乳腺癌小鼠动物模型中白细胞介素-17的表达及其意义

Objective To explore the impact of interleukin (IL) -17 expression on tumor growth in experimental models of murine mammary carcinoma and potential mechanisms. Methods Two murine cell lines, MA782/5S28102 and 4T1 were used to establish experimental models of murine mammary carcinoma. The IL-17 expression in tumor tissues derived from MA782-bearing mice or 4T1-bearing mice was detected in early and late stages of the tumor by Western blotting. The tumor cells and tumor-infiltrated-lymphocytes were separated from tumor tissues and cultured for 5 days with stimulation of PMA, anti-CD3 antibody and anti-CD28 antibody. The supernatants of culture media of stimulated tumor cells or tumor-infiltrated-lymphocytes were harvested and tested for IL-17 concentration by enzyme linked immunosorbent assay (ELISA). To evaluate the effect of IL-17 on the proliferation of tumor cells, 4T1 cells were culture in media with or without IL-17 and the cell number was counted on the day 5. For ire vivo assay, 4T1-bearing mice were injected with IL-17 or culture media via tail vein, and the tumor volume was measured. To assay the angiogenesis, the tumor tissues from 4T1-bearing mice with or without injection of IL-17 were stained with anti-CD31 antibody by immunohistochemistry. Results The IL-17 expression was significantly higher in late stage than in early stage of tumor in two experimental models. The tumor expression of IL-17 was secreted by tumor infiltrated lymphocytes (P ＜0.01). IL-17 could not increase the generation of tumor cells in vitro (1. 11 ±0. 11, 1. 28 ±0. 21 ,P ＞0. 05). But IL-17, injected into 4T1 -bearing mice, markedly enhanced in vivo tumor growth and significantly increased tumor vascularity (35. 79 ±9. 49, 13. 52 ±3. 55,P ＜0.01). Conclusion IL-17 in tumor tissue probably promotes tumor growth through enhancing angiogenesis.     

乳腺癌小鼠动物模型中白细胞介素-17的表达及其意义

Objective To explore the impact of interleukin (IL) -17 expression on tumor growth in experimental models of murine mammary carcinoma and potential mechanisms. Methods Two murine cell lines, MA782/5S28102 and 4T1 were used to establish experimental models of murine mammary carcinoma. The IL-17 expression in tumor tissues derived from MA782-bearing mice or 4T1-bearing mice was detected in early and late stages of the tumor by Western blotting. The tumor cells and tumor-infiltrated-lymphocytes were separated from tumor tissues and cultured for 5 days with stimulation of PMA, anti-CD3 antibody and anti-CD28 antibody. The supernatants of culture media of stimulated tumor cells or tumor-infiltrated-lymphocytes were harvested and tested for IL-17 concentration by enzyme linked immunosorbent assay (ELISA). To evaluate the effect of IL-17 on the proliferation of tumor cells, 4T1 cells were culture in media with or without IL-17 and the cell number was counted on the day 5. For ire vivo assay, 4T1-bearing mice were injected with IL-17 or culture media via tail vein, and the tumor volume was measured. To assay the angiogenesis, the tumor tissues from 4T1-bearing mice with or without injection of IL-17 were stained with anti-CD31 antibody by immunohistochemistry. Results The IL-17 expression was significantly higher in late stage than in early stage of tumor in two experimental models. The tumor expression of IL-17 was secreted by tumor infiltrated lymphocytes (P ＜0.01). IL-17 could not increase the generation of tumor cells in vitro (1. 11 ±0. 11, 1. 28 ±0. 21 ,P ＞0. 05). But IL-17, injected into 4T1 -bearing mice, markedly enhanced in vivo tumor growth and significantly increased tumor vascularity (35. 79 ±9. 49, 13. 52 ±3. 55,P ＜0.01). Conclusion IL-17 in tumor tissue probably promotes tumor growth through enhancing angiogenesis.     

Relations of transcription expression of IL—2 with nuclear factor of activated T cells as well as changes of C—Fos and C—Jun after trauma

Objective:To observe the relations among expression of interleukin-2(IL-2)in spleen lymphocytes,DNA binding activity of nuclear factor of activated T cells(NFAT)and expression of the partly family members C-Fos,C-Jun after trauma.Methods:A murine closed trauma model was used,animals were sacrificed6,12hours and 1,4,7,10,14days,respectively after injury,Spleen lymplocytes were isolated from injured mice and stimulated with concanavalin-A,The culture supernatants were harvested and assayed for IL-2activity,Total RNA was extracted from spleen lymphocytes and assayed for IL-2mRNA.Nuclear protein was extracted,and the DNA binding activity of NFAT was measured using an electrophoretic mobility shift assay(EMSA),the expressions of C-Fos,C-Jun protein determined by Western blot analysis.Results:The expressions of IL-2 activity and IL-2mRNA in spleen lymphocytes were decreased in injured mice compared with those in control mice,and the most obvious decrease appeared on the 4th day after injury,The DNA binding activity of NFAT decreased gradually and reached the minimum that was only41%of the control on the 4th day after injury,which was cloely associated with the decline of IL-2activity and IL-2mRNA.An decrease in the expression ofC-Fos on the lst and 4th day after injury,trauma had no significant effect on the C-Jun expression.Conclusions:These results suggest that the inhibition of IL-2 expression is partly due to the impairment in the activation of NFAT in injured mice;and the decline in the DNA binding activity of NFAT is partly due to trauma block in the C-Fos expression.     

携带γ-干扰素的溶瘤病毒CNHK300-mIFN-γ治疗肝癌

目的 观察鼠源γ-干扰素(mIFN-γ)基因插入溶瘤腺病毒CNHK300后,该病毒对不同的肝癌细胞株及其裸鼠移植瘤的增殖力及特异性杀伤作用.方法 用噻唑蓝(MTT)法观察CNHK300-mIFN-γ(简称CNHK300-Mγ)对人正常肝细胞WRL-68、人肝癌细胞SMMC-7721、HepG Ⅱ的特异性杀伤作用.裸鼠肝癌SMMC-7721模型观察CNHK300-Mγ、CNHK300和ONYX-015的抗肿瘤疗效,并用酶联免疫吸附试验(ELISA)检测血清中mIFN-γ的表达量.结果 CNHK300-Mγ能选择性地杀伤肝癌细胞,在肿瘤细胞中半数抑制浓度(IC50)＜1,而在正常肝细胞中为370.肝癌模型中,CNHK300-Mγ治疗组能表达mIFN-γ,治疗结束后第3天和第7天血清中的mIFN-γ分别为(231±101)ng/L和(1733±191)ng/L.CNHK300-Mγ具有明显的肿瘤生长抑制作用,与CNHK300、ONYX2015和对照组的差异均有统计学意义(P＜0.05).结论 mIFN-γ的插入使病毒对肿瘤细胞的杀伤能力提高,CNHK300-Mγ具有明显的肿瘤生长抑制作用.

Abstract：

Objective To study the effect of mouse interferon-γ (mIFN-γ) insertion on the selective replication and cytotoxicity of a tumor-specific oncolytic adenovirus CNHK300 in hepatocellular carcinoma (HCC) cells and HCC xenografts. Methods The cytotoxicity of CNHK300-mIFN-γ (CNHK300-Mγ) was examined by methyl thiazol tetrazolium (MTT) colorimetric assay in normal and HCC cells.Healthy nude BALB/C mice were injected with the SMMC-7221 cells. Forty mice with tumors of 5-8 mm in diameter were randomly divided into 4 groups of 10 mice: CNHK300-Mγ group (CNHK300-Mγ was injected into the tumor once every other day for 5 times) , CNHK300 group ( CNHK300 was injected) , ONYX-015 group (ONYX-015 was injected) , and control group (diluent of virus was injected). Seven, 14, 21, 28, 35, and 42 days after the initial injection the size of tumor was examined ( Tumor volume was estimated as a × b2 × 0. 5 , where a and b were the maximal and minimal diameters, respectively). Forty-eighth after the finish of the whole course of treatment, the mice were killed. Twenty-four mice with the same tumors were randomly divided into 3 groups of 8 mice: CNHK300-Mr group, CHNK300 group and control group.The mice were killed in half, 3, 7 days after the last injection. Enzyme linked immunosorbent assay (ELISA) was used to detect the expression of mIFN-γ in blood. Results CNHK300-Mγ could selectively kill tumor cell lines, whose 50% inhibitory concentration (IC50) was under 1 in tumor cells and 370 in normal liver cells. ELISA showed that the expression of mIFN-γ in the blood of the CNHK300-Mγ group was (231 ± 101) ng/L or (1733 ± 191) ng/L in 3 days or 7 days after virus infection, while no mIFN-γ could be detected in CHNK300 group and control group. The tumor size in the CNHK300-Mr group was significantly smaller than in the CNHK300 group, ONYX-015 group, and control group (P＜0. 05). Conclusion Being capable of specifically killed HCC cells and mediating effective expression of therapeutic gene in vivo, CNHK300-Mγ holds a splendid future as a potential antitumor agent.     

组织特异性胞嘧啶脱氨酶和5-氟胞嘧啶热化疗对裸鼠结肠癌肝脏转移的影响

Objective To investigate the effects of tissue specific cytosine deaminase/5-fluorocytosine (CD/5-FC) thermotherapy on hepatic metastasis of colonic carcinoma in nude mice. Methods Forty-five nude mice were randomly divided into control group, 5-FC group and 5-FC thermotherapy group according to the random number table (15 mice in each group). Mice models of hepatic metastasis of colonic carcinoma were established by portal vein injection of LoVo/CEACD cells. The hepatic metastasis rate and number of metastatic nodules of the 3 groups were compared by ehi-square test and one-way ANOVA. The pathological changes in tumor tissues and apoptotic index of tumor cells were observed. The expression of the CD gene in tumor tissues was detected by fluorescent quantitative RT-PCR and Western blot. Results The number of metastatic nodules and liver metas-tasis rate were 4.6±1.3 and 100.0% in control group, 2.2±1.0 and 60.0% in 5-FC group, 0.5±0.8 and 13.3% in 5-FC thermotherapy group, with statistical difference among the 3 groups (F=25.898, χ2=5.208, 19.548, 5.168, P<0.05). The mean apoptotic indexes of tumor cells of the 3 groups were 4.6%, 9.9% and 17.4%, respectively. Vacuolar degeneration, cell necrosis, cytolysis and apoptotic bodies were mostly observed in the 5-FC thermotherapy group. The expression of CD gene in tumor tissue was detected in all the groups. Conclusion Tissue specific CD/5-FC thermotherapy has inhibitory effects on the hepatic metastasis of LoVo cells transfected with CD gene.     

组织特异性胞嘧啶脱氨酶和5-氟胞嘧啶热化疗对裸鼠结肠癌肝脏转移的影响

Objective To investigate the effects of tissue specific cytosine deaminase/5-fluorocytosine (CD/5-FC) thermotherapy on hepatic metastasis of colonic carcinoma in nude mice. Methods Forty-five nude mice were randomly divided into control group, 5-FC group and 5-FC thermotherapy group according to the random number table (15 mice in each group). Mice models of hepatic metastasis of colonic carcinoma were established by portal vein injection of LoVo/CEACD cells. The hepatic metastasis rate and number of metastatic nodules of the 3 groups were compared by ehi-square test and one-way ANOVA. The pathological changes in tumor tissues and apoptotic index of tumor cells were observed. The expression of the CD gene in tumor tissues was detected by fluorescent quantitative RT-PCR and Western blot. Results The number of metastatic nodules and liver metas-tasis rate were 4.6±1.3 and 100.0% in control group, 2.2±1.0 and 60.0% in 5-FC group, 0.5±0.8 and 13.3% in 5-FC thermotherapy group, with statistical difference among the 3 groups (F=25.898, χ2=5.208, 19.548, 5.168, P<0.05). The mean apoptotic indexes of tumor cells of the 3 groups were 4.6%, 9.9% and 17.4%, respectively. Vacuolar degeneration, cell necrosis, cytolysis and apoptotic bodies were mostly observed in the 5-FC thermotherapy group. The expression of CD gene in tumor tissue was detected in all the groups. Conclusion Tissue specific CD/5-FC thermotherapy has inhibitory effects on the hepatic metastasis of LoVo cells transfected with CD gene.     

他克莫司对小鼠T_H17型细胞分化增殖的影响及其机制

目的 观察在T_H17型细胞极化环境下,他克莫司(Tac)对小鼠T_H17型细胞分化增殖的影响及机制.方法 将小鼠脾脏初始CD4+CD25+T淋巴细胞使用抗CD3抗体及抗CD28抗体进行活化,使用转化生长因子β_1,(TGF-β_1,)和白细胞介素6(IL-6)等细胞因子进行诱导,促使其向T_H17型细胞方向分化.将体外培养的T_H 17型细胞分为4组,分别用不同剂量的Tac进行干预,即:Tac0 ng/ml组(对照组)、Tac 0.1 ng/ml组、Tac 1 ng/ml组和Tae 10 ng/ml组.应用流式细胞术检测各组T_H 17型细胞亚群纯度,使用逆转录聚合酶链反应(RT.PCR)检测各组IL-17 mRNA相对表达量.结果 Tac可抑制小鼠T_H17型细胞亚群的分化增殖,降低IL-17 mRNA的表达量,且呈剂量依赖性,各组间比较,差异均有统计学意义(P<0.05).结论 Tac通过抑制T_H17型细胞胞浆内的钙调磷酸酶,抑制T_H17型细胞亚群的分化增殖,抑制IL-17 mRNA产生并减轻炎症进程,从而抑制排斥反应.     

Synergistic reversal of type 1 diabetes in NOD mice with anti-CD3 and interleukin-1 blockade: evidence of improved immune regulation

Inflammatory cytokines are involved in autoimmune diabetes: among the most prominent is interleukin (IL)-1β. We postulated that blockade of IL-1β would modulate the effects of anti-CD3 monoclonal antibody (mAb) in treating diabetes in NOD mice. To test this, we treated hyperglycemic NOD mice with F(ab')(2) fragments of anti-CD3 mAb with or without IL-1 receptor antagonist (IL-1RA), or anti-IL-1β mAb. We studied the reversal of diabetes and effects of treatment on the immune system. Mice that received a combination of anti-CD3 mAb with IL-1RA showed a more rapid rate of remission of diabetes than mice treated with anti-CD3 mAb or IL-1RA alone. Combination-treated mice had increased IL-5, IL-4, and interferon (IFN)-γ levels in circulation. There were reduced pathogenic NOD-relevant V7 peptide-V7(+) T cells in the pancreatic lymph nodes. Their splenocytes secreted more IL-10, had increased arginase expression in macrophages and dendritic cells, and had delayed adoptive transfer of diabetes. After 1 month, there were increased concentrations of IgG1 isotype antibodies and reduced intrapancreatic expression of IFN-γ, IL-6, and IL-17 despite normal splenocyte cytokine secretion. These studies indicate that the combination of anti-CD3 mAb with IL-1RA is synergistic in reversal of diabetes through a combination of mechanisms. The combination causes persistent remission from islet inflammation.     

血必净促进内毒素/脂多糖刺激调节性T淋巴细胞凋亡并介导辅助性T淋巴细胞漂移的作用

目的 了解血必净注射液促进LPS刺激CD4+CD25+调节性T淋巴细胞(Treg细胞)凋亡过程及介导辅助性T淋巴细胞(Th)漂移的调节作用.方法 免疫磁珠法分选获得大鼠脾脏CD4+CD25+Treg细胞,分为常规培养对照组、抗CD3/CD28组、抗CD3/CD28+LPS组、抗CD3/CD28+血必净组和抗CD3/CD28+LPS+血必净组,培养3 d后应用流式细胞术检测Treg细胞凋亡率及叉头翼状螺旋转录因子3(Foxp3)表达.将CD4+CD25+Treg细胞与CD4+CD25+T淋巴细胞1:1培养,伴刀豆球蛋白A刺激68 h,检测上清液中Th1分泌的γ干扰素(IFN-γ)、Th2分泌的IL-4、Th17分泌的IL-17水平.结果 抗CD3/CD28+LPS+血必净组Treg细胞凋亡率为(45.1±2.7)%,明显高于抗CD3/CD28+LPS组[(29.4±1.6)%,P＜0.01];2组Foxp3平均荧光强度分别为95±9、140±18,差异有统计学意义(P＜0.01).同时,抗CD3/CD28+LPS+血必净组IFN-γ分泌水平显著高于抗CD3/CD28+LPS组(P＜0.01),IL-4则呈相反变化(P＜0.05),抗CD3/CD28+LPS+血必净组IFN-γ/IL-4较对照组升高(P＜0.01);抗CD3/CD28+血必净组IL-17分泌水平较抗CD3/CD28组明显下降(P＜0.05).结论 CD4+CD25+Treg细胞活化介导了Th1向Th2功能性极化;血必净对LPS诱导的T淋巴细胞免疫功能有重要调节作用,可促进CD4+CD25+Treg细胞凋亡并介导Th2向Th1漂移,从而缓解细胞免疫抑制状态.     

NOD小鼠未成熟树突状细胞体外诱导淋巴细胞生成并扩增调节性T细胞的研究

目的 探索非肥胖糖尿病(non-obese diabetic,NOD)小鼠未成熟树突状细胞(immature dendritic cell,imDC)诱导并扩增调节性T细胞(regulatory T cells,Treg)的优化方案.方法 分离、培养NOD小鼠骨髓来源的imDC和淋巴细胞,并进行混合淋巴细胞培养,分别加...     

西罗莫司和环孢素A对Th17细胞和调节性T淋巴细胞分化影响的比较

目的 比较西罗莫司(SRL)和环孢素A(CsA)对体外诱导CD4+T淋巴细胞向Th17和调节性T淋巴细胞分化的影响.方法 使用抗CD3和抗CD28单克隆抗体(简称抗CD3单抗和抗CD28单抗)刺激CD4+T淋巴细胞,并在培养体系中分别加入转化生长因子β(TGF-β);TGF-β+白细胞介素6(IL-6);TGF-β+IL-6+SRL;TGF-β+IL-6+CsA.72 h后用流式细胞术检测细胞内Foxp3和IL-17的表达水平,观察CD4+T淋巴细胞的分化情况及SRL和CsA对CD4+T淋巴细胞体外分化的影响.结果 在经抗CD3单抗和抗CD28单抗刺激后,TGF-β可诱导Foxp3+细胞(调节性T淋巴细胞,Treg)的增殖与分化,而TGF-β+ID6可诱导Th17细胞的增殖与分化.在培养体系中加人SRL和CsA,均可使Th17细胞的增殖与分化减少;SRL可促进Treg的增殖与分化,而CsA抑制Treg的增殖与分化.结论 SRL可以促进CD4+T淋巴细胞向Treg增殖与分化,而抑制Th17细胞的增殖与分化;CsA既抑制Treg的增殖与分化,又抑制Th17细胞的增殖与分化.     

Anti-CD3-enhanced interleukin-2 immunotherapy of pulmonary metastases.

A newly developed monoclonal rat IgG 2b antibody which in vitro can activate both helper and cytolytic T-lymphocytes by binding to the CD3 epsilon subunit of the T-cell receptor complex was tested alone and in combination with Interleukin-2, a growth factor for activated T-cells, for ability to reduce established pulmonary metastases in a murine model. C57BL/6 mice injected iv with a tumor cell suspension of a weakly immunogenic fibrosarcoma, MCA106, were randomly assigned to 1 of 15 treatment groups for intraperitoneal injections with YCD3 (0, 0.1, 1, 10, or 100 micrograms) on Days 3, 5, 7, 10, 12, 17, and 19 or with IL-2 (0, 1000, or 50,000 units bid) on Days 3 through 7, 10 through 12, and 17 through 19. On Day 21 all mice were sacrificed for enumeration of metastases. Pooled splenocytes of three randomly selected mice from each group were assayed for surface expressions of T-cell markers Thy-1, Ly2, and L3T4. Results: High-dose IL-2 (50,000 units bid) in combination with low-dose YCD3 (1 microgram) reduced metastases 60% (P less than 0.005). YCD3 or IL-2 alone was ineffective. Combined high-dose IL-2 (50,000 units) and high-dose YCD3 (100 micrograms) resulted in 100% mortality. Phenotypically, YCD3 induced a dose-dependent depletion of T-cells from 25 to 2.4% (0.1 to 100 micrograms, respectively). These results suggest potential clinical applicability of low-dose anti-CD3 monoclonal antibody to enhance antitumor efficacy of high-dose IL-2. However, the toxicity of high-dose anti-CD3 and high-dose IL-2 cautions for care in selection of dose.     

瘤体内直接注射白细胞介素-7基因抗小鼠乳腺肿瘤免疫效应的研究

目的 观察瘤体内直接注射白细胞介素(IL)-7基因抗小鼠乳腺肿瘤免疫效应.方法 构建IL-7真核表达质粒(pcDNA3-IL-7);建立乳腺癌TM40D细胞BALB/C小鼠移植模型;瘤体内直接注射pcDNA3-IL-7,观察小鼠肿瘤体积变化;酶联免疫吸附试验(ELISA)法检测外周血干扰素(IFN)-γ含量;流式细胞仪检测胞内IFN-γ的分泌量;局部肿瘤经治疗后行常规病理分析.结果 成功构建pcDNA3-IL-7;与对照磷酸盐缓冲液(PBS)组(115.2±11.8) ng/L、pcDNA3组(133.6±9.4) ng/L比较,pcDNA3 -IL-7注射组(242.3±10.1)ng/L外周血IFN-γ明显增高;pcDNA3-IL-7明显抑制肿瘤生长(P＜0.05);流式细胞仪检测显示pcDNA3-IL-7显著促进CD4+T细胞、CD8+T细胞内IFN-γ的分泌量;常规病理显示pcDNA3 -IL-7注射组肿瘤组织大量坏死,炎性细胞和大量淋巴细胞浸润.结论 瘤体内直接注射pcDNA3-IL-7明显抑制小鼠乳腺肿瘤生长,显著促进IFN-γ分泌,增强了小鼠机体抗乳腺肿瘤的免疫效应.     

Susceptibility of thermally injured mice to cytomegalovirus infection

Thermally injured patients are very susceptible to infection with cytomegaloviruses. In this study a role of burn-associated type 2 T cell responses on the cytomegalovirus infection was examined in a mouse model of thermal injury. A predominance of type 2 T cell responses in splenic lymphocytes of thermally injured mice has been previously demonstrated. SCID mice inoculated with splenic T cells from thermally injured mice were susceptible to infection with a small amount (5 PFU/mouse) of murine cytomegalovirus (MCMV). Conversely, SCID mice inoculated with splenic T cells from normal mice were resistant to the same infection. High levels of IL-4 and IL-10, but not IFN-gamma and IL-2, were detected in sera of thermally injured mice (TI-mice) infected with MCMV when those were compared with sera of normal mice infected with MCMV. IL-4 and IL-10 (type 2 cytokines) were produced by splenic T cells from MCMV-infected TI-mice, when they were stimulated in vitro with anti-CD3 mAb. Type 1 cytokines (IFN-gamma and IL-2), however, were not produced by these T cells after the same stimulation. In contrast, splenic T cells from MCMV-infected normal mice produced type 1 cytokines by the stimulation with anti-CD3 mAb. These results suggest that the susceptibility of mice to MCMV infection is markedly influenced by burn-associated type 2 T cell responses.     

外源性IL-2对小鼠CD4^＋CD25^＋调节性T细胞影响的实验研究

目的：探讨IL-2对CD4＋CD25＋调节性T细胞（Tregs）的增殖及功能的影响。方法：提取B6小鼠脾脏细胞,流式细胞仪分离CD4＋CD25＋Tregs,将新鲜分离的CD4＋CD25＋Tregs与抗CD3单克隆抗体、同种同系抗原递呈细胞（APCs）及外源性IL-2共同培养,测定其增殖活性;并检测体外扩增后的CD4＋CD25＋Tregs的免疫抑制活性及其Foxp3的表达。结果：与外源性IL-2共同培养的CD4＋CD25＋Tregs增殖程度强烈,与对照组比较,差异有统计学意义（P〈0.05）;体外扩增的CD4＋CD25＋Tregs抑制CD4＋CD25-T细胞增殖活性的能力与新鲜分离的CD4＋CD25＋Tregs相似（P〉0.05）。体外扩增的CD4＋CD25＋Tregs的Foxp3表达与新鲜分离的CD4＋CD25＋Tregs亦相似（P〉0.05）。结论：外源性IL-2能够消除CD4＋CD25＋Tregs的无反应状态,且体外扩增的CD4＋CD25＋Tregs保持了其抑制活性。