【摘要】： 目的：短帚霉(Scopulariopsis brevicaulis)是一種腐生性條件致病真菌,通常引起甲真菌病,較少引起其他部位的感染,但目前國外已經有關于深部組織感染的報道。此菌分離于我科門診病人,經中國醫(yī)學科學院微生物研究所鑒定為Scopulariopsis brevicaulis。 本研究一方面通過對短帚霉在四種不同培養(yǎng)基上的培養(yǎng)情況進行觀察,以便尋找出適合其生長的培養(yǎng)基及溫度；另一方面,應用氟康唑、伊曲康唑和特比萘芬三種常用抗真菌藥物治療小鼠的短帚霉系統(tǒng)感染,通過對試驗小鼠生存率以及心、肝、脾、肺組織的真菌逆培養(yǎng)陽性率和腎臟組織載菌量的檢測來評價它們在小鼠體內抗短帚霉的活性,為臨床上治療短帚霉系統(tǒng)感染提供實驗室依據；并通過組織病理學檢查,為進一步了解此菌引起小鼠的病理形態(tài)學改變提供實驗室依據。 方法： 1形態(tài)學觀察試驗 1.1大體形態(tài) 取我科保存菌種常溫下復蘇,3天后轉種于沙堡氏瓊脂培養(yǎng)基(SDA)、馬鈴薯葡萄糖瓊脂(PDA)、米飯培養(yǎng)基和玉米吐溫80瓊脂上,分別置37℃和27℃溫箱培養(yǎng)2周,每日測量并記錄菌落直徑,觀察菌落形態(tài)。 1.2平皿培養(yǎng)的光鏡下觀察每日分別挑取37℃和27℃的四種培養(yǎng)基的菌落標本置于載玻片上,經乳酸酚棉藍染色,光鏡下觀察。 2生理學實驗 2.1尿素酶試驗將菌落接種于尿素瓊脂培養(yǎng)基上,室溫培養(yǎng)1周,觀察培養(yǎng)基變色情況。 3體內藥敏試驗 3.1短帚霉系統(tǒng)感染動物模型的制作： 取我科保存菌種復蘇轉種,用無菌生理鹽水制備成菌懸液,通過腹腔注射途徑接種至應用環(huán)磷酰胺免疫抑制的小鼠體內,每只小鼠腹腔注射菌懸液為0.5ml。 3.2生存觀察 接種后2h開始給藥,氟康唑、伊曲康唑、特比萘芬均為10mg/(kg.d),腹腔注射,每天1次,連續(xù)5天。連續(xù)觀察各組小鼠接種后的一般狀況及小鼠接種后21天內的自然死亡情況,記錄死亡日期和每天的死亡只數。 3.3組織逆培養(yǎng) 接種真菌懸液后的第6天各組分別處死組織載菌量部分小鼠,取出心、肝、脾、肺分別置于組織研磨器中,研碎后取少許組織分3點接種于改良沙堡氏固體培養(yǎng)基,置于27℃恒溫箱中培養(yǎng)。 3.4腎臟組織載菌量(CFU)的測定 分別取氟康唑組、伊曲康唑組、特比萘芬組和對照組中的15只小鼠,每組的15只小鼠各取一側的腎臟,組織研磨器研碎后,用無菌生理鹽水稀釋至1ml,取10微升組織懸液均勻涂布于平皿培養(yǎng)基表面,27℃恒溫箱培養(yǎng),48h后讀取菌落數。 3.5組織病理學檢查 取各臟器表面肉眼可見病理改變組織,用10%福爾馬林溶液常溫固定,常規(guī)脫水、透明、包埋、切片,HE和PAS染色,光鏡下觀察組織病理學變化。 結果： 1形態(tài)學觀察試驗 1.1繪制的生長曲線 在SDA和PDA兩種培養(yǎng)基上,短帚霉于培養(yǎng)的第1周生長較快,其生長速度與時間呈正比；在兩種溫度下,27℃較37℃生長快。在米飯培養(yǎng)基和吐溫80兩種培養(yǎng)基上生長速度始終較慢,27℃較37℃生長稍快。 1.2菌落直徑 同種培養(yǎng)基在不同溫度下其菌落直徑的比較結果顯示,于生長7天和14天時,各培養(yǎng)基在27℃的菌落直徑均比37℃菌落直徑大,有顯著性差異(P0.05)。 27℃不同培養(yǎng)基上,生長第7天和14天時,菌落直徑均以PDA最大,SDA次之,米飯培養(yǎng)基和吐溫80更小。生長第7天和14天時,PDA與SDA菌落差異無顯著性(P0.05),米飯培養(yǎng)基和吐溫80菌落差異無顯著性(P0.05),其余各培養(yǎng)基之間均有顯著性差異(P0.05)。 1.3大體菌落觀察培養(yǎng)3-4天,菌落開始生長,初為白色膜樣菌落,生長迅速,以后變?yōu)榛液稚?1.4光鏡下觀察可見分枝分隔菌絲,帚狀枝,分生孢子自環(huán)孢子梗長出一串,呈球形,壁厚,表面光滑或粗糙。 2生理學實驗 2.1尿素酶實驗呈陽性。 3生存率觀察 氟康唑組在給藥后21天的存活只數為17只,第6天開始死亡,第7天為死亡高峰；伊曲康唑組在給藥后21天的存活只數為16只,第6天開始死亡,第7天為死亡高峰；特比萘芬組在給藥21天的存活只數為15只,第6天開始死亡,第8天為死亡高峰；對照組在給藥后21天的存活只數為9只,第2天開始死亡,第7天為死亡高峰。 4真菌逆培養(yǎng)結果 氟康唑治療組、伊曲康唑治療組和特比萘芬治療組小鼠心肺組織真菌逆培養(yǎng)陽性率生理鹽水對照組低,有顯著性差異(P0.05)。余下各組的真菌逆培養(yǎng)陽性率比較均無差異(P0.05)。 5腎臟組織載菌量 伊曲康唑治療組、氟康唑治療組和特比萘芬治療組中小鼠的腎臟組織載菌量較生理鹽水對照組均有顯著減少(P0.05)；余下各組的腎臟組織載菌量比較均無顯著性差異(P0.05)。 6病理學變化蘇木精-伊紅染色早期為急性炎細胞浸潤,可見組織細胞壞死,后期可見組織細胞及多核細胞反應性增生。PAS染色可見紫紅色的菌絲和圓形孢子。 結論： 1沙堡氏瓊脂培養(yǎng)基(SDA)、馬鈴薯葡萄糖瓊脂(PDA)均適于短帚霉生長,米飯培養(yǎng)基和玉米吐溫80瓊脂不適于其生長；與37℃溫度相比較,短帚霉更適于在27℃下生長；短帚霉的尿素酶實驗為陽性。 2氟康唑、伊曲康唑和特比萘芬在小鼠體內均具有良好的抗短帚霉活性,在小鼠的心、肺組織中有較高的真菌清除率,小鼠的腎臟組織載菌量顯著減少,明顯提高了小鼠生存率。 3對于短帚霉感染的小鼠來說,氟康唑、伊曲康唑和特比萘芬的治療效果無明顯差異。
[Abstract]:Objective: Sopulariopsis brevicalis is one of the pathogenic fungi of saprochomycosis, which usually causes onychomycosis, and less causes the infection of other parts, but there are reports of deep tissue infection at present. The bacterium was isolated from the outpatients of our family and was identified as Sopulariopsis brevicalis by the Institute of Microbe of the Chinese Academy of Medical Sciences. one aspect of the present study is to find a culture medium and a temperature suitable for its growth, on the one hand, by observing the culture of the broom in four different media; on the other hand, the application The effect of the three common antifungal agents, such as fluconazole, ilaconic and tebiprofen, was used to treat the short-broom system infection in mice and to evaluate the survival rate of the test mice and the positive rate of the fungal reverse culture of the heart, the liver, the spleen, the lung tissue, and the detection of the amount of the tissue of the kidney. The activity of the invention is to provide a laboratory basis for the clinical treatment of the infection of the broom fungus system, and the pathological change of the mouse is provided by the histopathological examination to further understand the pathological change of the mouse caused by the bacteria. chamber basis . Method: 1 form The general morphology of the test 1.1 was first recovered at normal temperature, and three days later, it was transferred to the Sabouraud's agar medium (SDA), and the potato dextrose agar (P (DA), rice culture medium and corn Tween 80 agar, respectively, and incubated at 37 & deg; C and 27 & deg; C for 2 weeks. The colony diameter was measured and recorded daily, and the colony morphology was observed. The specimen is placed on the carrier glass. On-chip, stained with lactic acid phenol and cotton blue, and observed under light microscope. Inoculate the colonies to the urine The culture was cultured at room temperature for 1 week, and the culture was observed at room temperature. Color-raising and color-changing conditions. In-vivo drug sensitivity test: 3.1. 3.1 Production of an animal model of the infection of the short-broom-mold system: the preservation of the strain in the family is taken to restore the strain. transferring, preparing a bacterial suspension by using sterile physiological saline, and over-abdominal injection Inoculated to mice with immunosuppression with cyclic fosfamine, with a suspension of 0.5 ml for each mouse's intraperitoneal injection of bacteria. 3.2 Survival observation The administration was started at 2 h after the inoculation, and the dose of fluor-kang, irqu-kang and the specific ratio of fenofene was 10 mg/ (kg. d). The intraperitoneal injection was carried out once a day. 5 days. Continuous observation The general condition after vaccination in the group and the natural death in 21 days after the inoculation of the mice, the date of death and the number of deaths per day were recorded. The tissue-carrying amount of the tissue was sacrificed in the group, respectively. The mouse, the heart, the liver, the spleen and the lung were placed in the tissues, respectively. In the grinder, a small amount of tissue was taken and a small group of tissue was inoculated at 3 points in the modified Sabdberg solid culture medium and placed in an incubator at 27.degree. C., and the determination of the amount of bacterial carrier (CFU) in the kidney was determined separately 15 of the 15 mice in each group were taken from each group of 15 mice in the group consisting of the group consisting of the group consisting of the group consisting of a group consisting of a group consisting of a group consisting of a group consisting of a group consisting of a group consisting of a group consisting of a group consisting of a group of grinding of the kidney and the tissue of the tissue After crushing, dilute to 1 ml with sterile saline, and uniformly coat the 10 microliters of tissue suspension on the surface of the culture medium, culture at 27.degree. C., and read the number of colonies after 48 h. Neo-Confucianism Check and take all the dirty Visual and pathological changes of the surface of the device The tissues were fixed at normal temperature with 10% formalin, and the normal dehydration, transparency, embedding, slicing, HE and PAS staining were used to observe the pathological changes of the tissues under light microscope. Fruit:1 Morphological observation test 1.1 The growth curve plotted in SDA and P DA two media In the first week of culture, the growth rate of the short-broom was proportional to the time, and at the two temperatures, the growth rate was fast at 27 鈩，

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