三维立体培养法诱导去分化软骨细胞Ⅱ型胶原的重新表达  

Collagen type Ⅱ was redifferentiated with three dimensional culture in dedifferentiated chondrocytes

作　　者：何清义[1] 李强[1] 罗飞[1] 许建中[1] 

机构地区：[1]第三军医大学附属西南医院骨科,重庆400038

出　　处：《中华实验外科杂志》2007年第3期307-309,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30300357）;国家高技术研究“863”发展计划资助项目（2003AA205021）

摘　　要：目的用藻酸盐三维立体培养法及离心管聚集体诱导培养法促进去分化的永生化软骨细胞第50代（IHACs50）重新表达软骨细胞的Ⅱ型胶原标志性表型。方法利用藻酸盐三维立体培养法以及离心管聚集体诱导培养法分别诱导培养去分化的永生化人关节软骨细胞第50代，然后用Ⅰ、Ⅱ、Ⅲ型胶原的免疫组织化学染色、Ⅱ型胶原的逆转录-聚合酶链反应（RT-PCR）检测、激光共聚焦显微镜检测、胶原定量检测该永生化软骨细胞的Ⅱ型胶原表型表达情况。结果免疫组织化学染色、激光共聚焦显微镜检测、RT-PCR检测分别提示藻酸盐三维立体培养法和离心管聚集体诱导培养法使去分化永生化人关节软骨细胞的Ⅱ型胶原染色阳性以及621bp的Ⅱ型胶原mRNA表达，Western blot和^3H-脯氨酸标记SDS-PAGEⅡ型胶原定量检测发现前者促进Ⅱ型胶原分泌较后者多。结论去分化永生化人关节软骨细胞可以经不同的诱导方法而重新表达Ⅱ型胶原表型，藻酸盐三维立体培养法因与软骨细胞的生理环境相似，故促进Ⅱ型胶原重新表达较多。Objective The marked phenotype collagen type Ⅱ of chondrocyte in dedifferentiated irmnortalized human articular chondrocytes （IHACs P50） were inducted with alginate three dimensional culture and centrifuge aggregate culture. Methods Dedifferentiated IHACs P50 were induced with alginate three dimensional culture and centrifuge aggregate culture, then type Ⅰ , Ⅱ, Ⅲ collagen immunohistochemical stain and reverse transcriptional polymerase chain reaction of type Ⅱ collagen and laser confocal microscopy as well as collagen quantity were performed. Results Immunohistochemical stain and laser confocal microscopy as well as reverse transcriptional polymerase chain reaction revealed respectively that collagen type Ⅱ was stained positive and 621 base pair collagen type Ⅱ mRNA was expressed in alginate three dimensional culture and centrifuge aggregate culture, the former promoted more type Ⅱ collagen compared with the later confirmed with Western-blot and SDS-PAGE. Conclusion Various induced methods were able to redifferentiate type Ⅱ collagen in dedifferentiated IHACs. More type Ⅱ collagen was promoted with alginate three dimensional culture in dedifferentiated IHACs due to its similarity to the microenviroment of chondrocytes in vivo.

关 键 词：软骨细胞 诱导 表型 

分 类 号：R686[医药卫生—骨科学]