我国主要梨产区梨腐烂病菌培养表型与致病力差异的研究

[目的]旨在研究我国主要梨产区不同培养表型的梨腐烂病菌的致病力差异。[方法]从全国13个省、市、自治区采集梨腐烂病样品,通过组织分离法共分离鉴定出465株梨腐烂病菌,在PDA培养基上观察其培养表型并进行分类;挑选出128株菌株,采用打孔法接种菌丝块测定各菌株在‘库尔勒香梨’上的致病力。[结果]根据所分离的菌株在PDA培养基上的菌落颜色及形态不同粗分为5类,各类菌株均能产生多种类型的子座,菌株的不同培养性状与其地理来源并无关联;在梨果上,不同分离菌株的致病力存在明显分化,但是菌株的致病力差异与其地理来源及培养性状并无关联,且将菌株致病力进行强、中、弱划分后,发现中等致病力的菌株占64%,为优势菌株。[结论]我国的梨腐烂病菌存在多样的培养性状,且菌株的致病力分化明显,中等致病力的菌株占多数,但是菌株的培养性状、致病力及其地理来源这三者之间并无关联,因此要深入了解梨腐烂病菌,还需从菌株的致病基因入手,采用分子生物学手段对其进行研究。

Characterization of a novel rat cholangiocarcinoma cell culture model-CGCCA

AIM： TO characterize a culture model of rat CCA cells, which were derived from a transplantable TTA-induced CCA and designated as Chang Gung CCA （CGCCA）. METHODS： The CGCCA cells were cultured at in vitro passage 12 times on a culture dish in DMEM medium. To measure the doubling time, 103 cells were plated in a 96-well plate containing the growth medium. The cells were harvested 4 to 10 d after seeding, and a standard MTT assay was used to measure the growth. The phenotype of CACCA cell and xenograft was determined by immunohistochemical study. We also determine the chromosomal alterations of CGCCA, G-banding and spectral karyotyping studies were performed. The CGCCA cell line was transplanted into the nude mice for examining its tumorigenicity. 2-Deoxy-2-（18F）fluoro-D- glucose （FDG） autoradiography was also performed to evaluate the FDG uptake of the tumor xenograft. RESULTS： The doubling time for the CGCCA cell line was 32 h. After transplantation into nude mice, FDG autoradiography showed that the tumors formed at the cell transplantation site had a latency period of 4-6 wk with high FDG uptake excluding necrosis tissue. Moreover, immunohistochemical staining revealed prominent cytoplasmic expression of c-erb-B2, CK19, c-Met, COX-n, EGFR, MUC4, and a negative expression of K-ras. All data confirmed the phenotypic features of the CGCCA cell line coincide with the xenograft mice tumors, indicating cells containing the tumorigenicity of CCA originated from CCA. In addition, karyotypic band- ing analysis showed that the diploid （2n） cell status combines with ring and giant rod marker chromosomes in these clones; either both types simultaneously appeared or only one type of marker chromosome in a pair appeared in a cell. The major materials contained in the marker chromosome were primarily identified from chromosome 4. CONCLUSION： The current CGCCA cell line may be used as a non-K-ras effect CCA model and to obtain information and reveal novel pathways for CCA. Further applications regarding tumor markers or therapeutic targeting of CCA should be addressed accordingly.

艰难梭菌临床分离株的毒素检测和MLST分型

目的初步研究中日友好医院艰难梭菌临床分离株的毒素特征、核糖体分型和多位点序列分型情况．为进一步监测医院内艰难梭菌暴发流行奠定基础。方法收集2012年至2013年本院疑似艰难梭菌感染（Clostridium diffcile infection，CDI）的321例腹泻患者的标本，采用表型培养法进行菌株分离和鉴定，并对分离菌株进行毒素B的细胞毒试验，分析菌株的毒素基因（tcdA、tcdB、cdtA、cdtB），采用核糖体分型（ribotyping，RT）和多位点序列分型（muhiloeus sequence typing，MLST）分析菌株间的同源性；收集患者临床信息，分析不同患者人群的CDI发生率。结果共从46名腹泻患者中分离到48株艰难梭菌菌株，其中3株菌株分离自同一患者；CDI发生率为14．3％（46／321），其中门诊患者为12．8％（5／39）、住院患者为14．5％（41／282）；所有菌株的毒素B细胞毒试验均为阳性，其中STl型较其他ST型菌株毒素作用更强；毒素基因tcdA（-）／tcdB（＋）型为20．8％（10／48），主要是ST37和ST81：所有菌株的RT和MLST结果一致，均为相同的9个型别，其中以STl／RT027为主。占27．1％（13／48），这些STl／RT027菌株毒素基因检测均为tcdA（＋）／tcdB（＋）、二元毒素基因检测均为cdtA（＋）／cdtB（＋），其中中医呼吸病区占84．6％（11／13），提示该病区存在STl／RT027型菌株的小范围暴发流行的可能。结论CDI在我院主要以医院感染为主，分离株主要为毒素基因，tcdA（＋）/tcdB（＋）的菌株，监测STl／RT027高产毒性流行株引发的暴发流行尤为重要。