抗人B7-H1单克隆抗体的制备和鉴定

目的:采用杂交瘤技术制备抗人B7-H1单克隆抗体,并对其进行鉴定。方法:经抗原免疫的小鼠脾细胞与小鼠骨髓瘤细胞以常规方法融合;用间接ELISA法筛选分泌抗体的杂交瘤细胞株;阳性克隆用有限稀释法获得稳定分泌抗人B7-H1单克隆抗体的杂交瘤细胞株;扩增杂交瘤细胞注射进小鼠腹腔后制备腹水;纯化腹水中的单克隆抗体并对其亚型进行鉴定;用间接ELISA法测抗体效价;将肺癌组织制成石蜡切片,用抗人B7-H1抗体进行免疫组化染色。结果:获得1株稳定分泌抗人B7-H1单克隆抗体的杂交瘤细胞株,所分泌的单抗类型为IgG1;抗体效价为1×108,纯化后的抗体含量为6.76g/L;免疫组化实验中,单抗可与肺癌组织表面的B7-H1蛋白特异地结合。结论:制备了人B7-H1单克隆抗体,为B7-H1检测试剂盒的研制奠定了基础。     

聚乙二醇干扰素α-2a对慢性乙型肝炎患者树突状细胞B7-H1表达的影响

目的：观察聚乙二醇干扰素α-2a对慢性乙型肝炎患者外周血树突状细胞功能及B7-H1的影响,探讨慢性乙型肝炎病毒逃逸的的机制。方法：慢性乙型肝炎患者31例,给予聚乙二醇干扰素α-2a180txg抗病毒治疗52周,分别于0、12、26、52周检测肝功能、HBV-DNA;流式细胞术检测外周血mDC表面HLA-DR、CD80、CD86、CD83、CDla、B7一H1水平。根据患者HBV—DNA水平,将患者分为应答组（A组）、非应答组（B组）,10例健康志愿者作正常对照组（C组）。结果：慢性乙肝患者的树突状细胞膜表面分子HLA-DR、CD80、CD86、CD83、CDla的表达均降低。聚乙二醇干扰素α-2a治疗后应答组膜表面分子HLA-DR、CD80、CD86、CD83、cDla的表达高于非应答组65．3±6．2％VS44．2±5．5％,67．2±7．4％VS37.3±7．2％,68．4±3．6％VS42．5±7．3％,65．6±6．8％VS43．2±3．9％,49．4±9．5％VS37．5±7．9％,（P〈0．05）。应答组B7-H1表达水平较治疗前下降,非应答组B7-H1水平无明显变化12．73±3．8％VS25．24±2．92％,（P〈0．05）。结论：慢性乙型肝炎患者树突状细胞功能低下,聚乙二醇干扰素α-2a治疗可以提高树突状细胞功能,降低B7-H1表达,促进HBV-DNA的清除。树突状细胞功能低下及B7-H1高表达是乙型肝炎病毒免疫逃逸的因素之一。     

不同病毒载量慢性乙型肝炎患者树突状细胞B7-H1表达及对免疫功能的影响

目的：观察慢性乙型肝炎患者外周血树突状细胞表面共刺激分子B7-H1 的表达及对免疫功能的影响。方法：检测慢性乙型肝炎患者肝功能、HBV-DNA 水平,将患者分为高病毒载量高ALT 组（A 组）、高病毒载量低ALT 组（B 组）、低病毒载量组（C 组）及正常对照组（D 组）。流式细胞术检测各组患者外周血树突状细胞表面HLA-DR、CD80、CD86、CD83、CD1a、B7-H1 表达,酶联免疫吸附试验（ELISA）检测DC 培养上清液和混合淋巴细胞培养上清液中细胞因子IL-12、IL-10 水平。结果：慢性乙肝患者的树突状细胞膜表面分子HLA-DR、CD80、CD86、CD83、CD1a的表达均明显降低（A、B、C组与D组比较分别为42.3± 4.9 %、46.7± 7.0%、52.5± 6.3 %vs 94.5± 3.5 %;34.5± 5.3 %、39.9± 6.4 %、45.6± 5.2 %vs 90.6± 6.5 %;38.2± 8.6 %、36.1± 5.4 %、42.5± 6.8 % vs87.7± 5.1 %;28.3± 6.5 %、25.6± 3.4 %、33.5± 4.3% vs 82.6± 4.8 %;32.3± 5.8 %、29.3± 5.3 %、48.3± 4.9 % vs 68.2± 5.2 % P〈0.05）,B7-H1 表达水平明显升高（27.48± 21.4 %、21.83± 20.2 %、15.43± 10.32 %vs 4.23± 2.2%P〈0.05）。B7-H1 表达水平与ALT呈正相关,与IL-12 水平呈负相关。结论：慢性乙型肝炎患者树突状细胞功能低下,其机制可能与树突状细胞高表达B7-H1 有关。B7-H1 高表达抑制了淋巴细胞的功能,导致乙型肝炎病毒持续感染。     

共刺激因子B7-H1和B7-H3蛋白表达与胆囊癌病例特征相关

本研究选取2015年3月至2017年5月在我院保存的胆囊癌标本55例,同选取癌旁正常组织(距癌边缘>5 cm)作为对照,采用免疫组化染色检测B7-H1和B7-H3蛋白表达,分析B7-H1和B7-H3表达与胆囊癌临床病理特征和预后的关系,探讨共刺激因子B7-H1和B7-H3蛋白在胆囊癌中的表达及意义。研究结果表明:胆囊癌组织B7-H1和B7-H3蛋白阳性表达率分别为76.36%和63.63%,明显高于癌旁组织(p<0.05);B7-H1蛋白表达与胆囊癌TNM分期、淋巴结转移和侵袭深度有关(p<0.05);B7-H3蛋白表达与胆囊癌TNM分期、分化程度、淋巴结转移和侵袭深度有关(p<0.05);胆囊癌组织中B7-H1与B7-H3蛋白表达呈正相关(rs=0.516, p<0.05);B7-H1和B7-H3表达双阳性者和B7-H1或B7-H3单一表达阳性者中位总生存时间分别为20个月和21个月,明显低于B7-H1和B7-H3表达双阴性者组,差异比较有统计学意义(p<0.05)。本研究结论认为:B7-H1和B7-H3蛋白表达与胆囊癌病理特征有关系,两者间有一定相关性,且与患者预后有关。     

Human Recombinant B7-H3 Expressed in E.colt Enhances T Lymphocyte Proliferation and IL-10 Secretion in Vitro

Since it was proposed in 1970, the two-signal hypothesis for T lymphocyte activation became widely accepted, which elucidated T cells required two distinct signals for optimal T helper precursor cell expansion. This model hypothesized that peptides presen…     

小鼠B7-H4基因的真核表达及其对淋巴细胞增殖的影响

目的构建小鼠B7-H4胞外段的真核表达载体,观察其在体外对淋巴细胞增殖的影响,为深入研究B7-H4在T细胞活化及移植排斥反应中的作用提供实验材料。方法提取小鼠肺、脾脏总RNA,RT-PCR反转录cDNA,以此为模板,扩增B7-H4胞外段基因,将其导入pGEM-T Easy载体,构建TA-mB7-H4质粒。用XBaI和HindIII双酶切后琼脂糖凝胶电泳分析和测序鉴定。将测序证实的mB7-H4酶切后装入MYC-HIS-EGFP-N荧光表达载体中,构建B7-H4-EGFP真核表达载体,转化JM109感受态细菌,提取重组质粒,酶切后琼脂糖凝胶电泳分析和测序鉴定。同时构建control-EGFP载体。应用脂质体法将重组质粒转染CHO细胞,经G418筛选,获得稳定表达B7-H4-EGFP的CHO细胞株,用MTT分析其分别对BALB/c小鼠、C57小鼠淋巴细胞和二者混合淋巴细胞增殖的影响。结果经测序证实,所克隆的小鼠B7-H4 cDNA和构建的重组质粒基因序列正确;转染的CHO细胞能稳定地表达跨膜型重组蛋白B7-H4;表达的B7-H4对淋巴细胞增殖具有明显抑制作用。结论成功构建了B7-H4真核表达系统,能表达有生物学活性的B7-H4分子,为进一步探讨B7-H4在T细胞活化和移植排斥反应中的作用奠定了基础。     

卵巢癌细胞与巨噬细胞共培养对B7-H1表达的影响及机制

为了探讨卵巢癌细胞与巨噬细胞共培养后对B7．H1表达的影响及其可能机制,利用佛波酯（PMA）诱导THP-1或外周血单核细胞分化为巨噬细胞后,与人卵巢癌细胞株SKOV3体外非接触共培乔24h,qRT-PCR、Western blot以及流式细胞术分别检测SKOV3与巨噬细胞B7-H1的表达：进一步利用NF-KB、JAK2／STAT3、p38MAPK信号通路的抑制剂作用于共培养体系,检测B7-H1表达的变化,以探讨其机制。结果显示,共培养24h后,SKOV37L巨噬细胞B7-H1mRNA和蛋白的表达较非共培养组均显著升高（P〈0．05）,而阻断NF-κB、JAK2／STAT3、p38MAPK信号通路后,B7-H1的上调均明显被抑制（P〈0．05）。SKOV3与巨噬细胞共培养后B7-H1的表达升高伊〈0．05）,其机制可能涉及到NF—κB、JAK2／STAT3、p38MAPK信号通路的激活。     

聚乙二醇干扰素琢-2a 对慢性乙型肝炎患者树突状细胞 B7-H1 表达的影响*

摘要目的:观察聚乙二醇干扰素α-2a 对慢性乙型肝炎患者外周血树突状细胞功能及B7-H1 的影响,探讨慢性乙型肝炎病毒逃 逸的的机制。方法：慢性乙型肝炎患者31例,给予聚乙二醇干扰素琢-2a180 μg 抗病毒治疗52周,分别于0、12、26、52周检测肝功 能、HBV-DNA;流式细胞术检测外周血mDC表面HLA-DR、CD80、CD86、CD83、CD1a、B7-H1 水平。根据患者HBV-DNA 水平, 将患者分为应答组（A 组）、非应答组（B 组）,10 例健康志愿者作正常对照组（C 组）。结果：慢性乙肝患者的树突状细胞膜表面分 子HLA-DR、CD80、CD86、CD83、CD1a 的表达均降低。聚乙二醇干扰素α-2a 治疗后应答组膜表面分子HLA-DR、CD80、CD86、 CD83、CD1a 的表达高于非应答组65.3± 6.2 %vs 44.2± 5.5 %,67.2± 7.4%vs 37.3± 7.2 %,68.4± 3.6 %vs 42.5± 7.3 %,65.6± 6.8 %vs 43.2± 3.9 %, 49.4± 9.5 %vs 37.5± 7.9 %,(P <0.05)。应答组B7-H1 表达水平较治疗前下降,非应答组B7-H1 水平无明显变 化12.73± 3.8%vs 25.24± 2.92 %,(P<0.05)。结论：慢性乙型肝炎患者树突状细胞功能低下,聚乙二醇干扰素α-2a 治疗可以提高 树突状细胞功能,降低B7-H1 表达,促进HBV-DNA 的清除。树突状细胞功能低下及B7-H1 高表达是乙型肝炎病毒免疫逃逸的 因素之一。     

Blockade of the B7-H1/PD-1 pathway for cancer immunotherapy

The aim of cancer immunotherapy is to treat malignant disease by inducing or enhancing cancer specific immune responses. With the identification of tumor-associated antigens (TAAs) in the 1990s, cancer immunotherapy research largely focused on inducing immune responses against TAAs but achieved limited success. More recently, the underlying mechanisms and molecular pathways that cancers manipulate to subvert immune-mediated destruction have been identified, including a set of molecules with potent coinhibitory functions. Coinhibitory molecules are expressed on the surface of immune cells, cancer cells, and stromal cells and negatively regulate immune responses to cancer. In particular, one of these ligand-receptor coinhibitory interactions, B7-H1/PD-1, is critical for modulating immune responses to cancer. This knowledge led to the design of revolutionary new immunotherapeutics based on the manipulation of these molecular pathways. Monoclonal antibodies (mAbs) are the primary immunotherapeutic modality used to promote immune function via antagonism or agonism of inhibitory or stimulatory molecular pathways, respectively. Here, we review current knowledge on the function of the B7-H1/PD-1 pathway in mice and humans, its role in the subversion of immune responses in cancer, and clinical evidence that mAb targeting of this pathway results in profound immune anti-cancer effects.     

不同病毒载量慢性乙型肝炎患者树突状细胞 B7-H1 表达及对 免疫功能的影响 *

摘要 目的： 观察慢性乙型肝炎患者外周血树突状细胞表面共刺激分子 B7-H1 的表达及对免疫功能的影响。方法： 检测慢性乙型 肝炎患者肝功能、 HBV-DNA 水平, 将患者分为高病毒载量高 ALT 组 （ A 组） 、 高病毒载量低 ALT 组 （B 组） 、低病毒载量组 （C 组） 及正常对照组 （D 组）。 流式细胞术检测各组患者外周血树突状细胞表面 HLA-DR、 CD80、 CD86、 CD83、 CD1a、 B7-H1 表达, 酶联免 疫吸附试验 （ELISA ） 检测 DC 培养上清液和混合淋巴细胞培养上清液中细胞因子 IL-12、 IL-10 水平。结果： 慢性乙肝患者的树突 状细胞膜表面分子 HLA-DR、 CD80、 CD86、 CD83、 CD1a 的表达均明显降低 (A、 B、 C 组与 D 组比较分别为 42.3± 4.9%、 46.7± 7.0 %、 52.5 ± 6.3 % vs 94.5± 3.5%; 34.5 ± 5.3%、 39.9 ± 6.4%、 45.6 ± 5.2 % vs 90.6± 6.5%; 38.2 ± 8.6%、 36.1 ± 5.4%、 42.5 ± 6.8 % vs 87.7 ± 5.1%; 28.3 ± 6.5%、 25.6 ± 3.4%、 33.5 ± 4.3% vs 82.6 ± 4.8%; 32.3 ± 5.8%、 29.3 ± 5.3%、 48.3 ± 4.9 % vs 68.2 ± 5.2 % P< 0.05), B7-H1 表达水平明显升高(27.48± 21.4%、 21.83± 20.2%、 15.43± 10.32 % vs 4.23± 2.2 % P<0.05)。B7-H1 表达水平与 ALT 呈正相关, 与 IL-12 水平呈负相关。 结论： 慢性乙型肝炎患者树突状细胞功能低下, 其机制可能与树突状细胞高表达 B7-H1 有关。 B7-H1 高表达抑制了淋巴细胞的功能, 导致乙型肝炎病毒持续感染。     

B7-H1和B7-H4在口腔鳞状细胞癌组织中的表达及临床意义

目的:探讨口腔鳞状细胞癌(Oral Squamous Cell Carcinoma,OSCC)中B7-H1和B7-H4的表达及其临床意义,并为OSCC的临床诊断、治疗、判断预后及预防等提供依据。方法:采用免疫组织化学S-P法检测B7-H1及B7-H4在60例OSCC及20例非肿瘤患者正常口腔黏膜组织(NOM)中的表达情况,分析两者与OSCC临床病理特征的相关性。结果:B7-H1在OSCC组织中表达显著高于在NOM组织中表达(29例,48.3%v4例,20%,x~2=4.969,P0.05);B7-H4在OSCC组织中表达亦显著高于在NOM组织中表达(31例,51.7%v5例,25%,x~2=4.310,P0.05)。B7-H1与B7-H4在OSCC组织的表达都与TNM分期、淋巴结转移和肿瘤分化程度显著相关(P0.05),而与年龄、性别及肿瘤直径大小等无关。OSCC组织中B7-H1和B7-H4的高表达呈显著性正相关性(x~2=5.613 P0.05),60例组织中B7-H1和B7-H4共表达现象有11例(18.3%),NOM中未发现两者共表达现象。结论:B7-H1和B7-H4过表达与OSCC发生、发展及预后有关,可以作为预后指标。     

Autophagy regulates T lymphocyte proliferation through selective degradation of the cell-cycle inhibitor CDKN1B/p27Kip1

The highly conserved cellular degradation pathway, macroautophagy, regulates the homeostasis of organelles and promotes the survival of T lymphocytes. Previous results indicate that Atg3-, Atg5-, or Pik3c3/Vps34-deficient T cells cannot proliferate efficiently. Here we demonstrate that the proliferation of Atg7-deficient T cells is defective. By using an adoptive transfer and Listeria monocytogenes (LM) mouse infection model, we found that the primary immune response against LM is intrinsically impaired in autophagy-deficient CD8⁺ T cells because the cell population cannot expand after infection. Autophagy-deficient T cells fail to enter into S-phase after TCR stimulation. The major negative regulator of the cell cycle in T lymphocytes, CDKN1B, is accumulated in autophagy-deficient naïve T cells and CDKN1B cannot be degraded after TCR stimulation. Furthermore, our results indicate that genetic deletion of one allele of CDKN1B in autophagy-deficient T cells restores proliferative capability and the cells can enter into S-phase after TCR stimulation. Finally, we found that natural CDKN1B forms polymers and is physiologically associated with the autophagy receptor protein SQSTM1/p62 (sequestosome 1). Collectively, autophagy is required for maintaining the expression level of CDKN1B in naïve T cells and selectively degrades CDKN1B after TCR stimulation.     

Overexpression of B7-H1 (PD-L1) significantly associates with tumor grade and postoperative prognosis in human urothelial cancers

PURPOSE: The programmed death-1 (PD-1)/B7-H1 (also called PD-L1) pathway negatively regulates T cell activation and has been suggested to play an important role in regulating antitumor host immunity. To investigate the clinical significance of B7-H1 expression to the tumor grade and postoperative prognosis of patients with urothelial cancer, we analyzed the relationship between B7-H1 expression and various clinicopathological features and postoperative prognosis. EXPERIMENTAL DESIGN: Sixty-five urothelial cancer cases were examined. B7-H1 expression in tumors and the numbers and phenotypes of tumor-infiltrating lymphocytes were evaluated by immunohistochemistry and flow cytometry. RESULTS: A substantial expression of B7-H1 was observed in all urothelial cancers investigated. Tumor specimens from patients with higher WHO grade or primary tumor classifications showed significantly higher percentages of tumor-associated B7-H1. Tumor-associated B7-H1 expression was significantly associated with a high frequency of postoperative recurrence and poor survival rate. Furthermore, multivariate analysis indicated that tumor-associated B7-H1 was more significant prognostic factor than WHO grade. CONCLUSIONS: Our results demonstrate that the aberrant expression of B7-H1 in urothelial cancer is associated with aggressive tumors, suggesting a regulatory role of tumor-associated B7-H1 in antitumor immunity. Therefore, the manipulation of tumor-associated B7-H1 may become a beneficial target for immunotherapy in human urothelial cancer.     

A critical role for Romo1-derived ROS in cell proliferation

Low levels of endogenous reactive oxygen species (ROS) originating from NADPH oxidase have been implicated in various signaling pathways induced by growth factors and mediated by cytokines. However, the main source of ROS is known to be the mitochondria, and increased levels of ROS from the mitochondria have been observed in many cancer cells. Thus far, the mechanism of ROS production in cancer cell proliferation in the mitochondria is not well-understood. We recently identified a novel protein, ROS modulator 1 (Romo1), and reported that increased expression of Romo1-triggered ROS production in the mitochondria. The experiments conducted in the present study showed that Romo1-derived ROS were indispensable for the proliferation of both normal and cancer cells. Furthermore, whilst cell growth was inhibited by blocking the ERK pathway in cells transfected with siRNA directed against Romo1, the cell growth was recovered by addition of exogenous hydrogen peroxide. The results of this study suggest that Romo1-induced ROS may play an important role in redox signaling in cancer cells.     

A critical role of the KCa3.1 channel in mechanical stretch-induced proliferation of rat bone marrow-derived mesenchymal stem cells

Mechanical stimulation is an important factor regulating mesenchymal stem cell (MSC) functions such as proliferation. The Ca²⁺-activated K⁺ channel, K_Ca3.1, is critically engaged in MSC proliferation but its role in mechanical regulation of MSC proliferation remains unknown. Here, we examined the K_Ca3.1 channel expression and its role in rat bone marrow-derived MSC (BMSC) proliferation in response to mechanical stretch. Application of mechanical stretch stimulated BMSC proliferation via promoting cell cycle progression. Such mechanical stimulation up-regulated the K_Ca3.1 channel expression and pharmacological or genetic inhibition of the K_Ca3.1 channel strongly suppressed stretch-induced increase in cell proliferation and cell cycle progression. These results support that the K_Ca3.1 channel plays an important role in transducing mechanical forces to MSC proliferation. Our finding provides new mechanistic insights into how mechanical stimuli regulate MSC proliferation and also a viable bioengineering approach to improve MSC proliferation.     

骨髓间充质干细胞移植对大鼠低氧性肺动脉高压的影响

目的：探讨骨髓间充质干细胞（MSCs）移植对大鼠低氧性肺动脉高压（HPH）的影响。方法：体外分离、培养、鉴定SD大鼠骨髓MSCs、绿色荧光蛋白腺病毒标记MSCs细胞。将健康雄性SD大鼠随机分为4组：正常对照组（NC组）8只、低氧性肺动脉高压组（HPH组）8只,低氧性肺动脉高压同时骨髓间充质干细胞移植组（MSCs组）24只,低氧性肺动脉高压同时携带血管内皮生长因子（VEGF）的MSCs移植组（VEGF＋MSCs组）24只。采用常压间歇低氧法建立大鼠肺动脉高压模型,干细胞转染并行干细胞移植。观察大鼠平均肺动脉压力（mPAP）,计算右心室肥厚指数（RVHI）,显微镜下观察各组大鼠肺小动脉形态结构改变,并在荧光显微镜下观察干细胞移植7d,14d,28d时腺病毒转染荧光标记的MSCs在肺小动脉分布及表现。结果：NC组28d时mPAP（mmHg）为15．5±1．5,而HPH组、MSCs组及MSCs＋VEGF组分另q为26．1±1．9、21．6±2．7及20．1±2．9,均明显高于NC组（P〈0．01）,但MSCs组及MSCs＋VEGF组较HPH组明显下降（P〈0．01）,MSCs组与MSCs＋VEGF组无明显差别。NC组28d时RVHI为0．28±0．02,而HPH组、MSCs组及MSCs＋VEGF组RVHI分别为0．43±0．07、0．34±0．03及0．35±0．01,均明显高于NC组（P〈0．01）,但MSCs组及MSCs＋VEGF组较HPH组明显下降（P〈0．05）,MSCs组与MSCs＋VEGF组无明显差别。HPH组28d时,肺小动脉管壁明显增厚,管腔明显狭窄、闭塞,内皮细胞不完整,而MSCs组血管壁较HPH组变薄,管腔通畅,内皮细胞完整性改善,MSC8组及MSCs＋VEGF组的表现改变不明显。结论：骨髓间充质干细胞移植可改善肺小动脉血管重塑,从而部分逆转HPH的进程;而将VEGF与MSCs联合移植并未提高单纯MSCs移植的作用。     

Bone marrow-derived mesenchymal stem cells promote cell proliferation of multiple myeloma through inhibiting T cell immune responses via PD-1/PD-L1 pathway

Objectives: This study aims to explore the effect of bone marrow mesenchymal stem cells (BMSCs) on multiple myeloma (MM) development and the underlying mechanism.

Materials and Methods: BMSCs from C57BL/6 J mice were isolated and the third passage was used for subsequent experiments. Additionally, a series of in vitro transwell coculture assays were performed to explore the effects of BMSCs on the proliferation of MM cells 5TGM1 and CD4⁺ T cells. Furthermore, a 5TGM1-induced MM mice model was established. Moreover, PD-L1 shRNA was transfected into BMSCs to investigate whether PD-1/PD-L1 pathway involved in BMSCs-mediated regulation of T cells and MM growth.

Results: Data revealed that BMSCs significantly promoted 5TGM1 proliferation in a dose-dependent manner. Furthermore, BMSCs administration exerted stimulatory effects on MM development in terms of shortening the mouse survival rate, promoting tumor growth, and enhancing inflammatory infiltration in the MM model mice. Moreover, BMSCs decreased the percentage of Th1 and Th17 cells, whereas increased that of Th2 and Treg cells. Their corresponding cytokines of these T cell subsets showed similar alteration in the presence of BMSCs. Additionally, BMSCs significantly suppressed CD4⁺ T cell proliferation. We also found that PD-L1 shRNA inhibited 5TGM1 proliferation likely through activation of CD4⁺ T cells. Further in vivo experiments confirmed that PD-L1 inhibition attenuated BMSCs-induced MM growth, inflammation infiltration and imbalance of Th1/Th2 and Th17/Treg.

Conclusion: In summary, our findings demonstrated that BMSCs promoted cell proliferation of MM through inhibiting T cell immune responses via PD-1/PD-L1 pathway.