【作者】 朱海军；

【导师】 朱敬先； 高顺强；

【作者基本信息】 河北医科大学 ， 皮肤病与性病学， 2008， 硕士

【摘要】 目的:林生地霉(Geotrichum silvicola)为新近发现的一种罕见地霉,首次从巴西果蝇和印度柞蚕幼虫身上分离出来[1]。2002年,我科从一例脓癣患儿的皮损中分离得到一株林生地霉,首次证实该菌可以感染人类[2]。2005年,我科又自一例中毒性表皮坏死松解症患者的血液中分离出一株林生地霉,说明该菌亦可以引起系统性感染[3]。有关林生地霉的形态学、分子生物学、营养生理学等各方面均已作了详细的研究[2~5]。研究发现林生地霉血液分离株和皮损分离株在菌落形态与基因序列上略有差异。本研究采用动物实验的方法,将林生地霉血液分离株和皮损分离株分别接种于不同免疫状态的昆明小鼠皮下、腹腔及静脉,通过对实验小鼠的死亡率、28天内平均存活天数,心、肝、脾、肺、肾组织的真菌逆培养阳性率及各脏器真菌负荷量的检测,一方面来探讨机体免疫状态和感染途径对林生地霉血液分离株系统性感染的影响,另一方面来研究林生地霉血液分离株与皮损分离株在致病力方面有无差别。方法:1系统性林生地霉感染动物模型的制作将我科保存菌株活化后,接种于小鼠体内使其感染,解剖小鼠分离出恢复毒力的菌株。分离出的菌株用无菌生理盐水制成菌悬液,分别通过皮下、腹腔及静脉接种于不同免疫状态的昆明小鼠体内。2小鼠生存状况及死亡情况的观察连续观察生存保护实验各组小鼠接种后4周内的生存状况及死亡情况,并计算各组小鼠的死亡率及平均存活天数。3各脏器大体改变将分期处死的小鼠解剖,无菌条件下取其心脏、肝脏、脾脏、肺脏及肾脏,观察各脏器是否肿大或萎缩、有无颜色改变及其表面有无脓性感染灶。4组织真菌逆培养无菌条件下将各脏器研碎后分别接种于试管培养基中37℃培养,计数各组小鼠内脏逆培养阳性数。培养阳性的菌落分别移种至平皿培养基及小培养的培养基上培养,观察其菌落形态以及菌丝和孢子的形态。5脏器真菌负荷量测定无菌条件下取接种血液分离株小鼠的心脏、肝脏、脾脏、肺脏、肾脏和接种皮损分离株小鼠的肾脏,匀浆后分别进行组织的真菌平皿培养,计算各脏器的真菌负荷量。6组织病理学检查取接种血液分离株小鼠的心、肝、脾、肺、肾各脏器,用10%福尔马林溶液固定,常规脱水、透明、包埋、切片,进行HE和PAS染色,光镜下观察其组织病理学改变。结果:1小鼠感染后的生存状况免疫正常小鼠接种林生地霉后,其生活习性无明显改变。皮下接种组小鼠背部接种部位出现一结节。免疫抑制腹腔组及免疫抑制静脉组小鼠的进食量明显减少,并逐渐消瘦。2小鼠感染后的死亡情况免疫抑制腹腔组及免疫抑制静脉组在接种林生地霉后均有小鼠死亡,而其它组无死亡小鼠出现。接种血液分离株的免疫抑制腹腔组及免疫抑制静脉组小鼠的死亡率及平均存活天数与其它组比较,均有显著性差异(P <0.05);而其它各组间比较均无显著性差异(P>0.05)。在同一免疫状态和接种途径下,血液分离株与皮损分离株比较,其小鼠死亡率及平均存活天数均无显著性差异(P>0.05)。3各脏器大体改变小鼠解剖后,可见到不同程度呈紫红色的肝脾肿大及呈白色、粉色的肺脏萎缩。腹腔接种组个别小鼠脏器还可见粟粒大小脓肿。4组织真菌逆培养小鼠内脏组织培养3天后可见真菌生长,其菌落形态及镜下形态均与接种的菌株相同。免疫抑制皮下组小鼠内脏逆培养阳性率明显低于免疫抑制腹腔组及免疫抑制静脉组(P <0.05),而后两组比较其小鼠内脏逆培养阳性率无显著性差异(P >0.05);在同一组内,血液分离株与皮损分离株比较,无显著性差异(P >0.05)。平皿培养菌落初为白色粉末状,后逐渐变为酵母样,其边缘可见呈放射状的绒毛样菌丝。小培养可见大量分支分隔的菌丝及矩形、桶形关节孢子和圆形孢子。5脏器真菌负荷量接种血液分离株的各组小鼠肾脏真菌负荷量比较,有显著性差异(P <0.05);在小鼠免疫状态及接种途径相同的情况下,血液分离株与皮损分离株的小鼠肾脏真菌负荷量无显著性差异(P >0.05)。腹腔接种组及静脉接种组小鼠脾脏真菌负荷量明显高于其它脏器,差异有显著性(P <0.05)。6组织病理学检查HE染色可见组织细胞明显变性、坏死和大量急性、慢性炎细胞浸润,并可见组织细胞反应性增生。PAS染色组织内可见紫红色的菌丝、关节孢子及圆形孢子。结论:1通过皮下、腹腔及静脉接种林生地霉,均可致小鼠发生系统性感染。2林生地霉血液分离株为一种条件致病性真菌,其致病力与机体免疫状态密切相关。3小鼠的感染程度与接种途径相关,静脉途径较其它接种途径感染程度重。4无论何种接种途径,脾脏均为林生地霉血液分离株最易感染的器官。5林生地霉血液分离株与皮损分离株在致病力方面无显著性差别。更多还原

【Abstract】 Objective: Geotrichum silvicola, a new strain in Geotrichum, was firstly isolated from Brazil fly and Indian oak silkworm. In 2002, we isolated a strain from a child’s kerion-like lesions, which firstly confirmed that Geotrichum silvicola can infect human. Then it was isolated from blood of the patient suffered from toxic epidermal necrolysis in our department after three years, which demonstrated that this strain can cause systemic infection. Geotrichum silvicola was studied about morphology, molecular biology and physiology. Two strains show a little discrepancy in the colonial morphology and gene order.In this research,mice in different immune states were inoculated Geotrichum silvicola isolated from the skin lesion and blood through intradermal, intraperitoneal and intravenous pathways, respectively. By observing the death rate and mean survival time of experimented mice during 28 days , the retro-culture rate and fungi residual burden of heart, liver, spleen, lung, kidney, we want to investigate the influence of immune state and infected pathway on systemic infection with Geotrichum silvicola isolated from blood, and to compare the pathogenecity of two strains. Methods:1 Establition of animal model suffered from systemic infection of Geotrichum silvicola The preserved fungi were activated, then injected into the vena caudalis of mice. We dissected the infected mice and obtained the strain of recovered virulence, then made up conidial suspension and inoculated different immune mice through intradermal, intraperitoneal and intravenous pathways, respectively.2 Observation of mice’s survival states and death conditions We observed the survival states and death conditions of mice during four weeks, and calculated the death rate and mean survival time.3 The change of organs Dissected the sacrificed mice, took the heart, liver, spleen, lung and kidney under sterile conditions, and observed their changes of size, color, and so on.4 Fungal retro-culture The infected tissues were grinded and subcultivated onto the media of SDA. After cultured under 37℃for two weeks, we counted the positive numbers. The colonies were subcultivated onto the media of plate culture and microculture. We observed the colonial morphology and shape of hyphae, spores.5 Observation of fungi residual burden of organs The heart, liver, spleen, lung and kidney of mice that inoculated Geotrichum silvicola isolated from blood and the kidney of mice that inoculated Geotrichum silvicola isolated from the skin lesion were homogenated, then cultured on platiculture. The fungi residual burden of every organ was calculated.6 Histopathological Examination The organs of mice that inoculated Geotrichum silvicola isolated from blood were fixed, dehydrated, cleared, embedded, sliced, stained with HE and PAS respectively, then observed under light microscope.Results:1 The survival state of mice The life habits had no obvious change after inoculated with Geotrichum silvicola. The mice that inoculated fungi through intradermal pathway had a nodue on the back. The immunsuppressed mice that inoculated intraperitoneally and intravenously reduced food-intake obviously, and emaciated gradually.2 The death condition of mice The immunsuppressed groups that inoculated intraperitoneally and intravenously had dead mice, but other groups had no death. Comparing the immunsuppressed groups that inoculated intraperitoneally and intravenously with others, the death rate and mean survival time of mice had significant difference. Among other groups, they had no significant difference. In same immune state and inoculated pathway, two strains also had no significant difference.3 The change of organs Mauve intumescent liver and pink lung of the mice can be seen after dissection. The organs of mice inoculated intraperitoneally have some microabscess.4 Fungal retro-culture When visceras were cultured for three days, the fungi began to grow. The colonial morphology and shape of hyphae, spores were identical with that of inoculated strains. The fungal retro-culture rate of the immunsuppressed group that inoculated intradermally was obviously lower than the immunsuppressed groups that inoculated intraperitoneally and intravenously, while the latter groups had no significant difference. In the same group, two strains had no significant difference. In flat plate, the colony presented white power at first, and then became yeast-like type. On its periphery, there were radiate fuzz. In the microculture, there were a lot of branched septate hyphae, rectangular, tubby arthroconidia and round spores.5 Fungal residual burden of organs The kidney fungal residual burden of every group that inoculated Geotrichum silvicola isolated from blood was obviously different. The kidney fungal residual burden of mice that inoculated Geotrichum silvicola isolated from blood was similar to that of mice that inoculated Geotrichum silvicola isolated from the skin lesion. The fungal residual burden of spleen was more than other organs.6 Histopathological Examination HE dyeing showed cell degeneration and necrosis, acute or chronic inflammation, reactive hyperplasia of histocytes. PAS dyeing displayed mauve hypha, arthroconidia, round or oval spores in tela.Conclusion:1 Mice could develop systemic infection when they were injected Geotrichum silvicola through intradermal, intraperitoneal and intravenous pathways, respectively. 2 Geotrichum silvicola isolated from blood was a opportunistic fungus, its virulence was correlative with immune state.3 Infected degree of mice was relevant to inoculated pathway. The infected degree of mice injected intravenously was more serious than others.4 Spleen was the most common involved organs.5 Geotrichum silvicola from different origination showed the same pathogenicity.更多还原