本文選題：真菌性角膜炎 + 趨化因子��； 參考：《青島大學》2011年博士論文

【摘要】：目的 構建C57BL/6小鼠真菌性角膜炎模型并進行評價；通過C3基因敲除小鼠探討補體系統(tǒng)在真菌性角膜炎發(fā)病過程中的潛在作用；研究趨化因子在真菌性角膜炎中的表達及潛在作用。 方法 第一部分：補體系統(tǒng)在角膜真菌感染中的作用 選用近交系C57BL/6小鼠作為研究對象。將三種常見致病性真菌(茄病鐮刀菌、煙曲霉菌和白色念珠菌)配制成濃度為108CFU/ml菌液。采用表層鏡法構建真菌性角膜炎模型,即在刮除角膜上皮的小鼠角膜表面覆蓋由大鼠角膜制成的“表層鏡”,在其間注入菌液,瞼裂縫合24h后拆除縫線。在角膜接種菌液1d、3d、5d和7d時,觀察角膜潰瘍形態(tài)并進行臨床評分。將實驗動物分為正常對照組和C3基因敲除C57BL/6小鼠感染組(簡稱C3-/-組),采用表層鏡法構建茄病鐮刀菌性角膜炎模型。在角膜接種菌液1d、3d、5d和7d時,裂隙燈顯微鏡觀察角膜大體病理改變特點,并根據角膜混濁的面積、密度和表面的規(guī)則性對真菌性角膜炎的嚴重性進行臨床評分和組織病理學檢測。 第二部分：趨化因子在小鼠角膜真菌感染過程中的表達 采用基質注射法法分別構建茄病鐮刀菌和煙曲霉菌和白色念珠菌角膜炎模型�；|注射生理鹽水作為對照組。在角膜接種菌液6h、12h、24h和48h時,對模型臨床評分、真菌負荷、組織病理學進行研究,并通過RT-Q-PCR方法檢測CCL1、CCL2、CCL5、CCL11、CCL27、CXCL10、CXCL12和CCR7的mRNA表達水平。 結果 第一部分 裂隙燈顯微鏡檢查顯示,茄病鐮刀菌表面有有大量的苔被附著,煙曲霉菌潰瘍大多呈龕狀,白色念珠菌潰瘍形態(tài)不一。臨床評分顯示,菌液接種后病情呈加重趨勢,第3天時最重,之后病情開始減輕。不同真菌所致角膜大體病理改變的變化趨勢基本一致(F=3.065,P=0.103)。大體觀察和臨床評分顯示,C3-/-組茄病鐮刀菌感染后1d以內為輕度,第3d發(fā)展為中度,第5、7d惡化為重度。真菌培養(yǎng)菌落計數結果顯示,C3-/-組茄病鐮刀菌感染后3d內可見較多菌落生長,第5、7天菌落減少。正常對照組真菌負荷明顯小于C3-/-組(P0.05)組織病理學檢查顯示,正常對照組角膜潰瘍在第3天時最重,基質內可見大量炎性細胞浸潤,以多形核粒細胞為主；C3-/-組多形核粒細胞進行性增多。 第二部分 角膜接種兩種菌液后臨床評分進行升高。白色念珠菌接種后頭12h載菌量進行性升高,之后下降,然后再次升高；煙曲霉菌載菌量進行性下降。CCL2、CCL11、CCL27、CXCL10和CXCL12在接種后有升高,其中以CCL2升高最為明顯,CXCL10次之。在白色念珠菌性角膜炎中,CCL2在接種12h達到峰值,而CXCL1O在接種6h即達到峰值。在煙曲霉菌性角膜炎中,CCL2在接種24h達到峰值,而CXCL10是在接種12h達到峰值。CXCL12濃度雖有升高但遠低于CXCL10。 結論 通過表層鏡法可以成功建立C57BL/6小鼠常見致病真菌性角膜炎模型。當補體系統(tǒng)功能缺陷時,角膜真菌感染早期天然免疫防御功能明顯減弱,角膜容易發(fā)生壞死和穿孔,但感染早期角膜炎癥反應卻較補體功能正常組輕。趨化因子CCL2和CXCL10可能在真菌性角膜炎發(fā)病早期發(fā)揮重要作用。趨化因子CXCL10和CXCL12可能影響角膜新生血管的發(fā)生和發(fā)展。
[Abstract]:objective
The C57BL/6 mouse fungal keratitis model was constructed and evaluated. The potential role of the complement system in the pathogenesis of fungal keratitis was explored through C3 knockout mice, and the expression and potential effect of chemokines in fungal keratitis was studied.
Method
Part I: the role of complement system in corneal fungal infection
The C57BL/6 mice were used as the research object. Three common pathogenic fungi (Fusarium Solanum, Aspergillus fumigatus and Candida albicans) were prepared into the concentration of 108CFU/ml bacteria. The epiphytic keratitis model was constructed by the surface mirror method, which covered the "surface mirror" made from the cornea of the rat cornea on the cornea surface of the cornea that scraped the corneal epithelium. In the meantime, the bacterial fluid was injected and the eyelid cleft was sutured after 24h to dismantle the suture. When the cornea was inoculated with 1D, 3D, 5D and 7d, the corneal ulcer morphology was observed and the clinical score was observed. The experimental animals were divided into normal control group and C3 gene knockout C57BL/6 mice infection group (C3-/- group), and the Fusarium keratitis model of Solanum Solanum was constructed by the surface mirror method. At the time of 1D, 3D, 5D and 7d, the characteristics of corneal pathological changes were observed in the slit lamp microscope, and the severity of fungal keratitis was evaluated by clinical score and histopathology according to the area, density and surface regularity of corneal opacities.
The second part: expression of chemokine in mouse corneal fungal infection.
The matrix injection method was used to construct Fusarium Solanum, Aspergillus fumigatus and Candida albicans respectively. Matrix injection of saline was used as the control group. When the cornea was inoculated with 6h, 12h, 24h and 48h, the model clinical score, fungus load, histopathology were studied, and CCL1, CCL2, CCL5, CCL11, CCL27 were detected by RT-Q-PCR method. MRNA expression levels of CXCL10, CXCL12 and CCR7.
Result
Part one
The slit lamp microscopy showed that a large number of moss were attached to the Fusarium Solanum on the surface of Solanum, most of Aspergillus fumigatus ulcers were in niches, and the morphology of Candida albicans was different. The clinical score showed that the disease was aggravated after inoculation, and the condition was the heaviest at third days. The changes of the pathological changes of cornea caused by different fungi were changed. The trend was basically the same (F=3.065, P=0.103). The gross observation and clinical score showed that the infection of Fusarium Solanum in C3-/- group was mild in 1D, the development of 3D was moderate, and the 5,7d deteriorated to severe. The colony count results of fungus culture showed that more colonies could be seen in 3D, and the colony decreased at 5,7 after the infection of Fusarium Solanum in the C3-/- group, and the normal control was the normal control. The group fungal load was less than C3-/- group (P0.05) histopathological examination showed that the corneal ulcer was the heaviest in the normal control group at third days, a large number of inflammatory cells infiltrated in the matrix, and polymorphonuclear granulocyte was mainly in the C3-/- group, and the polymorphonuclear granulocyte in the group of C3-/- was increased.
The second part
The clinical score of the cornea was increased after inoculation of two kinds of bacteria. After inoculation of Candida albicans, the amount of 12h carrying bacteria increased, then decreased, and then increased again; the bacteria carrying amount of Aspergillus fumigatus decreased.CCL2, CCL11, CCL27, CXCL10 and CXCL12 increased after inoculation, among which CCL2 was the most obvious, CXCL10 time. In keratitis, CCL2 reached its peak at 12h inoculation, while CXCL1O reached its peak at 6h. In Aspergillus fumigatus keratitis, CCL2 reached its peak at 24h, while CXCL10 was higher but far lower than CXCL10. in the peak.CXCL12 concentration of 12h.
conclusion
The common pathogenic fungal keratitis model of C57BL/6 mice can be successfully established by the surface mirror method. When the function defect of the complement system, the early natural immune defense function of the corneal fungal infection is obviously weakened, the cornea is prone to necrosis and perforation, but the early keratitis response to the infection is lighter than the complement function normal group. Chemokine CCL2 and CXCL 10 may play an important role in the early stage of fungal keratitis. Chemokines CXCL10 and CXCL12 may affect the occurrence and development of corneal neovascularization.
【學位授予單位】：青島大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R772.21

【參考文獻】

相關期刊論文 前7條

1 劉敬,謝立信,史偉云,馬志中;建立兔淺層白色念珠菌性角膜炎模型[J];山西醫(yī)科大學學報;2004年04期

2 孫旭光,張巖,李然,王智群,羅時運,金秀英,張文華;Etiological analysis on ocular fungal infection in the period of 1989-2000[J];Chinese Medical Journal;2004年04期

3 王麗婭,張月琴,王印其,王剛生,盧嘉彪,鄧潔華;中國三地區(qū)真菌性角膜病致病菌種的調查[J];中華眼科雜志;2000年02期

4 張文華,潘志強,王智群,金秀英,羅士運,鄒洋,武宇影,李然;化膿性角膜潰瘍常見致病菌的變遷[J];中華眼科雜志;2002年01期

5 鐘文賢;謝立信;史偉云;孫士營;;真菌性角膜炎654例感染譜分析[J];中華醫(yī)學雜志;2006年24期

6 王麗婭,孫聲桃,張月琴,王印其,祝磊,李家臣,徐筠;河南地區(qū)真菌性角膜病致病菌種調查[J];中國實用眼科雜志;2003年03期

7 宋書華,林躍生,黎明,孫明霞,梁志光,陳龍山,陳家祺;真菌性角膜炎的病原學分析[J];中國實用眼科雜志;2005年05期

，

本文編號：2033443