胶原支架在骨髓间充质干细胞治疗中枢神经系统疾病中的应用

骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)已被证明能够改善中枢神经系统疾病的预后,但单独移植的BMSCs很难在损伤部位长期存活并分化为神经细胞。新近研究发现,胶原支架能够促进BMSCs的增殖、存活与分化,进而增强其修复缺损组织、重建神经功能的作用。现就BMSCs治疗中枢神经系统疾病的研究现状、胶原支架的应用和改性方式及其复合BMSCs治疗中枢神经系统疾病的研究进展进行综述。     

血管外膜成纤维细胞诱导BMSCs向平滑肌细胞定向分化

观察大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)与血管外膜成纤维细胞(ad-ventitial fibroblasts,AF)直接接触培养后,BMSCs向血管成分细胞分化的情况.将BMSCs(DAPI标记)与外膜成纤维细胞按一定的比例混合培养7 d,BMSCs单独培养作对照,显微镜下观察细胞形态变化;用免疫荧光染色检测BMSCs的血管平滑肌细胞表型标志物肌动蛋白(SMα-actin)表达的情况;RT-PCR检测SMα-actin mRNA表达的情况.BMSCs与血管外膜成纤维细胞共培养后可见细胞核蓝染的BMSCs与SMα-actin表达阳性(红色)的双标细胞出现,且随培养时间的延长,BMSCs的SMα-actin表达阳性率增高.结果可见:与血管外膜成纤维细胞直接接触有诱导BMSCs向血管平滑肌细胞分化的趋势.     

hUC-MSCs肝样细胞分化机制的研究进展

间充质干细胞(mesenchymal stem cells,MSCs)是来源于发育早期中胚层的一类多能干细胞,广泛分布于全身结缔组织和器官间质中,是一类具有自我更新、不断增殖和多向分化潜能的成体干细胞。随着组织工程和再生医学的研究和发展,MSCs成为一种治疗各种终末期肝脏疾病的潜在治疗手段。该文就人脐带间充质干细胞(human umbilicalcord mesenchymal stem cells,h UCMSCs)的生物学特性、体外诱导分化为肝样细胞(hepatocyte-like cells,HLCs)及其可能分化机制的研究进展予以综述。     

骨髓间充质干细胞在肿瘤发生及治疗作用中的研究进展

骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)主要存在于骨髓中,具有多向分化和增殖的能力。BMSCs与肿瘤关系密切,除了对肿瘤具有趋向性,还可以影响肿瘤细胞的生长增殖、侵袭转移和血管生成等,在肿瘤的发生、发展过程中具有重要调节作用。同时BMSCs在肿瘤基因治疗和免疫治疗中的应用也日趋受到重视,为肿瘤治疗拓展了研究思路。本文主要就BMSCs对肿瘤发生、发展的影响,及其在肿瘤治疗中的应用进行综述。     

新生猪骨髓间充质干细胞衍生细胞的分离、培养及生物学特性分析

利用骨髓间充质干细胞(Bone mesenchymal stem cells,BMSCs)治疗疾病已经逐渐成为现实,但是作为被移植的种子细胞,BMSCs体外传代能力非常有限,种子细胞来源极为贫乏。本研究通过差速贴壁筛选的方法分离出一种猪BMSCs的衍生细胞株,命名为猪骨髓间充质干细胞衍生细胞(Bone mesenchymal stem-derived cells,BMSDCs)。分别对BMSDCs与BMSCs细胞进行细胞生物学特性分析,探讨其体外诱导分化特性,并应用流式细胞术测定细胞表面标记物。结果表明,BMSC和BMSDCs细胞倍增时间分别为31.3 h和30.3 h,平均传代时间分别为3-5 d和2-3 d;两种细胞均阳性表达CD34、CD90,阴性表达CD44、CD45;经体外诱导后均可分化为成脂细胞和成肌细胞。在传代能力上,前者可传代15至20次,后者可长期传代(200次以上)且维持正常染色体特征。研究认为在适宜的实验条件下,体外培养的猪骨髓间充质干细胞的衍生细胞——BMSDCs能够稳定生存增殖并维持BMSCs多向分化潜能,可作为组织工程的理想种子细胞。     

大鼠BMSCs条件培养基对NSCs形态、生长及分化的影响

通过对SD大鼠骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)及新生鼠神经干细胞(neural stem cells,NSCs)进行体外分离、培养及鉴定后,观察BMSCs条件培养基对NSCs向神经细胞分化的影响。采用全贴壁培养法培养BMSCs,并采用流式细胞术鉴定其特异性表面抗原标记。无血清技术培养NSCs,采用免疫荧光技术鉴定其特异性抗原标记。BMSCs条件培养基(含10%胎牛血清的DMEM培养基)对NSCs诱导7 d后,镜下观察细胞形态和生长,并采用免疫荧光技术检测NSCs分化。BMSCs高表达CD90、CD29(90%),而CD45呈阴性表达。免疫荧光染色显示,NSCs标记蛋白Nestin、SOX2为阳性。NSCs经BMSCs培养液诱导分化7 d后经免疫荧光鉴定,MAP-2及GFAP呈阳性,阳性率分别为73.80%和50.47%;NSCs经胎牛血清(fetal bovine serum,FBS)诱导分化7 d后经免疫荧光鉴定,MAP-2及GFAP呈阳性,阳性率分别为42.14%和31.90%。BMSCs条件培养基可诱导NSCs分化为神经元及星形胶质细胞,其中BMSCs分泌的细胞因子在诱导分化中可能发挥重要作用。     

Expression of Pdx-1 in bone marrow mesenchymal stem cells promotes differentiation of islet-like cells in vitro

Bone marrow mesenchymal stem cells(BMSCs) have the ability of self-renewal and multi-directional differentiation.Recent reports showed that BMSCs could differentiate into endocrine cells of pancreas.However,the differentiation is not efficient enough to produce insulin-producing cells for the future therapeutic use.Pdx-1 is a crucial regulator for pancreatic development.Therefore we constructed a eukaryotic expression vector containing Pdx-1 to determine the effect of Pdx-1 ex-pression on differentiation of BMSCs in vitro.The results showed that BMSCs could self-assemble to form functional pancreatic islet-like structures after differentiation in vitro.The proportion of insu-lin-producing cells differentiated from Pdx-1 BMSCs was 28.23%±2.56%,higher than that from BMSCs transfected with vacant vector and Pdx-1-BMSCs(7.23%±1.56% and 4.08%±2.69% respec-tively) by flow cytometry.Immunocytochemical examination also testified the expression of multiple β-cells-specific genes such as insulin,glucagons,somatostatin in differentiated BMSCs.The results also revealed that the expressions of genes mentioned above in Pdx-1 BMSCs were higher than that in Pdx-1-BMSCs,which was confirmed by Western blotting analysis and RT-PCR.Glucose-induced insulin secretion from Pdx-1 BMSCs in 5mmol/L and 25mmol/L glocuse was(56.61±4.82) μU/mL and(115.29±2.56) μU/mL respectively,which were much higher than those from Pdx-1-BMSCs((25.53±6.49) μU/mL and(53.26±7.56) μU/mL respectively) .Grafted animals were able to maintain their body weight and survive for relatively longer periods of time than hyperglycemic sham-grafted controls,which demonstrated an overall beneficial effect of the grafted cells on the health of the animals.These findings thus suggested that exogenous expression of Pdx-1 should provide a promising approach for efficiently producing islet-like cells from BMSCs for the future therapeutic use in diabetic patients.     

诱导骨髓间充质干细胞向心肌细胞分化研究进展

骨髓间充质干细胞(one marrow mesenchymal stem cells, BMSCs)是骨髓内的一类非造血干细胞,具有自我更新和多向分化的潜能。心肌细胞本身不能再生,受损伤的心肌细胞无法通过自身的增殖和分化完成修复。BMSCs在体内外不同的诱导条件下可分化为心肌细胞,应用于缺血性心脏病的治疗,是目前心肌再生治疗的理想种子细胞。本文主要从外界干预诱导、缺氧条件影响、基因转染、临床自体移植方面将近年诱导BMSCs向心肌细胞分化的研究进行综述,以期为今后临床应用BMSCs治疗心肌梗死等心脏疾病提供理论依据。     

大鼠骨髓间充质干细胞培养、鉴定与成胰岛样β细胞诱导

为治疗糖尿病寻找新的胰岛β细胞替代物,该研究对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)进行了体外分离培养、鉴定及成胰岛样β细胞诱导。应用全骨髓离心贴壁培养法分离大鼠BMSCs,进行培养、传代、表面标志物检测,利用DMSO和高糖环境对BMSCs进行胰岛样β细胞诱导。结果显示,大鼠BMSCs体外培养细胞形态呈成纤维细胞样,可以稳定传代;CD13、CD44和CD106表达呈阳性,CD49d呈阴性。在成胰诱导条件下,BMSCs可形成胰岛样圆形细胞团,双硫腙染色呈棕红色,PDX1(pancreatic duodenal homeodomain 1)、CK19(cytokeratin 19)、巢蛋白、胰岛素免疫组化染色均呈阳性;经RT-PCR检测发现,成胰诱导后的BMSCs中表达胰岛素、PDX1和glucagon基因;经q-PCR检测发现,成胰诱导后的BMSCs中胰岛素m RNA水平是大鼠胰腺成体干细胞(pancreas adult stem cells,PASCs)的1.09倍(P0.05);同时,有相应量的胰岛素合成。结果表明,分离得到大鼠BMSCs体外生长稳定,能转分化为胰岛样细胞,该研究结果为糖尿病治疗提供了新的实验依据。     

银杏叶提取物联合骨髓间充质干细胞治疗(Ⅰ型)糖尿病大鼠疗效观察

目的:探讨银杏叶提取物联合骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)治疗(Ⅰ型)糖尿病大鼠的效果.方法:用腹腔内注射链脲酶菌素方法造模Ⅰ型糖尿病wistar大鼠,BMSCs治疗(1× 106/个)(DM)大鼠的同时,给予银杏叶提取物(Ginkgo biloba extract,GBE)治疗20天,同时设立单纯BMSCs治疗组,单纯GBE治疗组,糖尿病模型组以及正常对照组.分时段监测各组大鼠随机血糖、胰岛素、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)以及超氧化物歧化酶(SOD)水平.结果:与其它各组相比,与正常对照组相比DM组血糖水平明显高于GBE组和BMSCs组,BMSCs联合GBE治疗组的大鼠MDA水平明显降低于各治疗组,胰岛素、GSH-Px和SOD水平则明显高于其他对照治疗组.结论:BMSCs联合GBE治疗Ⅰ型DM大鼠,可以明显降低大鼠的血糖和氧化应激水平,其效果好于单独银杏叶提取物治疗或BMSCs治疗.     

骨髓间充质干细胞向肝细胞分化的相关细胞因子及信号通路的研究进展

骨髓干细胞包括造血干细胞（HSCs）和间充质干细胞（MSCs）,骨髓间充质干细胞（BMSCs）是一类具有自我更新、增殖和多向分化能力的细胞,具有不对称分裂和无限增殖的特点。在肝细胞生长因子（HGF）的作用下,BMSCs可以分化为肝细胞,参与诱导这一分化过程的相关信号通路包括NF-kB信号通路、Notch信号通路、MAPK信号通路、Wnt信号通路和STAT3信号通路。文章主要就BMSCs分化为肝细胞的相关信号通路进行了综述。     

骨髓间充质干细胞在组织工程研究中的应用新进展

骨髓间充质干细胞又称为骨髓源性间充质干细胞,是指存在于骨髓基质细胞系统中的一类干细胞,具有高度稳定的体外扩增能力和多向分化潜能等特点。骨髓间充质干细胞因其取材方便,易于分离和培养,以及在适当条件下可诱导分化为皮肤、骨骼、内脏、血液、神经等多种组织细胞的独特优势,目前被广泛应用于药物开发、免疫调节、组织修复、器官重建等多个研究领域。近年来,骨髓间充质干细胞作为种子细胞在组织工程领域有着非常诱人的潜在应用前景。本文就骨髓间充质干细胞在组织工程学研究中应用的最新进展作一综述。     

The comparition of biological characteristics and multilineage differentiation of bone marrow and adipose derived Mesenchymal stem cells

We compared the two sources of adipose and bone marrow-derived mesenchymal stem cells (BMSCs and AMSCs ) in multiple differentiation capacity and biological characteristics to provide a theoretical basis for stem cells transplantation. We isolated bone marrow- and adipose-derived mesenchymal stem cells and compared their phenotype,cell doubling time, the secretion of factors and their ability of multi-differentiation. We also compared their differences in T lymphocyte activation, proliferation and suppression. BMSCs and AMSCs were similar in cell phenotype and the differences existed only in the expression of CD106. On the proliferation rate, AMSCs were faster than BMSCs (doubling time 28 vs. 39?h). In addition, both of these two sources of cells were able to differentiate into bone, fat and cartilage that proved their stem cells properties and the number of stem cell progenitors (CFU-F) from adipose tissue were 10 times larger than those from bone marrow. But AMSCs showed a diminished capacity for suppressing T lymphocyte proliferation and activation compared to BMSCs. Cell origin and abundance were decisive factors in stem cells applications and, in the same volume, with the same premise of AMSCs and BMSCs, adipose tissue is a more promising source of stem cells.     

体外缺血缺氧条件下大鼠骨髓间充质干细胞凋亡发生的机制研究

目的:研究体外大鼠骨髓间充质干细胞(Bone marrow-derived mesenchymal stem cells,BMSCs)在缺血缺氧条件下发生凋亡的作用机制。方法:采取大鼠骨髓,以密度梯度离心分离出单个核细胞(MNCs),于体外培养并由牛垂体提取物(PEX)诱导扩增传代培养出骨髓间充质干细胞(MSCs)。经形态学和流式细胞仪检测MSCs表面标志物鉴定后,骨髓间充质干细胞(BMSCs)在缺血缺氧条件下培养,通过Annexin V/PI双染细胞凋亡检测比较不同组别细胞的凋亡率和蛋白印迹法(western blot)来观察细胞中蛋白的变化。结果:①经形态学观察和流式细胞仪检测MSCs表面标志物鉴定,提示骨髓间充质干细胞培养成功。②对照组(无缺血缺氧)与缺血缺氧组比较,缺血缺氧组的凋亡率显著性增加,而通过磷酸化Akt的表达量显著性增加提示PI3K(Phosphoinosi-tide-3kinase)/Akt(ProteinkinaseB,PKB)信号通路被激活(P<0.05);同时缺血缺氧组与缺血缺氧+PI3K/Akt抑制剂(LY294002)组比较,缺血缺氧+PI3K/Akt抑制剂(LY294002)组的凋亡率显著降低,而通过磷酸化Akt的表达量显著减少提示PI3K/Akt信号通路被抑制(P<0.05)。结论:PI3K/Akt信号通路对体外缺血缺氧条件下培养的骨髓间充质干细胞凋亡发生有关键性作用。     

M-CSF诱导人骨髓间充质干细胞分化为肝样细胞的分子机制

本研究主要目的是明确M-CSF诱导骨髓间充质干细胞分化为肝样细胞的分子机制,为临床中的肝移植和治疗肝病提供新思路。对取自于本院骨科治疗的患者的股骨骨髓间充质干细胞进行提取、分离、传代培养及鉴定。流式细胞仪检测BMSCs的表面表型。为了诱导BMSCs的肝分化,本研究将BMSCs加入到培养基中。骨髓间充质干细胞诱导21 d后,BMSCs表达了肝细胞特异性标志物a-蛋白(AFP)和细胞角蛋白18(CK18),通过免疫荧光染色证实了分化与为分化的BMSCs表达的差异性。分化的BMSCs还显示了肝细胞的体外功能特征,包括白蛋白产生、尿素分泌和糖原储存。本研究结果表明,BMSCs在M-CSF诱导下可分化为功能性肝细胞样细胞,可作为肝病治疗的细胞来源。     

The Methods and Mechanisms to Differentiate Endothelial-Like Cells and Smooth Muscle Cells from Mesenchymal Stem Cells for Vascularization in Vaginal Reconstruction

Endothelial cells and smooth muscle cells (SMCs) are important aspects of vascularization in vaginal reconstruction. Research has confirmed that mesenchymal stem cells could differentiate into endothelial-like cells and SMCs. But the methods were more complicated and the mechanism was unknown. In the current study, we induced the bone mesenchymal stem cells (BMSCs) to differentiate into endothelial-like cells and SMCs in vitro by differentiation medium and investigated the effect of Wnt/β-catenin signaling on the differentiation process of BMSCs. Results showed that the hypoxic environment combined with VEGF and bFGF could induce increased expression of endothelial-like cells markers VEGFR1, VEGFR2, and vWF. The SMCs derived from BMSCs induced by TGF-β1 and PDGF-AB significantly expressed SMC markers SMMHC11 and α-SMA. The data also showed that activation of Wnt/β-catenin signaling could promote the differentiation of BMSCs into endothelial-like cells and SMCs. Thus, we established endothelial-like cells and SMCs in vitro by more simple methods, presented the important role of hypoxic environment on the differentiation of BMSCs into endothelial-like cells, and confirmed that the Wnt/β-catenin signaling pathway has a positive impact on the differentiation of BMSCs into endothelial-like cells and SMCs. This is important for vascular reconstruction.     

Placenta- versus bone-marrow-derived mesenchymal cells for the repair of segmental bone defects in a rabbit model

Tissue-engineered bones (TEBs) constructed with bone-marrow-derived mesenchymal stem cells (BMSCs) seeded on biomaterial scaffolds have achieved good results for bone defect repair in both animal experiments and clinical trials. This has been limited, however, by the source and quantity of BMSCs. We here explored TEBs constructed by placenta-derived mesenchymal stem cells (PMSCs) and compared their effect for the repair of critical-sized segmental osteoperiosteal defects with TEBs constructed with BMSCs. PMSCs were isolated from rabbit placenta by gradient centrifugation and in vitro monolayer culturing, and BMSCs were isolated from the hindlimb bone marrow of newborn rabbit. Primary cultured PMSCs and BMSCs were uniformly in a spindle shape. Immunocytochemistry indicated that both types of cells are positive for CD44 and CD105, and negative for CD34 and CD40L, confirming that they are mesenchymal stem cells. BrdU-labeled PMSCs and BMSCs were respectively co-cultured with bio-derived bone materials to construct TEBs in vitro. Critical-sized segmental osteoperiosteal defects of radii were created in 24 rabbits by surgery. The defects were repaired with TEBs constructed with PMSCs and BMSCs. The results showed that TEBs constructed by both PMSCs and BMSCs could repair the osteoperiosteal defects in a 'multipoint' manner. Measurement of radiography, histology, immunohistochemistry, alkaline phosphatase activity, osteocalcin assaying and biomechanical properties have found no significant difference between the two groups at 2, 4, 8 and 12 weeks after the transplantation (P > 0.05). Taken together, our results indicate that PMSCs have similar biological characteristics and osteogenic capacity to BMSCs and can be used as a new source of seeding cells for TEBs.     

人骨髓间充质干细胞在精原细胞培养条件下生物学特征的观察

目的体外分离培养流产4h、八月龄男胎的骨髓间充质干细胞(BMSCs),并观察BMSC在精原细胞培养条件下的生物学特征,探讨人骨髓间充质干细胞诱导分化为精原细胞的可行性,为BMSCs作为"种子细胞"开辟新的途径。方法分离培养、鉴定人BMSCs,研究其在精原细胞培养条件下诱导培养BMSCs并观察其生物学特征。结果 BMSCs具有间充质细胞的特征;实验组细胞形态发生变化,免疫组化显示Oct-4和C-kit染色阳性;对照组细胞则无。结论人胎儿BMSCs有较强的增殖能力;人胎儿BMSCs在精原细胞培养条件下诱导培养,能表现出精原细胞的一些生物学特征。     

Osteogenic potential: comparison between bone marrow and adipose-derived mesenchymal stem cells

Bone tissue engineering(BTE) is now a promising re-search issue to improve the drawbacks from traditional bone grafting procedure such as limited donor sources and possible complications. Stem cells are one of the major factors in BTE due to the capability of self re-newal and multi-lineage differentiation. Unlike embry-onic stem cells, which are more controversial in ethical problem, adult mesenchymal stem cells are considered to be a more appropriate cell source for BTE. Bone marrow mesenchymal stem cells(BMSCs) are the ear-liest-discovered and well-known stem cell source using in BTE. However, the low stem cell yield requiring long expansion time in vitro, pain and possible morbidities during bone marrow aspiration and poor proliferation and osteogenic ability at old age impede its' clinical ap-plication. Afterwards, a new stem cell source coming from adipose tissue, so-called adipose-derived stemcells(ASCs), is found to be more suitable in clinical ap-plication because of high stem cells yield from lipoaspi-rates, faster cell proliferation and less discomfort and morbidities during harvesting procedure. However, the osteogenic capacity of ASCs is now still debated be-cause most papers described the inferior osteogenesis of ASCs than BMSCs. A better understanding of the osteogenic differences between ASCs and BMSCs is crucial for future selection of cells in clinical application for BTE. In this review, we describe the commonality and difference between BMSCs and ASCs by cell yield, cell surface markers and multiple-differentiation poten-tial. Then we compare the osteogenic capacity in vitro and bone regeneration ability in vivo between BMSCs and ASCs based on the literatures which utilized both BMSCs and ASCs simultaneously in their articles. The outcome indicated both BMSCs and ASCs exhibited the osteogenic ability to a certain extent both in-vitro and in-vivo. However, most in-vitro study papers verified the inferior osteogenesis of ASCs; conversely, in-vivo research reviews revealed more controversies in this issue. We expect the new researchers can have a quick understanding of the progress in this filed and design a more comprehensive research based on this review.     

Comparison of gingiva‐derived and bone marrow mesenchymal stem cells for osteogenesis

Presently, bone marrow is considered as a prime source of mesenchymal stem cells; however, there are some drawbacks and limitations. Compared with other mesenchymal stem cell (MSC) sources, gingiva‐derived mesenchymal stem cells (GMSCs) are abundant and easy to obtain through minimally invasive cell isolation techniques. In this study, MSCs derived from gingiva and bone marrow were isolated and cultured from mice. GMSCs were characterized by osteogenic, adipogenic and chondrogenic differentiation, and flow cytometry. Compared with bone marrow MSCs (BMSCs), the proliferation capacity was judged by CCK‐8 proliferation assay. Osteogenic differentiation was assessed by ALP staining, ALP assay and Alizarin red staining. RT‐qPCR was performed for ALP, OCN, OSX and Runx2. The results indicated that GMSCs showed higher proliferative capacity than BMSCs. GMSCs turned more positive for ALP and formed a more number of mineralized nodules than BMSCs after osteogenic induction. RT‐qPCR revealed that the expression of ALP, OCN, OSX and Runx2 was significantly increased in the GMSCs compared with that in BMSCs. Moreover, it was found that the number of CD90‐positive cells in GMSCs elevated more than that of BMSCs during osteogenic induction. Taking these results together, it was indicated that GMSCs might be a promising source in the future bone tissue engineering.