本文選題：煙曲霉 + KpsF基因��； 參考：《山西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景:煙曲霉是一種常見的腐生真菌,侵襲性曲霉病(Invasive aspergillosis,IA)約90%由煙曲霉引起,其病死率呈上升趨勢(shì),其治療成為當(dāng)前臨床常見的疑難問題。鈣調(diào)磷酸酶(Calcineurin,Ca N)在病原真菌的形態(tài)發(fā)生和毒性方面等的重要作用使其成為一個(gè)有吸引力的抗真菌靶點(diǎn)。在煙曲霉研究中,Ca N定位于菌絲的尖端和分隔處,對(duì)煙曲霉致病性有顯著的影響。研究表明Ca N發(fā)揮作用同Px Ix IT模序密切相關(guān),鈣調(diào)磷酸酶底物或靶蛋白中如Crz1、SIm1、SIm2、Hph1、Cbp A中都含有類似Px Ix IT模序,而在煙曲霉Kps F基因序列中也有類似模序存在。雖然鈣調(diào)磷酸酶抑制劑FK506和環(huán)孢素A在現(xiàn)代移植醫(yī)學(xué)應(yīng)用非常成功;但是與鈣調(diào)磷酸酶相關(guān)的系統(tǒng)毒副作用使其臨床抗真菌應(yīng)用受到極大限制。因此,研究煙曲霉Kps F基因功能,以期發(fā)現(xiàn)新的內(nèi)源性鈣調(diào)磷酸酶抑制劑,為侵襲性曲霉病的治療提供新的思路。目的:1.構(gòu)建煙曲霉Kps F基因缺陷株和Kps F基因過表達(dá)株,初步了解Kps F基因?qū)熐箯较蛏L和形態(tài)學(xué)的影響。2.通過體外抗真菌藥物實(shí)驗(yàn)初步明確Kps F基因?qū)ΤＳ每拐婢幬锩舾行杂绊憽?.初步明確Kps F基因與鈣調(diào)磷酸酶和鈣離子體內(nèi)平衡的相互關(guān)系。4.通過構(gòu)建綠色熒光定位株初步探究Kps F基因在菌絲生長中的定位情況。方法:1.使用原生質(zhì)體法構(gòu)建煙曲霉Kps F基因缺陷株,并用Southern blot驗(yàn)證。2.使用p UCGH質(zhì)粒作為載體,以潮霉素作為篩選標(biāo)記,并通過原生質(zhì)體法構(gòu)建Kps F基因過表達(dá)株。3.將對(duì)照菌株Ku80、△Kps F和Kps F過表達(dá)菌株這三種菌株配成不同濃度菌懸液,接種一定濃度于固體培養(yǎng)基上,分別于24 h,48 h,72 h,96 h,120 h記錄菌落直徑,觀察菌落生長狀況,并作統(tǒng)計(jì)分析;使用小培養(yǎng)的方法對(duì)菌株進(jìn)行形態(tài)學(xué)研究,培養(yǎng)48 h,滴加乳酸酚棉蘭染液進(jìn)行觀察。4.使用斑點(diǎn)法探究對(duì)照菌株Ku80、△Kps F和Kps F過表達(dá)菌株這三種菌株抗真菌藥物的敏感性,在加有抗真菌藥物的基礎(chǔ)培養(yǎng)基上點(diǎn)種菌懸液,分別于24 h,48 h,72 h,96 h,120 h記錄菌落直徑,觀察菌落生長狀況,并統(tǒng)計(jì)分析。5.RT-PCR檢測(cè)不同濃度Ca2+液體培養(yǎng)基條件下三種不同菌株的Cna A和Vcx A基因m RNA表達(dá)水平,并作統(tǒng)計(jì)分析。6.使用激光共聚焦顯微鏡觀察定位株Kps F-EGFP在菌絲生長中的定位情況。結(jié)果:1.成功構(gòu)建煙曲霉?Kps F,Kps F基因過表達(dá)菌株,Kps F基因綠色熒光定位株(實(shí)驗(yàn)室已經(jīng)構(gòu)建并保存)。2.煙曲霉?Kps F和Ku80徑向生長結(jié)果未見明顯差異(P0.05),過表達(dá)株徑向生長結(jié)果相對(duì)于對(duì)照菌株Ku80下降約45%。3.煙曲霉?Kps F和對(duì)照菌株Ku80形態(tài)差別不大,Kps F過表達(dá)株菌絲分枝較多,排列不整齊,其隔膜相對(duì)較少;分生孢子頭相對(duì)短小,頂囊發(fā)育不良,頂囊上的單層小梗不明顯。4.當(dāng)卡泊芬凈藥物濃度為1.0~5.0 ug/ml時(shí),?Kps F出現(xiàn)較小的矛盾生長變化,Ku80和Kps F過表達(dá)菌株未見明顯變化。?Kps F,Ku80和Kps F過表達(dá)菌株對(duì)伊曲康唑、伏立康唑、兩性霉素B和FK506四種抗真菌藥體外藥物敏感性,經(jīng)過多次重復(fù)測(cè)量方差分析統(tǒng)計(jì)未見明顯差異(P0.05)。5.在10 m M Ca Cl2濃度下,?Kps F的Cna A基因表達(dá)量顯著高于對(duì)照菌株Ku80和Kps F過表達(dá)株,?Kps F的Cna A表達(dá)量是Ku80的29.76倍(P0.01),Kps F過表達(dá)株是對(duì)照菌株Ku80的1.56倍(P0.05)。而在100 m M濃度下,三種菌株Cna A表達(dá)量差別不明顯。普通GMM培養(yǎng)基,?Kps F的Cna A表達(dá)量是Ku80的2.81倍,Kps F過表達(dá)株和Ku80無差別。6.在10 m M Ca Cl2濃度下,Kps F過表達(dá)株Vcx A基因表達(dá)量明顯高于Ku80和?Kps F,Kps F過表達(dá)株是Ku80的2.65倍,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。?Kps F和Ku80相比無明顯變化。而在普通GMM培養(yǎng)基和100 m M條件下三種菌株Vcx A基因表達(dá)差異無統(tǒng)計(jì)學(xué)意義。7.煙曲霉Kps F-EGFP的綠色熒光均勻分布在菌絲細(xì)胞膜處。結(jié)論:1.Kps F基因缺陷對(duì)于煙曲霉徑向生長沒有影響,Kps F基因過表達(dá)使煙曲霉生長受限。2.Kps F基因缺陷對(duì)煙曲霉生長形態(tài)沒有明顯影響,但是Kps F基因過表達(dá)影響菌絲排列,分枝的角度,其分生孢子頭相對(duì)短小,頂囊發(fā)育不良,頂囊上的單層小梗不明顯。3.Kps F基因缺陷與Kps F基因過表達(dá)均對(duì)體外抗真菌藥物敏感性無明顯影響。4.在低濃度Ca2+條件下,Kps F基因可能參與鈣調(diào)磷酸酶的負(fù)反饋調(diào)節(jié),并且在10 m M Ca2+條件下負(fù)反饋調(diào)節(jié)作用明顯,在高濃度Ca2+條件下該負(fù)反饋調(diào)節(jié)作用卻受到抑制;Kps F基因?qū)τ阝}離子體內(nèi)平衡有一定的影響。5.煙曲霉Kps F基因可能與菌絲的細(xì)胞膜有關(guān)。
[Abstract]:Background: Aspergillus fumigatus is a common saprophytic fungus. Invasive aspergillosis (IA) is caused by Aspergillus fumigatus, which is caused by Aspergillus fumigatus. The mortality of Aspergillus fumigatus is on the rise, and its treatment has become a common difficult problem. The important role of calcineurin (Calcineurin, Ca N) in the morphogenesis and toxicity of pathogenic fungi makes it become an important factor. In the study of Aspergillus fumigatus, Ca N is located at the tip and division of mycelium, which has a significant effect on the pathogenicity of Aspergillus fumigatus. The study shows that Ca N plays a close relationship with the Px Ix IT motif, and the calcineurin substrate or target protein, such as Crz1, SIm1, SIm2, Hph1, contains similar patterns in Cbp. Similar motif exists in the Kps F gene sequence of Aspergillus fumigatus. Although calcineurin inhibitor FK506 and cyclosporine A are very successful in modern transplant medicine, the systemic toxic side effects associated with calcineurin can greatly restrict the clinical antifungal application of Aspergillus fumigatus. Therefore, the study of the function of Aspergillus fumigatus Kps F gene is expected to be found. New endogenous calcineurin inhibitors provide new ideas for the treatment of invasive aspergillosis. Objective: 1. construction of Aspergillus fumigatus Kps F gene defect strain and Kps F gene overexpressed strain, preliminary understanding of the effect of Kps F gene on the radial growth and morphology of Aspergillus fumigatus,.2. through an in vitro antifungal drug test, preliminarily clarified the common resistance of Kps F gene to the common antifungal agent. The influence of fungal drug susceptibility on the interaction between Kps F gene and calcineurin and calcium ions in the body.4. by constructing a green fluorescent location plant to explore the location of Kps F gene in the growth of mycelium. Method: 1. using protoplast method to construct Aspergillus fumigatus Kps F gene defective strain, and use Southern blot to verify.2.. 1. P UCGH plasmid was used as a carrier, hygromycin was used as a screening marker, and the Kps F overexpression strain.3. was constructed by Protoplast.3., and the control strain Ku80, Delta Kps F and Kps F overexpressed strain were mixed with different concentration strains, and inoculated on solid medium, respectively, in 24 h, 48 h, 72, 96 and 120. Diameter, observe the colony growth condition, and make statistical analysis; use the small culture method to carry on the morphological study to the strain, culture 48 h, add the lactate cotton blue dye to observe.4. using dot method to explore the control strain Ku80, Delta Kps F and Kps F overexpressed strain the sensitivity of antifungal drugs, in addition to antifungal drugs 24 h, 48 h, 72 h, 96 h, and 120 h were recorded on the basic culture medium, and the colony diameter was recorded, and the growth status of the colony was observed. The expression level of Cna A and Vcx A gene of three different strains of Ca2+ liquid culture medium with different concentration Ca2+ was statistically analyzed, and a statistical analysis was made by laser confocal microscopy. Location of Kps F-EGFP in the growth of mycelium. Results: 1. the successful construction of Aspergillus fumigatus, Kps F, Kps F gene overexpressed strain, Kps F gene green fluorescent location strain (laboratory established and preserved).2. Aspergillus fumigatus, Kps F and Ku80 radial growth results, the results of overexpressed plant radial growth relative to the control bacteria The strain Ku80 decreased about 45%.3. Aspergillus fumigatus? Kps F and the control strain Ku80 had little difference in morphology, Kps F overexpressed mycelium branches, irregular arrangement and less septum; the spore head was relatively short, the top capsule was poorly developed, the single peduncle on the top capsule was not obviously.4. when the concentration of cappofinn was 1.0~5.0 ug/ml, and Kps F appeared smaller. The overexpressed strains of Ku80 and Kps F did not change significantly. The sensitivity of the four antifungal agents of Kps F, Ku80 and Kps F to itraconazole, voriconazole, amphotericin B and FK506 in vitro was no significant difference (P0.05) under the concentration of 10 The expression of a A gene was significantly higher than that of the control strain Ku80 and Kps F overexpressed strain, and the Cna A expression of Kps F was 29.76 times of Ku80 (P0.01), and the Kps expressed strain was 1.56 times as high as that of the control strain. The expression of F overexpressed strain and Ku80.6. at the concentration of 10 m M Ca Cl2, the Vcx A gene expression of Kps F overexpression strain was significantly higher than that of Ku80 and 2.65 times. The green fluorescence of Kps F-EGFP of Aspergillus fumigatus.7. was distributed uniformly at the membrane of mycelium cell. Conclusion: 1.Kps F gene defect has no effect on the radial growth of Aspergillus fumigatus. Kps F gene overexpression makes the F gene defect of Aspergillus fumigatus.2.Kps F gene has no obvious influence on the growth morphology of Aspergillus fumigatus, but the Kps F gene overexpression affects the growth of Aspergillus fumigatus. Mycelium arrangement, branching angle, its conidium head is relatively short, the top capsule is dysplastic, the single layer on the top capsule is not obvious.3.Kps F gene defect and Kps F gene overexpression have no obvious effect on anti fungal drug sensitivity in vitro.4. under the low concentration Ca2+ condition, Kps F basis may participate in the negative feedback regulation of calcineurin. Under the condition of 10 m M Ca2+, the negative feedback regulation is obvious, and the negative feedback regulation is inhibited under the high concentration of Ca2+, and the Kps F gene has a certain effect on the balance of calcium ions in the body of calcium ions, and the F gene of.5. Aspergillus fumigatus Kps may be related to the cell membrane of the mycelium.

【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R519

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