FK506对许旺细胞体外增殖和分泌NGF的影响

FK506 promoting proliferation of Schwann cells in vitro and NGF of Schwann cells secreted highly by itself

目的研究FK506促进许旺细胞体外增殖及对许旺细胞（SCs）分泌NGF的影响。方法将纯化的许旺细胞分6组：A组（空白对照组）：含10％胎牛血清的DMEM／F12；B组：0．1ng／mlFK506；C组：0．5ng／mlFK506；D组：1．0ng／mlFK506；E组：5．0ng／mlFK506；F组：10ng／mlFK506。将许旺细胞于倒置显微镜下观察并用S—100蛋白免疫组化染色鉴定；MTT法筛选FK506促SCs增殖的最佳作用浓度：流式细胞仪检测SCs周期；ELISA法检测培养72h后SCs的NGF的分泌量。结果MTT法筛选：0．5ng／mlFK506为促进SCs增殖的最佳作用浓度；当FK506浓度大于1．0ng／ml时，SCs的生长活性逐渐下降并随着FK506浓度的逐渐增高，SCs的生长活性受抑制作用逐渐加强。流式细胞仪检测：含10％胎牛血清的DMEM／F12培养24h、48h、72h，SCsS期百分比分别为：27．8％，39．3％和58．4％；0．5ng／mlFK506培养24h、48h、72h，SCsS期百分比分别为：54．2％、60．3％和94．6％。ELISA法检测FK506促SCs增殖后表达NGF的实验研究中发现：0．5ng／mlFK506作用72h后的SCs所分泌的NGF高达0．188ng／ml。结论FK506应用于体外培养的SCs初期就能促进SCs增殖并使其保持良好的活性而高分泌NGF。

Objective To explore on FK506 promoting proliferation of Schwann cells in vitro and NGF of Sehwann cells secreted highly by itself. Methods Purified Schwann cells divide into six groups：group A （control group） DMEM/F12 contained 10% calf bloodserum; group B contained 0.1 ng/ml FK506; group C contained 0.5 ng/ml FK506; group D contained FK506：1.0 ng/ml;group E contained FK506：5.0 ng/ml; group F contained FK506：10 ng/ml. Morphology of Schwann cells were obsyrved by invert microscope and evaluated Schwann cells in immunocytochemistry staining with anti-S-100. The best concentration of FK506 who promoted proliferation of Schwann cells by MTT. Cell cycle of Schwann cells were determined by flow cytometry. The level of NGF in the conditioned media was determined by an enzyme-linked immunoadsordent assay after 72 h. Results Group C was the best concentration which promoting proliferation of Schwann cells among 5 groups, when the concentration 〉 1.0 ng/ml FK506 to promote Schwann cell proliferation gradually weakened. Detected by flow cytometry showed that： containing 10% fetal DMEM/F12 bovine serum for 24 h,72 h and 48 h after Schwann cells in S phase percentage were 27.8%,39.3% and 58.4% in the 0.5 ng / ml FK506 for 24 h,72 h and 48 h after Schwann cells S percentage period were 54.2%,60.3% and 94.6%. S phase of the latter than the former in 24 h,72 h and 48 h, respectively higher： 26.4% and 21% and 36.2%. FK506 detected by ELISA promote Sehwann cell proliferation after the expression of NGF in the experimental study found： 0.5 ng/ml FK506 for 72 h after the Schwann cells secreted by the NGF as high as 0.188 ng/ml, respectively. Conclusion FKS06 can promote proliferation of Schwann cells at early time in vitro and Schwann cells＇ good situation is highly kept to secrete NGF.