本文選題：煙曲霉菌 + 真菌性角膜炎　； 參考：《青島大學(xué)》2010年博士論文

【摘要】： 目的：構(gòu)建RNA干擾質(zhì)粒載體抑制煙曲霉菌堿性絲氨酸蛋白酶(ALP)和磷脂酶B(PLB)基因,檢測(cè)ALP、PLB的表達(dá)及酶活性變化并篩選基因沉默菌株；建立鼠ALP、PLB基因沉默煙曲霉菌角膜炎動(dòng)物模型,觀(guān)察抑制煙曲霉菌ALP、PLB基因?qū)φ婢忠u及角膜炎癥過(guò)程的影響；利用醋酸鋰轉(zhuǎn)化介導(dǎo)的RNA干擾抑制侵襲性煙曲霉菌ALP、PLB基因,觀(guān)察其對(duì)真菌性角膜炎的實(shí)驗(yàn)性治療作用。方法：(1)分別設(shè)計(jì)合成針對(duì)煙曲霉菌ALP、PLB基因的dsRNA序列,構(gòu)建質(zhì)粒載體pALP、pPLB對(duì)煙曲霉菌進(jìn)行RNA干擾,利用RT-PCR、Western-blot等方法分別檢測(cè)煙曲霉菌ALP、PLB在轉(zhuǎn)錄和翻譯水平的變化,并利用特殊培養(yǎng)基鑒定其酶活性改變、篩選基因沉默菌株△ALP和△PLB；(2)采用劃痕法將△ALP2和△PLB1菌株接種至Wistar大鼠角膜建立ALP、PLB基因沉默煙曲霉菌角膜炎動(dòng)物模型,利用免疫組織化學(xué)染色、實(shí)時(shí)熒光定量PCR、Western-blot等方法檢測(cè)角膜組織中角膜組織基質(zhì)金屬蛋白酶2、9(MMP、2MMP-9)表達(dá)的變化,并通過(guò)組織病理學(xué)方法和臨床觀(guān)察檢測(cè)真菌侵襲及角膜炎癥的改變；(3)采用醋酸鋰轉(zhuǎn)化法介導(dǎo)pALP2、pPLB1單獨(dú)或聯(lián)合對(duì)鼠煙曲霉菌角膜炎動(dòng)物模型進(jìn)行RNA干擾實(shí)驗(yàn)性治療,采用ELISA檢測(cè)角膜組織中TNF-α、IL-1β表達(dá)的變化,Western-blot檢測(cè)角膜組織中MMP-9、MMP-2與組織基質(zhì)金屬蛋白酶抑制劑1、2(TIMP-1、TIMP-2)表達(dá)比例的變化,并進(jìn)行組織病理學(xué)方法與臨床觀(guān)察,分析RNA干擾實(shí)驗(yàn)性治療的效果。結(jié)果：(1)構(gòu)建的載體質(zhì)粒經(jīng)酶切鑒定及測(cè)序符合實(shí)驗(yàn)設(shè)計(jì)要求,對(duì)煙曲霉菌實(shí)施RNA干擾所獲基因沉默菌株中,ALP、PLB目的基因mRNA與蛋白表達(dá)量均顯著低于標(biāo)準(zhǔn)煙曲霉菌株(F=23.77-176.48,P0.01),其中△ALP2、△PLB1菌株抑制效果較好、酶活性顯著低于標(biāo)準(zhǔn)菌株(q=16.082、33.507,P0.01)。(2)接種煙曲霉菌后1-8天,各組鼠角膜組織中MMP-9 mRNA及蛋白表達(dá)顯著增高,而MMP-2 mRNA及蛋白表達(dá)僅于晚期出現(xiàn)增高�！鰽LP組、△PLB組角膜組織中MMP-9mRNA及蛋白表達(dá)顯著低于對(duì)照組(P0.01)；△PLB組角膜組織中MMP-2mRNA及蛋白表達(dá)均顯著低于對(duì)照組(P0.01),而△ALP組MMP-2 mRNA表達(dá)較對(duì)照組增高(P0.01)；組織病理學(xué)及臨床觀(guān)察顯示△ALP組、△PLB組真菌侵襲、角膜炎癥反應(yīng)明顯較輕。(3)對(duì)煙曲霉菌角膜炎實(shí)驗(yàn)性RNA干擾治療結(jié)果顯示,注射后6小時(shí)-7天對(duì)照組、pBC-hygro組(空質(zhì)粒載體)角膜組織中TNF-α、IL-1β、MMP-9/TIMP-1均顯著增高(P＜0.01), MMP-2/TIMP-2于晚期增高明顯(PO.05),但二者間差異不明顯(P0.05); pALP組、pPLB組、尤其是聯(lián)合治療組TNF-α、IL-1β、MMP-9/TIMP-1增高幅度均顯著低于對(duì)照組(P＜0.01), pPLB組、聯(lián)合治療組MMP-2/TIMP-2表達(dá)低于對(duì)照組,而pALP組MMP-2/TIMP-2則于晚期輕度增高；組織病理學(xué)及臨床觀(guān)察顯示pALP組、pPLB組尤其是聯(lián)合組真菌侵襲、角膜炎癥反應(yīng)明顯輕于對(duì)照組和pBC-hygro組。結(jié)論：(1)pALP、pPLB載體質(zhì)粒構(gòu)建成功,符合RNA干擾預(yù)期設(shè)計(jì)；(2)RNA干擾可有效抑制煙曲霉菌ALP和PLB的表達(dá),但并不足以完全抑制其酶活性,可能與RNA干擾的效率有關(guān)；(3)ALP、PLB通過(guò)促進(jìn)宿主角膜組織中MMP-2、MMP-9的表達(dá),在煙曲霉菌角膜炎發(fā)病機(jī)制中發(fā)揮重要作用；(4)通過(guò)載體質(zhì)粒注射、醋酸鋰轉(zhuǎn)化對(duì)角膜致病煙曲霉菌實(shí)施RNA干擾,單獨(dú)或聯(lián)合抑制其ALP、PLB基因表達(dá),可有效減少宿主角膜中前炎癥因子釋放、調(diào)節(jié)MMPs/TIMPs表達(dá)平衡,抑制真菌性角膜炎的發(fā)展,有可能成為治療角膜真菌感染的新途徑。
[Abstract]:Objective: to construct the RNA interference plasmid vector inhibition of Aspergillus fumigatus alkaline serine protease (ALP) and phospholipase B (PLB) gene, detection of ALP, changes of expression and activity of PLB and screening of gene silencing strains; establish rat ALP, PLB gene silencing of Aspergillus fumigatus keratitis animal model, to observe the inhibitory effect of Aspergillus fumigatus ALP, PLB gene on invasion and fungal keratitis process; RNA interference mediated transformation using lithium acetate inhibited the invasive Aspergillus fumigatus ALP, PLB gene and observe its therapeutic effect on experimental fungal keratitis. Methods: (1) respectively according to the design and synthesis of Aspergillus fumigatus ALP, dsRNA sequence of PLB gene, plasmid vector pALP pPLB RNA interference against Aspergillus fumigatus using RT-PCR, Western-blot and other methods were used to detect Aspergillus fumigatus ALP, PLB changes in the level of transcription and translation, and use the special culture medium identified the enzyme activity change, screening Gene silencing strains of delta ALP and delta PLB; (2) the scratch method of delta ALP2 and delta PLB1 strains were inoculated to Wistar rat corneal establishment of ALP, PLB gene silencing of Aspergillus fumigatus keratitis animal model, using immunohistochemical staining, real-time quantitative PCR Western-blot method detected the cornea tissue in metal matrix protease 2,9 (MMP, 2MMP-9) expression, and by histopathological method and clinical observation on detection of fungal keratitis and invasion changes; (3) using lithium acetate transformation method mediated by pALP2, alone or in combination with pPLB1 on rat animal model of Aspergillus fumigatus keratitis bacteria RNA interference experimental treatment with TNF- alpha ELISA cornea tissue detection, change the expression of IL-1, MMP-9, Western-blot and MMP-2 in the detection of corneal tissue, tissue inhibitor of matrix metalloproteinase 1,2 (TIMP-1, TIMP-2) level of proportion, and histopathology The clinical observation and study method, analysis of experimental treatment by RNA interference effect. Results: (1) vector plasmid by restriction enzyme digestion and sequencing experiments accord with the design requirements, the interference of RNA gene silencing strains, the implementation of Aspergillus fumigatus ALP, expression of PLB gene and mRNA protein were significantly lower than the standard smoke Aspergillus strains (F=23.77-176.48, P0.01), the delta ALP2, Delta PLB1 strains have good inhibition effect, the enzyme activity was significantly lower than that of standard strains (q=16.082,33.507, P0.01). (2) 1-8 days after inoculation of Aspergillus fumigatus, rats in corneal tissue expression of MMP-9 protein and mRNA increased significantly, while the MMP-2 mRNA and protein expression in later increased. ALP group, PLB group, the corneal tissue and protein expression of MMP-9mRNA was significantly lower than the control group (P0.01) were significantly lower than the control group; the expression of MMP-2mRNA and protein in corneal tissue in the PLB group (P0.01), and group MMP-2 mRNA ALP Was higher than that in control group (P0.01); clinical observation and histopathological examination showed a ALP group, PLB group of fungi invasion, the inflammatory reaction was significantly lighter. (3) display of Aspergillus fumigatus keratitis experimental RNA interference treatment results, 6 hours after injection -7 day pBC-hygro group (control group, empty plasmid vector) TNF- alpha, IL-1 beta in corneal tissue, MMP-9/TIMP-1 were significantly increased (P < 0.01), MMP-2/TIMP-2 (PO.05) increased significantly in the late stage, but the difference between the two is not obvious (P0.05); pALP group, pPLB group, especially the combined treatment group of TNF- alpha, IL-1 beta, MMP-9 increased /TIMP-1 amplitude were significantly lower than that of the control group (P < 0.01), pPLB group, combined treatment group MMP-2/TIMP-2 was lower than that in control group, pALP group and MMP-2/TIMP-2 in the late stage increased slightly and clinical observation of histopathology; pALP group, pPLB group, especially the combined group of fungal invasion, the inflammatory reaction was significantly lower than that in control group and pBC-hy Gro group. Conclusion: (1) pALP, pPLB vector plasmid was successfully constructed with RNA interference expected design; (2) RNA interference can effectively inhibit the expression of Aspergillus fumigatus ALP and PLB, but not enough to completely inhibit the enzyme activity, may be related to the efficiency of RNA interference; (3) ALP, PLB by promoting MMP-2 host in corneal tissue, the expression of MMP-9, play an important role in the pathogenesis of Aspergillus fumigatus keratitis; (4) by injection of the plasmid, the implementation of lithium acetate transformation of RNA interference on corneal pathogenic Aspergillus fumigatus, alone or in combination with the inhibition of ALP, PLB gene expression, which can effectively reduce the release of proinflammatory cytokines in the host membrane. Regulating the expression of MMPs/TIMPs, inhibit the development of fungal keratitis, may become a new way of treatment for corneal fungal infection.

【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R772.21

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