GasC基因在马尔尼菲青霉菌广西野生银星竹鼠寄生株与人感染株的差异表达
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摘要
目的初步探讨马尔尼菲青霉菌(Penicillium marneffei,PM)竹鼠寄生株和人感染株中GasC基因表达变化情况。
方法随机选取15株成年竹鼠体内分离PM和15株人感染株PM在SDA液基25℃震荡培养72h。采Trizol法提取PM菌株的总RNA。荧光定量PCR技术测出两种不同来源的菌株GasC基因的表达量。统计分析两种不同来源的菌株GasC基因的表达量是否具有差异性。
结果①15只成年广西野生银星竹鼠自然携带PM阳性率100%,30株菌株经在SDA液基25℃震荡培养72h后均产可溶性红色色素,但有程度差别,即色价不同。统计分析两种不同来源的PM在25℃时产色无差异(p＞0.05)。②30株菌株提取RNA并逆转录成cDNA,经PCR后电泳可见GasC基因的目的条带。③采用荧光定量PCR测出15株竹鼠寄生株PM和15株人感染株PM GasC基因的表达量,SPSS统计分析组间差异无统计学意义(p＞0.05)。④菌株的色价与GasC基因的表达量有统计学差异(p＜0.05),且色素色价越高,其菌株GasC基因表达水平就越高。
结论两种不同来源的PM25℃时产色无差异;GasC基因在PM寄生株和人感染株均有表达,其表达量不存在差异,提示两组PM菌株在活力和致病力上可能不存在差异;菌株色价与GasC基因的表达相关,色素色价越高,其菌株GasC基因表达水平就越高,但分泌色素多且GasC基因表达高的菌株致病力是否较强,需进一步动物实验验证。
Objectives To investigate the differential expression of the GasC gene in Guangxi Penicillium marneffei (PM) between the isolates obtained from Rhizomys pruinosus and those from Penicilliosis marneffei(PSM) patients
Methods Selected randomly 30 isolates separately obtained from Rhizomys pruinosus and those from PSM patients cultured in SDA liquid medium at 25℃after 72h. Total RNA was extracted with Trizol. Detect the expression of GasC gene in each isolates through the fluorescence quantitive PCR technic. Analysis the difference of the GasC expressed in the two group by SPSS.
Results①The natural positive carrier rate of the 15 was 100 %, 30 isolates can all produced the red pigment at 25°C after 72h cultured in SDA liquid medium,and they all produced the red pigment with different level,but no difference in statistics between the two group (p>0.05) .②we can see the goal gene through the gelelectrophoresis③Detect the expression of GasC gene in each isolates through the Real Time PCR and there is not difference in statistics between the two group (p>0.05) .④The expression of GasC gene is posertivly connect with the ability for producing the pigment of PM, and the deeper the pigment shows , the higher the level of GasC gene expressed.
Conclusions There is no difference in producing red pigment between the isolates obtained from Rhizomys pruinosus and those from Penicilliosis marneffei(PSM) patients; The expression of GasC gene is not difference in statistics between the two group which preliminary revealed the similarity in growth vitality and pathogenicity;The expression of GasC gene is connect with the ability for producing the pigment of PM, and the deeper the pigment shows , the higher the level of GasC gene expressed. It must be proved by the further experiment on animals whether the PM with deeper pigment and the high expression of GasC gene presents stronger pathogenicity.
方法随机选取15株成年竹鼠体内分离PM和15株人感染株PM在SDA液基25℃震荡培养72h。采Trizol法提取PM菌株的总RNA。荧光定量PCR技术测出两种不同来源的菌株GasC基因的表达量。统计分析两种不同来源的菌株GasC基因的表达量是否具有差异性。
结果①15只成年广西野生银星竹鼠自然携带PM阳性率100%,30株菌株经在SDA液基25℃震荡培养72h后均产可溶性红色色素,但有程度差别,即色价不同。统计分析两种不同来源的PM在25℃时产色无差异(p＞0.05)。②30株菌株提取RNA并逆转录成cDNA,经PCR后电泳可见GasC基因的目的条带。③采用荧光定量PCR测出15株竹鼠寄生株PM和15株人感染株PM GasC基因的表达量,SPSS统计分析组间差异无统计学意义(p＞0.05)。④菌株的色价与GasC基因的表达量有统计学差异(p＜0.05),且色素色价越高,其菌株GasC基因表达水平就越高。
结论两种不同来源的PM25℃时产色无差异;GasC基因在PM寄生株和人感染株均有表达,其表达量不存在差异,提示两组PM菌株在活力和致病力上可能不存在差异;菌株色价与GasC基因的表达相关,色素色价越高,其菌株GasC基因表达水平就越高,但分泌色素多且GasC基因表达高的菌株致病力是否较强,需进一步动物实验验证。
Objectives To investigate the differential expression of the GasC gene in Guangxi Penicillium marneffei (PM) between the isolates obtained from Rhizomys pruinosus and those from Penicilliosis marneffei(PSM) patients
Methods Selected randomly 30 isolates separately obtained from Rhizomys pruinosus and those from PSM patients cultured in SDA liquid medium at 25℃after 72h. Total RNA was extracted with Trizol. Detect the expression of GasC gene in each isolates through the fluorescence quantitive PCR technic. Analysis the difference of the GasC expressed in the two group by SPSS.
Results①The natural positive carrier rate of the 15 was 100 %, 30 isolates can all produced the red pigment at 25°C after 72h cultured in SDA liquid medium,and they all produced the red pigment with different level,but no difference in statistics between the two group (p>0.05) .②we can see the goal gene through the gelelectrophoresis③Detect the expression of GasC gene in each isolates through the Real Time PCR and there is not difference in statistics between the two group (p>0.05) .④The expression of GasC gene is posertivly connect with the ability for producing the pigment of PM, and the deeper the pigment shows , the higher the level of GasC gene expressed.
Conclusions There is no difference in producing red pigment between the isolates obtained from Rhizomys pruinosus and those from Penicilliosis marneffei(PSM) patients; The expression of GasC gene is not difference in statistics between the two group which preliminary revealed the similarity in growth vitality and pathogenicity;The expression of GasC gene is connect with the ability for producing the pigment of PM, and the deeper the pigment shows , the higher the level of GasC gene expressed. It must be proved by the further experiment on animals whether the PM with deeper pigment and the high expression of GasC gene presents stronger pathogenicity.
引文
1.Supparatpinyo K,Khamwan C,Baosoung V,et al.Disseminated Penicillium marneffei infection in southeast Asia.Lancet 1994,344(8915):110-113.
2.Singh PN,Ranjana K,Singh YI,et al.Indigenous disseminated Penicillium marneffei infection in the state of Manipur,India:report of four autochthonous cases.J Clin Microbiol.1999Aug;37(8):2699-702.
3.Drouhet E.Penicilliosis due to penicillium marneffei:a new emerging systemic mycosis in AIDS patients traveling or living in southeast Asia:review of 44 cases reported in HIV infected patients during the last 5 years compared to 44 cases of non AIDS patients reported over 20 years.J Mycol Med:1993;4:195-224.
4.Yuen,K.Y.,g.Pascal,et al.Exploring the Penicilliummarneffei genome.Arch.Microbiol.2003.179:339-353.
5.廖小梅,冉玉平.爱滋病合并播散性马尔尼菲青霉菌感染一例.中华医学杂志.2002,82(5):325-329.
6.郑文军.严煜林.艾滋病合并马尔尼菲青霉病五例.中华皮肤科杂志.2004,37(4):202.
7.尹松梅.吴裕丹.艾滋病合并播散性马尔尼菲青霉菌感染2例临床分析.中国实用内科杂志.2004,24(6):381-382.
8.李云.陈燕.李雪梅.艾滋病合并马尔尼菲青霉病一例.中华检验医学杂志.2004.27(9):548.
9.余江.粟军.爱滋病合并马尔尼菲青霉病1例与常见病原体的鉴别.四川医学.2005.26(1):85.
10.易建云,蒋月婷.爱滋病合并马尔尼菲青霉菌感染1例.临床检验杂志.2003.21(4):255.
11.宋伟南.曾泳而.陈万山.马尔尼菲青霉菌在艾滋病人中感染状况及药敏结果分析.2005.5(3)266-256.
12.王露霞,徐德兴.爱滋病患者感染马尔尼菲青霉菌一例报告.第一军医大学报.2001,21(5):371.
13.唐志荣,刘伟,陈杰,等.广西AIDS例合并马尔尼菲青霉菌病感染及其治疗[J].Applied Prev Med 2007,13(1):28-29
14.梁伶,李菊裳.马尔尼菲青霉的生态学研究.广西医科大学学报,1999,16(5):602-604.
15.李菊裳,邓卓霖,潘乐泉.广西银星竹鼠自然带马内青霉菌的真菌学研究报告.中国人兽共患病杂志,1986,2(2):5-7.
16.李山,邓卓霖,马韵.广西鼠类携带马尔尼菲青霉的研究.广西医学,1995,17(1):1-2.
17.Dixon,D.M.,Polak,A.& Szaniszlo,P.J.(1987).Pathogenicity and virulence of wild-type and melanin deficient Wangiella dermatitidis.J Med Vet Mycol 25,97-106.
18.Nosanchuk J.Clinical impact of fungal melanisation.The Congress Handbook & Abstract Book 2 of The 8th InternationalMy cological Congress in Cairns,Australia on August 20-25,2006:264
19.Zuber,S.,M.J.Hynes,and A.Andrianopoulos.2003.The g-protein -subunit GasC plays a major role in germination in the dimorphic fungus Penicillium marneffei,genetics 164:487-499
20.实时荧光定量PCR——一种科学准确的定量方法.AppliedBiosystems 公司
21.陈义光,彭德娇,田宏现.红曲及红曲霉的研究与应用[J].湖北农学院学 报(自然科学版),2000,20(2):188-190.
22.刘红芳、席丽艳、李希清,等.马尔尼菲青霉总RNA方法提取的比较.中华皮肤科杂志[J].2006,39(8):480-481
23.CooperCR Jr,McginnisMR.Pathology ofPenicillium marneffei.An emerging acquired immunodeficiency syndrome related pathogen.Arch PatholLabMed,1997,121(8):798-804
24.李菊裳、潘乐泉、邓卓霖.马内青霉菌病1例报告,临床皮肤科杂志,1985,1:24-26.
25.李利、李明、张保庆,等.马尔尼菲青霉病误诊为支气管肺炎伤寒1例,中国误诊学杂志,2001,10.15;1(10):1594.
26.Imwidthaya P.Update of Penicillosis marneffei in Thailand.Revie warticle.Mycopathologia 1994 Sep;127(3):135-7.
27.Louthrenoo W,Thamprasert K,Sirisanthana T.Osteoarticular penicilliosis marneffei.A report of eight cases and review of the literature.Br J Rheumatol 1994 Dec;33(12):1145-50.
28.Casadevall,A.,Rosas,A.L.,and Nosanchuk,J.D.(2000)Melanin and virulence in Cryptococcus neoformans.Curr Opin Microbiol 3:354-358.
29.吴易,梁伶,李菊裳.广西银星竹鼠与人马尔尼菲青霉病关系的研究.中国皮肤性病学杂志2004;18:196-198.
30.Kudeken N,Kawakami K,Saito A.CD4+ T cell mediated fatal hyperinflammatory reactions in mice infected with Penicillium marneffei[J].Clin Exp Immunol,1997,107(3):468-473.
31.Thiriach S,Cooper CR,Vanittanakom N.Phagocytosis and killing of human pathogenic Penicilliumm arneffei and Pen icillium spp.by mouse macrophage J774.1 cells.The Congress Handbook &Abstract Book 1 of The 8th InternationalMycological Congress inCairns,Australia on August 20225,2006:235
32.刘栋华,谭升顺,梁伶,等.经皮肤损伤感染马尔尼菲青霉菌致病力动物实验研究.中国皮肤性病学杂志.2006,20(6):324-326
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[17]刘栋华,谭升顺,梁伶,等.经皮肤损伤感染马尔尼菲青霉菌致病力动物实验研究.中国皮肤性病学杂志.2006,20(6):324-326
[18]Casadevall, A., Rosas, A. L., and Nosanchuk, J. D. (2000)Melanin and virulence in Cryptococcus neoformans. Curr Opin Microbiol 3: 354-358.
[19]Nosanchuk J. Clinical impact of fungal melanisation. The Congress Handbook & Abstract Book 2 of The 8th InternationalMy cological Congress in Cairns, Australia on August 20-25, 2006:264
[20]Zuber, S., M. J. Hynes, and A. Andrianopoulos. 2003. The G-protein -subunit GasC plays a major role in germination in the dimorphic fungus Penicillium marneffei. Genetics 164:487-499
[21]Thiriach S, Cooper CR, Vanittanakom N. Phagocytosis and killing of human pathogenic Penicillium marneffei and Pen icillium spp. by mouse macrophage J774. 1 cells. The Congress Handbook &Abstract Book 1 of The 8th International Mycological Congress inCairns,Australia on August 20225, 2006: 235
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2.Singh PN,Ranjana K,Singh YI,et al.Indigenous disseminated Penicillium marneffei infection in the state of Manipur,India:report of four autochthonous cases.J Clin Microbiol.1999Aug;37(8):2699-702.
3.Drouhet E.Penicilliosis due to penicillium marneffei:a new emerging systemic mycosis in AIDS patients traveling or living in southeast Asia:review of 44 cases reported in HIV infected patients during the last 5 years compared to 44 cases of non AIDS patients reported over 20 years.J Mycol Med:1993;4:195-224.
4.Yuen,K.Y.,g.Pascal,et al.Exploring the Penicilliummarneffei genome.Arch.Microbiol.2003.179:339-353.
5.廖小梅,冉玉平.爱滋病合并播散性马尔尼菲青霉菌感染一例.中华医学杂志.2002,82(5):325-329.
6.郑文军.严煜林.艾滋病合并马尔尼菲青霉病五例.中华皮肤科杂志.2004,37(4):202.
7.尹松梅.吴裕丹.艾滋病合并播散性马尔尼菲青霉菌感染2例临床分析.中国实用内科杂志.2004,24(6):381-382.
8.李云.陈燕.李雪梅.艾滋病合并马尔尼菲青霉病一例.中华检验医学杂志.2004.27(9):548.
9.余江.粟军.爱滋病合并马尔尼菲青霉病1例与常见病原体的鉴别.四川医学.2005.26(1):85.
10.易建云,蒋月婷.爱滋病合并马尔尼菲青霉菌感染1例.临床检验杂志.2003.21(4):255.
11.宋伟南.曾泳而.陈万山.马尔尼菲青霉菌在艾滋病人中感染状况及药敏结果分析.2005.5(3)266-256.
12.王露霞,徐德兴.爱滋病患者感染马尔尼菲青霉菌一例报告.第一军医大学报.2001,21(5):371.
13.唐志荣,刘伟,陈杰,等.广西AIDS例合并马尔尼菲青霉菌病感染及其治疗[J].Applied Prev Med 2007,13(1):28-29
14.梁伶,李菊裳.马尔尼菲青霉的生态学研究.广西医科大学学报,1999,16(5):602-604.
15.李菊裳,邓卓霖,潘乐泉.广西银星竹鼠自然带马内青霉菌的真菌学研究报告.中国人兽共患病杂志,1986,2(2):5-7.
16.李山,邓卓霖,马韵.广西鼠类携带马尔尼菲青霉的研究.广西医学,1995,17(1):1-2.
17.Dixon,D.M.,Polak,A.& Szaniszlo,P.J.(1987).Pathogenicity and virulence of wild-type and melanin deficient Wangiella dermatitidis.J Med Vet Mycol 25,97-106.
18.Nosanchuk J.Clinical impact of fungal melanisation.The Congress Handbook & Abstract Book 2 of The 8th InternationalMy cological Congress in Cairns,Australia on August 20-25,2006:264
19.Zuber,S.,M.J.Hynes,and A.Andrianopoulos.2003.The g-protein -subunit GasC plays a major role in germination in the dimorphic fungus Penicillium marneffei,genetics 164:487-499
20.实时荧光定量PCR——一种科学准确的定量方法.AppliedBiosystems 公司
21.陈义光,彭德娇,田宏现.红曲及红曲霉的研究与应用[J].湖北农学院学 报(自然科学版),2000,20(2):188-190.
22.刘红芳、席丽艳、李希清,等.马尔尼菲青霉总RNA方法提取的比较.中华皮肤科杂志[J].2006,39(8):480-481
23.CooperCR Jr,McginnisMR.Pathology ofPenicillium marneffei.An emerging acquired immunodeficiency syndrome related pathogen.Arch PatholLabMed,1997,121(8):798-804
24.李菊裳、潘乐泉、邓卓霖.马内青霉菌病1例报告,临床皮肤科杂志,1985,1:24-26.
25.李利、李明、张保庆,等.马尔尼菲青霉病误诊为支气管肺炎伤寒1例,中国误诊学杂志,2001,10.15;1(10):1594.
26.Imwidthaya P.Update of Penicillosis marneffei in Thailand.Revie warticle.Mycopathologia 1994 Sep;127(3):135-7.
27.Louthrenoo W,Thamprasert K,Sirisanthana T.Osteoarticular penicilliosis marneffei.A report of eight cases and review of the literature.Br J Rheumatol 1994 Dec;33(12):1145-50.
28.Casadevall,A.,Rosas,A.L.,and Nosanchuk,J.D.(2000)Melanin and virulence in Cryptococcus neoformans.Curr Opin Microbiol 3:354-358.
29.吴易,梁伶,李菊裳.广西银星竹鼠与人马尔尼菲青霉病关系的研究.中国皮肤性病学杂志2004;18:196-198.
30.Kudeken N,Kawakami K,Saito A.CD4+ T cell mediated fatal hyperinflammatory reactions in mice infected with Penicillium marneffei[J].Clin Exp Immunol,1997,107(3):468-473.
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