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Volume 4,Issue 2,1997 Table of Contents
1997, 4(2):81-84.
DOI: 10.3872/j.issn.1007-385X.1997.2.002
Abstract:
T lymphocytes, which play a very important role in tumor immunity, recognize the tumor antigenic peptide presented by MHC molecules on the surface of tumor cells. FBL-3 is a transplantable Friend virus-induced leukemia of B6 (H-2~(b) ) origin, which can induce the CTL against FBL-3 by immunizing B6 mice with attenuated FBL-3. The present paper was to elute immunogenic peptides ( CD8 T cell epitopes) from class I complexes at the cell surface of viable FBL-3 cells by acid treatment. The acid treated cells remained viable but lost sensitivity to be lysed by FBL-3 specific cytotoxic T lymphocytes (CTLs). The sensitivity of the acid-treated cell was restored to the FBL-3 specific CTLs by supplement of the acid-eluted cell-free supematants. Paptides were subsequently fractionated by reverse-phase high performance liquid chromatography (RP-HPLC) . The fractionated peptides in HPLC fractions 5~6 and 23 ~ 60 (tubes) showed the capacity to sensitize RAM-S cells, which could be lysised by FBL-3 specific CTLs. The experiment indicates that this method may be useful in the definition of petide epitopes relevant to tumor cells.
T lymphocytes, which play a very important role in tumor immunity, recognize the tumor antigenic peptide presented by MHC molecules on the surface of tumor cells. FBL-3 is a transplantable Friend virus-induced leukemia of B6 (H-2~(b) ) origin, which can induce the CTL against FBL-3 by immunizing B6 mice with attenuated FBL-3. The present paper was to elute immunogenic peptides ( CD8 T cell epitopes) from class I complexes at the cell surface of viable FBL-3 cells by acid treatment. The acid treated cells remained viable but lost sensitivity to be lysed by FBL-3 specific cytotoxic T lymphocytes (CTLs). The sensitivity of the acid-treated cell was restored to the FBL-3 specific CTLs by supplement of the acid-eluted cell-free supematants. Paptides were subsequently fractionated by reverse-phase high performance liquid chromatography (RP-HPLC) . The fractionated peptides in HPLC fractions 5~6 and 23 ~ 60 (tubes) showed the capacity to sensitize RAM-S cells, which could be lysised by FBL-3 specific CTLs. The experiment indicates that this method may be useful in the definition of petide epitopes relevant to tumor cells.
1997, 4(2):85-89.
DOI: 10.3872/j.issn.1007-385X.1997.2.003
Abstract:
本文用转染了人突变DHFR基因逆转录病毒载体的新一代产病毒细胞AM12-S31,研究了其耐药特性、产生的病毒滴度及DHFR基因在小鼠造血细胞的表达.MTT法显示AM12-S31细胞对G418和氨甲喋呤耐受,对照细胞AM12对G418和MTX敏感.产生的病毒滴度为7.8×10~4～4.2×10~5CFU/ml,且为无复制活性病毒.将AM12-S31上清转染小鼠骨髓造血细胞后进行体内、外研究.CFU-GM分析显示:于不同C418浓度均有阳性克隆,而AM12上清转染组则无耐G418克隆.将转染后骨髓细胞从尾静脉回输给经致死剂量(900 rad)照射小鼠,MTX筛选(第一周为4mg/kg,每周二次;以后各周10mg/kg,每周二次),结果:实验组小鼠白细胞计数和红细胞压积逐渐恢复正常;对照组小鼠 30天内全部死亡.PCR分析耐G418 CFU-GM克隆和实验组小鼠骨髓细胞,均检测到标志基因Neo~R的存在.因此,初步研究表明该产病毒细胞能够产生高滴度、安全有效的非复制型逆转录病毒,并能成功转染小鼠造血细胞、表达目的基因,使在MTX筛选条件下重建小鼠造血功能.该研究为进一步开展耐药基因治疗打下了基础.
本文用转染了人突变DHFR基因逆转录病毒载体的新一代产病毒细胞AM12-S31,研究了其耐药特性、产生的病毒滴度及DHFR基因在小鼠造血细胞的表达.MTT法显示AM12-S31细胞对G418和氨甲喋呤耐受,对照细胞AM12对G418和MTX敏感.产生的病毒滴度为7.8×10~4～4.2×10~5CFU/ml,且为无复制活性病毒.将AM12-S31上清转染小鼠骨髓造血细胞后进行体内、外研究.CFU-GM分析显示:于不同C418浓度均有阳性克隆,而AM12上清转染组则无耐G418克隆.将转染后骨髓细胞从尾静脉回输给经致死剂量(900 rad)照射小鼠,MTX筛选(第一周为4mg/kg,每周二次;以后各周10mg/kg,每周二次),结果:实验组小鼠白细胞计数和红细胞压积逐渐恢复正常;对照组小鼠 30天内全部死亡.PCR分析耐G418 CFU-GM克隆和实验组小鼠骨髓细胞,均检测到标志基因Neo~R的存在.因此,初步研究表明该产病毒细胞能够产生高滴度、安全有效的非复制型逆转录病毒,并能成功转染小鼠造血细胞、表达目的基因,使在MTX筛选条件下重建小鼠造血功能.该研究为进一步开展耐药基因治疗打下了基础.
1997, 4(2):90-94.
DOI: 10.3872/j.issn.1007-385X.1997.2.004
Abstract:
Our previous investigation have demonstrated that multiple genes alterations, such as deletion of Rb, p53 and p16 gene, point mutation of H-ras gene were detected in cell line and solid tumor of human gastric cancer. We have transfect-ed the independent construct containing Rb, p53, p16 and expression of H-ras antisense RNA respectively into human gastric cancer cell line, and we have analyzed the biological properties of several independent transfectant cell lines, which express exogenous Rb, p53, p16 and H-ras antisense RNA respectively. The cell growth ability was inhibited by introduction of p53 and H-ras antisense RNA, and tumorigenicity also suppressed significantly by p53, p16 and H-ras antisense RNA. These results indicated that alterations of p53, p16 and H-ras gene were involved in human gastric car-cinogenesis.
Our previous investigation have demonstrated that multiple genes alterations, such as deletion of Rb, p53 and p16 gene, point mutation of H-ras gene were detected in cell line and solid tumor of human gastric cancer. We have transfect-ed the independent construct containing Rb, p53, p16 and expression of H-ras antisense RNA respectively into human gastric cancer cell line, and we have analyzed the biological properties of several independent transfectant cell lines, which express exogenous Rb, p53, p16 and H-ras antisense RNA respectively. The cell growth ability was inhibited by introduction of p53 and H-ras antisense RNA, and tumorigenicity also suppressed significantly by p53, p16 and H-ras antisense RNA. These results indicated that alterations of p53, p16 and H-ras gene were involved in human gastric car-cinogenesis.
1997, 4(2):95-99.
DOI: 10.3872/j.issn.1007-385X.1997.2.005
Abstract:
以逆转录病毒载体(pLXSN)将人源γ-干扰素(huIFN-γ)基因转导入4种不同的人肝癌细胞株,经G418抗性筛选均获得了阳性克隆.PCR和RT-PCR结果均表明IFN-γ基因已在基因组DNA中整合并表达.IFN-γ生物活性检测结果表明,在基因修饰的4种不同个体人肝癌细胞株及同一细胞株的5种不同克隆中,分泌的IFN-γ活性有较大差别.流式细胞仪检测细胞表面HLAⅠ类分子,结果表明基因修饰肿瘤细胞表面HLAⅠ类分子表达有显著提高.同时还首次对HLAⅠ类分子专一位点A2表达进行了分析,结果表明经IFN-γ基因修饰后,A2表达增加与HLAⅠ类分子总体增加相一致,本实验为进行基因工程修饰的肿瘤疫苗研究奠定了基础.
以逆转录病毒载体(pLXSN)将人源γ-干扰素(huIFN-γ)基因转导入4种不同的人肝癌细胞株,经G418抗性筛选均获得了阳性克隆.PCR和RT-PCR结果均表明IFN-γ基因已在基因组DNA中整合并表达.IFN-γ生物活性检测结果表明,在基因修饰的4种不同个体人肝癌细胞株及同一细胞株的5种不同克隆中,分泌的IFN-γ活性有较大差别.流式细胞仪检测细胞表面HLAⅠ类分子,结果表明基因修饰肿瘤细胞表面HLAⅠ类分子表达有显著提高.同时还首次对HLAⅠ类分子专一位点A2表达进行了分析,结果表明经IFN-γ基因修饰后,A2表达增加与HLAⅠ类分子总体增加相一致,本实验为进行基因工程修饰的肿瘤疫苗研究奠定了基础.
1997, 4(2):100-103.
DOI: 10.3872/j.issn.1007-385X.1997.2.006
Abstract:
In order to investigate the feasibility of granulocyte-macrophage colony stimulating factor (GM-CSF) gene therapy, murine GM-CSF cDNA recombinant retrovirus pLXSN/GM was transfered into retrovirus-packaging cell line PA3 17 by electroporation, and the transfected cells were used to infect hematopoietic progenitor cells rich populations. Transfective efficiency of NeoR gene was detected by G418 resistant CFU-GM test, and the results showed 36% them. In the genome of the infected target cells, integrated NeoR gene and GM-CSF cDNA were identified successfully by PCR and Southern Blot analysis respectively. The recombinant plasmids were showed to be capable of expressing GM-CSF mRNA in hematopoietic cells by in situ hybridization. In Dexter culture system, the present of GM-CSF-producing transduced cells inscreased mature nonadherent cell numbers as compared to controls. These results demonstrated that recombinant plasmids were successfully transfected into hematopoietic progenitor cells, and expressed in the cells. Therefore, it provided a basis for further investigation of gene therapy.
In order to investigate the feasibility of granulocyte-macrophage colony stimulating factor (GM-CSF) gene therapy, murine GM-CSF cDNA recombinant retrovirus pLXSN/GM was transfered into retrovirus-packaging cell line PA3 17 by electroporation, and the transfected cells were used to infect hematopoietic progenitor cells rich populations. Transfective efficiency of NeoR gene was detected by G418 resistant CFU-GM test, and the results showed 36% them. In the genome of the infected target cells, integrated NeoR gene and GM-CSF cDNA were identified successfully by PCR and Southern Blot analysis respectively. The recombinant plasmids were showed to be capable of expressing GM-CSF mRNA in hematopoietic cells by in situ hybridization. In Dexter culture system, the present of GM-CSF-producing transduced cells inscreased mature nonadherent cell numbers as compared to controls. These results demonstrated that recombinant plasmids were successfully transfected into hematopoietic progenitor cells, and expressed in the cells. Therefore, it provided a basis for further investigation of gene therapy.
6 The Role of Recombinant p53 Adenovirus Vector in the Growth of Lung Adenocarcinoma Cell Line GLC-82
1997, 4(2):104-106.
DOI: 10.3872/j.issn.1007-385X.1997.2.007
Abstract:
为研究野生型p53基因对肺腺癌细胞株GLC-82细胞生长的作用,确立腺病毒介导的野生型p53基因在肿瘤治疗中的意义,应用分子克隆技术首先构建了野生型p53复制缺陷型腺病毒重组体,应用生化染色方法确定了重组体的转染效率,以免疫组化法及PCR技术分别确定了腺病毒载体介导的p53基因转染GLC-82细胞后p53蛋白和p53 cDNA的表达情况;最后应用细胞生物学方法检测了p53对GLC-82细胞扩增及细胞周期的影响.结果表明所构建的重组体可以高效、快速转染GLC82细胞,抑制GLC-82细胞扩增,使细胞合成期和分裂后期的量减少,出现细胞凋亡现象:提示野生型p53基因导入可作为治疗肺腺癌的途径之一
为研究野生型p53基因对肺腺癌细胞株GLC-82细胞生长的作用,确立腺病毒介导的野生型p53基因在肿瘤治疗中的意义,应用分子克隆技术首先构建了野生型p53复制缺陷型腺病毒重组体,应用生化染色方法确定了重组体的转染效率,以免疫组化法及PCR技术分别确定了腺病毒载体介导的p53基因转染GLC-82细胞后p53蛋白和p53 cDNA的表达情况;最后应用细胞生物学方法检测了p53对GLC-82细胞扩增及细胞周期的影响.结果表明所构建的重组体可以高效、快速转染GLC82细胞,抑制GLC-82细胞扩增,使细胞合成期和分裂后期的量减少,出现细胞凋亡现象:提示野生型p53基因导入可作为治疗肺腺癌的途径之一
Wang Baocheng
,
Guo Jun
,
Gu Guangyu
,
Shi Qiuli
,
Di Jianshi
,
Yao Changying
,
Zhi Xiaoyan
,
Xu Fang
1997, 4(2):107-110.
DOI: 10.3872/j.issn.1007-385X.1997.2.008
Abstract:
To reverse drug resistance mediated by the MDR1 gene product P-glycoprotein(P-gp) in tumor cells in a specific manner, a hammerhead ribozymes which can cleave the GUC sequence in codon 196 of MDR1 mRNA was designed and cloned into a recombinant retroviral vector pDOR-neo at BamH I restriction sites and packaged with packaging cell line PA317 cells.The viral supernatant was used to infect the multidrug-resistant human hepatocarcinoma cell line BEL-7402/DOX. After selection with G418,resistant colonies were obtained.Stable expression of retroviruses in both PA317 and infected BEL-7402/DOX cells was confirmed by Northern Blot hybridization. Down-regulation of P-gp and even of MDR1 mRNA was found in BEL-7402/DOX infected with ribozyme construct. The rate of BEL - 7402/DOX infected cell defected by flow cytometric analysis was 8.2 ~ 14.6% while in uninfected cell it was 93 .4 ~ 97.5% . The BEL-7402/ DOX cell infected with ribozyme construct was found back to drug-sensitivity to a series of drugs by MTT colorimetric assay . The results demonstrated that the recombinant retroviral vector expressing ribozyme transfecting human hepatocarcinoma BEL-7402/DOX could inhibit MDR1 gene expression and reverse tumor MDR phenotype back to drug-sensitive condition.
To reverse drug resistance mediated by the MDR1 gene product P-glycoprotein(P-gp) in tumor cells in a specific manner, a hammerhead ribozymes which can cleave the GUC sequence in codon 196 of MDR1 mRNA was designed and cloned into a recombinant retroviral vector pDOR-neo at BamH I restriction sites and packaged with packaging cell line PA317 cells.The viral supernatant was used to infect the multidrug-resistant human hepatocarcinoma cell line BEL-7402/DOX. After selection with G418,resistant colonies were obtained.Stable expression of retroviruses in both PA317 and infected BEL-7402/DOX cells was confirmed by Northern Blot hybridization. Down-regulation of P-gp and even of MDR1 mRNA was found in BEL-7402/DOX infected with ribozyme construct. The rate of BEL - 7402/DOX infected cell defected by flow cytometric analysis was 8.2 ~ 14.6% while in uninfected cell it was 93 .4 ~ 97.5% . The BEL-7402/ DOX cell infected with ribozyme construct was found back to drug-sensitivity to a series of drugs by MTT colorimetric assay . The results demonstrated that the recombinant retroviral vector expressing ribozyme transfecting human hepatocarcinoma BEL-7402/DOX could inhibit MDR1 gene expression and reverse tumor MDR phenotype back to drug-sensitive condition.
1997, 4(2):111-114.
DOI: 10.3872/j.issn.1007-385X.1997.2.009
Abstract:
The recombinant human fusion protein GM-CSF/LIF was used to study the effects of different doses rhGM-LIF, rhGM-CSF,rhLIF,and rhGM-CSF plus rhLIF on tumor cells in our paper. The results showed that rhGM-LIF induced myeloid leukemia cells differentiation and inhibited the growth of the cells significantly. Its effect was dose-related, more significantly than rhGM-CSF,rhLIF,and rhGM-CSF plus rhLIF in some dose range. It is important that rhGM-LIF can inhibit the growth of human liver cancer cell. The rhGM-LIF might be a useful recombinant protein for tumor thera-py.
The recombinant human fusion protein GM-CSF/LIF was used to study the effects of different doses rhGM-LIF, rhGM-CSF,rhLIF,and rhGM-CSF plus rhLIF on tumor cells in our paper. The results showed that rhGM-LIF induced myeloid leukemia cells differentiation and inhibited the growth of the cells significantly. Its effect was dose-related, more significantly than rhGM-CSF,rhLIF,and rhGM-CSF plus rhLIF in some dose range. It is important that rhGM-LIF can inhibit the growth of human liver cancer cell. The rhGM-LIF might be a useful recombinant protein for tumor thera-py.
Yu Xiaodan
,
Tang Peixian
,
Zhang Mingwei
,
Zhao Chunhua
,
Peng Shanyun
,
liu Yuanlin
,
Hou Chunmei
,
Mao Ning
1997, 4(2):115-117.
DOI: 10.3872/j.issn.1007-385X.1997.2.010
Abstract:
In order to examine the feasibility of cytokine fusion gene transfer into tumor cells, a retroviral vector (pLXSN) for human interleukin 6/interleukin 2 (IL6/IL2) fusion gene was constructed by using PCR and ligating the 2 genes with a synthesized flexible linker. The IL6/IL2 fused gene was introduced into B16 melanoma cell line mediated by retrovirus and the changes of adhesive and metastatic ability of fused gene-modified cells were detected. The B16-IL6/ IL2 cells showed efficient expression of both cytokines and decreased binding affinity with extracellular matrix (Laminin and Matrigel), accompanying with decreased experimental metastatic potentials. These data suggest that the mechanism involving the decreased metastases may relate with the changes of biological characteristics and immune stimulating activity of gene-modified cells.
In order to examine the feasibility of cytokine fusion gene transfer into tumor cells, a retroviral vector (pLXSN) for human interleukin 6/interleukin 2 (IL6/IL2) fusion gene was constructed by using PCR and ligating the 2 genes with a synthesized flexible linker. The IL6/IL2 fused gene was introduced into B16 melanoma cell line mediated by retrovirus and the changes of adhesive and metastatic ability of fused gene-modified cells were detected. The B16-IL6/ IL2 cells showed efficient expression of both cytokines and decreased binding affinity with extracellular matrix (Laminin and Matrigel), accompanying with decreased experimental metastatic potentials. These data suggest that the mechanism involving the decreased metastases may relate with the changes of biological characteristics and immune stimulating activity of gene-modified cells.
1997, 4(2):130-133.
DOI: 10.3872/j.issn.1007-385X.1997.2.014
Abstract:
建立杂交瘤单抗亲和层析纯化抗原、抗原体外致敏淋巴细胞和RT-PCR克隆人抗体基因及噬菌体呈现技术构建人源抗体库的策略.将亲和层析纯化的大肠癌相关抗原CA-Hb3经SDS-PAGE和免疫印迹鉴定后,与IL-2和丝裂原于体外致敏10个大肠癌病人各10ml外周血淋巴细胞(PBL),出现淋巴母细胞化和集落形成现象,提取总RNA并纯化mRNA,经RT-PCR扩增3种VH-CHI(γ)基因和5种VL-CL(κλ)基因,再经PCR克隆3种VH(γ)和8种VL(κλ)基因.通过(Gly_4Ser)_3相应的寡核苷酸连接序列将VH和VL基因不同组合连成24种单链抗体(ScFv)基因,经 SfiI酶切,将之克隆入fUSE 5RF,用电穿孔法将此表达载体转化MC1061,四环素抗性筛选得到10~6库容的初级抗体库,ScFv基因插入百分率为85%.该策略将可能普遍用于鼠源单抗人源化.
建立杂交瘤单抗亲和层析纯化抗原、抗原体外致敏淋巴细胞和RT-PCR克隆人抗体基因及噬菌体呈现技术构建人源抗体库的策略.将亲和层析纯化的大肠癌相关抗原CA-Hb3经SDS-PAGE和免疫印迹鉴定后,与IL-2和丝裂原于体外致敏10个大肠癌病人各10ml外周血淋巴细胞(PBL),出现淋巴母细胞化和集落形成现象,提取总RNA并纯化mRNA,经RT-PCR扩增3种VH-CHI(γ)基因和5种VL-CL(κλ)基因,再经PCR克隆3种VH(γ)和8种VL(κλ)基因.通过(Gly_4Ser)_3相应的寡核苷酸连接序列将VH和VL基因不同组合连成24种单链抗体(ScFv)基因,经 SfiI酶切,将之克隆入fUSE 5RF,用电穿孔法将此表达载体转化MC1061,四环素抗性筛选得到10~6库容的初级抗体库,ScFv基因插入百分率为85%.该策略将可能普遍用于鼠源单抗人源化.
Ye Qinong
,
Wang Hengliang
,
Han Zhaozhong
,
Su Guofu
,
Huang Cuifen
,
Yao Xiao
,
Zhang Yi
,
Zhou Tingchong
1997, 4(2):134-136.
DOI: 10.3872/j.issn.1007-385X.1997.2.015
Abstract:
Messenger RNA was isolated directly from lung cancer cell line A549 using magnetic particles. First strand synthesis from mRNA was driven by M-MLV(Moloney Murine Leukemia Virus) reverse transcriptase and random hexam-eric primer, followed by second strand synthesis using RNase H and DNA polymerase I After treatment with T4 DNA polymerase to flush the ends, the double-stranded cDNA was cloned into the plasmid expression vector digested with EcoRI and followed by removing cohesive end. The number of independent clones of the resulting cDNA library was about 9.0 x 105. The estimated percentage of colonies with inserts was about 85 % . The insert size ranges from 0.5 kb ~ 4 kb. The CPP32 gene coding for death protease was obtained by PCR with the cDNA library from lung cancer cells as a template for the first time. Construction of the cDNA library laid a foundation for screening other genes regulating death of lung cancer cells.
Messenger RNA was isolated directly from lung cancer cell line A549 using magnetic particles. First strand synthesis from mRNA was driven by M-MLV(Moloney Murine Leukemia Virus) reverse transcriptase and random hexam-eric primer, followed by second strand synthesis using RNase H and DNA polymerase I After treatment with T4 DNA polymerase to flush the ends, the double-stranded cDNA was cloned into the plasmid expression vector digested with EcoRI and followed by removing cohesive end. The number of independent clones of the resulting cDNA library was about 9.0 x 105. The estimated percentage of colonies with inserts was about 85 % . The insert size ranges from 0.5 kb ~ 4 kb. The CPP32 gene coding for death protease was obtained by PCR with the cDNA library from lung cancer cells as a template for the first time. Construction of the cDNA library laid a foundation for screening other genes regulating death of lung cancer cells.
联系方式
- 《中国肿瘤生物治疗杂志》
- 1994年创刊
- 主办单位：Chinese Society of Immunology, Chinese Anti-cancer Association
- 邮编：200433
- 电话：021-81871002-22
- 电子邮箱：cjcb@biother.cn
- 网址:http://www.biother.cn
- 刊号：ISSN 1007-385X
- CN 31-1725/R
- 国内定价： ￥20元/册
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Supported by:Beijing E-Tiller Technology Development Co., Ltd.