Abstract: Objective　To investigate the impact of Toxoplasma gondii type I, II and II rhoptry protein 16 (ROP16) on programmed cell death ligand 1 (PD⁃L1) expression in lung adenocarcinoma cells, and to examine the effects of T. gondii type IROP16 protein on the relative PD⁃L1 expression, the relative PD⁃L1 distribution on the cell membrane surface, and the binding of programmed cell death 1 (PD⁃1) to PD⁃L1 in lung adenocarcinoma cells. Methods　Lentiviral vectors overexpressing T. gondii type I, II and III ROP16 proteins were generated, and transfected into the human lung adenocarcinoma A549 cell line. A549 cells were used as a blank control group, and A549 cells transfected with an empty lentiviral expression vector were used as a negative control group, while A549 cells transfected with lentiviral vectors overexpressing T. gondii type I, II and III ROP16 proteins served as experimental groups. Stably transfected cells were selected with puromycin and verified using Western blotting, quantitative real⁃time PCR (RT⁃qPCR), and immunofluorescence assays. The PD⁃L1 expression was quantified at translational and transcriptional levels using Western blotting and RT⁃qPCR assays in A549 cells in the five groups, and the relative PD⁃L1 distribution was detected on the A549 cell membrane surface using flow cytometry. In addition, the effect of T. gondii type I ROP16 protein on the PD⁃1/PD⁃L1 binding was measured in A549 cells enzyme⁃linked immunosorbent assay (ELISA).   Results　The relative ROP16 protein expression was 0, 0, (1.546 ± 0.091), (1.822 ± 0.047) and (2.334 ± 0.089) in the blank control group, negative control group, and the T. gondii type I, II and III ROP16 protein overexpression groups (F = 1 339.00, P < 0.001), and the relative ROP16 mRNA expression was (2.153 ± 0.949), (2.436 ± 1.614), (14.343 ± 0.020), (12.577 ± 0.285) and (15.090 ± 0.420) in the blank control group, negative control group and the T. gondii type I, II and III ROP16 protein overexpression groups, respectively (F = 483.50, P < 0.001). The ROP16 expression was higher in the T. gondii type I, II and III ROP16 protein overexpression groups than in the blank control group at both translational and transcriptional levels (all P  values < 0.001). Immunofluorescence assay revealed that T. gondii type I, II and III ROP16 proteins were predominantly localized in A549 cell nuclei. Western blotting showed that the relative PD⁃L1 protein expression was (0.685 ± 0.109), (0.589 ± 0.114), (1.007 ± 0.117), (0.572 ± 0.151), and (0.426 ± 0.116) in the blank control group, negative control group, and the T. gondii type I, II and III ROP16 protein overexpression groups (F = 9.46, P < 0.05), and RT⁃qPCR assay quantified that the relative PD⁃L1 mRNA expression was (1.012 ± 0.190), (1.281 ± 0.465), (1.950 ± 0.175), (0.889 ± 0.251), and (0.230 ± 0.192) in the blank control group, negative control group, and the T. gondii type I, II and III ROP16 protein overexpression groups (F = 14.18, P < 0.05). The PD⁃L1 expression was higher in the T. gondii type I ROP16 protein overexpression group than in the blank control group at both translational and transcriptional levels (both P values < 0.05). Flow cytometry detected that the relative distributions of PD⁃L1 protein were (10.83 ± 0.60)%, (11.23 ± 0.20)%, and (14.61 ± 0.50)% on the A549 cell membrane surface (F = 28.31, P < 0.05), and the relative distribution of PD⁃L1 protein was higher in the T. gondii type I ROP16 protein overexpression group than in the blank control group and negative control group (both P values < 0.001). ELISA measured significant differences in the absorbance (A value) among the T. gondii type I ROP16 protein overexpression group, the blank control group and the negative control group if the concentrations of the recombinant PD⁃1 protein were 0.04 (F = 10.45, P < 0.05), 0.08 μg/mL (F = 11.68, P < 0.05) and 0.12 μg/mL (F = 52.68, P < 0.05), and the A value was higher in the T. gondii type I ROP16 protein overexpression group than in the blank control group and the negative control group (both P values < 0.05), indicating that T. gondii type I ROP16 protein promoted the PD⁃L1/PD⁃1 binding in A549 cells in a concentration⁃dose manner.   Conclusions　T. gondii type I ROP16 protein overexpression may up⁃regulate PD⁃L1 expression in A549 cells at both transcriptional and translational levels and the relative PD⁃L1 distribution on the A549 cell membrane surface, and affect the PD⁃1/PD⁃L1 binding in a concentration⁃dependent manner.  

Key words: Toxoplasma gondii, Rhoptry protein 16, Lung adenocarcinoma, Programmed cell death ligand 1, Programmed cell death protein 1

摘要： 目的　观察刚地弓形虫Ⅰ、Ⅱ、Ⅲ型棒状体蛋白16（rhoptry protein 16，ROP16）对肺腺癌细胞中程序性死亡配体⁃1（programmed cell death 1 ligand 1，PD⁃LI）表达的影响，探索刚地弓形虫I型ROP16蛋白对肺腺癌细胞PD⁃L1相对表达量、细胞膜表面相对分布量及程序性死亡蛋白1（programmed cell death protein 1，PD1）PD⁃1/PD⁃L1结合的影响。方法　 分别构建过表达刚地弓形虫Ⅰ、Ⅱ、Ⅲ型ROP16蛋白慢病毒载体，并转染A549细胞系。以A549细胞为空白对照组，A549细胞转染慢病毒空表达载体为阴性对照组，过表达刚地弓形虫Ⅰ、Ⅱ、Ⅲ型ROP16蛋白的A549细胞为实验组。通过嘌呤霉素筛选稳定过表达细胞株并通过Western blotting、实时荧光定量PCR（quantitative real⁃time PCR，RT⁃qPCR）及免疫荧光实验进行验证。利用Western blotting及RT⁃qPCR技术分别检测刚地弓形虫Ⅰ、Ⅱ、Ⅲ型ROP16蛋白过表达组及空白对照组、阴性对照组A549细胞中PD⁃L1表达水平，并通过流式细胞术对细胞膜表面PD⁃L1相对分布量进行检测。以酶联免疫吸附试验（enzyme⁃linked immunosorbent assay，ELISA）为原理设计PD⁃1/PD⁃L1表面结合实验，评估A549细胞内刚地弓形虫Ⅰ型ROP16蛋白对PD⁃1/PD⁃L1结合的影响。结果　空白对照组、阴性对照组及刚地弓形虫I、II、III型ROP16过表达组中ROP16蛋白相对表达量分别为0、0、（1.546 ± 0.091）、（1.822 ± 0.047）、（2.334 ± 0.089），ROP16 mRNA相对表达量分别为（2.153 ± 0.949）、（2.436 ± 1.614）、（14.343 ± 0.020）、（12.577 ± 0.285）、（15.090 ± 0.420），差异均有统计学意义（F = 1 339.00、483.50，P均＜ 0.001）；与空白对照组比较，刚地弓形虫I、II、III型ROP16过表达组中ROP16蛋白及mRNA表达水平均升高（P均＜ 0.001）。免疫荧光实验结果显示，刚地弓形虫I、II、III型ROP16蛋白主要定位于细胞核中。空白对照组、阴性对照组及刚地弓形虫I、II、III型ROP16过表达组PD⁃L1蛋白相对表达量分别为（0.685 ± 0.109）、（0589 ± 0.114）、（1.007 ± 0.117）、（0.572 ± 0.151）、（0.426 ± 0.116），PD⁃L1 mRNA相对表达量分别为（1.012 ± 0.190）、（1.281 ± 0.465）、（1.950 ± 0.175）、（0.889 ± 0.251）、（0.230 ± 0.192），差异均有统计学意义（F = 9.46、14.18，P均< 0.05）；与空白对照组比较，刚地弓形虫I型ROP16过表达组PD⁃L1蛋白及mRNA表达水平均升高（P均 < 0.05）。流式细胞术检测结果表明，空白对照组、阴性对照组和刚地弓形虫I型ROP16过表达组PD⁃L1蛋白在A549细胞膜表面相对分布量分别为（10.83 ± 0.60）%、（11.23 ± 0.20）%、（14.61 ± 0.50）%，差异有统计学意义（F = 28.31，P < 0.05）；与空白对照组及阴性对照组比较，刚地弓形虫Ⅰ型ROP16蛋白过表组中PD⁃L1蛋白在细胞膜表面的相对分布量上调（P均＜0.001）。ELISA检测结果表明，在重组PD⁃1蛋白浓度为0.04、0.08、0.12 μg/mL时，刚地弓形虫Ⅰ型ROP16过表达组、空白对照组、阴性对照组细胞吸光度（absorbance，A）值差异均有统计学差异（F = 10.45、11.68、52.68，P均＜ 0.05），其中刚地弓形虫Ⅰ型ROP16过表达组A值均高于空白对照组及阴性对照组（P均＜ 0.05），表明刚地弓形虫Ⅰ型ROP16蛋白以浓度依赖方式促进A549细胞中PD⁃L1与PD⁃1结合。结论　过表达刚地弓形虫Ⅰ型ROP16蛋白可在mRNA及蛋白水平上调A549细胞中PD⁃L1表达及在A549细胞膜表面的相对分布量，并以浓度依赖方式影响PD⁃1/PD⁃L1结合。

关键词: 刚地弓形虫, 棒状体蛋白16, 肺腺癌, 程序性死亡配体?1, 程序性死亡蛋白1