目的:构建PD-1(programmed death receptor 1)全长启动子及不同截短体的报告基因,并对其转录活性进行检测。方法:通过PCR及双酶切方法,从人全血基因组DNA中获得PD-1基因编码序列,包含不同长度碱基的PD-1启动子序列,分别克隆到报告基因pG...目的:构建PD-1(programmed death receptor 1)全长启动子及不同截短体的报告基因,并对其转录活性进行检测。方法:通过PCR及双酶切方法,从人全血基因组DNA中获得PD-1基因编码序列,包含不同长度碱基的PD-1启动子序列,分别克隆到报告基因pGL3-Basic载体上,构建8个不同长度PD-1启动子的报告基因;用脂质体转染法将8个报告基因分别转染至Jurkat细胞系;采用双萤光素酶报告基因系统评估PD-1启动子的活性。结果:经PCR方法扩增出大小分别为1650、1450、1250、1128、874、674、474和274 bp的不同长度的PD-1启动子序列,测序正确(与GenBank报道一致),酶切鉴定正确;瞬时转染Jurkat细胞系后经报告基因检测,8个启动子均具有转录活性。结论:构建了PD-1启动子的报告基因,并证实均有转录活性,且以pGL3-1128活性最高,为PD-1的核心启动子,为进一步研究PD-1的转录调控奠定了实验基础。展开更多
目的探讨肺腺癌程序性死亡分子-1(programmed death 1,PD-1)、程序性死亡分子配体-1(programmed death ligand 1,PD-L1)蛋白的表达与K-RAS基因突变的相关性。方法采用免疫组化EnVision两步法检测PD-1、PD-L1蛋白的表达,应用实时荧光定量...目的探讨肺腺癌程序性死亡分子-1(programmed death 1,PD-1)、程序性死亡分子配体-1(programmed death ligand 1,PD-L1)蛋白的表达与K-RAS基因突变的相关性。方法采用免疫组化EnVision两步法检测PD-1、PD-L1蛋白的表达,应用实时荧光定量PCR技术检测K-RAS基因的突变类型。结果肺腺癌组织中PD-1、PD-L1的阳性率均高于良性病变肺组织(P<0.01),PD-1、PD-L1蛋白表达与患者性别、年龄、吸烟史、分化程度、淋巴结转移及TNM分期均无相关性(P>0.05)。36例肺腺癌样本中发生K-RAS基因突变者8例(22.2%),K-RAS基因突变与患者性别、年龄、吸烟史、分化程度、淋巴结转移及TNM分期均无关(P>0.05)。相关分析显示PD-1、PD-L1蛋白表达与K-RAS突变无关(P>0.05)。结论 PD-1、PD-L1蛋白在肺腺癌中的表达明显高于良性病变肺组织,但两者表达程度与肺腺癌的临床病理特征及K-RAS基因突变无关。展开更多
Summary: By using semi-quantitative RT-PCR method, it was found that PD-L1 mRNA but not PD-L2 mRNA was expressed in H22 hepatoma cells and both PD-L1 and PD-L2 mRNAs were expressed in tumor tissues of tumor-bearing mi...Summary: By using semi-quantitative RT-PCR method, it was found that PD-L1 mRNA but not PD-L2 mRNA was expressed in H22 hepatoma cells and both PD-L1 and PD-L2 mRNAs were expressed in tumor tissues of tumor-bearing mice and upregulated as compared with muscle tissues in normal mice and H22 hepatoma cells. PD-L1 and PD-L2 were also expressed on the surface of the activated T cells. The soluble recombinant sPD-1 expressed from the constructed eukaryotic expression vector could enhance the lysis of tumor cells by lymphocytes stimulated specifically with antigen. The expresssion of sPD-1 by local gene therapy on the inoculation site of H22 hepatoma cells could inhibit the growth of tumor. The results of this study indicate that expression of soluble receptor of negative costimulatory molecules could reduce the inhibitory effect on T cells in tumor microenvironment and enhance the cytotoxicity of T cells on tumor cells. This possibly provides a new method of improving efficacy of tumor gene therapy.展开更多
文摘目的:构建PD-1(programmed death receptor 1)全长启动子及不同截短体的报告基因,并对其转录活性进行检测。方法:通过PCR及双酶切方法,从人全血基因组DNA中获得PD-1基因编码序列,包含不同长度碱基的PD-1启动子序列,分别克隆到报告基因pGL3-Basic载体上,构建8个不同长度PD-1启动子的报告基因;用脂质体转染法将8个报告基因分别转染至Jurkat细胞系;采用双萤光素酶报告基因系统评估PD-1启动子的活性。结果:经PCR方法扩增出大小分别为1650、1450、1250、1128、874、674、474和274 bp的不同长度的PD-1启动子序列,测序正确(与GenBank报道一致),酶切鉴定正确;瞬时转染Jurkat细胞系后经报告基因检测,8个启动子均具有转录活性。结论:构建了PD-1启动子的报告基因,并证实均有转录活性,且以pGL3-1128活性最高,为PD-1的核心启动子,为进一步研究PD-1的转录调控奠定了实验基础。
文摘目的探讨肺腺癌程序性死亡分子-1(programmed death 1,PD-1)、程序性死亡分子配体-1(programmed death ligand 1,PD-L1)蛋白的表达与K-RAS基因突变的相关性。方法采用免疫组化EnVision两步法检测PD-1、PD-L1蛋白的表达,应用实时荧光定量PCR技术检测K-RAS基因的突变类型。结果肺腺癌组织中PD-1、PD-L1的阳性率均高于良性病变肺组织(P<0.01),PD-1、PD-L1蛋白表达与患者性别、年龄、吸烟史、分化程度、淋巴结转移及TNM分期均无相关性(P>0.05)。36例肺腺癌样本中发生K-RAS基因突变者8例(22.2%),K-RAS基因突变与患者性别、年龄、吸烟史、分化程度、淋巴结转移及TNM分期均无关(P>0.05)。相关分析显示PD-1、PD-L1蛋白表达与K-RAS突变无关(P>0.05)。结论 PD-1、PD-L1蛋白在肺腺癌中的表达明显高于良性病变肺组织,但两者表达程度与肺腺癌的临床病理特征及K-RAS基因突变无关。
文摘Summary: By using semi-quantitative RT-PCR method, it was found that PD-L1 mRNA but not PD-L2 mRNA was expressed in H22 hepatoma cells and both PD-L1 and PD-L2 mRNAs were expressed in tumor tissues of tumor-bearing mice and upregulated as compared with muscle tissues in normal mice and H22 hepatoma cells. PD-L1 and PD-L2 were also expressed on the surface of the activated T cells. The soluble recombinant sPD-1 expressed from the constructed eukaryotic expression vector could enhance the lysis of tumor cells by lymphocytes stimulated specifically with antigen. The expresssion of sPD-1 by local gene therapy on the inoculation site of H22 hepatoma cells could inhibit the growth of tumor. The results of this study indicate that expression of soluble receptor of negative costimulatory molecules could reduce the inhibitory effect on T cells in tumor microenvironment and enhance the cytotoxicity of T cells on tumor cells. This possibly provides a new method of improving efficacy of tumor gene therapy.
