多孔纯钛表面掺锶羟基磷灰石涂层对骨髓间充质干细胞粘附的影响（英文）

目的:观察电化学沉积的纳米结构掺锶羟基磷灰石涂层表面对大鼠骨髓间充质干细胞早期粘附行为的影响。创新点:首次观察电化学沉积的掺锶羟基磷灰石涂层表面骨髓间充质干细胞的早期粘附行为,并对相关基因进行了检测。方法:纯钛表面经过喷砂和双重酸处理,形成多孔粗糙结构。用电化学方法在其粗糙表面沉积羟基磷灰石涂层(HA)和掺锶羟基磷灰石涂层(Sr-HA)。用贴壁法将4周大鼠股骨骨髓间充质干细胞分离进行原代培养后将细胞接种到多孔纯钛,HA和Sr-HA表面培养1、6和24小时。用场发射扫描电镜(SEM)观察细胞的形貌特点。用异硫氰酸荧光素(FITC)标记的鬼笔环肽进行免疫荧光染色标记细胞骨架,Hoechst 33258进行细胞核染色,激光共聚焦荧光显微镜进行拍照后使用Image J进行细胞的计数和图形分析。用实时荧光定量逆转录聚合酶链式反应(RT-q PCR)测定24、48和72小时不同实验组中骨髓间充质干细胞中FAK、vinculin、integrin β1和integrin β3的基因表达,并进行统计学分析。结论:SEM观察结果显示,骨髓间充质干细胞在三种表面都能正常黏附、生长和增殖。在Sr-HA组表面,细胞粘附、细胞活力和细胞骨架的铺展都较粗糙表面组和HA组有较显著的提高。RT-q PCR结果显示,在各个时间点Sr-HA组表面骨髓间充质干细胞的FAK、vinculin、integrin β1和integrin β3的基因表达与粗糙组有显著性差异,且在24小时后与HA组亦有显著性差异。综上所述,掺锶羟基磷灰石纳米涂层具有较好的生物相容性,可以促进大鼠骨髓间充质干细胞在纯钛表面的早期粘附。     

骨髓间充质干细胞释放miR-1180上调Wnt信号通路活性并促进卵巢癌细胞糖酵解和化疗耐药能力的研究（英文）

目的:已知骨髓间充质干细胞在癌症的发生发展中起有重要作用,本研究分析它们在增强卵巢癌化疗耐药能力中的具体作用。创新点:发现骨髓间充质干细胞可以通过释放微小RNA(micro RNA)影响卵巢癌化疗耐药能力,并确定了介导此作用的micro RNA分子和相关作用机制。方法:收集骨髓间充质干细胞条件培养基,以微阵列方法分析其中microRNA表达谱。针对所获高表达microRNA,分析它(们)对细胞内糖酵解及相关化疗耐药行为的影响。通过生物信息学方法查找所获microRNA的靶基因,分析信号作用机制。纳入59名卵巢癌患者,以Kaplan-Merier生存分析方法考察所获microRNA分子表达程度的临床预后意义。结论:迁移至卵巢癌组织内的骨髓间充质干细胞可释放mi R-1180分子。MiR-1180分子进入癌细胞后,识别并下调SFRP1蛋白(分泌型Wnt受体,起信号抑制作用),由此提高Wnt通路活性。活化后的Wnt信号通路可增强癌细胞内糖酵解水平(即Warburg效应),从而引起糖酵解依赖性化疗耐药行为。     

骨髓间充质干细胞防治心肌细胞凋亡的Bcl-2和Bax蛋白表达研究

论文通过文献资料法、逻辑分析法等对骨髓间充质干细胞作用于心肌细胞凋亡应用,以及心肌细胞凋亡与Bcl-2和Bax蛋白表达的关系进行研究,旨在探讨骨髓间充质干细胞调控Bcl-2、Bax基因蛋白表达的方式对心肌细胞凋亡的作用机制。研究发现,心肌细胞凋亡与Bcl-2、Bax基因蛋白的表达存在高度相关性,通过调控Bcl-2、Bax的基因表达能够在一定程度上防止心肌细胞凋亡的发生与发展,而骨髓间充质干细胞则可以通过自身诱导分化的特性有效的调控Bcl-2和Bax蛋白的表达,使损伤后的心肌功能得到改善。     

组织工程复合支架聚乳酸-羟基乙酸共聚物和羟基磷灰石纳米粒子接种自体骨髓间充质干细胞应用于骨再生（英文）

目的:对应用接枝的羟基磷灰石(g-HA)/聚乳酸-羟基乙酸共聚物(PLGA)纳米复合支架接种自体骨髓间充质干细胞(MSCs)和骨形态发生蛋白2(BMP-2)治疗重症骨缺损的新的治疗策略进行评估,并通过肌肉内移植研究人工骨的组织相容性和移植物在体内的矿化和缺损骨的愈合。创新点:改性的PLGA接种自体MSCs的组织工程骨加速了骨缺损的愈合,使临床重症骨缺损的治疗有了新的手段。方法:应用溶剂浇铸和粒子沥滤方法将PLGA和g-HA制备成复合支架g-HA/PLGA。在g-HA/PLGA支架上接种兔自体MSCs制成组织工程移植物。取宽0.3 cm长2.0 cm的上述移植物埋入兔背部肌肉内,8周后取出移植物,使用扫描式电子显微镜(SEM)检测人工骨的组织相容性(图3a),X射线能量色散谱(EDX)分析钙浓度。然后,用锯锯掉兔前肢桡骨骨干2.0 cm,取同样长度的上述移植物放置于骨缺损处(图4)。术后2、4、8周应用计算机X线摄影(CR)检测骨缺损愈合情况(图5),组织学分析愈合组织结构(图6),SEM检测人工骨与周围组织的相容性(图7),反转录聚合酶链式反应(RT-PCR)检测愈合组织Collagen I、Collagen II和Bmp-2基因的表达。结论:PLGA掺入g-HA主要改善了矿化作用,有益于骨相关基因的表达和骨形成。自体MSCs的应用增强了骨形成和PLGA支架的矿化作用,并加速了支架的吸收。     

大鼠骨髓间充质干细胞的分离、培养及生物学鉴定

目的:探讨成体大鼠的间充质干细胞(MSCs)的分离、体外培养,为其应用提供理论依据.方法:用密度梯度离心结合贴壁培养法分离纯化大鼠骨髓MSCs,传代扩增,对分离的细胞进行碱性磷酸酶的检测.结果:取得较高纯度的成体大鼠骨髓MSCs,并保持细胞的活性;成体大鼠骨髓MSCs在体外培养中为贴壁生长的单个核球形细胞,培养3～4d后开始大量增殖,并形成形态均一的细胞增殖集群,对分离后所获得的细胞进行AKP染色为强阳性.结论:采用密度梯度离心结合贴壁培养法可获得较高纯度的MSCs,是实用、便捷和可行的方法.并且在体外培养条件下能大量增殖,形成形态均一的细胞集落,可以成为进一步进行细胞扩增或其他实际应用的基础.     

体外培养经皮移植自体骨髓间充质干细胞治疗四肢骨不连

目的:探讨体外培养经皮移植自体骨髓间充质干细胞(MSCs)治疗四肢骨折骨不连的效果。方法:12例四肢骨折骨不连患者采用体外培养经皮移植自体MSCs治疗。结果:9例获得了骨性愈合,骨折愈合平均时间为5个月(4~7月),X线显示骨折线消失,骨痂形成。骨折愈合率为75%(9/12)。结论:体外培养经皮移植自体MSCs治疗四肢骨折骨不连创伤小、安全、并发症少,临床效果满意。     

增强型绿色荧光蛋白基因转染CHO细胞研究

目的：探讨增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）在中华仓鼠卵巢细胞（CHO-DHFR）细胞中的作用。方法：将增强型绿色荧光蛋白基因的真核表达载体pcDNA3．1（＋）-EGFP，转染至培养的中华仓鼠卵巢细胞（Chinese Hamster Ovary, CHO-DHFR^- ）中。结果：成功表达并产生绿色荧光。结论：证明EGFP是一种良好的报告基因和筛选标志．为进一步研究应用最广泛的哺乳动物细胞表达系统一CHO细胞表达系统奠定了基础。     

基于芯片数据的神经分化基因网络互作分析为神经系统疾病的细胞疗法提供基础（英文）

目的:通过筛选差异基因,获得控制骨髓间充质干细胞向神经细胞分化及神经发育的中心基因,为治疗神经系统疾病提供参考。方法:从基因表达综合数据库(Gene Expression Omnibus database)中获得芯片数据,利用生物信息学软件筛选差异基因,并对差异基因进行GO功能富集、蛋白互作网络分析和中心基因分析。结论:通过分析,初步推测Nrcam、Sema3a、Mapk8、Dlg4、Slit1、Creb1、Ntrk2、Cntn2和Pax6等中心基因在调控骨髓间充质干细胞向神经细胞的分化中发挥重要作用;Dcx、Nrcam、Sema3a、Cntn2、Slit1、Ephb1和Pax6等中心基因在神经发育过程中发挥作用;Fgf2、Tgfβ1、Vegfa、Serpine1、Il6和Stat1等中心基因在抑制神经分化过程中发挥作用。     

间充质干细胞的研究进展。

间充质干细胞（Mesenchymal stem cells,MSCs）具有低免疫原性、免疫调节作用及多向分化潜能,并且来源充足、可避免伦理学争议的优点,使其有望成为种子细胞,应用于临床干细胞移植治疗．目前通过体内外诱导等方法已能实现MSCs的扩增和定向分化,本文对MSCs的研究进展作一综述,希望对今后MSCs的相关研究与临床应用提供参考．     

骨髓间充质干细胞诱导分化为神经细胞的研究进展

近年来研究认为骨髓中存在骨髓间充质干细胞,具有多项分化潜能,体内实验证明其不但能分化成骨、软骨、脂肪等中胚层起源的细胞,而且能分化为神经元和神经胶质细胞等外胚层起源细胞.本文将对骨髓间充质干细胞诱导分化为神经细胞的研究进展作一综述.     

Osteogenic differentiation of mesenchymal stem cells promoted by overexpression of connective tissue growth factor

Objective: Large segmental bone defect repair remains a clinical and scientific challenge with increasing interest focusing on combining gene transfection with tissue engineering techniques. The aim of this study is to investigate the effect of connective tissue growth factor (CTGF) on the proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells (MSCs). Methods: A CTGF-expressing plasmid (pCTGF) was constructed and transfected into MSCs. Then expressions of bone morphogenesis-related genes, proliferation rate, alkaline phosphatase activity, and mineralization were examined to evaluate the osteogenic potential of the CTGF gene-modified MSCs. Results: Overexpression of CTGF was confirmed in pCTGF-MSCs. pCTGF transfection significantly enhanced the proliferation rates of pCTGF-MSCs (P<0.05). CTGF induced a 7.5-fold increase in cell migration over control (P<0.05). pCTGF transfection enhanced the expression of bone matrix proteins, such as bone sialo-protein, osteocalcin, and collagen type I in MSCs. The levels of alkaline phosphatase (ALP) activities of pCTGF-MSCs at the 1st and 2nd weeks were 4.0- and 3.0-fold higher than those of MSCs cultured in OS-medium, significantly higher than those of mock-MSCs and normal control MSCs (P<0.05). Overexpression of CTGF in MSCs enhanced the capability to form mineralized nodules. Conclusion: Overexpression of CTGF could improve the osteogenic differentiation ability of MSCs, and the CTGF gene-modified MSCs are potential as novel cell resources of bone tissue engineering.     

Human bone marrow-derived mesenchymal stem cells transplanted into damaged rabbit heart to improve heart function

INTRODUCTION Congestive heart failure is the end stage of manycardiovascular diseases. Myocardial infarction (MI)is a life-threatening event that may cause suddencardiac death and heart failure. Despite considerableadvances in diagnosis and treatment of heart disease,cardiac dysfunction after MI is still the majorworldwide cardiovascular disorder. Damaged myo-cardium after acute MI is gradually replaced by fi-brotic noncontractile cells to form scar tissue. Thedeveloping ventricul…     

Optimal time for mesenchymal stem cell transplantation in rats with myocardial infarction

Background:Bone marrow mesenehymal stem cell(MSC)transplantation is a promising strategy in the treatment of myocardial infarction(MI).However,the time for transplanting cells remains controversial.The aim of this study was to find an optimal time point for cell transplantation.Methods:MSCs were isolated and cultured from Sprague-Dawley(SD) rats.MI model was set up in SD rats by permanent ligation of left anterior descending coronary artery.MSCs were directly injected into the infarct berder zone at 1 h,1 week and 2 weeks after MI,respectively.Sham-operated and MI centrel groups received equal volume of phosphate buffered saline(PBS).At 4 weeks after MI,cardiac function Was assessed by echocardiography;vessel density Was analyzed on hematoxylin-eosin stained slides by light microscopy;the apoptosis of cardiomyocytes Was evaluated by terminal deoxynucleotidy1 transferase-mediated dUTP nick end-labeling(TUNEL) assay;the expressions of proteins were analyzed by Western blot.Results:MSC transplantation improved cardiac function.reduced the apoptosis of cardiomyocytes and increased vessel density.These benefits were more obvious in l-week group than in 1-h and 2-week groups.There are more obvious increases in the ratio of bc1-2/bax and the expression of vascular endothelial growth factor(VEGF)and more obvious decreases in the expression of cleaved-caspase-3 in 1-week group than those in other two groups.Conclusion:MSC transplantation was beneficial for the recovery of cardiac function.MSC transplantation at l week post-MI exerted the best effects on increases of cardiac function,anti-apoptosis and angiogenesis.     

Growth factors induce the improved cardiac remodeling in autologous mesenchymal stem cell-implanted failing rat hearts

Therapeutically delivered mesenchymal stem cells (MSCs) improve ventricular remodeling. However,the mechanism underlying MSC cardiac remodeling has not been clearly determined. Congestive heart failure (CHF) was induced in rats by cauterization of the left ventricular free wall. MSCs were cultured from autologous bone marrow and injected into the border zone and the remote myocardium 5 d after injury. Ten weeks later,when compared with sham operation,CHF significantly increased nucleus mitotic index,capilla...     

Ex vivo expansions and transplantations of mouse bone marrow-derived hematopoietic stem/progenitor cells

To examine the effects of co-culture with bone marrow mesenchymal stem cells on expansion of hematopoietic stem/progenitor cells and the capacities of rapid neutrophil engraftment and hematopoietic reconstitution of the expanded cells, we expanded mononuclear cells (MNCs) and CD34⁺/c-kit⁺ cells from mouse bone marrow and transplanted the expanded cells into the irradiated mice. MNCs were isolated from mouse bone marrow and CD34⁺/c-kit⁺ cells were selected from MNCs by using MoFlo Cell Sorter. MNCs and CD34⁺/c-kit⁺ cells were co-cultured with mouse bone marrow-derived mesenchymal stem cells (MSCs) under a two-step expansion. The expanded cells were then transplanted into sublethally irradiated BDF1 mice. Results showed that the co-culture with MSCs resulted in expansions of median total nucleated cells, CD34⁺ cells, GM-CFC and HPP-CFC respectively by 10.8-, 4.8-, 65.9- and 38.8-fold for the mononuclear cell culture, and respectively by 76.1-, 2.9-, 71.7- and 51.8-fold for the CD34⁺/c-kit⁺ cell culture. The expanded cells could rapidly engraft in the sublethally irradiated mice and reconstitute their hematopoiesis. Co-cultures with MSCs in conjunction with two-step expansion increased expansions of total nucleated cells, GM-CFC and HPP-CFC, which led us to conclude MSCs may create favorable environment for expansions of hematopoietic stem/progenitor cells. The availability of increased numbers of expanded cells by the co-culture with MSCs may result in more rapid engraftment of neutrophils following infusion to transplant recipients. Project supported by NIH-Blood, Heart & Lung (National Institute of Health, USA, IR 01 4L70593-01) and Zhejiang Provincial Science Foundation (No. 011103397), China     

Tongxinluo promotes mesenchymal stem cell tube formation in vitro

Objective  

To study whether Tongxinluo (TXL) can induce angiogenesis in bone marrow mesenchymal stem cells (MSCs), and to investigate the underlying mechanism.     

Tissue-engineered composite scaffold of poly(lactide-<Emphasis Type="Italic">co</Emphasis>-glycolide) and hydroxyapatite nanoparticles seeded with autologous mesenchymal stem cells for bone regeneration

Objective

A new therapeutic strategy using nanocomposite scaffolds of grafted hydroxyapatite (g-HA)/poly(lactide-co-glycolide) (PLGA) carried with autologous mesenchymal stem cells (MSCs) and bone morphogenetic protein-2 (BMP-2) was assessed for the therapy of critical bone defects. At the same time, tissue response and in vivo mineralization of tissue-engineered implants were investigated.

Methods

A composite scaffold of PLGA and g-HA was fabricated by the solvent casting and particulate-leaching method. The tissue-engineered implants were prepared by seeding the scaffolds with autologous bone marrow MSCs in vitro. Then, mineralization and osteogenesis were observed by intramuscular implantation, as well as the repair of the critical radius defects in rabbits.

Results

After eight weeks post-surgery, scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) revealed that g-HA/PLGA had a better interface of tissue response and higher mineralization than PLGA. Apatite particles were formed and varied both in macropores and micropores of g-HA/PLGA. Computer radiographs and histological analysis revealed that there were more and more quickly formed new bone formations and better fusion in the bone defect areas of g-HA/PLGA at 2–8 weeks post-surgery. Typical bone synostosis between the implant and bone tissue was found in g-HA/PLGA, while only fibrous tissues formed in PLGA.

Conclusions

The incorporation of g-HA mainly improved mineralization and bone formation compared with PLGA. The application of MSCs can enhance bone formation and mineralization in PLGA scaffolds compared with cell-free scaffolds. Furthermore, it can accelerate the absorption of scaffolds compared with composite scaffolds.