模拟微重力对K562细胞增殖和分化的影响

Impacts of simulated microgravity on proliferation and erythroid differentiation of K562 cells

目的在航天飞行条件下航天员会出现"航天飞行性贫血",其分子机制尚不明确,实验观察了美国国家航空航天局(NASA)旋转式细胞培养系统模拟的微重力环境对重组人红细胞生成素(Epo)诱导K562细胞增殖分化的影响。方法利用NASA旋转式细胞培养系统模拟的微重力环境,细胞计数分析细胞增殖情况,联苯胺染色法检测血红蛋白合成情况,应用免疫荧光和流式细胞术检测血型糖蛋白A(GPA)和转铁蛋白受体CD71在细胞表面的表达情况,应用荧光染料标记的鬼笔环肽和紫杉醇分别显示纤维型肌动蛋白和微管。结果旋转培养的K562细胞的细胞增殖速度显著低于地面对照组;旋转培养还显著抑制Epo诱导的血红蛋白合成,联苯胺阳性细胞从Epo处理常规培养组的(15.0±1.2)%降为旋转培养组的(9.0±0.9)%;旋转培养还抑制GPA和CD71在细胞表面的表达。此外,旋转培养还促进微管解聚。结论结果表明,旋转培养模拟微重力环境能抑制Epo诱导的K562细胞的增殖和分化;而模拟微重力环境使细胞骨架解聚可能使定位于细胞表面的蛋白(如GPA、CD71以及Epo受体等)在运输到细胞表面的过程发生障碍,从而降低对Epo的反应能力。

Objective The impact of simulated microgravity on proliferation,differentiation,and cytoskeleton of K562 cells induced by erythropoietin(Epo)in national aeronautics and space administration rotary cell culture system(RCCS)was observed.Methods Cell number was counted using a haemocytometer.The percentage of cells staining for hemoglobin was estimated by staining with benzidine.Erythroid antigens glycophorin A(GPA)and Transferrin receptor(CD71)on surface of K562 cells were labeled by direct immunofluorescence using the following conjugated antibodies:fluorescein isothiocyanate(FITC)-conjugated anti-CD71 antibodies and FITC-conjugated anti-GPA antibodies.Then flow cytometric analysis was performed to detect their expression levels on cell surface.Polymerized actin(F-actin)and microtubules in K562 cells were stained with Oregon Green 488-conjugated phalloidin and Oregon Green 488-conjugated paclitaxel respectively.Results Without Epo stimulation,cell density cultured in flask was(4.5±1.1)×10~5/ml,while cell density cultured in RCCS was(3.1±1.0)×10~5/ml.With Epo stimulation,cell density cultured in flask was(5.6±0.9)×10~5/ml,while cell density cultured in RCCS was(3.4±1.4)×10~5/ml.The hemoglobin-positive percentage of K562 cells cultured in flash without Epo treatment was 5.9%,while the benzidine-positive percentage in Epo-treated cells was 15%.Simulated microgravity significantly inhibited Epo-induced erythroid differentiation of K562 cells,with the benzidine-positive percentage 9%.Compared to the flask-cultured K562 cells,the expression levels of GPA-and CD71 on the surface of RCCS-cultured K562 cells decreased significantly.Compared to the flask-cultured K562 cells,the polymerized microtubule levels in RCCS-cultured K562 cells decreased significantly,whereas F-actin showed no any changes.Conclusion These results show that RCCS-simulated microgravity can induce the inhibition of proliferation and erythroid differentiation,down-regulation of surface erythroid antigens,and cytoskeleton changes in K562 cells.