他克莫司抑制外周血单个核细胞培养上清对瘢痕疙瘩成纤维细胞的作用

Tacrolimus Inhibits the Enhancing Effects of Peripheral Blood Mononuclear Cell Supernatant on Proliferation and Collagen Expression in Keloid Fibroblasts: Implication for New Therapeutic Approach

目的:研究他克莫司(FK506)抑制外周血单个核细胞(PBMC)培养上清对瘢痕疙瘩成纤维细胞的作用,探讨FK506在瘢痕疙瘩治疗中可能的的作用和机制。方法:用消化法原代培养人瘢痕疙瘩来源的成纤维细胞,梯度密度离心法分离培养人PBMC。将瘢痕疙瘩来源的成纤维细胞随机分组,给予PBMC培养上清处理,实验组同时给与不同浓度FK506处理。四甲基偶氮唑蓝法(MTT)检测瘢痕疙瘩成纤维细胞增殖活性,荧光实时定量PCR法检测Ⅰ型胶原表达。结果:单纯给予PBMC培养上清处理后,成纤维细胞的增殖活性与对照组相比明显增高(P<0.01),同时给予PBMC上清和FK506时发现FK506在20 ng/ml和100 ng/ml时能够抑制PBMC上清的促增殖作用(P<0.01),荧光实时定量PCR结果显示:单纯给予PBMC培养上清处理后,Ⅰ型胶原的表达与对照组相比明显增高(P<0.05),给予PBMC上清和FK506后,在FK506浓度为20 ng/ml和100 ng/ml时Ⅰ型胶原表达降低(P<0.01)。结论:FK506能够抑制PBMC培养上清对瘢痕疙瘩成纤维细胞的作用,因此,FK506可能通过抑制PBMC的作用来达到预防和治疗瘢痕疙瘩的作用。

Objective： To study the inhibition of tacrolimus upon the enhancing effects of peripheral blood mononuclear cell（PBMC） supernatant on keloid fibroblasts,and to investigate the role of tacrolimus in keloid thearapy and its mechanism.Methods： Keloid fibroblasts were harvested by digestion method.PBMCs were separated with density gradient centrifugation.Culture supernatant derived from the PBMC was added to keloid fibroblasts.The experimental group was treated with PBMC supernatant and different concentration tacrolimus at the same time.Proliferation of keloid fibroblasts activity was detected with [3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） method.TypeⅠcollagen expression was detected with Real-time PCR.Results： PBMC culture supernatant treatment increased fibroblast proliferation activity significantly（P 0.01）.The co-operation of PBMC supernatant and tacrolimus inhibited the proliferation on the concentration of tacrolimus in 20 ng/ml and 100 ng/ml.（P 0.01）.Real-time PCR showed that： The co-operation decreased collagen typeⅠexpression（P 0.01） with tacrolimus concentration of 20 ng/ml and 100 ng/ml.Conclusions： Tacrolimus can inhibit the effect of peripheral blood mononuclear cell culture supernatant on keloid fibroblasts.Tacrolimus may prevent the keloids and play an important role of keloids therapy.