Feng Xuetao, Tian Shaoqi, Sun Kang, Zhang Jihua, Zhang Cailong, Liu Shihai, Zhou Ming, Lü Jiangtao
Department of Orthopaedics, Affiliated Hospital of Medical College, Qingdao University, Qingdao Shandong 266000, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2011 Oct;25(10):1250-5.
To study the effect of platelet lysate (PL) on chondrogenic differentiation of human umbilical cord derived mesenchymal stem cells (hUCMSCs) in vitro.
Umbilical cords were voluntarily donated by healthy mothers. The hUCMSCs were isolated by collagenase digestion and cultured in vitro. The surface markers of the cells were detected by flow cytometer. According to different components of inductive medium, the cultured hUCMSCs were divided into 3 groups: group A [H-DMEM medium, 10% fetal bovine serum (FBS), and 10%PL]; group B [H-DMEM medium, 10%FBS, 10 ng/mL transforming growth factor beta1 (TGF-beta1), 1 x 10(-7) mol/L dexamethasone, 50 microg/mL Vitamin C, and 1% insulin-transferrin-selenium (ITS)]; and group C (H-DMEM medium, 10%FBS, 10 ng/mL TGF-beta1, 1 x 10(-7) mol/L dexamethasone, 50 microg/mL vitamin C, 1%ITS, and 10%PL). The hUCMSCs were induced in the mediums for 2 weeks. Toluidine blue staining was used to detect the secretion of chondrocyte matrix. Immunofluorescence method was used to identify the existence of collagen type II. The expressions of Aggrecan and collagen type II were detected by semiquantitative RT-PCR.
Flow cytometer results showed that the hUCMSCs did not express the surface markers of hematopoietic cell CD34, CD45, and human leukocyte antigen DR, but expressed the surface markers of adhesion molecule and mesenchymal stem cells CD44, CD105, and CD146. Toluidine blue staining and immunofluorescence showed positive results in group C, weak positive results in group B, and negative results in group A. Semiquantitative RT-PCR showed the expressions of Aggrecan and collagen type II at mRNA level in groups B and C, but no expression in group A. The mRNA expressions of Aggrecan and collagen type II were higher in group C than in group B (P < 0.05).
Only 10%PL can not induce differentiation of hUCMSCs into chondrocytes, but it can be a supplement to the induced mediums. PL can improve hUCMSCs differentiating into chondrocytes obviously in vitro. This study provides new available conditions for constructing tissue engineered cartilage.
研究血小板裂解液（PL）对人脐带间充质干细胞（hUCMSCs）体外成软骨分化的影响。
脐带由健康母亲自愿捐献。通过胶原酶消化法分离hUCMSCs并进行体外培养。采用流式细胞仪检测细胞表面标志物。根据诱导培养基的不同成分，将培养的hUCMSCs分为3组：A组[H-DMEM培养基、10%胎牛血清（FBS）和10%PL]；B组[H-DMEM培养基、10%FBS、10 ng/mL转化生长因子β1（TGF-β1）、1×10⁻⁷ mol/L地塞米松、50 μg/mL维生素C和1%胰岛素-转铁蛋白-硒（ITS）]；C组（H-DMEM培养基、10%FBS、10 ng/mL TGF-β1、1×10⁻⁷ mol/L地塞米松、50 μg/mL维生素C、1%ITS和10%PL）。将hUCMSCs在各培养基中诱导2周。采用甲苯胺蓝染色检测软骨细胞基质的分泌情况。采用免疫荧光法鉴定Ⅱ型胶原的存在。通过半定量RT-PCR检测聚集蛋白聚糖和Ⅱ型胶原的表达。
流式细胞仪结果显示，hUCMSCs不表达造血细胞CD34、CD45和人白细胞抗原DR的表面标志物，但表达黏附分子和间充质干细胞CD44、CD105和CD146的表面标志物。甲苯胺蓝染色和免疫荧光检测显示，C组结果为阳性，B组结果为弱阳性，A组结果为阴性。半定量RT-PCR显示，B组和C组在mRNA水平有聚集蛋白聚糖和Ⅱ型胶原的表达，而A组无表达。C组聚集蛋白聚糖和Ⅱ型胶原的mRNA表达高于B组（P < 0.05）。
仅10%PL不能诱导hUCMSCs分化为软骨细胞，但可作为诱导培养基的补充成分。PL能明显促进hUCMSCs体外向软骨细胞分化。本研究为构建组织工程软骨提供了新的可行条件。
