摘要
目的通过观察鹿茸多肽对白细胞介素1β(interleukin1β,IL-1β)诱导软骨表型化骨髓间充质干细胞(marrowmesenchymalstemcells,MSCs)凋亡的影响,以优化软骨组织工程的种子细胞。方法新西兰大白兔2只,抽取其骨髓;经密度梯度离心得到单核细胞,经体外分离、培养获得兔MSCs。用转化生长因子β1(transforminggrowthfactorβ1,TGF-β1)和碱性成纤维细胞生长因子(basicfibroblastgrowthfactor,bFGF)将其诱导分化软骨表型。将软骨表型化MSCs随机分成A组(空白对照组)5%胎牛血清的α-MEM培养液;B组(IL-1β组)5%胎牛血清的α-MEM培养液加入100ngIL-1β;C组(鹿茸多肽组)5%胎牛血清的α-MEM培养液加入10μg/ml鹿茸多肽作用3d后再加入100ngIL-1β;D组(TGF-β1组)5%胎牛血清的α-MEM培养液加入10ng/mlTGF-β1作用3d后再加入100ngIL-1β。分别于培养24、48和72h后取样,采用透射电镜观察细胞形态,AnnexinV法检测细胞凋亡率,RT-PCR技术分析Caspase-3mRNA表达水平,ELISA法检测Caspase-3蛋白酶活性。结果透射电镜观察B组24h后细胞核内染色质开始呈块状凝集,分布于核膜下,核膜不规则;48h后细胞核内染色质凝集加剧;72h后部分细胞内的核碎片凝集成凋亡小体。各时间点C、D组细胞结构改变相对滞后,且数量较少;A组细胞结构几乎无明显改变。各时间点B组MSCs凋亡率、Caspase-3mRNA表达及蛋白酶活性逐渐增高,与A组比较差异有统计学意义(P<0.01);C、D组与B组比较则逐渐下降,且差异有统计学意义(P<0.05)。结论Caspase-3参与MSCs凋亡,鹿茸多肽通过减少Caspase-3mRNA表达,并抑制其蛋白酶活性,阻止或逆转软骨表型化MSCs凋亡。
Objective To observe the effect of pilose antler polypeptides （PAP） on the apoptosis of rabbit marrow mesenchymal stem cells （MSCs） differentiated into chondrogenic phenotype by interleukin 1β （IL-1β） so as to optimize the seeding cells in cartilage tissue engineering. Methods The MSCs were separated from the nucleated cells fraction of autologus bone marrow by density gradient centrifuge and cultured in vitro. The MSCs were induced into ehondrogenie phenotype by transforming growth factor β1 （TGF-β1） and basic fibroblast growth factor （bFGF）. According to different medias, the MSCs were randomly divided into four groups： group A as black control group, group B（100 ng IL-1β）,group C（10 C（10μg/ml PAP＋100 ng IL-1β） and group D（100 ng/ml TGF-β1-100 ng IL-1β）. The samples were harvested and observed by morphology, flow cytometry analysis, RT-PCR and ELISA at 24, 48 and 72 hours. Results The intranuclear chromatin agglutinated into lump and located under nulear membranes which changed into irregular shape at 24 hours. The intranuclear chromatin agglutinated intensifily at 48 hours. Then the nucear fragments agglutinated into apoptosic corpuscles at 72 hours in group B. The structure change of cells in groups C and D was later than that in group B, and the number of cells changed shape was fewer than that in group B. The structure change of cells in group A was not significant. The apoptosic rate of cells, the mRNA expression of Caspase-3 and the enzymatic activity of Caspase-3 gradually increased in group B, and there were significant differences compared with groups A, C and D （P〈 0. 01 ）, Conclusion Caspase-3 is involved in aoptosis of the MSCs differentiated into chondrogenic phenotype cultured in vitro. PAP could prevent from or reverse apoptosis of these MSCs by decreasing the expression of Caspase-3 and inhibiting the activity of Caspase-3.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2006年第4期427-430,共4页
Chinese Journal of Reparative and Reconstructive Surgery
基金
福建省科技计划重大资助项目(2004Y018)~~
关键词
鹿茸多肽
骨髓间充质干细胞
软骨表型
凋亡
Pilose antler polypeptide Marrow mesenchymal stem cells Chondrogenic phenotype Apoptosis
