RNA干扰曲霉菌ALP、PLB基因表达治疗真菌性角膜炎的实验研究

发布时间：2018-04-11 13:23

本文选题：烟曲霉菌 + 真菌性角膜炎　；参考：《青岛大学》2010年博士论文

【摘要】： 目的：构建RNA干扰质粒载体抑制烟曲霉菌碱性丝氨酸蛋白酶(ALP)和磷脂酶B(PLB)基因,检测ALP、PLB的表达及酶活性变化并筛选基因沉默菌株；建立鼠ALP、PLB基因沉默烟曲霉菌角膜炎动物模型,观察抑制烟曲霉菌ALP、PLB基因对真菌侵袭及角膜炎症过程的影响；利用醋酸锂转化介导的RNA干扰抑制侵袭性烟曲霉菌ALP、PLB基因,观察其对真菌性角膜炎的实验性治疗作用。方法：(1)分别设计合成针对烟曲霉菌ALP、PLB基因的dsRNA序列,构建质粒载体pALP、pPLB对烟曲霉菌进行RNA干扰,利用RT-PCR、Western-blot等方法分别检测烟曲霉菌ALP、PLB在转录和翻译水平的变化,并利用特殊培养基鉴定其酶活性改变、筛选基因沉默菌株△ALP和△PLB；(2)采用划痕法将△ALP2和△PLB1菌株接种至Wistar大鼠角膜建立ALP、PLB基因沉默烟曲霉菌角膜炎动物模型,利用免疫组织化学染色、实时荧光定量PCR、Western-blot等方法检测角膜组织中角膜组织基质金属蛋白酶2、9(MMP、2MMP-9)表达的变化,并通过组织病理学方法和临床观察检测真菌侵袭及角膜炎症的改变；(3)采用醋酸锂转化法介导pALP2、pPLB1单独或联合对鼠烟曲霉菌角膜炎动物模型进行RNA干扰实验性治疗,采用ELISA检测角膜组织中TNF-α、IL-1β表达的变化,Western-blot检测角膜组织中MMP-9、MMP-2与组织基质金属蛋白酶抑制剂1、2(TIMP-1、TIMP-2)表达比例的变化,并进行组织病理学方法与临床观察,分析RNA干扰实验性治疗的效果。结果：(1)构建的载体质粒经酶切鉴定及测序符合实验设计要求,对烟曲霉菌实施RNA干扰所获基因沉默菌株中,ALP、PLB目的基因mRNA与蛋白表达量均显著低于标准烟曲霉菌株(F=23.77-176.48,P0.01),其中△ALP2、△PLB1菌株抑制效果较好、酶活性显著低于标准菌株(q=16.082、33.507,P0.01)。(2)接种烟曲霉菌后1-8天,各组鼠角膜组织中MMP-9 mRNA及蛋白表达显著增高,而MMP-2 mRNA及蛋白表达仅于晚期出现增高。△ALP组、△PLB组角膜组织中MMP-9mRNA及蛋白表达显著低于对照组(P0.01)；△PLB组角膜组织中MMP-2mRNA及蛋白表达均显著低于对照组(P0.01),而△ALP组MMP-2 mRNA表达较对照组增高(P0.01)；组织病理学及临床观察显示△ALP组、△PLB组真菌侵袭、角膜炎症反应明显较轻。(3)对烟曲霉菌角膜炎实验性RNA干扰治疗结果显示,注射后6小时-7天对照组、pBC-hygro组(空质粒载体)角膜组织中TNF-α、IL-1β、MMP-9/TIMP-1均显著增高(P＜0.01), MMP-2/TIMP-2于晚期增高明显(PO.05),但二者间差异不明显(P0.05); pALP组、pPLB组、尤其是联合治疗组TNF-α、IL-1β、MMP-9/TIMP-1增高幅度均显著低于对照组(P＜0.01), pPLB组、联合治疗组MMP-2/TIMP-2表达低于对照组,而pALP组MMP-2/TIMP-2则于晚期轻度增高；组织病理学及临床观察显示pALP组、pPLB组尤其是联合组真菌侵袭、角膜炎症反应明显轻于对照组和pBC-hygro组。结论：(1)pALP、pPLB载体质粒构建成功,符合RNA干扰预期设计；(2)RNA干扰可有效抑制烟曲霉菌ALP和PLB的表达,但并不足以完全抑制其酶活性,可能与RNA干扰的效率有关；(3)ALP、PLB通过促进宿主角膜组织中MMP-2、MMP-9的表达,在烟曲霉菌角膜炎发病机制中发挥重要作用；(4)通过载体质粒注射、醋酸锂转化对角膜致病烟曲霉菌实施RNA干扰,单独或联合抑制其ALP、PLB基因表达,可有效减少宿主角膜中前炎症因子释放、调节MMPs/TIMPs表达平衡,抑制真菌性角膜炎的发展,有可能成为治疗角膜真菌感染的新途径。
[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.

【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R772.21

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