自体富血小板血浆诱导兔脂肪干细胞成软骨分化的实验研究

[目的]体外分离、培养、鉴定兔脂肪干细胞,探讨富血小板血浆体外诱导脂肪干细胞成软骨分化潜能。[方法]取Ⅰ型胶原酶消化兔脂肪后,贴壁法分离培养脂肪干细胞,取第3代细胞分别予以成脂、成骨诱导,证实其多向分化潜能;同时取第3代细胞予以富血小板血浆诱导,2周后倒置显微镜观察细胞形态,行Ⅱ型胶原免疫荧光细胞化学染色、甲苯胺蓝染色和实时荧光定量PCR检测Ⅱ型胶原和聚集蛋白聚糖的表达。[结果]可以从兔脂肪中培养出脂肪干细胞,成脂、成骨诱导证实其多向分化潜能。经自体富血小板血浆诱导的脂肪干细胞,其Ⅱ型胶原免疫荧光细胞化学染色、甲苯胺蓝染色均为阳性。实时荧光定量PCR检测发现经自体富血小板血浆诱导的兔脂肪干细胞Ⅱ型胶原α1链基因和聚集蛋白聚糖基因表达明显高于对照组未经诱导的兔脂肪干细胞(P<0.01)。[结论]自体富血小板血浆可以有效诱导兔脂肪干细胞表达II型胶原和蛋白聚糖,可以诱导兔脂肪干细胞向软骨细胞方向分化。     

CD34阳性兔真皮干细胞的体外分离培养和鉴定

目的 探讨兔真皮干细胞的体外分离培养和鉴定方法,初步对其生长特性进行研究,并为生物组织工程提供一种新的种子细胞及成熟的分离鉴定方法.方法 以新生新西兰大白兔为研究对象,机械法直接分离得到真皮组织,采用酶消化法获取细胞,利用干细胞贴壁黏附生长的特性获取高克隆细胞群,并进行传代筛选.应用流式细胞技术检测细胞周期,成骨诱导检测其分化潜能,免疫细胞化学法检测细胞表面分子的表达.结果 原代培养的兔真皮细胞经过连续传代培养至第3代,细胞形态均一.流式细胞仪细胞周期分析显示,体外培养3 d时DNA合成前期细胞(G1期)为55.8%,DNA合成期细胞(S期)为34.6%.免疫细胞化学显示:细胞表面波形蛋白(vimentin)、CD34表达阳性,细胞角蛋白19(cytoKeratin19)、Ⅷ因子和巢蛋白(nestin)表达阴性.结论 新生新西兰大白兔真皮组织中存在多能干细胞,该细胞可向成骨细胞分化,具有多向分化潜能.     

兔胚胎关节软骨细胞体外培养的研究

目的 探讨兔胚胎软骨细胞体外培养的生物学特性。方法 对孕4周兔胚胎关节软骨用酶消化法分离培养细胞。观察细胞存活率、贴壁率、生长曲线和组织形态学改变。结果 兔胚胎软骨细胞可从胚胎软骨组织中消化分离出来，经鉴定具有软骨细胞的特性。原代兔胚软骨细胞存活率达97％以上，细胞贴壁率达80％以上，从原代到第4代都有高增殖力，到第8代时增殖力降低。到第12代时几乎丧失细胞增殖。结论 体外培养的胚胎软骨细胞前4代适合于作修复关节软骨缺损的组织工程细胞。     

骨髓间质干细胞体外定向肌样分化的研究

目的 研究兔骨髓间质干细胞经5—氮杂胞苷诱导，在体外定向肌样分化的情况。方法 取兔胫骨骨髓，分离并培养骨髓间质干细胞，用5—氮杂胞苷苦(10μmol/L)定向诱导，分别在培养的第7、14、2l、28天用免疫组织化学的方法检测细胞中的肌球蛋白重链，取未经5—氮杂胞苷诱导正常培养的细胞为对照组。结果 兔骨髓间质干细胞经5—氮杂胞苷诱导，在其培养的第21、28天免疫组织化学染色方法检测细胞中的肌球蛋白重链呈阳性，而在其培养的第7、14天以及对照组，免疫组织化学染色方法检测细胞中的肌球蛋白重链呈阴性。结论 骨髓间质干细胞是骨髓来源的具有多向分化潜能的干细胞，在5—氮杂胞苷的定向诱导下，可以肌样分化。     

胚胎干细胞分化为软骨细胞的研究现状与展望

胚胎干细胞具有多向分化潜能,在组织修复治疗方面具有广阔的应用前景.近年研究表明,胚胎干细胞在细胞因子、激素、微环境等作用下能够分化为软骨细胞.本文对近年来胚胎干细胞向软骨细胞定向分化中的各种因素进行综述.     

人诱导性多潜能干细胞向表皮样干细胞分化的实验研究

目的 探讨人诱导性多潜能干细胞( iPSC)向表皮样干细胞分化的可能性.方法 (1)以经过灭活处理的小鼠胚胎Fb株作为滋养层,将人iPSC株接种于其上,加入胚胎干细胞完全培养液培养,Ⅳ型胶原酶消化法传代,倒置相差显微镜下观察人iPSC形态及生长状况并行碱性磷酸酶(AKP)染色.以胚胎干细胞不完全培养液悬浮培养iPSC,观察拟胚体形成能力.(2)将人iPSC接种于铺有人羊膜的6孔培养板培养,设为诱导组;另于未铺羊膜的6孔培养板上培养iPSC,作为对照组.观察2组iPSC形态,免疫细胞化学染色法检测整合素β1和细胞角蛋白19( CK19)表达.结果 (1)人iPSC在胚胎干细胞完全培养液中呈典型的干细胞克隆状生长,边界清楚,增殖旺盛;AKP染色阳性.iPSC在无滋养层条件下悬浮培养可形成拟胚体.(2)诱导组iPSC经培养4d后形成干细胞克隆,部分细胞整合素β1和CK19表达阳性.对照组细胞大量死亡,未见整合素β1和CK19表达.结论 人iPSC在羊膜诱导下可定向分化为表皮样干细胞,有望成为皮肤组织工程新的种子细胞.     

骨髓间充质干细胞体外定向软骨细胞分化的研究

目的　研究体外培养的兔骨髓间充质干细胞在特定培养条件下定向软骨细胞分化的情况。方法　取兔髂骨骨髓 ,分离培养骨髓间充质干细胞 ,传代后以高糖DMEM无血清特定培养诱导 (转化生长因子 β1 2 0ug L ,地塞米松10 7mmol L ,维生素C 5 0ug L) ,细胞 爬片甲苯胺蓝染色检测蛋白多糖 ,免疫细胞化学染色法检测Ⅱ胶原 ,S 10 0蛋白分泌 ,常规培养的细胞作为对照组。结果　骨髓间充质干细胞经特定培养条件诱导第 14 ,2 1,2 8天甲苯胺蓝染色呈明显异染 ,Ⅱ胶原 ,S 10 0蛋白免疫细胞化学染色阳性 ,第 7天及对照组阴性。结论　骨髓间充质干细胞是骨髓来源的具有多向分化潜能的干细胞 ,在体外培养可定向分化为软骨细胞 ,并能分泌软骨特异性基质。     

体外利用慢病毒介导BMP-2基因诱导兔滑膜间充质干细胞向软骨细胞分化

目的使用慢病毒介导兔BMP-2基因转染兔滑膜间充质于细胞（SMSCs）,检测其安全性,并在体外定向诱导至软骨细胞。方法通过构建包含BMP-2基因的慢病毒载体,转染SMSCs后行安全性分析;各项检测和MicroRNA分析判断转染后的SMSCs是否进入软骨细胞分化谱系。结果贴块法获得的SMSCs在经分离纯化后,表现出间充质干细胞应有的结构和表面标志物,具有多向分化潜能。增殖动力学、核型分析、致瘤型分析等证实被慢病毒转染的SMSCs安全。Westernblot、免疫荧光等证实且能稳定表达BMP-2蛋白。14d后,免疫组化、MicroRNA检测等证实在不需外加外源性BMP-2的体外环境下SMSCs可自发向软骨细胞分化。结论经重组慢病毒载体感染的兔SMSCs足够安全,能稳定表达BMP-2,体外能自发向软骨细胞分化。     

大鼠骨髓间充质干细胞作为滋养层培养小鼠胚胎干细胞

目的:探讨大鼠骨髓间充质干细胞(mesenchymal stemcells,MSCs)作为滋养层体外培养小鼠胚胎干细胞(embryonic stemcells ESCs)的可能。方法:收集BALB/C小鼠3·5d胎龄的囊胚,接种于大鼠骨髓间充质干细胞的滋养层上,培养5~6d后挑取胚胎干细胞集落,胰酶消化传代。观察细胞集落生长情况,通过碱性磷酸酶染色、细胞核型分析对细胞生物学特性进行检测。结果:ES细胞呈集落性生长,碱性磷酸酶组织化学染色阳性,具有正常核型及多向分化潜能。结论:大鼠MSCs可以作为滋养层体外培养小鼠胚胎干细胞,并能保持未分化状态。     

Sox9诱导人脐血干细胞向软骨细胞分化的实验研究

目的 探讨Sox9基因对脐血干细胞向软骨细胞分化的影响。方法 体外分离培养人脐血干细胞,采用脂质体转染Sox9载体质粒,观察转染后细胞形态的变化,免疫组化染色观察collagenⅡ、aggrecan的表达,RT-PCR检测collagenⅠ、collagenⅡ、aggrecan的m RNA水平变化,Western blot检测collagenⅡ的表达变化。结果 Sox9对脐血干细胞形态无明显影响,与传统方法诱导组相比,Sox9转染后可明显促进脐血干细胞向软骨细胞分化。结论 Sox9对脐血干细胞的软骨分化有很强的调控作用,脐血干细胞可作为组织工程软骨一种良好的种子细胞。     

骨骺干细胞复合壳聚糖/纳米羟基磷灰石支架体外培养的实验研究

目的 探讨免疫磁珠分选的骨骺干细胞(PSCs)经诱导后向软骨细胞方向分化的性能,及其与壳聚糖/纳米羟基磷灰石(CS/nHA)支架材料复合培养构建组织工程化软骨的可行性.方法 用显微手术器械剪取新生24 h内的清洁级SD大鼠股骨下端骺板两端的La Croix环处组织,应用免疫磁珠分选系统分离纯化PSCs,免疫荧光染色观察PSCs成纤维细胞生长因子受体-3(FGFR-3)的表达情况.体外培养并诱导PSCs向软骨细胞特性方向分化,免疫细胞化学、甲苯胺兰及番红O染色检测诱导后细胞Ⅱ型胶原及软骨基质的表达情况.以诱导后接种于CS/nHA支架培养的PSCs为实验组,以诱导后未接种支架的细胞为对照组.扫描电镜观察细胞的黏附及形态学改变,MTT法检测细胞增殖情况,阿辛蓝法测定细胞合成的糖胺多糖(GAG)情况.对两组间不同培养时间的吸光度值及GAG进行比较.结果 免疫磁珠分选的PSCs体外培养增殖迅速,免疫荧光染色显示FGFR-3阳性表达.诱导后的PSCsⅡ型胶原免疫细胞化学染色、甲苯胺兰染色及番红O染色均旱阳性.细胞接种CS/nHA支架后1、4 d,实验组与对照组吸光度值比较差异无统计学意义(P>0.05),而7 d实验组与对照组吸光度值比较筹异有统计学意义(t=-2.786,P=0.024).培养14 d后培养液GAG含量为(89.66±6.52)μg/106个细胞,高于对照组[(78.62±4.63)μg/106个细胞)],差异有统计学意义(t=-3.084,P<0.05).结论 免疫磁珠分选的PSCs经诱导后可向软骨细胞功能方向分化,诱导后的PSCs与CS/nHA分层支架复合有望构建组织工程化软骨.     

Evaluation of the ability of natural and synthetic scaffolds in providing an appropriate environment for growth and chondrogenic differentiation of adipose-derived mesenchymal stem cells

Background:

Although progenitor cells have been observed in articular cartilage, this part has a limited ability to repair due to a lack of blood supply. Formerly, tissue engineering was mainly based on collecting chondrocytes from the joint surface, culturing them on resorbable scaffolds such as poly D, L-lactic glycolic acid (PLGA) and then autologous transplantation. In recent times, due to difficulties in collecting chondrocytes, most of the researchers are focused on stem cells for producing these cells. Among the important factors in this approach, is using appropriate scaffolds with good mechanical and biological properties to provide optimal environment for growth and development of stem cells. In this study, we evaluated the potential of fibrin glue, PLGA and alginate scaffolds in providing a suitable environment for growth and chondrogenic differentiation of mesenchymal stem cells (MSCs) in the presence of transforming growth factor-β3.

Materials and Methods:

Fibrin glue, PLGA and alginate scaffolds were prepared and MSCs were isolated from human adipose tissue. Cells were cultured separately on the scaffolds and 2 weeks after differentiation, chondrogenic genes, cell proliferation ability and morphology in each scaffold were evaluated using real time-polymerase chain reaction, MTT chondrogenic assay and histological examination, respectively.

Results:

Proliferation of differentiated adipose tissue derived mesenchymal stem cells (AD-MSCs) to chondrogenic cells in Fibrin glue were significantly higher than in other scaffolds. Also, Fibrin glue caused the highest expression of chondrogenic genes compared to the other scaffolds. Histological examination revealed that the pores of the Fibrin glue scaffolds were filled with cells uniformly distributed.

Conclusion:

According to the results of the study, it can be concluded that natural scaffolds such as fibrin can be used as an appropriate environment for cartilage differentiation.     

重组人骨形成蛋白2诱导脂肪成体干细胞向软骨分化的形态学研究

目的 探索分离培养兔脂肪成体干细胞，经重组人骨形成蛋白2（recombinant human bone morphogenetic protein2，rhBMP-2）定向诱导后，向软骨细胞分化的可能。方法 将4月龄新西兰兔脂肪组织机械分割，通过Ⅰ型胶原酶消化后得到脂肪成体干细胞（adipose-derived adult stem cells，ADASCs），采用微球方法进行培养，在rhBMP-2诱导组、重组人转化生长因子β1（recombinant human transforming growth factor β1，rhTGF-β1）诱导组和rhBMP-2＋rhTGF-β1（共同诱导组）干预6d后，各组诱导液中分别加入维生素C50μg／ml，再干预8d，应用HE、Alcian blue、Von kossa染色和Ⅱ型胶原免疫组织化学染色等组织学方法对诱导后的细胞进行鉴定。结果 连续诱导培养14d后，各诱导组HE染色显示软骨陷窝形成，对照组软骨陷窝形成不明显。Alcian blue染色见细胞基质中富含蛋白多糖，胞外基质分泌较多的Ⅰ型胶原；但Von kossa染色为阴性；对照组均为阴性。结论 在微球方法培养下，通过相关形态学研究表明，rhBMP-2具有诱导脂肪成体干细胞向软骨细胞分化的能力，将为采用ADASCs修复软骨损伤奠定理论基础。     

Intermittent Hydrostatic Pressure Enhances Growth Factor‐Induced Chondroinduction of Human Adipose‐Derived Mesenchymal Stem Cells

Hydrostatic pressure (HP) plays an essential role in regulating function of chondrocytes and chondrogenic differentiation. The objective of this study was to examine effects of intermittent HP on chondrogenic differentiation of human adipose‐derived mesenchymal stem cells (hASCs) in the presence or absence of chemical chondrogenic medium. Cells were isolated from abdominal fat tissue and confirmed for expression of ASC surface proteins and differentiation potential. Passage 3 pellets were treated with chemical (growth factor), mechanical (HP of 5 MPa and 0.5 Hz with duration of 4 h/day for 7 consecutive days), and combined chemical–mechanical stimuli. Using real‐time polymerase chain reaction, the expression of Sox9, collagen II, and aggrecan as three major chondrogenic markers were quantified among three experimental groups and compared to those of stem cells and human cartilage tissue. In comparison to the chemical and mechanical groups, the chemical–mechanical group showed the highest expression for all three chondrogenic genes close to that of cartilage tissue. Results show the beneficial role of intermittent HP on chondrogenic differentiation of hASCs, and that this loading regime in combination with chondrogenic medium can be used in cartilage tissue engineering.     

Effect of Cryopreservation and Cell Passage Number on Cell Preparations Destined for Autologous Chondrocyte Transplantation

Autologous chondrocyte transplantation (ACT) is an effective and safe therapy for repairing articular cartilage defects and requires cell preservation and subculture before transplantation. We compared the effects of cryopreservation and passaging on cell viability, proliferation, and maintenance of the function of chondrocytes and synovium-derived mesenchymal stem cells (MSCs) used as sources for ACT. These cells were isolated from the knee joints of rabbits and were cultured, passaged serially, and divided into 2 groups that were either cryopreserved or not. The morphology, viability, gene expression, and differentiation potential of the 2 groups were compared. Maintenance of the potential to undergo chondrogenic differentiation was determined with the use of a 3-dimensional culture method. Passaging and cryopreservation significantly affected the ability of chondrocytes to maintain their morphology, express chondrogenic genes, and differentiate. In contrast, synovium-derived cells were not affected by passaging and cryopreservation. Our results may serve as the foundation for the application of passaged and cryopreserved chondrocyte or other source cells of MSCs in ACT.     

MicroRNAs在骨髓间充质干细胞成软骨分化中的作用

软骨组织由细胞外基质与分散其间的软骨细胞共同构成，由于缺乏血管?神经和淋巴系统，损伤后自身修复能力差。目前各种促进软骨损伤修复的方法效果都不理想，诱导骨髓间充质干细胞向软骨细胞分化修复软骨损伤已成为当下研究的热点。多种MicroRNA参与并调控骨髓间充质干细胞成软骨分化过程，本文就MicroRNA调控骨髓间充质干细胞成软骨分化及其机制的研究进展做一综述。     

The effect of two- and three-dimensional cell culture on the chondrogenic potential of human adipose-derived mesenchymal stem cells after subcutaneous transplantation with an injectable hydrogel

Articular cartilage is an avascular tissue composed of chondrocytes, a unique cell type responsible for abundant matrix synthesis and maintenance. When damaged, it never heals spontaneously under physiological circumstances. Therefore, the delivery of mesenchymal stem cells using hydrogel has been considered for cartilage repair. This study aims at investigating the influence of in vitro chondrogenic differentiation of human adipose tissue-derived stem cells (hATSCs) on in vivo cartilage formation when associated with a cellulose-based self-setting hydrogel (Si-HPMC). hATSCs were characterized for their proliferation, surface marker expression, and multipotency. The in vitro chondrogenic potential of hATSCs cultured within Si-HPMC in control or chondrogenic medium was evaluated by measuring COL2A1, ACAN, SOX9, and COMP expression by real-time PCR. Alcian blue and type II collagen staining were also performed. To determine whether in vitro chondrogenically differentiated hATSCs may give rise to cartilage in vivo, cells differentiated as a monolayer or in pellets were finally associated with Si-HPMC and implanted subcutaneously into nude mice. Cartilage formation was assessed histologically by alcian blue and type II collagen staining. Our data demonstrate that hATSCs exhibited proliferation and self-renewal. hATSCs also expressed typical stem cell surface markers and were able to differentiate towards the adipogenic, osteogenic, and chondrogenic lineages. Real-time PCR and histological analysis indicated that Si-HPMC enabled chondrogenic differentiation of hATSCs in inductive medium, as demonstrated by increased expression of chondrogenic markers. In addition, histological analysis of implants showed that chondrogenically differentiated hATSCs (monolayers or pellets) have the ability to form cartilaginous tissue, as indicated by the presence of sulphated glycosaminoglycans and type II collagen. This study therefore suggests that an in vitro induction of hATSCs in 2D was sufficient to obtain cartilaginous tissue formation in vivo. Si-HPMC associated with autologous hATSCs could thus be a significant tool for regenerative medicine in the context of cartilage damage.     

Indomethacin induces differential effects on in vitro endochondral ossification depending on the chondrocyte's differentiation stage

Heterotopic ossification (HO) is the abnormal formation of bone in soft tissues and is a frequent complication of hip replacement surgery. Heterotopic ossifications are described to develop via endochondral ossification and standard treatment is administration of indomethacin. It is currently unknown how indomethacin influences heterotopic ossification on a molecular level; therefore, we aimed to determine whether indomethacin might influence heterotopic ossification via impairing the chondrogenic phase of endochondral ossification. Progenitor cell models differentiating in the chondrogenic lineage (ATDC5, primary human bone marrow stem cells and ex vivo periosteal agarose cultures) were treated with increasing concentrations of indomethacin and a decrease in gene‐ and protein expression of chondrogenic and hypertrophic markers (measured by RT‐qPCR and immunoblotting) as well as decreased glycosamino‐glycan content (by alcian blue histochemistry) was observed. Even when hypertrophic differentiation was provoked, the addition of indomethacin resulted in decreased hypertrophic marker expression. Interestingly, when mature chondrocytes were treated with indomethacin, a clear increase in collagen type 2 expression was observed. Similarly, when ATDC5 cells and bone marrow stem cells were pre‐differentiated to obtain a chondrocyte phenotype and indomethacin was added from this time point onward, low concentrations of indomethacin also resulted in increased chondrogenic differentiation. Indomethacin induces differential effects on in vitro endochondral ossification, depending on the chondrocyte's differentiation stage, with complete inhibition of chondrogenic differentiation as the most pronounced action. This observation may provide a rational behind the elusive mode of action of indomethacin in the treatment of heterotopic ossifications. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:847–857, 2017.