尾静脉注射牙龈卟啉单胞菌对野生大鼠海马神经干细胞和神经元标志分子表达的影响  

Effects of Porphyromonas gingivalis injected through tail vein on the expressions of biomarkers in neural stem cells and neurons of wild-type rats hippocampus

作　　者：庾靖君 雷双[1] 李福龙 陈双双 唐晓琳[1] Yu Jingjun;Lei Shuang;Li Fulong;Chen Shuangshuang;Tang Xiaolin(Department of Periodontics,School and Hospital of Stomatology,China Medical University,Liaoning Provincial Key Laboratory of Oral Diseases,Shenyang 110002,China)

机构地区：[1]中国医科大学口腔医学院·附属口腔医院牙周病科,辽宁省口腔疾病重点实验室,沈阳110002

出　　处：《中华口腔医学杂志》2022年第4期375-383,共9页Chinese Journal of Stomatology

基　　金：辽宁省教育厅2021年度科学研究经费项目(LJKZ0782)。

摘　　要：目的观察尾静脉注射牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)对野生成年大鼠海马神经干细胞和神经元标志分子表达水平的影响,初步探讨Pg入血对海马神经发生的作用。方法建立尾静脉注射Pg大鼠模型:18只雄性Sprague-Dawley(SD)大鼠按随机数字表法随机分为3组(每组6只)。低剂量组、高剂量组大鼠分别经尾静脉注射1.0×10^(3)和1.0×10^(8)菌落形成单位(colony forming unit,CFU)的Pg菌液200μl,假手术组大鼠注射等体积磷酸盐缓冲液(phosphate buffer saline,PBS),3组大鼠均每周注射3次,连续8周。行为学检测:应用莫里斯水迷宫(Morris water maze,MWM)定位航行实验、空间探索实验检测大鼠学习和记忆能力。免疫组织化学法检测各组大鼠海马颗粒下区(subgranular zone,SGZ)神经干细胞标志分子神经上皮干细胞蛋白(nestin)、神经母细胞和未成熟神经元标志分子双皮质素(doublecortin,DCX)、成熟神经元标志分子神经元核抗原(neuronal nuclei,NeuN)的阳性细胞分布。蛋白质印迹法检测各组大鼠海马组织nestin、DCX、NeuN的表达水平。结果学习和记忆能力:MWM定位航行实验结果显示,第5天到达平台时间高剂量组[22.83(16.00,38.34)s]显著长于假手术组[5.59(5.41,6.17)s](t=-11.17,P<0.001),低剂量组[9.85(8.75,21.01)s]与假手术组相比差异无统计学意义(t=-6.83,P=0.080);MWM空间探索实验中60 s内穿越原平台位置次数,高剂量组[1.50(1.00,2.00)次]显著少于假手术组[4.00(2.75,4.00)次](t=9.75,P=0.003),低剂量组[2.50(2.00,3.00)次]与假手术组相比差异无统计学意义(t=4.50,P=0.382)。免疫组织化学法结果显示,对于nestin阳性细胞密度,低剂量组[(35.36±4.32)个/mm^(2)]和高剂量组[(26.51±5.89)个/mm^(2)]均显著低于假手术组[(59.58±14.15)个/mm^(2)](t=24.21,P=0.018;t=33.07,P=0.005);DCX阳性细胞平均吸光度值,低剂量组(0.007±0.002)和高剂量组(0.006±0.002)均显著低于假手术组(0.011±0.001)(t=0.004,P=0.Objectives To study the effects of Porphyromonas gingivalis(Pg)injected through tail vein on the molecular expression levels of biomarkers of neural stem cells(NSC)and neurons in the hippocampus of wild-type adult rats,and the effects on hippocampal neurogenesis.Methods Eighteen male Sprague-Dawley(SD)rats were randomly divided into 3 groups based on the table of random numbers(n=6 in each group).In low-intensity group and high-intensity group,rats were injected intravenously through tail vein with 200μl Pg ATCC33277[1.0×10^(3) and 1.0×10^(8) colony forming unit(CFU),respectively]3 times per week for 8 weeks.In the sham group,200μl of phosphate buffer saline(PBS)was given instead.Behavioral tests:the navigation and the exploration tests using Morris water maze(MWM)were applied to evaluate learning and memory ability of rats.Immunohistochemistry was performed to detect cells positively expressing nestin,doublecortin(DCX)and neuronal nuclei(NeuN)in the subgranular zone(SGZ)of rats in each group.Western blotting was used to evaluate the expression levels of nestin,DCX and NeuN in rat hippocampus.Results Learning and memory abilities:on day 5 of navigation test,the lagency time was 22.83(16.00,38.34)s in the high-intensity group,significantly longer than the sham group[5.59(5.41,6.17)s](t=-11.17,P<0.001).There were no significant differences between the low-intensity group[9.85(8.75,21.01)s]and the sham group(t=-6.83,P=0.080).Results in the exploration test showed that,in the high-intensity group,the number of fime crossing over the previous platform area within 60 s was 1.50(1.00,2.00),significantly less than the sham group[4.00(2.75,4.00)](t=9.75,P=0.003);no significant differences between the low-intensity group[2.50(2.00,3.00)]and the sham one(t=4.50,P=0.382).Immunohistochemistry showed that the nestin+cell density in the low-intensity group[(35.36±4.32)cell/mm^(2)]and high-intensity group[(26.51±5.89)cell/mm^(2)]were significantly lower than the sham group[(59.58±14.15)cell/mm^(2)](t=24.21,P=0.018;t=33.07

关 键 词：紫单胞菌 龈 菌血症 大鼠 SPRAGUE-DAWLEY 海马 认知障碍 神经干细胞 成年海马神经发生 

分 类 号：R781.42[医药卫生—口腔医学]