骨髓间充质干细胞体外诱导为神经细胞的研究

本实验观察了大鼠MSCs向神经细胞方向的诱导分化情况,以期为MSCs在神经移植领域的临床应用提供理论基础。用含10 ng／ml bFGF+20％FBS的DMEM对MSCs进行预诱导24 h后,以含200μmol／L的BHA+2％DMSO的无血清DMEM对MSCs进行诱导,观察诱导后细胞的光、电镜形态学变化,通过免疫组织化学法对诱导后细胞进行神经细胞表型及神经递质合成酶鉴定。结果显示:MSCs经BHA和DMSO诱导后,80％以上的细胞表现出神经元样形态,胞浆内可见较多Nissl体,并表达nestin、NSE、NF、MAP、SYN,部分诱导后的细胞表达ChAT、TH、GAD;电镜下观察,诱导后细胞核大而圆,核仁明显,胞浆内细胞器发达,可见大量粗面内质网和游离核糖体。提示,MSCs体外可被诱导分化为神经元样细胞,诱导后的细胞有合成某些神经递质的能力并具有发育早期神经元的超微结构特点。     

脐血间充质干细胞静脉移植治疗新生大鼠缺氧缺血性脑损伤的时效性研究

目的探讨脐血间充质干细胞(MSCs)静脉移植治疗新生鼠缺氧缺血性脑损伤(HIBD)的可行性及其时效性。方法脐血MSCs移植使用前以4′,6-二脒基-2-苯吲哚盐酸(DAPI)体外标记。实验选用7日龄SD大鼠38只制备HIBD模型,死亡3只,余35只共分3组:空白对照组(n=11);移植1组(n=12),在HIBD后第2天经鼠尾静脉注入脐血MSCs;移植2组(n=12),在HIBD后1周开始移植。两组均于移植后第2天以及HIBD后2周分别随机将鼠处死、取脑,用于脑组织病理形态学观察,并取海马回区相同部位的缺血脑组织切片,荧光显微镜下观察DAPI阳性细胞数。结果移植2组,1周后缺血脑组织细胞外间隙缩小,细胞数明显增加,脑组织水肿已明显减轻,在大鼠病灶侧脑内,可见大量的DAPI阳性细胞向病灶区及周围迁移和扩散,没有明显的界限。而移植1组于移植后病灶侧脑内很少见到DAPI阳性脐血MSCs分布,其脑组织水肿程度及细胞外间隙的改善和细胞数目的增加也不明显。结论脐血MSCs移植治疗新生大鼠HIBD,能有效透过血脑屏障,在病灶脑组织周围迁移、扩散、整合;移植时间选择HIBD后1周时有良好疗效。移植治疗过程中未见植入反应和其他不良反应。     

Poly I:C primes the suppressive function of human palatine tonsil-derived MSCs against Th17 differentiation by increasing PD-L1 expression

It has been established that mesenchymal stem cells (MSCs) can have a suppressive effect on T cells, yet much remains unknown about the underlying mechanisms that support this effect. The T cell co-stimulatory pathway involving the programmed death-1 (PD-1) receptor and its ligand PD-L1 regulates T cell activation, tolerance, and subsequent immune-mediated tissue damage. In this study, human palatine tonsil-derived MSCs (T-MSCs) constitutively expressed PD-L1 and exhibited a suppressive activity that specifically targeted murine Th17 differentiation. Additionally, polyinosinic–polycytidylic acid (poly I:C), a Toll-like receptor 3 (TLR3) ligand, increased PD-L1 expression on T-MSCs. The elevated PD-L1 levels enhanced the suppressive functions of T-MSCs on Th17 differentiation. Therefore, pre-stimulation of T-MSCs with poly I:C may serve as an effective therapeutic priming step for modulating Th17-dominant immune responses.     

MSCs来源外泌体在创面修复中的研究进展

目的总结近年来 MSCs 来源外泌体（exosomes，EXOs）在创面修复中的研究进展。方法广泛查阅国内外有关 MSCs-EXOs 在创面修复中作用的文献，总结分析 MSCs-EXOs 在创面修复过程中的作用机制及其临床应用前景。结果MSCs-EXOs 在创面愈合过程中可抑制早期的炎性反应，促进血管新生和上皮细胞的增殖与迁移，后期调控胶原合成并抑制瘢痕增生。与 MSCs 相比，MSCs-EXOs 具有高稳定性、易于储存、不易致瘤、无需增殖、便于定量使用等优点，有着广阔的临床应用前景。结论MSCs-EXOs 能够促进创面修复，且有希望发展成为临床上一种促进急性或慢性创面修复的产品。     

Extracellular calcium and CaSR drive osteoinduction in mesenchymal stromal cells

Bone is the main store of calcium and progenitor cells in the body. During the resorption process, the local calcium concentration reaches 8–40 mM, and the surrounding cells are exposed to these fluctuations in calcium. This stimulus is a signal that is detected through the calcium sensing receptor (CaSR), which modulates chemotactic and proliferative G protein-dependent signaling pathways. The objective of the present work is to evaluate the roles of extracellular calcium ([Ca²⁺]_o) and the CaSR in osteoinduction. Rat bone marrow mesenchymal stromal cells (rBMSCs) were stimulated with 10 mM of Ca²⁺. Several experiments were conducted to demonstrate the effect of [Ca²⁺]_o on chemotaxis, proliferation and differentiation on the osteoblastic lineage. It was found that [Ca²⁺]_o induces rBMSCs to migrate and proliferate in a concentration-dependent manner. Real-time polymerase chain reaction and immunofluorescence also revealed that 10 mM Ca²⁺ stimulates overexpression of osteogenic markers in rBMSCs, including alkaline phosphatase (ALP), bone sialoprotein, collagen Ia1 and osteocalcin. Functional assays determining ALP activity and mineralization tests both corroborate the increased expression of these markers in rBMSCs stimulated with Ca²⁺. Moreover, CaSR blockage inhibited the cellular response to stimulation with high concentrations of [Ca²⁺]_o, revealing that the CaSR is a key modulator of these cellular responses.     

三维球体间充质干细胞移植对大鼠缺血再灌注损伤脑组织Nogo-A及NgR表达的影响

目的 探讨胎盘来源间充质干细胞移植对脑缺血再灌注后大鼠脑组织Nogo—A及其受体NgR的mRNA及蛋白表达的影响及意义。方法 实验动物随机分为假手术组(Sham组)及模型组,模型组再分为治疗组(MSCs组)以及溶剂对照组(Vehicle组),应用线栓法制备大鼠大脑中动脉缺血模型,缺血2h后拔除鱼线再灌注,1d后MSCs组注射间充质干细胞,Vehicle组注射等量培养基溶液,移植后1d,3d,7d应用RT-PCR法检测mRNA表达水平;Western blot法检测蛋白表达水平。结果 与Vehicle组相比,MSCs组大鼠7d神经运动功能有明显改善(P<0.05);MSCs组1,3,7dmRNA及蛋白水平均较Vehicle组降低(P<0.05)。结论 胎盘间充质干细胞移植可以改善脑缺血再灌注后神经运动功能,其机制可能与下调脑组织Nogo—A及其受体NgR水平有关。     

Changes of mesenchymal stromal cells mobilization and bone turnover in an experimental bone fracture model in ovariectomized mice

Objective: The aim of this study was to characterize the mesenchymal stromal cells (MSCs) and endothelial progenitor cells (EPCs) mobilization, and bone turnover in osteoporotic fracture healing in ovariectomized mice. Methods: In total, 112 female C57/BL mice were divided into two groups. The first group was sham-operated (SO), and the other group was ovariectomized (OVX). After three weeks, the right femora of the mice were fractured under anesthesia and internally fixed with steel pin. Peripheral blood and bone marrow were was collected for flow cytometry analysis, at 0 hours (h), 12 h, 24 h, 72 h and 168 h after fracture. MSCs and EPCs levels were assessed using cell surface antigens in different combinations (CD44+ CD34-CD45-, and CD34+ KDR+CD45-) by flow cytometry. At 0, 14, 28 and 42 days after fracture, sera were assayed for circulating levels of procollagen type I-N-terminal propeptide (P1NP) and C-terminal telopeptide of type I-collagen (CTX) by ELISA. Femurs were harvested at 2 weeks and 6 weeks after fracture for X-ray radiography, micro-computed tomography (micro-CT) and histology. Results: Our results showed that bone marrow and peripheral blood MSCs numbers of the OVX mice were significantly lower than the SO mice, at 12 h, 24 h and 72 h after fracture. In addition, circulating P1NP and CTX levels of the OVX mice were significantly higher than the SO mice, at 2 and 4 weeks. Conclusion: Results of the present study revealed disorders of bone marrow MSCs mobilization and bone turnover may partially account for the delay of osteoporotic fracture healing.     

Biosynthesis of collagen I,II, RUNX2 and lubricin at different time points of chondrogenic differentiation in a 3D in vitro model of human mesenchymal stem cells derived from adipose tissue

The first aim of the study was to identify the most appropriate time for differentiation of adipose tissue derived mesenchymal stem cells (MSCs) to chondrocytes, through the self-assembly process. For this purpose, the expression of some chondrocyte markers, such as collagen type I, collagen type II, RUNX2 and lubricin was investigated at different times (7, 14, 21 and 28 days) of chondrogenic differentiation of MSCs, by using immunohistochemistry and Western blot analysis. The second aim of the study was to demonstrate that the expression of lubricin, such as the expression of collagen type II, could be a possible biomarker for the detection of chondrocytes well-being and viability in the natural self-assembling constructs, called ‘cell pellets’. Histology (hematoxylin and eosin) and histochemistry (alcian blue staining) methods were used to assess the chondrogenic differentiation of MSCs. The results showed that after 21 days the differentiated chondrocytes, when compared with MSCs cultured without chondrogenic medium (CD44, CD90 and CD105 positive; CD45, CD14 and CD34 negative), were able to produce significant quantities of collagen type I, collagen type II, and lubricin, suggesting hyaline cartilage formation. During the differentiation phase, the cells showed a reduced expression of RUNX2, a protein expressed by osteoblasts. Our studies demonstrated that 21 days is the optimum time for the implantation of chondrocytes differentiated from adipose tissue-derived MSCs. This information could be useful for the future development of cell-based repair therapies for degenerative diseases of articular cartilage.     

MSCs-肝细胞共培养上清对体外培养L02细胞的影响

目的以体外培养L02细胞为实验对象,了解骨髓间充质干细胞(MSCs)与肝细胞共培养上清的生物活性。方法采用两步酶分离法、全骨髓贴壁筛选法分别分离肝细胞、MSCs,按比例直接共培养,收集不同时段的共培养上清,用于人肝细胞系-L02细胞的培养。分别以单独培养的肝细胞上清、MSCs上清为对照,观察共培养上清对L02细胞活力和总蛋白合成的影响;同时观察其对L02细胞损伤模型的AST、LDH及细胞凋亡的影响。结果肝细胞上清、MSCs上清以及共培养上清对L02细胞的活力和增殖均有显著促进作用,表现为L02增殖活跃,总蛋白合成量增加;加入上述细胞上清的酒精损伤L02细胞AST、LDH释放和细胞凋亡显著低于未加细胞上清的对照组,其中共培养上清效果最为显著(P<0.05)。结论共培养上清对L02有明显的刺激增殖和损伤保护作用。     

MAPKs通路在MSCs的增殖及向成骨细胞分化过程中的作用

间充质干细胞（MSCs）是一种多潜能成体干细胞,在体外诱导剂的作用下能向成骨细胞分化。在MSCs向成骨细胞分化过程中,受到MAPKs、BMPs、Notch和Wnt等多种信号通路的调控。其中MAPKs信号通路研究比较深入,近年来研究表明在MAPKs信号通路的五种途径中,ERKs、p38MAPK和JNKs途径参与了成骨细胞增殖和分化的信号转导。现对MAPKs通路与其参与的MSCs增殖和成骨分化过程简要综述。