体外诱导的人脂肪间充质干细胞源性软骨细胞体内成骨活性☆

周全，邓展生，朱勇，李保军，叶川，许宇霞

课题背景：课题背景：间充质干细胞向软骨细胞分化包括体内、体外两种诱导方式，前者主要是通过体内微环境诱导间充质干细胞向软骨分化，但这种诱导机制随机性大，且体内微环境极其复杂，难以进行监控和调整，所以目前诱导间充质干细胞向软骨细胞分化的研究多在体外进行。在体外诱导间充质干细胞向软骨细胞分化需要特定的诱导条件，其中转化生长因子家族和地塞米松是最重要的诱导因素，此外细胞密度也是体外成功诱导分化不可忽视的因素，可促进软骨形成。

偏倚或不足：实验发现在无支架离心管条件下，人脂肪来源的间充质干细胞在体外经定向诱导并未形成成熟的透明软骨，可能与体内环境受多因素共同影响而非相对单一的体外作用条件有关，需要进一步摸索调整诱导条件，以便在体外构建成熟的软骨组织。

术语解析：Aggrecan与Ⅱ型胶原蛋白是软骨细胞最特异的表面标记，Aggrecan单体上带有数量众多的硫酸软骨素和硫酸角质素等负电荷基团，这使它的大分子聚合物在Ⅱ型胶原纤维的网络架构中高度水合。Aggrecan与胶原纤维复合物的生物学特性赋予关节软骨组织承受外力，恢复弹性形变的能力。当Aggrecan的分解增加、合成减少时，糖胺聚糖基团上所携带的负电荷亦随之丢失，造成关节软骨不再具有正常的生理功能。

周全☆，男，1979年生，江苏省淮安市人，汉族，中南大学湘雅医学院在读博士，医师，主要从事骨与软骨组织工程方面的研究。
wuque1@yahoo.
com.cn

摘要
目的:目前对间充质干细胞向软骨细胞诱导分化主要包括体内诱导和体外诱导两种方式，前者依靠体内微环境，随机性大且难以进行监控和调整。本实验探讨体外诱导人脂肪间充质干细胞向软骨细胞分化，以及诱导后细胞在裸鼠体内的成软骨能力。
方法：实验于2006-08/2007-05在中南大学湘雅医院中心实验室完成。①对象与材料：皮下脂肪组织来源于股骨颈骨折手术患者6例，年龄25~64岁，对实验及治疗均签署知情同意书，实验经医院医学伦理委员会批准。清洁级裸鼠5只，体内成软骨实验过程中对动物的处置符合动物伦理学标准。离心管容量20 mL，由GeneRay公司生产。②实验方法：将皮下脂肪组织剪碎，I型胶原酶消化法分离培养脂肪间充质干细胞，待细胞铺满瓶底80%时胰酶消化传代。传至第6代时收集 5×106个细胞，用含1%新生牛血清、高糖DMEM、10 μg/L转化生长因子β1、37.5 mg/L维生素C、6.25 mg/L胰岛素、6.25 mg/L转铁蛋白、10-7 mol/L地塞米松的软骨细胞诱导剂重悬于无支架离心管内。③实验评估：诱导过程中观察细胞聚集状态。诱导2周后采用苏木精-伊红染色、RT-PCR、Western blot对细胞形态及功能变化进行检测。将诱导后细胞与纤维蛋白原凝胶混合，植入裸鼠皮下，3周后切取植入组织块，苏木精-伊红染色及II型胶原免疫组化染色观察体内成软骨情况。
结果：①诱导后细胞聚集状态：诱导2 d后脂肪间充质干细胞自行聚集为一条状细胞团，1周后细胞团聚集呈球状。②苏木精-伊红染色结果：未见有软骨陷窝形成。③RT-PCR检测结果：细胞团内Ⅱ型胶原蛋白和Sox9 mRNA均呈阳性表达。④Western blot检测结果：细胞团内有较强的Ⅱ型胶原蛋白和Aggrecan表达。⑤体内成软骨情况：植入裸鼠皮下的组织块内有大量软骨陷窝及II型胶原蛋白形成。
结论：①在无支架离心管内，脂肪间充质干细胞经软骨诱导剂作用后具有典型的软骨细胞表型，但未形成成熟软骨组织所具备的软骨陷窝。②诱导后细胞与凝胶复合种植于裸鼠体内，可形成具有典型软骨特征的组织。
关键词：脂肪间充质干细胞；软骨；组织学；生物医学工程

周全，邓展生，朱勇，李保军，叶川，许宇霞.体外诱导的人脂肪间充质干细胞源性软骨细胞体内成骨活性[J].中国组织工程研究与临床康复，2008，12(8):1460-1463 [www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1460(ps).pdf]

中图分类号: R394.2
文献标识码: A
文章编号: 1673-8225
(2008)08-01460-04

In vivo osteogenic activity of chondrocytes derived from in vitro induced human adipose tissue-derived mesenchymal stem cells

AIM:The induction method of mesenchymal stem cells into chondrocytes includes in vivo induction and in vitro induction. In vivo induction depends on in vivo microenvironment. It is randomized and difficult to monitor and regulate. This study investigated the chondrogenic differentiational induced condition of human adipose tissue-derived mesenchymal stem cells (ADMSCs) in vitro and the chondrogenic capability of induced cells in vivo and vitro.
METHODS: Experiments were performed at the Central Lab of Xiangya Hospital of Central South University between August 2006 and May 2007. ①Subcutaneous adipose tissue was attained from 6 subjects aged 25-64 years undergoing femoral neck fracture surgeries. This study was approved by the hospital’s committee of ethics and informed consent was obtained from all subjects. Five clean nude mice were used in this experiment, and animal experiments in the present study were performed in compliance with the guidelines of animal ethics. Centrifuge tube (20 mL volume) was produced by GeneRay. ②The adipose was sheared, and ADMSCs were isolated and cultured by using collagenase Ⅰ dissociation. When 80% confluence, cells were passaged. 5×106 the sixth passage of ADMSCs were suspended in chondrogenic media containing 1% fetal bovine serum (FBS), DMEM-high glucose, 10 μg/L transforming growth factor (TGF)β1, 37.5 mg/L vitamin C, 6.25 mg/L insulin, 6.25 mg/L transferrin and 10-7 mol/L dexamethasone in centrifuge tube. ③Cells aggregation was observed during inducing. 14 days later, haematoxylin-eosin (HE) staining, reserve transcriptase-polymerase chain reaction (RT-PCR) and Western blot were applied to detect the morphology and function of induced cells. Induced ADMSCs were admixed with fibrinogen gel and transplanted in nude mice. 3 weeks later, the compounds were harvested and detected by using HE staining and immunohistochemistric staining of collagen Ⅱ.
RESULTS: ①2 days later, the induced cells aggregated spontaneously, and then cells aggregated into a globular cell mass 1 weeks later. ②HE staining showed that no cartilage lacuna was found in the cell mass. ③RT-PCR demonstrated that collagen type Ⅱ and Sox9 mRNA were expressed in the induced ADMSCs.. ④Western blot showed that collagen Ⅱ and Aggrecan were expressed in the induced ADMSCs. ⑤After transplanted in nude mice, a lot of cartilage lacunas were formed and collagen Ⅱ was synthesized in compounds.
CONCLUSION: ①In centrifuge tube , the ADMSCs can be differentiated into chondrocytes, but cannot construct mature cartilage, ②while mature cartilage is constructed when ADMSCs are transplanted in nude mice with fibrinogen gel.

Zhou Q, Deng ZS, Zhu Y, Li BJ, Ye C, Xu YX.In vivo osteogenic activity of chondrocytes derived from in vitro induced human adipose tissue-derived mesenchymal stem cells.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(8):1460-1463(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1460(ps).pdf]

脂肪间充质干细胞因其取材方便、来源广泛、获得细胞多和供区损伤小的特点[1]而成为软骨组织工程种子细胞研究的热点。Kato等[2-3]报道通过体外离心管培养法构建生长板软骨及组织工程化关节软骨，但对在体外离心管内诱导间充质干细胞向软骨细胞分化并形成组织工程化软骨尚未见报道。Ⅱ型胶原蛋白和Aggrecan是软骨细胞最特异的表面标记，Sox9是成软骨的关键启动子，本实验探讨在无支架离心管内诱导脂肪间充质干细胞向软骨细胞分化。

设计：细胞观察。
单位：中南大学湘雅医院脊柱外科。
材料：实验于2006-08/2007-05在中南大学湘雅医院中心实验室完成。①对象：皮下脂肪组织来源于股骨颈骨折手术患者6例，年龄25~64岁，对实验及治疗均签署知情同意书，实验经医院医学伦理委员会批准。②动物：清洁级6周龄裸鼠5只（由中南大学动物学部提供，动物质量合格证号：SCXK（沪）2003-0003），体质量15~20 g，体内成软骨实验过程中对动物的处置符合动物伦理学标准。③主要试剂与材料：高糖DMEM，低糖DMEM，新生牛血清(Gibco，新西兰)；Ⅱ型胶原蛋白抗体，Aggrecan 抗体，转化生长因子β1（Santa Cruz，美国）；I型胶原酶，胰岛素，转铁蛋白，地塞米松，维生素C(Sigma，美国)；组织细胞总RNA提取试剂Trizol（MRC，美国）；20 mL离心管（GeneRay）；RT反应试剂盒，PCR反应试剂盒（MBI，美国）；细胞总蛋白提取试剂盒（天为生物，中国）。
设计、实施、评估者：设计及评估为第二作者，实施为第一作者，均经过系统培训，未使用盲法评估。
方法：
脂肪间充质干细胞的分离与培养：自股骨颈骨折手术患者的皮下获取脂肪组织，以磷酸盐缓冲液冲洗3次以上，用眼科剪去除肉眼可见的血管和致密结缔组织，剪碎，加入3倍体积的I型胶原酶，37 ℃震荡消化2 h，以200目滤网滤去未消化完全的大块脂肪组织， 2 000 r/min离心10 min，弃上清，加入含10%新生牛血清的低糖DMEM重悬沉淀细胞，移入培养瓶，置于 37 ℃、体积分数为0.05的CO2培养箱中培养，3 d换液一次，待细胞铺满瓶底80%时，用0.25%胰酶消化传代。
脂肪间充质干细胞向软骨细胞的定向诱导：取传至第6代的脂肪间充质干细胞，消化后收集5×106个细胞，用含1%新生牛血清、高糖DMEM、10 μg/L转化生长因子β1、37.5 mg/L维生素C、6.25 mg/L胰岛素、 6.25 mg/L转铁蛋白、10-7 mol/L地塞米松的软骨细胞诱导剂重悬于20 mL无支架离心管内，3 d更换一次诱导剂，观察细胞聚集状态。2周后取出细胞团进行检测。
苏木精-伊红染色：观察细胞团内细胞形态变化及是否有软骨陷窝形成。
RT-PCR检测：引物由上海生工生物工程技术服务有限公司合成，β-actin上游5’-ACTCTTCCAGCCTTCCTTCC-3’，下游5’- ACTCGTCATACTCCTGCTTGC -3’，313 bp；Ⅱ型胶原蛋白上游5’-TTCAGCTATGGAGATGACAATC-3’，下游5’-AGAGTCCTAGAGTGA CTGAG -3’，472 bp；Sox9上游5’-GAACGCACATCAAGAGGGAG-3’，下游5’-TCTCGTTGATTTCGCTGCTC-3’，631 bp。分别采用Trizol试剂盒提取细胞总RNA，RT试剂盒合成cDNA，根据设计引物进行PCR扩增合成cDNA，PCR产物行1.5％琼脂糖凝胶电泳鉴定。检测诱导前和诱导2周后脂肪间充质干细胞Ⅱ型胶原蛋白和Sox9 mRNA的表达。
Western blot检测：检测诱导前和诱导2周后脂肪间充质干细胞Ⅱ型胶原蛋白和Aggrecan的表达。
体内成软骨实验：将诱导后细胞吹散，与1×1010 L-1纤维蛋白原凝胶混合，植入裸鼠皮下，3周后切取植入组织块，固定、包埋、切片，苏木精-伊红及II型胶原蛋白免疫组化染色，观察体内成软骨情况。
主要观察指标：①诱导后细胞聚集状态观察。②苏木精-伊红染色结果。③RT-PCR检测结果。④Western blot检测结果。⑤体内成软骨情况。

2.1 诱导后细胞聚集状态观察向软骨细胞定向诱导 2 d后，脂肪间充质干细胞自行聚集为一条状细胞团，随着诱导时间的延长，细胞团逐渐聚集呈球状，见图1。

2.2 苏木精-伊红染色结果诱导2周后细胞团内细胞聚集紧密，但未见有软骨陷窝形成。
2.3 RT-PCR检测结果诱导前脂肪间充质干细胞无Ⅱ型胶原蛋白和Sox9 mRNA表达，诱导2周后Ⅱ型胶原蛋白和Sox9 mRNA均呈较高表达，见图2。

2.4 Western blot检测结果诱导前脂肪间充质干细胞不合成Ⅱ型胶原蛋白和Aggrecan，诱导2周后Ⅱ型胶原蛋白和Aggrecan均呈较强表达，见图3。

2.5 体内成软骨观察将诱导的细胞与1×1010 L-1纤维蛋白原凝胶混合后植入裸鼠皮下，3周后苏木精-伊红染色结果显示植入的组织块内有大量软骨陷窝形成（图4），II型胶原免疫组化染色显示复合物内有大量II型胶原蛋白合成（图5）。

由于软骨无血管及软骨内细胞成分少，关节软骨自我修复能力有限[4-5]，软骨受损后多通过纤维软骨来修复甚至完全不进行修复[6]。组织工程是修复软骨缺损最具光明前景的方法之一而被广泛应用于软骨修复的研究[7-9]，种子细胞的研究又是软骨组织工程研究的重点之一。通过自体软骨细胞移植修复关节软骨缺损已经应用于骨科临床[10]，但是软骨细胞来源有限及软骨细胞体外扩增后容易失去正常的生物学特性，限制其广泛应用[11]。因此，研究者将目光投向具有多向分化潜能的间充质干细胞。脂肪间充质干细胞自被发现以来，已经证实可以定向分化为成骨细胞、软骨细胞、脂肪细胞、肌细胞、神经细胞、内皮细胞等[1,12-14]。体外诱导脂肪间充质干细胞向软骨分化是多因素共同作用的复杂过程，探索合适的诱导条件，诱导脂肪间充质干细胞成功向脂肪间充质干细胞向具有形成正常关节软骨功能的细胞分化，以便应用于临床修复受损关节软骨是脂肪间充质干细胞研究的热点之一。
目前对间充质干细胞向软骨细胞诱导分化的研究中，主要包括体内诱导和体外诱导两种。体内诱导主要是通过体内微环境诱导间充质干细胞向软骨分化，但这种诱导机制随机性大，且体内微环境极其复杂，难以进行监控和调整。所以目前诱导间充质干细胞向软骨细胞分化的研究多在体外进行。
在体外诱导间充质干细胞向软骨细胞分化需要特定的诱导条件，其中转化生长因子家族和地塞米松是最重要的诱导因素，转化生长因子β家族在骨髓、脂肪、骨膜等来源的间充质干细胞向软骨细胞分化中起至关重要的作用[15-17]。转化生长因子β1为间充质干细胞进入软骨表型分化提供适宜的外部信号[18]，Johnstone等[19]发现含10μg/L转化生长因子β1的无血清诱导剂可以诱导全部间充质干细胞向软骨细胞分化，含10-7 mol/L地塞米松的无血清诱导剂可以诱导部分间充质干细胞向软骨细胞分化，且两者的作用有协同作用。细胞密度也是体外成功诱导间充质干细胞向软骨细胞分化的重要条件，提高细胞密度能够促进软骨形成[20]，实验中以诱导剂重悬脂肪间充质干细胞2 d后，细胞自行聚集，形成高密度细胞团，并随着诱导时间的延长，细胞聚集更为紧密。
Ⅱ型胶原蛋白和Aggrecan是软骨细胞最特异的表面标记，Sox9是成软骨的关键启动子。诱导2周后，脂肪间充质干细胞明显表达Ⅱ型胶原蛋白和Sox9 mRNA，Western blot结果显示诱导后间充质干细胞较强表达Ⅱ型胶原蛋白和Aggrecan，说明诱导后细胞具有典型的软骨细胞表型；但离心管内的细胞团并未形成成熟软骨组织具备的软骨陷窝，说明在本实验的诱导条件下，不能在体外构建成熟的软骨组织, 这可能与体内环境是多因素共同作用，而体外作用条件相对单一有关；而将诱导后细胞与凝胶复合后，种植于裸鼠体内3周后可以形成具有典型软骨特征的组织，则从另一个角度证实脂肪间充质干细胞已成功诱导分化为软骨细胞。

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