骨髓间质干细胞体外定向肌样分化的研究

目的 研究兔骨髓间质干细胞经5—氮杂胞苷诱导，在体外定向肌样分化的情况。方法 取兔胫骨骨髓，分离并培养骨髓间质干细胞，用5—氮杂胞苷苦(10μmol/L)定向诱导，分别在培养的第7、14、2l、28天用免疫组织化学的方法检测细胞中的肌球蛋白重链，取未经5—氮杂胞苷诱导正常培养的细胞为对照组。结果 兔骨髓间质干细胞经5—氮杂胞苷诱导，在其培养的第21、28天免疫组织化学染色方法检测细胞中的肌球蛋白重链呈阳性，而在其培养的第7、14天以及对照组，免疫组织化学染色方法检测细胞中的肌球蛋白重链呈阴性。结论 骨髓间质干细胞是骨髓来源的具有多向分化潜能的干细胞，在5—氮杂胞苷的定向诱导下，可以肌样分化。     

Mesenchymal, but not hematopoietic, stem cells can be mobilized and differentiate into cardiomyocytes after myocardial infarction in mice

Bone marrow (BM) cells are reported to contribute to the process of regeneration following myocardial infarction. The present study examined two independent clonal studies to determine the origin of bone marrow (BM)-derived cardiomyocytes. First, we transplanted single CD34(-)c-kit(+)Sca-1(+)lineage(-) side population cells (hematopoietic stem cells) from enhanced green fluorescent protein (EGFP)-transgenic mice into lethally irradiated mice, induced myocardial infarction, and treated them with G-CSF to mobilize stem cells. At 8 weeks, we could not find any EGFP(+) cardiomyocytes. In contrast, more than 5000 EGFP(+) cardiomyocytes were observed in whole BM cell-transplanted mice, suggesting that they were derived from non-hematopoietic cells. Next, clonally purified mesenchymal stem cells (MSC) that expressed EGFP in the cardiomyocyte-specific manner were transplanted directly into BM of lethally irradiated mice, and similar experiment was performed. EGFP(+) actinin(+) cells were observed in the ischemic myocardium, indicating that MSC had been mobilized and differentiated into cardiomyocytes. Together, these results suggest that the origin of the BM-derived cardiomyocytes is MSC.     

骨髓间充质干细胞分化为心肌细胞的实验研究

目的 探讨骨髓间充质干细胞(MsCs)定向分化为心肌细胞的可行性，建立一种以干细胞移植为主治疗心肌梗死的治疗策略。方法利用Percoll分离骨髓细胞培养获得MsCs；用5-氮杂胞苷(0．1、1、5、10、50和100μmol/L)定向诱导24h，分别在诱导培养的第14d和21d，检测细胞中的α-横纹肌肌动蛋白表达。将培养的MSCs，移植于急性梗死心肌组织内，4周后进行组织学和免疫组织化学染色，并用超声检测室壁运动和心功能改变。结果骨髓间充质干细胞经5-氮杂胞苷诱导，培养21d后，5和10μmol/L 5-氮杂胞苷诱导后的MSCs有一部分细胞表达α-横纹肌肌动蛋白，并一些细胞自发性跳动；细胞移植4周后，在缺血心肌有一部分MSCs与宿主心肌细胞形成连接并分化为具有典型的肌小节和表达α-横纹肌肌动蛋白阳性的心肌细胞。结论MsCs可以分化成心肌细胞。     

Development of regenerative cardiomyocytes from mesenchymal stem cells for cardiovascular tissue engineering

We have isolated a cardiomyogenic (CMG) cell line from murine bone marrow stroma. Stromal cells were immortalized, treated with 5-azacytidine, and spontaneous beating cells were repeatedly screened for. The cells showed a fibroblast-like morphology. However, this morphology changed after 5-azacytidine treatment in about 30% of the cells, which connected with adjoining cells after 1 week, formed myotube-like structures and began spontaneous beating after 2 weeks, and beat synchronously after 3 weeks. These cells expressed atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Electron microscopy revealed a cardiomyocyte-like ultrastructure including typical sarcomeres and atrial granules. They had sinus node-like or ventricular cell-like action potentials. Analysis of the isoform of contractile protein genes, such as myosin and alpha-actin, indicated that their phenotype was similar to fetal ventricular cardiomyocytes. These cells expressed Nkx2.5, GATA4, TEF-1, and MEF2-C mRNA before 5-azacytidine treatment, and expressed MEF2-A and MEF2-D after treatment. This new cell line provides a powerful model for the study of cardiomyocyte transplantation.     

脐血间质干细胞移植于梗死心肌后对心肌基本结构的保护作用

目的 探讨经诱导分化为肌源性干细胞的脐血间质干细胞（mesenchymal stem cell，MSC）移植于梗死心肌后对梗死心肌组织结构改变的影响。方法 将36只成年杂种犬随机分为脐血MSC移植组（移植组）和对照组，每组各18只。移植组：用5一氮杂胞嘧啶核苷（5-azacytidine，5-aza）诱导分化后的脐血MSC，经结扎的犬冠状动脉左前降支远端灌注移植入梗死区；对照组：给予等量含0．02％的4，6-联脒-2-苯基吲哚（DAPI）的IMDM培养液注射。于移植2周、4周和8周时用Nagar—Olsen染色法观察心肌组织基本结构改变，用免疫细胞化学染色法观察残存心肌细胞结蛋白（desmin）分布变化。结果 Nagar—Olsen染色结果显示：移植组梗死心肌组织内弹力纤维、胶原纤维排列整齐、规律；对照组排列较紊乱、无规律，部分发生融合。移植组梗死区域心肌细胞结蛋白表达明显高于对照组（P〈0．01），而两组正常区域心肌细胞结蛋白表达差别无统计学意义（P〉0．05）。结论 脐血MSC在体外经5-aza诱导分化为肌源性干细胞并移植入梗死心肌后，对梗死心肌组织结构具有保护作用。     

Differentiation "in vitro" of primary and immortalized porcine mesenchymal stem cells into cardiomyocytes for cell transplantation

Cell transplantation to regenerate injured tissues is a promising new treatment for patients suffering several diseases. Bone marrow contains a population of progenitor cells known as mesenchymal stem cells (MSCs), which have the capability to colonize different tissues, replicate, and differentiate into multilineage cells. Our goal was the isolation, characterization, and immortalization of porcine MSCs (pMSCs) to study their potential differentiation "in vitro" into cardiomyocytes. pMSCs were obtained from the aspirated bone marrow of Large-White pigs. After 4 weeks in culture, adherent cells were phenotypically characterized by flow cytometry and immunochemistry by using monoclonal antibodies. Primary pMSCs were transfected with the plasmid pRNS-1 to obtain continuous growing cloned cell lines. Fresh pMSCs and immortalized cells were treated with 5-azacytidine to differentiate them into cardiomyocytes. Flow cytometry analysis of isolated pMSCs demonstrated the following phenotype, CD90(pos), CD29(pos), CD44(pos), SLA-I(pos), CD106(pos), CD46(pos) and CD45(neg), CD14(neg), CD31(neg), and CD11b(neg), similar to that described for human MSC. We derived several stable immortalized MSC cell lines. One of these, called pBMC-2, was chosen for further characterization. After "in vitro" stimulation of both primary or immortalized cells with 5-azacytidine, we obtained different percentages (30%-50%) of cells with cardiomyocyte characteristics, namely, positive for alpha-Actin and T-Troponin. Thus, primary or immortalized pMSCs derived from bone marrow and cultured were able to differentiate "ex vivo" into cardiac-like muscle cells. These elements may be potentials tools to improve cardiac function in a swine myocardial infarct model.     

Regenerative medicine in cardiology

Emerging evidence indicates that cardiomyocytes can be regenerated from embryonic stem cells, adult mesenchymal stem cells, and cardiac side population cells (cardiac stem cells). When these cells were injected into diseased hearts, indices of cardiac function improved. There are ethical difficulties involved in the use of embryonic stem cells. The ratio of differentiation into cardiomyocytes is still relatively low at present. Transplantation of regenerated cardiomyocytes or stem cells, however, will become a future therapy for severe heart failure.     

脐血间质干细胞经肌源性诱导后细胞内游离钙的变化

目的研究3种不同的肌源性诱导方法对脐血间质干细胞（mesenchymal stem cells,MSCs）进行诱导后细胞内Ca2＋调控系统分化的影响。方法 2007年1月至2010年4月上海交通大学医学院附属新华医院采用5-氮杂胞嘧啶核苷（5-azacytidine,5-aza）、心肌裂解液和心肌诱导液分别诱导犬脐血MSCs。采用激光共聚焦显微镜检测5-aza、心肌裂解液和心肌诱导液诱导的细胞、心肌细胞和MSCs内Ca2＋荧光探针Fluo-3/AM结合的Ca2＋,每份细胞选取2~5个视野,每一种细胞观察记录30个视野,取每1 min摄得的10张图象的平均荧光强度值,以光密度值（OD）反映细胞内游离Ca2＋浓度;并连续监测细胞内游离Ca2＋浓度的变化。结果经3种方法诱导后细胞内游离Ca2＋浓度均高于心肌细胞内游离Ca2＋浓度（F=59.400,P=0.000）。5-aza、心肌裂解液和心肌诱导液诱导的细胞内游离Ca2＋浓度差异有统计学意义（F=18.988,P=0.000）。5-aza诱导的细胞内游离Ca2＋浓度与心肌裂解液诱导的细胞比较差异无统计学意义（OD 1 076.88±44.65 vs.1 040.90±37.48,P=0.186）,但其浓度高于心肌诱导液诱导的细胞（OD 1 076.88±44.65 vs.973.91±46.49,P=0.001）,心肌裂解液诱导的细胞内游离Ca2＋浓度高于心肌诱导液诱导的细胞（OD 1 040.90±37.48 vs.973.91±46.49,P=0.001）。3种诱导方法诱导后的细胞内游离Ca2＋呈自发性波动,与心肌细胞相似,但其频率和幅度有明显差异。5-aza、心肌裂解液诱导后的MSCs在KCl刺激下能使细胞内Ca2＋瞬时释放,心肌诱导液诱导后的MSCs对KCl也有反应,但不能诱发Ca2＋释放,而是使Ca2＋释放时间延长。结论 5-aza诱导、心肌裂解液和心肌诱导液诱导对MSCs的Ca2＋调控系统有分化作用,3种方法的效果不同,其产生差异的原因和效果的优劣需进一步实验阐明。     

脂肪组织来源的基质细胞研究进展

目的介绍近期国内外脂肪组织来源的基质细胞研究进展. 方法广泛查阅近期有关文献,归纳分析此种细胞的培养和分化的研究状况. 结果脂肪组织中存在一种多能的基质细胞,体外培养条件下这种细胞生长状态类似成纤维细胞,可长期增殖,细胞绝大多数为中胚层来源,混有少量的血管周细胞、内皮细胞和平滑肌细胞,在一定的诱导条件下能分化为脂肪细胞、软骨细胞、成骨细胞、成肌细胞和神经细胞. 结论脂肪组织来源的基质细胞可替代间充质干细胞作为组织工程的另一种干细胞.     

骨髓间充质干细胞体外转化为骨骼肌细胞的初步观察

目的观察骨髓间充质干细胞(mesenchymal stem cells，MSCs)经5．氮杂胞苷(5-Aza)诱导分化为成骨骼肌肌细胞的过程。方法分离纯化4～6周龄SD大鼠的骨髓MSCs，以5-Aza作用24h。于作用1周后的不同时相点，观察细胞形态变化，免疫组织化学染色观察细胞内结蛋白(desmin)、骨骼肌肌动蛋白(actin)、肌球蛋白重链(MHC)、肌生成素(myogenin)表达。结果经5-Aza诱导后14d，部分细胞表达desmin；诱导后24d，细胞表达myogenin、MHC、actin。细胞形态和排列方式发生明显变化，可见有肌管样细胞出现。结论MSCs经5-Aza诱导后可以向成骨骼肌细胞定向分化。     

Marrow stromal cells for cellular cardiomyoplasty: feasibility and potential clinical advantages

OBJECTIVES: Marrow stromal cells are mesenchymal stem cells able to differentiate into cardiomyocytes in vitro. We tested the hypothesis that marrow stromal cells, when implanted into myocardium, can undergo milieu-dependent differentiation and express cardiomyogenic phenotypes in vivo. METHODS: Isogenic adult rats were used as donors and recipients to simulate autologous transplantation. Marrow stromal cells isolated from donor leg bones were culture-expanded, labeled with 4;,6-diamidino-2-phenylindole, and then injected into the myocardium of the recipients. The hearts were harvested from 4 days to 12 weeks after implantation, and the implant sites were examined to identify the phenotypes of the labeled marrow stromal cells. RESULTS: Viable cells labeled with 4;, 6-diamidino-2-phenylindole can be identified in host myocardium at all time points after implantation. Implanted marrow stromal cells show the growth potential in a myocardial environment. After 4 weeks, donor cells derived from marrow stromal cells demonstrate myogenic differentiation with the expression of sarcomeric myosin heavy chain and organized contractile proteins. Positive staining for connexin 43 indicates the formation of gap junctions, which suggests that cells derived from marrow stromal cells, as well as native cardiomyocytes, are connected by intercalated disks. CONCLUSIONS: Different cell sources have been used as donor cells for cellular cardiomyoplasty. Our findings indicate that marrow stromal cells can also be used as donor cells. In an appropriate microenvironment they will exhibit cardiomyogenic phenotypes and may replace native cardiomyocytes lost by necrosis or apoptosis. Because marrow stromal cells can be obtained repeatedly by bone marrow aspiration and expanded vastly in vitro before being implanted or used as autologous implants, and because their use does not call for immunosuppression, the clinical use of marrow stromal cells for cellular cardiomyoplasty appears to be most advantageous.     

Lunate arthroplasty with autologous mesenchymal stem cells in a rabbit model

BACKGROUND: There is no ideal treatment for end-stage degenerative wrist disorders and subsequent carpal collapse. The purpose of this study was to investigate whether autologous cartilage constructs tissue-engineered from bone-marrow-derived mesenchymal stem cells can be effective for carpal bone reconstruction. METHODS: Total lunate excision was performed in twenty-seven adult New Zealand White rabbits. Mesenchymal stem cells were isolated from marrow and then were culture-expanded. Group-1 rabbits underwent excision only. Group-2 animals underwent excision followed by implantation of a scaffold consisting of gelatin and hyaluronan. Group-3 animals underwent excision followed by implantation of a mesenchymal stem cell-seeded scaffold that had been preincubated in chondrogenic medium. The group-1 animals were killed at six weeks, whereas the group-2 and group-3 animals were killed at six or twelve weeks. Tissues were harvested for radiographic and histologic analysis. RESULTS: Significant carpal collapse (a 5.4% +/- 2.8% reduction in the carpometacarpal index, p < 0.05) was observed in the group-1 animals by six weeks. In contrast, the carpal height was maintained in the group-2 and 3 animals. There was no radiographic evidence of ossification in the group-1 or 2 animals, whereas there was radiographic evidence of ossification in all six group-3 rabbits killed at the twelve-week time-point. Histologic sections from the group-3 animals showed filling of the lunate space with islands of cartilage with interspersed bone ossicles at six weeks. At twelve weeks, there was abundant bone formation as well as evidence of neovascularization. Osseous tissue was present in the central portions of the constructs while the periphery was lined with cartilage. In groups 1 and 2, the lunate space was filled with poorly organized fibrous tissue. CONCLUSIONS: Cartilaginous implants preformed from autologous mesenchymal stem cells seeded onto biodegradable scaffold can prevent carpal collapse. The newly formed osteochondral tissue appears to function as an adequate biologic lunate spacer for at least twelve weeks in this animal model.     

基因转染脂肪间充质干细胞作为软骨组织工程种子细胞的研究

目的 观察人转化生长因子(hTGF)β2基因转染诱导脂肪间充质干细胞向软骨细胞的定向分化能力,探讨脂肪间充质干细胞作为种子细胞和在基因增强的软骨组织工程中应用的可行性.方法 取3周龄Lewis大鼠的脂肪组织,消化法获得脂肪间充质干细胞,pcDNA 3.1(+)/hTGFD2通过脂质体介导转染脂肪间充质干细胞,用免疫化学染色、逆转录-聚合酶链反应(RT-PCR)和Western blot检测筛选的阳性克隆细胞中hTGFB2基因与软骨特异性蛋白-Ⅱ型胶原和蛋白多糖表达的情况;然后将基因转染的脂肪间充质干细胞与PLGA支架体外构建细胞-载体复合物,再将其植入裸鼠体内,12周后观察基因增强的组织工程软骨的形成情况.结果 从成体大鼠脂肪组织中培养出脂肪问充质干细胞,能大量稳定增殖传代.hTGFB2基因在脂肪间充质干细胞内能瞬时及稳定表达,并促使Ⅱ型胶原和蛋白多糖合成;细胞.载体复合物在裸鼠体内经12周的培养,可以形成形态、结构接近正常软骨的组织工程软骨.结论 PODNA 3.1(+)/hTGFl32成功转染脂肪间充质干细胞,诱导其向软骨细胞分化,脂肪间充质干细胞可作为基因增强的软骨组织工程较理想的种子细胞.     

脂肪间充质干细胞的成骨诱导分化

目的　研究脂肪间充质干细胞 (MSCs)在特定培养条件下向成骨细胞分化 ,探讨其作为骨组织工程的种子细胞的可行性。方法　取 3周龄Lewis大鼠的腹股沟脂肪垫 ,消化法获得脂肪MSCs,用成骨诱导培养基诱导其向成骨细胞分化 ,组织化学染色、免疫细胞化学染色和Westernblotting检测细胞分化的情况。结果　从成体大鼠脂肪组织中培养出脂肪MSCs,能大量稳定增殖传代。在地塞米松、抗坏血酸、β-甘油磷酸钠的诱导下 ,脂肪MSCs的ALP活性增高 ,VonKossa染色出现钙结节 ,OPN、BMP - 2免疫细胞化学染色阳性 ,Westernblotting检测到诱导后细胞OPN、BMP - 2的表达 ,且随诱导时间延长表达增强。结论　从脂肪组织中可获得具有多分化潜能的MSCs,并能在体外稳定增殖传代 ,经诱导后可分化为脂肪细胞和成骨细胞 ,有可能成为骨组织工程较理想的种子细胞之一     

脂肪间充质干细胞的基本生物学特性及向成骨细胞诱导分化的实验研究

目的　研究脂肪间充质干细胞的基本生物学特性以及在特定培养条件下向成骨细胞分化 ,探讨其作为骨组织工程的种子细胞的可行性。方法　取 3周龄Lewis大鼠的腹股沟脂肪垫 ,消化法获得脂肪间充质干细胞 ,分别用脂肪诱导培养基和成骨诱导培养基诱导其向脂肪细胞与成骨细胞分化 ,组织化学染色、免疫细胞化学染色和Westernblot检测细胞分化的情况。结果　从成体大鼠脂肪组织中培养出脂肪间充质干细胞 ,原代脂肪间充质干细胞能自发分化为脂肪细胞 ,传代细胞在胰岛素和呋塞米的作用下生成脂滴 ,过氧化物酶体增殖物激活受体 (PPAR)γ表达增强 ,向脂肪细胞分化 ;在呋塞米、抗坏血酸、β 甘油磷酸钠的诱导下 ,脂肪间充质干细胞的碱性磷酸酶(ALP)活性检测显示诱导组与对照组差异有显著性 (P <0 .0 1) ,vonKossa染色出现钙结节 ,骨桥蛋白 (OPN)、骨形态发生蛋白 (BMP) 2免疫细胞化学染色阳性 ,Westernblot检测到诱导后细胞OPN、BMP2的表达。结论　从脂肪组织中可获得具有多分化潜能的间充质干细胞 ,经诱导后可分化为脂肪细胞和成骨细胞 ,有可能成为骨组织工程较理想的种子细胞之一