【目的】探明贵州黑山羊(Guizhou Black Goats)干扰素-epsilon基因结构与功能,为羊传染病的防治及开发新型抗病毒药物提供理论依据。【方法】提取贵州黑山羊肝脏总RNA,通过RT-PCR扩增贵州黑山羊IFN-ε基因(GZHSY-IFN-ε)并测序,采用生...【目的】探明贵州黑山羊(Guizhou Black Goats)干扰素-epsilon基因结构与功能,为羊传染病的防治及开发新型抗病毒药物提供理论依据。【方法】提取贵州黑山羊肝脏总RNA,通过RT-PCR扩增贵州黑山羊IFN-ε基因(GZHSY-IFN-ε)并测序,采用生物信息学分析基因序列和预测蛋白结构。【结果】GZHSY-IFN-ε基因编码区长度为582 bp,编码194个氨基酸,贵州黑山羊与山羊、绵羊、羚牛、大羚羊IFN-ε的核苷酸序列同源性依次为99.7%、99.1%、99.1%、97.9%,氨基酸序列同源性依次为99.0%、97.9%、97.9%、94.8%;重组表达质粒p ET-28a-IFN-ε的双酶切结果正确;阳性菌株经诱导后,重组蛋白大小约39 ku,以可溶性蛋白形式表达,与抗His标签鼠单克隆抗体发生特异性反应;IFN-ε蛋白为不稳定亲水蛋白,有信号肽,无跨膜结构,亚细胞定位主要为胞外。IFN-ε蛋白存在16个磷酸化位点,无糖基化位点。二级结构主要为α-螺旋,与IL10RB、IFNAR^(2)、IFNAR1等蛋白存在相互作用。【结论】首次成功克隆贵州黑山羊IFN-ε基因序列,构建pET-28a-IFN-ε重组质粒并成功表达,具有良好的免疫原性。展开更多
目的:利用生信分析探讨细胞因子IL-12及IFN-γ对乳腺癌患者免疫微环境的影响,并结合临床病理特征,对其评估新辅助化疗疗效的价值进行研究。方法:在基因表达综合(GEO)数据库中下载GSE163882,比较IL-12相对应基因IL12B及IFN-γ相对应基因I...目的:利用生信分析探讨细胞因子IL-12及IFN-γ对乳腺癌患者免疫微环境的影响,并结合临床病理特征,对其评估新辅助化疗疗效的价值进行研究。方法:在基因表达综合(GEO)数据库中下载GSE163882,比较IL-12相对应基因IL12B及IFN-γ相对应基因IFNG在病理完全缓解(pCR)患者和残留病灶(RD)患者中的表达。收集于我院接受新辅助化疗的50例乳腺癌患者的临床资料,使用卡方检验分析临床病理特征与新辅助化疗疗效的相关性;通过ROC曲线评估血清IL-12、IFN-γ的预测价值,并且确定最佳截断值;采用二元Logistic回归进行多因素分析。通过ssGSEA免疫浸润分析IL12B和IFNG与免疫细胞浸润的相关性;采用GO和KEGG功能富集分析,探讨相关基因潜在生物学功能及信号通路。结果:在GEO数据库GSE163882数据集中IL12B和IFNG在pCR患者中呈现高表达。于我院收治的临床患者中pCR患者化疗前血清IL-12和IFN-γ水平明显高于非pCR组(P γ表达状态显著相关(P γ水平是新辅助化疗疗效的独立影响因素。免疫浸润分析显示,活化CD4 T细胞、活化CD8 T细胞、活化B细胞和自然杀伤细胞在pCR患者中高度浸润。结论:血清IL-12和IFN-γ表达状态与新辅助化疗效果具有相关性,化疗前血清IFN-γ高表达是pCR的独立预测因子。Objective: To investigate the effects of cytokines IL-12 and IFN-γ on the immune microenvironment of breast cancer patients by means of Bioinformatics analysis, and to study their value in evaluating the efficacy of neoadjuvant chemotherapy combined with clinicopathological characteristics. Methods: GSE163882 was downloaded from the Gene Expression Omnibus database, and the expressions of IL-12 corresponding gene IL12B and IFNG corresponding gene IFN-γ were compared in pathologic complete response patients and residual lesion patients. The clinical data of 50 breast cancer patients who received neoadjuvant chemotherapy at The Affiliated Hospital of Qingdao University were collected. Chi-square test was used to analyze the correlation between clinicopathological features and the efficacy of neoadjuvant chemotherapy. The predictive value of serum IL-12 and IFN-γ was evaluated by ROC curve. Multivariate analysis was performed by binary Logistic regression. The correlation between IL12B and IFNG and immune cell infiltration was analyzed by ssGSEA immunoinfiltration. GO and KEGG functional enrichment analysis were used to explore the potential biological functions and signaling pathways of related genes. Results: IL12B and IFNG were highly expressed in pCR patients in the GSE163882 dataset. The serum levels of IL-12 and IFN-γ in pCR patients before chemotherapy were significantly higher than those in non-PCR group (P γ expression after neoadjuvant chemotherapy (P γ level before chemotherapy was an independent factor influencing the efficacy of neoadjuvant chemotherapy. Immunoinfiltration analysis showed that activated CD4 T cells, activated CD8 T cells, activated B cells, and natural killer cells were highly infiltrated in pCR patients. Conclusion: The expression of serum IL-12 and IFN-γ is correlated with the effect of neoadjuvant chemotherapy, and the high expression of serum IFN-γ before chemotherapy is an independent predictor of pCR.展开更多
目的研究细胞程序性死亡配体-1(programmed cell death protein ligand-1,PD-L1)功能抑制调控免疫活化影响ApoE^(-/-)小鼠动脉粥样硬化发生发展的机制。方法将24只ApoE^(-/-)小鼠随机分为正常组,高脂组和高脂^(+)抗PD-L1单抗组,通过高...目的研究细胞程序性死亡配体-1(programmed cell death protein ligand-1,PD-L1)功能抑制调控免疫活化影响ApoE^(-/-)小鼠动脉粥样硬化发生发展的机制。方法将24只ApoE^(-/-)小鼠随机分为正常组,高脂组和高脂^(+)抗PD-L1单抗组,通过高胆固醇饲料喂养建立动脉粥样硬化(atherosclerosis)模型。实验动物饲养70 d后,分离各组实验动物血管(主动脉根部至腹主动脉)及肝脏组织,进行油红O染色;HE染色检测肝组织病理改变;ELISA检测血清中总胆固醇(CHO)、甘油三酯(TG)、高密度脂蛋白(HDL-c)、低密度脂蛋白(LDL-c)和炎症因子(IFN-γ、TNF-α、IL-1β)含量。流式细胞计数检测肝脏淋巴细胞(CD4^(+)、CD8^(+)、CD4^(+)IFN-γ^(+)和CD8^(+)IFN-γ^(+)T细胞)。RT-PCR检测肝脏组织IFN-γ、TNF-α、IL-1β、CD4和CD8表达。结果与高脂组比较,给予抗PD-L1单抗后促血管壁及肝脏脂质累积并上调血清及肝组织CHO、TG、LDL-c和HDL-c含量。高脂饲养条件下给予抗PD-L1单抗促血清和肝组织谷丙转氨酶(GPT)和谷草转氨酶(GOT)含量升高,但是对碱性磷酸酶(AKP)含量没有影响。高脂饲养条件下给予抗PD-L1单抗促血清和肝脏组织IFN-γ、TNF-α和IL-1β含量升高。高脂饲养条件下给予抗PD-L1单抗抑制CD4表达及促CD8表达。高脂饲养条件下给予抗PD-L1单抗促肝脏CD8^(+)T和CD8^(+)IFN-γ^(+)T细胞活化,但是对CD4^(+)IFN-γ^(+)T细胞活化没有影响。结论高脂饲养条件下给予抗PD-L1单抗通过活化肝脏CD8^(+)IFN-γ^(+)T细胞损伤肝脏功能加重动脉粥样硬化。展开更多
文摘目的:利用生信分析探讨细胞因子IL-12及IFN-γ对乳腺癌患者免疫微环境的影响,并结合临床病理特征,对其评估新辅助化疗疗效的价值进行研究。方法:在基因表达综合(GEO)数据库中下载GSE163882,比较IL-12相对应基因IL12B及IFN-γ相对应基因IFNG在病理完全缓解(pCR)患者和残留病灶(RD)患者中的表达。收集于我院接受新辅助化疗的50例乳腺癌患者的临床资料,使用卡方检验分析临床病理特征与新辅助化疗疗效的相关性;通过ROC曲线评估血清IL-12、IFN-γ的预测价值,并且确定最佳截断值;采用二元Logistic回归进行多因素分析。通过ssGSEA免疫浸润分析IL12B和IFNG与免疫细胞浸润的相关性;采用GO和KEGG功能富集分析,探讨相关基因潜在生物学功能及信号通路。结果:在GEO数据库GSE163882数据集中IL12B和IFNG在pCR患者中呈现高表达。于我院收治的临床患者中pCR患者化疗前血清IL-12和IFN-γ水平明显高于非pCR组(P γ表达状态显著相关(P γ水平是新辅助化疗疗效的独立影响因素。免疫浸润分析显示,活化CD4 T细胞、活化CD8 T细胞、活化B细胞和自然杀伤细胞在pCR患者中高度浸润。结论:血清IL-12和IFN-γ表达状态与新辅助化疗效果具有相关性,化疗前血清IFN-γ高表达是pCR的独立预测因子。Objective: To investigate the effects of cytokines IL-12 and IFN-γ on the immune microenvironment of breast cancer patients by means of Bioinformatics analysis, and to study their value in evaluating the efficacy of neoadjuvant chemotherapy combined with clinicopathological characteristics. Methods: GSE163882 was downloaded from the Gene Expression Omnibus database, and the expressions of IL-12 corresponding gene IL12B and IFNG corresponding gene IFN-γ were compared in pathologic complete response patients and residual lesion patients. The clinical data of 50 breast cancer patients who received neoadjuvant chemotherapy at The Affiliated Hospital of Qingdao University were collected. Chi-square test was used to analyze the correlation between clinicopathological features and the efficacy of neoadjuvant chemotherapy. The predictive value of serum IL-12 and IFN-γ was evaluated by ROC curve. Multivariate analysis was performed by binary Logistic regression. The correlation between IL12B and IFNG and immune cell infiltration was analyzed by ssGSEA immunoinfiltration. GO and KEGG functional enrichment analysis were used to explore the potential biological functions and signaling pathways of related genes. Results: IL12B and IFNG were highly expressed in pCR patients in the GSE163882 dataset. The serum levels of IL-12 and IFN-γ in pCR patients before chemotherapy were significantly higher than those in non-PCR group (P γ expression after neoadjuvant chemotherapy (P γ level before chemotherapy was an independent factor influencing the efficacy of neoadjuvant chemotherapy. Immunoinfiltration analysis showed that activated CD4 T cells, activated CD8 T cells, activated B cells, and natural killer cells were highly infiltrated in pCR patients. Conclusion: The expression of serum IL-12 and IFN-γ is correlated with the effect of neoadjuvant chemotherapy, and the high expression of serum IFN-γ before chemotherapy is an independent predictor of pCR.
文摘目的研究细胞程序性死亡配体-1(programmed cell death protein ligand-1,PD-L1)功能抑制调控免疫活化影响ApoE^(-/-)小鼠动脉粥样硬化发生发展的机制。方法将24只ApoE^(-/-)小鼠随机分为正常组,高脂组和高脂^(+)抗PD-L1单抗组,通过高胆固醇饲料喂养建立动脉粥样硬化(atherosclerosis)模型。实验动物饲养70 d后,分离各组实验动物血管(主动脉根部至腹主动脉)及肝脏组织,进行油红O染色;HE染色检测肝组织病理改变;ELISA检测血清中总胆固醇(CHO)、甘油三酯(TG)、高密度脂蛋白(HDL-c)、低密度脂蛋白(LDL-c)和炎症因子(IFN-γ、TNF-α、IL-1β)含量。流式细胞计数检测肝脏淋巴细胞(CD4^(+)、CD8^(+)、CD4^(+)IFN-γ^(+)和CD8^(+)IFN-γ^(+)T细胞)。RT-PCR检测肝脏组织IFN-γ、TNF-α、IL-1β、CD4和CD8表达。结果与高脂组比较,给予抗PD-L1单抗后促血管壁及肝脏脂质累积并上调血清及肝组织CHO、TG、LDL-c和HDL-c含量。高脂饲养条件下给予抗PD-L1单抗促血清和肝组织谷丙转氨酶(GPT)和谷草转氨酶(GOT)含量升高,但是对碱性磷酸酶(AKP)含量没有影响。高脂饲养条件下给予抗PD-L1单抗促血清和肝脏组织IFN-γ、TNF-α和IL-1β含量升高。高脂饲养条件下给予抗PD-L1单抗抑制CD4表达及促CD8表达。高脂饲养条件下给予抗PD-L1单抗促肝脏CD8^(+)T和CD8^(+)IFN-γ^(+)T细胞活化,但是对CD4^(+)IFN-γ^(+)T细胞活化没有影响。结论高脂饲养条件下给予抗PD-L1单抗通过活化肝脏CD8^(+)IFN-γ^(+)T细胞损伤肝脏功能加重动脉粥样硬化。
