SHP-2对人胃癌细胞克隆增殖的影响

目的研究SHP-2对人胃癌细胞SGC-7901细胞克隆增殖的影响。方法采用重组腺病毒Ad-GFP(GFP)、Ad-GFP-SHP-2(WT)转染SGC-7901细胞;采用蛋白印迹技术(Western blot)检测SHP-2蛋白的表达;同时进行集落形成实验检测SGC-7901细胞克隆形成能力。另外,观察SHP-2抑制剂NSC-87877作用后,对SGC-7901集落形成的影响。结果当胃癌细胞感染复数为150时,转染成功的细胞数占总细胞数的0.9。WT组SHP-2蛋白过表达。WT组的平板克隆数明显高于SGC-7901亲本细胞组(Control 1)和GFP组,P<0.05。SHP-2抑制剂组(NSC-87877)的平板克隆数及软琼脂克隆数分别明显低于SGC-7901亲本细胞组(Control2),P<0.05。结论 SHP-2对SGC-7901细胞克隆增殖有明显促进作用。     

链霉素月桂基硫酸钠亚碲酸钾琼脂平板(双洗平板)配制过程中的重要环节的探讨

目的:通过对使用不同容器,在不同温度下加入1%亚碲酸钾溶液和链霉素溶液配制成的链霉素月桂基硫酸钠亚碲酸钾琼脂平板(双洗平板)进行分析比较,重申在配制双洗平板过程中把握好几个关键环节的重要性.方法:先用玻璃容器配制成的双洗平板(不加入1%亚碲酸钾溶液和链霉素溶液)作空白对照平板,再分别用玻璃容器和金属容器(非不锈钢)盛放并煮沸双洗琼脂各两份,一份在高温80度左右加入1%亚碲酸钾溶液和链霉素溶液制成平板,另一份在低温55度左右加入1%亚碲酸钾溶液和链霉素溶液制成平板.结果:用玻璃容器配制的,且在55度左右加入1%亚碲酸钾溶液和链霉素溶液的双洗平板颜色最接近空白对照平板的颜色,其它均与空白对照平板颜色相差很大.结论:配制双洗平板时要使用玻璃容器,且须在低温55度左右加入1%亚碲酸钾溶液和链霉素溶液,才能确保双洗平板质量的可靠性.     

琼脂扩散细胞毒性检查方法研究

目的: 摸索琼脂扩散法样品本身与样品浸提液之间的等量关系。方法: 选用0.5%琼脂浓度,直径为60 mm细胞培养皿中每皿加入3 mL琼脂与含血清细胞培养液混合液制备固化琼脂层,以镀银敷料30～100μL浸提液筛选与样品本身产生相同毒性的浸提液的剂量,最后以5批不同的样品进行验证。结果: 镀银敷料100μL浸提液与样品本身产生的毒性反应最相近,5批不同样品验证结果基本一致。结论: 在样品本身不适于直接进行琼脂扩散法细胞毒性检查时,可选用其100μL浸提液进行试验。     

头孢吡肟对革兰阴性杆菌的体外敏感性分析及方法评价

目的调查昆明医学院第一附属医院2007年8月至12月301株临床常见革兰阴性杆菌对第4代头孢菌素头孢吡肟的耐药状况,评价采用的MICROSCAN细菌鉴定药物敏感系统中快速接种法的准确性。方法用琼脂稀释法、纸片扩散法、标准浊度法和MICROSCAN快速接种法测定头孢吡肟对301株细菌的体外抑菌活性。以琼脂稀释法为标准参考方法,比较其他3种方法与其结果的一致性。结果琼脂稀释法测定301株革兰阴性杆菌对头孢吡肟的体外总敏感率为78.07%;纸片扩散法、标准浊度法和MICROSCAN快速接种法与标准参考方法的一致率分别为99.00%,98.34%和95.35%。纸片扩散法、标准浊度法检查结果与琼脂稀释法无统计学差异（P〉0.05）,MICROSCAN快速接种法检查结果与琼脂稀释法有统计学差异（P〈0.05）。结论头孢吡肟对大部分临床常见革兰阴性杆菌具有良好的体外抗菌活性,纸片扩散法及标准浊度法结果准确性较高,MICROSCAN快速接种法的准确性有待进一步探讨。     

Prevalence and phenotypic evaluation of Candida dubliniensis in pregnant women with vulvovaginal candidosis in a university hospital in Ankara

Candida dubliniensis is very similar to Candida albicans in terms of genotypic and phenotypic characteristics. As the hormonal milieu of the vagina during pregnancy, characterised by a lack of maternal cell-mediated immunity, enhances Candida colonisation and serves as a risk factor for symptomatic expression, investigation into the isolation of C. dubliniensis in vaginal discharges of pregnant women with vulvovaginal candidosis was made. A total of 77 Candida isolates obtained from 60 patients positive for vulvovaginal candidosis collected from 218 pregnant women were investigated for C. dubliniensis subsistence. In total 41 Candida species phenotypically identified as C. albicans on the basis of a positive germ tube test and carbohydrate assimilation tests were screened for the presence of C. dubliniensis. Phenotypic tests for differentiation of C. dubliniensis from C. albicans, such as growth at 42 and 45 degrees C on Sabouraud dextrose agar, appearance on CHROMagar and colony morphology on Cornmeal-Tween-80 agar and Staib agar were carried out. Only one strain (2.43%) was phenotypically identified as C. dubliniensis. According to our study, a combination of at least five phenotypic methods is necessary for an exact diagnosis of C. dubliniensis. Large-scale studies of pregnant women are required to discover the aetiological importance of this yeast.     

Comparison of different culture media and growth conditions for recognition of Arcanobacterium haemolyticum

Detection of Arcanobacterium haemolyticum is based upon typical beta-hemolysis and colony morphology, but it may go undetected if only conventional sheep blood agar media for detection of beta-hemolytic streptococci are used. The influence of different culture media, atmospheres, and times of incubation for the recognition of colonies of 47 isolates of A. haemolyticum was studied. After 48 h of incubation, trypticase soy agar with 5% horse blood in 5% CO(2) was the best medium.     

电泳法检测非浓缩尿蛋白的临床应用

①目的 探讨电泳法检测尿蛋白组分变化的临床意义。②方法 采用法国SEBIAMG-300半自动电泳分析仪，对217例肾脏疾病病人进行尿蛋白组分分析。③结果 尿蛋白电泳显示，150例为肾小球性蛋白尿，32例为混合性蛋白尿，30例为生理性蛋白尿，5例为肾小管性蛋白尿。40例经肾活组织检查确诊的病人中，共检出蛋白尿33例，其中中、高分子型15例，中分子型12例，混合型6例。④结论 本法具有操作简便、易于量化、无创伤性、灵敏度高等优点，且其蛋白组分变化与肾脏活组织检查结果一致。     

介绍一种简便、快速的回收DNA片段的方法

我们采用国产 DF-17型电洗脱槽回收了限制性内切酶酶切的 DNA 片段,证明该方法是一种简便、快速、可靠的回收核酸片段的方法.回收的 DNA 片段可进一步用于 DNA 克隆、探针标记和 DNA 分析.     

马甲子叶抑菌有效部位的筛选

目的:探讨马甲子叶不同提取部位的抑菌作用,并筛选其有效部位。方法:系统溶剂法将马甲子叶提取为石油醚、三氯甲烷、乙酸乙酯、正丁醇和水5个部位后,用纸片法考察不同溶剂部位对金黄色葡萄球菌、枯草芽孢杆菌、大肠埃希菌、沙门氏菌的体外抑菌活性,采用琼脂平板涂布法测定有明显抑菌作用的有效部位的最小抑菌浓度(MIC)。结果:石油醚、三氯甲烷和水部位无抑菌效果。乙酸乙酯、正丁醇部位对受试病原菌有不同程度的抑菌效果,其中正丁醇部位抑菌活性达显著性差异,对金黄色葡萄球菌、枯草芽孢杆菌、大肠埃希菌、沙门氏菌的MIC值分别为0.714,0.714,0.357,0.357 g·L-1。乙酸乙酯部位对4种细菌均具有明显抑制效果。综合抑菌效果比较:正丁醇部位乙酸乙酯部位水部位。结论:正丁醇、乙酸乙酯部位是马甲子叶的有效抑菌部位,是寻找新抑菌活性成分的基础,为后期开发利用提供理论依据。     

Differentiation of murine male germ cells to spermatozoa in a soft agar culture system

Establishment of an in vitro system that allows the development of testicular germ cells to sperm will be valuable for studies of spermatogenesis and future treatments for male infertility. In the present study, we developed in vitro culture conditions using three-dimensional agar culture system (SACS), which has the capacity to induce testicular germ cells to reach the final stages of spermatogenesis, including spermatozoa generation. Seminiferous tubules from testes of 7-day-old mice were enzymatically dissociated, and intratubular cells were cultured in the upper layer of the SACS in RPMI medium supplemented with fetal calf serum (FCS). The lower layer of the SACS contained only RPMI medium supplemented with FCS. Colonies in the upper layer were isolated after 14 and 28 days of culture and were classified according to their size. Immunofluorescence and real-time PCR were used to analyse specific markers expressed in undifferentiated and differentiated spermatogonia (Vasa, Dazl, OCT-4, C-Kit, GFR-α-1, CD9 and α-6-integrin), meiotic cells (LDH, Crem-1 and Boule) and post-meiotic cells (Protamine-1, Acrosin and SP-10). Our results reveal that it is possible to induce mouse testicular pre-meiotic germ cell expansion and induce their differentiation to spermatozoa in SACS. The spermatozoa showed normal morphology and contained acrosomes. Thus, our results demonstrate that SACS could be used as a novel in vitro system for the maturation of pre-meiotic mouse germ cells to post-meiotic stages and morphologically-normal spermatozoa.