养、鉴定、标记和心脏归巢
选体重约100g左右、5周龄的Wistar大鼠,提取股骨、胫骨的骨髓,利用MSCs贴壁生长的特点分离和培养骨髓间充质干细胞；观察其形态改变,取3代MSCs通过流式细胞仪测定其表面抗原CD44、CD45和CD34,证实体外培养的细胞为骨髓间充质干细胞。静脉移植前,用5-溴脱氧脲嘧啶(Brdu)标记MSCs并通过Brdu免疫组织化学法检测体外Brdu标记的MSCs在心、肝、肾的分布。
3. rhGH联合MSCs静脉移植在阿霉素性心肌病心力衰竭大鼠中治疗的研究
将实验动物分为心衰组(HF组)30只、MSCs静脉移植治疗组(MSCs组)30只和rhGH+MSCs移植联合治疗组(GH+MSC组)30只。MSCs组和GH+MSCs组动物通过尾静脉注射MSCs(细胞密度为1 X 109个／ml)。GH+MSCs组皮下注射rhGH,2mg/kg、每日1次,连续10天。心衰组动物不作任何处理。治疗前和治疗后4周留取静脉血标本测定内皮素-1(ET-1)和脑利钠肽(BNP)的浓度。同时通过心脏彩色多普勒超声测定LVIDd、LVIDs、LVEF和LVFS;经右颈总动脉插管入左心室,测定LVSP.+dp/dt max、LVEDP及-dp/dt max;留取心脏标本,采用免疫组织化学双染的方法同时检测Brdu(细胞核)和肌球蛋白重链(MHC)(细胞浆)及Brdu(细胞核)和肌动蛋白(Actin)(细胞浆)证实MSCs进入体内分化为心肌细胞,并观察其在心肌内的存活和分布情况。用Brdu免疫组化观察血管内皮细胞形成状况；经HE染色观察血管数目。RT-PCR法测定各组的心肌IGF-1mRNA相对含量。通过VG染色和心肌细胞凋亡指数,观察心室重塑的改善。
4. rhGH联合骨髓动员在阿霉素性心肌病心力衰竭大鼠中治疗的研究
将实验动物分为心衰组(HF组)20只、骨髓动员组(MNC组)30只和GH+骨髓动员组(GH+MNC组)20只。给MNC组和GH+MNC组动物皮下注射rhG-GSF, 50ug/Kg、每日1次、连续5天。心衰组动物给相同剂量生理盐水皮下注射5天。给GH+MNC组皮下注射rh-GH,2mg/kg、每日1次、连续10天。所有大鼠均给予腹腔注射Brdu,连续4周。G-GSF治疗第6天留取静脉血,测定血常规和用流式细胞仪测定CD34,其中10只处死,留取心脏标本测定CD34。治疗前和治疗后4周留取静脉血标本测定ET-1和BNP的浓度；心脏彩色多普勒超声测定LVIDd、LVIDs、LVEF和LVFS;经右颈总动脉插管入左心室,测定LVSP、+dp/dt max、LVEDP及-dp/dt max;留取心脏标本,采用免疫组织化学双染法同时检测Brdu(细胞核)和MHC(细胞浆)及Brdu(细胞核)和Actin(细胞浆)证实MNC进入体内分化为心肌细胞,并观察在心肌内的存活和分布情况。用Brdu免疫组化观察血管内皮细胞形成状况,经HE染色观察血管数目。RT-PCR法测定各组的心肌IGF-1 mRNA相对含量。通过VG染色和心肌细胞凋亡指数,观察心室重塑的改善。
结果：
1.阿霉素性心肌病心力衰竭大鼠模型的选择
模型1组LVIDd和LVIDs较正常组增加(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);模型2组大鼠的LVIDd和LVIDs较正常组增加(5.55±0.36mm vs 3.91±0.23mm,3.67±0.44mm vs 0.97±0.21mm,P<0.05),模型1组和模型2组大鼠的EF和FS均较正常组降低(76.79±8.56%、70.74±7.86%vs 98.51±3.61%；44.52±3.51%、35.11±3.67%vs76.77±1.90%P<0.051；LVSP、+dp/dtmax和-dp/dtmax均较正常组降低(13.25±0.88 Kpa、12.55±0.78 Kpa vs 16.21±0.24 Kpa; 590.67±73.40 Kpa/s、489.12±53.4 Kpa/s vs 861.67±39.96 Kpa/s; 534.00±59.22 Kpa/s、499±45.22 Kpa/s vs 632.00±45.19 Kpa/s, P<0.05), LVEDP较正常组升高(0.97±0.13 Kpa.2.20±0.40vs-0.08±0.17 Kpa,P<0.05)；体重(BW)较正常组减轻(200.33±8.43g.218.11±8.64g vs 240.33±7.11g,P<0.05),HW／BW和LW／BW较正常组增加(3.68±0.29g/kg、3.70±0.22 g／kg vs 2.73±0.44 g／kg；2.84±0.18 g／kg.2.92±0.22g/kg vs2.15±0.10g/kg,P<0.05)；大体标本显示模型组的心腔扩大,室壁组变薄；心肌HE染色显示模型组大鼠心肌细胞不同程度的变性,模型1组可见坏死。模型1组肝、肾损害较模型2组重。综上述情况模型2组适合研究使用。
2.间充质干细胞(MSCs)的体外分离、培养、鉴定、标记和心脏归巢
体外培养的MSCs呈贴壁生长,大多数细胞为梭形细胞；细胞表面抗原CD45.CD34呈阴性表达，CD44呈阳性表达。MSCs组心肌HE染色可见类似单个核细胞的细胞,Brdu免疫组化染色可见棕色核,而心衰组缺如；MSCs组肝、肾Brdu免疫组化染色未见棕色核。本实验证实体外培养的细胞是MSCs,主要归巢到心脏损伤部位。
3.rhGH联合MSCs静脉移植在阿霉素性心肌病心力衰竭模型中治疗研究
3.1心脏超声心功能测定
MSCs组和GH+MSCs组与心衰组比较,LVIDd,LVIDs均明显降低(3.41±0.72 mm、2.50土0.44 mm vs 5.50土0.34 mm；1.51土0.32mm、1.06±0.33 mm vs3.61±0.56 P<0.001)；EF,FS均明显升高(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)。本实验从超声上证实MSCs静脉移植及加用GH都能明显改善心功能。
3.2血液动力学
MSCs组和GH+MSCs组的LVSP.+dp／dpmax和-dp/dpmax与心衰组比较,均升高(14.88±0.78 Kpa.15.66±0.21 Kpa vs 12.55±0.88 Kpa;790.67±73.88 Kpa／s.827.67±33.96 Kpa／s vs 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);MSCs组和GH+MSCs组的LVEDP与心衰组比较均明显降低(-0.01±0.11 Kpa、-0.03±0.13 Kpa vs 2.20±0.40 Kpa／s,均P<0.05)。本实验从血液动力学上证实MSCs静脉移植及加用GH都能明显改善心功能。
3.3 BNP.ET-1
MSCs组和GH+MSCs组的BNP治疗后较治疗前明显降低(210.12土14.44pg／ml vs390.62土11.98 pg／ml;177.34土21.33pg／ml vs401.33土22.12 pg／ml P<0.001).心衰组治疗前后BNP水平比较,没有统计学意义(408.25土31.45pg／ml vs388.12土9.52pg／ml p>0.05). MSCs组和GH+MSCs组的ET-1治疗后较治疗前明显降低(2.56土0.34 ng／ml vs4.92土0.59 ng／ml;1.88土0.21 ng／ml vs5.01土0.77 ng／ml,P<0.001).心衰组治疗前后ET-1水平比较没有统计学意义(5.22±0.30 ng／ml vs 5.12土0.36 ng／ml,p>0.05).本实验从血清学指标上证实MSCs静脉移植及加用GH都能明显改善心功能。
3.4心肌切片HE、免疫组化及心肌IGF-1 mRNA相对含量
MSCs组和GH+MSCs组心肌Brdu免疫组织化染色可见血管内皮细胞有棕色核,血管数目明显增多(16.33±1.41/0.2mm、18.44±1.17/0.2mm VS 9.90±1.60/0.2mmP<0.001),加用GH后,这种细胞和血管数明显增多。MSCs组和GH+MSCs组心脏免疫组织化学双染可见红色核和棕色胞浆的心肌样细胞,加用GH后,这种细胞明显增多。MSCs组和GH+MSCs组心肌IGF-1 mRNA相对含量较心衰组明显升高(0.7118±0.0106,1.0361±0.0157 vs0.5017±0.0121 P<0.05)：GH+MSCs组心肌IGF-1 mRNA相对含量较MSCs组明显升高(1.0361±0.0157 vs0.7118±0.0106 P<0.05)。本实验证实MSCs在心脏损伤部位可转化心肌样细胞和血管内皮细胞,加GH能增加转换比率;MSCs治疗可提高心肌组织IGF-1 mRNA的相对含量,加GH其含量明显增多,提高MSCs的存活和分化。
3.5 VG染色和凋亡指数
MSCs组和GH+MSCs组与心衰组比较,胶原纤维明显减少,胶原容积分数明显减少(15.20±5.40%、14.33±5.11%vs 19.88±2.78%,P<0.05)。MSCs组和GH+MSCs组与心衰组比较能降低凋亡指数(17.44±6.78%、12.34±2.98%vs 27.33±7.98%,P<0.05)。本实验证实MSCs静脉移植及加用GH都能降低凋亡心肌细胞数目和胶原纤维,改善心室重塑。
4 rhGH联合骨髓动员在阿霉素性心肌病心力衰竭模型中治疗研究
4.1MNC数目和CD34定性
G-CSF能增加MNC数目,MNC组和GH+MNC组MNC数目治疗后较治疗前明显增高(15.79 X 109/L vs 5.36 X109/L; 15.99 X 109 vs6.30 X 109/L, P＜0.05)。心衰组MNC数目治疗前后比较,MNC无差异性(4.92 X 109/L vs 5.39 X 109/L P>0.05)。MNC组和GH+MNC组血液中MNC流式细胞仪CD34定性为阳性、心脏免疫荧光CD34定性为阳性；心衰组血液中MNC流式细胞仪CD34定性为阴性、心脏免疫荧光CD34定性为阴性。本实验证实骨髓动员可动员出CD34+MNC并归巢至心脏受损部位。
4.2心脏超声结果
MNC组和GH+MNC组与心衰组比较,LVIDd, LVIDs均明显降低(2.91±0.66mm、2.77±0.77mm vs5.50±0.34 mm；1.51±0.55 mm、1.45±0.26mm vs3.61±0.56 mmP<0.001)；EF,FS均明显升高(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)。本实验从超声上证实骨髓动员及加用GH都能明显改善心功能。
4.3血液动力学结果
MNC组和GH+MNC组的LVSP、+dp／dpmax和-dp／dpmax与心衰组比较,均升高(14.33±0.44 Kpa.15.22±0.33 Kpa vs 12.55±0.88 Kpa；767.44±71.47 Kpa／s.803.67±56.63 Kpa／svs 489.12±53.40 Kpa／s;601.33±42.33Kpa／s.603.00±46.29 Kpa／s vs 499±45.22 Kpa／s, P<0.05):LVEDP与心衰组比较均明显降低(-0.08±0.11 Kpa.-0.11±0.17 Kpa vs 2.20±0.40 Kpa／s,均P<0.05)。本实验从血液动力学上证实骨髓动员及加用GH都能明显改善心功能。
4.4 BNP、内皮素-1结果
MNC组和GH+MNC组的BNP治疗后较治疗前明显降低(207.55±18.56pg／ml vs377.12±21.38 pg／ml；189.14土27.63pg／ml vs389.43±24.16 pg／ml,P<0.001).心衰组治疗前后BNP水平比较没有统计学意义(399.77±21.75pg/ml vs393.12±19.62pg／ml,p>0.05).MNC组和GH+MNC组的ET-1治疗后较治疗前明显降低(2.11±0.44 ng／ml vs4.99±0.67 ng／ml；1.89±0.17ng／ml vs5.01±0.87ng／ml,P<0.001).心衰组治疗前后ET-1水平比较,没有统计学意义(5.67±0.43ng／ml vs5.70±0.56 ng／ml,p>0.05)本实验从血清学指标上证实骨髓动员及加用GH都能明显改善心功能。
4.5心肌切片HE、免疫组化及心肌IGF-1 mRNA相对含量结果：
MNC组和GH+MNC组心脏Brdu免疫组织化染色可见血管内皮细胞有棕色核,血管数目明显增多(14.67±1.33／0.2mm.15.65±1.21／0.2mm VS 9.47±1.21／0.2mm,P<0.001),加用GH后,这种细胞和血管数明显增多。MNC组和GH+MNC组心脏免疫组织化学双染可见红色核和棕色胞浆的心肌样细胞,加用GH后,这种细胞明显增多。MNC组和GH+MNC组心肌IGF-1 mRNA相对含量较心衰组明显升高(0.7034±0.0113,0.978±0.013 vs 0.497±0.0141P<0.05);GH+MNC组心肌IGF-1 mRNA相对含量较MNC组明显升高(0.978±0.013vs0.7034±0.0113 P<0.05)。本实验证实MNC在心脏损伤部位可转化心肌样细胞和血管内皮细胞,加GH能增加转换比率；骨髓动员可提高心肌组织IGF-1 mRNA的相对含量,加GH其含量明显增多,提高MNC的存活和分化。
4.6 VG染色和凋亡指数：
MNC组和GH+MNC组与心衰组比较,胶原纤维明显减少,胶原容积分数明显减少(13.20±4.40.12.89±4.65 VS 21.21±4.33,P<0.05).MNC组和GH+MNC组与心衰组比较,能降低凋亡指数(8.44±6.34%、6..34±5.98%VS 27.33±7.33,P<0.05)。本实验证实骨髓动员及加用GH都能降低凋亡心肌细胞数目和胶原纤维,改善心室重塑。
结论：
1.模型2符合治疗研究使用。
2.体外培养的细胞是MSCs,主要归巢到心脏损伤部位,加GH能增加MSCs定居。
3.MSCs在心脏损伤部位可转化心心肌样细胞和血管内皮细胞,加GH能增加转换比率。
4.MSCs治疗可提高心肌组织IGF-1 mRNA的相对含量,加GH其含量明显增多,提高MSCs的存活和分化。
5.MSCs静脉移植及加用GH都能明显改善心功能。
6.MSCs静脉移植及加用GH都能降低凋亡数目和胶原纤维,改善心室重塑。
7.骨髓动员可动员出CD34+MNC,并归巢到心脏损伤部位,加GH能增加CD34+MNC定居。
8.CD34+MNC在心脏损伤部位可转化心肌样细胞和血管内皮细胞,加GH能增加转换比率。
9.骨髓动员治疗可提高心肌组织IGF-1 mRNA的相对含量,加GH其含量明显增多,提高MNC的存活和分化。
10.骨髓动员及加用GH均明显改善心功能。
11.骨髓动员及加用GH均能降低凋亡数目和胶原纤维,改善心室重塑。
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 cardiomyopathy
LVIDd 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 heart
MSCs 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 cardiomyopathy
3.1 cardiac echocardiography results
Compared 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 results
LVSP,+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 results
After 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 mRNA
we 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 apoptosis
After 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 results
Compared 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 results
LVSP,+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 results
After 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 mRNA
We 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 apoptosis
After 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 cardiomyopathy
6. 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 cardiomyopathy
11. MNC and GH+MNC therapy could decrease the number of collagen and apoptosis and improve ventricular remodeling.
骨髓干细胞是具有自我复制和多向分化潜能的多能干细胞,可修复损伤的心肌细胞和血管内皮细胞。目前骨髓间充质干细胞(mesenchymal stem cells, MSCs)和骨髓动员出CD34+单个核细胞(CD34+mononuclear Cell CD34+MNC)在扩张型心肌病(DCM)中的研究仍处于起步阶段,尚缺乏理论证据。本研究分为两部分,为重组人生长激素(rhGH)联合MSCs静脉移植对阿霉素性心肌病心力衰竭大鼠的治疗研究和rhGH联合骨髓动员对阿霉素性心肌病心力衰竭大鼠的治疗研究。首先确定MSCs和CD34+MNC是否归巢到心脏受损部位,探讨重组人生长激素(recombinant human growth hormone, rhGH)对MSCs和CD34+MNC的存活和分化的影响,从心功能和心室重塑两方面观察其疗效,并推测其可能的机制,试图寻找一种干细胞移植更为简便有效的方法,为临床治疗扩张性心肌病所致心力衰竭提供理论依据。
方法：
1.阿霉素性心肌病心力衰竭大鼠模型的选择
通过腹腔注射阿霉素的方法制作阿霉素性心肌病心力衰竭大鼠模型。模型1组采用2.5 mg/kg、3次／周、用2周、共计6次,总剂量15 mg/kg。末次注射完成后观察2周。模型2组采用2.5 mg/kg、1次／周、用6周、共计6次、总剂量15 mg/kg。末次注射完成后观察2周。通过心脏彩色多普勒超声和血液动力学检测评价其心功能指标。留取心脏标本,测定体重、心脏重量和左心室重量,并观察心腔大小。留取左心室和肝、肾标本用HE和电镜观察病理变化,确定最佳制作模型。
2.间充质干细胞(MSCs)的体外分离、培养、鉴定、标记和心脏归巢
选体重约100g左右、5周龄的Wistar大鼠,提取股骨、胫骨的骨髓,利用MSCs贴壁生长的特点分离和培养骨髓间充质干细胞；观察其形态改变,取3代MSCs通过流式细胞仪测定其表面抗原CD44、CD45和CD34,证实体外培养的细胞为骨髓间充质干细胞。静脉移植前,用5-溴脱氧脲嘧啶(Brdu)标记MSCs并通过Brdu免疫组织化学法检测体外Brdu标记的MSCs在心、肝、肾的分布。
3. rhGH联合MSCs静脉移植在阿霉素性心肌病心力衰竭大鼠中治疗的研究
将实验动物分为心衰组(HF组)30只、MSCs静脉移植治疗组(MSCs组)30只和rhGH+MSCs移植联合治疗组(GH+MSC组)30只。MSCs组和GH+MSCs组动物通过尾静脉注射MSCs(细胞密度为1 X 109个／ml)。GH+MSCs组皮下注射rhGH,2mg/kg、每日1次,连续10天。心衰组动物不作任何处理。治疗前和治疗后4周留取静脉血标本测定内皮素-1(ET-1)和脑利钠肽(BNP)的浓度。同时通过心脏彩色多普勒超声测定LVIDd、LVIDs、LVEF和LVFS;经右颈总动脉插管入左心室,测定LVSP.+dp/dt max、LVEDP及-dp/dt max;留取心脏标本,采用免疫组织化学双染的方法同时检测Brdu(细胞核)和肌球蛋白重链(MHC)(细胞浆)及Brdu(细胞核)和肌动蛋白(Actin)(细胞浆)证实MSCs进入体内分化为心肌细胞,并观察其在心肌内的存活和分布情况。用Brdu免疫组化观察血管内皮细胞形成状况；经HE染色观察血管数目。RT-PCR法测定各组的心肌IGF-1mRNA相对含量。通过VG染色和心肌细胞凋亡指数,观察心室重塑的改善。
4. rhGH联合骨髓动员在阿霉素性心肌病心力衰竭大鼠中治疗的研究
将实验动物分为心衰组(HF组)20只、骨髓动员组(MNC组)30只和GH+骨髓动员组(GH+MNC组)20只。给MNC组和GH+MNC组动物皮下注射rhG-GSF, 50ug/Kg、每日1次、连续5天。心衰组动物给相同剂量生理盐水皮下注射5天。给GH+MNC组皮下注射rh-GH,2mg/kg、每日1次、连续10天。所有大鼠均给予腹腔注射Brdu,连续4周。G-GSF治疗第6天留取静脉血,测定血常规和用流式细胞仪测定CD34,其中10只处死,留取心脏标本测定CD34。治疗前和治疗后4周留取静脉血标本测定ET-1和BNP的浓度；心脏彩色多普勒超声测定LVIDd、LVIDs、LVEF和LVFS;经右颈总动脉插管入左心室,测定LVSP、+dp/dt max、LVEDP及-dp/dt max;留取心脏标本,采用免疫组织化学双染法同时检测Brdu(细胞核)和MHC(细胞浆)及Brdu(细胞核)和Actin(细胞浆)证实MNC进入体内分化为心肌细胞,并观察在心肌内的存活和分布情况。用Brdu免疫组化观察血管内皮细胞形成状况,经HE染色观察血管数目。RT-PCR法测定各组的心肌IGF-1 mRNA相对含量。通过VG染色和心肌细胞凋亡指数,观察心室重塑的改善。
结果：
1.阿霉素性心肌病心力衰竭大鼠模型的选择
模型1组LVIDd和LVIDs较正常组增加(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);模型2组大鼠的LVIDd和LVIDs较正常组增加(5.55±0.36mm vs 3.91±0.23mm,3.67±0.44mm vs 0.97±0.21mm,P<0.05),模型1组和模型2组大鼠的EF和FS均较正常组降低(76.79±8.56%、70.74±7.86%vs 98.51±3.61%；44.52±3.51%、35.11±3.67%vs76.77±1.90%P<0.051；LVSP、+dp/dtmax和-dp/dtmax均较正常组降低(13.25±0.88 Kpa、12.55±0.78 Kpa vs 16.21±0.24 Kpa; 590.67±73.40 Kpa/s、489.12±53.4 Kpa/s vs 861.67±39.96 Kpa/s; 534.00±59.22 Kpa/s、499±45.22 Kpa/s vs 632.00±45.19 Kpa/s, P<0.05), LVEDP较正常组升高(0.97±0.13 Kpa.2.20±0.40vs-0.08±0.17 Kpa,P<0.05)；体重(BW)较正常组减轻(200.33±8.43g.218.11±8.64g vs 240.33±7.11g,P<0.05),HW／BW和LW／BW较正常组增加(3.68±0.29g/kg、3.70±0.22 g／kg vs 2.73±0.44 g／kg；2.84±0.18 g／kg.2.92±0.22g/kg vs2.15±0.10g/kg,P<0.05)；大体标本显示模型组的心腔扩大,室壁组变薄；心肌HE染色显示模型组大鼠心肌细胞不同程度的变性,模型1组可见坏死。模型1组肝、肾损害较模型2组重。综上述情况模型2组适合研究使用。
2.间充质干细胞(MSCs)的体外分离、培养、鉴定、标记和心脏归巢
体外培养的MSCs呈贴壁生长,大多数细胞为梭形细胞；细胞表面抗原CD45.CD34呈阴性表达，CD44呈阳性表达。MSCs组心肌HE染色可见类似单个核细胞的细胞,Brdu免疫组化染色可见棕色核,而心衰组缺如；MSCs组肝、肾Brdu免疫组化染色未见棕色核。本实验证实体外培养的细胞是MSCs,主要归巢到心脏损伤部位。
3.rhGH联合MSCs静脉移植在阿霉素性心肌病心力衰竭模型中治疗研究
3.1心脏超声心功能测定
MSCs组和GH+MSCs组与心衰组比较,LVIDd,LVIDs均明显降低(3.41±0.72 mm、2.50土0.44 mm vs 5.50土0.34 mm；1.51土0.32mm、1.06±0.33 mm vs3.61±0.56 P<0.001)；EF,FS均明显升高(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)。本实验从超声上证实MSCs静脉移植及加用GH都能明显改善心功能。
3.2血液动力学
MSCs组和GH+MSCs组的LVSP.+dp／dpmax和-dp/dpmax与心衰组比较,均升高(14.88±0.78 Kpa.15.66±0.21 Kpa vs 12.55±0.88 Kpa;790.67±73.88 Kpa／s.827.67±33.96 Kpa／s vs 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);MSCs组和GH+MSCs组的LVEDP与心衰组比较均明显降低(-0.01±0.11 Kpa、-0.03±0.13 Kpa vs 2.20±0.40 Kpa／s,均P<0.05)。本实验从血液动力学上证实MSCs静脉移植及加用GH都能明显改善心功能。
3.3 BNP.ET-1
MSCs组和GH+MSCs组的BNP治疗后较治疗前明显降低(210.12土14.44pg／ml vs390.62土11.98 pg／ml;177.34土21.33pg／ml vs401.33土22.12 pg／ml P<0.001).心衰组治疗前后BNP水平比较,没有统计学意义(408.25土31.45pg／ml vs388.12土9.52pg／ml p>0.05). MSCs组和GH+MSCs组的ET-1治疗后较治疗前明显降低(2.56土0.34 ng／ml vs4.92土0.59 ng／ml;1.88土0.21 ng／ml vs5.01土0.77 ng／ml,P<0.001).心衰组治疗前后ET-1水平比较没有统计学意义(5.22±0.30 ng／ml vs 5.12土0.36 ng／ml,p>0.05).本实验从血清学指标上证实MSCs静脉移植及加用GH都能明显改善心功能。
3.4心肌切片HE、免疫组化及心肌IGF-1 mRNA相对含量
MSCs组和GH+MSCs组心肌Brdu免疫组织化染色可见血管内皮细胞有棕色核,血管数目明显增多(16.33±1.41/0.2mm、18.44±1.17/0.2mm VS 9.90±1.60/0.2mmP<0.001),加用GH后,这种细胞和血管数明显增多。MSCs组和GH+MSCs组心脏免疫组织化学双染可见红色核和棕色胞浆的心肌样细胞,加用GH后,这种细胞明显增多。MSCs组和GH+MSCs组心肌IGF-1 mRNA相对含量较心衰组明显升高(0.7118±0.0106,1.0361±0.0157 vs0.5017±0.0121 P<0.05)：GH+MSCs组心肌IGF-1 mRNA相对含量较MSCs组明显升高(1.0361±0.0157 vs0.7118±0.0106 P<0.05)。本实验证实MSCs在心脏损伤部位可转化心肌样细胞和血管内皮细胞,加GH能增加转换比率;MSCs治疗可提高心肌组织IGF-1 mRNA的相对含量,加GH其含量明显增多,提高MSCs的存活和分化。
3.5 VG染色和凋亡指数
MSCs组和GH+MSCs组与心衰组比较,胶原纤维明显减少,胶原容积分数明显减少(15.20±5.40%、14.33±5.11%vs 19.88±2.78%,P<0.05)。MSCs组和GH+MSCs组与心衰组比较能降低凋亡指数(17.44±6.78%、12.34±2.98%vs 27.33±7.98%,P<0.05)。本实验证实MSCs静脉移植及加用GH都能降低凋亡心肌细胞数目和胶原纤维,改善心室重塑。
4 rhGH联合骨髓动员在阿霉素性心肌病心力衰竭模型中治疗研究
4.1MNC数目和CD34定性
G-CSF能增加MNC数目,MNC组和GH+MNC组MNC数目治疗后较治疗前明显增高(15.79 X 109/L vs 5.36 X109/L; 15.99 X 109 vs6.30 X 109/L, P＜0.05)。心衰组MNC数目治疗前后比较,MNC无差异性(4.92 X 109/L vs 5.39 X 109/L P>0.05)。MNC组和GH+MNC组血液中MNC流式细胞仪CD34定性为阳性、心脏免疫荧光CD34定性为阳性；心衰组血液中MNC流式细胞仪CD34定性为阴性、心脏免疫荧光CD34定性为阴性。本实验证实骨髓动员可动员出CD34+MNC并归巢至心脏受损部位。
4.2心脏超声结果
MNC组和GH+MNC组与心衰组比较,LVIDd, LVIDs均明显降低(2.91±0.66mm、2.77±0.77mm vs5.50±0.34 mm；1.51±0.55 mm、1.45±0.26mm vs3.61±0.56 mmP<0.001)；EF,FS均明显升高(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)。本实验从超声上证实骨髓动员及加用GH都能明显改善心功能。
4.3血液动力学结果
MNC组和GH+MNC组的LVSP、+dp／dpmax和-dp／dpmax与心衰组比较,均升高(14.33±0.44 Kpa.15.22±0.33 Kpa vs 12.55±0.88 Kpa；767.44±71.47 Kpa／s.803.67±56.63 Kpa／svs 489.12±53.40 Kpa／s;601.33±42.33Kpa／s.603.00±46.29 Kpa／s vs 499±45.22 Kpa／s, P<0.05):LVEDP与心衰组比较均明显降低(-0.08±0.11 Kpa.-0.11±0.17 Kpa vs 2.20±0.40 Kpa／s,均P<0.05)。本实验从血液动力学上证实骨髓动员及加用GH都能明显改善心功能。
4.4 BNP、内皮素-1结果
MNC组和GH+MNC组的BNP治疗后较治疗前明显降低(207.55±18.56pg／ml vs377.12±21.38 pg／ml；189.14土27.63pg／ml vs389.43±24.16 pg／ml,P<0.001).心衰组治疗前后BNP水平比较没有统计学意义(399.77±21.75pg/ml vs393.12±19.62pg／ml,p>0.05).MNC组和GH+MNC组的ET-1治疗后较治疗前明显降低(2.11±0.44 ng／ml vs4.99±0.67 ng／ml；1.89±0.17ng／ml vs5.01±0.87ng／ml,P<0.001).心衰组治疗前后ET-1水平比较,没有统计学意义(5.67±0.43ng／ml vs5.70±0.56 ng／ml,p>0.05)本实验从血清学指标上证实骨髓动员及加用GH都能明显改善心功能。
4.5心肌切片HE、免疫组化及心肌IGF-1 mRNA相对含量结果：
MNC组和GH+MNC组心脏Brdu免疫组织化染色可见血管内皮细胞有棕色核,血管数目明显增多(14.67±1.33／0.2mm.15.65±1.21／0.2mm VS 9.47±1.21／0.2mm,P<0.001),加用GH后,这种细胞和血管数明显增多。MNC组和GH+MNC组心脏免疫组织化学双染可见红色核和棕色胞浆的心肌样细胞,加用GH后,这种细胞明显增多。MNC组和GH+MNC组心肌IGF-1 mRNA相对含量较心衰组明显升高(0.7034±0.0113,0.978±0.013 vs 0.497±0.0141P<0.05);GH+MNC组心肌IGF-1 mRNA相对含量较MNC组明显升高(0.978±0.013vs0.7034±0.0113 P<0.05)。本实验证实MNC在心脏损伤部位可转化心肌样细胞和血管内皮细胞,加GH能增加转换比率；骨髓动员可提高心肌组织IGF-1 mRNA的相对含量,加GH其含量明显增多,提高MNC的存活和分化。
4.6 VG染色和凋亡指数：
MNC组和GH+MNC组与心衰组比较,胶原纤维明显减少,胶原容积分数明显减少(13.20±4.40.12.89±4.65 VS 21.21±4.33,P<0.05).MNC组和GH+MNC组与心衰组比较,能降低凋亡指数(8.44±6.34%、6..34±5.98%VS 27.33±7.33,P<0.05)。本实验证实骨髓动员及加用GH都能降低凋亡心肌细胞数目和胶原纤维,改善心室重塑。
结论：
1.模型2符合治疗研究使用。
2.体外培养的细胞是MSCs,主要归巢到心脏损伤部位,加GH能增加MSCs定居。
3.MSCs在心脏损伤部位可转化心心肌样细胞和血管内皮细胞,加GH能增加转换比率。
4.MSCs治疗可提高心肌组织IGF-1 mRNA的相对含量,加GH其含量明显增多,提高MSCs的存活和分化。
5.MSCs静脉移植及加用GH都能明显改善心功能。
6.MSCs静脉移植及加用GH都能降低凋亡数目和胶原纤维,改善心室重塑。
7.骨髓动员可动员出CD34+MNC,并归巢到心脏损伤部位,加GH能增加CD34+MNC定居。
8.CD34+MNC在心脏损伤部位可转化心肌样细胞和血管内皮细胞,加GH能增加转换比率。
9.骨髓动员治疗可提高心肌组织IGF-1 mRNA的相对含量,加GH其含量明显增多,提高MNC的存活和分化。
10.骨髓动员及加用GH均明显改善心功能。
11.骨髓动员及加用GH均能降低凋亡数目和胶原纤维,改善心室重塑。
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 cardiomyopathy
LVIDd 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 heart
MSCs 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 cardiomyopathy
3.1 cardiac echocardiography results
Compared 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 results
LVSP,+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 results
After 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 mRNA
we 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 apoptosis
After 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 results
Compared 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 results
LVSP,+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 results
After 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 mRNA
We 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 apoptosis
After 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 cardiomyopathy
6. 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 cardiomyopathy
11. MNC and GH+MNC therapy could decrease the number of collagen and apoptosis and improve ventricular remodeling.
引文
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