弓形虫Chinese1基因型虫株诱导巨噬细胞偏移应答的研究

发布时间：2018-03-15 22:34

本文选题：刚地弓形虫　切入点：ROP16　出处：《安徽医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的研究中国流行的优势基因型弓形虫Chinese1型虫株TgCtwh3与type I型RH株在诱导小鼠巨噬细胞偏移/极化能力上的差异性，并分析其可能存在的机制。方法（1）RH、TgCtwh3速殖子的复苏、传代：RH、TgCtwh3速殖子由本实验室保种，复苏后按每只106速殖子腹腔接种约6-8周龄的BALB/C小鼠；接种后密切观察，待小鼠出现明显活动减少、弓背、竖毛等症状时，脱颈处死小鼠。置于75%酒精中约5min后，将其四肢固定于蜡板，用消毒过的剪刀、镊子打开小鼠腹腔，，注入无菌生理盐水冲洗腹腔再进行收集，并对腹水中的速殖子进行计数后待用;待传代3次后，弓形虫活力已经完全稳定时，可进行下一步实验；（2）虫株毒力实验：将40只BALB/C小鼠分2组，每组分别腹腔接种101、102、103和104个TgCtwh3和RH速殖子，同样剂量同时注射接种5只小鼠，观察记录小鼠的发病和死亡时间；（3）提取TgCtwh3速殖子的总RNA并按照逆转录试剂盒说明书逆转录合成cDNA，设计引物进行TgCtwh3的棒状体蛋白ROP16PCR扩增，酶切鉴定后送生工测序；（4）RAW264.7细胞的培养和传代；按弓形虫：细胞为3:1的比例并分别感染TgCtWh3和RH虫株速殖子，待培养24小时后先收集上清液再按照试剂盒要求分别收取细胞的总蛋白、细胞核蛋白及提取细胞的总RNA；（5）对收集的标本分别进行蛋白印迹检测M1/M2偏移相关转录因子和细胞因子stat3、pstat3、stat6、pstat6、iNOS、Arg-1、NF-κB和IκBα；荧光定量PCR检测IL-10、IL-12p40、iNOS、Arg-1等mRNA水平；收集的上清的进行NO的检测。结果（1）研究结果发现，Chinese1基因型弓形虫TgCtwh3株的ROP16的氨基酸序列与RH株在503位点处是相同的，均为亮氨酸（L503）；（2）TgCtwh3株的虫株毒力稍弱于type I型的RH株；（3）TgCtwh3株和RH株感染RAW264.7细胞24h后，前者高表达iNOS和Arg-1；而后者高表达Arg-1；TgCtwh3株诱导巨噬细胞产生的NO浓度稍高于正常组和RH株感染组；（4）TgCtwh3株和RH株感染RAW264.7细胞24h后，细胞均高表达IL-12，低表达IL-10。结论（1）TgCtwh3株与RH株在对巨噬细胞的诱导活化上存在差异，TgCtwh3株同时高表达iNOS和Arg 1，在表达程度上更倾向于iNOS，而RH株仅高表达Arg 1；（2）在作用RAW264.7细胞24小时后，TgCtwh3株和RH株均处在先天性保护性免疫状态（M1），高表达IL 12，低表达IL 10。
[Abstract]:Objective to study the difference of the migration / polarization ability of mouse macrophages induced by TgCtwh3 and type I RH, a dominant genotype of Toxoplasma gondii (Toxoplasma gondii) in China, and to analyze its possible mechanism. Methods the resuscitation of TgCtwh3 TgCtwh3 tachyzoites was carried out in our laboratory. After resuscitation, TgCtwh3 tachyzoites were inoculated intraperitoneally with each 106-8 week old BALB/C mice. After placed in 75% alcohol for about 5 minutes, the limbs were fixed to the wax plate, and the disinfected scissors, tweezers and tweezers were used to open the abdominal cavity of the mice and inject sterile saline to wash the abdominal cavity and collect them. After counting the tachyzoites in ascites and after three passages, when the activity of Toxoplasma gondii was completely stable, the next step was to test the virulence of Toxoplasma gondii strain: 40 BALB/C mice were divided into 2 groups. Each group was intraperitoneally inoculated with 101,102,103 and 104 TgCtwh3 and RH tachyzoites respectively, and 5 mice were inoculated with the same dose at the same time. The total RNA of TgCtwh3 tachyzoites was extracted and synthesized by reverse transcription according to the instructions of reverse transcription kit. Primers were designed to amplify the rodlike protein ROP16PCR of TgCtwh3. The culture and passage of RAW264.7 cells were identified by enzyme digestion and sequenced. Toxoplasma gondii were infected with Toxoplasma gondii at the ratio of 3: 1 and infected with Tachyzoites of TgCtWh3 and RH strains, respectively. After 24 hours of culture, the supernatant was collected and then the total protein of the cell was collected according to the kit requirements. Nuclear protein and total RNAs of extracted cells were detected by Western blotting, and the levels of mRNA such as IL-10IL-12p40iNOSN Arg-1 were detected by fluorescence quantitative PCR and no was detected in the supernatant of the collected supernatant by Western blotting. Results 1) the results showed that the amino acid sequence of ROP16 of Toxoplasma gondii TgCtwh3 strain of Chinese1 genotype was the same as that of RH strain at 503locus, and the virulence of TgCtwh3 strain was slightly weaker than that of RH type I strain RH strain 3TgCtwh3 strain and RH strain RH strain after 24 hours of infection with RAW264.7 cell, the results showed that the amino acid sequence of Toxoplasma gondii strain TgCtwh3 was the same as that of RH strain. The former overexpressed iNOS and Arg-1, while the latter overexpressed Arg-1TgCtwh3 strain induced macrophage no concentration slightly higher than normal group and RH strain infected with TgCtwh3 strain and RH strain infected with RAW264.7 cells for 24 hours, the expression of IL-12 was higher than that of RH strain, and the expression of IL-10 was lower than that of RH strain. Conclusion there is a difference between TgCtwh3 strain and RH strain in inducing activation of macrophages. TgCtwh3 strain has high expression of both iNOS and Arg 1, and RH strain is more inclined to express iNOS1, while RH strain only has high expression of TgCtwh3 strain and RH strain of RH strain after 24 hours of treatment with RAW264.7 cell line TgCtwh3 and RH strain. All of them were in congenital protective immunity, high expression of IL ~ (12) and low expression of IL ~ (10).
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R382.5

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