体外诱导人骨髓间充质干细胞分化为软骨细胞

背景:自体软骨细胞来源困难是束缚软骨细胞移植和软骨工程学发展的主要问题之一。骨髓间充质干细胞具有多向分化潜能,在不同诱导条件下,骨髓间充质干细胞能分化形成多种组织细胞,如软骨细胞、成骨细胞、成肌细胞及神经细胞等。胰岛素样生长因子-I是一种对肢体和软骨的形成及发育起重要调控作用的生长因子。目的:观察胰岛素样生长因子-I和软骨细胞培养液是否能在体外诱导骨髓间充质干细胞向软骨细胞分化。设计:开放性实验。单位:中山大学附属第二医院骨科和医学研究中心。材料:实验于2003-03/11在中山大学附属第二医院医学研究中心完成。人骨髓间充质干细胞取自因健康原因需终止妊娠的4～6月龄水囊引产的胚胎。方法:采用Percoll分离液分离培养人胚骨髓间充质干细胞,体外扩增,用流式细胞仪测定骨髓间充质干细胞的CD44,CD71,CD34,CD45表达;在第4代骨髓间充质干细胞的培养基中加入100μg/L胰岛素样生长因子-I和软骨细胞培养液,采用倒置显微镜观察、Ⅱ型胶原免疫组织化学、细胞内蛋白多糖含量测定等方法判断诱导细胞的形态变化和表达软骨基质的能力。主要观察指标:通过检测细胞CD34,CD44,CD45的表达,进行骨髓间充质干细胞表型鉴定。通过观测诱导后细胞Ⅱ型胶原免疫组织化学及细胞分泌蛋白多糖能力的变化判断其是否向软骨细胞分化。结果:①倒置显微镜观察结果:体外培养的骨髓间充质干细胞呈现成纤维细胞形态。②骨髓间充质干细胞表面抗原鉴定:流式细胞仪结果显示细胞均一性较好,第4代骨髓间充质干细胞阳性表达CD44,阴性表达CD34,CD45,说明分离获得的细胞符合骨髓间充质干细胞的特点。③光镜下观察骨髓间充质干细胞诱导为软骨细胞的形态变化:加入软骨细胞条件培养液和胰岛素样生长因子-I共培养,在培养过程中见骨髓间充质干细胞逐渐变圆,15d后可见部分细胞呈短梭形或多角形,突起短,呈现软骨细胞的特征。④Ⅱ型胶原免疫组织化学染色:胰岛素样生长因子组细胞在培养后第15天约72.5%的细胞可见棕黄色的颗粒分布于胞浆内,呈弱阳性或较强阳性。对照组骨髓间充质干细胞第15天的Ⅱ型胶原免疫组织化学为阴性。⑤蛋白多糖含量测定:胰岛素样生长因子-I和软骨细胞培养液共培养组培养15d后,细胞内蛋白多糖含量为(8.92±0.91)μg/L,高于单纯骨髓间充质干细胞培养组(2.56±0.26)μg/L,P<0.05],但低于软骨细胞组(13.69±1.51)μg/L,P<0.05]。结论:胰岛素样生长因子-I和软骨细胞培养液能诱导骨髓间充质干细胞向软骨细胞分化。

Human bone marrow-derived mesenchymal stem cells differentiate into chondrocytes in vitro

BACKGROUND:How to obtain sufficient autogeneic chondrocytes is a problem which must be answered as soon as possible in both the transplantation of chondrocytes and the development of cartilage engineering.Bone marrow-derived mesenchymal stem cells have the potential of multidirectional differentiation. Under different induced conditions, they can differentiate into multiple tissue cells, such as chondrocyte, osteoblasts , sarcoblast, nerve cells and so on. Insulin-like growth factor-I plays an important role in regulating the formation and development of limb and cartilage.OBJECTIVE:To observe the effect of the insulin-like growth factor-I and culture solution of chondrocyte on inducing bone marrow-derived mesenchymal stem cells to differentiate into chondrocytes in vitro.DESIGN:Open experiment.MATERIALS:This experiment was carried out at the Medical Study Center, Second Hospital Affiliated to Sun Yat-sen University from March to November 2003.Human bone marrow-derived mesenchymal stem cells were harvested from 4 to 6-month old embryos, all of which were from pregnant women who needed to terminate of pregnancy by induction delivery with water bag for health.METHODS:Human embryonic mesenchymal stem cells were isolated with Percoll separating medium. Subsequently, the cells were amplified in vitro,and the expression of surface makers of bone marrow-derived mesenchymal stem cells, such as CD44, CD71, CD34 and CD45 were measured with flow cytometer to identify the cells in our experiment. 100 μg/L insulin-like growth factors and culture solution of chondrocytes were added in the culture medium of bone marrow-derived mesenchymal stem cells of the fourth generation. The morphological changes of induced cells were observed with an inverted microscope. The expression of type Ⅱ cartilage matrix was observed by collogen immunohistochemistry. The proteoglycan level in the cells was detected, too.MAIN OUTCOME MEASURES:Phenotype of bone marrow-derived mesenchymal stem cells was identified through detecting the expressions of CD34, CD44 and CD4.Type Ⅱ collagen immunohistochemistry and the change of cellular ability to secrete proteoglycan after induction were observed to determine whether bone marrow-derived mesenchymal stem cells can differentiate into chondrocytes.RESULTS: ① Being observed under an inverted microscope,the bone marrow-derived mesenchymal stem cells presented a morphology like fibroblasts when they were cultured in vitro. ②Identification of surface antigen of bone marrow-derived mesenchymal stem cells: These cells were detected to have good homogenicity with flow cytometer. The fourth generation of bone marrow-derived stem cells positively expressed CD44, negatively expressed CD34 and CD45, suggesting these cells had the characteristics of bone marrow-derived mesenchymal stem cells. ③Observation of the morphological change of chondrocytes induced by bone marrow-derived mesenchymal stem cells under optical microscope: chondrocyte condition culture solution and insulin-like growth factors-Ⅰ were added to co-culture.During the process of culture, bone marrow-derived mesenchymal stem cells were seen to have a shape of round gradually. Fifteen days later, some cells presented a shape of short-shuttle or polygon with short mutations,which were the shape characteristics of chondrocytes. ④Immunohistochemical staining of Type Ⅱ collagen: In the insulin-like growth factor group,72.5% cells had many brown granules in cytoplasm, which were weakly positive or strongly positive expression of Type Ⅱ collagen. In the control group, bone marrow-derived mesenchymal stem cells was negative expression of type Ⅱ collagen on the 15th day. ⑤ Measurement of proteoglycan level: After co-culture with insulin-like growth factor-Ⅰ and chondrocyte culture solution for 15 days, proteoglycan was higher in the cells of co-culture group (8.92±0.91) μg/L ] than in culture group (2.56±0.26) μg/L,P ＜ 0.05], but lower than in the chondrocyte group(13.69±1.51) μg/L, P＜ 0.05].CONCLUSION:Insulin-like growth factor-Ⅰ and chondrocyte culture solution can induce bone marrow-derived mesenchymal stem cells to differentiate into chondrocytes.