人芽囊原虫体外纯培养法的改良及形态观察
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摘要
以琼脂取代蛋白对传统的洛克氏液-鸡蛋-血清(Locke’s egg serum,LES)双相培养基进行改良,联合采用LES双相培养基、改良LES双相培养基(m LES双相培养基)、IMDM液相培养基和IMDM固相培养基建立人芽囊原虫的体外纯培养的标准流程,并观察虫体在不同培养基中的形态特征和生长情况。结果显示,人芽囊原虫在4种培养基中均以空泡型和颗粒型的形态为主,培养后镜下未见细菌生长。在mLES双相培养基中的生长速度最快,培养后第2天原虫数为3.09×10~6,达到中对数期。原虫在LES双相培养基、mLES双相培养基和IMDM液相培养基中的生长对数期的分裂时间分别为(49.72±1.35)、(24.16±2.53)和(36.3±1.5)h。提示联合采用LES双相培养基、mLES双相培养基、IMDM液相培养基和IMDM固相培养基可建立人芽囊原虫的无菌纯培养体系。
The Locke 's egg serum(LES) biphasic medium was modified by agar replacement of protein. A sterile in vitro culture system for Blastocystis hominis was then established using LES, modified LES(m LES) and liquid-state and solid-state Iscove 's Modified Dulbecco 's Medium(IMDM). Morphological and growth characteristics of the parasites in different media were observed. Blastocystis hominis showed vacuolar and granular morphologies in all types of media and had the most rapid growth in the m LES, in which the log phase was achieved on day 2 of culture(3.09 × 10~6 parasites). The doubling times were(49.72 ± 1.35) h in LES,(24.16 ± 2.53) h in mLES, and(36.30 ± 1.50) h in IMDM. The sterile in vitro culture system for Blastocystis hominis is established using LES,mLES, and liquidus and solid-state IMDM.
The Locke 's egg serum(LES) biphasic medium was modified by agar replacement of protein. A sterile in vitro culture system for Blastocystis hominis was then established using LES, modified LES(m LES) and liquid-state and solid-state Iscove 's Modified Dulbecco 's Medium(IMDM). Morphological and growth characteristics of the parasites in different media were observed. Blastocystis hominis showed vacuolar and granular morphologies in all types of media and had the most rapid growth in the m LES, in which the log phase was achieved on day 2 of culture(3.09 × 10~6 parasites). The doubling times were(49.72 ± 1.35) h in LES,(24.16 ± 2.53) h in mLES, and(36.30 ± 1.50) h in IMDM. The sterile in vitro culture system for Blastocystis hominis is established using LES,mLES, and liquidus and solid-state IMDM.
引文
[1]Horiki N,Maruyama M,Fujita Y,et al.Epidemiologic survey of Blastocystis hominis infection in Japan[J].Am J Trop Med Hyg,1997,56(4):370-374.
[2]Chandramathi S,Suresh K,Kuppusamy UR.Solubilized antigen of Blastocystis hominis facilitates the growth of human colorectal cancer cells,HCT116[J].Parasitol Res,2010,106(4):941-945.
[3]Chan KH,Chandramathi S,Suresh K,et al.Effects of symptomatic and asymptomatic isolates of Blastocystis hominis on colorectal cancer cell line,HCT116[J].Parasitol Res,2012,110(6):2475-2480.
[4]Albrecht H,Stellbrink HJ,Koperski K,et al.Blastocystis hominis in human immunodeficiency virus-related diarrhea[J].Scand J Gastroenterol,1995,30(9):909-914.
[5]田利光,陈家旭,程国金,等.安徽阜阳HIV阳性者合并人芽囊原虫感染及其影响因素分析[J].中国血吸虫病防治杂志,2012,24(3):303-306,310.
[6]Ramírez-Miranda ME,Jiménez-González DE,Rodríguez-Campa ME,et al.Irritable bowel syndrome:frequency and phylogenetic relationship of Blastocystis sp.from Mexican patients[J].Rev Gastroenterol Mex,2011,76(4):309-315.
[7]Ragavan ND,Kumar S,Chye TT,et al.Blastocystis sp.in irritable bowel syndrome(IBS)-detection in stool aspirates during colonoscopy[J].PLo S One,2015,10(9):e0121173.
[8]Su FH,Chu FY,Li CY,et al.Blastocystis hominis infection in long-term care facilities in Taiwan:prevalence and associated clinical factors[J].Parasitol Res,2009,105(4):1007-1013.
[9]Wawrzyniak I,Poirier P,Viscogliosi E,et al.Blastocystis,an unrecognized parasite:an overview of pathogenesis and diagnosis[J].Ther Adv Infect Dis,2013,1(5):167-178.
[10]Yoshikawa H,Koyama Y,Tsuchiya E,et al.Blastocystis phylogeny among various isolates from humans to insects[J].Parasitol Int,2016,65(6 Pt B):750-759.
[11]Stensvold CR,Suresh GK,Tan KS,et al.Terminology for Blastocystis subtypes--a consensus[J].Trends Parasitol,2007,23(3):93-96.
[12]Boeck WC,Drbohlav J.The Cultivation of Endamoeba Histolytica[J].Proc Natl Acad Sci USA,1925,11(5):235-238.
[13]Zhang X,Qiao J,Wu X,et al.In vitro culture of Blastocystis hominis in three liquid media and its usefulness in the diagnosis of blastocystosis[J].Int J Infect Dis,2012,16(1):e23-e28.
[14]姚繁荣,乔继英,杨珺华,等.人芽囊原虫在RPMI1640及LES培养中的比较[J].陕西师范大学学报(自然科学版),2003,31(S2):259-264.
[15]Singh M,Ho LC,Yap AL,et al.Axenic culture of reptilian Blastocystis isolates in monophasic medium and speciation by karyotypic typing[J].Parasitol Res,1996,82(2):165-169.
[16]Tan KS,Ng GC,Quek E,et al.Blastocystis hominis:A simplified,high-efficiency method for clonal growth on solid agar[J].Exp Parasitol,2000,96(1):9-15.
[17]Pomroy AJ.Direct counting of bacteria preserved with lugol iodine solution[J].Appl Environ Microbiol,1984,47(5):1191-1192.
[18]Bearzi C,Rota M,Hosoda T,et al.Human cardiac stem cells[J].Proc Natl Acad Sci USA,2007,104(35):14068-14073.
[19]乔继英,薛弘燮,吐尔逊艾.人芽囊原虫形态学的观察[J].中国寄生虫学与寄生虫病杂志,1998,16(2):124-125.
[2]Chandramathi S,Suresh K,Kuppusamy UR.Solubilized antigen of Blastocystis hominis facilitates the growth of human colorectal cancer cells,HCT116[J].Parasitol Res,2010,106(4):941-945.
[3]Chan KH,Chandramathi S,Suresh K,et al.Effects of symptomatic and asymptomatic isolates of Blastocystis hominis on colorectal cancer cell line,HCT116[J].Parasitol Res,2012,110(6):2475-2480.
[4]Albrecht H,Stellbrink HJ,Koperski K,et al.Blastocystis hominis in human immunodeficiency virus-related diarrhea[J].Scand J Gastroenterol,1995,30(9):909-914.
[5]田利光,陈家旭,程国金,等.安徽阜阳HIV阳性者合并人芽囊原虫感染及其影响因素分析[J].中国血吸虫病防治杂志,2012,24(3):303-306,310.
[6]Ramírez-Miranda ME,Jiménez-González DE,Rodríguez-Campa ME,et al.Irritable bowel syndrome:frequency and phylogenetic relationship of Blastocystis sp.from Mexican patients[J].Rev Gastroenterol Mex,2011,76(4):309-315.
[7]Ragavan ND,Kumar S,Chye TT,et al.Blastocystis sp.in irritable bowel syndrome(IBS)-detection in stool aspirates during colonoscopy[J].PLo S One,2015,10(9):e0121173.
[8]Su FH,Chu FY,Li CY,et al.Blastocystis hominis infection in long-term care facilities in Taiwan:prevalence and associated clinical factors[J].Parasitol Res,2009,105(4):1007-1013.
[9]Wawrzyniak I,Poirier P,Viscogliosi E,et al.Blastocystis,an unrecognized parasite:an overview of pathogenesis and diagnosis[J].Ther Adv Infect Dis,2013,1(5):167-178.
[10]Yoshikawa H,Koyama Y,Tsuchiya E,et al.Blastocystis phylogeny among various isolates from humans to insects[J].Parasitol Int,2016,65(6 Pt B):750-759.
[11]Stensvold CR,Suresh GK,Tan KS,et al.Terminology for Blastocystis subtypes--a consensus[J].Trends Parasitol,2007,23(3):93-96.
[12]Boeck WC,Drbohlav J.The Cultivation of Endamoeba Histolytica[J].Proc Natl Acad Sci USA,1925,11(5):235-238.
[13]Zhang X,Qiao J,Wu X,et al.In vitro culture of Blastocystis hominis in three liquid media and its usefulness in the diagnosis of blastocystosis[J].Int J Infect Dis,2012,16(1):e23-e28.
[14]姚繁荣,乔继英,杨珺华,等.人芽囊原虫在RPMI1640及LES培养中的比较[J].陕西师范大学学报(自然科学版),2003,31(S2):259-264.
[15]Singh M,Ho LC,Yap AL,et al.Axenic culture of reptilian Blastocystis isolates in monophasic medium and speciation by karyotypic typing[J].Parasitol Res,1996,82(2):165-169.
[16]Tan KS,Ng GC,Quek E,et al.Blastocystis hominis:A simplified,high-efficiency method for clonal growth on solid agar[J].Exp Parasitol,2000,96(1):9-15.
[17]Pomroy AJ.Direct counting of bacteria preserved with lugol iodine solution[J].Appl Environ Microbiol,1984,47(5):1191-1192.
[18]Bearzi C,Rota M,Hosoda T,et al.Human cardiac stem cells[J].Proc Natl Acad Sci USA,2007,104(35):14068-14073.
[19]乔继英,薛弘燮,吐尔逊艾.人芽囊原虫形态学的观察[J].中国寄生虫学与寄生虫病杂志,1998,16(2):124-125.