In Vitro Chondrogenic Phenotype Differentiation of Bone Marrow-derived Mesenchymal Stem Cells

In order to study the chondrogenic phenotype differentiation of adult sheep bone marrowderived mesenchymal stem cells (MSCs) in a defined medium as potential seed cells for cartilage tissue engineering, MSCs were isolated by density centrifugation with Percoll solution from bone marrow aspirated from sheep iliac crest. The third passage of MSCS were induced with H-DMEM containing TGF-β3 .IGF-I, Dexamethasone and VitC. The shape and ultrastructure of cells were observed, toluidine blue stain for GAG and immunohistochemistry for type II collagen were applied for chondrogenic phenotype identification. After 14 days of induction, MSCs changed from a spindlelike appearance to a polynal shape, a large amount of endoplasmic reticulum, Golgi complex and mitochondria were observed, and the differentiation of MSCs chondrogenic phenotype was verified by positive staining of toluidine blue and immunohistochemistry. MSCs derived from bone marrow can differentiate to chondrogenic phenotype when induced in vitro and can be used as optimal seed cells for cartilage tissue engineering.     

SH-SY5Y human neuroblastoma cell line: in vitro cell model of dopaminergic neurons in Parkinson’s disease

Objective To evaluate the human neuroblastoma SH-SY5Y cell line as an in vitro model of dopaminergic （DAergic） neurons for Parkinson＇s disease （PD） research and to determine the effect of differentiation on this cell model. Date sources The data of this review were selected from the original reports and reviews related to SH-SY5Y cells published in Chinese and foreign journals （Pubmed 1973 to 2009）. Study selection After searching the literature, 60 articles were selected to address this review. Results The SH-SY5Y cell line has become a popular cell model for PD research because this cell line posses many characteristics of DAergic neurons. For example, these cells express tyrosine hydroxylase and dopamine-13-hydroxylase, as well as the dopamine transporter. Moreover, this cell line can be differentiated into a functionally mature neuronal phenotype in the presence of various agents. Upon differentiation, SH-SY5Y cells stop proliferating and a constant cell number is subsequently maintained. However, different differentiating agents induce different neuronal phenotypes and biochemical changes. For example, retinoic acid induces differentiation toward a cholinergic neuronal phenotype and increases the susceptibility of SH-SY5Y cells to neurotoxins and neuroprotective agents, whereas treatment with retinoic acid followed by phorbol ester 12-O-tetradecanoylphorbol-13-acetate results in a DAergic neuronal phenotype and decreases the susceptibility of cells to neurotoxins and neuroprotective agents. Some differentiating agents also alter kinetics of 1-methyl-4-phenyl-pyridinium （MPP~） uptake, making SH-SY5Y cells more similar to primary mesencephalic neurons. Conclusions Differentiated and undifferentiated SH-SY5Y cells have been widely used as a cell model of DAergic neurons for PD research. Some differentiating agents afford SH-SY5Y cells with more potential for studying neurotoxiclty and neuroprotection and are thus more relevant to experimental PD research.     

Stem cells in infantile hemangioma

Background:Infantile hemangioma(IH)is the most common tumor of infancy and the pathogenesis is still unclear.Recent new evidences have been shown that IH arises from stem cells.Data sources:Based on recent original publications from Pub Med,Elsevier and Google Scholar,a large number of articles about pathogenesis and treatment of IH were selected by their titles and abstracts.Results:The hemangioma-derived stem cells expressed stem cell-specific marker CD133 and mesenchymal markers CD29,CD44,and comprised between 0.1%and 1%of the cells in proliferating-phase IH.During the proliferative phase,stem cells differentiated into large amounts of endothelial cells and pericytes;while during the involuting phase,stem cells became less and predominantly differentiated toward adipocytes.Signaling pathways like VEGF/VEGFR,Notch signaling,were found to be related to these processes.Corticosteroids,Rapamycin and propranolol had a significant effect on stem cells by inhibiting the cell growth or differentiation,or participating in maintaining the cell stability.Conclusions:Stem cells derived from hemangioma play an important role in the pathogenesis of IH,and may be important targets of therapy.     

Small interfering RNA-mediated knockdown of Notch1 in lung

Background The immunologic response to allergens mediated by T lymphocytes is an incipient key element in the pathogenesis of asthma, and Thl/Th2 balance is regarded as the core of asthma pathogenesis. Notch is a single-pass transmembrane receptor protein that regulates differentiation, proliferation and apoptosis in a broad range of cells. It is considered that the Notch signal pathway works in every stage of T cell development and differentiation. Whether the pathway of asthma pathogenesis is related to Notch1 remains unknown. This study is aimed to investigate whether the pathway of asthma pathogenesis is related to Notch1 by examining the effect of knockdown of the Notch1 gene by small interfering RNA on T cell differentiation. Methods An OVA-induced asthma mouse model was established. The expression of Notch1 in the tissue and T cells of the lung from asthmatic mice was detected by RT-PCR and Western blotting. The expression of Notch1 and cytokine interleukin （IL）-4 and interferon （IFN）-γ in activated lung T cells was detected by RT-PCR and enzyme-linked immunosorbent assay after blocking Notch1 by small interfering RNA. Results The mRNA and protein expression of Notch1 increased significantly both in the lung tissue and lung T cells of asthmatic mice （both P 〈0.05）. IL-4 decreased and IFN-y increased significantly in active lung T cells after Notch1 was blocked by Notchl-specific small interfering RNA （IL-4： （2.51±0.51） pg/ml vs 0.64±0.27） pg/ml protein; IFN-γ： （21.72±4.24） pg/ml vs （39.79±4.09） pg/ml protein, P 〈0.05）. Conclusion This study demonstrated that the Notch1 signal might play a role in the pathogenesis of asthma by its involvement in Thl/Th2 differentiation.     

Central tendon splitting combined with SutureBridge double-row technique as a surgical treatment for insertional Achilles tendinopathy

Background Surgical treatment of insertional Achilles tendinopathy should be considered when a variety of conservative measures fail. To achieve a satisfactory outcome, thorough debridement of the Achilles tendon is critical, besides excision of the bursitis and the calcaneal exostosis. Central tendon-splitting provides straightforward access to the calcified or degenerative tissue within the Achilles tendon. For Achilles tendon reconstruction if detachment is present, several surgical techniques have been reported. Controversy surrounds the technique can provide maximum security for reattachment of the Achilles tendon. The SutureBrid~e double-row construct, initially used in rotator cuff repair, is Drobablv a aood choice.     

Role of Wnt/β-catenin Signaling Pathway in the Mechanism of Calcification of Aortic Valve简

Aortic valve calcification is a common disease in the elderly, but its cellular and molecular mechanisms are not clear. In order to verify the hypothesis that Wnt/β-catenin signaling pathway is involved in the process of calcification of aortic valve, porcine aortic valve interstitial cells（VICs） were isolated, cultured and stimulated with oxidized low density lipoprotein（ox-LDL） for 48 h to induce the differentiation of VICs into osteoblast-like cells. The key proteins and genes of Wnt/β-catenin signaling pathway, such as glycogen synthase kinase 3β（GSK-3β） and β-catenin, were detected by using Western blotting and real-time polymerase chain reaction（PCR）. The results showed that the VICs managed to differentiate into osteoblast-like cells after the stimulation with ox-LDL and the levels of proteins and genes of GSK-3β and β-catenin were increased significantly in VICs after stimulation for 48 h（P0.05）. It is suggested that Wnt/β-catenin signaling pathway may play a key role in the differentiation of VICs into osteoblast-like cells and make great contribution to aortic valve calcification.     

Rapamycin Modulates the Maturation of Rat Bone Marrow-derived Dendritic Cells

The purpose of the study was to observe the effect of rapamycin （RAPA） on the differentiation and maturation of rat bone marrow-derived dendritic cells （BMDCs） in vitro. BMDCs from Wistar rats were cultured with granulocyte-macrophage colony-stimulating factor plus interleukin-4 in the presence or absence of RAPA （20 ng/mL）, and stimulated with lipopolysaccharide （LPS） for 24 h before cells and supernatants were collected. Surface phenotype of BMDCs was flow-cytometrically detected to determine the expression of maturation markers, MHC class Ⅱ and CD86. Supernatants were analyzed for the production of IL-12 and IFN-γ cytokines by using ELISA. BMDCs were co-cultured with T cells from Lewis rats and mixed lymphocyte reaction was assessed by MTT method. The morphology of BMDCs stimulated with LPS remained immature after RAPA pretreatment. RAPA significantly decreased the CD86 expression, impaired the IL-12 and IFN-γ production of BMDCs stimulated with LPS, and inhibited the proliferation of allogeneic T cells. In conclusion, RAPA can inhibit the maturation of BMDCs stimulated with LPS in terms of the morphology, surface phenotype, cytokine production, and ability of BMDCs to stimulate the proliferation of allogeneic T cells in vitro.     

Effect of Autophagy Over Liver Diseases

Abstract In recent years, increasingly evidences show that autophagy plays an important role in the pathogenesis and development of liver diseases, and the relationship between them has increasingly become a focus of concern. Autophagy refers to the process through which the impaired organelles, misfolded protein, and intruding microorganisms is degraded by lysosomes to maintain stability inside cells. This article states the effect of autophagy on liver diseases (hepatic fibrosis, fatty liver, viral hepatitis, and liver cancer), which aims to provide a new direction for the treatment of liver diseases.     

Study of cultured bovine capsular bag in pure ocular tissue

The proliferation, differentiation and fibrosis of lens epithelia cells （LECs） is mainly responsible for posterior capsular opacification （PCO）. From the primary culture of LECs to the culture of lens capsular bag, the models of posterior capsular opacification have been developed. At present, the most commonly used model is cell culture in medium with serum. But the culture in pure ocular tissue has not been reported. Therefore, we established a new model of posterior capsular opacification-culturing bovine lens capsular bag in pure ocular tissue to exclude the role of serum. Our study established a new culture method to investigate the proliferation, differentiation and apoptosis of lens epithelia cells in the aqueous humor with or without lens cortex and vitreous humor. The purpose of the study is to model posterior capsular opacification in vivo as closely as possible and to discuss the influence of ocular tissue on posterior capsular opacification.     

Millimeter-wave exposure promotes the differentiation of bone marrow stromal cells into cells with a neural phenotype

This study investigated the ability of millimeter-wave （MMW） to promote the differentiation of bone marrow stromal cells （BMSCs） into cells with a neural phenotype. The BMSCs were primarily cultured. At passage 3, the cells were induced by β-mercaptoethanol （BME） in combination with MMW or BME alone. The expressions of nucleostemin （NS） and neuron-specific enolase （NSE） were detected by immunofluorescent staining and Western blotting respectively to identify the differentiation. The untreated BMSCs predominately expressed NS. After induced by BME and MMW, the BMSCs exhibited a dramatic decrease in NS expression and increase in NSE expression. The differentiation rate of the cells treated with BME and MMW in combination was significantly higher than that of the cells treated with BME alone （P〈0.05）. It was concluded that MMW exposure enhanced the inducing effect of BME on the differentiation of BMSCs into cells with a neural phenotype.     

Does erroneous differentiation of tendon-derived stem cells (TDSCs) contribute to the pathogenesis of calcifying tendinopathy?

Calcifying tendinopathy is a common tendon disorder with calcium deposits in the mid-substance presented with chronic activity-related pain, tenderness, local edema and various degrees of incapacitation. Most of current treatments are neither effective nor evidence-based because its underlying pathogenesis is poorly understood and treatment is usually symptomatic. Understanding the pathogenesis of calcifying tendinopathy is essential for its effective evidence-based management. One of the key histopathological features of calcifying tendinopathy is the presence of chondrocyte phenotype which surrounds the calcific deposits, suggesting that the formation of calcific deposits was cell-mediated. Although the origin of cells participating in the formation of chondrocyte phenotype and ossification is still unknown, many evidences have suggested that erroneous tendon cell differentiation is involved in the process. Recent studies have shown the presence of stem cells with self-renewal and multi-differentiation potential in human, horse, mouse and rat tendon tissues. We hypothesized that the erroneous differentiation of tendon-derived stem cells (TDSCs) to chondrocytes or osteoblasts leads to chondrometaplasia and ossification and hence weaker tendon, failed healing and pain, in calcifying tendinopathy. We present a hypothetical model on the pathogenesis and evidences to support this hypothesis. Understanding the key role of TDSCs in the pathogenesis of calcifying tendinopathy and the mechanisms contributing to its erroneous differentiation would provide new opportunities for its management. The re-direction of the differentiation of resident TDSCs to tenogenic or supplementation of MSCs programmed for tenogenic differentiation may be enticing targets for the management of calcifying tendinopathy in the future.     

Effect of growth and differentiation factor 6 on the tenogenic differentiation of bone marrow-derived mesenchymal stem cells

Background Recent studies showed that bone marrow-derived mesenchymal stem cells (BMSCs) had risk of ectopic bone formation.In this study,we aimed to investigate the effect of growth and differentiation factor 6 (GDF-6) on the tenogenic differentiation of BMSCs in vitro,and then combined with small intestine submucous (SIS) to promote tendon regeneration in vivo.Methods The BMSCs were isolated from the green fluorescent protein (GFP) rats,and were characterized by multi-differentiation assays following our previous study protocol.BMSCs cultured with different concentrations of GDF-6,without growth factors served as control.After 2 weeks,mRNA expression and protein expression of tendon specific markers were examined by qRT-PCR and Western blotting to define an optimal concentration of GDF-6.Mann-Whitney U-test was used to compare the difference in relative mRNA expression among all groups; P ≤0.05 was regarded as statistically significant.The GDF-6 treated BMSCs combined with SIS were implanted in nude mice and SD rat acute patellar tendon injury model,the BMSCs combined with SIS served as control.After 12 and 4 weeks in nude mice and tendon injury model,the samples were collected for histology.Results After the BMSCs were treated with different concentration of GDF-6 for 2 weeks,the fold changes of the specific markers (Tenomodulin and Scleraxis) mRNA expression were significantly higher in GDF-6 (20 ng/ml) group (P ≤0.05),which was also confirmed by Western blotting result.The BMSCs became parallel in orientation after GDF-6 (20 ng/ml) treatment,but the BMSCs in control group were randomly oriented.The GDF-6 (20 ng/ml) treated BMSCs were combined with SIS,and were implanted in nude mice for 12 weeks,the histology showed neo-tendon formation.In the SD rat patellar tendon window injury model,the histology also indicated the GDF-6 (20 ng/ml) treated BMSCs combined with SIS could promote tendon regeneration.Conclusions GDF-6 has tenogenic effect on the tenogenic differentiation of BMSCs,and GDF-6 (20 ng/ml) has better tenogenic effect compared to other concentrations.The GDF-6 (20 ng/ml) treated BMSCs combined with SIS can form neo-tendons and promote tendon regeneration.     

骨髓间充质干细胞生物学特性的研究

骨髓间充质干细胞(BM-MSC)是一种来自中胚层发育的早期干细胞。它具有多向分化潜能的特性,在特定的条件下,可以分化为骨细胞、软骨细胞、肌腱细胞和脂肪细胞等。近年来研究发现,BM-MSC不仅具有多向分化和支持造血的功能,而且具有免疫抑制和诱导免疫耐受的作用,因此具有广阔的临床应用潜能。撰文就BM-MSC的分离、表面标志、鉴定及多向分化潜能予以综述。     

超声定位下针刀治疗髌腱腱病的初步研究

目的 探讨超声定位下针刀治疗髌腱腱病的临床价值.方法 超声定位下对18例髌腱腱病患者髌腱内高密度强回声区组织进行针刀治疗,治疗后4周随访.对比治疗前后髌腱内高密度强回声区组织的弹性应变率和11点数字疼痛评分（11-point numerical radng scales,NRS）变化.结果 治疗后高密度强回声区组织弹性应变率和NRS评分均显著低于治疗前（P＜0.01）.结论 超声定位下针刀治疗可降低髌腱内瘢痕组织的硬度,是一种安全有效的治疗方法.     

Central tendon splitting combined with suture bridge double-row technique as a surgical treatment of insertional Achilles tendinopathy

Backgrounds Surgical treatment of insertional Achilles tendinopathy should be considered when a variety of conservative measures failed. In order to achieve a satisfactory outcome, thorough debridement of the Achilles tendon is critical besides excision of the bursitis and the cacalneal exostosis. Central tendon-splitting approach provides a most straightforward access to the calcified or degenerative tissue within the Achilles tendon. For the reconstruction of Achilles tendon if detachment exists, a number of surgical techniques had been reported up to now. It has been controversial about which technique can provide maximum security for the reattachment of the Achilles tendon. Suture bridge double-row construct, initially used in rotator cuff repair, is probably a good choice.     

骨髓间充质干细胞构建组织工程半月板软骨种子细胞

目的使用特定细胞因子刺激诱导分离扩增后的兔骨髓间充质干细胞（Mesenchynml stem cells,MSCs）,在体外试验中使其分化为软骨细胞,为后续构建工程化半月板研究提供种子细胞,探讨兔MSCs重建半月板组织的可行性和有效性。方法用碱性成纤维细胞生长因子（Basic fibroblast growth factor,bFGF）和转化生长因子β1（Transforming growth factor-βl,TGF-β1）协同刺激已分离并经体外传代培养扩增的兔MSCs,通过倒置细胞显微镜观察及免疫组化检测,证明其已经向软骨细胞系分化。结果用bFGF和TGF-β1协同刺激已分离并经体外扩增的兔MSCs后,细胞生长速度增快,免疫组织化学检测提示向软骨细胞系分化。结论兔MSCs可向软骨细胞分化;bFGF和TGF-β1能加快MSCs的体外增殖并促使其向软骨细胞系分化,可为构建工程化半月板提供理想的自体来源种子细胞。     

脑肿瘤干细胞相关研究进展

颅内恶性肿瘤浸润生长、复发快,是当前神经外科治疗的难点。近年来研究表明,肿瘤组织中存在少数"种子细胞"——脑肿瘤干细胞,其不仅可增殖、分化为肿瘤细胞,同时也主导肿瘤的血管生成、侵袭与转移。脑肿瘤干细胞的研究对于阐释脑部肿瘤的发病和发展机制有重要作用。该文就脑肿瘤干细胞的起源、分化、标志物以及治疗等问题予以综述。     

人脂肪间充质干细胞与软骨细胞接触式共培养的实验研究

目的探讨接触式细胞共培养条件下,人软骨细胞对脂肪间充质干细胞的诱导分化效应。方法用4,6-联脒-2-苯基吲哚(PKH26)荧光标记脂肪间充质干细胞,与软骨细胞进行接触式细胞共培养,细胞比例为1:1,单独培养的脂肪间充质干细胞为对照组。共培养2周后,应用流式细胞仪分选荧光标记阳性的脂肪间充质干细胞,提取细胞总RNA,采用Real-Time PCR检测Ⅱ型胶原和蛋白多糖基因的相对表达改变。结果成功分离提取脂肪间充质干细胞;Real-timePCR检测结果显示,共培养组脂肪间充质干细胞Ⅱ型胶原和蛋白多糖基因相对表达较对照组显著增加(P0.05),蛋白多糖上调307倍,Ⅱ型胶原上调584倍。结论与软骨细胞接触式共培养后,人脂肪间充质干细胞能够被诱导分化为软骨细胞。     

组织微环境对肿瘤发生发展的影响

肿瘤微环境与肿瘤发生发展密切相关.最近的研究证实,肿瘤形成可能是干细胞分化异常导致.而微环境是干细胞稳态调节的关键,干细胞微环境的失调在肿瘤发生中扮演了重要角色.而在肿瘤发展过程中,适宜的微环境将促进肿瘤快速增殖,改变这些特异的微环境可以抑制肿瘤的恶性表型.同时.在肿瘤迁移过程中,微环境也起到了重要作用.这些机制的研究有助于加深对肿瘤的认识,为肿瘤的预防和治疗提供新的理论基础.