[Purpose] To construct TNFR1 recombinant plasmids of TNFR1 and its related mutants,and to study the relationship between the TNFR1 cytoplasm domains and the anti-tumor biological function of sTNF-α.[Methods] The TNFR1-pEGFP-N1,Y236A-TNFR1-pEGFP-N1,ΔNSD-TNFR1-pEGFP-N1 and ΔDD-TNFR-pEGFP-N1 recombinant plasmids were constructed by RT-PCR and overlapping PCR.TNFR1 internalization was observed with fluorescence microscope.The TNFR1 expression rate was detected by flow cytometry and cytotoxicity was detected by MTT colorimetric.[Results] Recombinant plasmids of TNFR1 full length gene and the related mutants were successfully obtained without point mutation,frame-shift or deletion mutation.With these vectors as tools,the results indicated that TNFR1 wildtype can mediate sTNF-α internalization whereas Y236A-TNFR1 can't.It was shown that ΔNSD-TNFR1 and ΔDD-TNFR1 also affected the cytotoxicity of sTNF-α to target cell.[Conclusion] TNFR1 internalization domain,NSD and DD domain are essential for the cytotoxicity of sTNF-α against tumor cells.This study will help us to further understand the relationship between the TNFR1 and sTNF-α mediated tumor cytotoxicity,it will also provide the experimental tools to elucidate the mechanism of tmTNF-α cytotoxicity against tumor cells,so as to provide the novel target for tumor gene therapy.

Catalytic site deleted tmTNF-α mutants,Δ1-12 tmTNF-α,and Δ1-9,K11E tmTNF-α,were constructed by overlapping PCR technology,and then inserted into vector pcDNA3.0. ELISA,MTT colorimetric assay and Western blotting were used to detect the biological function of recombined plasmids. It was found that the mutants with deletion at the sites of 1 to 12 lost the ability to be catalyzed,and their cytotoxicity and NF-κB were also affected. The mutant with deletion of the sites of 1 to 9 and with mutation in the site of 11 from K to E could inhibit the function of TACE,while its cytotoxicity and NF-κB remained the same as wild-type tmTNF-α. In other words,the Δ1-9,K11E tmTNF-α mutant not only possessed the cytotoxic effect of the wild type tmTNF-α,but also could induce the activation of NF-κB to transport signals forwardly and reversely. Compared with the Δ1-12 tmTNF-α,this mutant seems to be a better tool to study the bio-function of tmTNF-α.

To study the relationship between lipid rafts and tmTNF-α,we established overlapping PCR methods for constructing a mutant named cav-tmTNF-αwhich locate only in lipid rafts together with another mutant C-47A-tmTNF-αwhich only out of lipid rafts. And then the mutants were inserted into vector pIRES2-EGFP.It was confirmed that there were no other point mutations,frame-shift or deletion mutations but the expected ones.For analysis of the function,recombined plasmids were detected by confocal microscope,sucrose density gradient centrifugation,and MTT colorimetric.The results showed that part of tmTNF-αwere localized in lipid rafts,and were related to ICAM-1.One the other hand,whatever tmTNF-αwas inside or outside of the lipid rafts,the cytotoxicity to target cell was unaffected. But destruction of the target cell lipid rafts resulted in declined cytotoxicity of tmTNF-α.This study might provide potential information on the bio-function for tmTNF-α.

Objective To investigate the effects of the bi-directional regulation of TM-TNF-α mutant at site-28(Δ-28mTM-TNF-α)on proinflammatory cytokine expression in U937.Methods COS-7 cells were transiently transfected with Δ-28mTM-TNF-α plasmid,and the expressed Δ-28mTM-TNF-α was used to induce U937 cells.NO release assay was performed to determine the influence of the TM-TNF-α mutant on U937.U937 cells were also transiently transfected with Δ-28mTM-TNF-α plasmid.After pre-stimulation with sTNFR1,the IL-8 and IL-1 β mRNA was tested by RT-PCR to evaluated the reverse signal via the TM-TNF-α mutant.Results TM-TNF-α mutant failed to induce U937 produce NO.Reverse signal via TM-TNF-α mutant could be triggered,which up-regulated pro-inflammatory cytokine IL-1 β and IL-8 mRNA transcription.Conclusion Forward signaling is abrogated and reverse signaling is maintained in TM-TNF-a mutant,indicating that transmembrane domain plays critical role for initiating forward signaling.

Our previous study showed that transmembrane TNF-α (TM-TNF-α) had broader tumoricidal spectrum than secretory TNF-α (s-TNF-α). This study examined the difference between the two kinds of TNF-α in inducing cells and the relationship between the apoptosis induced by TM-TNF-α and the cell cycle. Bioassay was employed to compare the cytotoxic effect of two kinds of TNF-α on cell lines L-929 and HepG2. TUNEL was used to detect apoptosis and the TdT and PI co-staining were used for determining the phase of apoptotic cells. Our results showed that TM-TNF-α could kill not only s-TNF-sensitive L929 cells but also s-TNF-tolerant HepG2 cells. TM-TNF-α predominantly induced apoptosis while s-TNF could induce both apoptosis and necrosis. The apoptosis of L-929 cells induced by TM-TNF-α mainly occurred in S phase and the apoptosis of HepG2 predominantly took place in G1 phase. It is concluded that the cytotoxic effects of the two TNF differ substantially. Since TM-TNF-α works locally, mainly induces apoptosis and has broader anti-tumor spectrum, it may be more effective for the treatment of tumor than s-TNF.

[Objective] To establish the osteosarcoma cell line MG63 transfected by two types of TNFα gene (wild TNFα and mutant of transmembrane TNFα ), and to study the biologic effects of TNFα expressed by the transfected cell lines. [Methods] Human osteosarcoma cell line MG63 was transfected separately with two types of retrovirus vector containing wild TNFα gene and mutant of transmembrane TNFα. The transfected cells were further identified for TNFα gene expression and its activity by FCM, ELISA and biological activity assay. [Results] The osteosarcoma cells transfected with TNFα gene could express high levels of TNFα and its mutant, and all of them had biological activities. [Conclusion] The establishment of human osteosarcoma cell MG63 expressing human TNFα gene and its mutant lays the foundation for further studying the biological function of TNFα gene and gene therapy for osteosarcoma.

目的观察跨膜型TNF-α介导的反向信号对NK细胞杀伤功能的影响。方法用sTNFRI激活NK92细胞的TM-TNF-α反向信号;MTT比色法检测NK92细胞对靶细胞的杀伤作用;β-己糖氨酶释放实验检测NK92细胞在杀伤靶细胞时的胞吐作用;ELISA实验检测NK92细胞sTNF-α的分泌;流式细胞术检测NK92细胞FasL的表达。结果预先激活TM-TNF-α反向信号,可以促进NK92细胞对靶细胞的杀伤功能;增强NK92细胞的胞吐;促进FasL的表达和sTNF-α的分泌。结论TM-TNF-α反向信号可能通过促进NK92细胞胞吐、sTNF-α分泌及FasL的表达而促进NK92细胞对靶细胞的杀伤。

Objective:To prepare TM-TNF-α specific monoclonal antibody, identify their specificity and function.Methods:Using antigen anticipation software, the TM-TNF-α segment composing of 20 amino acids residues as antigenic peptide was determined, conjugated with carrier protein and immunized to BALB/c mice. Hybridoma technique was used to prepare monoclonal antibody. ELISA, flow cytometry and Western blot were performed to identify their specificity. The effect of monoclonal antibody on the apoptosis rate of Jurkat cells was observed in the model of AICD.Results:6 monoclonal antibodies against TM-TNF-α were successfully prepared. The results of ELISA revealed that 6 monoclonal antibodies could all specifically interact with the peptide, but not cross-react with S-TNF-α and other cytokines. It was confirmed by using flow cytometry that 6 monoclonal antibodies could all combine to TM-TNF-α on cell surface and could discriminate human TM-TNF-α from mouse TM-TNF-α. Furthermore, these 6 monoclonal antibodies were found by Western blot to prefer interaction with 26 kD TM-TNF-α rather than 17 kD S-TNF-α. In the model of AICD, monoclonal antibodies were shown no effect on the apoptosis rate of Jurkat cells.Conclusion:6 of hTM-TNF-α specific monoclonal antibodies have been successfully prepared and shown no interference with the binding of TNF-α and TNF receptor, which may be a useful tool for further explore the roles of TM-TNF-α in related diseases.

Objective To establish the MCF-7 cell line which express transmembrane (TM)-TNF-α intracellular domain (TNF-ICD) protein stably and highly, and to investigate the reverse signaling of TM-TNF-α. Methods The vector of pIRES2-EGFP, which contained human TNF-ICD cDNA, was transduced into a human breast tumor cell line MCF-7 with Lipofectamine 2000. The positive cell clone expressing TNF-ICD protein was detected with indirect immunofluorescence method and flow cytometry. The cytotoxicity of TNF-α against the MCF-7 cells expressing TNF-ICD was determined by bioassay. NO production and NF-κB activity were detected by ELISA method. Results The MCF-7 cells transfected with the TNF-ICD gene could express high lever of TNF-ICD protein, which was confirmed by immunofluorescence and FACS. Bioassay shown that the MCF-7 cells expressing TNF-ICD was resisted to the cytotoxicity of TNF-α. The NO production and NF-κB activity of the transfected cells increased significantly. PDTC, a NF-κB inhibitor, could restore efficiently the sensibility of TNF-ICD-transfected MCF-7 cells to TNF-α. Conclusion The establishment of tumor cell line MCF-7 expressing TNF-ICD gene provide a good model for further studying reverse signaling of TM-TNF-α. TM-TNF-α intracellular domain (-76-1) may be an active domain which involved in the reverse signaling of TM-TNF-α, stimulating NF-κB, and resisting to the cytotoxicity of TNF-α.

The evolution of hepatocellular carcinoma (HCC) is a compound process which involves many kinds of genes and transductional pathways. The expression of the peptidyl-proplyl-isomerase PIN1 gene, the mutation in exon 3 of β-catenin and its correspondent abnormal expression and their roles in the hepatocellular carcinogeneisis were investigated. Among 29 pair cases of HCC and non-carcinoma tissues, the expression of PIN1 gene was detected by immunochemical staining. Mutations in exon 3 of β-catenin gene and differential expression of β-catenin gene were investigated by the methods of PCR-SSCP, direct sequencing and immunohistochemical technique as well. The results indicated: (1) 44.8% (13/29) cases of HCC presented higher level of PIN1 gene expression than non-cancerous tissues (x 2=32.63, P<0.05), especially in cytoplasm and nucleus, while there was lower level of PIN1 expression in non-cancerous tissues; (2) 58.6% (17/29) HCC tissues showed β-catenin protein accumulation in cytoplasm and nucleus. 46.2% (6/13) HCC tissues indicated β-catenin protein accumulation with higher level of PIN1 expression, while 53.8% (7/13) HCC tissues indicated β-catenin protein accumulation with lower level or trace of PIN1 expression (x 2=0.00, P>0.05); (3) 24.1% (7/29) of primary tumor lesions carried gene mutations in exon 3 of β-catenin, and accompanied by β-catenin protein accumulation. There was no mutation in non-cancerous tissues. All the mutation presented in tissues with low level of PIN1 expression. There was no mutation of β-catenin gene in tissues with high PIN1 expression level (x 2=58.12, P<0.05). So it was postulated that the increase of PIN1 gene expression could promote hepatocellular carcinogenesis via a way different from β-catenin gene mutation.

The killing effects of herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) approach by the addition of several commonly clinical chemotherapeutic agents on hormone refractory prostate cancer (HRPC) cells PC-3m were investigated. After transferring of the HSV-tk gene into PC-3m cells, mRNA and protein expression of HSV-tk was detected by reverse-transcript polymerase chain reaction (RT-PCR) and strept avidin-biotin complex (SABC) immunohistochemical method. The killing effect of GCV, cisplatin (CDDP), etoposide (VP-16), vincristine (VCR), methotrexate (MTX), 5-fluorouracil (5-Fu), and suramin on PC-3m cells was evaluated by morphological assessment analysis, trypan blue exclusion assay and MTT assay respectively. Additionally, the cooperative effect of HSV-tk/GCV system combined with the above agents on the target cancer cells was determined by MTT. Furthermore, apoptosis and necrosis induced by GCV plus 5-Fu or suramin was analyzed by flow cytometry (FCM). The results showed that that there was HSV-tk mRNA and protein expression in pDR2-tk plasmid transduced PC-3m cell. Combination of GCV with VP-16, VCR, 5-Fu or suramin led to an enhanced cellular killing effect, but with CDDP resulted in a reduced one and with MTX in an approximate one. FCM revealed that synergistic use of GCV and 5-fu or suramin resulted in a rather large proportion of apoptosis and necrosis with the apoptosis index being 36.38% and 35.51%, and the proportion of necrosis being 33.05% and 28.87%, respectively. In conclusion, HSV-tk/CGV approach by addition of certain clinical available chemotherapeutic drugs brings on statistically significant enhanced cell killing over single-agent treatment. Our results highlight the potential for such new combination therapies for future treatments of HRPC.

Objective:To obtain TM-TNF-α mimic peptide. Methods:The peptides displayed on phages were prepared by phage amplification and PEG/NaCl precipitation. Their cytotoxcities were measured with MTT method, the mode of cell death was evaluated by apoptosis detection kit. The effect of peptides displayed on phages on SODD mRNA level and the translocation of NF-κB was e valuated by RT-PCR and Western blot. Results:Peptides-presenting phage P18 and P12 could mimic the TM-TNF-α and S-TNF-α biological activities,respectively. Conclusion:TM-TNF-α mimic peptide displayed on phages has been obtained and provided a potential therapeutic agent for cancer patients.

To clone and construct eukaryotic expression plasmid containing mouse 4-IBB extra-membrane encoding region and human IgG Fc fusion gene, express 4-1BB-Fc fusion protein with high biological activity, and study its biological effect in vitro. Mouse full-length 4-1BB cDNA was cloned from total RNA of the mouse spleen with RT-PCR technique, ligated to the pGEM-T Easy vector and identified by sequence scanning. Its extra-membrane encoding region was cloned, digested by restrictive enzyme and inserted into the eukaryotic expression plasmid pcDNA3.1 together with human IgG1 Fc cDNA. The recombinant plasmid, pcDNA3.1-4-1BB-Fc, was transfected into COS-7 and CHO cells by using LipofectAMINE~(TM)2000, and the CHO cell lines stably expressing the fusion protein was obtained through G418 selection. The 4-1BB-Fc protein was purified by protein A affinity chromatography column. The expression of 4-1BB-Fc was identified by sandwich ELISA and Western blot. The binding of 4-1BB-Fc protein to 4-1BBL expressed in DC cell line DC2.4 was determined by flow cytometry (FACS). The suppression effect of 4-1BB-Fc on T lymphocyte proliferation in vitro was tested by MTT and CFSE-labeling method. The ORF and linking sequence of 4-1BB-Fc gene was coincident with what was expected. ELISA and Western blot confirmed protein expression in CHO cells and the purified 4-1BB-Fc protein was proved to suppress T lymphocyte proliferation in vitro. The 4-1BB-Fc eukaryotic expression vector was successfully constructed and a purified recombinant 4-1BB-Fc protein with biological activity was obtained. This lays the experimental foundation for further studies on the role of 4-1BB-Fc in transplant rejection and other biological functions.

Objective To study the effect of soluble murine B and T lymphocyte attenuator (mBTLA) and human IgG1 Fc fusion protein (mBTLA-hIg) on the expression of B7-1 and B7-2 on dendritic cells (DC). Methods An eukaryotic expression vector (pmBTLA-hIg), which expressed soluble mBTLA-hIg fusion protein, was constructed. Then the plasmid was transfected into a mouse dendritic cell line, DC2.4, origining from C57BL/ 6 (B6) mouse, by LipofectAMINETM 2000. The expression of mBTIA mRNA and mBTLA-hIg fusion protein were determined by RT-PCR, ELBA and Western blot. The effect of mBTLA-hIg fusion protein on the expression of costimulatory molecules, B7-1 (CD80) and B7-2 (CD86), on DC was analyzed by flow cytometry. Results The eukaryotic expression vector (pmBTLA-hIg) was constructed and confirmed by restriction endonucleases digestion and DNA sequencing. pmBTLA-hIg transfected DC2.4 could express mBTLA mRNA and release a 43.3×103 fusion protein. The fusion protein could binding to DC, and then up-regulated the expression of B7-1 (CD80), but down-regulated the expression of B7-2 (CD86), on DC2.4. Moreover the change of the expression of B7-1 and B7-2 could block by the addition of anti-GST-mBTLA serum. Conclusion Soluble mBTLA-hIg fusion protein can regulate the co-stimulatory molecules expression, which might be resulted from the reverse signal of BTLA.

Objective To explore the effects of CD40Ig gene transfer in vitro on cardiac allograft survival in mice. Methods The recombinant adenovirus vector carrying murine CD40 extracellular domain and human IgG Fc fusion gene (AdCD40Ig) was constructed. Using BALB/c mice as donors and C57BL/6 mice as recipients, the model of mice abdomen heterotopical heart transplantation was set up. In the experimental group, the isolated donor heart was transfected with AdCD40Ig, then transplanted to recipient. The other groups included empty vector transfected control group, untransfected control group, and syngeneic control group (the donors and the recipients were C57BL/6 mice). The survival time of donor hearts and the infiltration of inflammatory cells in heart allograft were observed. The expression of CD40Ig fusion protein in recipient was identified by Sandwich ELISA; The IFN-γ producing cells in recipient was determined by FACS. Results The survival time of the cardiac allograft in experimental group was ( 15.8± 0.7) days, significantly longer than empty vector transfected control group and untransfected control group (P 0.01). In the second day after transplantation, ELISA confirmed that the expression of CD40Ig fusion protein in recipients was highest, but decreased rapidly after one week; On the 7th day after transplantation, the infiltration of inflammatory cells of allograft in experimental group was less than empty vector transfected control group and untransfected control group. The IFN-γ producing CD4~ + and CD8~ + T cells in experimental group were ( 2.18± 0.16) % and ( 10.82± 0.74) %, respectively, which was close to syngeneic control group, but signi- ficantly lower than empty vector transfected control group and untransfected control group (P 0.01). Conclusion Donor heart transfected with AdCD40Ig in vitro could sufficiently inhibit the proliferation of alloreactive T cells in recipients and prolong the murine cardiac allograft survival.

目的制备特异的能区分人跨膜型肿瘤坏死因子(TM-TNF-α)和分泌型肿瘤坏死因子(sTNF-α)的多克隆抗体.方法借助抗原肽预测软件对TM-TNF-α的抗原表位进行预测,合成TM-TNF-α特异的20个氨基酸的多肽,将之与匙孔碱血蓝蛋白(KLH)耦联,BALB/c小鼠皮背部多点注射,收集分离血清,采用酶联免疫吸附试验(ELISA)和流式细胞术鉴定其特异性.结果成功免疫BALB/c小鼠,ELISA证实该抗血清与免疫多肽有良好的结合,而与sTNF-α、白细胞介素-2(IL-2)和γ-干扰素(IFN-γ)无交叉反应,流式细胞术显示该抗血清可与细胞系U937表达的TM-TNF-α发生特异性结合.结论成功制备了TM-TNF-α特异性多克隆抗体,为制备TM-TNF-α特异性单克隆抗体奠定了基础,为区分TM-TNF-α和sTNF-α的生物学作用提供了有力的工具.

目的建立非何杰金氏淋巴瘤来源的NK92细胞杀伤异种细胞的细胞模型.方法常规培养NK92细胞和猪内皮细胞(PEC),利用MTT法检测NK92细胞对PEC细胞的杀伤效应,通过β-hexosaminidase释放实验显示杀伤过程中,NK92细胞的胞吐量.结果效靶比为10:1时作用时间6h,NK92细胞的胞吐量最大,对PEC的杀伤率为100%.结论NK92细胞杀伤异种细胞的细胞模型建立成功.

目的用sTNFRⅠ预先激活TM-TNF-α介导的反向信号,观测其对sTNF-α激活单核细胞系U937细胞的影响. 方法预先用sTNFRⅠ与U937细胞作用30 min,洗涤后加入sTNF-α作用不同时间,观测预激活反向信号对sTNF-α刺激U937产生活性氧、胞内促炎细胞因子TNF-α、IL-1β、IL-8 mRNA的转录及IκB-α水平(Western blot)的影响. 结果单独用sTNFRⅠ激活的反向信号并不影响U937细胞的功能,而预激活反向信号能协同增强sTNF-α刺激U937细胞产生活性氧,且呈sTNFRⅠ浓度、sTNF-α浓度依赖关系;预激活反向信号协同增强sTNF-α刺激U937炎性细胞因子mRNA(TNF-α、IL-1β、IL-8等)的转录水平;并促进U937细胞IκB-α的降解. 结论预先激活TM-TNF-α介导的反向信号协同增强sTNF-α对U937的激活作用,提示反向信号可能参与机体对早期炎症反应的调节.

目的增强肿瘤坏死因子受体(TNFR)封闭肽的生物学效应.方法通过计算机模建,模拟并推测提高TNFR封闭肽生物学效应需置换的位点和氨基酸;合成原封闭肽和突变肽;受体竞争抑制结合实验比较原肽与突变肽对绿色荧光蛋白-肿瘤坏死因子融合蛋白(GFP-TNF)结合TNFR的竞争抑制效应;细胞毒作用抑制实验比较原肽与突变肽对TNFR的封闭效应.结果受体竞争抑制结合实验结果显示:两种肽均可与GFP-TNF竞争结合U937细胞表面的TNFR.原肽的半数抑制浓度(IC50)为140 μg/ml,而突变肽为100 μg/ml,提示突变肽的竞争抑制效应高于原肽;细胞毒抑制实验显示:突变肽对于TNF-α杀伤U937细胞的细胞毒作用的抑制率为57%,高于原肽的40%(P<0.01).结论突变肽的生物学效应强于原肽.