抗菌肽基因在E.Coli JM109中的克隆表达

合成5条50～70 bp的DNA片段,用"片段拼凑"的方法通过三步PCR扩增得到目的基因,天蚕素基因被克隆到PUC19载体上,再转化大肠杆菌JM109.电泳分析重组质粒,确定天蚕素基因已转化到JM109上.摇床发酵转化子,IPTG诱导表达目的肽,最后提纯表达产物.SDS-PAGE电泳分析表明有特异性带出现,荧光色谱分析表明表达肽与目的肽组成一致,最后确定天蚕素在JM109中得到表达.抑菌圈活性实验表明,表达肽具有一定抗菌活性.     

Validation and reproducibility of computerised cell-viability analysis of tissue slices

Background

The identification of live cells using membrane integrity dyes has become a frequently used technique, especially with articular cartilage and chondrocytes in situ where tissue slices are used to assess cell recovery as a function of location. The development of a reproducible computerised method of cell evaluation would eliminate many variables associated with manual counting and significantly reduce the amount of time required to evaluate experimental results.

Methods

To validate a custom computerised counting program, intra-person and inter-person cell counts of nine human evaluators (three groups – unskilled, novice, and experienced) were compared with repeated pixel counts of the custom program on 15 digitised images (in triplicate) of chondrocytes in situ stained with fluorescent dyes.

Results

Results indicated increased reproducibility with increased experience within evaluators [Intraclass Correlation Coefficient (ICC) range = 0.67 (unskilled) to 0.99 (experienced)] and between evaluators [ICC = 0.47 (unskilled), 0.85 (novice), 0.93 (experienced)]. The computer program had perfect reproducibility (ICC = 1.0). There was a significant relationship between the average of the experienced evaluators results and the custom program results (ICC = 0.77).

Conclusions

This study demonstrated that increased experience in cell counting resulted in increased reproducibility both within and between human evaluators but confirmed that the computer program was the most reproducible. There was a good correlation between the intact cell recovery determined by the computer program and the experienced human evaluators. The results of this study showed that the computer counting program was a reproducible tool to evaluate intact cell recovery after use of membrane integrity dyes on chondrocytes in situ. This and the significant decrease in the time used to count the cells by the computer program advocate its use in future studies because it has significant advantages.

     

骨骼肌缺血再灌注损伤及发病机制初探

目的：探讨骨骼肌缺血再灌注损伤过程的微循环变化。组织学改变以及多肽含量变化和意义。方法：42只雄性大鼠随机分为留伴行神经组和去伴行神经组,建立标准骨骼肌缺血再灌注模型,采用激光多谱勒及显微放大分析系统,组织学方法以及凝胶电泳方法等观察缺血再灌注损伤变化。结果：缺血再灌注损伤后的骨骼肌微血管管径在20分钟时恢复率基本达到高峰约60％,此后为平台期：90分钟最高峰,为75％,主干血管流速率亦在20分钟基本达到上限,病理检查显示：缺血的骨骼肌纤维呈空泡状,核形态增大,染色加深,红细胞严重聚集,凝胶蛋白电泳提示：缺血骨骼肌中分子量20KD左右的多肽显著增加。结论：骨骼肌缺血再灌注损伤发生后,骨骼肌的微循环发生不同程度的破坏,及因之导致骨骼结构损伤和异常的20KD多肽含量明显增加,以及红细胞凝聚,白细胞的改变,这些共同构成了缺血再灌注损伤机制。     

罗哌卡因、芬太尼用于硬膜外分娩镇痛的效果和结局

目的:评价0.125%的罗哌卡因与每毫升含5μg芬太尼的混合液硬膜外注射用于分娩镇痛的效果与结局。方法:180例初产妇人工破膜后行硬膜外镇痛,先注入1.5%的利多卡因4m l试验剂量后,注入0.125%的罗哌卡因与每毫升含5μg芬太尼的混合液15 m l。再根据病人的要求可给予补充剂量的上述混合液,每次8 m l。另160例初产妇作为对照组未接受镇痛。结果:两组间VAS疼痛分级差异有统计学意义,镇痛组第一产程活跃期明显比对照组,且催产素的使用率高于对照组,差异均有统计学意义。而两组间第二产程、器械助产率和剖宫产率、羊水胎粪污染率、Apgar评分、新生儿体重及产后出血量等差异均无统计学意义。结论:0.125%罗哌卡因与每毫升含5μg芬太尼的混合液硬膜外注射用于病人分娩镇痛具有极好的镇痛效果,不影响母婴结局,不增加器械助产率和剖宫产率。     

维持性血液透析患者血清胱抑素C浓度与左心室肥厚的相关分析

比较64例维持性血液透析(MHD)患者和20例健康对照者一般临床资料和临床常用血生化指标;采用乳胶颗粒增强免疫透射比浊法检测血清胱抑素C浓度,超声心动图测定心脏腔径及心功能参数.结果 示MHD患者随透析时间延长,血清胱抑素C浓度逐渐增加,且左心室肥厚发生率显著增高.左心室肥厚者收缩压、左心室重量指数和血胱抑素C浓度明显高于无左心室肥厚者.胱抑素C与左心室重量指数、收缩压相关(r=0.633,0.397,均P<0.01).提示MHD患者血清胱抑素C变化可能与左心室肥厚密切相关,并可能是透析患者远期心血管并发症的影响因素.     

雌激素受体β基因多态性与原因不明月经过少的关系

目的:探讨西南地区雌激素受体β(ER-β)基因多态性与原因不明月经过少的关系。方法:选择西南地区100名原因不明月经过少患者为实验组,100名正常月经者作为对照组。应用分子生物学的方法研究ER-β基因RsaI和AluI限制性片段长度多态性和5号内含子的高变区CA重复序列多态性在实验组与对照组中分布。结果:RsaI和AluI限制性片段长度多态性在两组中均呈多态性分布。分离出9种CA重复序列等位基因,正常组和实验组的等位基因的分布频率差异无显著性。以重复次数n≤20作为SS型,n>20为LL型,再比较两组病人SS型和LL型等位基因的分布频率,差异有显著性。结论:ER-β基因多态性与原因不明月经过少有关,R等位基因可能是其保护因素,SS型等位基因可能是原因不明月经过少的危险因素。     

原因不明月经过少雌激素受体α基因多态性及其表达的研究

目的:探讨原因不明月经过少雌激素受体α(estrogen receptor α,ERα)基因多态性及其与表达的关系。方法:选择100名原因不明月经过少患者为实验组,100名月经正常人群为对照组。应用分子生物学的方法分析ERa基因PvuⅡ,XbaⅠ限制性片段长度多态性。通过逆转录-多聚酶链反应(RT-PCR)和Western印迹法分析ERα表达。结果:P基因型频率实验组为47.5%,对照组为30.5%,OR=2.062。X基因型频率实验组为20.5%,对照组为30.5%,OR=0.588。PvuⅡ和XbaⅠ限制性片段长度多态性在两组中均呈多态性分布。实验组ERα的mRNA和蛋白质表达均比对照组低(P<0.05)。结论:ERα基因多态性与原因不明月经过少有关,P等位基因可能是其危险因素,X等位基因可能是其保护因素。ERα在子宫内膜中原因不明月经过少中的表达低于月经量正常子宫内膜,且可能与月经量多少有关。     

金雀异黄素及二个结构改造物与抗肿瘤、抗血小板作用的关系

目的;合成金雀异黄水溶性衍生物并研究它们在抗肿瘤,抗血小板作用中的影响。方法：通过浓硫酸酯化金雀异黄素（Ｇ）,加入饱和ＮａＣｌ溶液,获得二个水溶性衍生物;金雀异黄素－４’－硫酸酯钠（Ｇ１）的金雀异黄素－７,４‘－二硫酸酯二钠（Ｇ２）。通过