茂丹通脉片含药血清体外诱导 S 分M化C为 内皮细胞的作用

目的：观察芪丹通脉片含药血清体外诱导大鼠骨髓间充质干细胞（MSCs）向内皮细胞分化的作用。方法：灌胃法制备芪丹通脉片含药血清和对照血清。采用密度梯度离心法分离和培养大鼠MSCs,取第三代MSCs,采用10wg／LVEGF预诱导24h后,分别加入15％芪丹通脉片含药血清与对照血清体外时MSCs诱导分化,至第7天,利用相差显微镜观察细胞形态改变,透射电镜观察细胞超微结构。免疫荧光方法检测内皮细胞特异性表面标志CD31、Ⅷ因子的表达。结果：至第7天,合15％芪丹通脉片合药血清组诱导后的MSCs形态发生明显改变,呈“卵石样”改变,透射电镜下细胞胞浆内可见Weible-Palade小体,共聚焦显微镜下可见CD31、Ⅷ因子阳性细胞。对照血清组MSCs形态仍呈长梭型,电镜下胞浆内无Weible-Palade小体,共聚焦显微镜下无CD31、Ⅷ因子阳性细胞。结论：益气活血复方芪丹通脉片含药血清具有体外诱导大鼠MSCs向内皮细胞定向分化的作用。     

大鼠严重烧伤早期牛磺酸对心肌的保护效应

目的:明确牛磺酸对烧伤后心肌组织具有保护作用.方法:健康雄性S-D大鼠32只,随机分为正常组,假伤组、烧伤组和牛磺酸处理组,其中烧伤组和牛磺酸处理致成30%TBSA Ⅲ°烧伤,于伤后6h以多道生理信号采集处理系统检测心肌力学指标等(SDP,DBP,LVSP,LVEDP,+LVdp/dtmax,-LVdp/dtmax).结果:烧伤后6 h LVSP,+LVdp/dtmax,-LVdp/dtmax较对照组均明显降低(P＜0.01),LVEDP明显增高(P＜0.01;而加入牛磺酸后均较烧伤组明显减轻.结论:牛磺酸可以明显改善烧伤后心肌功能,发挥其保护效应.     

Ang(1-7)和依那普利拉对大鼠严重烧伤早期心脏保护作用的研究

目的:观察Ang(1-7)和依那普利拉对烧伤早期心功能及心肌损伤的保护作用.方法:健康雄性Wistar大鼠24只,随机分为正常组、烧伤组、Ang(1-7)组和依那普利拉组.于烧伤后6H检测血流动力和心肌力学指标(SBP,DBP,LVSP,LVEDP, LVdp/dt-max,-LVdp/dtmax),血清cTnI,及血清和心肌组织中AngⅡ含量变化.结果:ACEi组和Ang(1-7)组SBP,DBP,LVSP, LVdp/dt-max,-LVdp/dtmax较烧伤组均明显增高(P＜0.01),LVEDP和血清cTnI明显降低(P＜0.01),ACEi组血清和心肌中AngⅡ较烧伤组明显降低(P＜0.01).结论:Ang(1-7)和ACEi均能有效的改善大鼠烧伤早期心功能,减轻心肌损伤.     

骨髓间充质干细胞和内皮祖细胞移植促进血流重建的比较

目的:比较骨髓间充质细胞(Bone Marrow Mesenchymal Stem Cells,BM/MSC)和骨髓源内皮祖细胞(Bone Marrow Endothelialprogenitor cells,BM/EPC)移植促进血流重建的效果,为进一步优化骨髓干细胞移植治疗肢体缺血提供理论基础。方法:获取Lewis大鼠骨髓单个核细胞,在体外培养分化为MSC和EPC。采用Lewis大鼠建立单侧后肢缺血模型。在模型建立后3天,将0.8mlD-Hanks液注入大鼠缺血侧后肢,为对照组(n=6);将8×106个骨髓MSC植入大鼠缺血侧后肢,为MSC组(n=6);将体外培养的8×106个EPC植入大鼠缺血侧后肢,为EPC组(n=6)。细胞移植后3周行缺血大鼠后肢动脉造影,检测缺血侧后肢侧支血管数;获取缺血侧后肢腓肠肌,分别行CD31和α-SMA免疫组化染色,计算毛细血管密度和小动脉密度。结果:MSC组与EPC组侧支血管数无显著性差异,二者均高于对照组;EPC组毛细血管密度明显高于MSC组,二者均高于对照组;MSC组与EPC组小动脉密度无显著性差异,二者均高于对照组。结论:骨髓间充质干细胞移植和内皮祖细胞移植均能够明显促进血流重建,而且骨髓间充质干细胞在治疗肢体缺血性疾病中的优势应该受到重视。     

骨髓间质干细胞向大鼠损伤心肌组织的迁移

实验旨在动态观察骨髓间充质干细胞（mesenchymal stem cells,MSCs）向不同微环境下心肌组织的迁移特点,明确组织损伤在干细胞迁移中的作用,为提高干细胞治疗的靶向性和高效性奠定初步试验基础。分离纯化雄性Sprague-Dawley（SD）大鼠的骨髓MSCs,输注入雌性SD大鼠。实验分为4组：正常大鼠＋MSCs移植组,假手术＋MSCs移植组,心肌缺血＋MSCs移植组,心肌缺血对照组（心肌缺血＋培养基移植）。结扎冠状动脉前降支制造心肌缺血模型,将相等数量的雄性MSCs经尾静脉注射移植入前3组雌性大鼠体内,对照组注射等体积培养基,分别于移植后1周及8周取心脏组织标本,采用荧光原位杂交方法（fluorescence in situ hybridization,FISH）检测大鼠Y染色体雄性鉴别基因sty片段的表达,用透射电镜观察大鼠心肌组织超微结构改变。结果发现,移植后1周和8周,正常大鼠移植组和对照组大鼠的心肌组织中均未见sry基因的表达,但假手术移植组和心肌缺血移植组的心肌组织中均可见sty基因的表达,心肌缺血移植组的Y染色体sty基因阳性细胞数量在两个时间点均显著高于假手术移植组（P〈0．01）。分别比较心肌缺血移植组和假手术组在移植后1周和8周的Y染色体sry基因阳性细胞的数量,两个时间点无明显差异。心肌组织的超微结构观察发现心肌缺血移植组大鼠的心肌梗死周边区域可见一些细胞,其形态类似于体外培养的MSCs。研究结果提示MSCs具有向损伤心肌组织迁移的特性,迁移的高峰期可能在组织损伤1周左右,组织损伤及其程度在干细胞迁移中起重要作用。     

骨髓间充质干细胞移植治疗大鼠肝损伤的实验研究

目的:了解肝损伤大鼠在输注骨髓间充质干细胞后肝功能生化指标和肝脏组织病理变化情况,为临床应用提供实验依据。方法:将大鼠随机分为正常组和造模组。造模组采用腹腔注射四氯化碳的方法构建,然后将造模组随机分为干细胞移植治疗组、模型对照组。干细胞移植组经门静脉输注标记的骨髓间充质干细胞。3周后处死大鼠。然后检测大鼠肝功能、肝脏病理改变分析干细胞移植治疗肝损伤效果。结果:干细胞移植治疗3周后,大鼠的肝脏与对照组比较,明显恢复,但是并没有恢复到正常水平。结论:骨髓间充质干细胞对肝损伤的大鼠有治疗作用。     

芪丹通脉片对大鼠慢性脑缺血损伤大鼠学习记忆的影响

目的:研究芪丹通脉片对慢性脑缺血所致学习记忆障碍的治疗作用及其可能机制.方法:采用双侧颈动脉结扎方法复制慢性脑缺血模型.将健康雄性SD大鼠随机分为空白对照组、模型组、假手术组、芪丹通脉片低剂量组、芪丹通脉片中剂量组、芪丹通脉片高剂量组、阳性对照尼莫地平组,应用Morris水迷宫检测大鼠学习记忆能力,HE染色观察海马神经元形态学改变.结果:与对照组比较,模型组大鼠可见显著学习记忆障碍,并可见海马神经元呈现出典型的神经病理性改变,海马区神经细胞数量减少、固缩等改变.芪丹通脉片可显著减轻慢性脑缺血所致学习记忆能力,并减其轻海马神经元损伤,且有显著剂量依赖性.结论:本实验证实芪丹通脉片可显著减轻慢性脑缺血所致学习记忆障碍,其可能机制是通过减轻海马损伤来改善学习记忆能力.     

芪丹通脉片对大鼠慢性脑缺血损伤大鼠学习记忆的影响

目的:研究芪丹通脉片对慢性脑缺血所致学习记忆障碍的治疗作用及其可能机制。方法:采用双侧颈动脉结扎方法复制慢性脑缺血模型。将健康雄性SD大鼠随机分为空白对照组、模型组、假手术组、芪丹通脉片低剂量组、芪丹通脉片中剂量组、芪丹通脉片高剂量组、阳性对照尼莫地平组,应用Morris水迷宫检测大鼠学习记忆能力,HE染色观察海马神经元形态学改变。结果:与对照组比较,模型组大鼠可见显著学习记忆障碍,并可见海马神经元呈现出典型的神经病理性改变,海马区神经细胞数量减少、固缩等改变。芪丹通脉片可显著减轻慢性脑缺血所致学习记忆能力,并减其轻海马神经元损伤,且有显著剂量依赖性。结论:本实验证实芪丹通脉片可显著减轻慢性脑缺血所致学习记忆障碍,其可能机制是通过减轻海马损伤来改善学习记忆能力。     

基质衍生因子(SDF-1)对骨髓间充质干细胞定向迁移的影响

目的:研究基质细胞衍生因子-1(SDF-1)/CXCR4轴在骨髓间充质干细胞迁徙到受损胰腺中的作用。方法:密度梯度离心、贴壁培养骨髓间充质干细胞,建立STZ诱导糖尿病模型并制备正常和受损胰腺组织提取液,利用Transwell小室体外迁移体系观察不同浓度SDF-1和不同组织提取液对骨髓间充质干细胞的趋化作用,及SDF-1/CXCR4特异抑制剂AMD3100对骨髓间充质干细胞迁移的影响。结果:成功培养了骨髓间充质干细胞并建立了糖尿病大鼠模型。SDF-l对骨髓间充质干细胞有剂量依赖性的趋化作用,造模1周的胰腺组织提取液对骨髓间充质干细胞有明显的趋化作用,而这种作用可部分被SDF-1受体CXCR4的抑制剂AMD3100抑制。结论:受损胰腺组织提取液对骨髓间充质干细胞有明显的趋化作用,SDF-1/CXCR4轴可能在组织提取液趋化骨髓间充质干细胞迁移中起主要的作用。     

microRNA499 慢病毒载体转染诱导大鼠骨髓间充质干细胞 向心肌样细胞分化*

目的:探讨microRNA 499(miR-499)慢病毒转染对诱导大鼠骨髓来源间充质干细胞(BM-MSCs)向心肌样细胞分化的作用。方法:取第四代Wistar大鼠骨髓来源间充质干细胞进行流式细胞检测,鉴定干细胞表面特异标记物。使用符合干细胞鉴定标准的细胞批次用于后续实验。实验设置miR499慢病毒转染、慢病毒空白转染2个处理组,分别于处理后即日、1d,3d,5d,7d收集细胞进行下列实验:实时荧光定量PCR检测心肌重要转录因子GATA4、NKx2.5和MEF2C的mRNA表达,western-blot检测心肌特异蛋白I(cTnI)的表达。结果:培养第四代Wistar大鼠骨髓来源间充质干细胞表达干细胞表面特异标记物,可用于实验。大鼠骨髓来源间充质干细胞microRNA 499慢病毒载体转染后microRNA 499表达明显升高,且转染后1d,3d,5d,7d,GATA4、NKx2.5和MEF2C的mRNA表达逐渐增强。慢病毒空白转染组未见明显变化。western-blot检测自第3天开始可见cTnI阳性表达条带,慢病毒空白转染组未检测到明显阳性表达条带。结论:microRNA 499可诱导大鼠骨髓来源间充质干细胞向心肌样细胞分化。     

Intravenous administration of mesenchymal stem cells improves cardiac function in rats with acute myocardial infarction through angiogenesis and myogenesis

Mesenchymal stem cells (MSCs) are pluripotent cells that differentiate into a variety of cells, including cardiomyocytes and endothelial cells. However, little information is available regarding the therapeutic potency of systemically delivered MSCs for myocardial infarction. Accordingly, we investigated whether intravenously transplanted MSCs induce angiogenesis and myogenesis and improve cardiac function in rats with acute myocardial infarction. MSCs were isolated from bone marrow aspirates of isogenic adult rats and expanded ex vivo. At 3 h after coronary ligation, 5 x 10(6) MSCs (MSC group, n=12) or vehicle (control group, n=12) was intravenously administered to Lewis rats. Transplanted MSCs were preferentially attracted to the infarcted, but not the noninfarcted, myocardium. The engrafted MSCs were positive for cardiac markers: desmin, cardiac troponin T, and connexin43. On the other hand, some of the transplanted MSCs were positive for von Willebrand factor and formed vascular structures. Capillary density was markedly increased after MSC transplantation. Cardiac infarct size was significantly smaller in the MSC than in the control group (24 +/- 2 vs. 33 +/- 2%, P <0.05). MSC transplantation decreased left ventricular end-diastolic pressure and increased left ventricular maximum dP/dt (both P <0.05 vs. control). These results suggest that intravenous administration of MSCs improves cardiac function after acute myocardial infarction through enhancement of angiogenesis and myogenesis in the ischemic myocardium.     

Increasing donor age adversely impacts beneficial effects of bone marrow but not smooth muscle myocardial cell therapy

We evaluated the impact of donor age on the efficacy of myocardial cellular therapy for ischemic cardiomyopathy. Characteristics of smooth muscle cells (SMC), bone marrow stromal cells (MSCs), and skeletal muscle cells (SKMCs) from young, adult, and old rats were compared in vitro. Three weeks after coronary ligation, 3.5 million SMCs (n = 11) or MSCs (n = 9) from old syngenic rats or culture medium (n = 6) were injected into the ischemic region. Five weeks after implantation, cardiac function was assessed by echocardiography and the Langendorff apparatus. In the in vitro study, the numbers and proliferation of MSCs from fresh bone marrow and SKMCs from fresh tissue but not SMCs were markedly diminished in old animals (P < 0.05 both groups). SKMCs from old animals did not reach confluence. After treatment with 5-azacytidine (azacitidine), the myogenic potential of old MSCs was decreased compared with young MSCs. In the in vivo study, both SMC and MSC transplantation induced significant angiogenesis compared with media injections (P < 0.05 both groups). Transplantation of SMCs but not MSCs prevented scar thinning (P = 0.03) and improved ejection fraction and fractional shortening (P < 0.05). Load-independent indices of cardiac function in a Langendorff preparation confirmed improved function in the aged SMC group (P = 0.01) but not in the MSC group compared with the control group. In conclusion, donor age adversely impacts the efficacy of cellular therapy for myocardial regeneration and is cell-type dependent. SMCs from old donors retain their ability to improve cardiac function after implantation into ischemic myocardium.     

Tanshinone IIA and astragaloside IV promote the migration of mesenchymal stem cells by up-regulation of CXCR4

Mesenchymal stem cells (MSCs) have a therapeutic potential to treat cardiovascular diseases. However, a significant barrier to MSC therapy is insufficient MSC engraftment in ischemic myocardium after systemic administration. Here, we investigated the modulatory effects of tanshinone IIA and astragaloside IV on the migration of MSCs and further defined the underlying mechanisms. CXCR4 expression in MSCs was determined by using flow cytometry, real-time PCR, and western blotting. The results showed that CXCR4 expression was significantly higher in tanshinone IIA- and astragaloside IV-stimulated MSCs than that of the control. MSC migration toward stromal cell-derived factor-1α (SDF-1α) was studied using a transwell system. MSCs treated with tanshinone IIA and astragaloside IV showed stronger migration than that of the control. Moreover, this enhanced migration ability was abrogated by a CXCR4 inhibitor. In a rat acute myocardial infarction model, MSCs stimulated with tanshinone IIA and astragaloside IV were stained with Dio and injected into model rats via the tail vein. Dio-labeled cells in myocardium sections were observed by fluorescence microscopy. Tanshinone IIA- and astragaloside IV-stimulated MSCs showed enhanced capacities to home to ischemic myocardium sites. In addition, there was no significant difference in the SDF-1α expression among groups. These data suggest that tanshinone IIA and astragaloside IV regulate MSC mobilization, at least partially via modulation of the CXCR4 expression.     

Implanted bone marrow-derived mesenchymal stem cells fail to metabolically stabilize or recover electromechanical function in infarcted hearts

Mesenchymal stem cells (MSCs) have been used with success in several clinical applications for clinical treatment of ischemic hearts. However, the reported effects of MSC-based therapy on myocardial infarction (MI) are inconsistent. In particular, the preventive effects of MSC-based therapy on arrhythmic sudden death and metabolic disorders after infarction remain controversial. Here, we investigated the effects of MSCs on reverse remodeling in an infarcted myocardium, and found that MSC-therapy failed to achieve the complete regeneration of infarcted myocardium. Histological analyses showed that although infarct size and interstitial fibrosis induced by MI recovered significantly after MSC treatment, these improvements were marginal, indicating that a significant amount of damaged tissue was still present. Furthermore, transplanted MSCs had slight anti-apoptotic and anti-inflammatory effects in MSC-implanted regions and no significant improvements in cardiac function were observed, suggesting that naïve MSCs might not be the right cell type to treat myocardial infarction. Furthermore, small ion profiling using ToF-SIMS revealed that the metabolic stabilization provided by the MSCs implantation was not significant compared to the sham group. Together, these results indicate that pretreatment of MSCs is needed to enhance the benefits of MSCs, particularly when MSCs are used to treat arrhythmogenicity and metabolically stabilize infarcted myocardium.     

Impact of repeated intravenous bone marrow mesenchymal stem cells infusion on myocardial collagen network remodeling in a rat model of doxorubicin-induced dilated cardiomyopathy

Bone marrow mesenchymal stem cells (MSCs) transplantation improved cardiac function and reduced myocardial fibrosis in both ischemic and non-ischemic cardiomyopathies. We evaluated the effects of repeated peripheral vein injection of MSCs on collagen network remodeling and myocardial TGF-β1, AT1, CYP11B2 (aldosterone synthase) gene expressions in a rat model of doxorubicin (DOX)-induced dilated cardiomyopathy (DCM). Thirty-eight out of 53 SD rats survived at 10 weeks post-DOX injection (2.5 mg/kg/week for 6 weeks, i.p.) were divided into DCM blank (without treatment, n = 12), DCM placebo (intravenous tail injection of 0.5 mL serum-free culture medium every other day for ten times, n = 13), and DCM plus MSCs group (intravenous tail injection of 5 × 10⁶ MSCs dissolved in 0.5 mL serum-free culture medium every other day for 10 times, n = 13). Ten untreated rats served as normal controls. At 20 weeks after DOX injection, echocardiography, myocardial collagen content, myocardial expressions of types I and III collagen, TGF-β1, AT1, and CYP11B2 were compared among groups. At 20 weeks post-DOX injection, 8 rats (67 %) survived in DCM blank group, 9 rats (69 %) survived in DCM placebo group while 13 rats (100 %) survived in DCM plus MSCs group. Left ventricular end-diastolic diameter was significantly higher and ejection fraction was significantly lower in DCM blank and DCM placebo groups compared to normal control rats, which were significantly improved in DCM plus MSCs group (all p < 0.05 vs. DCM blank and DCM placebo groups). Moreover, myocardial collagen volume fraction, types I and III collagen, myocardial mRNA expressions of TGF-β1, AT1, CYP11B2, and collagen I/III ratio were all significantly lower in DCM plus MSCs group compared to DCM blank and DCM placebo groups (all p < 0.05). Repeated intravenous MSCs transplantation could improve cardiac function by attenuating myocardial collagen network remodeling possibly through downregulating renin–angiotensin–aldosterone system in DOX-induced DCM rats.     

A natural compound induced cardiogenic differentiation of endogenous MSCs for repair of infarcted heart

An intra-myocardial injection of a cardiogenic factor (cardiogenin) was reported to induce myocardial regeneration of exogenous mesenchymal stem cell (MSCs) origin. In this study, replacement of the dangerous intra-myocardial injection with a safe method and whether the endogenous MSCs contribute to the cardiogenin-mediated myocardial regeneration were investigated. Bone marrow transplantation with labeled MSCs was performed in rats, which were subsequently subject to a permanent ligation of left anterior descending coronary artery one week after the transplantation. The rats were then treated with the cardiogenin through oral administration for 2 weeks. We not only demonstrated the substantial therapeutic effects of cardiogenin on myocardial infarction through an oral administration, but also provided direct evidences that the bone marrow derived endogenous MSCs are the major cellular source of the regenerating myocardium. Preliminary mechanistic studies suggested that miR-9 and its target E-cadherin may be required for intercalated disc formation.     

Paracrine effect of CXCR4‐overexpressing mesenchymal stem cells on ischemic heart injury

It has been reported that CXCR4‐overexpressing mesenchymal stem cells (MSC^CX4) can repair heart tissue post myocardial infarction. This study aims to investigate the MSCCX4‐derived paracrine cardio‐protective signaling in the presence of myocardial infarction. Mesenchymal stem cells (MSCs) were divided into 3 groups: MSC only, MSC^CX4, and CXCR4 gene‐specific siRNA‐transduced MSC. Mesenchymal stem cells were exposed to hypoxia, and then MSCs‐conditioned culture medium was incubated with neonatal and adult cardiomyocytes, respectively. Cell proliferation–regulating genes were assessed by real‐time polymerase chain reaction (RT‐PCR). In vitro: The number of cardiomyocytes undergoing DNA synthesis, cytokinesis, and mitosis was increased to a greater extent in MSC^CX4 medium‐treated group than control group, while this proproliferative effect was reduced in CXCR4 gene‐specific siRNA‐transduced MSC–treated cells. Accordingly, the maximal enhancement of vascular endothelial growth factor, cyclin 2, and transforming growth factor‐β2 was observed in hypoxia‐exposed MSC^CX4. In vivo: MSCs were labeled with enhanced green fluorescent protein (EGFP) and engrafted into injured myocardium in rats. The number of EGFP and CD31 positive cells in the MSC^CX4 group was significantly increased than other 2 groups, associated with the reduced left ventricular (LV) fibrosis, the increased LV free wall thickness, the enhanced angiogenesis, and the improved contractile function. CXCR4 overexpression can mobilize MSCs into ischemic area, whereby these cells can promoted angiogenesis and alleviate LV remodeling via paracrine signaling mechanism.     

阿司匹林对体外培养骨髓基质细胞成骨性分化的影响

目的:探讨阿司匹林对骨髓基质细胞成骨性分化的影响。方法:培养SD大鼠骨髓基质细胞(BMSCs),传代3次后进行成骨诱导分化,诱导培养基中加入不同浓度阿司匹林(0.5、1、2、5、10mmol/L),同时设立对照组。采用cck-8法分析细胞增殖情况。比较阿司匹林组与对照组在细胞碱性磷酸酶(ALP)活性、骨钙素(OC)分泌量、钙结节染色等方面的成骨性差异。结果:阿司匹林无促进细胞增殖活性,而高浓度阿司匹林能够强烈抑制细胞增殖。0.5、1、2mmol/L浓度阿司匹林可促进BMSCs的成骨性分化,中低浓度组碱性磷酸酶含量、骨钙素分泌量在不同阶段显著高于对照组。14天茜素红染色可见中低浓度组钙结节数量高于对照组。结论:中低浓度阿司匹林作用于骨髓基质细胞可促进其成骨细胞特性表达,这表明阿司匹林有促进骨代谢合成的作用。     

The influence of mesenchymal stem cell transplantation time on myocardial reparation in rat experimental heart failure

Mesenchymal stem cells (MSCs) have an ability to migrate in the organism to injured tissue to exert influence on inflammation and reparation in these regions. The aim of this study was to determine the optimal time of MSCs transplantation for myocardial reparation in rat experimental heart failure. The experiments were carried out on inbred line Wistar-Kyoto rats. Myocardial experimental infarction (EI) was induced by ligation of the left descending coronary artery. MSCs were isolated from bone marrow, cultivated in vitro and injected into the tail vein on the day of experimental infarction operation. It was shown that the time of MSCs transplantation exerted an essential influence on angiogenesis in a damaged myocardium, on ventricular dilatation and morphological structure of the scar. The best time for MSCs transplantation was determined within two days before EI, and seven days after EI. As a result, the overload of the border zone of infarct region decreased, and no features of infarction relapse were shown in the border zone.