摘要
目的观察体外坏死股骨头骨组织对犬骨髓间充质干细胞（BMSC）增殖分化的影响。方法用液氮冷冻法制作犬股骨头坏死模型，采用分离液分离法结合贴壁法获取犬BMSC，利用Transwell培养板将BMSC与不同骨组织共培养，依据培养条件不同分3组，空白组：10％胎牛血清＋DMEM培养基，对照组：10％胎牛血清＋DMEM培养基＋正常股骨头骨组织，实验组：10％胎牛血清＋DMEM培养基＋坏死股骨头骨组织。观察细胞生长状况，采用噻唑蓝（MTY）比色法测各组细胞相对数，作细胞内碱性磷酸酶染色并检测培养液上清中碱性磷酸酶浓度，茜素红染色、油红O染色分别观察钙结节、脂滴的形成。结果对照组与实验组BMSC增殖率高于空白组，在1、3、5d差异有统计学意义（P〈0．05），实验组增殖率在5、7d时大于对照组（P〈0．05）；7d时细胞内碱性磷酸酶染色示对照组与实验组均为阳性，第7、9天培养液上清中碱性磷酸酶浓度按以下顺序依次减小，对照组〉实验组〉空白组（20．4±1．2）U／L比（60．5±4．5）U／L，P〈0．05。21d时茜素红染色示各组均有钙结节形成，数量按以下顺序依次减小，对照组〉实验组〉空白组，21d时油红O染色示各组均未见有脂滴形成。结论正常与坏死股骨头骨组织对BMSC增殖均有促进作用，并且均可诱导BMSC向成骨细胞分化而未向脂肪细胞分化，坏死股骨头骨组织对BMSC增殖的促进作用稍强；而对BMSC的成骨诱导能力弱于正常股骨头骨组织。
Objective To examine the effects of bone tissue from osteonecrosis of femoral head on the proliferation and differentiation of canine bone marrow mesenchymal stem ceils in vitro culture. Methods A canine model of femoral head osteonecrosis was induced by liquid nitrogen freezing. BMSC were isolated from dog ilium bone marrow by a combination of gradient centrifugation and adherent wall culture. Differem bone tissues and BMSC were cultivated indirectly in vitro by co-cultured in Transwell plate. According to the culture media, 3 groups were established： blank group （10% FBS/DMEM）, control group （10% FBS/ DMEM ＋ bone tissue from natural femoral head） and experimental group（ 10% FBS/DMEM ＋ bone tissue from osteonecrosis of femoral head）. Cell proliferation was measured by methylthiazol tetrazolium （MTT） method. Cell differentiation was examined by alkaline phosphatase （ALP） staining and its concentration examined. Alizarin red staining method was used to study the calcification effects and Oil red O staining method was used to detect if there was fat emergence. Results As compared with the blank group, the proliferation in the control and experiment groups were significantly promoted after culturing for Days 1, 3 and 5 （P 〈 0. 05）. The proliferation of the experiment group was higher than the control group at Day 5 and 7 day （P 〈 0. 05 ）. After a 7-day co-culturing, ALP staining was positive in the control and experiment groups. At Day 7 and 9, the ALP activity in culture fluid was in this order： control group 〉 experiment group 〉blank group（P 〈0. 05）. Alizarin red staining show control group had the most calcium nodules（ 12. 17 ±2.48, P 〈 0. 05） and the number of calcium nodules in the experiment group was more than the blank group （P 〈0. 05）. Oil red O staining show there was no fat emergence after 21 days in every group. Conclusion Both natural and osteonecrotic bone tissue of femoral head could promoted the proliferation of canine BMSC and induces th
出处
《中华医学杂志》
CAS
CSCD
北大核心
2013年第11期856-859,共4页
National Medical Journal of China
基金
国家自然科学基金（30772194）
关键词
股骨头坏死
骨髓
干细胞
Femoral head necrosis
Bone marrow
Stem cells
