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。

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]

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

Abstract

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]