可溶性CD40配体对肺癌细胞A549的生物学作用及其机制研究

Biological Effects of Soluble CD40 Ligand on Lung Cancer Cell Line A549 and Its Mechanism

背景与目的:尽管对CD40分子在B细胞中的功能已有深入的研究,但CD40在肺癌细胞中的功能目前知之甚少。本研究旨在探讨可溶性CD40配体(solubleCD40ligand,sCD40L)对肺癌细胞株A549(CD40表达阳性细胞株)的生物学作用及其相关机制。方法:采用四氮唑盐(MTT)比色、3H标记胸腺脱氧嘧啶核苷(3H-TdR)掺入法检测sCD40L对A549细胞增殖的影响,免疫荧光标记和流式细胞术测定细胞表型及细胞周期的改变,流式细胞术、逆转录聚合酶链式反应(RT-PCR)及Westernblot分析测定sCD40L对A549细胞凋亡的影响及Bcl-2、Bax基因表达的变化。结果:(1)sCD40L可抑制A549细胞的增殖(与对照组比较P<0.05)。(2)sCD40L作用72h后,A549细胞表面CD49e、CD54、TNFRⅠ及CD95L的表达犤分别为(61.2±4.8)%,(31.2±6.1)%,(42.7±5.9)%,(38.2±3.4)%犦较对照组犤分别为(34.7±2.1)%,(7.1±1.6)%,(15.2±4.1)%,(10.1±2.3)%犦明显升高,而TNFRⅡ的表达犤(8.7±0.8)%犦较对照组犤(58.1±3.6)%犦下降。(3)sCD40L作用72h后,A549细胞G1期细胞比例犤(76.0±9.1)%犦较对照组犤(56.7±6.9)%犦增加,S期细胞比例犤(10.3±5.7)%犦较对照组犤(32.7±5.5)%犦减少。(4)sCD40L在短期(72h)内并不引起A549细胞明显凋亡,但可上调Bax的表达。

BACKGROUND &OBJECTIVE: Although the roles of CD40 in B cells have been intensively studied, little is known on the function of CD40 in lung cancer cell lines. This study was to investigate biological effects of soluble CD40 ligand (sCD44L) on lung cancer cell line A549 (CD40 positive), and its possible mechanism. METHODS: A549 cells were co incubated with sCD40L, cell proliferation was detected by MTT assay and 3H TdR incorporation method. Immunofluorescence technique and flow cytometry (FCM) were used to evaluate changes in cell phenotypes and cell cycle. Cell apoptosis, and expression changes of Bcl 2 and Bax were observed by FCM, reverse transcriptase polymerase chain reaction (RT PCR), and Western blot. RESULTS: Compared with control cells, proliferation of A549 cells co incubated with sCD40L was inhibited (P< 0.05). Positve rates of cell surface molecules, CD49e,CD54,TNFRⅠ, and CD95L, in A549 cells co incubated with sCD40L for 72 h were (61.2±4.8)%, (31.2±6.1)%,(42.7±5.9)%, and (38.2±3.4)%, respectively, while those in control cells were (34.7±2.1)%, (7.1±1.6)%, (15.2±4.1)%, and (10.1±2.3)%,respectively (P< 0.05). However, positive rate of TNFRⅡin A549 cells co incubated with sCD40L[(8.7±0.8)%] was lower than that in control cells [(58.1±3.6)%] (P< 0.05). G1 phase of A549 cells treated with sCD40L for 72 h was (76.0±9.1)%, more than that of control cells [(56.7±6.9)%], while S phase of sCD40L treated A549 cells [(10.3±5.7)%] was less than that of control cells [(32.7±5.5)%]. No significant apoptosis of A549 cells was observed after co incubated with sCD40L for 72 h, but Bax expression was up regulated. CONCLUSION: sCD40L may inhibit cell proliferation, cause changes in phenotype and cell cycle of A549 cells, and alter expression of apoptosis associated gene, such as Bax.