肺特异性X蛋白转染表达对A549肺癌株生物学功能的影响

Anti - proliferation and inducing apoptosis effects of lung - specific X protein on A549 lung cancer cell lines

目的观察肺特异性x蛋白（LUNX）转染表达对A549肺癌细胞株的增殖凋亡和生物学功能的影响。方法应用慢病毒基因转染表达方法检测LUNX表达对A549肺癌细胞株的抗增殖作用和生物学功能的影响；通过免疫组织化学法和实时定量聚合酶链反应（Real-timePCR）技术检测LUNX一小干扰RNA（siRNA）在A549肺癌株中的表达水平，Real—timePCR测定转染后A549肺癌细胞中增殖指标溴化脱氧尿嘧啶核苷（BrdUrd），碱性磷酸酶（AKP）和活性标志物表面活性蛋白C（SPC）的mRNA水平表达变化，并通过显微镜、苏木素．伊红（HE）染色、透射电镜等观察凋亡细胞的形态学改变。结果Real-timePCR和免疫细胞化学证实转染后A549肺癌细胞中有LUNX稳定表达；Real．timePCR结果示：A组AKP、SPC和BrdUrd密度为（0．73±0．14）、（0．53±0．12）、（O．31±0．10）U／L，B组为（1．64±0．38）、（1．47±0．25）、（1．28±0．21）U／L，C组为（1．71±0．45）、（1．53±0．32）、（1．33±0．26）U／L。与B、C对照组比较，A组AKP、SPC和BrdUrd扩增产物条带吸光度（A）值均显著降低（P〈0．01）。噻唑蓝（MTr）比色法表明：随着LUNX—siRNA与A549肺癌细胞效靶比增加及作用时间延长，抑制率明显增强（P〈0．05）。相同作用时间不同比例之间差异也均有统计学意义（P〈0．05），同一比例不同作用时间之间差异有统计学意义（P〈0．01）。LUNX-siRNA作用于肺癌细胞48h后，形态学观察肺癌细胞发生凋亡或坏死，与对照组比较，差异有统计学意义（P〈0．05）。结论体外制备的LUNX—siRNA对肺癌A549细胞具有抑制增殖和促进凋亡作用，其可能是通过通过负性调控信号通路降低A549肺癌细胞株中AKP、SPC和BrdUrd的表达。

Objective To study the anti - proliferation and inducing apoptosis effects of lung - specific X protein （LUNX） on A549 lung cancer cell lines. Methods The anti - proliferation and the cyto- toxicity of LUNX on A549 lung cancer cell line were detected by methyl thiazol tetrazolium （MTY） assay. To observe LUNX expression in A549 lung cancer cell lines, and the mRNA level were evaluated with real - time quantitative polymerase chain reaction （Real- time PCR）. The morphological changes of the apeptosis cell were observed by invert microscope, hematoxylin and eosin （HE） stain, transmission electron micro- scope and acridine orange / ethidium bromide （AO/EB） double fluorescent staining. To analyze by using SPSS 14. 0 statistical software. Results The LUNX expression in Asg9 cells transfected with LUNX - small interfering RNA （siRNA） was confirmed by Real -time PCR and immunocytochemical method, respectively. Alkaline phosphatase （AKP）, surfactant protein C （SPC） and bromodeoxyuridine （BrdUrd） in group A were （0.73 ±0. 14）, （0.53 ±0. 12） and （0.31 ±0. 10） U/L, in group B were （1.64 ±0.38）, （1.47 ± 0. 25） and （ 1.28 ±0. 21） U/L, in group C were （1.71±0. 45）, （1.53 ±0. 32） and （ 1.33±0. 26） U/L. Compared with that in the control group, AKP, SPC and BrdUrd mRNA level decreased significantly in PEFLUNX transfected A549 cells （P 〈 0. 05 ）. MTT assay showed that the inhibitory rate enhanced obvi- ously with the addition of effect/target rate and extension of time （ P 〈 0. 05 ）. Lung cancer cell apoptosis or necrosis were morphologically observed after lung cancer cells were induced by LUNX for 48 hours （ P 〈 0. 05 ）. Conclusion LUNX are highly effective protein which have a stronger anti - proliferation and inducing apoptosis effects on lung cancer ceils. LUNX protein might inhibit the proliferation of A549 cells by decreasing the expression of AKP, SPC and BrdUrd regulate pathway negatively.