An Investigation of the Effects of B7-H4 Gene rs10754339 and miR-125a Gene rs12976445 on Cancer Susceptibility

Objective To investigate the effects of the B7-H4 gene rs10754339 and miR-125a gene rs12976445 on cancer susceptibility through a case-control study and meta-analysis.Methods A total of 1,490 cancer patients(lung/gastric/liver/:550/460/480)and 800 controls were recruited in this case-control study.The meta-analysis was performed by pooling the data from previous related studies and the present study.Results The results of this study showed that in the Hubei Han Chinese population,the rs10754339gene was significantly associated with the risk of lung and gastric cancer but not liver cancer,and the rs12976445 gene was significantly associated with the risk of lung cancer but not liver or gastric cancer.The meta-analysis results indicated that rs10754339 and rs12976445 contributed to cancer susceptibility in the Chinese population and also revealed a significant association between rs10754339and breast cancer risk,as well as between rs12976445 and lung cancer risk.Conclusion The B7-H4 gene rs10754339 and miR-125a gene rs12976445 may be the potential genetic markers for cancer susceptibility in the Chinese population,which should be validated in future studies with larger sample sizes in other ethnic populations.

口腔鳞状细胞癌组织TRIM14和HOXB7蛋白表达与患者临床病理特征及预后的关系

目的 探讨三结构域蛋白14(TRIM14)、同源异型盒基因B7(HOXB7)蛋白在口腔鳞状细胞癌(OSCC)患者癌组织中的表达水平与患者临床病理特征及预后的关系。方法 收集2016年1月至2018年1月行手术治疗的OSCC患者148例的癌组织与癌旁正常组织,免疫组化法分析组织中TRIM14、HOXB7表达情况;Kaplan-Meier生存曲线分析OSCC患者癌组织TRIM14、HOXB7表达水平与生存率之间的关系;COX回归分析OSCC患者预后不良的影响因素。结果 OSCC癌组织TRIM14、HOXB7高表达率均高于癌旁正常组织(P<0.05)。OSCC癌组织TRIM14、HOXB7表达与肿瘤T分期、M分期、淋巴结转移、周围组织浸润、分化程度相关(P<0.05)。OSCC患者5年生存率为49.32%(73/148),Kaplan-Meier生存曲线表明TRIM14、HOXB7高表达患者生存率低于TRIM14、HOXB7低表达患者(χ^(2)=14.480、24.841,P<0.001)。多因素COX分析结果显示,T3-T4分期、M1分期、淋巴结转移、周围组织浸润、癌组织TRIM14、HOXB7高表达均是OSCC患者预后不良的危险因素(P<0.05)。结论 OSCC患者癌组织TRIM14与HOXB7蛋白表达水平均上调,其表达与患者临床病理特征和5年生存预后明显相关。

Therapeutic antitumor response to cervical cancer in mice immunized with U14 vaccines transfected with costimulatory B7 gene

Objective To investigate the effect of U14 vaccine transfected with the B7 gene in inducing antitumor immune response to murine cervical carcinoma in Chinese 615-strain mice.Methods A recombinant retroviral plasmid vector expressing mouse B7-1 gene (pLNSX-mB7) was transfected into 615-strain mouse cervical carcinoma cell line No. 14 (U14) by electroporation to set up a highly-expressed mB7-1 U14 cell clonal strain (B7+U14). In vivo experiments: (1) B7+U14 vaccine was primed to protect the 615-strain mice against U14 re-challenge. (2) B7+U14 vaccine was injected into tumor-bearing mice with different tumor sizes. Lifetimes and tumor sizes were recorded. In vitro cytotoxicity assay: Mice were immunized with B7+U14 or U14 vaccine and 2 weeks later, spleen cells of those mice were cultured for 2 days. The cytotoxicity of these cells against U14 was detected by 5-diphenyl tetrazolium bromide assay.Results We obtained several B7-1 high expression clonal U14 lines. In vivo experiment, we did not find tumor growing in 3 of the 6 mice primed by B7+U14 vaccine during their entire life after re-challenge with U14. The other 3 mice developed tumors and their average survival time was longer than that of the control group (P<0.01). All 6 mice grew tumors in the control group. When the transplanted tumors became palpable, the mice were randomly divided into 3 groups to be injected with B7+U14 vaccine. It was effective for tumor-bearing mice only when the tumor diameters were <3?mm. When the diameters were ≥3?mm, it was not efficacious to inject B7+U14 vaccine (P<0.05). In vitro cytotoxicity assay, cytotoxic T lymphocytes induced by B7+U14 vaccine had a higher cytotoxicity against U14 than that induced by U14 vaccine (F=310.8, P<0.001).Conclusions Vaccines of cervical cancer cells transfected with the costimulatory molecule B7 gene can induce antitumor immune protection in host mice against U14 re-challenge. This treatment may cure part of the tumor-bearing mice but be restricted by tumor size. The results suggest that transfecting the B7 gene into cervical cancer as a cell vaccine may be an efficient supplementary method to treat cervical cancer after operation.

外阴鳞癌组织中DCTN2和HOXB7水平表达及与预后的价值研究

目的 探讨外阴鳞癌组织中动力蛋白激活蛋白2(dynactin2,DCTN2)和同源异型盒基因B7(homeobox gene B7,HOXB7)水平表达及与预后的价值研究。方法 收集2008年1月~2017年12月在江油市人民医院行手术治疗的132例外阴鳞癌患者的癌组织与癌旁健康组织,采用免疫组织化学法检测组织中DCTN2和HOXB7表达情况;采用Kaplan-Meier法分析外阴鳞癌患者癌组织DCTN2和HOXB7表达与五年生存率之间的关系;采用COX回归分析外阴鳞癌患者五年后生存情况的影响因素。结果 外阴鳞癌组织中DCTN2阳性表达低于癌旁健康组织(31.06%vs 71.21%),HOXB7阳性表达高于癌旁健康组织(65.91%vs 37.12%),差异具有统计学意义(χ^(2)=42.583,21.899,均P <0.05)。外阴鳞癌组织DCTN2和HOXB7表达与肿瘤FIGO分期、淋巴结转移、淋巴结转移数量、分化程度和腹股沟淋巴结情况相关(χ^(2)=16.998, 19.144;6.983, 5.800;6.595, 7.244;6.076, 5.665;5.864, 6.493,均P <0.05)。Kaplan-Meier生存曲线表明DCTN2阳性表达外阴鳞癌患者生存率高于阴性表达患者(χ^(2)=14.878,P <0.001),HOXB7阳性表达患者生存率低于阴性表达患者(χ^(2)=14.824,P <0.001)。多因素COX分析结果显示,FIGO分期、淋巴结转移、淋巴结转移数量、分化程度和HOXB7表达均是导致外阴鳞癌患者五年死亡的危险因素(P <0.05),DCTN2表达为保护因素(P <0.05)。结论 外阴鳞癌患者癌组织DCTN2表达下调,HOXB7表达升高,其表达与患者五年生存预后密切相关。

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.

Construction of Eukaryotic Expression Vector Containing B7-1/GFP Gene and Its Expression in Osteosarcoma Cell Line

Objective： To construct eukaryotic expression vector containing B7-1/GFP geneand study its expression in osteosarcoma cell line LM8. Methods： By using gene cloning technique, eukaxyotic expression vector pEGFP-C1 was used to construct the murine B7-1 recombinant plasmid （pEGFP-C1/B7）. Recombinant plasmid was transfected into LM8 cells with liposome and was confirmed by restriction endonuclease digestion and DNA sequencing. The expression of the fusion protein was detected using fluorescence microscope and Western blot analysis. Results： The recombinant eukaryotic expression plasmid pEGFP-C1/B7 was successfully constructed, which was confirmed by DNA sequencing, RT-PGR and restriction enzymes analysis. The green fluorescent protein could be detected in the transfected LM8 with fluorescence microscope. The expected B7-1 and green fluorescent protein （GFP） fusion protein was detected by RT-PCR and Western blot. Conclusion： The eukaryotic expression vector containing B7-1/GFP gene was constructed successfully, and it could be expressed in LM8 after transfection.

Cloning and expression of HLA-B7 gene

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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.

Construction of B7x Gene Overexpression Lentiviral-based Vector

To construct overexpression lentiviral-based vector carrying rat B7x gene,B7x gene precursor sequences amplified by polymerse chain reaction（PCR） were ligated with pLVTHM to generate pLVTHM-B7x gene expression lentiviral-based vector.The positive clones were selected to be submitted to DNA sequencing.HEK293T cells were co-transfected with pLVTHM-B7x and two packaging plasmids psPAX2 and pMD2G to produce lentivirus which express B7x gene.The mRNA expression levels of B7x gene and virus titer were detected by real-time PCR in HEK293T cells.The B7x protein levels were detected by Western blot analysis in HEK293T cells.The identification of restriction enzyme digestion and DNA sequencing confirmed that pLVTHM-B7x lentiviral-based vector was successfully constructed.Green fluorescence was observed in HEK293T packaging cells by means of an inverted fluorescence microscope and the virus titer measured was 2×108 TU/mL.It will establish the foundation for studing deeply the biological function of B7x to construct successfully B7x expression lentiviral-based vector.

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.

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.

B7基因修饰的肿瘤细胞诱导抗大肠癌主动免疫

目的 :研究B7基因修饰的肿瘤细胞诱导抗大肠癌CTL的活化。方法 :采用电击法将B7基因导入小鼠大肠癌细胞CMT93 ,G418筛选阳性克隆 ,免疫组化显示B7分子有高效表达 ,B7 +CMT93(CMT93 -B7)接种于C57BL/6小鼠背部皮下 ,其致瘤性显著下降 (P<0 01) ;CMT93 -B7致敏的小鼠对野生型瘤细胞具有免疫保护作用(P<0 05) ;用CMT93和CMT93 -B7细胞分别经腹腔免疫小鼠 ,得到腹腔浸润淋巴细胞及致敏脾细胞 ,MTT法进行体外杀伤实验。结果 :CMT93 -B7诱导的CTL(cytotoxicTlymphocyte)对CMT93的杀伤活性显著高于野生型CMT93诱导的CTL对相同靶细胞的杀伤活性(P<0 05) ,并且CMT93 -B7诱导的CTL对CMT93 -B7的杀伤率显著高于对野生型CMT93的杀伤率 (P<0 05)。结论 :B7分子有效地促进抗大肠癌CTL的活化 。

人B7-H3基因转染及其生物学功能的研究

B7 H3是新近发现的B7家族新成员。为探讨其体外生物学功能及单克隆抗体的研制构建了B7 H3基因转染细胞。利用PCR的方法扩增出B7 H3基因 ,继而插入逆转录病毒载体pGEZ Term ,重组逆转录病毒载体与两个辅助病毒载体脂质体法共转染包装细胞 2 93T ,经用含有完整病毒颗粒的 2 93T细胞的培养上清感染L92 9细胞 ,Zeocin筛选获得B7 H3的基因转染细胞。RT PCR、Westernblot和流式细胞仪表型分析等方法鉴定表明 ,B7 H3/L92 9基因转染细胞能稳定表达人B7 H3蛋白。继而基因转染细胞对T细胞体外培养试验显示该基因转染细胞与T细胞共培养可抑制其体外增殖。ELISA法分析表明该基因转染细胞抑制活化T细胞IFN γ及IL 10的分泌。CD2 8信号可以逆转B7 H3对活化T细胞的抑制效应。综上结果证实 ,B7 H3对体外活化的T细胞具有负性调控作用。

沉默B7-H3基因表达对人肝癌细胞侵袭能力的影响

目的研究靶向沉默B7-H3基因表达对HepG2细胞侵袭能力的影响及可能机制。方法设计针对B7-H3基因的shRNA沉默质粒,转染HepG2细胞,下调B7-H3的表达。划痕修复实验检测转染前后HepG2细胞移行能力的变化,Transwell实验检测基因沉默对细胞侵袭能力的影响,MST-1法和ELISA凋亡试剂盒分别检测细胞增殖和凋亡水平变化。通过Western blot实验和明胶酶谱实验检测侵袭相关分子MMP-2、MMP-9的表达和活性变化。结果成功构建B7-H3 shRNA沉默质粒并转染HepG2细胞。与对照质粒转染组和未转染组比较,沉默B7-H3基因表达后,B7-H3 shRNA转染组HepG2细胞的移行(24 h:P=0.001;48 h:P<0.001;72 h:P<0.001)和侵袭(P<0.001)能力受到显著抑制,细胞的增殖和凋亡水平没有明显变化(P>0.05)。MMP-2、MMP-9的蛋白表达(MMP-2:P<0.001;MMP-9:P=0.007)和活性(MMP-2:P<0.001;MMP-9:P<0.001)均显著下降。结论通过B7-H3 shRNA沉默质粒靶向沉默B7-H3的基因表达能抑制HepG2细胞的侵袭能力,其机制可能与抑制MMP-2、MMP-9的表达和活性有关。

B7-1与CD40L在淋巴瘤免疫治疗中的协同作用

为了研究B7-1与CD40L共刺激途径在淋巴瘤的免疫治疗中的作用,探讨治疗淋巴瘤疫苗有效的作用方式,将A20淋巴瘤细胞株接种在BALB/c小鼠身上以构建荷瘤小鼠模型,将B7-1与CD40L表达载体单独或联合导入肿瘤内,以PBS和空载体作为对照,观察肿瘤生长的情况;应用肿瘤病理切片和HE染色技术观察肿瘤组织形态及淋巴细胞浸润情况,采用CCK-8试剂盒检测荷瘤小鼠脾CTL杀伤效应。结果显示,瘤内注射B7-1或CD40L表达载体可导致肿瘤生长延缓或体积缩小,两者联合注射较对照组和单独注射组的肿瘤消退效应明显增强。肿瘤形态学观察表明,治疗小鼠肿瘤局部有炎性细胞浸润并伴有大面积的坏死,肿瘤局限化;CCK8试剂盒检测显示小鼠脾CTL杀伤能力增强。结论:B7-1与CD40L疫苗对淋巴瘤具有治疗效应,两者联合治疗的效果优于单一治疗。质粒载体瘤内注射可作为一种安全有效的肿瘤疫苗作用方式。

人B7-1/Sart3融合基因的克隆和表达

目的 构建人共刺激分子CD80 (B7 1)、T细胞识别的鳞状细胞癌抗原 3(Sart3)融合基因真核表达载体 ,并分别在原核和人成纤维细胞中表达。方法 采用RT PCR技术 ,从人胎盘组织抽提的总RNA中克隆Sart3基因的部分cDNA序列 (第 1～ 12 81bp) ,此段序列包括已报道的能诱导HLA限制性细胞毒性T淋巴细胞的抗原表位。然后将不含终止密码子的B7 1cDNA和Sart3cDNA共克隆入真核表达载体 pEGFP N1,测定核苷酸序列确定重组成功后 ,分别转染大肠杆菌和人成纤维细胞 ,利用流式细胞仪和Westernblot检测B7和Sart3的表达。结果 构建人B7、Sart3融合基因真核表达载体B7Sart3/pEGFPN。测序结果与GenBank的B7、Sart3相应序列 (NM_0 0 5 191、NM_0 14 70 6 )一致 ,阅读框无改变。流式细胞仪测定发现在转染的人成纤维细胞表面有明显的荧光增强 ,免疫印迹发现融合蛋白可以与抗B7抗体反应 ,融合蛋白分子量与B7 Sart3 EGFP的预测分子量相当。结论 成功构建真核表达载体B7Sart3/pEGFPN ,并在人成纤维细胞中表达。为下一步研究将SART3蛋白作为肿瘤疫苗应用于骨肉瘤的生物治疗打下基础。

趋化因子MCP-3协同B7诱导小鼠抗宫颈癌的主动免疫效果

目的 :探讨趋化因子MCP 3能否增强B7基因诱导小鼠抗宫颈癌主动免疫的效果。方法 :实验组 :将pCMV/MCP 3以基因药物的方式肌注 615小鼠 ,同时用B7+U14细胞疫苗在同一部位免疫小鼠 ,再将野生型U14移植入已免疫的小鼠。对照组A :用B7+U14细胞疫苗 +生理盐水同时免疫 615系小鼠 ;对照组B :用pCMV/MCP 3 +野生型U14免疫小鼠 ;其它处理同治疗组。观察 3组小鼠的成瘤情况、生存期。用以上 3种方案分别处理小鼠 ,2周后分别取脾T淋巴细胞用MTT法体外检测其对U14的杀伤率。结果 :经方差分析比较 3组在移植U14后不同时程瘤体平均体积有显著性差异 (F =91.86,P <0 .0 0 1)。3组平均生存期分别为 10 0 ,63 ,3 7d ,经Kaplan Meier曲线比较 ,差异具显著性 (P <0 .0 1)。体外检测经MCP 3和B7+U14疫苗处理的小鼠 ,其T淋巴细胞在不同效靶比对U14的杀伤效率均明显高于经MCP 3和U14疫苗处理者 (F =4 0 62 ,P <0 .0 0 1)。结论 :趋化因子MPC 3能增强B7基因修饰的U14细胞疫苗的抗肿瘤效果 。

共刺激分子B7-2对小鼠肝癌治疗作用的实验研究

目的 :应用转染有小鼠B7- 2基因片段的肝癌细胞 ,建立小鼠肝癌模型 ,观察小鼠肿瘤生长和消退情况 ,研究共刺激分子B7- 2对肿瘤的免疫治疗作用。方法 :建立BALB/c小鼠转B7- 2基因肝癌细胞株H2 2 /B7- 2 ,细胞计数法测定肿瘤细胞体外增殖能力 ;BALB/c鼠皮下接种H2 2 /B7- 2及野生型H2 2细胞H2 2 /Wt,以空载体转染细胞H2 2 /neo为对照 ,建立小鼠肝癌模型 ,观察小鼠成瘤期、荷瘤小鼠存活期及肿瘤结节大小 ;同源淋巴细胞肿瘤细胞混合培养 (MTLCs)后测定淋巴细胞增殖指数和CTLs活性 ,同时测定培养上清IL -2、IFNγ ,研究B7- 2分子的抗肿瘤免疫效果。结果 :细胞在体外增殖能力一致 (P =0 .782 ) ;当接种不同肿瘤细胞后 ,三组动物都发生肿瘤 ,接种H2 2 /B7- 2组肿瘤形成有迟发性 ,并在接种 18d后肿瘤都开始缩小 ,但最终不会完全消失 ;接种H2 2 /Wt和H2 2 /neo组动物肿瘤呈进行性生长。H2 2 /B7- 2在体外刺激淋巴细胞增殖和诱导CTLs的能力明显强于对照细胞 (P <0 .0 5 )。结论 :共刺激分子B7- 2能增强肝癌细胞的免疫原性 ,它在抗肿瘤早期发挥作用。用其治疗肝癌是有效的 ,但不能介导完全和长期的抗肿瘤效应。

共表达人p53、GM-CSF和B7-1基因的重组腺病毒的构建

为开展肿瘤的复合基因治疗 ,构建以串联方式携带人野生型p53、GM CSF和B7 1基因的重组腺病毒穿梭质粒pBB 1 0 2 .将pBB 1 0 2与腺病毒包装质粒GT40 50共转染 2 93细胞 ,通过细胞内同源重组获得重组腺病毒BB 1 0 2 .在 2 93细胞中扩增病毒 ,并通过氯化铯密度梯度超速离心纯化病毒 ,获得高滴度和高纯度的病毒 .分别经免疫组织化学分析、ELISA和流式细胞分析 ,检测BB 1 0 2介导的人野生型p53、GM CSF和B7 1基因在喉癌细胞Hep 2中的表达 .结果表明 ,BB 1 0 2能够有效地将其所携带的目的基因导入Hep 2细胞并使其在细胞中高效表达 ,表达高峰期为转染后 2～ 4d ,此后随时间递减 ,可持续 1 0d以上 .

鼠抗人B7-2单克隆抗体的研制及生物学特性研究

以天然高表达膜型B7-2分子的人恶性B淋巴瘤经胞株Daudi和B7-2基因转染细胞株L929-B7-2为免疫原及检测细胞株,采用B细胞杂交瘤技术建立了1株稳定分泌抗人B7-2单抗的杂交瘤,命名为6A5。快速定性试纸法鉴定6A5属小鼠IgG1亚类。经体内诱生腹水法制备单抗,小鼠腹水形成的阳性率为80%,腹水的收获量平均为5.9 ml/只小鼠。经免疫亲和层析法纯化,腹水中抗体蛋白的得率为1.1 mg/ml。采用间接免疫荧光法及流式细胞仪分析,单抗6A5与L929-B7-2、PBTC、Raji和Daudi的阳性结合率分别为99.9%、4.6%、90.6%和99.1%。将6A5(终浓度为5μg/ml)加入到Daudi细胞的培养体系中,经显微镜观察及MTT法分析,6A5对Daudi细胞的生长在24 h内具有抑制作用(P<0.05)。以丝裂霉素处理的L929-B7-2为刺激细胞,PBTC为反应细胞,加入6A5共同培养。经MTT法分析,6A5能阻断B7-2介导的协同刺激信号,抑制PBTC增殖(P<0.05)。提示6A5是一株功能性抗体,在B7-2分子相关的基础和应用研究中具有重要的价值。