Synergetic anticancer effect of combined quercetin and recombinant adenoviral vector expressing human wild-type p53,GM-CSF and B7-1 genes on hepatocellular carcinoma cells in vitro

AIM: This study investigated the anti-cancer effect ofcombined quercetin and a recombinant adenovirus vectorexpressing the human p53, GM-CSF and B7-1 genes(designated BB-102) on human hepatocellular carcinoma(HCC) cell lines in vitro.METHODS: The sensitivity of HCC cells to anticancer agentswas evaluated by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The viability of cells infectedwith BB-102 was determined by trypan blue exclusion. Theexpression levels of human wild-type p53, GM-CSF and B7-1genes were determined by Western blot, enzyme-linkedimmunosorbent assay (ELISA) and flow cytometric analysis,respectively. The apoptosis of BB-102-infected or quercetin-treated HCC cells was detected by terminal deoxynucleotidyltransferase (TdT) assay or DNA ladder electrophoresis.RESULTS: Quercetin was found to suppress proliferation ofhuman HCC cell lines BEL-7402, HUH-7 and HLE, with peaksuppression at 50 μmol/L quercetin. BB-102 infection wasalso found to significantly suppress proliferation of HCC celllines. The apoptosis of BB-102-infected HCC cells was greaterin HLE and HUH-7 cells than in BEL-7402 cells. Quercetin didnot affect the expression of the three exogenous genes inBB-102-infected HCC cells (P＞0.05), but it was found to furtherdecrease proliferation and promote apoptosis of BB-102-infected HCC cells.CONCLUSION: BB-102 and quercetin synergeticallysuppress HCC cell proliferation and induce HCC cell apoptosis,suggesting a possible use as a combined anti-cancer agent.

Prevention of hepatocellular carcinoma in mice by IL-2 and B7-1 genes co-transfected liver cancer cell vaccines

AIM: To study the immunoprotective effect of liver cancer vaccine with co-transfected IL-2 and B7-1 genes on hepatocarcinogenesis in mice.METHODS: The murine liver cancer cell line Hepal-6 was transfected with IL-2 and/or B7-1 gene via recombinant adenoviral vectors and the liver cancer vaccines were prepared. C57BL/6 mice were immunized with these vaccines and challenged with the parental Hepal-6 cells afterwards.The immunoprotection was investigated and the reactive T cell line was assayed.RESULTS: The immunoprotection of the tumor vaccine was demonstrated. The effect of IL-2 and B7-1 genes cotransfected Hepal-6 liver cancer vaccine (Hep6-IL2/B7vaccine) on the onset of tumor formation was the strongest.When attacked with wild Hepal-6 cells, the median survival period of the mice immunized with Hep6-IL2/B7 vaccine was the longest (68 days, χ2=7.70-11.69, P＜0.05) and the implanted tumor was the smallest (z =3.20-44.10, P＜0.05).The effect of single IL-2 or B7-1 gene-transfected vaccine was next to the IL2/B7 gene co-transfected group, and the mean survival periods were 59 and 54 days, respectively.The mean survival periods of wild or enhanced green fluorescence protein gene modified vaccine immunized group were 51 and 48 days, respectively. The mice in control group all died within 38 days and the implanted tumor was the largest (z=3.20-40.21, P＜0.05). The cellular immunofunction test and cytotoxicity study showed that the natural killer (NK) cell, lymphokine activated killer (LAK) cell and cytotoxic T lymphocyte (CTL) activities were significantly increased in mice immunized with the Hep6-IL2/B7 vaccine, (29.5±2.5%,65.0±2.9%, 83.1±1.5% respectively, compared with other groups, P＜0.05).CONCLUSION: The Hep6-IL2/B7 liver cancer vaccines can induce the mice to produce activated and specific CTL against the parental tumor cells, and demonstrate stronger effect on the hepatocarcinogenesis than single gene modified or the regular tumor vaccine. Therefore, the vaccines may become a novel potential therapy for recurrence and metastasis of HCC.

ANTITUMOR IMMUNITY AND VACCINE EFFECT INDUCED BY IL-12 SYNERGIZES B7-1 GENE TRANSFECTED CELLS

Objective: To study the synergic effects of IL-12 and B7-1 transfectant on antitumor immunity in vivo. Methods: The retrovirus vector encoding mIL-12 and mB7-1 gene was tranfected into EL-4 thymic lymphoma cells respectively.The cells were used as tumor vaccine and the therapeutic effect was observed. Results: In contrast to the miceimmunized with EL-4/Wt or EL-4/Neo groups, thetumorigenicity of EL-4/IL-12 transfectant was decreased(P<0.001). The EL-4/IL-12 and EL-4/B7-1 cells irradiatedwith 60Co showed significant systematic protective effectsagainst the rechallenge of EL-4/Wt. 60Co irradiatedEL-4/IL-12 cells delayed the occurrence of tumor andprolonged the survival period of tumor bearing mice.Combination of the vaccines of EL-4/IL-12 and EL-4/B7-1 resulted in the enhanced therapeutic effect compared witheach single transfectant group (P<0.001). Conclusion: The results showed that IL-12 transduced cells could enhancethe antitumor immunity of host as cancer vaccine.Combination of the EL-4/IL-12 and EL-4/B7-1 transfectant could improve immunity of host and is a prospect cancervaccine.

GM-CSF GENE OR B7-1 GENE MODIFIED MURINE EL-4 CELLS VACCINE

Objective: To study the vaccine potency of gene-modified tumor cells. Methods: The EL-4 lymphoma was transduced with recombinant retrovirus containing the murine GM-CSF gene or B7-1 gene. The effect of gene transduction on antitumor immunity was investigated. Results: Flow cytometry analysis showed that expression of their surface marker between wild-type EL-4 cells and gene transduced tumor cells was the same except for CD80 positive in B7-1 gene transduced cells. GM-CSF gene or B7-1 gene transduced EL-4 cells resulted in remarkable loss of tumorigenicity in syngenetic mice. The systemic protective immunity was induced against the challenge with EL-4/wt cells. Therapeutic vaccine with EL-4/GM-CSF or EL/7-1 cells could retard the growth of established early-stage EL-4/wt tumor significantly, but not retard the growth of late-stage EL-4/wt tumor. Irradiated GM-CSF gene transduced EL-4 cells showed strong vaccine effect against EL-4 cell challenge, but irradiated B7-1 gene transduced EL-4 cells showed weak vaccine effect. Remarkable cooperative antitumor effect against EL-4 cell challenge was observed when both irradiated EL-4/GM-CSF and EL-4/B7-1 were inoculated together. Conclusion: GM-CSF gene or B7-1 gene transduced BL-4 cells can be used as a good tumor vaccine. The combination of the two kinds of vaccine may have potential application value in human cancer treatment.

STUDY OF ENHANCED IMMUNOGENECITY OF B7-1 GENE TRANSFECTED HUMAN HELA CELL LINE

This work was supposed by CMB (No. 96—635) This is one of papers of the special issue on gene therapy research (Chin J Cancer Res Vol. 9 No. 4 December, 1997). Although cervical carcinoma cells may express the human papillomavirus protein E6 and E7, they fail to induce an effective specific cytotoxic T lymphocyte response. Recent studies suggest that expression of CD 80 (B7 1) on tumor cells is effective to induce antitumor immune responses. 1,2 In our study, CD 80 gene was transfected into human Hela cell line with a CD 80 expression plasmid (B7 1 +pcDNA 3) by electroporation, then the immunogenecity of the modified Hela cell was tested in TLMC (tumor lymphocyte mixed culture) system. Thymidine lymphocyte proliferation assays showed that the response of human peripheral blood lymphocytes (PBLS) to CD 80 positive Hela cells demonstrated a substantial increase in cell proliferation compared to the response to control cells. Cocultivation of allogeneic PBLs with CD 80 positive tumor cells for three days can induce an increased secretion of IL 2. Our results demonstrate an immunostimulatory effect of CD 80 expression on cervical cancer cells, which provides a basis for the development of a therapeutic tumor vaccine.

Experimental Study on the Antitumor Effect of Mouse B7-1 Gene

Summary: Mouse B7 1 cDNA was cloned by RT PCR from BALB/C mouse splenic cells and inserted into pcDNA3 to construct an eukaryotic expression vector. This constructor was named pCD mB7 1, in which the B7 1 cDNA was identified to be consistent with the data from other researchers. pCD mB7 1 plasmid was transfected into B16(F0) cells, and effective expression of mB7 1 in these tumor cells could be detected till the 6th month by RT PCR and RNA hybridization. Specific cytotoxity assay of lymphocytes was conducted after culturing with tumor cells and the results demonstrated that B16 cells transfected with B7 1 gene were more effective than B16 wt and B16 neo in inducing specific cytotoxity of lymphocytes against B16 wt cells. It is suggested that expression of B7 1 gene in tumor cells could enhance the immunogenicity and induce the effective antitumor immunity.

Induction of anti-hepatoma immunity by recombinant retrovirus expressing B7-1 /B7-2 costimulatory molecules

Objective: To construct recombinant R7-l/B7-2 retrovirus vectors and observe the effects of B7-l/R7-2 gene expression on in ho and in for immune response against against murine hepatoma. Methods: The recombinant retrovirus vectors expressing B7-1/B7-2 were constructed by gene cloning technology to produce retrovirus-infected PE501 and PA317 cell lines and murine hepatoma Hepal-6. The expression of R7-l/B7-2 was detected by fluorescence activated cell soning analysis (FACS). B7-l/B7-2 positive Hepal-6 Cell lines were used in inducing anti-hepatoma immunity in ho and in the. Results: In contrast to the excessive growth of parental Hemal-6 tumor, the growth of B7-l/B7-2-positive Hepal-6 inoculated into syngenic mice regressed. B7-1/R7-2-positive or cytokine-treated Hepal-6 alone could only induce mild cytototicity; in contrast, B7-1/B7-2-positive Hemal-6 treated with cytokine-stimulated spleen cells and activated the cytotoxicity effectively. Immunity in mice with R7-1/B7-2-positive tumor cells or cytokine-beated Hepal-6 only provided partial protection against parental Hepa1-6 tumor, whereas pretreatment of the transfected tumor cells with IFN-r and TNF-a induced complete immunity protection in vivo. Mice receiving inoculation of cytokine-treated B7-l/R7-2-positive Hemal-6 cells presented regression of the establoshed pental tUmor and survived for more than l00 d, while those untreated mice died within 40 d. Conclu sions: B7-l/R7-2 expression is necessary but not sufficient in inducing anti-hepatoma immune response, whereas it is efficient when combined with the beatment of IFN-γ and TNF-a.

Losartan Protects Podocytes against High Glucose-induced Injury by Inhibiting B7-1 Expression

The role of B7-1 in podocyte injury has received increasing attention.The aim of this study was to investigate whether losartan protects podocytes of patients with diabetic kidney disease(DKD)by regulating B7-1 and the underlying mechanisms.Rats with streptozotocin-induced DKD were treated with losartan for 8 weeks.Biochemical changes in blood and urine were analyzed.Kidneys were isolated for electron microscopy,immunofluorescence,real-time quantitative PCR(RT-PCR),and Western blot analysis.Immortalized mouse podocyte cells were cultured in normal or high glucose medium in the presence or absence of losartan for 48 h,and then the cells were collected for immunofluorescence,PCR,Western blotting and monolayer permeability detection.The phosphatidylinositol 3-kinase(PI3K)110a subunit and angiotensin II type 1 receptor(AT1R)plasmids were transfected into podocytes,respectively,and then Western blotting was performed to assess the expression of B7-1 protein.The results showed that losartan ameliorated podocyte structure and function in the rat model of DKD,and reduced the expression of B7-1 protein.Overexpression of PI3K 110a subunit in podocytes attenuated the inhibitory effect of losartan on B7-1 expression in high glucose-stimulated podocytes.The expression of B7-1 was significantly increased by overexpression of ATI R and significantly reduced by blocking PI3K 110a subunit.We conclude that losartan protects podocytes against high glucose-induced injury by inhibiting AT1R-mediated B7-1 expression.This effect is dependent on the AT1R-PI3K 110a subunit pathway.

抗奥合剂通过p38 MAPK/NF-κB信号通路和ACE2/Ang1-7/Mas轴缓解急性肺损伤研究

目的探讨抗奥合剂(KAHJ)治疗小鼠急性肺损伤(ALI)的作用及机制,为其可能作为缓解新型冠状病毒(COVID-19)感染后症状的药物提供依据。方法采用网络药理学方法预测KAHJ治疗ALI的主要活性成分、潜在靶点和相关信号通路。将C57BL/6J小鼠随机分为对照组、LPS组和LPS+KAHJ组。LPS+KAHJ组小鼠灌胃KAHJ(4.76 g·kg^(-1)·d^(-1),8.8 mL·kg^(-1)·d^(-1)),其余组小鼠灌胃生理盐水(8.8 mL·kg^(-1)·d^(-1))。14 d后,腹腔注射LPS(5 mg·kg^(-1))诱导ALI模型。收集小鼠血清和肺组织,通过组织病理学观察肺组织的病理变化。采用Western blot、qPCR、ELISA和IHC等方法评估KAHJ对ALI的改善作用。结果通过网络药理学筛选出疾病和药物共同的70个核心靶基因,并显示与多个信号通路密切相关,如MAPK、NF-κB、Apoptosis、COVID-19和肾素-血管紧张素系统(Ras)信号通路等。此外,通过实验验证发现KAHJ能改善小鼠ALI后的炎症和细胞凋亡,减少肺损伤和肺水肿,抑制肺纤维化。同时,KAHJ的作用机制与p38 MAPK和NF-κB的磷酸化以及ACE2/Ang1-7/Mas轴的调控也有着密切关系。结论KAHJ可能通过抑制p38 MAPK/NF-κB信号通路和调控ACE2/Ang1-7/Mas轴缓解ALI,为缓解COVID-19感染后症状提供了补充和替代药物。

血清Pannexin1、TSP-1、sB7-H3水平与急性脑梗死患者丁苯酞治疗效果的关系

目的探讨血清泛连接蛋白1(Pannexin1)、血小板反应蛋白-1(TSP-1)、可溶性共刺激分子B7-H3(sB7-H3)水平与急性脑梗死(ACI)患者丁苯酞治疗效果的关系。方法选取2021年3月至2022年3月新乡医学院第一附属医院收治的140例急性脑梗死患者作为ACI组,同期行健康体检的健康志愿者100例作为健康组。检测所有研究对象血清Pannexin1、TSP-1、sB7-H3表达水平,分析三者与ACI患者临床特征的关系。ACI患者入院后均使用丁苯酞治疗,分析治疗前后血清Pannexin1、TSP-1、sB7-H3表达差异,并根据治疗效果不同分为治疗有效组(n=91)与治疗无效组(n=49),比较两组血清Pannexin1、TSP-1、sB7-H3水平,明确与患者治疗效果的关系及预测价值。结果ACI组血清Pannexin1、TSP-1、sB7-H3表达水平均高于健康组,差异有统计学意义(P<0.05)。疾病重度、大面积梗死、颈动脉重度狭窄ACI患者血清Pannexin1、TSP-1、sB7-H3处于较高水平(P<0.05)。治疗后,ACI患者使用丁苯酞血清Pannexin1、TSP-1、sB7-H3表达水平均低于治疗前,差异有统计学意义(P<0.05)。治疗无效组患者血清Pannexin1、TSP-1、sB7-H3表达水平高于治疗有效组,差异有统计学意义(P<0.05)。血清Pannexin1、TSP-1、sB7-H3水平与ACI患者丁苯酞治疗效果均表现为正相关关系,差异有统计学意义(r=0.453、0.425、0.466,P<0.05)。Logistic回归分析显示,除受疾病程度、梗死面积、颈动脉狭窄程度的影响外,Pannexin1、TSP-1、sB7-H3同样是影响ACI患者丁苯酞治疗效果的危险因素,差异有统计学意义(P<0.05)。ROC曲线显示,血清Pannexin1、TSP-1、sB7-H3三项联合曲线下面积为0.934显著高于三项单独预测,且敏感度、特异度均较高。结论Pannexin1、TSP-1、sB7-H3在ACI患者血清中表达升高,且与患者丁苯酞治疗效果有关,临床可通过早期监测血清Pannexin1、TSP-1、sB7-H3水平早期预测治疗效果,以改善患者预后。

槐耳颗粒联合S-TACE对中晚期肝癌的近期疗效及对血清TGF-β1与B7-H3的影响

目的:探讨槐耳颗粒联合超选择性肝动脉化疗栓塞术(S-TACE)对中晚期肝癌的近期疗效及对血清转化生长因子-β1(TGF-β1)与血清协同刺激分子B7家族3(B7-H3)的影响。方法:选取唐山市人民医院2018年7月至2019年12月收治的76例中晚期肝癌患者,随机分为观察组和对照组,各38例。对照组患者给予S-TACE进行治疗,观察组患者在对照组的基础上联合槐耳颗粒进行治疗,比较两组患者临床疗效和治疗前后肝功能指标、甲胎蛋白(AFP)、凝血酶原时间(PT)、KPS评分、血清TGF-β1和B7-H3水平及不良反应发生情况。结果:治疗后,两组患者临床症状均得到显著改善,且观察组治疗总有效率92.11%显著优于对照组的73.68%(P<0.05)。治疗后,观察组患者AST、ALT、TBil、AFP、异常凝血酶原(DCP)、碱性磷酸酶(ALP)、谷氨酰转肽酶(GGT)、TGF-β1、B7-H3等指标水平较治疗前显著降低,且低于同期对照组;对照组患者AST、ALT、PT水平较治疗前显著升高(P<0.05)。观察组患者KPS评分高于对照组(P<0.05)。治疗后,观察组患者不良反应总发生率10.53%,与对照组(7.89%)相当(P>0.05)。结论:槐耳颗粒联合S-TACE可有效控制中晚期肝癌靶病灶进展情况,改善肝功能,促进血清TGF-β1、B7-H3水平下降,控制疾病进展,提高患者生存质量,安全性较好。

负性共刺激分子B7-H3和B7-H4在子宫内膜癌发生发展中的作用机制研究

目的:探究负性共刺激分子B7-H3和B7-H4在子宫内膜癌发生发展中的作用机制。方法:选择我院2020年1月—2022年1月收治的60例子宫内膜癌患者开展研究,作为A组。另取同期收治的50例不典型增生子宫内膜患者作为B组。再取40例因子宫内膜病变切取正常子宫内膜组织患者作为C组。分别进行三组组织芯片的制作,并以免疫组织化学法检测B7-H3、B7-H4及白细胞介素-10(IL-10)、转化生长因子-β1(TGF-β1)表达情况。通过Spearman相关性分析明确B7-H3、B7-H4与IL-10、TGF-β1水平的相关性。此外,分析B7-H3、B7-H4表达与子宫内膜癌患者临床病理特征的关系。结果:A组B7-H3、B7-H4与IL-10、TGF-β1阳性率高于B组及C组(均P<0.05)。经Spearman相关性分析发现:B7-H3、B7-H4与IL-10、TGF-β1表达均呈正相关(均P<0.05)。病理分级G2+G3、临床分期Ⅲ~Ⅳ期及淋巴结转移子宫内膜癌患者B7-H3阳性率高于病理分级G1、临床分期Ⅰ~Ⅱ期及无淋巴结转移患者(均P<0.05)。病理分级G2+G3、临床分期Ⅲ~Ⅳ期及淋巴结转移子宫内膜癌患者B7-H4阳性率高于病理分级G1、临床分期Ⅰ~Ⅱ期及无淋巴结转移患者(均P<0.05)。结论:负性共刺激分子B7-H3和B7-H4在子宫内膜癌发生、发展过程中起着促进作用,其作用机制可能和调控IL-10、TGF-β1表达有关。

超声血流参数变化与卵巢癌患者临床分期及血清B7H4、TK-1的关系

目的分析超声血流参数变化与卵巢癌患者临床分期及血清B7同源体4(B7H4)、胸苷激酶-1(TK-1)的关系。方法选取2020年10至2022年10月在武汉市汉阳医院和武汉亚心总医院接受治疗的86例卵巢癌患者作为卵巢癌组,根据国际妇产科联盟(FIGO)卵巢癌分期标准分期:Ⅰ+Ⅱ期40例,Ⅲ+Ⅳ期46例;选取同期同一医院43例卵巢良性肿瘤患者作为良性组。患者均行彩色多普勒超声检查,获得血管搏动指数(PI)、收缩期峰值流速(PSV)、阻力指数(RI)、舒张末期流速(EDV)参数;采用酶免疫点印迹化学发光法检测血清TK-1水平;采用酶联免疫吸附试验法检测血清B7H4水平。采用Pearson相关性分析卵巢癌患者血清B7H4、TK-1水平与PI、RI、PSV、EDV之间的相关性。结果卵巢癌组血清B7H4、TK-1水平高于良性组,超声血流参数PI和RI低于良性组,PSV和EDV高于良性组(P<0.05)。Ⅲ+Ⅳ期卵巢癌患者血清B7H4、TK-1水平高于Ⅰ+Ⅱ期卵巢癌患者(P<0.05),PI和RI低于Ⅰ+Ⅱ期卵巢癌患者,PSV和EDV高于Ⅰ+Ⅱ期卵巢癌患者(P<0.05)。Pearson相关分析显示,卵巢癌患者血清B7H4、TK-1水平与PI、RI均呈负相关(P<0.05),与PSV、EDV均呈正相关(P<0.05)。结论血清B7H4、TK-1水平在卵巢癌患者中高表达,其表达水平与超声血流参数有关,可用于评估患者病情发展程度。

肝癌患者血清中sB7-H3、GP73、QSOX-1的表达水平及临床诊断价值研究

目的 探讨肝癌患者血清可溶性B7-H3(sB7-H3)、高尔基体蛋白73(GP73)、血清清巯基氧化酶-1(QSOX-1)的表达水平及临床诊断价值。方法 选取在2018年2月至2020年2月期间来我院治疗原发性肝癌患者310例(观察组)及同期健康体检人员100例(对照组)作为研究对象。采用双抗体夹心法检测两组受试者血清中sB7-H3、GP73、QSOX-1的表达水平,并进行对比分析。结果 观察组受试者的sB7-H3、GP73、QSOX-1水平显著高于对照组,差异有统计学意义(P<0.05)。血清sB7-H3、GP73、QSOX-1水平在患者的性别、年龄上差异不显著,无统计学意义(P>0.05);Ⅰ~Ⅱ期组患者的血清sB7-H3、GP73、QSOX-1水平显著低于Ⅲ~Ⅳ期组,而GP73水平却显著高于Ⅲ~Ⅳ期组,差异有统计学意义(P<0.05);肿瘤直径≥3 cm组患者血清GP73、QSOX-1水平显著高于肿瘤直径<3 cm组,差异有统计学意义(P<0.05);有远处转移组的肝癌患者血清sB7-H3、GP73、QSOX-1水平显著高于无远处转移组,差异有统计学意义(P<0.05)。采用ROC曲线分析血清sB7-H3、GP73、QSOX-1在两组受试者的表达情况,结果显示sB7-H3、GP73、QSOX-1的曲线下面积(AUC)分别为0.930、0.820及0.817,最佳临界值分别为2 870.58 ng/mL、115.68 ng/mL及68.715 pg/mL,灵敏度分别为74.2%、73.2%、57.1%,特异度分别为93.0%、98.0%、93.0%。若sB7-H3、GP73、QSOX-1联合诊断,即其中之一为阳性即诊断为肝癌,其敏感度为84.5%,特异度为98.9%。结论血清sB7-H3、GP73、QSOX-1在原发性肝癌血清中高表达,表达水平与肿瘤直径、临床分期及是否发生远处转移有关,且sB7-H3、GP73、QSOX-1可作为原发性肝癌早期诊断的良好指标,sB7-H3、GP73、QSOX-1联合检测更能提高原发性肝癌的诊断的准确性。

血清B7-H3、PCT和sICAM-1水平对呼吸机相关性肺炎的评价研究

目的:探究血清B7同系物3蛋白(B7-H3)、降钙素原(PCT)和可溶性细胞间黏附分子-1(SICAM-1)水平对呼吸机相关性肺炎(VAP)的预测价值。方法:选取2019年6月~2020年6月在界首市人民医院接受机械通气患者76例作为研究对象,根据患者是否发生VAP将其分为感染组(n=35)和未感染组(n=41),采用多因素Logistic回归模型分析VAP与B7-H3、PCT、sICAM-1的关系。结果:35例肺部感染患者检测出病原菌85株,革兰阳性菌∶革兰阴性菌株∶真菌株∶其他病原菌为28∶41∶14∶2。与未感染组相比,感染组患者的临床肺部感染评分(CPIS)、B7-H3、PCT以及sICAM-1水平较高(P<0.05)。Logistic多因素回归分析显示,B7-H3、PCT、sICAM-1是机械通气患者发生VAP的独立危险因素(P<0.05)。ROC曲线显示,血清B7-H3、PCT、sICAM-1对诊断VAP的曲线下面积(AUC)分别为0.743、0.854、0.845,对应灵敏度分别为82.43%、65.13%、73.26%,特异度分别为68.59%、94.11%、88.24%。三者联合诊断的AUC为0.933,灵敏度和特异度分别为83.72%和94.13%。结论:血清B7-H3、PCT、sICAM-1对VAP有一定的诊断价值,三者联合诊断效能更好。

Anti-gastric cancer active immunity induced by FasL/B7-1 gene-modified tumor cells

AIM: To study the activation of cytotoxic T lymphocytes (CTLs) against gastric cancer cells induced by FasL/B7-1 (FB-11) gene-modified tumor cells, and to explore whether co-expression of FasL and B7-1 in SGC-7901 tumor cells could initiate synergistic antitumor effect. METHODS: FasL and B7-1 genes were transfected into human SGC-7901 gastric cancer cells with adenovirus vectors. The positive clones were selected by G418. FasL and B7-1 genes were detected by flow cytometry and RT-PCR. Abdominal infiltrating lymphocytes and sensitized spleen cells were obtained from mice that were immunized with SGC-7901/FB-11 or wild type SGC-7901 cells intraperitoneally, and cytotoxicity of these CTLs against tumor cells was determined by MTT assay. RESULTS: Flow cytometry and RT-PCR showed that FasL and B7-1 genes were highly expressed. FasL and B7-1 transfected cancer cells had a high apoptosis index. DNA laddering suggested that FasL and B7-1 genes induced gastric cancer cell apoptosis. FasL+/B7-1+SGC-7901 cells (SGC-7901/FB-11) were inoculated subcutaneously in the dorsal skin of C57BL/6 mice and then decreased their tumorigenicity greatly (z = 2.15-46.10, P＜0.01). SGC- 7901/FB-11 cell-sensitized mice obtained protective immune activity against the rechallenge of wild type SGC 7901 cells (z = 2.06-44.30, P＜0.05). The cytotoxicity of CTLs induced by SGC-7901/FB-11 cells against SGC-7901 was significantly higher than that of CTLs activated by wild-type SGC-7901 cells (84.1±2.4% vs30.5±2.3%,P＜0.05).CONCLUSION: FasL and B7-1 genes can effectively promote the activity of CTLs against gastric cancer cells. FasL/B7-1 molecules play an important role in CTL cytotoxicity.

鼠抗人B7-1分子功能性单克隆抗体的制备及生物学特性

目的 :制备鼠抗人B7 1分子的功能性单克隆抗体 ,研究其对高表达相应配基分子细胞的生物学效应。方法 :用两株转人B7 1基因细胞株XG7 B7和L B7分别作为免疫原及检测细胞株 ,利用B淋巴细胞杂交瘤技术制备单克隆抗体。以快速定性试纸分析法鉴定单抗所属的小鼠IgG亚类 ,采用竞争抑制及间接免疫荧光法分析单抗的特异性和亲和力 ,以高表达B7 1分子的恶性淋巴瘤细胞Raji和Daudi为靶细胞 ,分析单抗对其生长的影响。以人多发性骨髓瘤细胞转入B7 1基因细胞株XG1 B7为刺激细胞 ,以人外周血单个核细胞 (PBLs)为反应细胞 ,用MTT法分析单抗的中和活性。结果 :成功地获得了 1株鼠抗人B7 1的功能性单克隆抗体 (克隆 4E5 ) ,属于小鼠IgG1亚类 ,经流式细胞仪分析 ,4E5与PBLs、体外人工诱导的树突状细胞(DCs)、Raji和Daudi的阳性结合率分别为 10 2 %、95 1%、92 7%及 89 2 % ;能完全阻断标准抗人B7 1单抗与相应抗原的结合。同时还发现 ,单抗 4E5能显著地抑制恶性淋巴瘤细胞Raji和Daudi的生长繁殖 ,并能阻断B7分子介导的协同刺激信号的传导。结论 :单克隆抗体 4E5是一株抗人B7 1分子的功能性单抗 ,具有重要的研究和应用价值。

IL-12协同B7-1增强实验小鼠的抗肿瘤免疫

目的：我们应用逆转录病毒构建了ＩＬ－１２，Ｒ７－１和ＧＭ－ＣＳＦ表达载体，以研究基因修饰的肿瘤细胞的癌疫苗作用。方法：将３种表达载体分别转染ＥＭ胸腺瘤细胞并研究了该基因导入细胞的抗肿瘤免疫效果。结果：当接种了ＥＬ－４／ＩＬ－１２细胞后，在Ｃ５７ＢＬ／６同系鼠中其基因导入细胞的肿瘤原性比较ＥＬ－４和ＥＬ－４／Ｎｅｏ组明显减少（Ｐ＜０．０１）。在ＥＬ－４／ＩＬ－１２被排斥后，体内试验中诱发了实验动物抗 ＥＬ－４／Ｗｔ的系统性、保护性免疫，５１Ｃｒ释放测定中，获得一个较强的抗ＥＬ－４／Ｗｔ和一个较弱的抗同系Ｌｅｗｉｓ肿瘤细胞的ＣＴＬ活性，体内淋巴细胞消除分析的结果提示减少的肿瘤原性主要依赖于ＣＤ４＋，ＣＤ８＋和ＮＫ细胞。用ＥＬ－４／ＩＬ－１２细胞进行疫苗治疗比较用ＥＬ－４／Ｎｅｏ细胞能有效地延缓已建立的ＥＬ－４／Ｗｔ肿瘤的生长（Ｐ＜０．００５），ＥＬ－４／ＩＬ－１２和ＥＬ－４／Ｂ７－１联合比用单一的转基因细胞增强了治疗效果（Ｐ＜０．００５）。结论：提示应用ＩＬ－１２进行血液肿瘤的治疗是有效的，ＩＬ－１２和Ｂ７－１联合使用在未来人类癌症的治疗中亦可有一定的应用前景。

SEA和B7-1基因真核共表达载体的构建及在B16细胞的表达

目的构建葡萄球菌肠毒素A(SEA)和小鼠B71基因真核共表达载体。方法采用PCR和RTPCR方法分别克隆了带B71跨膜区的SEA(SEAB7tm)和小鼠B71基因,中间通过内部核糖体进入位点(Internalribosomeentrysite,IRES)序列的连接克隆至真核表达载体pcDNA3.1+。利用阳离子脂质体将重组质粒转染B16细胞,间接免疫荧光法检测B71和SEA分子在B16细胞膜表面的表达情况。结果测序结果与Genebank中公布的SEA、小鼠B71cDNA序列相符,双标记间接免疫荧光检测结果表明B71、SEA同时在转染的B16细胞膜上表达。结论成功构建了SEA和小鼠B71真核共表达载体,为进一步研究SEA和B71联合应用抗肿瘤免疫治疗及其免疫机理奠定了基础。

miR-129-5p通过HMGB1调控乳腺癌MCF-7细胞对紫杉醇的敏感性

目的:探讨miR-129-5p通过调控高迁移率族蛋白B1基因(high mobility group box 1,HMGB1)影响乳腺癌MCF-7细胞对紫杉醇(paclitaxel,PTX)的敏感性。方法:采用脂质体转染技术将miR-129-5p mimics、HMGB1小干扰RNA(si-HMGB1)分别转染入MCF-7细胞,用PTX刺激培养细胞后,用实时荧光定量PCR检测转染后MCF-7细胞miR-129-5p和HMGB1 m RNA的表达,Western blotting检测转染后MCF-7细胞HMGB1蛋白的表达,CCK-8增殖实验检测转染后PTX对MCF-7细胞增殖的影响,流式细胞术检测转染后对PTX诱导MCF-7细胞凋亡的影响。结果:转染miR-129-5p mimics后,MCF-7细胞中miR-129-5p的表达水平明显高于阴性对照组细胞(P<0.01);过表达miR-129-5p后可明显增强PTX抑制MCF-7细胞的增殖和诱导细胞凋亡的能力(均P<0.05),并显著抑制HMGB1 m RNA和蛋白的表达(均P<0.05)。转染si-HMGB1后,显著降低MCF-7细胞HMGB1 m RNA和蛋白的表达(均P<0.05);干扰HMGB1表达进一步促进PTX抑制MCF-7细胞的增殖并诱导细胞凋亡(均P<0.05)。结论:miR-129-5p通过下调HMGB1的表达增强乳腺癌MCF-7细胞对PTX的敏感性。