人芽囊原虫体外纯培养法的改良及形态观察

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。     

3种培养基中芽囊原虫生长状况观察

目的观察芽囊原虫在不同培养基中的生长状况,筛选合适的芽囊原虫培养方法。方法将10份芽囊原虫阳性粪便分别接种至Jone’s液、vitro液、1640 3种培养基中培养,选取生长情况良好、虫密度高的一份粪便传代培养后等量接种至3种不同的培养基中,每24 h计数虫体数量一次,连续观察10 d,并观察芽囊原虫的形态学变化和生长情况。结果10份粪便分别接种至3种培养基中培养,48 h后发现1640和Jone’s液中的虫密度高于vitro液。同一份粪便接种至3种不同培养基中连续观察10 d,结果表明3种培养基中虫体生长呈规律变化,均在接种后3、6、9 d出现生长高峰;Jone’s液中芽囊原虫密度最高。vitro液中观察到的虫体形态最清晰,且富有活力;Jone’s液中能观察到虫体多种繁殖状态。结论Jone’s液适于芽囊原虫的生长繁殖,可作为首选培养基;vitro液可作为观察芽囊原虫生长情况的首选培养基。     

人芽囊原虫在不同培养基中生长状况的观察

目的筛选培养人芽囊原虫的最适培养基。方法将同一株人芽囊原虫阳性粪便标本以2×105细胞/管接种至RPMI1640、199和LES培养基中,加入20%小牛血清及青、链霉素,pH值为7.5,放置厌氧罐中于37℃恒温培养,每24h计数,每6d转种1次。观察人芽囊原虫在3种培养基中的存活时间、虫体密度和虫体形态。结果人芽囊原虫在RPMI1640培养基中存活时间最长、虫体密度最高,虫体以空泡型多见;在LES培养基中存活时间最短、虫体密度最低,但虫体形态清晰、规则;在199培养基中存活时间和虫体密度均介于前两者之间。结论RPMI1640培养基适宜人芽囊原虫的生长繁殖,为人芽囊原虫体外培养的首选培养基;LES培养基中虫体形态清晰、规则,可用于人芽囊原虫的形态学研究;199培养基也可用于人芽囊原虫的体外培养,但不作为首选。     

mLES法培养人芽囊原虫的实验观察

目的建立一种简便、灵敏的可用于人芽囊原虫(Blastocystis hominis,Bh)感染诊断实验室方法。方法比较改良洛氏-鸡蛋-血清双相培养法(modified Locke-egg serum medium culture method,mLES培养法)与碘液直接涂片法对Bh的检出率;比较普通洛氏-鸡蛋-血清双相培养基(Locke-egg serum medium culture method,LES培养法)与mLES培养法中Bh形态、最低检出限、生长曲线及厌氧条件形成时间。结果检查397份临床粪便标本,mLES培养法Bh阳性率为5.54%(22/397),碘染法阳性率为1.01%(4/397),差异有统计学意义(P0.01)。中央空泡型是Bh存在两种培养基中的主要形态。mLES培养法48hBh最低检出限(10~2/5ml)优于LES培养法(10~3/5ml),重复测量方差分析表明组内Bh密度在不同时间点有差异、时间与分组交互作用及培养基种类的差异有统计学意义(P0.05);除了第6d,其余各时间点两种培养基中人芽囊原虫生长密度差异均有统计学意义(P0.05),mLES培养法前3dBh密度高于LES培养法。mLES培养法先于普通培养基达到厌氧环境。结论 mLES培养法简便、灵敏,可用于诊断Bh的体外培养检查。     

人芽囊原虫在腹泻患者中的感染状况及繁殖方式

目的　观察人芽囊原虫在腹泻患者中的感染状况及繁殖方式 ,为进一步研究其生理生化特性及致病机理奠定基础。　方法　调查腹泻患者临床表现 ;用生理盐水涂片、碘液染色和铁苏木素染色法在光镜下观察其在粪便标本和培养物及动物模型肠内容物中形态、结构及繁殖方式。　结果　健康人群和腹泻人群粪便标本中人芽囊原虫检出率分别为 0 .94% (2 2 13 )和 2 4.73 % (4 6 186) ;人芽囊原虫与其它肠道寄生原虫有合并感染 ;有二分裂、内二芽生殖和裂体增殖 3种繁殖方式。　结论　人芽囊原虫在腹泻患者中有较高的感染率 ;易与结肠内阿米巴和其他非致病阿米巴合并感染 ;以3种无性生殖方式繁殖。     

人芽囊原虫在腹泻患者中的感染状况及繁殖方式

目的观察人芽囊原虫在腹泻患者中的感染状况及繁殖方式，为进一步研究其生理生化特性及致病机理奠定基础。方法调查腹泻患者临床表现；用生理盐水涂片、碘液染色和铁苏木素染色法在光镜下观察其在粪便标本和培养物及动物模型肠内容物中形态、结构及繁殖方式。结果健康人群和腹泻人群粪便标本中人芽囊原虫检出率分别为0．94％(2／213)和24．73％(46／186)；人芽囊原虫与其它肠道寄生原虫有合并感染；有二分裂、内二芽生殖和裂体增殖3种繁殖方式。结论人芽囊原虫在腹泻患者中有较高的感染率；易与结肠内阿米巴和其他非致病阿米巴合并感染；以3种无性生殖方式繁殖。     

人芽囊原虫感染大鼠的方式改良及病理切片观察

目的建立人芽囊原虫感染大鼠动物模型。方法将30只雄性SD大鼠随机分为对照组和4个感染组(10~5、 10~6、 10~7和10~8),每组6只。4个感染组分别灌服人芽囊原虫滋养体10~5、 10~6、 10~7和10~8个/鼠,对照组灌服等量PBS。感染后第3天开始收集粪样,于洛克氏-鸡蛋-血清(LES)培养基中培养72 h后镜下观察,PCR和测序确认感染前后虫株的基因型。感染后第3～18天连续收集5次大鼠粪样培养观察,记录阳性鼠感染情况。感染后第21天将大鼠全部无痛处死,分别收集十二指肠、空肠、回肠、结肠和盲肠等肠段的内容物培养72 h后观察,计算其最终感染数和感染密度;并取不同部位的组织进行切片,苏木精-伊红(HE)染色后观察肠道内是否存在虫体。采用皮尔森相关性分析比较各肠段寄生数目的相关关系。结果 10~5、 10~6、 10~7和10~8感染组中确定感染人芽囊原虫的大鼠数目分别为2、 3、 5和6只。其中10~5感染组感染后第6天检出阳性结果,其余感染组第3天检出阳性,对照组均为阴性。PCR及序列比对证实,感染大鼠分离的人芽囊原虫与感染所用虫株均为ST7型。肠道内容物培养结果显示,回肠、盲肠和结肠的内容物均成功培养出人芽囊原虫,且盲肠与结肠内的虫数之间呈正相关,盲肠与结肠内容物培养获得的人芽囊原虫数呈正相关性(r=0.541, P 0.01),其余肠段未发现人芽囊原虫。HE染色结果显示,盲肠有人芽囊原虫寄生并存在黏蛋白,未发现虫体入侵、炎细胞浸润、黏膜糜烂等病理损伤;其余肠段未发现人芽囊原虫寄生。结论采用人芽囊原虫滋养体可成功建立大鼠感染模型。灌服10~5个人芽囊原虫滋养体可成功感染大鼠, 10~8个滋养体的感染效果稳定,各组随着感染人芽囊原虫数量的增加,感染的大鼠数也增加。     

人芽囊原虫在199单相培养基中的体外培养

目的观察199单相培养基对人芽囊原虫(Blastocystishominis)的体外培养情况。方法采用199单相培养基对B.hominis进行体外连续培养,比较不同pH值、血清种类及接种量等条件下虫体生长繁殖情况及影响因素。结果B.hominis在199培养基中最适培养条件为:10%~20%小牛、人或马血清;接种量不小于1×105原虫/管;青、链霉素于37℃条件下厌氧培养;pH为7.0、7.5及8.0时,B.hominis繁殖高峰分别为第4、8天,第3、6、9天和第2、4、7、9天。结论使用199单相培养基可以对B.hominis进行体外长期培养。     

不同环境中人芽囊原虫包囊的结构观察

目的　观察人芽囊原虫包囊在不同环境中的光学和超微结构的形态特点。　方法　从粪便分离人芽囊原虫 ,转种至改良的Jones培养基培养 72h后 ,再转种至混合纤维素脂微孔滤膜成囊培养基和Suresh成囊培养基中培养。采用光学和电子显微镜对培养基中与粪便中的包囊进行光学和超微结构的观察。　结果　光镜下观察 ,新鲜粪便中的包囊微小圆球形 ,具有折光性 ,不同的个体大小悬殊 ,HE染色可显示囊壁有 3层。用Suresh成囊培养基培养的包囊圆形或椭圆形 ,中心体色淡 ,内有大小不等的孢子体。混合纤维素脂微孔滤膜培养的包囊均较大 ,有空泡型、颗粒型、薄壁与厚壁包囊之分。透射电镜下观察 ,无纤维层的裸露包囊形态大都是圆形或卵圆形。包囊直径为 14 μm。包囊呈典型的人芽囊原虫核 ,其线粒体较多 ,胞质中糖原呈大小不一块状。　结论　包囊在不同环境中的形态基本相同、大小不一、结构多型。     

不同利什曼原虫分离株在培养基中生长繁殖观察

目的　比较我国不同类型内脏利什曼病流行区利什曼原虫前鞭毛体在不同培养基中的生长繁殖情况,为选择合适培养基用于利什曼原虫培养提供实验依据。方法　将3 ×105个KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体分别接种至1 mL NNN培养基、1 mL M199 + 20%胎牛血清培养基、1 mL M199 + 20%马血清培养基及1 mL 脑心浸液培养基（含血红素）中,22 ℃温箱中无菌静置培养,显微镜下连续观察计数8 d,绘制3株利什曼原虫前鞭毛体的生长曲线。 结果 KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体均能在NNN培养基、M199 + 20%胎牛血清培养基和M199 + 20%马血清培养基中生长繁殖,在NNN培养基中培养不同时间后的3株利什曼原虫前鞭毛体计数均显著高于M199 + 20%胎牛血清培养基和M199 + 20%马血清培养基（P均 < 0.05）,在这3种培养基中培养不同时间后的KS?2株利什曼原虫前鞭毛体计数均显著高于Cy和JIASHI?5株（P均 < 0.05）。KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体均不能在脑心浸液培养基中生长繁殖。结论　分离自我国不同类型内脏利什曼病流行区的利什曼原虫在同一培养基中生长增殖速度有差异,同一利什曼原虫分离株在不同培养基中的生长繁殖速度亦有差异。NNN培养基是最适合我国内脏利什曼病流行区利什曼原虫分离株的培养基。     

乙型肝炎患者骨髓间充质干细胞培养方法的优化和生物学特性

目的比较乙型肝炎患者骨髓间充质干细胞（MSCs）多种体外培养方法的效果，确定乙型肝炎患者MSCs的最佳体外培养方案；并对其生物学特性进行初步研究。方法比较2种接种方案和5种不同成分培养液的乙型肝炎患者MSCs体外培养成功率。比较5种不同成分培养液和4种换液方案的乙型肝炎患者MSCs生长曲线。进行乙型肝炎患者MSCs的形态观察和表面分子鉴定，传代培养以初步了解其生长特性。结果密度梯度离心接种法的体外培养成功率（88％）高于全骨髓接种法（76％），但差异无统计学意义。5种培养液的体外培养成功率差异有统计学意义（P〈0．01），其中患者自体血清培养液的培养成功率最高（100％），3种FBS培养液的培养成功率次之，健康成人血清培养液的最低（56％）。患者自体血清培养液的MSCs生长曲线最高，健康成人血清培养液的生长曲线最低，3种FBS培养液的MSCs生长曲线集中位于上述两者之间。以2d或者3d一次全量换液的MSCs生长曲线较高。乙型肝炎患者MSCs与正常人MSCs形态相同，表面分子表达一致，生长特性近似。结论乙型肝炎患者MSCs的体外培养方案以密度梯度离心法分离后，自体血清培养液进行培养，2～3d全量换液为佳，可以较快获得纯度较高的MSCs。乙型肝炎患者MSCs的生物学特性和正常人的近似。     

结肠小袋纤毛虫体外培养观察

本文对猪结肠小袋纤毛虫 (Balantidiumcoli)在体外培养的条件进行了试验并观察其生长情况。试验结果表明 :小袋纤毛虫在外界环境中接触空气 1h左右由滋养体形成包囊 ,滋养体在相对厌氧环境下的深层粪便中 ,或RSS培养液(Ringer液—血清—淀粉 )中能存活并繁殖。在 2 8.0～ 32 .3℃的夏季室温下小袋纤毛虫滋养体在 4cm深层粪便中存活至10 8h ,在RSS培养液中滋养体存活时间最长达 192h ,比在 37℃培养时间延长 96～ 12 0h。培养基pH值 6 .2～ 6 .4与 pH7.0～7.2相比未见明显差异 ;米粉用量过多加速滋养体死亡。本试验尚为摸索阶段 ,以上结论还需进一步验证     

人型支原体液体培养中pH与活菌浓度关系研究

目的探讨入型支原体液体培养过程中,培养时间、培养基pH与菌浓度的关系,研究人型支原体液体培养时的生长规律。方法用精密pH计定时测定人型支原体培养过程中培养基pH值,同时观察培养基颜色变化;采H{微量法测定菌浓度,每3h测定一次,连续测定48h。结果不同浓度的人型支原体在液体培养时呈现相同的生长规律,有类似于细菌的牛长曲线,最大菌体浓度（CCU）10^6／ml;人型支原体生长与培养基pH相关,pH6．8时,菌体活性最高．培养是呈橙红色;pHi〉7．0时,菌体活性逐渐下降,pH7．2时,培养基旱深红色,不冉发生明显的颜色改变,人型芰味体活菌量迅速降低。结论人型支原体液体培养基培养时,培养基pH6．8、液体颜色橙红时,可扶取较佳生长活性的高菌含量培养物。     

Prevalence of Blastocystis hominis infection in asymptomatic individuals from Bangkok, Thailand

Fresh stool examination was performed from 2,230 participants who enrolled in annual check-up programs of the Faculty of Medical Technology, Mahidol University in 1999-2000 and 2004. In this study, Blastocystis hominis infection was diagnosed by culturing in Jones' media. A total of 21% of fecal specimens (in 1999-2000) and 13.7% (in 2004) were positive for B. hominis. The vacuolated form was the predominant form found in culture solution after 48 hours of incubation. The distribution of infection was highest between the ages of 21-30 years (p<0.05). There was no significant difference in infection between male and female groups. Other parasites, eg Giardia lamblia, Entamoeba histolytica, Entamoeba coli, Endolimax nana, Trichomonas hominis, Strongyloides stercoralis, Opisthorchis viverrini and Taenia species, were also found by fresh stool examination.     

Polypeptides associated with in vitro cyst formation of Blastocystis hominis

The objective of this study was to characterize the polypeptides associated with cysts of Blastocystis hominis. This form is believed to be infective and plays a role in parasite resistance to anti-B. hominis drugs currently used for treatment of Blastocystis associated diarrhea. Cysts were induced through in vitro culture of the parasite in complete medium supplemented with bacterial extract with trypticase, metronidazole or doxycycline. SDS-PAGE analysis showed almost similar polypeptide patterns of parasite extracts obtained from in vitro cultured parasites before and after exposure with the three supplements. Polypeptide bands at 76, 58.5, 48, 45, 40, 38, 32, 25 and 22 kDa were constantly seen in all antigenic preparations and no specific cyst-associated polypeptide was present. However, on immunoblot analysis, 3 out of 16 blastocystosis human sera identified a cyst-associated polypeptide at 60 kDa in all parasite extracts prepared from cultures with the three supplements. In addition, there were associated morphological changes detected in these parasites stained with acridine orange and observed under fluorescence microscopy. Metronidazole induced cyst forms (reddish cells) as early as 12 hours post-exposure; more cyst production (with stronger immunoblot bands) occurred after 24 hours exposure. However, cysts rupture with release and destruction of B. hominis daughters cells occurred after 48 hours exposure. Doxycycline induced less cyst-like forms at 24 hours (weaker 60 kDa band) and less destruction of the cysts (60 kDa band still present at 72 hours post exposure). Bacterial extract and trypticase also induced cysts at 12 hours with increasing numbers up to 72 hours exposure (corresponding increase in intensity of 60 kDa band from samples harvested at 12 to 72 hours post exposure) without any sign of deleterious effect on the parasite.     

In-vitro cultivation: a sensitive method for detecting Blastocystis hominis

Currently, the detection of human infection with Blastocystis hominis is usually based on the examination under a light microscope of faecal samples, either directly, as 'simple smears', or after some form of concentration. Whether short-term, in-vitro cultivation would increase the sensitivity of such detection remains a matter of controversy. Over 900 fresh stool specimens, from soldiers in the Royal Thai Army, were each checked for the parasite using three methods: simple smears; formalin-ethyl-acetate concentration; and cultivation in Jones' medium. Although 334 of the samples were found to be culture-positive, the parasites were only detected in 142 of the simple smears, and faecal concentration led to an even lower sensitivity (64 positive samples). In-vitro cultivation does seem worthwhile in the detection of B. hominis carriage in field studies.     

Effect of blood culture media on the in vitro recovery of Mycoplasma hominis

Despite the prevalence of Mycoplasma hominis few cases of septicaemia due to this organism have been reported. The ability of various blood culture media to sustain the growth of an inoculum of M. hominis was therefore studied. Of the media tested, an 'in-house' Hartley's digest broth with 0.1% glucose was the most efficient. The investigation also demonstrated that growth of M. hominis is adversely affected by the concentration of liquoid (sodium polyanetholsulphonate) present in blood culture media.     

A study of three blood culture media for isolating genital mycoplasmas from obstetrical and gynaecological patients

Mycoplasma species are often found colonising the female genital tract. Their ability to become invasive and pathogenic, however, is often ignored, since attempts may not be made to culture these organisms from the bloodstream. We have investigated the ability of three types of blood culture media to support the growth of genital mycoplasmas. The media studied included brain-heart infusion broth, brain-heart infusion broth supplemented with 30% V/V sucrose and fastidious anaerobe broth. Genital mycoplasmas were cultured from the latter medium only. Since this was the sole medium which was liquoid-free, the inhibitory effects of liquoid on Mycoplasma spp. is discussed. This study comprised an investigation of 75 patients in obstetric and gynaecological wards with postpartum or post-operative fever. Genital mycoplasmas were isolated from five (6.7%) patients, four with Ureaplasma urealyticum and one with M. hominis. The value of considering these organisms in the differential diagnosis of fever in 'at risk' patients and of including appropriate media for their isolation is emphasised.     

Isolation and characterization of an RNA-proteolipid complex associated with the malignant state in humans.

An RNA-proteolipid complex was isolated from sera of patients with a variety of malignant disorders as well as from culture media of malignant cell lines. The complex, characterized by a relatively constant composition, contains 27S poly(A)+ RNA and Mr 1250 oligopeptide(s) and is rich in phospholipids and glycosphingolipids. Serum lipoproteins (low and high density) differ from this complex in density, chemical composition, and immunological reactivity. The complex was detected in 94 of 96 cases of malignancy tested but not in any of 58 patients with nonmalignant disorders or in 46 healthy individuals.