基因修复关节软骨缺损的可行性研究:外源性Ad-TGF-β_1转染修饰兔骨髓间充质干细胞

目的利用 Ad5/CMV－ TGF－β 1转染体外培养兔骨髓间充质干细胞,测定 TGF－β 1的表达产物为关节软骨缺损的基因治疗提供实验基础。方法采用体外培养法对兔骨髓间充质干细胞进行原代培养,应用已构建好的 Ad5/CMV－ TGF－β 1真核载体对其进行转染,并用免疫组织化学方法对转染细胞进行表达产物测定。结果采用腺病毒转染技术能够成功的实现骨髓间充质干细胞的外源性基因修饰,没有发生明细的细胞毒性反应; Ad5/CMV－ TGF－β 1对骨髓间充质干细胞的转染率为 95％左右;转染 8周后仍能观察到 TGF－β 1的表达。结论采用真核表达载体 Ad5/CMV－ TGF－β 1可以成功转染体外培养的骨髓间充质干细胞并可获得 TGF－β 1的高表达。     

骨髓间充质干细胞的基因转染

1人血管内皮生长因子165基因转染兔骨髓间充质干细胞的蛋白表达,2011年15卷10期1769页。2体外培养条件下人骨髓间充质干细胞碱性成纤维细胞生长因子基因的转染与鉴定,见2010年14卷19期3507页。3碱性成纤维细胞生长因子基因转染兔骨髓间充质干细胞,见2009年13卷49期9687页。     

转化生长因子β1转染骨髓间充质干细胞向软骨细胞的分化

目的:通过转化生长因子β1基因转染大鼠的骨髓间充质干细胞,观察目的基因的分泌情况及干细胞向软骨细胞分化效果,为构建新型的软骨组织工程学种子细胞提供新思路。方法:实验于2005-10/2006-09在中国医科大学细胞生物实验室完成。实验材料:6周龄Wistar大鼠10只,质粒pcDNA3.1-TGF-β1(由吉林大学徐莘香教授惠赠),转化生长因子β1、Ⅱ型胶原小鼠抗大鼠单克隆抗体(Neomaker公司)。实验分组:分为未经转染的骨髓间充质干细胞(未转染组)、转染空载的骨髓间充质干细胞(转染空载体组)和转染转化生长因子β1的骨髓间充质干细胞(转染转化生长因子β1组)。实验方法:①密度梯度离心法和贴壁分离法相结合分离、纯化大鼠骨髓间充质干细胞,免疫荧光法检测细胞表面标志。②质粒pcDNA3.1-TGF-β1扩增、提取、鉴定。③转化生长因子β1基因转染骨髓间充质干细胞。实验评估:①四甲基偶氮唑盐法测定筛选后细胞的增殖活性。②Westernblot检测转染后细胞转化生长因子β1和Ⅱ型胶原蛋白表达。结果:①免疫荧光法检测骨髓间充质干细胞:CD29和CD44表达阳性,CD34和CD45表达阴性。②pcDNA3.1-TGF-β1真核表达载体的酶切鉴定:基因测序与GeneBank cDNA序列相符。③转染基因对骨髓间充质干细胞增殖活性的影响:基因转染后骨髓间充质干细胞的增殖力变强,转染组的细胞增殖力高于未转染组和转染空载体组。④转化生长因子β1表达:转染后的骨髓间充质干细胞较未转染和转染空载体的骨髓间充质干细胞分泌转化生长因子β1蛋白增加。⑤Ⅱ型胶原的蛋白表达:在转化生长因子β1目的基因转染后的骨髓间充质干细胞Ⅱ型胶原蛋白表达较未染和转染空载体的骨髓间充质干细胞增强。结论:应用基因转染技术将转化生长因子β1目的基因导入骨髓间充质干细胞中,在蛋白质水平鉴定转染成功,且骨髓间充质干细胞成软骨分化所需的特异性细胞外基质Ⅱ型胶原明显升高,使骨髓间充质干细胞在保持很强的体外传代增殖能力同时,又能产生、表达转化生长因子β1。     

骨髓间充质干细胞的基因转染

1人血管内皮生长因子165基因转染兔骨髓间充质干细胞的蛋白表达,2011年15卷10期1769页。2体外培养条件下人骨髓间充质干细胞碱性成纤维细胞生长因子基因的转染与鉴定,见2010年14卷19期3507页。3碱性成纤维细胞生长因子基因转染兔骨髓间充质干细胞,见2009年13卷49期9687页。     

自体骨髓间充质干细胞修复兔关节软骨缺损的实验研究

目的通过对受损关节软骨进行未经诱导的兔自体骨髓间质干细胞移植,探讨组织工程移植修复兔关节软骨缺损的效果。方法取青紫蓝兔股骨骨髓分离间充质干细胞进行培养,采用变性处理及透明质酸盐修饰后的吸收性明胶海绵为载体,实验组用骨髓间充质干细胞修复自体股骨髁关节软骨缺损,对照组进行单纯吸收性明胶海绵移植。结果移植24周后,肉眼下实验组移植物与正常软骨组织难以区分,软骨表面光滑,对照组移植物为白色的疏松组织。光镜下修复组织类似周围正常软骨组织结构,甲苯胺蓝异染与正常软骨组织无区别,为透明软骨组织。对照组24周关节软骨缺损区被纤维软骨样组织修复。结论自体骨髓间质干细胞移植于受损关节软骨处,可促进关节缺损的修复,恢复软骨组织的结构功能。     

ECSOD体外转染恒河猴骨髓间充质干细胞的实验研究

本研究旨在探讨载带超氧化物歧化酶基因的腺病毒（AD-ECSOD）感染恒河猴骨髓间充质干细胞（R-BMMSC）生物学特性的影响。用含有ECSOD和报告基因EGFP的腺病毒感染经梯度密度离心和贴壁培养法分离、培养并纯化的R-BMMSC,用倒置荧光显微镜及流式细胞仪观察检测感染效率,ELISA法检测细胞培养上清中的ECSOD蛋白表达水平;流式细胞仪检测感染后R-BMMSC的表面抗原（CD34,CD29,CD45,CD90,mA-DR）;油红0和茜素红染色观察R-BMMSC的成骨及成脂诱导分化能力;MTT法检测AD-ECSOD转染对R-BMMSC增殖能力的影响。结果表明,不同滴度AD-ECSOD（感染滴度为每个细胞500,1000,1500和2000pfu）对R-BMMSC的感染效率均〉95％。转染后24h即可在细胞上清液中检测到ECSOD蛋白表达,且明显高于对照组（P〈0．01）,转染后48h蛋白表达量最高。AD-ECSOD转染后R-BMMSC的增殖能力及其表面抗原表达和多向分化能力与未转染组比较无统计荤差异fP〉0．05、．结论：AD-ECSOD能够高效转染R-BMMSC．对萁生物荤特性无明显影响．     

自体骨髓间充质干细胞修复兔关节软骨缺损的实验研究

目的 通过对受损关节软骨进行未经诱导的兔自体骨髓间质干细胞移植，探讨组织工程移植修复兔关节软骨缺损的效果。方法 取青紫蓝兔股骨骨髓分离间充质干细胞进行培养，采用变性处理及透明质酸盐修饰后的吸收性明胶海绵为载体，实验组用骨髓间充质干细胞修复自体股骨保关节软骨缺损。对照组进行单纯吸收性明胶海绵移植。结果 移植24周后，肉眼下实验组移植物与正常软骨组织难以区分，软骨表面光滑，对照组移植物为白色的疏松组织。光镜下修复组织类似周围正常软骨组织结构，甲苯胺蓝异染与正常软骨组织无区别，为透明软骨组织。对照组24周关节软骨缺损区被纤维软骨样组织修复。结论 自体骨髓间质干细胞移植于受损关节软骨处，可促进关节缺损的修复，恢复软骨组织的结构功能。     

软骨源形态发生蛋白基因转染自体骨髓间充质细胞修复关节软骨缺损

背景:以支架或干细胞在一定程度上能够修复软骨缺损,但是研究中发现较大块的缺损修复效果还达不到令人满意的程度.基因转染后的干细胞能够不断的释放生长因子有可能够为软骨缺损研究带来突破.目的:进一步验证软骨源形态发生蛋白转染的骨髓间充质干细胞是否会促进体内兔软骨修复.方法:从兔骨髓内分离获得骨髓间充质干细胞,脂质体感染的方法将软骨源形态发生蛋白基因转染到骨髓间充质干细胞中.实验组移植转染后的细胞;单纯骨髓间充质干细胞组移植未转染的骨髓间充质干细胞;空白对照组缺损区不移植细胞.术后行组织学检测及组织学评分分析修复情况.结果与结论:体内修复过程中,软骨源形态发生蛋白转染的骨髓间充质干细胞促进了软骨再生.透明软骨填充了软骨缺损区,并且深部区域显示了软骨下成骨.透明软骨的重建区域优于单纯骨髓间充质干细胞移植组.组织学评分实验组高于骨髓间充质干细胞对照组和空白组.提示转染软骨源形态发生蛋白基因的骨髓间充质干细胞能够促进和提高关节软骨的改建和修复.     

兔骨髓间充质干细胞经Ad-TGFβ1重组腺病毒载体转染定向分化为软骨细胞

目的:通过软骨组织工程与基因治疗相结合的技术,利用腺病毒载体将转化生长因子β1基因导入兔骨髓间充质干细胞,使其诱导靶细胞定向分化为软骨细胞。方法:实验于2004-03/2006-03在徐州医学院完成。①实验方法:兔麻醉后自胫骨上端抽取骨髓,体外分离培养骨髓间充质干细胞。取第2代细胞,以1×104/cm2接种,达70%融合时分为3组:Ad-TGFβ1转染组采用H-DMEM无血清培养基,加入刚解冻的Ad-TGFβ1液30μL,地塞米松100nmol/L,维生素C50mg/L;Ad-GFP对照组采用H-DMEM无血清培养基,加入Ad-GFP腺病毒,地塞米松100nmol/L,维生素C50mg/L;空白对照组采用H-DMEM完全培养基,不外加任何试剂或药品。②实验评估:倒置显微镜逐日观察细胞生长情况。分别于转染后7,14,21,28d检测细胞内蛋白多糖、转化生长因子β1、Ⅱ型胶原的表达。结果:①细胞形态学观察:Ad-TGFβ1转染后7d,集落中的细胞呈放射状向周围扩展,均一性好,呈长梭形,紧密排列似漩涡状,生长速度明显增快,细胞密度明显增高。②转染后细胞内蛋白多糖的检测:Ad-TGFβ1转染后14d胞浆呈紫蓝色,异染性明显,胞核清晰,可见核仁。转染后28d细胞明显老化,但胞浆中仍可见紫蓝色异染。其余两组均未见明显的异染性。③转染后转化生长因子β1的表达:Ad-TGFβ1转染组细胞中转化生长因子β1呈强阳性表达,其余两组几乎检测不到转化生长因子β1的表达。④转染后细胞内Ⅱ型胶原的表达:Ad-TGFβ1转染后7d胞浆中Ⅱ型胶原呈弱阳性表达,转染后14,21d呈强阳性表达,转染后28d表达有所降低。其余两组几乎不表达Ⅱ型胶原。结论:经Ad-TGFβ1重组腺病毒载体转染,兔骨髓间充质干细胞可定向分化为软骨细胞,是获得软骨组织工程种子细胞的方法之一。     

Cell transplantation therapy for the treatment of articular cartilage defect

Articular cartilage destruction is a major problem in rheumatoid arthritis patients. However, there is no treatment that is widely accepted to regeneratively repair the lesion. When the joint cartilage is destructed progressively and activity of daily life is worsened, joint arthroplasty is the most common treatment to relieve joint pain though it has limited survivorship and sometimes severe complications. Recently in order to repair cartilage defect, new method with cell transplantation; autologous cultured chondrocyte transplantation has been put into clinical practice. And we also have reported autologous cultured-expanded bone marrow mesenchymal cell transplantation. These methods have possibility to repair cartilage defect with good quality histologically, biochemically and biomechanically.     

Targeted RNases: a feasibility study for use in HIV gene therapy.

A targeted RNase would be ideal for gene therapy of several acquired and inherited disorders. Such an RNase may be engineered to contain a ribonucleolytic domain and a specific target RNA binding domain. To demonstrate the feasibility of this approach, an RNase targeted against human immunodeficiency virus (HIV) RNA--Tev-RNase T1--was designed and tested for its use in HIV-1 gene therapy. A human CD4+ T lymphoid (MT4) cell line and human peripheral blood lymphocytes (PBLs) were transduced with retroviral vectors lacking or expressing the tevT1 gene. Expression of enzymatically functional Tev-RNase T1 protein and its lack of toxicity was demonstrated in stable MT4 transductants. Compared with control cells lacking this protein, both transduced MT4 cells and PBLs expressing Tev-RNase T1 delayed HIV-1 replication. Tev-RNase T1 was shown to act after integration, since HIV-1 proviral DNA could be detected, but the amount of HIV-1 RNA produced in MT4 cells and PBLs was significantly decreased. This study demonstrates the feasibility of a targeted RNase strategy for therapeutic use.     

Chitosan composite scaffolds for articular cartilage defect repair: a review

Articular cartilage (AC) defects lack the ability to self-repair due to their avascular nature and the declined mitotic ability of mature chondrocytes. To date, cartilage tissue engineering using implanted scaffolds containing cells or growth factors is the most promising defect repair method. Scaffolds for cartilage tissue engineering have been comprehensively researched. As a promising scaffold biomaterial for AC defect repair, the properties of chitosan are summarized in this review. Strategies to composite chitosan with other materials, such as polymers (including collagen, gelatin, alginate, silk fibroin, poly-caprolactone, and poly-lactic acid) and bioceramics (including calcium phosphate, calcium polyphosphate, and hydroxyapatite) are presented. Methods to manufacture three-dimensional porous structures to support cell attachment and nutriment exchange have also been included.

Properties of chitosan/polymer and chitosan/bioceramic composite scaffolds for articular cartilage defect repair are reviewed.     

关节软骨缺损修复对功能重建的意义

关节软骨缺损的治疗目前方法众多,其软骨的生物治疗有:软骨膜和骨膜移植、自体软骨细胞种植、自体或异体关节软骨移植,这些技术的现状及存在的问题和进展对缺损修复后功能重建具有重要意义。     

骨形态发生蛋白基因治疗骨缺损

目的:总结骨形态发生蛋白在骨组织工程基因治疗骨缺损中的进展。资料来源:应用计算机检索PubMed数据库1990-01/2007-01相关骨形态发生蛋白基因治疗骨缺损方面的文献,检索词"BMP,tissue engineering,gene therapy,bone defect",限定文献语言种类为English。资料选择:选取包括骨形态发生蛋白基因治疗骨缺损的文献,阅读全部文章的文题和大部分文章的摘要。纳入标准:文章所述涉及骨形态发生蛋白及其基因治疗骨缺损。排除标准:重复性研究和Meta分析类文章。资料提炼:共检索到99篇关于骨形态发生蛋白及基因治疗骨缺损的文献,最终纳入30篇符合标准的文献。资料综合:骨组织工程是组织工程学的重要组成部分,它是指利用组织工程学进行骨组织缺损的修复或重建,包括种子细胞、特定的细胞因子和载体。骨形态发生蛋白是一种疏水性酸性蛋白质,在临床实践中,已证实其能促进骨愈合,被认为是目前最强有力的一种骨诱导因子。基因治疗在许多方面都代表了一种增进骨再生方法的理念。其允许特异的基因产物转移至精确的解剖学部位。运用骨形态发生蛋白基因于骨组织工程领域以治疗骨缺损是目前新的热点。骨组织中的骨形态发生蛋白以及骨形态发生蛋白的表达和功能由于他们显著的生物学作用而被长时间的研究。结论:应用骨形态发生蛋白基因疗法可以成功诱导骨和软骨的发生,为骨缺损的治疗开辟了新的解决途径。     

膝关节负荷改变对关节软骨的影响

目的:观察一侧膝关节固定的条件下对侧膝关节软骨的影响。方法:6月龄,体质量2.5kg新西兰白兔10只,分实验组和对照组。实验组分a,b两部分,a组3只、b组5只。用石膏固定一侧膝关节5.5周。a组去除固定后即刻取对侧膝关节软骨标本。b组去除固定4周后取对侧膝关节软骨标本。通过光镜及扫描电镜观察膝关节负荷改变对关节软骨的影响。对照组2只,膝关节不做任何处理。结果:兔一侧膝关节固定5.5周,对侧膝关节由于承重增加,在去除固定后即刻,对侧膝关节软骨即出现退行性改变。去除固定4周,对侧膝关节软骨尚未恢复正常。结论:①一侧膝关节固定,对侧膝关节由于负荷的增加可导致退行性变化。②在相同时间段,关节固定对软骨的影响大于负荷改变对软骨的影响。     

Transgene persistence and cell turnover in the diarthrodial joint: implications for gene therapy of chronic joint diseases.

Local gene therapy for chronic joint diseases requires prolonged transgenic expression, but this has not been reliably achieved in animal models. Using normal and immunocompromised animals, we examined the capacity of various cell types in joint tissues to maintain and express exogenous transgenes after direct intra-articular gene delivery. We found that transgenic expression could persist for the lifetime of the animal but required precise immunological compatibility between the vector, transgene product, and host. It was not dependent on vector integration or promoter origin. We identified two phenotypically distinct sub-populations of genetically modified cells within the joint: (i) transient cells, with a half-life of a few weeks, and (ii) stable cells that reside in the joint tissues indefinitely. Contrary to the prevailing assumption, the transient sub-population was composed almost exclusively of synovial fibroblasts, indicating that the synovium is not an appropriate tissue upon which to base a long-term therapy. Instead, fibroblasts in the ligaments, tendons, and capsule emerged as the primary cell types capable of sustained therapeutic transgene expression. This study sheds new light on the cellular dynamics of articular tissues and suggests that cell turnover and immune reactivity are the key determinants in achieving sustained transgenic expression intra-articularly.     

The genodermatoses: candidate diseases for gene therapy

Tremendous progress has been made in understanding the genetic basis of different forms of genodermatoses, a group of heritable diseases displaying a spectrum of phenotypic manifestations and clinical severity. The information about the underlying mutations in the candidate gene/protein systems has provided the basis for initial development of cutaneous gene therapy, and these heritable conditions appear to serve as appropriate candidate diseases for such efforts. Because of its accessibility and the fact that resident skin cells, such as epidermal keratinocytes and dermal fibroblasts, can be readily propagated in culture, skin serves as an appropriate target tissue for gene therapy. Various strategic considerations, including the use of in vivo or ex vivo approaches, gene replacement versus gene repair, utilization of different delivery systems, etc., require careful prioritization depending on the type of mutations and their pathogenetic consequences at the mRNA and protein levels.     

骨缺损的基因治疗

目的：创伤、肿瘤及人工关节翻修手术等均可造成骨缺损。传统的治疗方法包括自体骨、异体骨和人工骨移植，但每种方法均存在一定的局限性，难以完全满足临床需要。骨缺损的基因治疗克服了传统治疗方法的局限，具有重要研究意义。资料来源：应用计算机检索Pubmed数据库1990-01／2005-04有关骨缺损的基因治疗文章，检索词“bone，gene therapy，bone morphogenetic protein”，限定文章语言种类为English。同时计算机检索中国期刊全文数据库、万方数据1994-01／2005-04期间的相关文章，检索词“骨缺损，基因治疗，骨形成蛋白，组织工程”，限定文章语言种类为中文。资料选择：对资料进行初审，选取符合研究要求的有关文章，并开始查找全文。纳入标准：①有关骨缺损的基因治疗研究。②有关骨组织工程研究。排除标准：①重复研究：②综述文章。资料提炼：共收集到168篇有关骨缺损的基因治疗及组织工程骨方面的文章，排除重复或类似的同一研究，28篇符合研究要求。资料综合：①基因治疗骨缺损的技术选择：人骨形态发生蛋白基因是目前骨缺损基因治疗中应用最多的目的基因，已在兔、鼠、羊和狗模型中证实其对骨缺损的修复作用。各种研究中，靶细胞多采用骨髓基质细胞，载体多选用腺病毒。②基因治疗骨缺损研究进展：基因治疗与组织工程相结合以及多基因联合治疗骨缺损是该领域研究的方向。③问题与展望：目前的研究大多处于实验阶段，要应用于临床还有很多工作需要改进。结论：多种细胞生长因子具有促进骨愈合的作用，通过骨缺损的基因治疗方法可保证细胞生长因子局部的靶向释放，最大限度地增加局部治疗效果，减少全身副作用，被认为是维持骨缺损局部生长因子有效治疗浓度最有希望的一种方法。     

The feasibility of measuring joint angular velocity with a gyro-sensor

Arai T, Obuchi S, Shiba Y, Omuro K, Nakano C, Higashi T. The feasibility of measuring joint angular velocity with a gyro-sensor.

Objectives

To determine the reliability of an assessment of joint angular velocity using a gyro-sensor and to examine the relationship between ankle angular velocity and physical functions.

Design

Cross-sectional.

Setting

Kinesiology laboratory.

Participants

Twenty healthy young adults (mean age, 22.5y) and 113 community-dwelling older adults (mean age, 75.1y).

Interventions

Not applicable.

Main Outcome Measures

Maximal ankle joint velocity was measured using a gyro-sensor during heel-rising and jumping with knee extended. The intraclass correlation coefficient (ICC) was used to determine the intertester and intratester reliability. The Pearson correlation coefficient was used to examine the relationships between maximal ankle joint velocity and isometric muscle strength and isokinetic muscle power in young adults and also to examine the relationships between maximal ankle joint velocity and functional performance measurements such as walking time in older adults.

Results

High reliability was found for intertester (ICC=.96) and intratester reliability (ICC=.96). The data from the gyro-sensor highly correlated with muscle strength (r range, .62−.68; P<.01) and muscle power (r range, .45−.79; P range, .01−.05). In older subjects, mobility functions significantly correlated with the angular velocity of ankle plantarflexion.

Conclusions

Measurement of ankle angular velocity using a gyro-sensor is both reliable and feasible, with the results representing a significant correlation to muscle power and performance measurements.