N-cadherin is not essential for limb mesenchymal chondrogenesis.

The cell adhesion molecule N-cadherin is implicated in many morphogenetic processes, including mesenchyme condensation during limb development. To further understand N-cadherin function, we characterized a new N-cadherin allele containing the lacZ reporter gene under the regulation of the mouse N-cadherin promoter. The reporter gene recapitulates the expression pattern of the N-cadherin gene, including expression in heart, neural tube, and somites. In addition, strong expression was observed in areas of active cellular condensation, a prerequisite for chondrogenic differentiation, including the developing mandible, vertebrae, and limbs. Previous studies from our laboratory have shown that limb buds can form in N-cadherin-null embryos expressing a cardiac-specific cadherin transgene, however, these partially rescued embryos do not survive long enough to observe limb development. To overcome the embryonic lethality, we used an organ culture system to examine limb development ex vivo. We demonstrate that N-cadherin-deficient limb buds were capable of mesenchymal condensation and chondrogenesis, resulting in skeletal structures. In contrast to previous studies in chicken using N-cadherin-perturbing antibodies, our organ culture studies with mouse tissue demonstrate that N-cadherin is not essential for limb mesenchymal chondrogenesis. We postulate that another cell adhesion molecule, possibly cadherin-11, is responsible for chondrogenesis in the N-cadherin-deficient limb.     

Analysis of N-cadherin function in limb mesenchymal chondrogenesis in vitro.

During embryonic limb development, cartilage formation is presaged by a crucial mesenchymal cell condensation phase. N-Cadherin, a Ca2+ -dependent cell-cell adhesion molecule, is expressed in embryonic chick limb buds in a spatiotemporal pattern suggestive of its involvement during cellular condensation; functional blocking of N-cadherin homotypic binding, by using a neutralizing monoclonal antibody, results in perturbed chondrogenesis in vitro and in vivo. In high-density micromass cultures of embryonic limb mesenchymal cells, N-cadherin expression level is high during days 1 and 2, coincident with active cellular condensation, and decreases upon overt chondrogenic differentiation from day 3 on. In this study, we have used a transfection approach to evaluate the effects of gain- and loss-of-function expression of N-cadherin constructs on mesenchymal condensation and chondrogenesis in vitro. Chick limb mesenchymal cells were transfected by electroporation with recombinant expression plasmids encoding wild-type or two mutant extracellular/cytoplasmic deletion forms of N-cadherin. Expression of the transfected N-cadherin forms showed a transient profile, being high on days 1-2 of culture, and decreasing by day 3, fortuitously coincident with the temporal profile of endogenous N-cadherin gene expression. Examined by means of peanut agglutinin (PNA) staining for condensing precartilage mesenchymal cells, cultures overexpressing wild-type N-cadherin showed enhanced cellular condensation on culture days 2 and 3, whereas expression of the deletion mutant forms (extracellular/cytoplasmic) of N-cadherin resulted in a decrease in PNA staining, suggesting that a complete N-cadherin protein is required for normal cellular condensation to occur. Subsequent chondrogenesis was also affected. Cultures overexpressing the wild-type N-cadherin protein showed enhanced chondrogenesis, indicated by increased production of cartilage matrix (sulfated proteoglycans, collagen type II, and cartilage proteoglycan link protein), as well as increased cartilage nodule number and size of individual nodules, compared with control cultures and cultures transfected with either of the two mutant N-cadherin constructs. These results demonstrate that complete N-cadherin function, at the levels of both extracellular homotypic binding and cytoplasmic linkage to the cytoskeleton by means of the catenin complex, is required for chondrogenesis by mediating functional mesenchymal cell condensation.     

Caveolin expression during chondrogenesis in the avian limb.

Caveolin is the principal component and critical structural and functional element of caveolae, omega-shaped plasmalemmal invaginations, which have been implicated in a wide range of cellular processes in several different tissues. In the present study, we have investigated both the spatial and temporal expression of caveolin proteins during chondrogenesis in the avian tibiotarsus at days 10-20 of embryonic development. By using semiquantitative Western blotting, we found that caveolin-1 was clearly expressed in developing avian cartilage. The positive expression of caveolin-1 in cartilage showed an upward trend of accumulation temporally, with the highest levels of expression at 20 days of development. By using immunocytochemistry, we detected all three caveolin proteins in the cells of the outer fibrous articular surface, although caveolin-1 demonstrated the strongest and most consistent reactivity. In all cases, however, immunoreactivity appeared to be concentrated in cells facing the articular cavity. In the epiphyseal cartilage, immunocytochemistry revealed that caveolin-1 was present in the majority of chondrocytes within all layers of the cartilage and at all stages examined. A discrete, intense band of caveolin-1 immunoreactivity was apparent within the layer of flattened cells immediately underlying the proliferating rounded chondrocytes and suggests that caveolin-1 might be involved in regulating the progression of cells through these gradually maturing cell layers. In contrast to the results for caveolin-1, in the case of caveolin-2 and -3, chondrocytes were devoid of immunoreactivity in all regions of the epiphyseal cartilage. Overall, this study demonstrates that caveolin-1, -2, and -3 are expressed during chondrogenesis in the developing avian limb, although the patterns of expression are restricted both spatially and temporally throughout the differentiating cell layers of the cartilage. The results suggest that caveolin proteins might play a differentiation-dependent role during avian chondrogenesis.     

Role of N-cadherin in the sorting-out of mesenchymal cells and in the positional identity along the proximodistal axis of the chick limb bud.

Mesenchymal cells from different stages of chick limb buds sort out in monolayer culture, suggesting the presence of different cell affinities dependent on their positions along the proximodistal axis. However, it is still not clear which molecules are responsible for the sorting-out. Here, we propose that N-cadherin, a cell-adhesion molecule, is involved in the sorting-out and is likely to be a component of the mechanism of proximodistal patterning in the developing limb. N-cadherin proteins accumulate in the distal region of the chick limb bud as limb development proceeds. In monolayer culture of distal mesenchymal cells, the stage-dependent levels of N-cadherin proteins are maintained during cell sorting. The results of this study have also demonstrated that an anti-N-cadherin monoclonal antibody, NCD-2, clearly inhibits the cell sorting. Moreover, removal of the apical ectodermal ridge or retinoic-acid treatment of distal cells, which results in a change in the pattern of sorting-out, inhibits the accumulation of N-cadherin proteins, suggesting that the distribution of these proteins is related to the positional identity that gives rise to the different shape and number of cartilage elements along the proximodistal axis.     

Differential effect of hypoxia on human mesenchymal stem cell chondrogenesis and hypertrophy in hyaluronic acid hydrogels

Photocrosslinked hyaluronic acid (HA) hydrogels provide a conducive 3-D environment that supports the chondrogenesis of human mesenchymal stem cells (hMSCs). The HA macromer concentration in the hydrogels has a significant impact on the chondrogenesis of the encapsulated MSCs due to changes in the physical properties of the hydrogels. Meanwhile, hypoxia has been shown to promote MSC chondrogenesis and suppress subsequent hypertrophy. This study investigates the combinatorial effect of tuning HA macromer concentration (1.5–5% w/v) and hypoxia on MSC chondrogenesis and hypertrophy. To decouple the effect of HA concentration from that of crosslinking density, the HA hydrogel crosslinking density was adjusted by varying the extent of the reaction through the light exposure time while keeping the HA concentration constant (5% w/v at 5 or 15 min). It was found that hypoxia had no significant effect on the chondrogenesis and cartilaginous matrix synthesis of hMSCs under all hydrogel conditions. In contrast, the hypoxia-mediated positive or negative regulation of hMSC hypertrophy in HA hydrogels is dependent on the HA concentration but independent of the crosslinking density. Specifically, hypoxia significantly suppressed hMSC hypertrophy and neocartilage calcification in low HA concentration hydrogels, whereas hypoxia substantially enhanced hMSC hypertrophy, leading to elevated tissue calcification in high HA concentration hydrogels irrespective of their crosslinking density. In addition, at a constant high HA concentration, increasing hydrogel crosslinking density promoted hMSC hypertrophy and matrix calcification. To conclude, the findings from this study demonstrate that the effect of hypoxia on hMSC chondrogenesis and hypertrophy is differentially influenced by the encapsulating HA hydrogel properties.     

Induction of mesenchymal stem cell chondrogenesis by polyacrylate substrates

Mesenchymal stem cells (MSCs) can generate chondrocytes in vitro, but typically need to be cultured as aggregates in the presence of transforming growth factor beta (TGF-β), which makes scale-up difficult. Here we investigated if polyacrylate substrates modelled on the functional group composition and distribution of the Arg-Gly-Asp (RGD) integrin-binding site could induce MSCs to undergo chondrogenesis in the absence of exogenous TGF-β. Within a few days of culture on the biomimetic polyacrylates, both mouse and human MSCs, and a mesenchymal-like mouse-kidney-derived stem cell line, began to form multi-layered aggregates and started to express the chondrocyte-specific markers, Sox9, collagen II and aggrecan. Moreover, collagen II tended to be expressed in the centre of the aggregates, similarly to developing limb buds in vivo. Surface analysis of the substrates indicated that those with the highest surface amine content were most effective at promoting MSC chondrogenesis. These results highlight the importance of surface group functionality and the distribution of those groups in the design of substrates to induce MSC chondrogenesis.     

The effect of type II collagen coating of chitosan fibrous scaffolds on mesenchymal stem cell adhesion and chondrogenesis

The biocompatibility of chitosan and its similarity to glycosaminoglycans (GAG) make it attractive for cartilage tissue engineering. We have previously reported improved chondrogenesis but limited cell adhesion on chitosan scaffolds. Our objectives were to produce chitosan scaffolds coated with different densities of type II collagen and to evaluate the effect of this coating on mesenchymal stem cell (MSC) adhesion and chondrogenesis.Chitosan fibrous scaffolds were obtained by a wet spinning method and coated with type II collagen at two different densities. A polyglycolic acid mesh served as a reference group. The scaffolds were characterized by Fourier-transform infrared spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and type II collagen content. Constructs were analyzed after MSCs seeding via live/dead assay, weight and DNA evaluations, SEM, and TEM. Constructs were cultured in chondrogenic medium for 21 days prior to quantitative analysis (weight, DNA, and GAG), SEM, TEM, histology, immunohistochemistry, and quantitative real time polymerase chain reaction. The cell attachment and distribution after seeding correlated with the density of type II collagen. The cell number, the matrix production, and the expression of genes specific for chondrogenesis were improved after culture in collagen coated chitosan constructs.These findings encourage the use of type II collagen for coating chitosan scaffolds to improve MSCs adhesion and chondrogenesis, and confirm the importance of biomimetic scaffolds for tissue engineering.     

The involvement of TGFβ1 in early avian development: gastrulation and chondrogenesis

We examined the effects of transforming growth factor-β1 (TGFβ1) and a neutralizing monoclonal antibody on two phases of early chick embryo development: gastrulation and chondrogenesis. We carried out experiments in vivo and in vitro on mesoderm cells from the gastrulating embryo at day 1, and on sclerotome cells from day 3 embryos, having previously shown that this factor is present among these cells at these stages of development. Addition of the antibody to cultures of these cells produced a dose-dependent decrease in cell outgrowth and spreading and concomitantly reduced fibronectin deposition. In vivo studies of the effects of TGFβ1 on mesoderm during gastrulation were carried out by grafting beads carrying this agent into gastrulating embryos. We used beads of ion-exchange resin as well as hydrolysed polyacrylamide, and found that the grafts produced an accumulation of mesoderm cells around the implant and, at later stages, the formation of enlarged somites. There was no effect on embryonic axis formation. Studies of bromodeoxyuridine (BrdU) incorporation indicated that the mesoderm accumulation was due, at least in part, to an increase in cell proliferation. However, examination of the effect of TGFβ1 on BrdU incorporation by mesoderm during gastrulation and sclerotome cells in vitro indicated in inhibition of cell proliferation, an inconsistency explained in terms of the variation between the in vivo and in vitro conditions. We conclude that TGFβ1 is both appropriately located, and is able, to influence cell proliferation among the mesodermal cell populations during early development, and that this effect contributes to the overall control of mesodermal morphogenesis. Chondrogenesis was studied in vitro using micromass cocultures of sclerotome cells with notochordon a permeable substratum. Under these conditions, the addition of TGFβ1 caused an increase in the deposition of Alcian blue-stainable material, indicating a stimulation of chondrogenesis. We suggest that this result, coupled with the previous demonstration that TGFβ1 is present among the sclerotome cells in the embryo at this time, supports the contention that this factor exerts a regulatory effect on sclerotome cell differentiation.     

Effect of chitosan scaffold microstructure on mesenchymal stem cell chondrogenesis

Although numerous biomaterials have been investigated as scaffolds for cartilage tissue engineering, the effect of their microstructure on final construct characteristics remains unclear. The biocompatibility of chitosan and its similarity with glycosaminoglycans make it attractive as a scaffold for cartilage engineering. Our objective was to evaluate the effect of chitosan scaffold structure on mesenchymal stem cell proliferation and chondrogenesis. Chitosan fibrous scaffolds and chitosan sponges were seeded with mesenchymal stem cells in a chondrogenic medium. Constructs were analyzed 72 h after seeding via scanning electron microscopy (SEM), weight measurements and DNA quantification. Constructs were cultured for 10 or 21 days prior to confocal microscopy, SEM, histology, quantitative analysis (weight, DNA and glycosaminoglycan (GAG)), and quantitative real-time polymerase chain reaction. Mesenchymal stem cells maintained a viability above 90% on all chitosan scaffolds. The cell numbers in the constructs were similar at 72 h, 10 days and 21 days. However, matrix production was improved in chitosan fibrous constructs based on the GAG quantification and collagen II mRNA expression. Chondrogenesis on chitosan scaffolds is superior on microfibers compared to macroporous sponges.     

Enhanced chondrogenesis in co-cultures with articular chondrocytes and mesenchymal stem cells

In this work, articular chondrocytes (ACs) and mesenchymal stem cells (MSCs) with 1:1 and 1:3 cell ratios were co-cultured in order to evaluate if a majority of primary ACs can be replaced with MSCs without detrimental effects on in vitro chondrogenesis. We further used a xenogeneic culture model to study if such co-cultures can result in redifferentiation of passaged ACs. Cells were cultured in porous scaffolds for four weeks and their cellularity, cartilage-like matrix formation and chondrogenic gene expression levels (collagen I and II, aggrecan) were measured. Constructs with primary bovine ACs had ~1.6 and 5.5 times higher final DNA and glycosaminoglycan contents, respectively, in comparison to those with culture expanded chondrocytes or MSCs harvested from the same animals. Equally robust chondrogenesis was also observed in co-cultures, even when up to 75% of primary ACs were initially replaced with MSCs. Furthermore, species-specific RT-PCR analysis indicated a gradual loss of MSCs in bovine-rabbit co-cultures. Finally, co-cultures using primary and culture expanded ACs resulted in similar outcomes. We conclude that the most promising cell source for cartilage engineering was the co-cultures, as the trophic effect of MSCs may highly increase the chondrogenic potential of ACs thus diminishing the problems with primary chondrocyte harvest and expansion.     

Estimation of creatine phosphokinase and hydroxyproline in the developing limb: its use in evaluating the effect of teratogens on myogenesis and chondrogenesis

Forelimbs of mouse fetuses were examined for tissue-specific, drug-induced alterations in their biochemical composition. The activity of the enzyme creatine phospholinase (CPK; to estimate myogenesis) and the content of hydroxyproline (HP; to estimate chondrogenesis) were compared in homogenates of control and treated mouse-fetus forelimbs on day 14 of gestation. In addition, the content of DNA, RNA, and protein was also measured. Injection of 6-aminonicotinamide (6-AN) (15 mg/kg) on day 10 resulted in an overall growth retardation in day 14 fetuses and all biochemical parameters tested were reduced. The ratio of PH:CPK was unaffected by 6-AN treatment. Retinoic acid (vitamin A acid; 100 mg/kg), administered to pregnant female mice on day 10, produced severe forelimb defects and resulted in a signific 10 resulted in an overall growth retardation in day 14 fetuses and all biochemical parameters tested were reduced. The ratio of PH:CPK was unaffected by 6-AN treatment. Retinoic acid (vitamin A acid; 100 mg/kg), administered to pregnant female mice on day 10, produced severe forelimb defects and resulted in a signific 10 resulted in an overall growth retardation in day 14 fetuses and all biochemical parameters tested were reduced. The ratio of PH:CPK was unaffected by 6-AN treatment. Retinoic acid (vitamin A acid; 100 mg/kg), administered to pregnant female mice on day 10, produced severe forelimb defects and resulted in a significant reduction in day 14 forelimb HP and RNA content, without altering CPK, DNA, or protein; thus, the HP:CPK ratio was decreased. These results indicated that 1) 6-AN nonspecifically retards growth and cyto-differentiation in limbs; 2) retinoic acid inhibits synthesis of collagen and RNA; 3) retinoic acid has a differential effect upon chondrogenic and myogenic tissues of the limb, and 4) the comparison of HP content and CPK activity in tissue homogenates is an acceptable method of evaluating teratogenic compounds for selective effects.     

线粒体自噬与骨髓间充质干细胞成软骨分化的关联

背景:已有研究发现线粒体自噬在软骨缺损修复过程中发挥重要作用,因此有必要研究线粒体自噬在骨髓间充质干细胞成软骨分化过程中的作用及其调控方法,为进一步阐明软骨诱导机制并为理解生物材料如何调控骨髓间充质干细胞分化命运奠定基础。目的:建立并验证线粒体状态和自噬程度的表征方法,观察线粒体自噬与软骨细胞发育、骨髓间充质干细胞成软骨分化的关系。方法:分离培养新生乳兔、1月龄兔、18月龄兔软骨细胞和新生乳兔骨髓间充质干细胞,按照试剂盒说明方法进行线粒体标记Mito Tracker Red染色。选取第2代骨髓间充质干细胞,用完全软骨诱导培养基培养,在培养第1,3,7天按照试剂盒说明方法进行线粒体标记Mito Tracker Red染色。选取第2代骨髓间充质干细胞,用完全软骨诱导培养基培养24 h后,诱导组加入10μmol/L自噬诱导剂雷帕霉素干预10 h,抑制组加入5μmol/L自噬抑制剂氯喹干预10 h,此后换用完全软骨培养基培养,在培养第1,3,7天用线粒体自噬试剂盒Mitophagy Detection Kit染色并观察线粒体自噬情况。结果与结论:①不同兔龄软骨细胞线粒体染色:低兔龄软骨细胞中线粒体数目较高兔龄软骨细胞多;②骨髓间充质干细胞和幼兔软骨细胞线粒体染色:软骨细胞的线粒体形态呈点状,而骨髓间充质干细胞的线粒体形态呈线状;③骨髓间充质干细胞分化过程中线粒体染色:随诱导培养天数的增加,骨髓间充质干细胞中线粒体的形态由最初的网状变为点状,骨髓间充质干细胞骨架中促进自噬形成的微丝结构也逐渐减少;④线粒体自噬对成软骨分化的影响:雷帕霉素促进了线粒体自噬途径,进而促进骨髓间充质干细胞的成软骨分化。     

间质标志物巢蛋白在鼻咽癌中的表达及其临床意义

目的检测巢蛋白(N-cadherin)在鼻咽癌(nasopharyngeal carcinoma,NPC)组织中的表达及与患者临床病理特征的关系。方法采用免疫组化SP法检测122例NPC组织中N-cadherin的表达水平,以30例鼻咽慢性炎作为正常对照。结果 N-cadherin蛋白在NPC组织中的高表达阳性率为59.0%(72/122),明显高于其在慢性炎组织的表达(3.33%,1/30)(P<0.001)。N-cadherin蛋白高表达与NPC患者性别、N分期、临床分期以及复发密切相关;然而,N-cadherin蛋白表达与患者年龄、组织学类型、T分期、M分期无相关性(P>0.05)。此外,N-cadherin蛋白高表达与E-cadherin低表达呈明显负相关(rs=-0.198,P=0.029)。单因素分析显示,N-cadherin蛋白高表达组患者总体生存期明显低于低表达组(P=0.006)。多因素分析显示,N-cadherin蛋白表达不是影响患者预后的独立因素(P=0.296)。结论 N-cadherin在NPC中异常表达升高,且与淋巴结转移、临床分期以及复发密切相关,可作为判断NPC恶性生物学行为的指标。     

Lacrimo-auriculo-dento-digital syndrome: evidence for lower limb involvement and severe congenital renal anomalies.

We describe a 3-generation family with lacrimo-auriculo-dento-digital syndrome (LADD). In addition to the well described abnormalities of ears, teeth, lacrimal apparatus and digits, the patients exhibit several previously undescribed anomalies, including minor facial anomalies (broad forehead, telecanthus, bulbous nasal tip, full jaw, ptosis and flared nostrils), involvement of the first and second toes, and congenital renal disease causing death in the neonatal period in 2 cases.     

Enhanced chondrogenesis of mesenchymal stem cells in collagen mimetic peptide-mediated microenvironment

A new type of synthetic hydrogel scaffold that mimics certain aspects of structure and function of natural extracellular matrix (ECM) has been developed. We previously reported the conjugation of collagen mimetic peptide (CMP) to poly(ethylene oxide) diacrylate (PEODA) to create a polymer-peptide hybrid scaffold for a suitable cell microenvironment. In this study, we showed that the CMP-mediated microenvironment enhances the chondrogenic differentiation of mesenchymal stem cells (MSCs). MSCs were harvested and photo-encapsulated in CMP-conjugated PEODA (CMP/PEODA). After 3 weeks, the histological and biochemical analysis of the CMP/PEODA gel revealed twice as much glycosaminoglycan and collagen contents as in control PEODA hydrogels. Moreover, MSCs cultured in CMP/PEODA hydrogel exhibited a lower level of hypertrophic markers, core binding factor alpha 1, and type X collagen than MSCs in PEODA hydrogel as revealed by gene expression and immunohistochemisty. These results indicate that CMP/PEODA hydrogel provides a favorable microenvironment for encapsulated MSCs and regulates their downstream chondrogenic differentiation.     

Ultrastructural localization of cholinesterase uring chondrogenesis and myogenesis in the chick limb bud

Summary Cholinesterase (ChE) is transiently expressed in undifferentiated embryonic cells. In the chick limb bud ChE-activity was found in the apical ectodermal ridge and in the subridge mesenchyme. The reaction was localized in the perinuclear cisterna, in an extensive network of narrow profiles of endoplasmic reticulum (ER), and in the Golgi complexThe chondroblasts emerging from the subridge mesenenyme, also showed strong ChE-activity. During differentiation the enzyme first disappeared from the Golgi zone. Then, the narrow ChE-positive ER was successively replaced by ChE-negative extended rough ER characteristic for the differentiated chondrocyte.The myoblasts showed weak ChE-activity with the same ultrastructural localization as in other mesenchymal cells. After fusion the myotubes exhibited strong ChE-activity in the perinuclear cisterna and the developing sarcoplasmic reticulum. In later stages of myogenesis the myoblasts were closely attached to the myotubes and had lost their ChE-activity.During mitosis of ChE-positive cells, ChE-activity was retained in fragments of perinuclear cisterna and ER. In ChE-active mesenchymal cells and chondroblasts we observed specialized contact zones between ER and plasma membrane. ChE-active cisternae of ER run parallel to the plasma membrane with a gap of approximately 10–15 nm. We discuss a possible function of a cholinergic system during morphogenesis.     

软骨细胞上清液诱导滑膜间充质干细胞微团培养成软骨

背景：滑膜间充质干细胞在体外具有多向分化的能力,有望成为软骨组织工程中治疗软骨缺损的种子细胞,在其向软骨细胞分化过程中,合适的生长因子起了重要作用。 目的：利用富含生长因子的软骨细胞上清液诱导滑膜间充质干细胞向软骨细胞分化,并对其鉴定。 方法：采用消化法分别获得SD大鼠滑膜间充质干细胞、软骨细胞。收集软骨细胞上清液离心、过滤冻存备用。培养滑膜间充质干细胞至第3代后离心成微团,并用软骨细胞上清液进行成软骨诱导分化,通过形态学观察、免疫组织化学法、RT-PCR检测进行鉴定。 结果与结论：滑膜间充质干细胞使用软骨细胞上清液成软骨诱导21 d后,微团可见似软骨样组织。免疫组化法进行Ⅱ型胶原鉴定,基质能被Ⅱ型胶原染色,细胞染色呈现棕黄色。RT-PCR结果显示诱导后的微团表达软骨特异性基因Ⅱ型胶原和蛋白聚糖。证实软骨细胞分泌的可溶性因子可以诱导大鼠滑膜间充质干细胞向软骨方向分化。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;组织工程全文链接：     

神经型钙黏蛋白对小鼠脂肪间充质干细胞的成神经作用

目的:探讨神经型钙黏蛋白(N-cadherin)在小鼠脂肪间充质干细胞(ADMSCs)成神经分化中的作用。方法;用差速培养法获取小鼠ADMSCs,传至第5代,经成神经诱导液诱导ADMSCs 7 d后,免疫荧光检测胶质纤维酸性蛋白(GFAP)、神经元特异性烯醇化酶(NSE)和微管相关蛋白2(MAP2)的表达,半定量PCR检测细胞中N-cadherin mRNA的表达;采用N-cadherin对细胞进行基因修饰,免疫荧光检测神经丝蛋白(NF)和GFAP的表达。结果:ADMSCs经成神经诱导7 d后表达GFAP、NSE和MAP2,半定量PCR结果显示,与对照组相比成神经诱导后的细胞表达N-cadherin显著增高。转染N-cadherin的N-cadherin细胞培养24 h后发出细长的突起,与相邻细胞之间形成网状连接。免疫荧光结果显示,转染后的细胞NF和GFAP表达阳性。结论:ADMSCs在体外多种作用下,具有向神经细胞分化的潜能。N-cadherin转染后能改变ADMSCs的形态,并且在ADMSCs向神经分化中发挥一定的作用。