口服结核Ag85A-CD226 DNA疫苗在小鼠脾与肠道的表达水平检测

目的了解结核Ag85A-CD226 DNA疫苗经灌胃方式接种小鼠后在脾淋巴细胞与肠道的表达情况。方法将构建的pcDNA3.1-Ag85A-CD226、pcDNA3.1-Ag85A和pcDNA3.1-CD226真核表达质粒转化DH5α感受态大肠杆菌,扩增并提取纯化质粒,用脂质体包裹制成DNA疫苗。经灌胃方式将制备的DNA疫苗接种C57BL/6小鼠,设置Ag85A-CD226疫苗组、Ag85A疫苗组、CD226疫苗组、pcDNA3.1质粒组和生理盐水对照组。采用间接免疫荧光法检测Ag85A和CD226在肠道的表达。采用流式细胞术检测脾CD4+T细胞、CD8+T细胞和NK细胞的CD226表达。结果 CD226在Ag85A-CD226疫苗组脾脏CD4+T细胞和NK细胞的表达均明显强于Ag85A疫苗组、CD226疫苗组、pcDNA3.1质粒组和生理盐水对照组,差异具有统计学意义(P0.01);CD226在Ag85A-CD226疫苗组脾脏CD8+T细胞的表达明显强于Ag85A疫苗组、pcDNA3.1质粒组和生理盐水对照组,差异具有统计学意义(P0.01),与CD226疫苗组相比虽有所增加,但差异无统计学意义(P0.05)。CD226在Ag85A-CD226疫苗组小肠派氏淋巴结表达明显强于Ag85A疫苗组、CD226疫苗组、pcDNA3.1质粒组和生理盐水对照组,差异具有统计学意义(P0.01)。Ag85A只在Ag85A-CD226疫苗组和CD226疫苗组小肠固有层表达,且在Ag85A-CD226疫苗组的表达明显强于CD226疫苗组,差异具有统计学意义(P0.01)。结论 Ag85A-CD226 DNA疫苗接种后,CD226在脾淋巴细胞和肠道表达增强,Ag85A在肠道表达增强,CD226的表达增加会增强Ag85A在肠道的表达水平。     

CD39的临床意义及其与肝癌浸润T细胞耗竭的相关性分析

摘要 目的：探索CD39分子（编码基因ENTPD1）在原发性肝细胞肝癌（Hepatocellular carcinoma, HCC）中的表达、临床意义及其与HCC中免疫浸润和T细胞耗竭的相关性。方法：TIMER、GEPIA、Kaplan-Meier Plotter、TCGA等数据库分析CD39在肝癌中的差异性表达、与免疫浸润的关系、相关基因的表达及其与肝癌患者预后的关系。免疫荧光染色、细胞测序和流式检测验证临床HCC患者的癌及癌旁组织中CD39的差异性表达及其和CD8⁺T细胞耗竭特性的相关性。结果：（1）生物信息学分析结果显示：CD39在多种肿瘤组织中表达上调（包括HCC）（P＜0.05）；且其表达水平与HCC的临床预后等显著相关（P＜0.05）；与CD39低表达组相比，HCC中CD39高表达组患者无复发生存期（relapse-free survival, RFS）（P=0.025）和无进展生存期（progression-free survival, PFS）（P=0.026）较短；CD39与HCC肿瘤微环境中CD8⁺T、CD4⁺T、巨噬细胞等免疫细胞浸润水平均有明显正相关。（2）临床HCC样本验证：免疫荧光和流式结果显示CD39在癌组织中的表达水平高于癌旁正常组织，与耗竭相关分子LAYN、TIM3、CTLA4等共表达，且在耗竭CD8⁺T细胞中表达比例显著高于非耗竭细胞。结论：CD39在HCC肿瘤组织及浸润的CD8⁺T细胞中高表达，与HCC的RFS、PFS、免疫细胞浸润等紧密相关，且参与CD8⁺T细胞耗竭。     

The role of CD4 T cell help for CD8 CTL activation

CD4 T cells play an important role in the initiation and persistence of CD8 T cells responses. In this review, we report on and evaluate the mechanisms by which CD4 T cells contribute to activation of CD8 T cells and the signal pathways of the down-streaming events after CD4 T cell help.     

三种淋巴瘤细胞CD52抗原呈现稳定性研究及其应用

评估淋巴瘤细胞MC/CAR、HUT78和RAMOS的CD52抗原呈现稳定性,并比较三种细胞用于抗CD52单抗活性检测中的优劣性。采用免疫荧光法检测淋巴瘤细胞MC/CAR、HUT78和RAMOS的CD52抗原呈现率,分析MC/CAR、HUT78和RAMOS传代培养5~20代CD52抗原呈现稳定性。分别以MC/CAR、HUT78和RAMOS作为抗CD52单抗结合活性和补体依赖细胞毒性的靶细胞进行检测,并比较其优劣性。结果显示,MC/CAR、RAMOS和HUT78的CD52抗原呈现率分别为95.5%、63.2%和38.3%。MC/CAR传代培养5~20代CD52抗原呈现率均大于90%。RAMOS传代培养5~13代CD52抗原呈现率介于60%~67%,第14~15代CD52抗原呈现率介于50%~60%,第16~20代细胞CD52抗原呈现率介于40%~50%。HUT78传代培养5~20代CD52抗原呈现率介于26.7%~38.9%。淋巴瘤细胞MC/CAR在抗CD52单抗的结合活性检测中呈现出更好的剂量依赖曲线。淋巴瘤细胞RAMOS在抗CD52单抗的补体依赖细胞毒性检测中呈现出更好的剂量效应曲线。CD52抗原呈现率方面MC/CAR>RAMOS>HUT78。结果表明,MC/CAR更适用于抗CD52单抗的结合活性的检测,RAMOS适用于抗CD52单抗的补体依赖细胞毒性检测。     

CD11b表达与人工髋关节置换术后无菌性松动的相关性分析

目的:分析髋关节周围滑膜组织CD11b表达水平与人工髋关节置换术后无菌性松动的相关性。方法:以2006年5月至2016年5月于西京医院接受人工髋关节置换的患者为研究对象,对其髋关节周围滑膜组织进行CD11b免疫组化染色,并随访术后5年和10年无菌性松动的发生情况,通过单因素分析及logistic回归分析讨论CD11b表达与无菌性松动之间的相关性。结果:共300例患者纳入研究,全部获得随访,CD11b表达阳性患者163例,阳性率为54.33%;术后5年松动患者29例,术后5年无菌性松动发生率为9.67%;术后10年无菌性松动患者49例,术后10年无菌性松动发生率为16.33%;单因素分析结果表明CD11b表达阳性患者5年及10年松动率均高于CD11b表达阴性患者(P0.05);logistic回归分析结果表明CD11b过表达是髋关节置换术后无菌性松动发生的危险因素。结论:髋关节周围滑膜组织CD11b过表达是人工髋关节置换术后无菌性松动发生的危险因素,该分子或可作为无菌性松动的辅助诊断指标及潜在治疗靶点。     

CD3ε和PMA对fas基因转录调控作用的研究

研究了ＣＤ３ε和ＰＭＡ对细胞凋亡基因ｆａｓ的转录调控作用．根据已知的人ｆａｓ基因５＇上游序列设计引物,用ＰＣＲ法从人胸腺细胞基因组ＤＮＡ中扩增出ｆａｓ５＇上游长为４４６ｂｐ的启动子片段．将此片段定向克隆到以虫荧光素酶为报告基因的真核细胞表达载体ｐＸＰ２中．经限制性内切酶酶切鉴定及序列测定分析表明此重组表达质粒ＤＮＡ的结构和序列正确．用此质粒（ｐＸＰ２－ｆａｓｕｐ）瞬时转染人ＪｕｒｋａｔＴ淋巴细胞,分析报告基因的表达水平．结果表明ｆａｓ基因上游－５０６到－６０的区域有弱启动子活性,约是阴性对照的１．５倍．抗ＣＤ３ε抗体和ＰＭＡ处理均可增强ｆａｓ启动子的活性,促进报告基因的表达,其虫荧光素酶活性分别是未经处理的ｐＸＰ２－ｆａｓｕｐ转染细胞的１．７倍和３．３倍,但二者没有协同作用．ＰＫＣ的抑制剂ｓｔａｕｒｏｓｐｏｒｉｎｅ和ＰＴＫ的抑制剂ｈｅｒｂｉｍｙｃｉｎＡ可抑制ＰＭＡ诱导的ｆａｓ基因转录,使虫荧光素酶基因的表达降至未用ＰＭＡ处理的水平．但ＰＴＫ的另一种抑制剂ｇｅｎｉｓｔｅｉｎ可与ＰＭＡ发挥协同作用,上调ｆａｓ基因的转录,使虫荧光素酶活性增加到５倍．这些结果为阐明ＣＤ３ε及ＰＭＡ介导Ｔ淋巴细胞激活和凋亡的分子机制     

应用水动力转染技术使人低密度脂蛋白受体、CD81和浸入诱导蛋白L同时在小鼠体内表达

为建立人低密度脂蛋白受体（LDLR）、CD81和浸入诱导蛋白L（Sip—L）等多个HCV感染相关分子共表达的转基因小鼠,首先分别构建了白蛋白启动子调控的人LDLR、CD81和Sip-L基因的小鼠肝脏组织特异性表达载体,将3种质粒同时采用水动力转染技术导入小鼠体内,FIT—PCR和免疫组化技术检测转入体内基因的表达及持续时间。结果表明,转染8h后即可检测到3种基因在体内转录,人LDLR和CD81在转染1～4d后可在50％-90％的肝细胞中高效表达,7d后检测不到目的基因表达。为了进一步延长转染基因在小鼠体内的表达时间,又分别构建了人LDLR、CD81和Sip—L的染色体整合型表达载体,将它们与噬菌体ФC31的整合酶表达载体同时水动力转染小鼠,3种基因在体内表达时间可持续到转染后25d。以上结果表明建立了多个HCV感染相关分子共表达的转基因小鼠,为确定这些分子能否使小鼠感染HCV奠定了基础。     

Is CD36 gene polymorphism in region encoding lipid-binding domain associated with early onset CAD?

CD36 is a fatty acid translocase in striated muscle cells and cardiomyocytes. Some study suggested that alterations in CD36 gene may be associated with coronary artery disease (CAD) risk. The aim of the current study was to compare the frequency of CD36 variants in region encoding lipid-binding domain in Caucasian patients with early-onset CAD, no-CAD adult controls and neonates. The study group comprised 100 patients with early onset CAD. The genetic control groups were 306 infants and 40 no-CAD adults aged over 70 years. Exons 4, 5 and 6 including fragments of flanking introns were studied using the denaturing high-performance liquid chromatography technique and direct sequencing. Changes detected in analyzed fragment of CD36: IVS3-6 T/C (rs3173798), IVS4-10 G/A (rs3211892), C311T (Thr104Ile, not described so far) in exon 5, G550A (Asp184Asn, rs138897347), C572T (Pro191Leu, rs143150225), G573A (Pro191Pro, rs5956) and A591T (Thr197Thr, rs141680676) in exon 6. No significant differences in the CD36 genotype, allele and haplotype frequencies were found between the three groups. Only borderline differences (p = 0.066) were found between early onset CAD patients and newborns in the frequencies of 591T allele (2.00% vs 0.50%) and CGCGCGT haplotype (2.00% vs 0.50%) with both IVS3-6C and 591T variant alleles. In conclusion, CD36 variants: rs3173798, rs3211892, rs138897347, rs5956, rs143150225 rs141680676 and C311T do not seem to be involved in the risk of early-onset CAD in Caucasian population.     

抑制T细胞增殖的抗CD45单克隆抗体的建立

目的寻找能调节T细胞功能的相关分子,进行与T细胞介导的自身免疫性疾病相关的研究。方法从BALB/c小鼠骨髓中收集树突状细胞,免疫Wistar大鼠,进行细胞融合,建立杂交瘤细胞系。筛选得到很多株能调节T细胞功能的杂交瘤细胞系,对其中一株最能抑制T细胞增殖的杂交瘤细胞系进行了进一步的深入研究。结果显示其目标分子是CD45,同时增殖实验结果显示该抗体能显著抑制T细胞增殖反应。结论抗CD45单克隆抗体能有效抑制T细胞增殖,有望将本抗体用于T细胞介导的自身免疫性疾病的相关预防及治疗中。     

Biotechnological challenges of bioartificial kidney engineering

With the world-wide increase of patients with renal failure, the development of functional renal replacement therapies have gained significant interest and novel technologies are rapidly evolving. Currently used renal replacement therapies insufficiently remove accumulating waste products, resulting in the uremic syndrome. A more preferred treatment option is kidney transplantation, but the shortage of donor organs and the increasing number of patients waiting for a transplant warrant the development of novel technologies. The bioartificial kidney (BAK) is such promising biotechnological approach to replace essential renal functions together with the active secretion of waste products. The development of the BAK requires a multidisciplinary approach and evolves at the intersection of regenerative medicine and renal replacement therapy. Here we provide a concise review embracing a compact historical overview of bioartificial kidney development and highlighting the current state-of-the-art, including implementation of living-membranes and the relevance of extracellular matrices. We focus further on the choice of relevant renal epithelial cell lines versus the use of stem cells and co-cultures that need to be implemented in a suitable device. Moreover, the future of the BAK in regenerative nephrology is discussed.