人结肠直肠癌组织中B7-H1的表达及其临床意义

探讨共刺激分子B7-H1在人结肠直肠癌组织中的表达及其意义。运用免疫组织化学SP法检测B7-H1分子在60例结肠直肠癌组织中的表达,并对其表达水平与结肠直肠癌临床病理参数之间的关系进行相关性分析。结果显示,B7-H1分子在结肠直肠癌组织中的阳性表达显著高于正常结肠直肠组织(P<0.01);B7-H1在结肠直肠癌组织中阳性表达率为60%,与患者性别、年龄、肿瘤组织的类型、分级和分期无关(P>0.05),而与淋巴结是否转移显著相关(P<0.01)。B7-H1在结肠直肠癌组织中的高表达,可能在肿瘤的发生、发展中起到一定作用,有望成为结肠直肠癌免疫治疗的一个新靶点。     

B7-H1/PD-1共刺激途径与病毒感染

B7-H1及其受体PD-1是共刺激分子B7-CD28家族的重要成员.B7-H1在淋巴组织及外周非淋巴组织广泛诱导性表达,PD-1则主要表达在活化的T细胞、B细胞及髓系细胞表面.B7-H1/PD-1共刺激途径主要作用是负性调节T、B细胞的免疫反应,参与维持外周组织的免疫耐受.病毒感染可以上调B7-H1及PD-1的表达,抑制病毒特异性T细胞的免疫功能,B7-H1/PD-1途径是病毒逃避免疫监视,引发慢性感染的重要通路,因而阻断B7-H1/PD-1共刺激途径能够恢复病毒特异性T细胞的功能,清除病毒感染,这对于病毒感染的免疫治疗,尤其是病毒慢性感染的免疫治疗具有重要意义.     

B7-H1阻断对CD3AK细胞体外生物学活性的影响

目的: 探讨B7-H1阻断对CD3AK细胞增殖活化及其抗肿瘤免疫效应的影响.方法: 利用CD3单克隆抗体(mAb)刺激健康人外周血淋巴细胞诱导产生CD3AK细胞,然后利用B7-H1阻断型抗体阻断B7-H1通路,3H-TdR渗入法检测阻断后CD3AK细胞的增殖能力,ELISA法检测阻断后CD3AK细胞分泌IFN-γ、 TNF-α和IL-10的水平,同时将CD3AK细胞作用于膀胱肿瘤BIU-87细胞,MTT法检测阻断后CD3AK细胞的杀伤活性.结果: B7-H1阻断后,CD3AK细胞的增殖能力明显增强,体外存活时间明显延长;其分泌IFN-γ、 TNF-α的水平明显提高,而分泌IL-10的水平明显下降;同时CD3AK细胞对BIU-87细胞的杀伤活性亦明显升高.结论: 阻断B7-H1通路可以促进和维持CD3AK细胞的增殖和活化,并增强其抗肿瘤的免疫效应.阻断B7-H1通路将有望成为肿瘤免疫治疗的新策略.     

B7-H1 and B7-H4 expression in colorectal carcinoma: Correlation with tumor FOXP3 regulatory T-cell infiltration

B7-H1 and B7-H4 are newly discovered members of the B7-CD28 family. They can inhibit T cell activation and proliferation and regulate T cell immune response negatively. Both B7-H1 and B7-H4 are expressed in many tumors and are classified as co-inhibitors of cell-mediated immunity. FOXP3⁺ regulatory T cells (Tregs) play an important role in the maintenance of tumor immunity tolerance. However, the implication of B7-H1 and B7-H4 expression and their interaction with Tregs infiltration in colorectal cancer are unknown. The present study aimed to determine the expression of B7-H1 and B7-H4 as well as Tregs infiltration in colorectal cancer and to explore the clinical and pathological implication of suppressor immune cells and molecules. Frozen sections and immunohistochemical assay were undertaken to assess B7-H1, B7-H4 expression and Tregs infiltration in fresh specimens collected from 56 patients with colorectal carcinoma. The results showed that expression of B7-H1 and B7-H4 in colorectal carcinoma tissues was significantly higher than in adjacent normal mucosa (P < 0.001). B7-H1 expression was positively correlated to the infiltration depth, lymph node metastasis and advanced Duke's stage (P < 0.05, P < 0.05 and P < 0.05, respectively), whereas B7-H4 expression was positively related to the infiltration depth and lymph node metastasis (P < 0.01 and P < 0.05, respectively). Furthermore, Tregs infiltration was more frequent in tumor tissue than in adjacent normal mucosa and was associated with poor differentiation and positive lymph node metastasis (P < 0.01, and P < 0.01, respectively). The statistical analysis indicated a significant correlation between Tregs infiltration and B7-H1 or B7-H4 expression respectively. These results suggest that over-expression of B7-H1 and B7-H4 has stronger prognostic significance and promote tumor tolerance, and they might contribute to Tregs development in the colorectal carcinoma tolerogenic milieu.     

小胶质细胞表达B7-H1及LPS刺激对其表达影响的研究

目的 了解共刺激分子B7-H1在小鼠中枢神经系统小胶质细胞上的表达,及LPS刺激后的表达变化情况,探讨小胶质细胞在大脑炎症状态下的免疫功能变化。方法利用小鼠小胶质细胞株N9,以LPS刺激其活化,应用RT-PCR、定量PCR、流式细胞术、免疫组化检测B7-H1在刺激前后的表达变化规律;分离培养小鼠原代小胶质细胞并进行GSA-IB4染色鉴定。应用免疫组化检测原代小胶质细胞在LPS刺激前后的表达变化。结果①小鼠小胶质细胞株N9在未刺激状态下表达B7-H1的mRNA和蛋白分子;LPS刺激细胞活化后释放肿瘤坏死因子α,一氧化氮增加,同时B7-H1的mRNA和蛋白表达水平增加。②分离培养的小鼠原代小胶质细胞经GSA-IB4染色鉴定纯度在95％以上,免疫组化显示原代小胶质细胞组成性表达B7-H1,LPS刺激后表达明显上调。结论小鼠小胶质细胞株N9小胶质细胞组成性表达共刺激分子B7-H1,LPS刺激后表达上调,提示其参与了中枢神经系统免疫应答的调节。     

LPS调节U937细胞上B7-H1表达的初步研究

目的了解脂多糖（LPS）刺激后U937细胞上B7-H1的表达变化情况。方法培养U937细胞,用荧光半定量实时PCR和流式细胞仪技术,分别观测未刺激和用LPS刺激的U937细胞B7-H1mRNA与蛋白的表达情况。结果未经刺激的U937细胞组成性表达B7-H1,LPS刺激可显著增强B7-H1基因mRNA的转录和蛋白的表达。结论LPS在转录与翻译两个环节均可上调U937细胞B7-H1的表达水平。     

B7-H1阻断对未成熟树突状细胞免疫功能的影响

目的：探讨B7-H1阻断对未成熟树突状细胞(DCs)的免疫刺激活性的影响。 方法： 以细胞因子GM-CSF和IL-4体外诱导人单核细胞来源的树突状细胞，流式细胞仪检测在诱导过程中B7-H1的表达，并以B7-H1单抗阻断处理，分别以流式细胞仪、MTT法、ELISA、ELISPOT法检测B7-H1阻断对DCs的成熟和内吞能力、刺激淋巴细胞增殖、IL-12的分泌、诱导淋巴细胞分化的影响。 结果： DCs在诱导过程中B7-H1表达逐渐增强，第7 d时的阳性表达率为54.12％，以TNF-α诱导成熟后阳性表达率为83.64％；B7-H1阻断对DCs成熟和内吞能力无影响，但可使未成熟DCs刺激淋巴细胞增殖的能力和IL-12的分泌明显增强，并诱导淋巴细胞向分泌IFN-γ的Th1/Tc1的分化。 结论： B7-H1阻断可增强未成熟DCs的免疫刺激活性，利用B7-H1阻断的DCs瘤苗激发抗肿瘤免疫的方案值得进一步探讨。     

B7-H1协同刺激淋巴细胞增殖并诱导产生抑制性T细胞

目的：探讨B7-H1分子在抗原诱导的免疫反应中的作用。方法：在可溶性抗原PPD诱导淋巴细胞增殖体系中,加入中和抗体检测B7-H1的协同刺激作用。转染表达B7-H1的ECV304细胞,观测其对PPD诱导的淋巴细胞增殖、细胞因子分泌及淋巴细胞亚群比例的影响。将刺激后的淋巴细胞过继至自体或同种异体增殖体系中,检测其功能。结果：抗B7-H1中和抗体可降低PPD诱导的淋巴细胞增殖反应和细胞因子分泌;转染B7-H1的ECV304细胞可协同刺激抗原诱导的淋巴细胞增殖,促进细胞因子IL-10的表达,并改变T细胞亚群分布,增加CD4^＋T细胞亚群比例;自体或同种异体过继实验显示,B7-H1刺激后的淋巴细胞具有显著的免疫抑制功能。结论：B7-H1分子可协同刺激淋巴细胞增殖并诱导产生具有免疫抑制功能的T细胞亚群。     

Increased B7-H1 and B7-H4 Expressions on Circulating Monocytes and Tumor-Associated Macrophages are Involved in Immune Evasion in Patients with Gastric Cancer

Expression of the costimulatory molecule B7-H4, a member of the inhibitory B7 family, has been reported to be upregulated on tumor-associated macrophages, and this overexpression may be involved in immune evasion in cancer patients. The present study was designed to investigate B7-H4 expression on monocytes/macrophages and its relationship with immune evasion in gastric cancer patients. B7-H4 expression on circulating monocytes and tumor-associated macrophages was evaluated by multicolor flow cytometry. Carboxyfluorescein succinimidyl ester proliferation assays and quantitative interferon-gamma enzyme-linked immunosorbent assays were carried out to determine the inhibitory effect of B7-H4+ monocytes on CD4+ T cells. B7-H4 expression on circulating monocytes was upregulated and these B7-H4+ monocytes showed immunosuppressive properties. B7-H4 expression was closely related to the depth of invasion, as well as the presence of lymphatic and venous invasion. There were significant correlations between B7-H4 expression and B7-H1 or HLA-DR expression on monocytes/macrophages in gastric cancer patients. B7-H4 expression was remarkably upregulated in gastric cancer tissues compared with peripheral blood samples. Complete surgical removal of the tumor decreased B7-H4 expression on circulating monocytes from gastric cancer patients. Cocultures of gastric cancer cell lines and monocytes led to upregulation of B7-H4 expression on monocytes. Increased B7-H4 expression on circulating monocytes and tumor-associated macrophages may be one of the key mechanisms responsible for immune evasion by tumors in gastric cancer.     

共刺激分子B7-H1在胃癌中表达上调

目的了解胃癌患者胃黏膜组织中共刺激分子B7-H1的表达情况及其与肿瘤转移和预后的关系。方法应用流式细胞技术、免疫化学染色、免疫荧光染色与Western blot检测胃癌细胞系SGC-7901与新鲜切除的胃癌组织、癌旁组织及其远端正常胃黏膜组织中B7-H1的表达,并对相关数据进行相应的统计学分析,确定B7-H1表达水平与患者临床病理指标的关系。结果SGC-7901细胞中有B7-H1蛋白表达,主要分布在细胞膜,细胞质中少量;胃癌组织中B7-H1阳性表达率为(13/21)62%,癌旁组织中为(7/21)33%,而正常胃黏膜组织中没有B7-H1表达。统计分析显示,胃癌组织中B7-H1表达与肿瘤的浸润深度、周围淋巴结转移、远处转移及pTNM分期有关(P<0.05)。结论B7-H1分子可作为胃癌早期诊断与预后判断的新指标。     

膜型B7-H1分子诱导产生的T抑制细胞及其免疫学特性的初步研究

为了探讨B7-H1分子诱导产生的T抑制细胞免疫生物学特性,我们首先构建了人B7-H1基因,筛选出细胞膜表面稳定表达B7-H1蛋白的人ECV304细胞系,并在体外建立了由这种非专职抗原提呈细胞系刺激诱导纯化的CD4+T细胞,并使其具有抑制功能的新方法。通过检测其增殖效应和上清细胞因子水平的变化,观察到这群细胞对同种异体混合淋巴细胞反应(MLC),具有明显抑制作用,该作用与大量产生IL-10、TGF-β细胞因子有关。实验表明,ECV304/B7-H1可有效诱导具有免疫抑制的功能T细胞,为其进一步应用于临床治疗打下基础。     

Induction of B7-H1 receptor by bacterial cells fractions of Porphyromonas gingivalis on human oral epithelial cells: B7-H1 induction by Porphyromonas gingivalis fractions

The immune-regulatory B7-H1 receptor, also known as programmed death-ligand 1 (PD-L1), plays an important role in cell-mediated immune response. It is a co-signaling molecule that mediates regulation of T cell activation and tolerance and is able to negatively regulate activated T cell functions and survival. High expression of B7-H1 in host cells may contribute to the chronicity of inflammatory disorders and represents a possible mechanism of immune evasion. Porphyromonas gingivalis is regarded as a keystone pathogen in periodontitis and is able to invade host cells and disposes a variety of virulence factors including lipopolysaccharide (LPS), fimbriae and proteases such as gingipains. Based on previous studies that demonstrated the capability of P. gingivalis to induce up-regulation of PD-L1 in malignant and non-malignant oral epithelial cells, the aim of the present work was to analyse the potential of various cellular components of P. gingivalis to induce the PD-L1 receptor. Human squamous carcinoma cells and primary gingival keratinocytes were stimulated with total, inner and outer membrane fractions, cytosolic proteins, as well as LPS and peptidoglycans. PD-L1 protein expression was investigated by Western blot analysis and RT-PCR. It was demonstrated that the total membrane fraction induced the highest up-regulation in B7-H1 expression, followed by the outer and inner membrane, whereas cytosolic proteins and LPS did not. In conclusion, we provide evidence that the membrane fraction of P. gingivalis is responsible for up-regulation of the immune-regulatory receptor PD-L1 in squamous carcinoma cells and gingival keratinocytes, and thus may support immune evasion of oral carcinomas.     

负性协同刺激分子B7-H4在小鼠树突状细胞上的表达及其生物学意义

目的 探讨B7-H4分子在小鼠树突状细胞分化发育过程中的表达及其在T细胞活化中的作用.方法 采用GM-CSF和IL-4联合方案体外诱导小鼠髓系树突状细胞(DCs);采用流式细胞术检测未成熟DCs、成熟DCs以及IL-10诱导的DCs表面B7-H4分子的表达;采用3H-TdR掺入试验和抗B7-H4单抗(McAb)阻断试验分析DCs表达的B7-H4分子对T淋巴细胞的共刺激效应.结果 经GM-CSF和IL-4联合方案体外诱导的未成熟DCs较高水平表达B7-H4,在IL-10作用下B7-H4表达进一步上调,经TNF-α刺激成熟后,B7-H4的表达显著下调,不同功能状态下DCs表面B7-H4分子均可抑制T细胞的增殖,但以未成熟DCs、IL-10诱导的DCs表面B7-H4分子的抑制作用更为显著.结论 不同功能状态下的DCs均有B7-H4分子的表达,处于抑制状态下的DCs通过高表达B7-H4介导免疫不应答效应.     

B7-1/B7-H1相对比例变化对Hsp70-肽复合物抗肿瘤免疫效应的影响

目的：探讨Hsp7肽复合物对B7-1／B7-H1相对比例的影响及真核表达可溶性PD-1(sPD-1)对Hsp70-肽复合物抗肿瘤作用的影响。方法：通过RT-PCR和半定量PCR技术检测Hsp70-肽复合物体外刺激和体内免疫对小鼠脾细胞正调控共刺激分子B7-1和抑制性共刺激分子B7-H1及其受体PD-1表达的影响；体内转染表达sPD-1后，观察Hsp70-肽复合物免疫小鼠的肿瘤生长以及脾淋巴细胞毒性的变化。结果：基因表达检测表明．Hsp70-肽复合物体外刺激的小鼠脾细胞B7-1 mRNA和B7-H1 mRNA的水平随时问而变化，B7-1／B7-H1比值随刺激时间而增高；Hsp70-肽复合物体内免疫小鼠后期脾细胞B7-1表达下降，B7-H1及其受体PD-1表达上调，B7-1／B7-H1比值逆转；体内表达sPD-1可显著增强和延长Hsp70-肽复合物的抑瘤效果；体内表达sPD-1可提高Hsp70-肽复合物免疫的荷瘤小鼠脾细胞的杀伤率。结论：Hsp70-肽复合物的刺激引起共刺激分子B7-1和B7-H1表达的变化，B7-1／B7-H1的比例与激活效应相关，sPD-1通过阻抑B7-H1／PD-1途径、上调B7-1／B7-H1比例，可增强免疫应答，提高Hsp70-肽复合物特异性免疫治疗肿瘤的效应。     

B7-H4与肿瘤关系的研究进展

T细胞介导的免疫应答在肿瘤免疫中起到重要作用,而T细胞的活化过程又依赖于双信号和细胞因子,其中活化的第二信号来自协同刺激分子,协同刺激分子中B7/CD28家族成员在肿瘤免疫中发挥着重要作用.B7家族新成员B7-H4是T淋巴细胞免疫应答的负性调控因子,可抑制T细胞的增殖和细胞因子的产生,其与肿瘤的发生发展有着重要的关系,可作为肿瘤诊断和治疗的新靶点.     

鼠抗人B7-H4单克隆抗体的制备及其生物学特性鉴定

目的:研制鼠抗人B7-H4分子的单克隆抗体(mAb)并鉴定其生物学特性。方法:以高表达人B7-H4分子的转基因细胞L929/B7-H4为免疫原,常规免疫BALB/c小鼠,采用B淋巴瘤杂交技术,经免疫荧光标记和流式细胞术分析、反复筛选、多次克隆化培养,获得两株可特异分泌鼠抗人B7-H4分子mAb的杂交瘤细胞株。采用快速定性试纸分析法鉴定mAb所属的Ig亚类。用Dot-blot、Western blot鉴定mAb的特异性,竞争结合抑制实验鉴定mAb所识别的抗原结合位点,T细胞增殖抑制阻断实验对mAb的生物学功能进行初步的鉴定。结果:获得两株持续、稳定地分泌抗B7-H4 mAb的杂交瘤细胞,分别命名为1F10和2B2。Dot-blot的结果显示,两株mAb均能特异性识别B7-H4分子。Westernblot检测显示,mAb 2B2可以特异性识别B7-H4分子,而mAb 1F10则不能识别。位点竞争实验表明,两株mAb之间,mAb 1F10与商品化的mAb H74之间识别位点不同,mAb 2B2与商品化mAb识别位点相近。T细胞增殖抑制阻断试验显示,这两株mAb均能一定程度地阻断B7-H4对T细胞的抑制作用。结论:成功地获得两株抗人B7-H4分子的mAb,为进一步研究B7-H4分子的生物学功能提供了有效的工具。     

B7-H3 and its role in bone cancers

Most bone cancers have a high risk of metastasis, recurrence, and poor prognosis. Although conventional treatments are still the most important therapy, disadvantages still exist. Therefore, there is an unmet need to develop effective strategies. Immunotherapy is a promising therapy. Immunotherapies targeting checkpoints have proven to be successful, but B7-H3 (CD276, clusters of differentiation protein 276), a member of the B7-family of co-stimulatory molecules, is not being widely studied in bone cancers. This review summarized the studies on B7-H3 in bone cancers. 4 studies investigated B7-H3 expression in osteosarcoma, but there is no study on B7-H3 expression in chondrosarcoma. Two studies investigated the possibility to treat Ewing`s sarcoma through targeting the B7-H3 CAR (chimeric antigen receptors) T-cells or using anti-B7-H3 antibody. A study observed the growth of myeloma in B7-H3-deficient mice and the therapeutic effect of B7-H3 antibody and a study invested B7-H3 expression in myeloma patients. One study reported B7-H3 expression in osteoclastomas and one study investigated B7-H3 expression in chordoma tumor tissues. Two clinical trials are conducting on the therapy of osteosarcoma and myeloma using B7-H3 as a target. In conclusion, B7-H3 could be a target of bone cancers.     

卵巢上皮-间质肿瘤中B7-H3、B7-H4的表达及临床病理意义

目的探讨B7-H3、B7-H4在卵巢上皮-间质肿瘤中的表达及其临床病理意义。方法用RT-PCR技术及免疫组化PV9000两步法研究86例卵巢恶性上皮-间质肿瘤(恶性组),40例卵巢交界性上皮-间质肿瘤(交界组)和40例卵巢良性上皮-间质肿瘤(良性组)B7-H3与B7-H4mRNA及蛋白表达情况,并结合临床病理参数进行分析。结果 B7-H3、B7-H4mRNA在3组卵巢肿瘤组织中均有表达,且各自的3组卵巢肿瘤组织的mRNA光密度值差异均有统计学意义(P<0.05);B7-H3与B7-H4蛋白在恶性组中均呈细胞质和(或)细胞膜表达,其中B7-H3阳性率为81.4%(70/86),明显高于交界组22.5%(9/40)和良性组5.0%(2/40)的表达,差异有统计学意义(P<0.05);B7-H4阳性率为77.9%(67/86),明显高于交界组35.0%(14/40)和良性组5.0%(2/40)的表达,差异有统计学意义(P<0.05)。恶性组的卵巢癌组织中B7-H3蛋白在非黏液性卵巢癌中较黏液性卵巢癌高表达,差异有显著性(P<0.01),与临床分期、病理分级、患者年龄、腹水细胞学和淋巴结转移差异均无统计学意义(P>0.05)。B7-H4蛋白表达与组织病理学类型、临床分期、病理分级、患者年龄、腹水细胞学和淋巴结转移差异均无统计学意义(P>0.05)。结论 B7-H3与B7-H4在卵巢恶性上皮-间质肿瘤表达提示其可能与肿瘤的发生、发展有关,可为卵巢恶性肿瘤的诊断及治疗提供新的依据。     

LPS诱导胰腺癌细胞Panc-1表达B7-H1

目的: 研究胰腺癌细胞株Panc-1在应用脂多糖(LPS)刺激前后B7-H1表达的变化,并探讨其可能的分子机制。方法: LPS刺激以及丝裂原活化蛋白激酶(MAPKs)的特异性抑制剂处理Panc-1细胞前后,应用Western blotting检测MAPKs信号通路中p38丝裂原活化蛋白激酶(p38)、细胞外信号调节蛋白激酶(ERK)和c-Jun氨基端激酶(JNK)磷酸化水平的变化,real-time PCR和Western blotting检测B7-H1 mRNA和蛋白的表达变化。结果: LPS刺激后B7-H1的表达显著上调,p38、ERK和JNK的磷酸化水平也明显上调,加入MAPKs特异性的抑制剂后,LPS诱导的p38、ERK和JNK的磷酸化被抑制,并且在p38和ERK的抑制剂处理后,B7-H1的表达也明显被抑制,而在JNK的抑制剂处理后B7-H1的表达没有显著变化。结论: LPS可以诱导胰腺癌细胞株Panc-1表达B7-H1,并且p38和ERK的活化在LPS诱导的B7-H1的表达过程中起着重要作用。     

原发性胆汁性肝硬化患者共刺激分子B7-H4的检测及临床意义

目的 探讨原发性胆汁性肝硬化(primary biliary cirrhosis,PBC)患者共刺激因子B7-1t4的表达及其与疾病发病机制的关系.方法 分别采用荧光实时定量PCR法(real-time PCR)、酶联免疫吸附试验(ELISA)及流式细胞术(FCM)检测65名PBC患者外周血单个核细胞(PBMC)B7-H4mRNA表达水平、血清IL-2水平以及CD4+、CD8+ T细胞亚群和T细胞表面B7-H4表达百分率,同时监测抗线粒体抗体(anti-mitochondrial antibody,AMA)及临床各项生化指标的关系.结果 (1)PBC患者PBMC B7-144 mRNA水平及T细胞表面B7-H4表达百分率显著低于非PBC肝硬化组及健康对照组(P<0.01).(2)活化72 h后各实验组及对照组IL-2含量及CD4+、CD8+、CD4+ CD8+T淋巴细胞表达水平均低于活化前,以非PBC肝硬化组与健康对照组降低显著(P<0.05);PBC组IL-2含量及CD4+、CD4+ CD8+ T淋巴细胞表达水平高于非PBC肝硬化组与健康对照组(P<0.01).(3)AMA-M2阳性患者血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)及γ-谷氨酰转肽酶(GGT)水平升高,其中ALP及GGT升高显著(P<0.05);AMA-M2阳性患者与阴性患者T细胞表面B7-H4表达百分率差异无统计学意义(P>0.05).结论 共刺激因子B7-H4对PBC患者体内T细胞活化增殖及细胞因子分泌的抑制作用减弱,为研究PBC的发生发展和阐明PBC的发病机制提供了试验资料.