骨髓间充质干细胞移植治疗大鼠扩张型心肌病

1泸州医学院附属医院急诊科,四川省泸州市 646000;2哈尔滨医科大学附属第二医院心内科,黑龙江省哈尔滨市  150000 背景：扩张型心肌病所致的心肌纤维化是心力衰竭的病理基础,目前药物治疗、介入治疗和外科手术均不能替代坏死心肌和彻底改善心脏功能。 目的：观察异体骨髓间充质干细胞移植对大鼠扩张型心肌病心脏功能的作用和心肌纤维化的影响。 方法：40只Wistar大鼠随机数字表法分为细胞移植组(n=15)、对照组(n=15)和空白组(n=10),前2组建立大鼠扩张型心肌病模型。造模成功4周后细胞移植组注射骨髓间充质干细胞悬液150 μL(含3×106个细胞),对照组和空白组注射等量培养液。 结果与结论：与空白组相比,细胞移植组和对照组移植前左室收缩末期内径增加,射血分数和缩短分数明显下降(P < 0.01);移植后4周,细胞移植组超声心动图检查和移植前相比,左室收缩末期内径下降、射血分数和缩短分数明显升高(P < 0.01)。细胞移植组心脏胶原的表达低于对照组(P < 0.05)。与对照组相比,其他2组基质金属蛋白酶2及基质金属蛋白酶9表达明显下降(P < 0.05)。提示骨髓间充质干细胞移植后可改善心肌纤维化及扩张型心肌病鼠的心脏功能。      

血红素氧合酶1修饰骨髓间充质干细胞多点注射梗死灶周围对梗死后心肌结构重塑的影响

背景：研究表明血红素氧合酶1的过表达有抗炎症反应的作用,将血红素氧合酶1修饰的骨髓间充质干细胞(HO-1-BMSCs)移植入梗死心脏周围是否可调节基质金属蛋白酶/组织金属蛋白酶抑制剂的比例而明显改善梗死心肌重构呢？ 目的：观察血红素氧合酶1修饰的骨髓间充质干细胞移植对心肌梗死后心肌胶原的调节及心肌重塑的逆转作用。 方法：利用血红素氧合酶1腺病毒转染体外培养扩增的骨髓间充质干细胞。结扎大鼠左前降支动脉制造心肌梗死模型,1 h后,分别将HO-1-BMSCs、BMSCs多点注射到大鼠心脏梗死区四周,对照组注射等量磷酸盐缓冲液。 结果与结论：Adv-hHO-1转染BMSCs后获稳定表达;hHO-1-mRNA仅在HO-1-BMSCs移植组表达;与对照组相比,HO-1-BMSCs移植组基质金属蛋白酶2/9的表达显著减少(P < 0.05),Null-BMSCs组基质金属蛋白酶2/9的表达虽然减少,但差异无显著性意义(P > 0.05);与对照组相比,细胞移植组金属蛋白酶组织抑制剂2/3的表达显著增加,尤以HO-1-BMSCs组明显(P < 0.05);但金属蛋白酶组织抑制剂1无明显变化(P > 0.05)。金属蛋白酶组织抑制剂2/基质金属蛋白酶2和金属蛋白酶组织抑制剂3/基质金属蛋白酶9的比例在细胞移植的心脏中明显上升。与对照组比较,细胞移植组心室中的胶原蛋白沉积减少,心室腔内径显著缩小。结果表明血红素氧合酶1修饰的骨髓间充质干细胞移植可使基质金属蛋白酶/组织金属蛋白酶抑制剂的比例正常化,并逆转心肌细胞外基质的重构。     

人骨髓间充质干细胞培养上清与肝星状细胞相关酶的分泌

背景：研究证实骨髓间充质干细胞移植治疗能改善甚至逆转肝纤维化,但其具体机制尚不清楚。 目的：检测人骨髓间充质干细胞培养上清对肝星状细胞中基质金属蛋白酶2及组织抑制基质金属蛋白酶1表达的影响。 方法：采用密度梯度离心法分离人骨髓间充质干细胞,收集原代培养7 d的骨髓间充质干细胞培养上清,加入肝星状细胞中培养作为实验组,并设置单独肝星状细胞组、单独骨髓间充质干细胞组为对照。培养24,48 h后,检测各组细胞上清中基质金属蛋白酶2蛋白与组织抑制基质金属蛋白酶1蛋白的表达。 结果与结论：与单独肝星状细胞组、单独骨髓间充质干细胞组比较,实验组培养24,48 h后肝星状细胞基质金属蛋白酶2及组织抑制基质金属蛋白酶1表达减少(P < 0.05或P < 0.01)。表明骨髓间充质干细胞培养上清抑制肝星状细胞中基质金属蛋白酶2、组织抑制基质金属蛋白酶1的表达。     

骨髓间充质干细胞移植对心衰大鼠心肌组织中MMP-2、MMP-9和TIMP-1的影响

 目的：探讨骨髓间充质干细胞（BMSCs）移植对心衰大鼠心肌组织中基质金属蛋白酶2（MMP-2）、MMP-9和金属蛋白酶组织抑制剂1（TIMP-1）的影响及机制。方法：采用异丙肾上腺素(ISO)皮下注射的方法建立大鼠的心力衰竭模型,随机分成3组,每组8只,心肌内分别注射BMSCs（BMSCs组）、BMSCs条件培养液（BMSCs-CM组）及生理盐水（NS组）。RT-PCR检测3组心肌组织MMP-2、MMP-9和TIMP-1 mRNA的表达,Western blotting检测心肌组织MMP-2、MMP-9和TIMP-1蛋白,胞浆和胞核内核因子κB（NF-κB）的p65和p50,以及总蛋白中NF-κB抑制物（I-κB）和磷酸化NF-κB抑制物（p-IκB）蛋白表达水平。结果：和NS组比较,BMSCs组和BMSCs-CM组MMP-2和MMP-9 mRNA和蛋白表达明显减弱（P<0.05）,TIMP-1 mRNA和蛋白表达明显增强（P<0.05）;而与BMSCs-CM组比较,BMSCs组MMP-2和MMP-9 mRNA和蛋白表达明显减弱（P<0.05）,TIMP-1 mRNA和蛋白表达明显增强（P<0.05）。胞核中p65和p50蛋白表达,NS组明显多于BMSCs组和BMSCs-CM组(P<0.05);而胞浆中p65和p50蛋白表达,NS组明显少于BMSCs组和BMSCs-CM组(P<0.05)。和BMSCs-CM组比较,BMSCs组胞核中p65和p50蛋白表达明显升高(P<0.05);而胞浆中p65和p50蛋白表达,BMSCs组明显少于BMSCs-CM组(P<0.05)。NS组心肌组织总蛋白中p-IκB/IκB最高,明显多于BMSCs组和BMSCs-CM组(P<0.05),BMSCs组最低(P<0.05)。结论：BMSCs及其在缺氧条件下的培养液均可通过改变NF-κB的抑制蛋白IκB的活性,从而改变心衰心肌细胞胞核中NF-κB含量,影响有关基因的转录,使得MMPs水平升高和TIMPs水平降低。     

骨髓间充质干细胞在肝纤维化中的研究进展

肝纤维化是所有慢性肝病的共同病理基础,严重影响人类健康,大量肝病患者因缺乏理想、有效的治疗手段而死亡。骨髓间充质干细胞因具有可塑性、取材方便、不存在免疫排斥问题以及易于外源基因的转染和表达等优点而被广泛用于疾病研究,为肝纤维化的有效治疗带来希望。就骨髓间充质干细胞的概况和其在肝纤维化中的研究进行阐述。     

microRNA-1诱导大鼠骨髓间充质干细胞向心肌样细胞分化

目的研究microRNA-1(miRNA-1)能否诱导骨髓间充质干细胞(MSCs)向心肌样细胞分化。方法构建大鼠miRNA-1表达载体,分离扩增培养及鉴定大鼠MSCs。脂质体法转染大鼠第4代MSCs,实时定量RT-PCR(qRT-PCR)检测转染miRNA-1质粒后MSCs的miRNA-1表达水平。分别于转染2、4和6 d后用RT-PCR检测心肌重要转录因子GATA4、NKx2.5和MEF2C的mRNA表达,免疫荧光检测心肌特异蛋白I(cTnI)的表达。结果 90%以上的MSCs表达MSCs重要标志物CD29、CD44;未检测到造血前体细胞标志抗原CD34、白细胞标志抗原CD45的表达。转染miRNA-1质粒后,miRNA-1表达水平明显上调。转染miRNA-1质粒2、4和6 d后,GATA4、NKx2.5和MEF2C的mRNA表达逐渐增强。第4和6天后可见cTnI阳性表达细胞。结论 miRNA-1能诱导大鼠MSCs向心肌样细胞分化。     

金属蛋白酶组织抑制因子转染骨髓间充质干细胞静脉输入治疗缺血性心肌病

背景：金属蛋白酶组织抑制因子1可能通过抑制基质金属蛋白酶活性降低干细胞侵袭能力,提高其归巢修复能力。 目的：观察转染金属蛋白酶组织抑制因子1基因骨髓间充质干细胞移植修复损伤心肌的能力。 方法：开胸结扎SD大鼠冠状动脉前降支建立心肌梗死模型,1周后随机分组：空白组经尾静脉内注射DMEM悬液;干细胞组经尾静脉内注射含1×10⁷骨髓间充质干细胞的DMEM悬液;绿色荧光蛋白-干细胞组经尾静脉内注射转染带有绿色荧光蛋白基因慢病毒空载体的骨髓间充质干细胞(1×10⁷)DMEM悬液;TIMP-1-shRNA-干细胞组经尾静脉内注射转染TIMP-1-shRNA的骨髓间充质干细胞(1×10⁷)DMEM悬液。 结果与结论：造模后,4组大鼠心脏功能受损,收缩能力下降,治疗4周后心脏功能均有不同程度改善：与空白组比较,干细胞组、绿色荧光蛋白-干细胞组、TIMP-1-shRNA-干细胞组心脏收缩功能明显提高(P < 0.05),心肌梗死面积百分比明显缩小(P < 0.05),梗死区毛细血管密度明显增加(P < 0.05),且TIMP-1-shRNA-干细胞组改善程度优于干细胞组、绿色荧光蛋白-干细胞组(P < 0.05)。说明转染金属蛋白酶组织抑制因子1基因的骨髓间充质干细胞移植能够明显改善损伤心肌功能,修复缺血性心肌病。     

心肌细胞共培养诱导骨髓间充质干细胞向心肌样细胞分化

目的在体外以新生大鼠心肌细胞（CM）与骨髓间充质干细胞（MSCs）共同培养的方式模拟心肌微环境,研究MSCs分化为心肌细胞的机制。方法分离大鼠MSCs在体外培养纯化后进行细胞标记,将已标记的MSCs分别与搏动的CM、停止搏动的CM以及心肌细胞条件培养液混合培养。分别在共培养后第4、5天用免疫荧光染色检测MSCs细胞中的心肌特异性肌钙蛋白T（Troponin T）。结果与搏动的CM共培养后第4天MSCs出现自发收缩,与CM同步搏动并表达Troponin T,而在抑制心肌细胞搏动或缺乏与心肌直接接触的情况下MSCs未出现上述变化。结论说明在与CM直接接触的前提下,CM对MSCs的机械牵拉刺激为诱导MSCs分化为心肌细胞的必须条件。单纯的心肌细胞条件培养液则非关键因素。了解MSCs分化为心肌细胞的机制对于寻找适宜的细胞移植条件有重要指导意义。     

骨髓间充质干细胞经尾静脉移植治疗肝纤维化

BACKGROUND: Liver fibrosis is the early stage of terminal liver diseases. Effective treatment for liver fibrosis can prevent the occurrence of terminal liver diseases. Bone marrow mesenchymal stem cell transplantation is a promising method to treat liver fibrosis. OBJECTIVE: To study the therapeutic effect of bone marrow mesenchymal stem cells on liver fibrosis in rats. METHODS: Eighteen Sprague-Dawely rats were randomized into three groups: control, model and cell transplantation groups. Animal models of carbon tetrachloride-induced liver fibrosis were made in the latter two groups. After modeling, 1 mL bone marrow mesenchymal stem cells (5×105) or the same volume of normal saline was injected via the tail vein into the rats in the cell transplantation and model groups, respectively. Rats in the control group were given no treatment. Degree of liver fibrosis, liver function, histological changes of the liver were detected and observed in the three groups at 4 weeks after treatment. RESULTS AND CONCLUSION: In the control group, the liver tissues had normal structure with no fibrosis; in the model group, proliferation of fibrous tissues in the portal area of the liver, inflammatory cell infiltration, vacuolar degeneration and irregular arrangement of liver cells, and tissue structure damage were observed; in the transplantation group, liver tissue damage was severer than the control group but milder than the model group. Levels of serum hyaluronidase, type IV collagen and procollagen III were significantly lower in the cell transplantation group than the model group (P < 0.05). These findings indicate that bone marrow mesenchymal stem cell transplantation can alleviate liver fibrosis and improve liver function in rats with carbon tetrachloride-induced liver fibrosis.  中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程      

心肌组织裂解液诱导骨髓间充质干细胞向心肌样细胞的分化

背景：干细胞具有“环境依赖性分化”特性,以心肌组织裂解液模拟心肌微环境诱导骨髓间充质干细胞向心肌细胞方向分化的研究甚少。 目的：观察心肌组织裂解液对大鼠骨髓间充质干细胞向心肌细胞方向分化的影响。 方法：选用生长良好的第2代骨髓间充质干细胞,加入诱导液连续培养5周,连续观察细胞形态学特征的变化规律,采用α-Actin进行免疫细胞化学染色观察干细胞是否向肌细胞方向分化,收集诱导后的细胞行苏木精-伊红染色观察是否存在闰盘,并通过电镜观察其超微结构。 结果与结论：加入诱导液后大部分细胞呈“竹节样”,α-Actin免疫细胞化学染色阳性,表明间充质干细胞向肌细胞方向分化;苏木精-伊红染色可见闰盘,透射电镜下见排列整齐明暗相间的肌丝,扫描电镜下可见胞浆内有纤维状结构,说明“竹节样”细胞具有心肌细胞的典型形态结构特点。连续诱导12 d多个区域出现成片的、具有自律性搏动的多核肌管结构。心肌组织裂解液可模拟心肌细胞的微环境,诱导骨髓间充质干细胞分化为具有典型形态结构特征和相应功能活动的心肌样细胞。     

Bone marrow mesenchymal stem cells protect against bleomycin-induced pulmonary fibrosis in rat by activating Nrf2 signaling

Pulmonary fibrosis is a progressive and lethal disorder. Although the precise mechanisms of pulmonary fibrosis are not fully understood, oxidant/antioxidant may play an important role in many of the processes of inflammation and fibrosis. Keap1-Nrf2-ARE pathway represents one of the most important cellular defense mechanisms against oxidative stress. Mesenchymal stem cells (MSC) are in clinical trials for widespread indications including musculoskeletal, neurological, cardiac and haematological disorders. One emerging concept is that MSCs may have paracrine, rather than a functional, roles in lung injury repair and regeneration. In the present study, we investigated bone marrow mesenchymal stem cells (BMSCs) for the treatment of bleomycin-induced pulmonary fibrosis. Our results showed that BMSCs administration significantly ameliorated the bleomycin mediated histological alterations and blocked collagen deposition with parallel reduction in the hydroxyproline level. The gene expression levels of NAD(P)H: quinine oxidoreductase 1 (NQO1), gama-glutamylcysteine synthetase (γ-GCS), heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2), attenuated by bleomycin, were increased up to basal levels after BMSCs transplantation. BMSCs significantly increased superoxide dismutase (SOD) activity and inhibited malondialdehyde (MDA) production in the injured lung. The present study provides evidence that BMSCs may be a potential therapeutic reagent for the treatment of lung fibrosis.     

Nuclear factor kappa B signaling initiates early differentiation of neural stem cells

Inflammatory mediators, many of which activate the signaling of nuclear factor kappa B (NFκB), have received increasing attention in the field of neurogenesis. NFκB signaling regulates neurite outgrowth and neural plasticity as well as the proliferation/apoptosis and terminal differentiation of neural stem cells (NSCs). Early neurogenesis from NSCs produces identical progeny through symmetric division and committed daughter cells through asymmetric division. Here, we show that NFκB signaling is required for NSC initial differentiation. The canonical IKKβ/IκBα/p65 pathway is activated during the initial stages of neural differentiation induced by treatment with TNFα or withdrawal of epidermal growth factor/basic fibroblast growth factor. NSC-specific inhibition of NFκB in transgenic mice causes an accumulation of Nestin(+) /Sox2(+) /glial fibrillary acidic protein(+) NSCs. Inhibition of NFκB signaling in vitro blocks differentiation and asymmetric division and maintains NSCs in an undifferentiated state. The induction of initial differentiation and asymmetry by NFκB signaling occurs through the inhibition of C/EBPβ expression. Our data reveal a novel function of NFκB signaling in early neurogenesis and provide insight into the molecular mechanisms underlying neurodevelopmental disorders and neurodegenerative diseases.     

Trypanosoma cruzi infection and nuclear factor kappa B activation prevent apoptosis in cardiac cells

Studies of cardiac pathology and heart failure have implicated cardiomyocyte apoptosis as a critical determinant of disease. Recent evidence indicates that the intracellular protozoan parasite Trypanosoma cruzi, which causes heart disease in chronically infected individuals, impinges on host apoptotic pathways in a cell type-dependent manner. T. cruzi infection of isolated neuronal cells and cardiomyocytes protects against apoptotic cell death, whereas apoptosis is triggered in T cells in T. cruzi-infected animals. In this study, we demonstrate that the ability of T. cruzi to protect cardiac cells in vitro from apoptosis triggered by a combination of tumor necrosis factor alpha and serum reduction correlates with the presence of intracellular parasites and involves activation of host cell NF-kappaB. We further demonstrate that the apoptotic block diminishes activation of caspase 3. The ability of T. cruzi to prevent apoptosis of infected cardiomyocytes is likely to play an important role in establishment of persistent infection in the heart while minimizing potential damage and remodeling that is associated with cardiomyocyte apoptosis in cardiovascular disease.     

肝纤维化大鼠骨髓间充质干细胞体外诱导为类肝细胞

目的 观察正常、肝纤维化模型组大鼠骨髓间充质干细胞（MSCs）体外诱导分化为类肝细胞的差异。方法 Wistar大鼠随机分为对照组和肝纤维化模型组。用皮下注射CCl4乳剂建立肝纤维化模型。用密度梯度离心法和贴壁法分离大鼠的MSCs,经培养传代获得纯化的MSCs。用肝细胞生长因子（HGF）和成纤维细胞生长因子-4（FGF-4）诱导培养纯化的MSCs。留取15、21和27 d细胞培养液进行白蛋白（Alb）、甲胎蛋白（AFP）检测;于27 d收集细胞爬片,进行糖原染色和免疫细胞化学染色检测MSCs中CK-18、CK-19。结果 于诱导15、21和27 d,2组经诱导培养的MSCs AFP水平均高于未诱导的MSCs（P<0.01）,其中21 d AFP水平最高;而白蛋白水平21和27 d 2组经诱导培养的MSCs均高于未诱导的MSCs（P<0.01）,15 d无差异,27 d最高。27 d 2组经诱导培养的MSCs糖原染色和CK-18、CK-19免疫细胞化学染色均阳性,未诱导的MSCs糖原染色、CK-18、CK-19均阴性。从AFP、白蛋白水平综合比较,正常和肝纤维化模型组大鼠MSCs诱导效果无明显差别。结论 HGF、FGF-4可在体外诱导肝纤维化大鼠的MSCs分化为具有肝细胞样细胞表型和功能的类肝细胞。     

Bone marrow-derived mesenchymal stem cells prevent the apoptosis of neuron-like PC12 cells via erythropoietin expression

Hippocampal cholinergic neurostimulating peptide (HCNP) induces the synthesis of acetylcholine in medial septal nucleus in vitro and in vivo. HCNP precursor protein (HCNP-pp) is a multifunctional protein that participates in a number of signaling pathways, including MAPK/extracellular signal and G-protein-coupled receptor kinase 2. We recently demonstrated that the amount of collapsin response mediator protein-2 (CRMP-2) is increased in hippocampus of HCNP-pp transgenic mice. To clarify the interaction between HCNP/HCNP-pp and CRMP-2 and its role in synaptic function, we investigated whether HCNP-pp is localized to the synapse and if it affects protein expression. Here, we demonstrate that HCNP-pp co-localizes with CRMP-2 at presynaptic terminals. Furthermore, HCNP-pp overexpression increases synaptophysin levels. These findings suggest that HCNP-pp, in association with CRMP-2, plays an important role in presynaptic function in the hippocampus.     

1,25-二羟维生素D3负性调控被动致敏人气道平滑肌细胞中的NF-κB信号通路

 目的：探讨1,25-二羟维生素D3［1,25-(OH)2D3］对被动致敏人气道平滑肌细胞(HASMCs)中核因子κB（NF-κB）信号通路的影响。方法：原代培养HASMCs并使之被动致敏,以1,25-(OH)2D3作为干预因素。EMSA法检测NF-κB的DNA结合活性;免疫细胞化学染色技术观察NF-κB p65的核易位情况;Western blotting法检测核因子κB抑制蛋白α(IκBα)及p-IκBα蛋白的表达水平;实时荧光定量PCR检测维生素D受体(VDR)、维生素D 24-羟化酶(CYP24)和IκBα mRNA的表达水平;放线菌素D处理实验检测IκBα mRNA的表达。结果：(1) 1,25-(OH)2D3显著削弱被动致敏HASMCs中NF-κB的DNA结合活性及其亚单位 p65的核易位;(2) 1,25-(OH)2D3能通过增加被动致敏HASMCs中IκBα的mRNA稳定性及减少其蛋白磷酸化水平2个途径显著上调细胞中IκBα的表达;(3) 1,25-(OH)2D3显著上调被动致敏HASMCs中VDR的mRNA表达并诱发其功能性反应。结论：1,25-(OH)2D3能通过上调被动致敏HASMCs中IκBα的表达抑制细胞NF-κB信号通路,且这一作用与VDR有关,这可能是其调控被动致敏HASMCs的重要作用机制。