Constitutive and modulated cytokine expression in two permanent human bone marrow stromal cell lines.
Constitutive and modulated cytokine expression in two permanent human bone marrow stromal cell lines.
复制标题
两种永久性人骨髓基质细胞系中的组成型和调节型细胞因子表达。
作者：
K. Thalmeier;P. Meissner;G. Reisbach;L. Hültner;B. Mortensen;A. Brechtel;R. Oostendorp;P. Dörmer
我们详细分析了L87/4和L88/5这两个永久的人骨髓基质细胞系的细胞因子表达模式。这些细胞系是我们实验室以前建立的，具有高度的辐射耐受性而不会分离细胞，并支持长期培养富含Cd(34+)的人脐血细胞。对22种不同的细胞因子或细胞因子受体mRNAs的RT-PCR分析表明，与原代人右旋糖型基质相比，两种基质细胞系中的表达模式几乎相同。由于长期培养中使用的基质饲养层通常是在含有皮质类固醇的培养液中照射和生长的，我们通过RT-PCR、Northern印迹分析、生物测定和RIA分析了照射和地塞米松对两种细胞系细胞因子产生的影响。RT-PCR分析显示，c-kit、G-CSF、GM-CSF、IL-1β、IL-6、IL-7、IL-8、IL-11、Kit配体(KL)、LIF、M-CSF、MIP-1α、TGF-β和TNF-α在两种细胞系中均有表达，其中L87/4细胞比L88/5细胞产生更强的细胞因子。Northern印迹结果显示，照射和IL-1α处理后，两种细胞系GM-CSF、IL-1β和LIF的mRNA水平均升高。地塞米松可降低IL-1α诱导的GM-CSF、IL-1β、IL-6、IL-11和LIF的mRNA水平，而地塞米松对IL-1α诱导的G-CSF mRNA水平无影响。L87/4和L88/5细胞表现出地塞米松依赖的KL基因表达增加，而IL-1α不能刺激KL基因的表达。
We present a detailed analysis of cytokine expression patterns of the two permanent human bone marrow stromal cell lines, L87/4 and L88/5. These cell lines, previously established in our laboratory, are highly radiotolerant without cell detachment and support long-term cultures of CD(34+)-enriched human cord blood cells. RT-PCR analysis of 22 different cytokines or cytokine receptor mRNAs showed an almost identical expression pattern in the two stromal cell lines compared to primary human Dexter-type stroma. Since stromal feeder lines employed in long-term cultures usually are irradiated and grown in media containing corticosteroids, we analyzed the impact of irradiation and dexamethasone on cytokine production in the two cell lines by RT-PCR, Northern blot analysis, bioassays, and RIAs. By RT-PCR analysis, constitutive mRNA expression of c-kit, G-CSF, GM-CSF, IL-1 beta, IL-6, IL-7, IL-8, IL-11, Kit ligand (KL), LIF, M-CSF, MIP-1 alpha, TGF-beta, and TNF-alpha was demonstrated in both cell lines, with L87/4 a more potent cytokine producer than L88/5. Northern blot data showed an increase in mRNA levels for GM-CSF, IL-1 beta, and LIF by irradiation and IL-1 alpha treatment in both cell lines. IL-1 alpha-induced GM-CSF, IL-1 beta, IL-6, IL-11, and LIF mRNA levels were reduced by the addition of dexamethasone, whereas dexamethasone had no influence on the amounts of IL-1 alpha-induced G-CSF mRNA. L87/4 and, to a lower extent, L88/5 cells showed dexamethasone-dependent increases in KL mRNA, while KL mRNA levels were not stimulated by IL-1 alpha.