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  本文選題：剛地弓形蟲　切入點：ROP16　出處：《安徽醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的研究中國流行的優(yōu)勢基因型弓形蟲Chinese1型蟲株TgCtwh3與type I型RH株在誘導(dǎo)小鼠巨噬細胞偏移/極化能力上的差異性，并分析其可能存在的機制。 方法（1）RH、TgCtwh3速殖子的復(fù)蘇、傳代：RH、TgCtwh3速殖子由本實驗室保種，復(fù)蘇后按每只106速殖子腹腔接種約6-8周齡的BALB/C小鼠；接種后密切觀察，待小鼠出現(xiàn)明顯活動減少、弓背、豎毛等癥狀時，脫頸處死小鼠。置于75%酒精中約5min后，將其四肢固定于蠟板，用消毒過的剪刀、鑷子打開小鼠腹腔，，注入無菌生理鹽水沖洗腹腔再進行收集，并對腹水中的速殖子進行計數(shù)后待用;待傳代3次后，弓形蟲活力已經(jīng)完全穩(wěn)定時，可進行下一步實驗；（2）蟲株毒力實驗：將40只BALB/C小鼠分2組，每組分別腹腔接種101、102、103和104個TgCtwh3和RH速殖子，同樣劑量同時注射接種5只小鼠，觀察記錄小鼠的發(fā)病和死亡時間；（3）提取TgCtwh3速殖子的總RNA并按照逆轉(zhuǎn)錄試劑盒說明書逆轉(zhuǎn)錄合成cDNA，設(shè)計引物進行TgCtwh3的棒狀體蛋白ROP16PCR擴增，酶切鑒定后送生工測序；（4）RAW264.7細胞的培養(yǎng)和傳代；按弓形蟲：細胞為3:1的比例并分別感染TgCtWh3和RH蟲株速殖子，待培養(yǎng)24小時后先收集上清液再按照試劑盒要求分別收取細胞的總蛋白、細胞核蛋白及提取細胞的總RNA；（5）對收集的標(biāo)本分別進行蛋白印跡檢測M1/M2偏移相關(guān)轉(zhuǎn)錄因子和細胞因子stat3、pstat3、stat6、pstat6、iNOS、Arg-1、NF-κB和IκBα；熒光定量PCR檢測IL-10、IL-12p40、iNOS、Arg-1等mRNA水平；收集的上清的進行NO的檢測。 結(jié)果（1）研究結(jié)果發(fā)現(xiàn)，Chinese1基因型弓形蟲TgCtwh3株的ROP16的氨基酸序列與RH株在503位點處是相同的，均為亮氨酸（L503）；（2）TgCtwh3株的蟲株毒力稍弱于type I型的RH株；（3）TgCtwh3株和RH株感染RAW264.7細胞24h后，前者高表達iNOS和Arg-1；而后者高表達Arg-1；TgCtwh3株誘導(dǎo)巨噬細胞產(chǎn)生的NO濃度稍高于正常組和RH株感染組；（4）TgCtwh3株和RH株感染RAW264.7細胞24h后，細胞均高表達IL-12，低表達IL-10。 結(jié)論（1）TgCtwh3株與RH株在對巨噬細胞的誘導(dǎo)活化上存在差異，TgCtwh3株同時高表達iNOS和Arg 1，在表達程度上更傾向于iNOS，而RH株僅高表達Arg 1；（2）在作用RAW264.7細胞24小時后，TgCtwh3株和RH株均處在先天性保護性免疫狀態(tài)（M1），高表達IL 12，低表達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).
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R382.5

【參考文獻】

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1 周憲賓;姚成芳;;巨噬細胞M1/M2極化分型的研究進展[J];中國免疫學(xué)雜志;2012年10期

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