α_1—抗胰蛋白酶异质体的测量新方法及其临床意义的探讨

本文对a_1-抗胰蛋白酶异质体的检测方法(小扁豆凝集素亲合交叉免疫电泳法)进行改进。改进后的方法操作流程短、成本低、结果清晰、操作简便、不需预测a_1-抗胰蛋白酶总浓度,适合于临床广泛应用。本文用该法对原发性肝癌、转移性肝癌,其它器官癌肿、肝硬化及正常人血清中的a_1-抗胰蛋白酶异质体进行分析,发现小扁豆凝集素结合型a_1-抗胰蛋白酶百分比(LCA-R-AAT％)原发性肝癌组明显高于其它各组,以LCA-R-AAT(％)>25％为阳性标准,则原发性肝癌诊断的敏感性、特异性、准确性分别为80.6％、86.2％和84.3％,与小扁豆凝集素结合型甲胎蛋白联合检测,可使原发性肝癌诊断的敏感性提高至100％(P<0.05),特异性和准确性分别为82.8％和89.9％(P均>0.05),表明LCA-R-AAT(％)的检测对原发性肝癌的诊断及鉴别诊断有一定的价值。     

肺腺癌和癌旁组织蛋白质表达谱差异的研究

通过比较肺癌患者癌组织和癌旁组织的二维凝胶电泳图谱(two-dimensional electrophoresis,2DE),以寻找肺癌相关蛋白、肺癌患者癌组织和癌旁组织,采用固相pH梯度(immobilized pH gradient,IPG)2DE分离总蛋白质,凝胶经银染显后,ImageMaster 2D图像分析软件进行比较分析、识别差异表达的蛋白质并进行质谱鉴定获取肺癌相关蛋白信息。结果分析得到肺癌组织蛋白点709个,癌旁组织蛋白点722个,选择表达量差异达3倍以上的40个,进行胶内酶解,基质辅助电离解析飞行时间质谱(MALDI TOF MS)获得肽质量指纹图谱(PMF),搜寻蛋白质数据库,得到7个肺癌标志物的候选蛋白,但这些差异蛋白点在肺癌发生发展中的作用需要进一步研究。     

Reciprocal Interaction Between Intestinal Microbiota and Mucosal Lymphocyte in Cynomolgus Monkeys After Alemtuzumab Treatment

It has been known that the gut microbiota plays a central role in shaping normal mucosal immunity, however, little information is available whether the variability of mucosal lymphocytes impacts the commensal flora. Here, we applied a cynomolgus monkey model to characterize the structure and composition of the gut microbiota in response to lymphocyte depletion and to determine their potential association. Molecular profiling of 16S rDNA showed that the intestinal microbiota composition was perturbed after the depletion of mucosal lymphocytes and were recovered following the repopulation. Some specific bacteria from the orders Lactobacillales, Enterobacteriales and Clostridiales, and the genus Prevotella and Faecalibacterium, were primarily responsible for the variations of the gut microbiota after lymphocyte depletion. Interestingly, the species richness of the ileal mucosal microbiota was associated the proportions of TCRαβ⁺ or TCRγδ⁺ T cells (p < 0.01). We demonstrate for the first time the feature of intestinal microbiota composition after lymphocyte depletion and provide novel evidence that the perturbation of gut microbiota is associated with lymphocyte depletion. It may contribute to understand the relationship between gut commensal microbiota and mucosal immune system. Study results provide insight into biological activity of alemtuzumab in intestinal barrier in organ transplantation.     

乙酸纤维膜电泳法快速分离磷酸肌酸激酶同功酶

采用乙酸纤维膜(涤沦基型)电泳法对CPK同功酶进行分离。所用缓冲液为Tris-巴比妥—巴比妥钠缓冲液,pH8.8,离子强度0.029M。电泳电压300V,时间10min。该方法不仅操作简便,分离效果好,重复性强,更重要的是省时价廉,适合临床急诊工作的需要。     

上海市宝山区2015—2018年腹泻病人沙门菌血清分型和分子分型

目的 揭示上海市宝山区腹泻病人分离沙门菌的血清型和分子分型分布特征。方法 对2015—2018年分离的118株沙门菌进行血清分型和脉冲场凝胶电泳（pulsed-field gel electrophoresis,PFGE）分型,通过构建聚类树揭示不同血清型菌株的分子分型特征。结果 118株沙门菌分为25种血清型,前三位为肠炎沙门菌（45株,38.1%）、鼠伤寒沙门菌（26株,22.0%）、伦敦沙门菌（11株,9.3%）。其余22种血清型分别只包含1~3株菌。118株沙门菌通过PFGE分为71个PFGE带型,每个带型分别包含1~19株菌。45株肠炎沙门菌分为8个PFGE带型,相似度为75.0%~100.0%,在聚类树上形成一个簇;26株鼠伤寒沙门菌分为21个PFGE带型,相似度为48.0%~100.0%,在聚类树上形成两个簇;11株伦敦沙门菌分为8个PFGE带型,相似度为80.0%~100.0%,在聚类树上形成一个簇。结论 肠炎沙门菌和鼠伤寒沙门菌是上海市宝山区腹泻病人沙门菌优势血清型,但近年伦敦沙门菌有增多的趋势。肠炎沙门菌在本地区传播流行过程中的遗传变异相对比较保守,鼠伤寒沙门菌基因型多态性大,而且存在两个克隆。     

一种脲梯度电泳的简便方法

目的与方法：介绍了了种应用国产ＤＹＹ－Ⅲ小型电泳槽进行脲梯度电泳的方法。结果与结论：该法操作简便,普适性好,能获得很好的样品迁移图谱。可用于蛋白质变性／复性的研究及原基因工程产品复性工艺的探索。     

死精子症精子相关蛋白的蛋白质组学研究

目的:运用双向电泳(2-DE)和质谱技术比较1例死精子症患者和正常人精子的蛋白质组成差异。方法:用2-DE分离了10例正常生育者30份精子标本和1例死精子症3份精子标本的蛋白质,分别获得其参考图谱并进行比较,用质谱技术鉴定其中部分差异蛋白点。结果:在正常精子图谱和死精子症患者精子图谱分别分离出(905±57)和(792±28)个蛋白质点,死精子症患者精子图谱上有178个点未能匹配。对其中6个在患者图谱中缺失的蛋白质点进行了鉴定。结论:死精子症患者图谱上缺失的6个蛋白,可能与死精子症的形成有关。     

异氟醚预处理兔缺血再灌注心肌蛋白质组双向凝胶电泳分析

目的研究异氟醚预处理对缺血再灌注心肌保护的相关蛋白的变化。方法将8只新西兰大白兔随机分为心肌缺血再灌注对照组(C组)和异氟醚预处理组(I组),每组4只。两组均接受左冠状动脉前降支阻断40 m in,开放再灌注120 m in。I组在缺血前24 h前吸入1MAC异氟醚2 h。分别取各组缺血区心肌进行二维凝胶电泳,利用Im ageM aster 2D软件分析实验结果。结果与C组对比,I组有13个蛋白表达发生了显著变化,其中表达明显增强的有10个蛋白,表达明显降低的有3个蛋白。结论差异表达的蛋白可能在异氟醚对心肌缺血再灌注损伤的保护中发挥作用。     

应用双向电泳技术初步建立人精子头部蛋白质图谱

目的 :利用人类精子蛋白质组分析的双向蛋白电泳 (2 DE)技术建立正常人精子头部蛋白质图谱。　方法 :先用固相pH梯度 SDS电泳技术 (IPG DALT)对全精子和精子头部蛋白质抽提物进行蛋白质分离 ,然后用图像分析软件比较精子头部蛋白质图像与精子蛋白质图像的蛋白质斑点组成差异。其中 ,全精子蛋白质的抽提比较了硫脲 /尿素 /盐酸胍和Kit/盐酸胍两种抽提方法。　结果 :硫脲 /尿素 /盐酸胍法与Kit/盐酸胍法所得的全精子 2 DE图像蛋白质斑点分别为 80 2个和 797个 ,其中相同的有 4 92个 ,将两种方法获得的图像整合而得到的全精子蛋白质图像有1 1 0 7个蛋白质斑点 ;精子头部图像蛋白质斑点有 4 2 8个 ,经匹配 ,全部来源于全精子蛋白质图像。　结论 :综合采用两种蛋白质提取方法初步建立了精子头部蛋白质图谱。     

Immunochemical comparison between worm extracts of Onchocerca volvulus from savanna and rain forest

Summary Crude phospate-buffered extracts of adult Onchocerca volvulus from savanna (Mali) and rain forest (Cameroon) areas were comparatively analysed using biochemical and immunological methods. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing revealed only minor differences between the two extracts. Out of 42 bands detectable by SDS-PAGE at least 21 were identified as glycoproteins by their affinity to concanavalin A. High resolution analysis using two dimensional gel electrophoresis (2D-G) showed marked differences in the polypeptide patterns of the two extracts. Some of the over 100 polypeptides demonstrable by Coomassie blue staining (especially at pIs between 4·3 and 5·6 and mol. wts over 64kD) were clearly different when the two extracts were compared. Antigenic differences between the two extracts could be detected by crossed immunoelectrophoresis using a rabbit anti- O. volvulus hyperimmune serum. The comparison by tandem crossed immunoelectrophoresis demonstrated clearly the existence of at least three antigenic differences, four partial identities and 13 antigenic identities between the extracts. For the identification of O. volvulus antigens serologically recognized by infected patients, we combined the 2D-G with an immunoblotting technique using a pool of highly reactive onchocerciasis sera from Mali. IgG binding antigens were then identified by incubating the blot membrane with this serum pool and with ¹²⁵I-labelled protein A followed by autoradiography. IgE binding antigens were detected using a ¹²⁵I-labelled anti-human IgE antiserum. Whilst the overall antigenic patterns were similar, there were, however, clear differences between the antigen preparations which gives further evidence for antigenic diversity of O. volvulus from savanna and rain forest areas.