摘要
目的探讨转化生长因子β1(TGF-β1)基因转染对间充质干细胞(MSCs)增殖、向成软骨方向定向分化等生物学行为的调控作用及其机制。方法体外将具有促进MSCs增殖分化及抑制多种炎性介质活性等多重生物学效应的TGF-β1基因以不同剂量转入MSCs,通过免疫组化、RT-PCR、水貂肺上皮细胞生长抑制法、3H-TdR、Na235SO4掺入法、流式细胞仪、Ⅱ型胶原原位杂交及透射电镜等系列方法检测TGF-β1基因转染的瞬时和稳定表达情况,分析TGF-β1基因转染对MSCs增殖和向成软骨方向定向分化的调控及其作用机制。结果TGF-β1基因转入MSCs能获得瞬时及稳定表达,表达产物具有生物活性;3μl脂质体介导1μgTGF-β1基因转染能获得最佳促MSCs增殖及蛋白多糖、Ⅱ型胶原合成效应;TGF-β1基因转染能显著抑制IL-1对羟脯氨酸合成的降解作用。结论MSCs能作为基因治疗的受体细胞并可稳定高效表达具有生物学活性的TGF-β1;通过增强增殖细胞核抗原表达来提高S期细胞DNA含量,促进软骨特异性细胞外基质合成,从而促进MSCs增殖并调控其向成软骨方向定向分化、抑制多种炎性介质生物学活性以保护关节软骨,使提高关节软骨缺损的修复质量成为可能。
Objective The purpose of the present was to investigate the effect of transforming growth factorβ 1 (TGF-β 1 )gene transfection on the proliferation and differentiation of bone marrow-derived mes-enchymal stem cells(MSCs)into articular cartilage,and provide a basis for accelerating articular cartilage reparation using gene enhanced tissue engineering technology.Methods In the current study,the prin ciples of tissue engineering were combined organically with the principles of gene therapy to produce cul tured MSCs transduced with the full-length rat TGF-β 1 cDNA in vitro,in which TGF-β 1 had multiple biological efficacies to accelerate the proliferation and differentiation of MSCs and inhibited the activities of inflamma-tory factors.The MSCs were harvested from2month New-Zealand rabbits and separated by centrifugation according to Friedenstein method.The transient and long-lasting expressions of transfected TGF-β 1 gene in MSCs were measured by immunohistochemistry,RT-PCR,3 H-TdR,flow cytometry and transmission electron microscope.The transfected MSCs were observed for proliferation and determined differentiation into chon-droblasts by prime hybridization for detection of typeⅡcollagen.Results The transient and long-lasting expressions of TGF-β 1 gene transduced into MSCs were obtained,and its products displayed biological activ-ities.The best promotion of proliferation of MSCs and synthesis of typeⅡcollagen and specific extracellular matrix were achieved when the amount of TGF-β 1 cDNA was1μg by3μl lipofectamine induction.Trans-duction of MSCs with TGF-β 1 gene was also able to inhibit the activity of IL-1on the degradation of hy-droxyprolines significantly.Conclusion Transfer of gene encoding TGF-β 1 to MSCs increases its prolifera-tion and synthesis of hyaline cartilage specific extracellular matrix while reducing the bioactivity of inflam-matory factors such as IL-1.This new molecular tissue engineering approach could be of potential benefit to enhance the repair of damaged articular cartilage,especially those caused by diseases during the advanced stages.
出处
《中华骨科杂志》
CAS
CSCD
北大核心
2003年第7期427-433,共7页
Chinese Journal of Orthopaedics
基金
国家自然科学基金资助项目(30170270
30200063)
