异鼠李素激活p38信号促进鼠间充质干细胞成骨分化

目的 探究异鼠李素对骨髓间充质干细胞成骨分化的影响.方法 3周龄C57BL/6J小鼠骨髓间充质干细胞在成骨诱导液下诱导成骨分化.细胞计数试剂盒(CCK-8)检测不同浓度的异鼠李素对成骨细胞毒性影响并行t检验以分析最低有效浓度.碱性磷酸酶(ALP)及茜素红染色观察有效浓度的异鼠李素对骨髓间充质干细胞成骨分化及矿化的影响....     

Wnt3 a信号分子对大鼠骨髓间充质干细胞成骨分化 过程中端粒酶活性的影响

目的探索Wnt3a信号分子对体外培养的大鼠骨髓间充质干细胞成骨分化过程中端粒酶活性的影响。方法采用密度梯度离心联合贴壁筛选法分离培养SD大鼠股骨骨髓间充质干细胞。传代后通过形态学和流式细胞学检测细胞表面标志物CD29、CD44、CD45,筛选并鉴定细胞。采用成骨诱导培养基联合不同浓度的wnt3a(5 ng/mL、25 ng/mL、100 ng/mL)诱导大鼠骨髓间充质干细胞分化,以碱性磷酸酶活性检测评价其成骨分化能力。通过TRAP-ELISA端粒酶活性检测法检测在不同浓度的wnt3a干预下诱导骨髓间充质干细胞向成骨细胞分化过程中端粒酶活性的表达。结果骨髓间充质干细胞传至第3代后可获得均一性较高的细胞,流式细胞学检测细胞表面标志物CD29、CD44高表达,CD45低表达。成骨诱导培养基联合不同浓度的wnt3a诱导大鼠BMSCs 7 d后,碱性磷酸酶(Alkaline Phosphatase,ALP)活性检测结果:与普通成骨诱导培养基相比,含有5 ng/mL及25 ng/mL wnt3a的成骨诱导培养基可显著增加其碱性磷酸酶(ALP)活性(P0.05),而含有100 ng/mL wnt3a的成骨诱导培养基则明显抑制其碱性磷酸酶(ALP)活性(P0.05)。与普通成骨诱导培养基相比,联合诱导培养基诱导骨髓间充质干细胞成骨分化过程中,端粒酶活性的改变无明显的统计学差异。结论小剂量(5 ng/mL、25 ng/mL)的wnt3a分子能够促进体外培养的骨髓间充质干细胞成骨分化,而大剂量(100 ng/mL)的wnt3a分子则抑制其成骨分化。骨髓间充质干细胞具有较弱的端粒酶活性,成骨分化过程中其活性逐渐减弱,直至消失;wnt3a信号分子刺激并不能有效激活其端粒酶活性。     

不同组织来源间充质干细胞体外成骨分化能力的比较研究

目的比较成人脂肪间充质干细胞(ASCs)、脐带间充质干细胞(UC-MSCs)和成人骨髓间充质干细胞(BMSCs)的成骨能力,选择优势干细胞种类作为应用于骨组织工程治疗骨缺损的种子细胞。方法采用含10%胎牛血清的DMEM/Ham’s F-12培养液培养3种MSCs。取3种MSCs的P3代,通过CCK8方法检测其增殖能力;通过流式细胞仪进行鉴定;通过碱性磷酸酶(ALP)和茜素红染色检测骨分化蛋白ALP的分泌和矿化钙结节的沉积,并对钙结节进行定量分析;通过实时荧光定量PCR(RT-q PCR)方法检测骨再生相关基因的表达。结果 3种P3代MSCs在3~5d之间增殖均处于对数生长期;流式鉴定3种细胞的表面标志物阳性率:CD44、CD90和CD105均高于97%,阴性率:CD14、CD34和CD45均低于1%;ALP染色结果显示3种MSCs成骨诱导9d时,细胞内均表达ALP,茜素红染色结果显示成骨诱导18d时,均呈现较好的矿化能力,BMSCs和UC-MSCs成骨诱导后形成的钙结节无显著性差异;RT-q PCR结果显示3种MSCs成骨诱导组相比较于对照组,成骨再生相关基因Osterix、ALP、I型胶原(COL1)和骨钙素(OCN)均显著性高表达;3种MSCs成骨诱导9d时,UC-MSCs实验组的COLI基因表达显著性高于BMSCs,成骨诱导18d时,ASCs实验组的Osterix基因表达显著性高于BMSCs。结论 ASCs和UC-MSCs具有一定的成骨矿化能力,有望成为骨组织工程治疗骨缺损的种子细胞。     

大鼠牙髓干细胞与骨髓间充质干细胞分化成骨样细胞能力对比研究

目的：以骨髓间充质干细胞作为参考,讨论牙髓干细胞作为骨组织修复种子细胞的可行性。方法：通过酶消化法获得大鼠牙髓干细胞,离心法获得骨髓间充质干细胞,贴壁培养,通过倒置光学显微镜观察二者形态差异;流式细胞技术鉴定骨髓间充质干细胞的间质细胞表面标志物表达;MMT法检测细胞生长曲线;特定的成骨诱导液诱导干细胞成骨分化,免疫荧光法检测成骨细胞表面标志物表达。结果：分离的大鼠骨髓间充质干细胞与牙髓干细胞形态学以及生长特性具有相似性,且符合间充质干细胞特性,牙髓干细胞第4天进入对数生长期,第7天进入平台期,骨髓间充质干细胞第4天进入对数生长期,第8天进入平台期;经过成骨诱导后二者都具备成骨样细胞分化能力,牙髓干细胞在成骨诱导后的骨钙素表达上与骨髓间充质细胞有着相似的能力;牙本质泌涎蛋白表达阳性。结论：牙髓干细胞具有与骨髓间充质干细胞都具有成骨分化能力,但牙髓干细胞细胞成分相对复杂,增殖能力较强。     

婴幼儿血管瘤来源的间充质干细胞的体外多向分化实验

目的 观察婴幼儿血管瘤来源的间充质干细胞是否具有多向分化潜能.方法 应用贴壁筛选法从增生期血管瘤中分离间充质干细胞.将人骨髓中分离的间充质干细胞与切除的儿童包皮中分离的成纤维细胞作为对照.采用特异的诱导培养基,在体外诱导这3种细胞向脂肪细胞、骨细胞和软骨细胞分化.结果 在合适的诱导条件下,血管瘤来源的间充质干细胞和骨髓来源的间充质干细胞均可发生成脂肪、成骨和成软骨分化,而成纤维细胞没有表现出任何分化现象.结论 应用贴壁筛选法从血管瘤分离的间充质干细胞具有多向分化潜能.     

大鼠骨髓间充质干细胞成骨分化过程中降钙素基因相关肽受体的表达

[目的]探讨大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)向成骨细胞分化过程中降钙素基因相关肽(calcitonin gene-related peptide,CGRP)受体的表达变化。[方法]采用全骨髓法体外分离、培养大鼠骨髓间充质干细胞,分为诱导成骨组与未诱导组,在传代培养的不同时期(1、2、3周),采用免疫细胞化学、Western Blot对骨髓间充质干细胞的成骨诱导分化进行鉴定,采用RT-PCR、Western Blot检测各组细胞CGRP受体mRNA、蛋白的表达情况。[结果]RT-PCR结果显示同一时间点诱导组CGRP受体mRNA表达量高于未诱导组,诱导组CGRP受体mRNA表达以时间依赖性的方式不断增高;Western Blot结果显示同一时间点诱导组CGRP受体的蛋白水平高于未诱导组,诱导组CGRP受体蛋白水平以时间依赖性的方式增高。[结论]本实验从mRNA、蛋白水平证实大鼠骨髓间充质干细胞表达CGRP受体,随着成骨诱导的不断进行,CGRP的表达量随之上升。     

miR-26a促骨再生机制及其应用研究进展

诱导自身骨再生是修复大面积骨缺损的理想手段。miRNAs作为一种转录后水平的调控因子,能够调控一系列靶基因的表达。研究表明,miRNA-26a(miR-26a)能够同时促进与成骨-成血管相关的多个关键生长因子的表达,进而促进间充质干细胞成骨分化和骨再生。阐明miR-26a在干细胞成骨分化中的具体分子机制,有助于开发促进骨组织再生修复的新靶点。本文就目前miR-26a促进间充质干细胞成骨分化的调控机制和应用进展进行综述。     

人瘢痕疙瘩间充质样干细胞向成骨细胞分化过程中Notch信号表达的研究

目的研究人瘢痕疙瘩间充质样干细胞向成骨细胞分化过程中,Notch 1~4受体的表达情况,探讨Notch信号通路是否参与调控人瘢痕疙瘩间充质样干细胞的成骨分化。方法经患者知情同意后,分离培养人瘢痕疙瘩间充质样干细胞,采用流式细胞术检测CD29、CD34、CD44、CD45及CD90的表达;免疫细胞化学法检测Oct 4的表达;体外诱导其向成骨细胞分化,诱导3周后行茜素红染色;于诱导前及诱导后2、3周,采用实时定量-多聚酶链反应(RT-PCR)及免疫蛋白印迹法(western blot),分别检测细胞Notch 1~4受体mRNA及蛋白的表达。结果流式细胞术结果表明,分离培养的细胞高表达间充质干细胞表型CD29(98.76%)、cD44(98.11%)、CD90(97.86%),不表达造血干细胞表型CD34(0.05%)、CD45(0.03%);免疫细胞化学法检测多能干细胞标志物Oct4呈阳性表达;向成骨细胞体外诱导3周后,茜素红染色可见明显的钙盐结节。RT-PCR和western blot结果显示,随着诱导时间的延长,Notch 1~4受体mRNA和蛋白的相对表达量逐渐减少,差异有统计学意义(P0.05)。结论在人瘢痕疙瘩间充质样干细胞向成骨细胞诱导分化过程中,Notch受体信号的表达逐渐减弱,低水平的Notch信号激活可能有利于瘢痕疙瘩间充质样干细胞的成骨分化。     

BMP-2/Smads信号通路促进骨代谢失衡大鼠骨髓间充质干细胞的成骨分化

目的 本研究旨在探讨BMP-2/Smads信号通路对骨代谢失衡大鼠骨髓间充质干细胞(rBMSCs)成骨分化的影响.方法 切除雌性SD大鼠双侧卵巢,建立大鼠骨代谢失衡模型,即骨质疏松(osteoporosis,OP)模型.通过成骨诱导鉴定rBMSCs的成骨分化.采用脂肪诱导和流式细胞术(FCM)检测脂肪分化和rBMSC表...     

DKK1在大鼠骨髓间充质干细胞成骨、成脂分化早期的差异性表达

目的：分析DKK1在大鼠骨髓间充质干细胞成脂、成骨分化早期的表达,探讨其调节机制。方法 Real－time PCR检测骨髓间充质干细胞在成脂、成骨诱导培养早期DKK1基因表达的水平;检测激素处理骨髓间充质干细胞DKK1基因的表达水平。结果成脂组与正常对照组,在成脂诱导6、12、24、48时DKK1基因表达水平较对照组增高,差异有统计学意义, P＜0．01。成骨组与正常对照组,0．5、6两个时间点DKK1基因表达量较对照组明显降低,差异有统计学意义,P＜0．05。骨髓间充质干细胞激素处理3、6、12、24时检测,结果显示激素作用下,骨髓间充质干细胞DKK1表达明显增加,差异有统计学意义,P＜0．05。结论 DKK1在骨髓间充质干细胞成脂、成骨分化早期可能起重要的调节作用,激素可能通过调节DKK1的表达而发挥调节成脂、成骨分化的作用。     

The osteogenic potential of adipose-derived stem cells for the repair of rabbit calvarial defects

INTRODUCTION: Bone replacement is often necessary during reconstruction of craniofacial anomalies or trauma. Adipose-derived stem cells (ASCs) possess osteogenic potential and are a promising cell source for bone tissue engineering. The present study was designed to assess the osteogenic potential and utility of using ASCs to regenerate bone in a rabbit calvarial defect model. METHODS: Rabbit ASCs were seeded on gelatin foam (GF) scaffolds and induced in osteogenic medium containing bone morphogenetic protein (BMP)-2. Thirty-four 8-mm calvarial defects were randomly treated with autograft, no treatment, GF scaffold, GF + ASCs, or GF + osteoinduced ASCs. After 6 weeks, calvaria were harvested and underwent histologic and radiologic analyses to compare healing between the treatment groups. RESULTS: Defects treated with autograft underwent complete healing. Radiologically, there were no significant (P > 0.05) differences in healing among empty defects, and those treated with GF alone or GF plus osteoinduced ASCs. Osteoinduced ASCs exhibited significantly (P < 0.05) greater healing than noninduced ASCs. CONCLUSION: Preimplantation osteoinduction of ASCs enhances their osteogenic capacity. Lack of a significant osteogenic effect of ASCs on calvarial healing at 6 weeks may be secondary to use of noncritical-sized defects. Larger defects would likely demonstrate the osteogenic potential of ASCs more definitively.     

Characterization and multilineage potential of cells derived from isolated microvascular fragments

Background

A number of therapies are being developed that use microvessels isolated from adipose tissue (microvascular fragments [MVFs]) to improve tissue perfusion and implant survival. Because it has been demonstrated that stem cells are associated with microvessels, the purpose of these studies was to gain further insight into the stem cells associated with MVFs to better understand their therapeutic potential.

Materials and methods

Cells derived from MVF explants were compared with adipose-derived stem cells (ASCs) based on the expression of cell surface proteins for mesenchymal stem cells and their capacity for angiogenic, neurogenic, adipogenic, and osteogenic differentiation.

Results

The expression of cell surface proteins for mesenchymal stem cell markers was similar between MVF-derived cells and ASCs; however, the increase in markers consistent with endothelial cells and pericytes was accompanied by an improved ability to form capillary-like networks when cultured on matrigel. MVF-derived cells had increased neuregulin, leptin, and osteopontin expression compared with ASCs when exposed to neurogenic, adipogenic, and osteogenic induction media, respectively.

Conclusions

The stem cell functionality of cells derived from MVFs is retained after their isolation. This helps to explain the ability of MVFs to improve tissue perfusion and has implications for the use of MVFs as a means to deliver stem cells within their niche.     

Comparison of Effects of Mechanical Stretching on Osteogenic Potential of ASCs and BMSCs

Mechanical forces play critical roles in the development and remodeling processes of bone. As an alternative cell source for bone engineering, adipose-derived stem cells (ASCs) should be fully investigated for their responses to mechanical stress. Similarly, the osteogenic potential, stimulated by mechanical stress, should be compared with bone marrow stromal cells (BMSCs), which have been clinically used for bone tissue engineering. In this study, ASCs and BMSCs were osteogenic-induced for 48 hours, and then subjected to uniaxial mechanical stretching for 2 or 6 hours. Cell orientation, osteogenic regulatory genes, osteogenic genes and ALP activities were measured and compared between ASCs and BMSCs. ASCs could align in a perpendicular way to the direction of stretching stress, while BMSCs did not present a specific alignment. Both 2 and 6 hours mechanical stretching could enhance the mRNA expression of Osx and Runx2 in BMSCs and ASCs, while OCN mRNA only increased in ASCs after 6 hours mechanical loading. Mechanical stretching enhanced the BMP-2 mRNA expression in ASCs, while only after 6 hours of mechanical loading significantly increased the BMP-2 gene expression in BMSCs. Significant differences only exist between ASCs and BMSCs loaded at 2 hours of mechanical stretching. It is concluded that ASCs are more rapid responders to mechanical stress, and have greater potential than BMSCs in osteogenesis when stimulated by mechanical stretching, indicating their usefulness for bone study in a rat model.     

Stem cells in burn eschar

This study compares mesenchymal cells isolated from excised burn wound eschar with adipose-derived stem cells (ASCs) and dermal fibroblasts in their ability to conform to the requirements for multipotent mesenchymal stem cells (MSCs). A population of multipotent stem cells in burn eschar could be an interesting resource for tissue engineering approaches to heal burn wounds. Cells from burn eschar, dermis, and adipose tissue were assessed for relevant CD marker profiles using flow cytometry and for their trilineage differentiation ability in adipogenic, osteogenic, and chondrogenic conditions. Although the different cell types did not differ significantly in their CD marker expression, the eschar-derived cells and ASCs readily differentiated into adipocytes, osteoblasts, and chondrocytes, while dermal fibroblasts only exhibited some chondrogenic potential. We conclude that the eschar-derived mesenchymal cells represent a population of multipotent stem cells. The origin of the cells from burn eschar remains unclear, but it is likely they represent a population of adult stem cells mobilized from other parts of the body in response to the burn injury. Their resemblance to ASCs could also be cause for speculation that in deep burns the subcutaneous adipose tissue might be an important stem cell source for the healing wound.     

骨髓间充质干细胞成骨分化的研究与进展

超临界大面积或大段骨缺损仍是临床面临的难题,研究者们致力于研发人工骨材料,但为了解决人工骨材料成骨效应不良的问题,人们越来越关注骨髓间充质干细胞在骨组织工程中的应用。本综述以骨髓间充质干细胞、成骨分化、成骨细胞、临床应用"Bone marrow mesenchymal stem cells,osteogenesis differentiation,osteoblasts cell,clinical application"为检索词,应用计算机搜索CNKI数据库、万方数据库、维普数据库、PUBMED数据库,全面归纳总结骨髓间充质干细胞的分离培养、骨髓间充质干细胞鉴定方法、成骨诱导方法、成骨分化鉴定及其在临床中的应用,为证实其作为种子细胞治疗骨组织疾病提供理论依据。学者们初步研究了骨髓间充质干细胞结合移植、组织工程等技术来治疗骨和软骨缺损、骨关节炎、股骨头坏死等疾病,均取得了较好的临床疗效。但是骨髓间充质干细胞会有一定的优缺点,其临床试验及远期疗效的验证还需进一步深入研究。     

补肾中药对骨髓间充质干细胞成骨分化的作用研究

骨髓间充质干细胞作为骨损伤后,骨修复、重建的种子细胞,越来越成为骨组织工程学中的研究热点。骨修复、重建的关键核心是如何能够提高骨髓间充质干细胞的高效增殖和成骨定向分化。以"肾藏精,主骨,生髓"的中医理论为依据,同时实验和临床也证实,补肾中药可以有效地促进骨髓间充质干细胞增殖、以及定向成骨分化。为骨损伤之后的修复和重建提供新的治疗途径。笔者就近年补肾中药促进骨髓间充质干细胞成骨分化的研究作一综述。     

Stammzellen aus dem Fettgewebe

While stem cells derived from the bone marrow are well-known in clinical medicine, fatty tissue as a source of mesenchymal stem cells is still the subject of recent research. However, adipose-derived stem cells (ASC) are not only harvested less invasively, i.e. via minimally invasive liposuction, but also yield higher numbers of multipotent stem cells. Due to cell-cell interactions and also because of the very favorable secretion profile of growth factors and cytokines ASCs displayed an extraordinary regenerative potential in recent preclinical and clinical applications and achieved a significantly better healing in ischemic muscle, heart, and brain insults and in impaired wound healing. ASCs enhanced regeneration in skeletal tissues such as cartilage or bone. They also revealed immunomodulatory effects and improved the clinical status in immunological diseases. In conclusion ASCs are comparable to bone marrow-derived stem cells concerning possible applications in clinical medicine.     

外泌体非编码RNA调控骨髓间充质干细胞成骨分化的研究进展

骨髓间充质干细胞是一种具有自我更新和多项分化潜能的干细胞,针对其成骨分化作用的研究对骨缺损、骨再生等骨组织工程有重大意义。外泌体内含miRNA、lncRNA、circRNA等非编码RNA,可通过多种途径调控骨髓间充质干细胞的成骨分化,其研究成果可以为促进成骨分化、治疗骨质疏松症等疾病提供新的靶点与方法。本文就外泌体中miRNA、lncRNA以及circRNA对BMSCs成骨分化作用机制的研究进展做一综述。     

真皮间充质干细胞成骨分化的研究进展

骨组织工程技术修复骨组织的缺损显示了广阔的应用前景.真皮间充质干细胞(Dermal mesenchymal stem cells,DMSCs)来源于皮肤组织,易大量获取,对供区损伤极小,具有包括成骨分化在内的多向分化潜能,有望成为骨组织工程研究合适的种子细胞.我们就真皮间充质干细胞成骨分化的研究进展进行综述.