Detergent fractionation with subsequent subtractive suppression hybridization as a tool for identifying genes coding for plasma membrane proteins

Abstract:  The identification of tumor-specific proteins located at the plasma membrane is hampered by numerous methodological pitfalls many of which are associated with the post-translational modification of such proteins. Here, we present a new combination of detergent fractionation of cells and of subtractive suppression hybridization (SSH) to gain overexpressed genes coding for membrane-associated or secreted proteins. Fractionation of subcellular components by digitonin allowed sequestering mRNA of the rough Endoplasmatic reticulum and thereby increasing the percentage of sequences coding for membrane-bound proteins. Fractionated mRNAs from the cutaneous T-cell lymphoma (CTCL) cell line HuT78 and from normal peripheral blood monocytes were used for SSH leading to the enrichment of sequences overexpressed in the tumor cells. We identified some 21 overexpressed genes, among them are GPR137B, FAM62A, NOMO1, HSP90, SLIT1, IBP2, CLIF, IRAK and ARC. mRNA expression was tested for selected genes in CTCL cell lines, skin specimens and peripheral blood samples from CTCL patients and healthy donors. Several of the detected sequences are clearly related to cancer, but have not yet been associated with CTCL. qPCR confirmed an enrichment of these mRNAs in the rough endoplasmic reticulum fraction. RT-PCR confirmed the expression of these genes in skin specimens and peripheral blood of CTCL patients. Western blotting verified protein expression of HSP90 and IBP2 in HuT78. GPR137B could be detected by immunohistology in HuT78 and in keratinocytes of dysplastic epidermis, but also in sweat glands of healthy skin. In summary, we developed a new technique, which allows identifying overexpressed genes coding preferentially for membrane-associated proteins.     

免疫荧光检测人乳头状瘤病毒新技术

利用过氧化氢的氧化作用参与抗原抗体反应,使细胞的膜通透性增强,利于荧光标记抗体染液渗入,从而更快速、广泛地接触抗原,更有效的抗原抗体的嵌合交联。新技术的应用,增强了染色效果,缩短了实验时间,简化了操作,提高了阳性检出率,使实验准确性提高,为临床确诊、疗效观察及预后提供了可靠依据。     

The Sperm Acrosome: Immunological Analysis using Specific Polyclonal and Monoclonal Antibodies Directed Against the Outer Acrosomal Membrane of Boar Spermatozoa

An antiserum to the purified porcine outer acrosomal membrane (OAM) was raised in female Balb/c mice and was characterized by means of an indirect ELISA. The hyperimmune serum reacted selectively with the acrosomal cap of the sperm head and showed an extremely good cross reactivity with bull and human spermatozoa when assayed by indirect immunofluorescence. Immunoelectron microscopy using the protein A-gold method further confirmed the specificity of the anti-OAM-antiserum for the OAM. In an effort to identify the OAM antigens recognized by the hyperimmune serum and to analyse the extent of cross reactivity on a molecular level, the SDS-extractable proteins were separated by SDS-PAGE, transblotted and immunoprinted using an 125J-conjugated anti-mouse-antibody. To facilitate functional and structural analysis of distinct OAM-proteins monoclonal antibodies were generated by hybridization of mouse myeloma cells with the splenocytes of female Balb/c mice immunized with the purified OAM. One fusion resulted in about 100 anti-OAM-antibodies secreting hybridoma cultures, of which about 30% showed cross reaction with human and bull spermatozoa. Four stable cell lines were selected for this study secreting antibodies directed against the outer acrosomal membrane of boar spermatozoa. Whereas the polyclonal immune mouse serum stained the entire acrosomal cap, the four hybridoma antibodies generated a patch-work-like immunofluorescence pattern over the acrosome. HPLC-ELISA of the solubilized OAM revealed first information on the nature of the corresponding membrane antigen.     

孕足月胎膜早破与头位难产的相关性

目的：探讨足月妊娠胎膜早破与难产的关系。方法：选择262例足月妊娠胎膜早破患者作为观察组,另随机抽取本院同期262例无胎膜早破的健康孕妇作为对照组,比较两组难产率的发生情况。结果：观察组难产率为45.0%,明显高于对照组的22.9%（P〈0.01）。两组的难产因素差异无统计学意义（P〉0.05）。结论：胎膜早破与难产有相关性,应及早处理,防止母儿并发症的发生。     

组织工程化表皮膜片构建及其在供皮区的应用研究

目的构建组织工程化表皮细胞膜片并应用于供皮区创面治疗。方法利用患者少量自体或同种异体正常皮肤分离、培养、扩增的表皮细胞,接种于壳聚糖明胶膜构建成表皮细胞膜片;将膜片移植于治疗组供皮区创面、适度加压,同时设立对照组:空白材料对照以及传统油纱布对照覆盖创面。于术后5～10d、30d、90d行大体观察、组织学检查、广谱角蛋白、外皮蛋白、层粘连蛋白、免疫组织化学检测以及Ⅰ、Ⅲ型胶原比值测定(偏光显微镜、RTPCR)。结果利用自体及异体表皮细胞和壳聚糖明胶膜能够构建出人表皮细胞膜片,应用于临床供皮区创面治疗15例,经过3个月随访,疗效肯定。移植膜片创面愈合时间(8.1±1.3)d,空白材料对照区为(16.2±3.8)d,空白油纱布对照区为(23.0±5.8)d。移植膜片存活良好,结构较完整、术后30d及90d观察:12例无明显增生,3例有轻度增生(20.0%),而空白油纱布对照区11例遗留增生性瘢痕(74.4%,χ2=8.127,P<0.01)。结论表皮细胞-壳聚糖明胶膜片能促进供皮区创面早期愈合并减少后期瘢痕增生,具有良好治疗效果。     

低血容量性休克大鼠心肌细胞Ca2+浓度及膜电位的变化

目的 旨在探讨低血容量性休克大鼠心肌细胞Ca2 浓度及其膜电位的变化.方法 选用Wistar大鼠84只,随机分成休克高渗复苏组(HES组)、生理盐水复苏组(NS组).建立休克模型,按7个时相(休克前、休克、复苏后5、15、30、60、90min)处死大鼠.取心室肌细胞培养传代,用Fluo-4/AM为游离钙荧光探针、JC-1荧光染色,流式细胞仪分别检测不同时相心肌细胞Ca2 浓度及线粒体膜电位.结果 HES组在休克、复苏后5、15、30min各时间点心肌细胞Ca2 浓度较休克前明显升高,其膜电位较休克前显著降低(P＜0.01);在复苏后60、90min心肌细胞Ca2 浓度及线粒体膜电位与休克时比较差异有显著性意义(P＜0.01);NS组在休克、复苏后各个时相与休克前比较心肌细胞Ca2 浓度均明显升高,其膜电位明显下降(P＜0.01);复苏后各时间点与休克时比较差异无显著性意义(P＞0.05);HSE组和NS组在休克后60、90min心肌细胞Ca2 浓度及其膜电位差异有显著性意义(P＜0.01).结论 低血容量性休克可诱发大鼠心肌细胞Ca2 浓度升高,线粒体膜电位下降,导致心肌细胞电生理活动障碍;高渗盐溶液不但可改善低血容量休克时心肌细胞Ca2 浓度,而且能有效地稳定其线粒体膜电位;而生理盐水对心肌细胞Ca2 浓度及其膜电位的作用不显著.     

复方壳多糖组织工程皮肤的组织学特性

目的评价制备的复方壳多糖组织工程皮肤在组织学方面的特性，为其修复皮肤缺损创面提供实验依据。方法通过苏木精-伊红（HE）染色、高碘酸-希夫（PAS）染色、免疫组织化学染色及电镜，观察组织工程皮肤的组织学特征，包括表皮、真皮及两者的连接结构基底膜。结果制备的复方壳多糖组织工程皮肤表皮细胞增殖活跃，分层分化良好，厚约150μm，真皮层细胞生长正常，排列有序。PAS染色示真表皮间有均匀红染的条带出现，免疫组织化学染色结果显示Ⅳ型胶原、Ⅶ型胶原、层黏蛋白反应阳性，在电镜下可见角质形成细胞间大量桥粒，基底层内侧较多半桥粒，与机体皮肤结构相似。结论复方壳多糖组织工程皮肤组织结构良好，符合新型皮肤替代物在治疗皮肤缺损时的组织学要求。     

离体模式下地氟醚、异氟醚和氟烷通过氧合器应用的药代动力学研究

目的研究在离体模式下地氟醚、异氟醚和氟烷通过氧合器应用的药代动力学。方法选择成人型膜式氧合器，预充生理盐水2000ml，连接动静脉端形成环路。将预先配制在钢瓶内的2．4％地氟醚、0．46％异氟醚及0．308％氟烷混合气体输送至氧合器，气体流量3Umin，泵流量4Umin，温度30℃。在摄入及排出的0、1、2、4、8、16、32min采集氧合器入气口、排气口及动脉端样本，测定吸入麻醉药分压。结果在摄入阶段及排出阶段，动脉端溶液中三种吸入麻醉药分压迅速上升或下降，用药后8min时，三种药物的动脉端样本分压与吸入气分压之比(Pa／Pi)均达50％以上，停药后8min动脉端样本分压与动脉端样本分压峰值之比(Pa／Pa0)均降至10％以下。三种药物之间在同一时间点Pa／Pi及Pa／17aO均有显著性差异(P＜0．05)。各吸入麻醉药动脉端样本与氧合器排气口中分压之间呈线性相关关系(r=0．99)。结论(1)Bentley膜式氧合器具有快速转运吸入麻醉药的性能；(2)地氟醚、异氟醚及氟烷通过氧合器应用后摄取和排出速率随着药物的水／气分配系数的增高而减慢；(3)通过监测氧合器排气口中吸入麻醉药分压可以快速、准确地估计液相中吸入麻醉药分压。     

CPAP对化疗后单肺通气时氧合及肺内分流的影响

[目的] 探讨应用一种新的持续气道正压 (CPAP)系统,对肺癌化疗后病人单肺通气时氧合和肺内分流的影响.[方法] 择期行肺癌手术病人 22例,按美国麻醉医师协会( ASA)分级, Ⅰ～Ⅲ级;随机分为对照组 (A组 )和 CPAP组 (B组 ),每组各 11例.A组在单肺麻醉期间非通气侧肺的支气管导管直接开口于大气中; B组单肺通气期间非通气侧持续给予 CPAP( P= 0.2 kPa),仰卧双肺通气 20 min, 仰卧单肺通气 20 min,侧卧单肺通气 30 min, 60 min,于关闭胸腔双肺通气时 ,分别采取动脉血样作血气分析,计算肺内分流率 (Qs/ Qt).[结果] 于单肺通气后 30 min和 60 min时 ,B组氧合明显高于 A组 (P < 0.01), B组分流率 (Qs/ Qt)明显低于 A组 (P < 0.05).[结论] 化疗后病人单肺通气期间 , 非通气侧持续 CPAP,有助于提高氧合 ,减少肺内分流 ,减少低氧血症的发生率.