Wistar大鼠胰岛细胞体外分离、纯化及鉴定

目的 探索Wistar大鼠胰岛分离纯化的最佳条件.方法 采用肝胰管内灌注胶原酶消化分离胰岛及Ficoll 400密度梯度离心纯化.纯化后的胰岛经组织学染色、电镜及放射免疫法鉴定其特异性和活力.结果 组织学染色显示纯化后胰岛的活力和纯度分别在95%和85%以上.电镜显示纯化后的胰岛形态完整,包膜清晰,分泌颗粒丰富.放射免疫结果表明,低糖组和高糖组分泌胰岛素的浓度有显著差异,证明胰岛功能良好.结论 肝胰管内灌注胶原酶消化法是一种好的消化方法.影响胰岛收获量的因素很多,例如胰腺的充分扩张,胶原酶的浓度和活性以及消化的时间等.     

利什曼原虫K26序列应用于我国利什曼原虫分离株鉴定的价值分析

目的评价利什曼原虫K26序列应用于我国利什曼原虫分离株鉴定的价值。方法收集我国不同流行区的利什曼原虫16个分离株和6个国际参照株,培养后收集前鞭毛体,提取DNA,扩增K26序列并测序,应用ClustalX2进行序列比对,从GenBank下载同源性较高的利什曼原虫分离株的K26序列,构建系统进化树,分析我国不同流行区的利什曼原虫分离株的亲缘关系。结果我国16个利什曼原虫分离株和国际参照株DD8均扩增出单一条带,而5个非杜氏利什曼原虫复合体虫株均未扩增出任何条带。序列分析结果表明,我国人源型利什曼病流行区的3个分离株801、KS-2和KS-6均扩增出920 bp片段,自然疫源型利什曼病流行区的4个分离株XJ771、JIASHI-1、JIASHI-2和JIASHI-5均扩增出491 bp片段,人犬共患型利什曼病流行区4个分离株Cy、SC6、Hebei-Lv和Shanxi-Yang均扩增出404 bp片段,皮肤利什曼病流行区的5个分离株KXG-Xu、KXG-Liu、KXG-65、KXG-918和KXG-927均扩增出449 bp片段,且各流行区内虫株间的序列一致性均为100%。各流行区之间的虫株间的序列一致性较低,为40.2%~91.4%。系统进化树分析结果显示,我国16个利什曼原虫分离株聚集成2个群3个亚群,其中KXG-Xu、KXG-Liu、KXG-65、KXG-918、KXG-927、XJ771、JIASHI-1、JIASHI-2、JIASHI-5分离株聚集为A亚群,Cy、SC6、Hebei-Lv、Shanxi-Yang与婴儿利什曼原虫聚集为B亚群,A亚群和B亚群聚集为Ⅰ群;801、KS-2和KS-6分离株与杜氏利什曼原虫聚集为C亚群,进一步与国际参照株DD8聚集为Ⅱ群。结论利什曼原虫K26序列可应用于我国利什曼原虫分离株的鉴定。     

医院流行性多重耐药鲍曼不动杆菌的耐药及同源性分析

目的了解重症监护病房爆发流行的多重耐药鲍曼不动杆菌的耐药谱特征及其同源性。为临床防治提供依据。方法4株多重耐药鲍曼不动杆菌分离自外科重症监护病房（SICU）的感染惠者，采用E—TEST检测药物的MIC值，肠杆菌科基因组内重复一致序列聚合酶链反应（ERIC—PCR）进行克隆株的DNA分型。结果4株菌除对头孢哌酮／舒巴坦复合制剂的MIC值较低外，对头孢菌素类、氨基糖甙类和氟喹喏酮类等抗菌素均显示出了较高水平的多重耐药性；DNA分型证实为同一克隆株。结论本组鲍曼不动杆菌为多重耐药株，同一克隆株在不同感染个体间的相互传播导致了本次医院内感染的流行。     

大鼠气管-支气管上皮细胞的分离、鉴定和培养

目的探讨大鼠气管-支气管上皮细胞的分离、鉴定和培养的方法.方法采用链酶蛋白酶消化联合细胞刷刷洗气管-支气管分离上皮细胞,激光共聚焦显微镜下鉴定,于无血清完全F12培养基中培养.结果实验中可得到大约106个细胞/鼠,并且有较高的成活率和细胞纯度,在无血清完全F12培养基中细胞生长良好.结论消化后刷洗法是一种实用的大鼠气管-支气管上皮细胞提取方法,无血清完全F12培养基是一种可行的培养体系.     

Comparison of the complete genome sequence between C1 and G4 isolates of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus

The complete nucleotide sequence of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus isolate C1 (HearSNPV-C1) was determined and analyzed by comparing with the genome of HearSNPV-G4 isolate. C1 and G4 isolates occurred in the same host species and geographic location but showed different virulence. The HearSNPV-C1 genome consisted of 130,759 bp and 137 putative open reading frames larger than 150 nucleotides were identified. The two genomes shared 98.1% nucleotide sequence identity, with a total number of 555 bp substitutions, 1354 bp deletions, and 710 bp insertions in HearSNPV-C1. Comparison of ORFs and homologous repeat (hr) regions of the two genomes showed that there were four highly variable regions hr1, hr4, hr5, and bro-b, all in repeat regions. These results suggest that baculovirus strain heterogeneity may be often caused by SNPs and changes in the hrs and bro genes.     

Tenacity of Exogenous Human Papillomavirus DNA in Sperm Washing

Purpose: Sperm cells have been shown to take up exogenous DNA readily. The hypothesis was that sperm washing would remove exogenous viral DNA infecting sperm cells. The objective was to compare three types of sperm washing procedures for their capacity to remove exogenous human papillomavirus (HPV) DNA from infected sperm. Methods: Prewashed sperm were equally divided and sperm in one portion were exposed to L1 HPV DNA fragments for 30 min at 37°C. Untreated washed sperm served as the control. After transfection, the sperm were washed by either centrifuge, two-layer Isolate colloid wash, or test-yolk buffer procedures. Sperm parameters were measured on a Hamilton Thorn HTM-C analyzer. Sperm DNA were extracted and polymerase chain reaction (PCR) was carried out targeting the L1 consensus gene of HPV and the designated sentinel gene, 17q21 spanning the D17S855 gene. Amplified products were analyzed in 2% agarose gel electrophoresis. Results: PCR analyses detected the consensus L1 HPV gene in sperm after they were processed through either of the three procedures. Controls were negative for the L1 gene. Extracted DNA were verified by PCR amplification of 17q21 spanning the D17S855 gene. Transfected sperm had higher percentages of total motility and progression compared with the control. Centrifuged, washed, transfected sperm exhibited a greater curvilinear velocity and hyperactivation. Conclusions: The data showed that washing would not remove exogenous HPV DNA from sperm cells. The viral DNA was tenaciously bound to the sperm, suggesting an internalization into the sperm. The viral DNA also increased the motility of the sperm by affecting the velocity and progression of the sperm, which suggested either an increase in metabolism, an enhancement of the calcium-regulated motility mechanism, or an artifact of PCR reagents. More studies are needed to elucidate the mechanism of DNA stimulated sperm motility.     

精液的两种不同处理方式对体外受精-胚胎移植的影响

目的：探讨精液的两种不同处理方式对体外受精-胚胎移植的影响。方法：对73例继发不育的IVF—ET患者的结局进行回顾性分析。根据进入周期精液处理方式的不同将患者分为2组：梯度离心法处理组（A组）42例;梯度离心上游法联合应用组（B组）31例。结果：两组的促性腺激素用量、促排天数、获卵数、受精率、卵裂率、优质胚胎率间差异无统计学意义（P〉0．05）,胚胎种植率和临床妊娠率A组低于B组,A组分别为18．2％（16／88）,26．2％（11／42）;B组2．3（20／62）,51．6％（16／31）,两组的种植率和临床妊娠率相比较有统计学意义（P〈0．05）。结论：梯度离心法和上游法联合应用处理精液比单纯应用梯度离心法处理精液有助于提高IVF—ET患者的种植率和临床妊娠率,可改善妊娠结局。     

多药耐药鲍氏不动杆菌基因同源性分析及流行病学调查

目的 了解重症监护病房(ICU)流行的多药耐药鲍氏不动杆菌(MDR-AB)耐药谱特征及其同源性,为临床防治提供依据.方法 MDR-AB分离自2007年5月,4株系住ICU肺部感染患者的痰标本,4株系病房物体表面;按<全国检验技术操作规程>要求操作,用全自动微生物分析仪进行细菌鉴定及药敏试验;用脉冲场凝胶电泳(PFGE)对8株鲍氏不动杆菌进行基因分型,明确其是否为同一菌株的克隆.结果 4株痰标本的鲍氏不动杆菌对包括碳青酶烯类在内的多种抗菌药物耐药,经DNA分型同为A型,另4株物体表面的鲍氏不动杆菌经DNA分型亦同为A型,8株有高度同源性,证实为同一克隆株.结论 鲍氏不动杆菌为多药耐药株,同一克隆株在不同感染个体间的相互传播导致了ICU内感染的流行;1号株可能是交叉感染的源头,医护人员标准预防未到位,尤其是手卫生问题是可能的传播途径.     

吉祥草中杀灭钉螺化合物的提取分离

目的从吉祥草中提取分离杀灭钉螺活性高和对鱼毒性低的化合物.方法使用吉祥草200孔/25.4 mm原粉,经甲醇浸提,正丁醇萃取,再经硅胶柱层析和HPLC纯化,分离得到目标化合物.结果吉祥草原粉浸杀钉螺:浓度在17.5 mg/L,经72 h和120 h其死亡率分别达到86.0%和100.0%;浓度在130 mg/L,经168 h鱼死亡率为0.原粉经提取分离得到A 组分浓度在10 mg/L浸杀钉螺,经144 h其死亡率为84.4%,显示A组分对钉螺有较强的毒杀效应.A组分经HPLC分离纯化得到99.98%纯度的RC-I,API 2000质谱仪LC/MS联机测定其分子量,结合其他光谱分析推定RC-Ⅰ属甾体化合物.结论吉祥草中有杀螺作用较强的化合物.     

四个旋毛虫地理株肌幼虫虫体抗原和排泄—分泌抗原的免疫学分析

目的分析4个旋毛虫地理株肌幼虫的虫体抗原、排泄-分泌抗原的蛋白组成和免疫学特性。方法昆明小鼠16只,每只感染旋毛虫肌幼虫300条,45 d后人工消化法收集旋毛虫肌幼虫制备虫体抗原,幼虫体外培养获得排泄-分泌抗原。用聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis,SDSPAGE)分析虫体抗原和排泄-分泌抗原的组分,并用感染小鼠血清和旋毛虫病、血吸虫病、华支睾吸虫病、棘球蚴虫病、裂头蚴病、鞭虫病、卫氏并殖吸虫病、囊尾蚴病、钩虫病患者血清与抗原进行免疫印迹反应,观察阳性反应抗原条带。结果经SDS-PAGE分析,4个旋毛虫地理株虫体可溶性抗原和排泄-分泌抗原均有8条主带,相对分子质量(M_r)分别为100000、66000、49000、45000、43000、30000、18000、12000和M_r 66000、53000、49000、43000、36000、34000、30000、16000。免疫印迹结果显示,旋毛虫肌幼虫虫体可溶性抗原与旋毛虫病患者和感染小鼠血清的反应条带以M_r 95000、72000、53 000、49 000、43 000为主,排泄-分泌抗原反应条带以M_r 53 000、49 000、43 000、40 000为主。结论各地理株不同抗原的组分和免疫学反应存在一定的差异,其中虫体可溶性抗原的M_r 95 000、72 000成分和排泄-分泌抗原的M_r 40 000成分是进一步研究的重点。