人类肠道菌群DNA提取影响因素的研究

目的研究提取人类粪便中细菌基因组DNA的影响因素。方法采用溶菌酶和十二烷基磺酸钠(SDS)+石英砂+酚-氯仿抽提法提取粪便标本中细菌DNA,用PCR扩增细菌16SrDNA。比较不同粪便量,不同放置时间,不同放置温度下的粪便细菌DNA浓度及纯度改变;用Chao index评测高通量测序的结果。结果采用20mg粪便量提取出的细菌DNA的浓度最高;常温下放置3h后,提取的细菌DNA的浓度开始下降;在-20℃放置12h后,细菌DNA的浓度开始下降;-70℃放置48h后细菌DNA的浓度开始下降;样品纯度均在1.8以上;Chao index曲线趋于平缓表明测序数据量足够大。结论提取肠道细菌基因组DNA时,粪便标本取样量以20mg为宜,常温下粪便标本放置不超过2h,本研究所使用的方法所提取的DNA浓度可以达到高通量测序的样本要求。     

不同保存方法对柽柳基因组总DNA产量和质量的影响

通过比较不同保存方法对柽柳(Tamarix chinensis)基因组总DNA提取效果的影响,确定合适的野外采集植物样品的保存方法。采用改良CTAB法提取了室温风干、4℃、-20℃、-80℃和硅胶干燥5种保存方法分别保存24 h、1周和1月后的柽柳基因组总DNA,结果表明:4℃保存1月后DNA产量最低,为60.35μg/g,-80℃条件下保存24 h的材料中提取的DNA产量最高,为246.92μg/g,保存效果最好。但在野外仪器条件不具备的情况下,柽柳可采用室温风干或硅胶干燥保存的方法,1月内材料中DNA降解程度较轻,可以扩增出所需目的片段。     

3种粪便标本保存方法对肠道菌群检测结果影响的比较研究

比较3种粪便标本保存方法对肠道菌群检测结果的影响,为采集粪便标本后短期保存方法的选择提供参考。收集10名志愿者的新鲜粪便标本,对每份标本分别采用4℃、室温(25℃)、无水乙醇保存4 h,然后统一放入-80℃低温冰箱冻存。随后进行细菌DNA提取,并对16S rRNA V4区基因进行扩增,应用Illumina MiSeq平台进行高通量测序,比较3种保存方法下肠道菌群多样性和组成差异。结果表明,3种保存方法下,肠道菌群在Alpha多样性方面无统计学差异(P0.05)。肠道菌群属水平比较,毛螺旋菌属(Lachnospira)在无水乙醇组中相对丰度高于4℃组和室温组(P0.05),萨特菌属(Sutterella)在无水乙醇组中相对丰度高于4℃组(P0.05),而真杆菌属(Eubacterium)在无水乙醇组中相对丰度低于室温组(P0.05)。PCoA分析发现,当UniFrac距离加权后,4℃和无水乙醇保存同一受试者粪便标本,菌群结果较为接近,而室温保存会导致个别受试者菌群出现离散现象。无水乙醇短期保存粪便标本,肠道菌群多样性、组成以及结构保持稳定,能满足不同研究的需要。此外,无水乙醇具备价格低廉、便于携带等优点。因此,在新鲜粪便标本不能立即低温冻存时,无水乙醇保存可以作为首要选择。     

支原体菌株低温保存活力稳定性研究

目的研究低温冻存时间及温度对支原体菌种保存稳定性和菌种活力的影响。方法用固体平板计数法计算不同冻存时间、温度下的口腔支原体、肺炎支原体菌落数差异,分析这2个因素对口腔支原体、肺炎支原体菌种活力的影响。结果口腔支原体在生长48 h后达到生长高峰,肺炎支原体生长较为缓慢,在103 h到达生长高峰。口腔支原体在-70℃冻存1个月时,菌落数由10¹² cfu/mL下降至10¹¹ cfu/mL,冻存1个月的菌落数和冻存3个月、12个月的菌落数比较,差异均无统计学意义(P>0.05)。肺炎支原体在-70℃的冻存条件下,菌落数一直处于稳定水平;比较-35℃与-70℃的冻存条件下肺炎支原体的菌落数,差异无统计学意义(P>0.05),口腔支原体在-70℃冻存条件下的菌种活力明显高于-35℃(P<0.05)。结论口腔支原体生长迅速,但对于储存温度的敏感性较高,肺炎支原体相较口腔支原体生长缓慢,停滞期较长,菌株对冻存温度和时间的敏感度较低,稳定性高。菌种冻存温度应选择较低温度,冻存周期在1年内较为稳定。     

不同时间下超低温保存肝癌组织细胞RNA完整性的差异分析

目的研究肝癌组织细胞样本于-80℃超低温保存条件下的质量控制,探讨肝癌组织细胞样本在超低温保存中的时效性。方法随机抽取超低温新鲜冻存(24 h内)、保存3个月、6个月和1年不同时长的肝癌和对应的癌旁组织细胞各12对,HE染色评估标本取材的准确性,采用Promega自动提取核酸仪提取肝脏细胞RNA,利用Agilent 2100生物分析仪进行RIN值及28S/18S比值分析。对新鲜冻存、保存3个月、6个月以及1年的组织细胞样本的RIN值和28S/18S比值进行组间非配对t检验。结果 HE染色镜下观察发现切片组织细胞中肿瘤细胞比例达到80﹪以上,为生物样本库的合格样本。生物分析仪检测结果显示超低温新鲜冻存、保存3个月、6个月和1年的癌和癌旁组织细胞的RIN均值均7.0,平均28S/18S比值均1.5,组间非配对t检验显示,保存1年的肝癌组织细胞相比于新鲜冻存(7.442±0.674 vs 8.617±0.769,P=0.001)、保存3个月(7.442±0.674 vs 8.275±0.617,P=0.005)和6个月(7.442±0.674 vs 8.175±0.970,P=0.043)的组织细胞RIN值显著下降,提示存在一定程度的RNA降解,而其余各组之间差异均无统计学意义(P均0.05)。结论在超低温条件下新鲜冻存、保存3个月、6个月的肝癌及癌旁组织细胞,其质量均可以获得有效保证,而保存1年的肝癌组织细胞的RNA虽然存在一定程度的降解,但能够满足后续临床科研的要求。     

不同储存条件对大麻化学稳定性的影响

为探讨光照和温度对大麻植物中大麻酚类稳定性的影响,该研究将大麻植物检材以固体粉末和甲醇提取溶液的形式分别在室温(22±2)℃见光、室温(22±2)℃避光、4℃避光、-20℃避光条件下储存20 d后,采用超高效液相(UPLC-PDA)检测分析样本中Δ9-四氢大麻酚(Δ9-THC)、大麻二酚(CBD)和大麻酚(CBN)的含量变化情况。结果表明:3种大麻酚类在不同化学表型大麻中的含量变化趋势相同,固体粉末样本的Δ9-THC、CBD含量在室温光照条件下显著下降,CBN含量基本不变;甲醇提取样本中Δ9-THC、CBN和CBD含量在室温光照条件下均显著下降。避光条件下的室温(22±2)℃及低温(4℃、-20℃)可稳定保存两种形式的大麻样本。大麻中的精神活性成分Δ9-THC的降解满足一级反应动力学规律,光照是影响Δ9-THC降解的重要因素,如果在室温避光条件下储存,大麻或其甲醇提取物可稳定保存,可以更好地指导司法实践活动中短期内大麻检材的取证、运送、保存及鉴定。     

人体蠕形螨的DNA提取与随机引物PCR检测

【目的】探索人体毛囊蠕形螨和皮脂蠕形螨DNA的提取方法。【方法】采用液氮反复冻融研磨法破碎螨体细胞, 选用改良小昆虫DNA提取法、碱裂解法和试剂盒提取法, 分别提取冻存时间在5个月内和8~10个月的毛囊蠕形螨和皮脂蠕形螨基因组DNA, 并用随机引物PCR方法进行检测。【结果】蛋白核酸测定仪检测结果显示, 试剂盒法提取的DNA纯度较高、量较多, 明显优于改良小昆虫法和碱裂解法。随机引物扩增结果显示清晰的DNA指纹图谱, 两种人体蠕形螨DNA指纹具有明显差异。蠕形螨冻存时间影响DNA提取的量, 但对DNA提取的纯度和RAPD指纹图谱影响较小。不同DNA提取方法提取的同一种蠕形螨DNA指纹图谱基本相似, 试剂盒法和改良小昆虫法提取的DNA样本条带多而清晰, 碱裂解法提取的样本条带少而模糊。【结论】液氮反复冻融研磨法破碎蠕形螨细胞是有效的, 蠕形螨冻存时间不宜超过6个月, 试剂盒提取法是提取蠕形螨DNA的好方法。RAPD技术可以用于这两种人体蠕形螨DNA分子水平上的检测和分类。     

基于NGS方法评估不同保存条件对粪便样本中菌群含量的影响

目的评估粪便样本的不同保存条件对肠道微生态研究结果的影响。方法设计相关实验,比较7种不同的保存方法,基于高通量测序技术比对不同时间、不同贮存温度对粪便样本DNA质量、菌群多样性及病原菌检出等结果之间的差异。结果证实了对于粪便样本采集仍建议采取即刻提取核酸或-20℃保存的方法。同时比较多种保存方法后发现,采用不同样本保存方法受到影响的菌属集中在低丰度菌属(≤0.01%),对应较高丰度的病原菌检测结果的可靠性影响较小,样本中丰度超过千分之一的病原菌检测率超过95%。结论对于肠道微生态研究建议对粪便样本采取即刻提取核酸或-20℃保存的方法。对于由于其他原因未能妥善保存的粪便样本(如常温保存48 h),仍可对丰度超过千分之一的病原菌进行检测。以上结果对于肠道菌群研究中异地所采集的粪便样本运输和贮存具有一定的指导意义。     

11种灭活型样本保存液对病毒核酸保护效果的比较

收集11种不同厂家的灭活型样本保存液,将甲型流感病毒加入保存液和生理盐水中,在4℃、25℃和37℃条件下保存0 h、2 h、4 h、6 h和24 h后,分别提取各组的病毒核酸,并通过逆转录实时荧光PCR检测病毒载量变化,以探究不同样本保存液对病毒核酸保护效果是否存在差异。结果发现不同样本保存液中的病毒载量随保存时间和温度变化存在显著差异。据此可将11种灭活型样本保存液的保存效果分为四类：(1)优秀产品：在任一温度（4℃、25℃、37℃）条件下,即使保存24h时病毒核酸都未发生明显降解,占比27.2%(3/11);(2)良好产品：低温（4℃）和室温（25℃）情况下核酸未出现降解,但在高温（37℃）条件下6 h后保存效果明显下降,占比27.2%(3/11);(3)合格产品：低温（4℃）条件下对病毒核酸具有较好的保护效果,但在室温（25℃）和高温（37℃）情况下随保存时间延长其保护效果显著下降,占比18.1%(2/11);(4)不合格产品：任一保存条件下对病毒核酸的保护效果都较差,甚至加入病毒后立即导致病毒核酸发生快速降解,占比27.2%(3/11)。以上结果说明不同厂家样本保存液对病毒核酸...     

重组鼠疫耶尔森菌LcrV抗原原液性质研究

目的进行重组鼠疫耶尔森菌LcrV抗原原液二聚体含量及性质研究,确定LcrV原液的相关质控标准。方法在不同缓冲体系(0.85%NaCl(NS)、20 mmol/L PBS),不同蛋白浓度(2.0、1.5、1.0、0.5、0.1 mg/mL)及不同保存温度(4℃、-20℃、-70℃)条件下保存LcrV抗原,采用SDS-PAGE和HPSEC方法定期检测LcrV二聚体含量及纯度。将连续三批检定合格的LcrV抗原原液进行质谱相对分子质量测定、等电点测定、N末端氨基酸序列测定、圆二色(CD)谱、HPLC肽谱及氨基酸组成分析,研究LcrV抗原的相关性质。结果随着保存时间的延长LcrV抗原二聚体含量增加,低温保存时二聚体不易大量形成。在-20℃和-70℃条件下,NS保存的LcrV抗原比PBS体系保存稳定,而在4℃条件下NS保存的LcrV抗原容易降解。LcrV抗原高浓度保存容易发生聚合。LcrV抗原在低质量浓度(0.1 mg/mL)保存时免疫学活性明显下降。质谱检测到LcrV单体和二聚体共同存在,且与理论相对分子质量一致。LcrV原液检测等电点范围为4.6～6.3。N末端测序、CD谱、HPLC肽谱图及氨基酸组成分析与理论结果一致。结论 LcrV抗原原液保存条件确定为:NS体系,蛋白质量浓度1.0～2.0 mg/mL,-20℃以下冻存。制备的LcrV抗原各项检测结果与理论结果一致,抗原性质稳定。     

Characteristics of the membranes of the pellicle of the ciliatePseudomicrothorax dubius

Summary The membranes of the pellicle of the ciliatePseudomicrothorax dubius are investigated using thin section electron microscopy and freeze-fracture replicas. The plasma membrane is covered by a surface coat and is connected to the outer alveolar membrane by short, sometimes branched, bridges. The inner alveolar membrane is coated on both sides. The epiplasm lies in intimate contact with the cytoplasmic surface of this membrane, and there is a corresponding deposit on the other surface. This deposit is regularly striated.The epiplasmic layer and the alveoli are interrupted at sites of cytotic activity,e.g., the attachment sites of trichocysts, the cytoproct, and the parasomal sacs. The striated deposit ends where the epiplasm ends, indicating a direct relationship between these two epimembranous layers.There is a deposit along the sides of the first part of the tip of the trichocysts, and in this region the trichocyst membrane is free of intramembranous particles.The membrane of the parasomal sacs has a coat on both surfaces. That on the extraplasmic surface is similar to the surface coat of the plasma membrane. The origin of the cytoplasmic coat is unknown. The cytotic activity of these sacs is indicated by their highly irregular profiles.     

The early development of the tail and the transformation of the shape of the nucleus of the spermatid of the domestic fowl,Gallus gallus

Summary The differentiation of the spermatid, especially in reference to the formation of the flagellum, and transformation of the shape of the nucleus was investigated in the domestic fowl.In the early stage of the spermatid, a prominent Golgi apparatus appears around the centrioles. The Golgi vesicles then surround the axial-filament complex which develops from the distal centriole. These vesicles fuse to form continuous membrane at the earliest stage of flagellar formation, and in the succeeding stage Golgi lamellae are attached to the plasma membrane of the developing flagellum. From these observations, it is assumed that Golgi apparatus may be a source of the membrane system of the flagellum.The microtubules distributed around the nucleus form the circular manchette. The anterior region of the nucleus with the manchette is cylindrical in shape and the posterior region without it remains irregular in shape. When the circular manchette has been completed, the whole nucleus acquires a slender cylindrical shape. The circular manchette then changes into the longitudinal manchette. The nuclei of spermatids without a longitudinal manchette are abnormal in shape. In view of these observations it is assumed that the nuclear shaping of the spermatid may be accomplished by circular manchette and the maintenance of shape of the elongated nucleus by longitudinal manchette.The authors wish to thank Mr. Takayuki Mori for his helpful suggestions and technical advices     

Characterization of the composition of the aqueous humor and the vitreous body of the eye of the frog Rana temporaria L

This study aimed to analyze the aqueous humor (AH) and the vitreous body (VB) of the eye of the adult frog Rana temporaria L. as a representative species of amphibians, which lead a semi-terrestrial life. The presence of collagen, albumin, uric acid and electron donors was shown in both media; however, there are slight differences in their concentrations. To determine collagen, a spectral-fluorescent probe, cyanine dye, was used. The presence of collagen in AH of the frog was found at the first time. The total content of electron donors (ascorbic and uric acids, tryptophan, and tyrosine) in VB and HA was roughly estimated at ~ 1.5 × 10^− 4 mol/L. Both VB and AH absorb light in similar UV regions. The total protein and albumin contents in AH were found to be somewhat higher than those in VB. The uric acid content was at an equally low level in both intraocular media. It is supposed that the similarity of VB and AH compositions shown in this work is due to some exchange between VB and AH contents in the course of accommodation. The role of intraocular fluids in physiological functions of the eye and in protecting the retina against UV light is discussed.     

Observations on the permeability of the choriocapillaris of the eye

Summary The choriocapillaris is a fenestrated capillary bed located posterior to the retinal pigment epithelium. It serves as the main source of supply to the photoreceptors, retinal pigment epithelium, and other cells of the outer retina. The permeability of these capillaries to intravenously injected ferritin (MW — approx. 480,000; mol. diam. 11 nm) was examined in the mouse, rabbit, and guinea pig, each of which is characterized by a different type of retinal vascularization. In all three species, the bulk of the ferritin remained in the capillary lumina, where it appeared to be blocked at the level of the diaphragmed fenestrae. Some ferritin was present in endothelial cell vacuoles. The results confirm previous work on the rat choriocapillaris and indicate that the barrier function of the choriocapillary endothelium is present even among species in which the retinal circulation differs significantly.Supported by NIH grant EY03418