免疫抑制小鼠阿萨希丝孢酵母菌皮肤感染差异表达基因的初步筛选
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摘要
目的 观察免疫抑制小鼠和健康小鼠在遭遇条件致病真菌——阿萨希丝孢酵母菌初期，皮肤组织基因表达的差异；筛选出宿主抵抗该真菌感染的免疫相关基因，并探讨这些基因及其表达产物在防御阿萨希丝孢酵母菌感染发生过程当中的作用，从真菌感染免疫学角度提高我们对于阿萨希丝孢酵母菌所导致的局部感染及系统性感染——播散性毛孢子菌病发病机制的认识。
方法 12周龄、BCLB／C、雄性小鼠，共12只，随机分成试验组和对照组，在接种条件致病真菌阿萨希丝孢酵母菌前3天，试验组小鼠给予环磷酰胺腹腔注射(200 mg／kg)，使该组小鼠的免疫功能受到抑制，对照组小鼠保持正常的免疫功能。3天后，选取小鼠腹部皮肤，皮内接种阿萨希丝孢酵母菌细胞混悬液0.2ml(浓度为1×10~5CFU／ml，其中孢子比例＞95％)。接种后24h，处死全部小鼠，解剖获取皮损组织，提取总RNA，，用不同的荧光染料Cy5(红色)、Cy3(绿色)分别对实验组、对照组RNA经逆转录所获的cDNA进行标记，将标记后的cDNA与同一小鼠基因表达谱芯片进行杂交，洗片后用不同波长的激光对芯片进行照射，来源于试验组与对照组的cDNA因标记荧光染料的不同而分别产生红色或绿色荧光，用scan array 4000扫描仪对芯片杂交荧光信号进行扫描，并用Gene Pix3.0图像处理软件对所获信号强度进行分析。通过对两种荧光信号相对强度的分析，得到实验、对照两组基因表达的差异情况，利用Genebank及Pubmed等数据库对所获全部差异表达基因的功能进行分析，从而筛选出免疫功能相关的差异表达基因，并对这些基因的表达产物在宿主抗阿萨希丝孢酵母菌感染过程中所发挥的作用进行讨论。
结果 在芯片所包含的4096条小鼠基因中，试验组与对照组之间共有243条基因的表达存在差异，经过对这些基因表达产物功能的全面分析，共筛选出11条与小鼠抗阿萨希丝孢酵母菌感染免疫相关的差异表达基因，其中试验组编码C2、CD22、CD53、CD209a、
第三军医大学硕士学位论文
CD79A结合蛋白lb、EP4受体、淋巴细胞抗原6复合体、肿瘤坏死因子可诱导蛋白cg12一1、
干扰素诱导蛋白PU以一G、工gG的FC高亲和力受体1的基因表达下调，编码结合珠蛋白
的基因表达上调。
结论本试验尝试性的利用基因表达谱芯片分析技术，对免疫抑制小鼠和健康小鼠
在感染阿萨希丝抱酵母菌24小时后，皮损局部基因的表达情况进行了初步的比较性研
究，共筛选出编码CZ、CD53、结合珠蛋白等11条与免疫相关的差异表达基因，这些基
因表达的变化，导致了其编码产物数量的改变，使宿主对阿萨希丝抱酵母菌的正常免疫
应答功能发生障碍，而导致该菌感染的发生及播散。如免疫抑制组补体CZ基因的显著
下调，将导致补体系统激活障碍，使膜攻击复合体的形成减少，对阿萨希丝抱酵母菌的
溶细胞作用减弱，而C3b等具有调理作用的补体片段生成减少，将减弱巨噬细胞及中性
粒细胞对该菌的吞噬作用;免疫抑制组CD53基因表达下调，使巨噬细胞在氧化应激状
态下的自我保护能力降低，致使巨噬细胞的功能、数量发生改变，使宿主对阿萨希丝抱
酵母菌的清除能力降低;编码EP4受体、CDZOga基因的下调将导致皮肤组织局部
Langethans及其它树突状细胞游出、移行发生障碍、减弱了该类细胞对阿萨希丝抱酵母
菌的抗原提呈能力;试验组编码结合珠蛋白的基因表达上调，一方面可以防止过度急性
炎症反应的产生，而另一方面也可能为阿萨希丝抱酵母菌存活及播散提供了有利的条
件;筛选出的其它功能尚不明确的差异表达基因，可能具有潜在的研究价值，可作为进
一步研究阿萨希丝抱酵母菌感染发生机制的候选基因。
Objective To screen out the differential expressed genes between the immunosuppressed and healthy mice after intradermal inoculation of Trichosporon asahii. Finding out the potential function of those genes in the interaction between the mice and the fungus, and the potential pathways lead to the local fungal infection onset and further dissemination.
Methods twelve BALB/C mice randomly divided into two groups. Three days before the inoculation of the fungi, mice in the test group were treated with cyclophosphamide, so as to result in the suppression of their immunological function, and the mice in the control group keep in the normal immunological condition. 24 hours after intradermal inoculation of 0.2ml of the fungal preparation which contain the Trichosporon asahii (concentration is 1 X 105CFU/ml,and the percentage of spore >95%) , then all the mice were killed and autopsied. Total RNA of the murine inoculated local cutaneous were extracted, reverse transcripted, the cDNA of the test group and the control were respectively labelled with fluorochrome dyes Cy5(red) or Cy3(green), then hybridized with one DNA chips which contained 4096 murine genes, then the signal on the chips scanned and analyzed.
Results 243 genes which were differentially expressed between the test group and the control were screened out, through the analyzing of the function of all those genes, we fixed our attention on 11 genes which thought to be playing an important role in the event of the skin infectious process of T.asahii. They are ten down-regulated genes in the test group, respectively encoding C2, CD22, CD53,CD209a, CD79A binding protein Ib, Fcgrl (Fc receptor, IgG, high affinity I), interferon-induced protein with tetratricopeptide repeats 3, lymphocyte antigen 6 complex, TNF-inducible protein cg12-1, prostaglandin E receptor 4, and one up-regulated gene encoding haptoglobin.
Conclusions The immune function of the immunosuppressed mice was inhibited in many important aspects, such as complement activation, phagocytosis of the macrophage, emigration of the dendritic cell, inflammatory reaction etc. These changes may play an important role in the process of T.asahii infection. Many genes screened out in our experiement, which with unclear function can be considered as candidate genes for the further study of the pathogenesis of the Trichosporonosis.
方法 12周龄、BCLB／C、雄性小鼠，共12只，随机分成试验组和对照组，在接种条件致病真菌阿萨希丝孢酵母菌前3天，试验组小鼠给予环磷酰胺腹腔注射(200 mg／kg)，使该组小鼠的免疫功能受到抑制，对照组小鼠保持正常的免疫功能。3天后，选取小鼠腹部皮肤，皮内接种阿萨希丝孢酵母菌细胞混悬液0.2ml(浓度为1×10~5CFU／ml，其中孢子比例＞95％)。接种后24h，处死全部小鼠，解剖获取皮损组织，提取总RNA，，用不同的荧光染料Cy5(红色)、Cy3(绿色)分别对实验组、对照组RNA经逆转录所获的cDNA进行标记，将标记后的cDNA与同一小鼠基因表达谱芯片进行杂交，洗片后用不同波长的激光对芯片进行照射，来源于试验组与对照组的cDNA因标记荧光染料的不同而分别产生红色或绿色荧光，用scan array 4000扫描仪对芯片杂交荧光信号进行扫描，并用Gene Pix3.0图像处理软件对所获信号强度进行分析。通过对两种荧光信号相对强度的分析，得到实验、对照两组基因表达的差异情况，利用Genebank及Pubmed等数据库对所获全部差异表达基因的功能进行分析，从而筛选出免疫功能相关的差异表达基因，并对这些基因的表达产物在宿主抗阿萨希丝孢酵母菌感染过程中所发挥的作用进行讨论。
结果 在芯片所包含的4096条小鼠基因中，试验组与对照组之间共有243条基因的表达存在差异，经过对这些基因表达产物功能的全面分析，共筛选出11条与小鼠抗阿萨希丝孢酵母菌感染免疫相关的差异表达基因，其中试验组编码C2、CD22、CD53、CD209a、
第三军医大学硕士学位论文
CD79A结合蛋白lb、EP4受体、淋巴细胞抗原6复合体、肿瘤坏死因子可诱导蛋白cg12一1、
干扰素诱导蛋白PU以一G、工gG的FC高亲和力受体1的基因表达下调，编码结合珠蛋白
的基因表达上调。
结论本试验尝试性的利用基因表达谱芯片分析技术，对免疫抑制小鼠和健康小鼠
在感染阿萨希丝抱酵母菌24小时后，皮损局部基因的表达情况进行了初步的比较性研
究，共筛选出编码CZ、CD53、结合珠蛋白等11条与免疫相关的差异表达基因，这些基
因表达的变化，导致了其编码产物数量的改变，使宿主对阿萨希丝抱酵母菌的正常免疫
应答功能发生障碍，而导致该菌感染的发生及播散。如免疫抑制组补体CZ基因的显著
下调，将导致补体系统激活障碍，使膜攻击复合体的形成减少，对阿萨希丝抱酵母菌的
溶细胞作用减弱，而C3b等具有调理作用的补体片段生成减少，将减弱巨噬细胞及中性
粒细胞对该菌的吞噬作用;免疫抑制组CD53基因表达下调，使巨噬细胞在氧化应激状
态下的自我保护能力降低，致使巨噬细胞的功能、数量发生改变，使宿主对阿萨希丝抱
酵母菌的清除能力降低;编码EP4受体、CDZOga基因的下调将导致皮肤组织局部
Langethans及其它树突状细胞游出、移行发生障碍、减弱了该类细胞对阿萨希丝抱酵母
菌的抗原提呈能力;试验组编码结合珠蛋白的基因表达上调，一方面可以防止过度急性
炎症反应的产生，而另一方面也可能为阿萨希丝抱酵母菌存活及播散提供了有利的条
件;筛选出的其它功能尚不明确的差异表达基因，可能具有潜在的研究价值，可作为进
一步研究阿萨希丝抱酵母菌感染发生机制的候选基因。
Objective To screen out the differential expressed genes between the immunosuppressed and healthy mice after intradermal inoculation of Trichosporon asahii. Finding out the potential function of those genes in the interaction between the mice and the fungus, and the potential pathways lead to the local fungal infection onset and further dissemination.
Methods twelve BALB/C mice randomly divided into two groups. Three days before the inoculation of the fungi, mice in the test group were treated with cyclophosphamide, so as to result in the suppression of their immunological function, and the mice in the control group keep in the normal immunological condition. 24 hours after intradermal inoculation of 0.2ml of the fungal preparation which contain the Trichosporon asahii (concentration is 1 X 105CFU/ml,and the percentage of spore >95%) , then all the mice were killed and autopsied. Total RNA of the murine inoculated local cutaneous were extracted, reverse transcripted, the cDNA of the test group and the control were respectively labelled with fluorochrome dyes Cy5(red) or Cy3(green), then hybridized with one DNA chips which contained 4096 murine genes, then the signal on the chips scanned and analyzed.
Results 243 genes which were differentially expressed between the test group and the control were screened out, through the analyzing of the function of all those genes, we fixed our attention on 11 genes which thought to be playing an important role in the event of the skin infectious process of T.asahii. They are ten down-regulated genes in the test group, respectively encoding C2, CD22, CD53,CD209a, CD79A binding protein Ib, Fcgrl (Fc receptor, IgG, high affinity I), interferon-induced protein with tetratricopeptide repeats 3, lymphocyte antigen 6 complex, TNF-inducible protein cg12-1, prostaglandin E receptor 4, and one up-regulated gene encoding haptoglobin.
Conclusions The immune function of the immunosuppressed mice was inhibited in many important aspects, such as complement activation, phagocytosis of the macrophage, emigration of the dendritic cell, inflammatory reaction etc. These changes may play an important role in the process of T.asahii infection. Many genes screened out in our experiement, which with unclear function can be considered as candidate genes for the further study of the pathogenesis of the Trichosporonosis.
引文
1. R.Yang, J.Ao, W.Wang, et al. Disseminated trichosporonosis in China. Mycoses, 2003;46(11-12): 519-523.
2. Bryant PA, Venter D, Robins-Browne R, Curtis N. Chips with everything: DNA microarrays in infectious diseases. Lancet Infect Dis. 2004 Feb; 4(2): 100-11.
3. Wang WL, Li RY, Zhu X J, Wang DL. Differentially expressed genes in immunosuppressive and healthy mice after intranasal inoculation of Aspergillus fumigatus. Zhonghua Yi Xue Za Zhi. 2003 May 10; 83(9): 787-90. Chinese.
4. Yamagata E, Kamberi P, Yamakami Y, Hashimoto A, Nasu M. Experimental model of progressive disseminated trichosporonosis in mice with latent trichosporonemia. J Clin Microbiol. 2000 Sep; 38(9): 3260-6.
5. Faustino Mollinedo, Gumersindo Fomta N, et al. Recurrent Infectious Diseasea in Human CD53 Deficiency. Clinical And Diagnistic Laboratory Immunology. 997; 4:229-231.
6. Kim TR, Yoon JH, Kim YC, et al. Mol Cells. LPS-induced CD53 expression: a protection mechanism against oxidative and radiation stress. 2004 Feb 29; 17(1): 125-31.
7. Kabashima K, Sakata D, Nagamachi M, et al. Prostaglandin E2-EP4 signaling initiates skin immune responses by promoting migration and maturation of Langerhans cells. Nat Med. 2003 Jun; 9(6):744-9.
8. Colmenares M, Corbi AL, Turco SJ, Rivas L. The dendritic cell receptor DC-SIGN discriminates among species and life cycle forms of Leishmania. J Immunol. 2004 Jan 15;172(2): 1186-90.
9. Wang Y, Kinzie E, Berger FG, Lim SK, Baumann H. Haptoglobin, an inflammation-inducible plasma protein. Redox Rep 2001; 6(6): 379-85
10. Arred ouani M, Matthys P, Kasran A, Baumann H, Ceuppen JL.Haptoglobin and the Th1/Th2 balance: hints from in vitro and in vivo studies. Redox Pep 2001;6(6):369-71.
11．徐志伟，李文静．B细胞表面的抑制性受体及其作用机理．国外医学免疫学分册，2000，23(6)：319-321．
2. Bryant PA, Venter D, Robins-Browne R, Curtis N. Chips with everything: DNA microarrays in infectious diseases. Lancet Infect Dis. 2004 Feb; 4(2): 100-11.
3. Wang WL, Li RY, Zhu X J, Wang DL. Differentially expressed genes in immunosuppressive and healthy mice after intranasal inoculation of Aspergillus fumigatus. Zhonghua Yi Xue Za Zhi. 2003 May 10; 83(9): 787-90. Chinese.
4. Yamagata E, Kamberi P, Yamakami Y, Hashimoto A, Nasu M. Experimental model of progressive disseminated trichosporonosis in mice with latent trichosporonemia. J Clin Microbiol. 2000 Sep; 38(9): 3260-6.
5. Faustino Mollinedo, Gumersindo Fomta N, et al. Recurrent Infectious Diseasea in Human CD53 Deficiency. Clinical And Diagnistic Laboratory Immunology. 997; 4:229-231.
6. Kim TR, Yoon JH, Kim YC, et al. Mol Cells. LPS-induced CD53 expression: a protection mechanism against oxidative and radiation stress. 2004 Feb 29; 17(1): 125-31.
7. Kabashima K, Sakata D, Nagamachi M, et al. Prostaglandin E2-EP4 signaling initiates skin immune responses by promoting migration and maturation of Langerhans cells. Nat Med. 2003 Jun; 9(6):744-9.
8. Colmenares M, Corbi AL, Turco SJ, Rivas L. The dendritic cell receptor DC-SIGN discriminates among species and life cycle forms of Leishmania. J Immunol. 2004 Jan 15;172(2): 1186-90.
9. Wang Y, Kinzie E, Berger FG, Lim SK, Baumann H. Haptoglobin, an inflammation-inducible plasma protein. Redox Rep 2001; 6(6): 379-85
10. Arred ouani M, Matthys P, Kasran A, Baumann H, Ceuppen JL.Haptoglobin and the Th1/Th2 balance: hints from in vitro and in vivo studies. Redox Pep 2001;6(6):369-71.
11．徐志伟，李文静．B细胞表面的抑制性受体及其作用机理．国外医学免疫学分册，2000，23(6)：319-321．