Effects of dendritic cells transfected with full-length wild-type p53 and stimulated by gastric cancer lysates on immune response

AIM: To investigate the effects of dendritic cells (DCs) transfected with full-length wild-type p53 and stimulated by gastric cancer lysates on immune response. METHODS: The wild-type p53 was transduced to DCs with adenovirus, and the DCs were stimulated by gastric cancer lysates. The surface molecules (B7-1, B7-2, MHC-I, MHC-II) of all DCs were detected by FACS, and the ability of the DCs to induce efficient and specific immunological response in anti-51Cr-labeled target cells was studied. BALB/c mice injected with DCs and Mk28 were established, and CTL response in mice immunized with Lywt-p53DC was evaluated. Tumor-bearing mice were treated with Lywt-p53DC. RESULTS: The surface molecules of Lywt-p53DC had a high expression of B7-1 (86.70 +/- 0.07%), B7-2 (18.77 +/- 0.08%), MHC-I (87.20 +/- 0.05%) and MHC-II (56.70 +/- 0.07%); T lymphocytes had a specific CTL lysis ability induced by Lywt-p53DC; the CTL lysis rate was as high as 81%. The immune protection of Lywtp-53DC was obvious, the tumor diameter in Lywtp-53DC group was 3.10 +/- 0.31 mm, 2.73 +/- 0.23 mm, 3.70 +/- 0.07 mm on d 13, 16 and 19, respectively, which were smaller than control, DC, wtp53DC and LyDC group (P<0.05). Tumor growth rate in Lywtp53DC group was slower than that in other groups (P<0.05).CONCLUSION: DCs transfected with wild-type p53 and stimulated by gastric cancer lysates have specific CTL killing activity.     

CpG ODN对食管癌细胞RNA转染的脐血树突状细胞诱导CTL抗肿瘤功能的影响

目的探讨食管癌细胞RNA转染脐血树突状细胞（DCs）对细胞毒性T淋巴细胞（CTL）特异性抗肿瘤作用的影响,以及CpG寡脱氧核糖核苷酸（CpG ODN）的免疫佐剂作用。方法分离脐血单个核细胞,经SCF、GM-CSF和IL-4诱导和食管癌RNA转染形成成熟DCs,加入CpG ODN,检测DCs表面标志及DCs诱导的T细胞增殖、CTL杀伤活性。结果食管癌RNA转染后DCs高表达抗原提呈分子、协同刺激分子和黏附分子,对T细胞的促增殖作用和CTL杀伤活性显著增强（P〈0.01）,CpG ODN能显著促进DCs功能。结论CpG ODN具有增强食管癌细胞RNA转染的DCs对T细胞的促增殖作用和促CTL杀伤活性的作用。     

Antitumor effects of vaccine consisting of dendritic cells pulsed with tumor RNA from gastric cancer

AIM: To investigate the immunotherapeutic potential of vaccine consisting of dendritic cells (DCs) pulsed with total RNA from MFC gastric cancer cells. METHODS: DCs were prepared from the spleens of strain 615 mice by magnetic cell sorting (MACS). After culture for 24 h, DCs were pulsed with total RNA from MFC gastric cancer cells. Mice of one group were immunized with tumor RNA pulsed DC (RNA/DC) at the dosage of 1X10(6) on d 14 and 7 by s c inoculation before tumor implantation. Mice of another group were immunized with unpulsed DC (UDC) at the same dosage on days as the RNA/DC group. The third group of control mice was untreated. On d 0, all the mice were challenged with s c injections of 5X10(5) MFC gastric cancer cells. After inoculation, the mice were monitored closely with respect to tumor growth. Activities of NK cells in PBL and splenocytes and CTL were tested. RESULTS: On d 21 after tumor cell inoculation, the mice of control group manifested the largest tumors with volume at a mean of 2.6323+/-1.1435 cm(3), followed by the UDC and RNA/DC groups with mean volumes at 0.7536+/-0.3659 cm(3) and 0.3688+/-0.6571 cm(3), respectively. The activities of NK cells in PBL and splenocytes in RNA/DC group were 66.2% and 65.4%, respectively, higher than that in the control group. The tumor specific CTL activity in RNA/DC group was 49.5%, higher than that in the control group. CONCLUSION: The tumor vaccine with DCs pulsed with total RNA from gastric cancer cells possesses the ability to stimulate tumor specific CTL activity and to establish anti-tumor immunity when administered in vivo.     

肝癌细胞裂解物致敏的树突状细胞瘤苗体外诱导特异性抗肝癌免疫

目的 探讨肝癌患者肿瘤细胞裂解物致敏的树突状细胞(DC)瘤苗体外诱导自体T淋巴细胞特异性抗肝癌免疫效应。 方法 从肝癌患者外周血单个核细胞中诱导D C,用重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)和白细胞介素-4(rhIL-4)刺激活化,经自体肝癌细胞裂解物致敏。用流式细胞仪检测D C细胞表面分子的表达,酶联免疫吸附法检测T淋巴细胞培养上清液中干扰素(I F N)γ和白细胞介索-12(IL-12)的含量,液体闪烁计数仪测定肝癌细胞裂解物致敏的D C刺激自体T淋巴细胞增殖效应,四甲基偶氮唑盐法检测肝癌细胞裂解物致敏D C诱导的细胞毒T淋巴细胞对自体肝癌细胞的特异性杀伤作用。 结果 肝癌细胞裂解物致敏的DC瘤苗可上调DC表面CD1 a、CD40、CD86和人类白细胞抗原-DR分子表达水平,其与T淋巴细胞共培养产生的IFN γ、IL-12的浓度明显高于未致敏的D C组(t值分别为2.30、2.14,P<0.05),肝癌细胞裂解物组(t值分别为14.01、15.40,P<0.01)和对照组(t值分别为14.85、16.87,P<0.01)。同时肝癌细胞裂解物致敏的瘤苗可明显诱导T淋巴细胞的增殖,其诱导的细胞毒性T淋巴细胞对自体肝癌细胞的杀伤率(81.72％±9.49％)显著高于对HepG2的杀伤率(49.37％±11.21％)和人鼻咽癌肿瘤细胞的杀伤率(17.14％±5.65％),P<0.01。 结论 肝癌细胞     

Induction of lymphokine-activated cytotoxic T lymphocytes stimulated by dendritic cells and autologous tumor from a patient with gastric cancer and their effects in vitro

BACKGROUND/AIMS: The purpose of the study was to generate lymphokine-activated cytotoxic T lymphocytes stimulated by dendritic cells (DC) and autologous tumor from a patient with gastric cancer and to clarify their cytotoxic effects in vitro. METHODOLOGY: DC was induced by interleukin-4 (IL-4) and granulocyte-macrophage-colony-stimulating factor (GM-CSF) from the peripheral blood mononuclear cells (PBMC). Then, PBMC was incubated with mitomycin C-treated tumor cells and DC, and following that was activated with IL-2 and anti-CD3. Induction of DC and cytotoxic T cells (CTL) were confirmed by the analyses of the cell surface antigens, killing activities, and blocking tests. RESULTS: Induction of DC and cytotoxic T cells (CTL) was confirmed by the analyses of the cell surface antigens, killing activities, and blocking tests. In vitro study demonstrated that lymphokine-activated lymphocytes pulsed by DCs and autologous tumor contained the largest population of CTLs, the greatest production of IFN-gamma, and the greatest ATK activity. CONCLUSIONS: Those results indicated that CTLs could be generated in vitro from a patient with gastric cancer more successfully by this method than by conventional methods, suggesting the possibility of a new immunotherapy for the treatment of gastric cancer.     

An effective vaccine against colon cancer in mice： Use of recombinant adenovirus interleukin-12 transduced dendritic cells

AIM： To investigate the effect of a vaccine with recombinant adenovirus interleukin-12 （AdVIL-12） transduced dendritic cells （DCs） against colon cancer in mice. METHODS： DCs and AdVIL-12 were incubated together at different time intervals and at different doses. Supernatant was collected and tested for IL-12 by enzyme-linked immunosorbent assay （ELISA）. In order to determine whether tumor cell lysate-pulsed （TP） AdVIL-12/DCs enhance therapeutic potential in the established tumor model, CT26 colon tumor cells were implanted subcutaneously （s.c.） in the midflank of naive BALB/c mice. Tumor-bearing mice were injected with a vaccination of CT26 TP AdVIL-12/DCs on d 3 and 10. As a protective colon tumor model, naive BALB/c mice were immunized s.c. in their abdomens with CT26 TP AdVIL-12/DCs twice at seven day intervals. After the immunization on d 7, the mice were challenged with a lethal dose of CT26 tumor cells and survival times were evaluated. Subsequently, cytotoxic T lymphocyte （CTL） activity and interferon gamma （IFNy） secretion was evaluated in the immunized mice, and assayed CTL ex vivo. RESULTS： Murine DCs were retrovirally transduced with AdVIL-12 efficiency, and the AdVIL-12 transduced DCs secreted a high level of IL-12 （AdVIL-12/DCs, 615.27 ± 42.3 pg/mL vs DCs, 46.32 ± 7.29 pg/mL, P 〈 0.05）. Vaccination with CT26 TP AdVIL-12/DCs could enhance anti-tumor immunity against CT26 colon tumor in murine therapeutic models （tumor volume on d 19：CT26 TP AdVIL-12/DCs 107 ± 42 mm^3 vs CT26 TP DCs 383± 65 mm^3, P 〈 0.05） and protective models. Moreover, the CT26 TP AdVIL-12/DC vaccination enhances tumor-specific CTL activity, producing high levels of IFN7 in immunized mice. Ex vivo primed T cells with AdVIL-12/DCs were able to induce more effective CTL activity than in primed T cells with CT26 TP/DCs （E：T = 100：1, 69.49% ± 6.11% specific lysis vs 37.44% ＋ 4.32% specific lysis, P 〈 0.05）.CONCLUSION： Vaccination with recombinant AdVIL-12 transduced DC p     

Construction of pETNF-P16 plasmid and its expression properties in EC9706 cell line induced by X-ray irradiation

AIM: Recombined plasmid pETNF-P16 was constructed to investigate its expression properties in esophageal squamous carcinoma cell line EC9706 induced by X-ray irradiation and the feasibility of gene-radiotherapy for esophageal carcinoma. METHODS: Recombined plasmid pETNF-P16 was constructed and transfected into EC9706 cells with lipofectamine. ELISA,Western blot, and immunocytochemistry were performedto determine the expression properties of pETNF-P16 in EC9706 after transfection induced by X-ray irradiation. RESULTS: Eukaryotic expression vector pETNF-P16 was successfully constructed and transfected into EC9706 cells. TNFα expressions were significantly increased in the transfected cells after different doses of X-ray irradiation than in those after 0Gy irradiation (1 192.330-2 026.518 pg/mL,P&lt;0.05-0.01), and the TNFα expressions and P16 were significantly higher 6-48 h after 2 Gy X-ray irradiation (358.963-585.571 pg/mL, P&lt;0.05-0.001). No P16 expression was detected in normal EC9706 cells. However, there was strong expression in the transfected and irradiation groups. CONCLUSION: X-ray irradiation induction could significantly enhance TNFα and P16 expression in EC9706 cells transfected with pETNF-P16 plasmid. These results may provide important experimental data and therapeutic potential for gene-radiotherapy of esophageal carcinoma.     

Expression of heparanase mRNA in anti-sense oligonucleotide-transfected human esophageal cancer EC9706 cells

AIM: To investigate the effects of anti-sense oligonudeotides (ASODNs) on mRNA expression of heparanase in human esophageal cancer EC9706 cells. METHODS: One non-sense oligonucleotide (N-ODN) and five ASODNs against different heparanase mRNA sites were transfected into EC9706 cells, then the expression of heparanase mRNA in EC9706 cells was studied by in situ hybridization. RESULTS: The expression of heparanase mRNA could be inhibited by ASODNs.There was no significant difference among five ASODNs (P>0.05), but there was a significant difference between ASODNs and N-ODN or non-transfected group (ASODN1: 2.25±0.25, ASODN2: 2.21±0.23, ASODN3: 2.23±0.23, ASODN4: 2.25±0.24 vs N-ODN: 3.47±2.80 or non- transfected group: 3.51±2.93 respectively,P<0.05). CONCLUSION: The expression of heparanase mRNA in EC9706 cells can be inhibited by ASODNs in vivo, and heparanase ASODNs can inhibit metastasis of esophageal squamous cell carcinoma or other tumors by inhibiting the expression of heparanase.     

GM-CSF分泌型肝癌疫苗对荷瘤鼠脾血细胞毒性T淋巴细胞杀伤活性的影响

目的观察粒细胞巨噬细胞集落刺激因子(GM-CSF)分泌型肝癌疫苗对移植性肝癌小鼠细胞毒性T淋巴细胞(CTL)杀伤活性的影响。方法取小鼠肝癌细胞株H22细胞1×106/只注入小鼠腹腔内,接种7d形成腹水瘤后再在小鼠体内传3代。取生长旺盛且无血性的腹水,在无菌条件下制成2×107/ml的细胞悬液,以2×106细胞/0.1ml/只接种于小鼠右前肢皮下。将肝癌细胞移植瘤动物分成3组。4天后,在右侧背部皮下进行免疫治疗,即制备GM-CSF分泌型H22肝癌瘤苗并免疫ICR小鼠(H22-GM-CSF组,n=5),同时设立无GM-CSF基因修饰H22肝癌瘤苗组(H22组,n=5)和PBs对照组(PBS组,n=5),测量各组小鼠肿瘤体积;采用细胞增殖计数法检测小鼠脾血CTL杀伤活性。结果随着效/靶比增加,各组CTL杀伤活性均增强。在效/靶比为50∶1时,GM-CSF-H22组CTL杀伤活性为60±6.1%,明显高于H22组(17.4±0.9%)和PBS组(12.2±0.6%,P<0.01);GM-CSF分泌型肝癌细胞瘤苗明显抑制H22荷瘤小鼠肿瘤生长。在21天时,H22-GM-CSF组、H22组和PBS组小鼠肿瘤体积分别为0.63±0.05mm3、1.47±0.75mm3和1.79±0.34mm(3P<0.01)。结论 GM-CSF分泌型肝癌细胞瘤苗可抑制肿瘤细胞生长,增强CTL杀伤活性。