目的 了解长链非编码RNA102796（lncRNA102796）在刚地弓形虫慢性感染小鼠脑组织中的差异表达及其作用机制。 方法 取SPF级雌性BALB/c小鼠构建弓形虫慢性感染小鼠模型。感染后2个月,取感染小鼠（10只）和健康对照小鼠（10只）脑组织,提取总RNA以及神经细胞的细胞质和细胞核RNA,采用实时定量PCR（qRT-PCR）检测小鼠脑组织中lncRNA102796的表达和在神经细胞中的定位。生物信息学分析预测lncRNA102796的靶基因为阿片受体1（opioid receptor delta 1,oprd1）基因。取感染小鼠和健康对照小鼠脑组织,提取总RNA和细胞蛋白,qRT-PCR检测脑组织中oprd1 mRNA相对转录水平,蛋白质免疫印迹（Western blotting）检测脑组织中OPRD1的表达情况。构建lncRNA102796的干扰质粒（干扰lncRNA102796）和过表达质粒（过表达lncRNA102796）,分别以未干扰lncRNA102796的质粒pGPU6/GFP/Neo和未过表达lncRNA102796的质粒pcDNA3.1+为干扰对照和过表达对照。将质粒转染至神经小胶质细胞（BV-2）,提取细胞内RNA,qRT-PCR检测lncRNA102796的干扰和过表达对oprd1 mRNA相对转录水平的影响;提取蛋白质,Western blotting检测lncRNA102796的干扰和过表达对OPRD1表达的影响。取干扰和过表达质粒及2种对照质粒转染后的BV-2细胞,细胞计数试剂盒（CCK-8）实验检测lncRNA102796对细胞增殖的调节作用。构建过表达正义和过表达反义lncRNA102796质粒,提取2种质粒转染的BV-2细胞的RNA和蛋白质,RNA-蛋白沉降（RNA pull down）和Western blotting分析与lncRNA102796结合的蛋白。 结果 qRT-PCR检测结果显示,弓形虫慢性感染小鼠脑组织中lncRNA102796相对转录水平为0.303 ± 0.054,较健康小鼠下调（69.7 ± 6.7）%（t = 18.12,P < 0.05）;lncRNA102796在细胞核内的表达[（85.04 ± 9.41）%]高于细胞质[（14.95 ± 9.41）%]（t = 7.45,P < 0.05）。qRT-PCR检测结果显示,感染小鼠脑组织中oprd1 mRNA相对转录水平为0.170 ± 0.040,较健康对照小鼠下调了（83.0 ± 5.3）%（t = 27.17,P < 0.05）;Western blotting检测结果显示,感染小鼠脑组织中的OPRD1蛋白表达量较健康对照小鼠减少。干扰lncRNA102796后,lncRNA102796相对转录水平为0.311 ± 0.054,较对照组下调了（68.9 ± 6.6）%（t = 18.00,P < 0.05）,oprd1 mRNA相对转录水平为0.175 ± 0.040,较对照组下调（82.5 ± 5.1）%（t = 28.08,P < 0.05）,OPRD1蛋白的表达量较对照组减少;过表达lncRNA102796后,lncRNA102796相对转录水平为8.220 ± 1.192,较对照组上调（722.0 ± 146.0）%（t = 8.56,P < 0.05）,oprd1 mRNA相对转录水平为2.533 ± 0.365,较对照组上调（153.3 ± 44.7）%（t = 5.95,P < 0.05）,OPRD1蛋白表达量较对照组增加。CCK-8实验结果显示,干扰lncRNA102796可抑制神经小胶质细胞的增殖,培养48和72 h后,A₄₅₀值分别为0.272 ± 0.021、0.508 ± 0.014,低于对照组的0.473 ± 0.024、0.816 ± 0.014（t = 6.35、46.77,P < 0.05）;过表达lncRNA102796可促进小胶质细胞的增殖,培养48、72 h后,A₄₅₀值分别为0.621 ± 0.038、1.026 ± 0.114,高于对照组的0.365 ± 0.010、0.530 ± 0.147（t = 10.55、5.17,P < 0.05）。RNA pull down后,经Western blotting检测结果显示,OPRD1可以与lncRNA102796结合。 结论 在弓形虫慢性感染的小鼠脑组织中,lncRNA102796通过与靶基因oprd1结合抑制小胶质细胞的增殖,影响细胞周期,从而导致神经细胞的损伤。

Objective To investigate the differential expression and mechanism of long non-coding RNA102796 (lncRNA102796) in the brain of mice with chronic Toxoplasma gondii infection. Methods The model for chronic T. gondii infection was established using specific-pathogen-free female BALB/c mice. Two months after infection, the brains of infected mice (n = 10) and healthy mice (n = 10) were collected two months after infection. The expression of lncRNA102796 in mice chronically infected with T. gondii was detected by qRT-PCR, using the total RNA extracted from the brains of infected mice. The location of lncRNA102796 was determined by qRT-PCR using RNA which is extracted from the nucleus and cytoplasm of mice brains. The target gene of lncRNA102796 was predicted as opioid receptor delta 1 (oprd1) gene by bioinformatics analysis. The expression of oprd1 was further determined by qRT-PCR using the total RNA from the brains of infected mice and healthy mice. The expression of OPRD1 was further determined by Western blotting, using the brains of the infected mice. The interference and overexpression plasmids of lncRNA102796, the uninterfered lncRNA102796 plasmid pGPU6/GFP/Neo and non-overexpressed lncRNA102796 pcDNA3.1+ plasmid were used as control. The plasmids were transfected into BV-2 cells. The regulation of lncRNA102796 expression on oprd1 was investigated by qRT-PCR, using the RNA extracted from BV-2 cells. The regulation of lncRNA102796 expression on OPRD1 was investigated by western blotting, using protein extracted from BV-2 cells. The regulation of lncRNA102796 expression on cell proliferation was investigated by cell counting kit-8 assay, using BV-2 cells transfected with constructed plasmids. Constructed overexpression plasmids of anti-sense lncRNA102796 and sense lncRNA102796. The protein binding to lncRNA102796 was determined by RNA pull down and Western blotting, using the RNA and protein extracted from the overexpression BV-2 cells. Results qRT-PCR results confirmed that the expression of lncRNA102796 in the brain of mice infected chronically with T. gondii was 0.303 ± 0.054, which was down-regulated by (69.7 ± 6.7)% (t = 18.12, P < 0.05) compared to uninfected mice. The expression of lncRNA102796 was higher in the nucleus (85.04 ± 9.41)% than that in the cytoplasm (14.95 ± 9.41)% of BV-2 cells (t = 7.45, P < 0.05). The results from qRT-PCR suggested that the expression of oprd1 mRNA was 0.170 ± 0.040 in the infected mice brain, which was down-regulated (83.0 ± 5.3)% (t = 27.17, P < 0.05) compared to that of the normal mouse brain. Results from Western blotting suggested the expression of OPRD1 protein was down-regulated in the infected mice brain compared to that of normal mouse brain; Upon interfering lncRNA102796, the expression of lncRNA102796 was 0.311 ± 0.054, which was down-regulated by (68.9 ± 6.6)% compared to the control group (t = 18.00, P < 0.05); the expression of oprd1 was 0.175 ± 0.04, which was down-regulated by (82.5 ± 5.1)% compared to the control group (t = 28.08, P < 0.05); the expression of oprd1 protein also reduced. Upon overexpressing lncRNA102796, the expression of lncRNA102796 was 8.220 ± 1.192, which was up-regulated by (722.0 ± 146.0)% compared to the control group (t = 8.56, P < 0.05); the expression of oprd1 was 2.533 ± 0.365, which was up-regulated by (153.3 ± 44.7)% compared to the control group (t = 5.95, P < 0.05); the expression of oprd1 protein also increased. Results from CCK-8 assay revealed that interfered lncRNA102796 inhibited the proliferation of microglia. After 48 and 72 hours, the A₄₅₀ of BV-2 cells were 0.272 ± 0.021, 0.508 ± 0.014, respectively, which are lower than the A₄₅₀ of the control group 0.473 ± 0.024, 0.816 ± 0.014 (t = 6.35, 46.77, P < 0.05). Overexpression of lncRNA102796 can accelerate the proliferation of microglia. After 48, 72 h, the A₄₅₀ of BV-2 cells were 0.621 ± 0.038 and 1.026 ± 0.114, which were higher than the A₄₅₀ of control group 0.365 ± 0.010 and 0.530 ± 0.147 (t=10.55, P < 0.05). Results from Western blotting showed that RNA pull down confirmed that sense lncRNA102796 binds to OPRD1. Conclusion In the brain of mice chronically infected with T. gondii, lncRNA102796 suppresses microglial cell proliferation, affecting the cell cycle through binding to the target gene oprd1, thereby, causing neurological cell impairment.