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Volume 3,Issue 3,1996 Table of Contents
1996, 3(3):168-173.
DOI: 10.3872/j.issn.1007-385X.1996.3.002
Abstract:
A hammerhead RZ DNA was designed and synthesized, which can specifically cleave the bcl-2 mRNA. After demonstration of right sequences by sequencing and cleavage activity of RZ by in vitro cleaving experiment, The RZ DNA was recombinated into the pDOR - neo vector to form the recombinant pDOR - RZ. Using lipofectin - mediated DNA transfectionpDOR-RZ was successfully introduced into HL - 60 cells. The RZ expression was observed by Southern, RNA dot blot hybridization and flow cytometry (FCM) . The results demonstrated that (a) the RZ was expressed in 72 hours after transfection; (b) the synthesis of Bel - 2 protein was inhibited by the expression of RZ; (c) apoptotic peak appeared in FCM.
A hammerhead RZ DNA was designed and synthesized, which can specifically cleave the bcl-2 mRNA. After demonstration of right sequences by sequencing and cleavage activity of RZ by in vitro cleaving experiment, The RZ DNA was recombinated into the pDOR - neo vector to form the recombinant pDOR - RZ. Using lipofectin - mediated DNA transfectionpDOR-RZ was successfully introduced into HL - 60 cells. The RZ expression was observed by Southern, RNA dot blot hybridization and flow cytometry (FCM) . The results demonstrated that (a) the RZ was expressed in 72 hours after transfection; (b) the synthesis of Bel - 2 protein was inhibited by the expression of RZ; (c) apoptotic peak appeared in FCM.
1996, 3(3):174-177.
DOI: 10.3872/j.issn.1007-385X.1996.3.003
Abstract:
HSU17714 gene is a novel human colorectal carcinoma related gene isolated by subtractive hybridization method. The recombinant antisense expressive plasmid of HSU17714 gene was constructed by inserting the partial sequence (2612 bp) of this gene into the multiple cloning site of the vector pREP9 inversely. Then, it was transferred into human colon adenocarcinoma cells (SW1116) through liposome mediation. Obtained from the two-layer soft agarose culture test, flowcytometry test and cell growth rate detection, the data indicated that the antisense HSU17714 could inhibit the growth of SW1116 cells at different degrees. So, a conclusion could be drawn that the antisense HSU17714 gene expressive vector plays a role in the inhibition of SW1116 cell growth.
HSU17714 gene is a novel human colorectal carcinoma related gene isolated by subtractive hybridization method. The recombinant antisense expressive plasmid of HSU17714 gene was constructed by inserting the partial sequence (2612 bp) of this gene into the multiple cloning site of the vector pREP9 inversely. Then, it was transferred into human colon adenocarcinoma cells (SW1116) through liposome mediation. Obtained from the two-layer soft agarose culture test, flowcytometry test and cell growth rate detection, the data indicated that the antisense HSU17714 could inhibit the growth of SW1116 cells at different degrees. So, a conclusion could be drawn that the antisense HSU17714 gene expressive vector plays a role in the inhibition of SW1116 cell growth.
1996, 3(3):178-182.
DOI: 10.3872/j.issn.1007-385X.1996.3.004
Abstract:
To study the molecular properties of polypeptide core of human mucin antigens, a polypeptide core of mucin (apomucin) was isolated from human pancreatic cancer cell line SW1990.The tumor mucin was isolated by a combination of molecular sieve chromatography and CsCl density gradient ultracentrifugation. Anhydrous hydrogen flouride was employed to remove carbohydrate units from purified mucin molecules. The purity of mucin and apomucin were identified by SDS-PAGE, the amino acids of apomucin were analized and confirmed by Western Blot using anti-apomucin antibodies.A bunch of monoclonal antibodies were generated against this apomucin. The results showed that this isolated apomucin consisted of many different cDNA sequences peptides, included MUC-l,MUC-2 and MUC-3. The monoclonal antibodies against this apomucin reacted with peptides of molecular weight from 28 to >90 KD.A11 the five monoclonal antibodies against this apomucin reacted with cancer cell lines and cancer tissues,while only one also reacted with normal tissues.
To study the molecular properties of polypeptide core of human mucin antigens, a polypeptide core of mucin (apomucin) was isolated from human pancreatic cancer cell line SW1990.The tumor mucin was isolated by a combination of molecular sieve chromatography and CsCl density gradient ultracentrifugation. Anhydrous hydrogen flouride was employed to remove carbohydrate units from purified mucin molecules. The purity of mucin and apomucin were identified by SDS-PAGE, the amino acids of apomucin were analized and confirmed by Western Blot using anti-apomucin antibodies.A bunch of monoclonal antibodies were generated against this apomucin. The results showed that this isolated apomucin consisted of many different cDNA sequences peptides, included MUC-l,MUC-2 and MUC-3. The monoclonal antibodies against this apomucin reacted with peptides of molecular weight from 28 to >90 KD.A11 the five monoclonal antibodies against this apomucin reacted with cancer cell lines and cancer tissues,while only one also reacted with normal tissues.
1996, 3(3):183-185.
DOI: 10.3872/j.issn.1007-385X.1996.3.005
Abstract:
人肿瘤相关抗原可诱导宿主的细胞免疫反应已被证实。一些肿瘤粘蛋白也证实可诱发T细胞细胞毒作用。本研究是观察从胰腺癌细胞株提取的粘蛋白核芯肽诱导人肿瘤引流区淋巴结细胞的抗肿瘤免疫作用。4例乳腺癌、3例胃癌、3例结肠癌的肿瘤引流区淋巴结手术切除后立即在体外培养于含50μg/ml胰癌核芯肽及50单位IL-2的培养基中,观察其对多种癌细胞的杀伤效应(MMT法)。结果表明体外经胰癌核芯肽刺激后增殖的淋巴结细胞对多种肿瘤细胞有杀伤作用,如结肠癌、胃癌、胰腺癌及白血病细胞K562。本研究结果为以肿瘤粘蛋白核芯肽制备疫苗用于主动特异性免疫治疗或过继免疫治疗打下基础。
人肿瘤相关抗原可诱导宿主的细胞免疫反应已被证实。一些肿瘤粘蛋白也证实可诱发T细胞细胞毒作用。本研究是观察从胰腺癌细胞株提取的粘蛋白核芯肽诱导人肿瘤引流区淋巴结细胞的抗肿瘤免疫作用。4例乳腺癌、3例胃癌、3例结肠癌的肿瘤引流区淋巴结手术切除后立即在体外培养于含50μg/ml胰癌核芯肽及50单位IL-2的培养基中,观察其对多种癌细胞的杀伤效应(MMT法)。结果表明体外经胰癌核芯肽刺激后增殖的淋巴结细胞对多种肿瘤细胞有杀伤作用,如结肠癌、胃癌、胰腺癌及白血病细胞K562。本研究结果为以肿瘤粘蛋白核芯肽制备疫苗用于主动特异性免疫治疗或过继免疫治疗打下基础。
1996, 3(3):186-190.
DOI: 10.3872/j.issn.1007-385X.1996.3.006
Abstract:
通过RT-PCR克隆到含有全部编码序列的人IL-2 cDNA,并经核苷酸测序加以证实。将其克隆至逆转录病毒载体pLXSN,构建成人IL-2的重组逆转录病毒表达载体pLXSN-hIL2,体外经CRIP细胞包装后病毒滴度达7.6×10~5CFU/ml。NIH3T3小鼠成纤维细胞感染hIL-2重组逆转录病毒后,分泌IL-2水平达118.2U/ml;PCR从其基因组DNA中扩增到NeoR基因片段,提示重组逆转录病毒载体己整合至宿主细胞的基因组DNA中。本研究为开展人IL-2基因治疗奠定了基础。
通过RT-PCR克隆到含有全部编码序列的人IL-2 cDNA,并经核苷酸测序加以证实。将其克隆至逆转录病毒载体pLXSN,构建成人IL-2的重组逆转录病毒表达载体pLXSN-hIL2,体外经CRIP细胞包装后病毒滴度达7.6×10~5CFU/ml。NIH3T3小鼠成纤维细胞感染hIL-2重组逆转录病毒后,分泌IL-2水平达118.2U/ml;PCR从其基因组DNA中扩增到NeoR基因片段,提示重组逆转录病毒载体己整合至宿主细胞的基因组DNA中。本研究为开展人IL-2基因治疗奠定了基础。
1996, 3(3):191-194.
DOI: 10.3872/j.issn.1007-385X.1996.3.007
Abstract:
The Ad-IL-2 and/or Ad-IL-3 were i. p. injected into experimental leukemic mice after high dose chemotherapy. When Ad - LacZ was i. p. injected into leukemic mice, there were leukemic cells in bone marrow, vessal, liver and spleen. After i. p. injection of Ad-IL-2 and/or Ad-IL-3, the leukemia - bearing mice showed significant inhibition of tumor growth. As i.p. injection of Ad-IL-2, the splenic NK, CTL activity increased; as i. p. injection of Ad - IL - 3, the number and cytotoxicity of peritoneal macrophages were significantly higher than that of the control groups. After i. p. injection of Ad - IL - 2 and Ad - IL - 3 at the same time, the anti - leukemia activity was the best. Though there were less leukemic cells in the bone marrow, there were no leukemic cells in the liver and spleen. Our results suggested that i. p. injection of Ad - IL - 2 and Ad - IL - 3 could significantly enhance the anti - leukemia activity of chemotherapy.
The Ad-IL-2 and/or Ad-IL-3 were i. p. injected into experimental leukemic mice after high dose chemotherapy. When Ad - LacZ was i. p. injected into leukemic mice, there were leukemic cells in bone marrow, vessal, liver and spleen. After i. p. injection of Ad-IL-2 and/or Ad-IL-3, the leukemia - bearing mice showed significant inhibition of tumor growth. As i.p. injection of Ad-IL-2, the splenic NK, CTL activity increased; as i. p. injection of Ad - IL - 3, the number and cytotoxicity of peritoneal macrophages were significantly higher than that of the control groups. After i. p. injection of Ad - IL - 2 and Ad - IL - 3 at the same time, the anti - leukemia activity was the best. Though there were less leukemic cells in the bone marrow, there were no leukemic cells in the liver and spleen. Our results suggested that i. p. injection of Ad - IL - 2 and Ad - IL - 3 could significantly enhance the anti - leukemia activity of chemotherapy.
1996, 3(3):199-202.
DOI: 10.3872/j.issn.1007-385X.1996.3.009
Abstract:
In the present study,the effects of fibroblast-mediated IL-3 gene therapy on immune reconstitution was observed after syngeneic bone marrow transplantation in the mice which had received high-dose chemotherapy. After the treatment,the proliferation of bone marrow cells stimulated by ConA, the cytotoxicity of macrophages, the levels of IL-1, TNF induced from macrophage and IL-2 induced from the splenocyte of experimental mice were augmented significantly. The results suggest that the fibroblast-mediated IL-3 gene therapy could be used to accelerate the process of immune reconstitution and reduce the complication after bone marrow transplantation.
In the present study,the effects of fibroblast-mediated IL-3 gene therapy on immune reconstitution was observed after syngeneic bone marrow transplantation in the mice which had received high-dose chemotherapy. After the treatment,the proliferation of bone marrow cells stimulated by ConA, the cytotoxicity of macrophages, the levels of IL-1, TNF induced from macrophage and IL-2 induced from the splenocyte of experimental mice were augmented significantly. The results suggest that the fibroblast-mediated IL-3 gene therapy could be used to accelerate the process of immune reconstitution and reduce the complication after bone marrow transplantation.
1996, 3(3):203-206.
DOI: 10.3872/j.issn.1007-385X.1996.3.010
Abstract:
将可诱导表达的细小病毒非结构蛋白基因转染入人低分化胃腺癌细胞株MKN-45,该基因诱导表达后部分宿主细胞死亡,成活细胞生长特性发生显著改变,如细胞间粘附力增强,倍增时间延长,裸鼠体内成瘤能力下降。RNA斑点印迹显示非结构蛋白基因的表达能明显增加胃癌细胞白细胞介素-1α,白细胞介素-1β和白细胞介素6核因子表达,而对白细胞介素6表达无影响。提示细小病毒的抗肿瘤作用部分可能是由其非结构蛋白影响肿瘤细胞的细胞因子表达所介导的。
将可诱导表达的细小病毒非结构蛋白基因转染入人低分化胃腺癌细胞株MKN-45,该基因诱导表达后部分宿主细胞死亡,成活细胞生长特性发生显著改变,如细胞间粘附力增强,倍增时间延长,裸鼠体内成瘤能力下降。RNA斑点印迹显示非结构蛋白基因的表达能明显增加胃癌细胞白细胞介素-1α,白细胞介素-1β和白细胞介素6核因子表达,而对白细胞介素6表达无影响。提示细小病毒的抗肿瘤作用部分可能是由其非结构蛋白影响肿瘤细胞的细胞因子表达所介导的。
1996, 3(3):207-210.
DOI: 10.3872/j.issn.1007-385X.1996.3.011
Abstract:
Tumor infiltrating lymphocytes(TILs) were isolated by enzymatic digestion and discontinuous gradient centrifugation from 8 human advanced tumors (4 stomach carcinoma, 2 liver cancer, 1 non-small-cell lung carcinoma and 1 colon cancer). These cells were cultured in complete RPMI 1640 medium supplemented with l000U/ml of rhIL2 for 4-6 weeks, till the cell number reach over l09/total, reinfused to the same patients i.v. meanwhile, the patients received 105U of rhIL2 i.m for 5 days. One week before and one month after TIL infusion periphery blood from the patients was collected and the mononuclear cells were isolated. Cytotoxicity against a panel of tumor cell targets by MTT colorometric assay and lymphocyte phenotype by two-color flow cytometry were mornitored. The results showed that there was significant increase in the killing ability to the tested tumor targets to different extent, especially the killing to the target cells which shared the same histological type with the patients tumor. (43 against 1249 lytic units p<0.05), an enhancement of fluoreceint intensity on CD4 and CD8 cell subsets. ( T4 1.92 to 3.98,4.45 to 7.2 p<0.05) All the findings indicate that TILs plus lower dose rhIL2 are able to up-regulate the cellular immunological state in patients through immunological regulatory effect by cytokine cascade addition to the direct cytotoxicity.
Tumor infiltrating lymphocytes(TILs) were isolated by enzymatic digestion and discontinuous gradient centrifugation from 8 human advanced tumors (4 stomach carcinoma, 2 liver cancer, 1 non-small-cell lung carcinoma and 1 colon cancer). These cells were cultured in complete RPMI 1640 medium supplemented with l000U/ml of rhIL2 for 4-6 weeks, till the cell number reach over l09/total, reinfused to the same patients i.v. meanwhile, the patients received 105U of rhIL2 i.m for 5 days. One week before and one month after TIL infusion periphery blood from the patients was collected and the mononuclear cells were isolated. Cytotoxicity against a panel of tumor cell targets by MTT colorometric assay and lymphocyte phenotype by two-color flow cytometry were mornitored. The results showed that there was significant increase in the killing ability to the tested tumor targets to different extent, especially the killing to the target cells which shared the same histological type with the patients tumor. (43 against 1249 lytic units p<0.05), an enhancement of fluoreceint intensity on CD4 and CD8 cell subsets. ( T4 1.92 to 3.98,4.45 to 7.2 p<0.05) All the findings indicate that TILs plus lower dose rhIL2 are able to up-regulate the cellular immunological state in patients through immunological regulatory effect by cytokine cascade addition to the direct cytotoxicity.
1996, 3(3):211-213.
DOI: 10.3872/j.issn.1007-385X.1996.3.012
Abstract:
The result showed that CD3AK induced and expanded in vitro could kill MHC I class - negative K562 (NK - sensitive) and Daudi (NK - resistant) tumor cells in a MHC - nonrestricted manner. Induction of necrosis and / or apoptosis of target cells were responsible for the tumorlytic effect mediated by CD3AK.
The result showed that CD3AK induced and expanded in vitro could kill MHC I class - negative K562 (NK - sensitive) and Daudi (NK - resistant) tumor cells in a MHC - nonrestricted manner. Induction of necrosis and / or apoptosis of target cells were responsible for the tumorlytic effect mediated by CD3AK.
1996, 3(3):219-221.
DOI: 10.3872/j.issn.1007-385X.1996.3.015
Abstract:
运用逆转录病毒作为外源基因导入的载体,在实际临床运用时需要检测包装细胞在表达目的基因的同时,是否会产生有复制能力的逆转录病毒。本实验运用了Marker Rescue Assay(补救分析)和RT/PCR(逆转录PCR)两种方法。补救分析可检测到6×10~2CFU/ml(Colony Forming Units/ml)的有复制能力的逆转录病毒,RT/PCR的灵敏度为1CFU/10~3ml。这两种检测方法的建立,为逆转录病毒载体用于临床基因治疗的安全性提供一定的保证。
运用逆转录病毒作为外源基因导入的载体,在实际临床运用时需要检测包装细胞在表达目的基因的同时,是否会产生有复制能力的逆转录病毒。本实验运用了Marker Rescue Assay(补救分析)和RT/PCR(逆转录PCR)两种方法。补救分析可检测到6×10~2CFU/ml(Colony Forming Units/ml)的有复制能力的逆转录病毒,RT/PCR的灵敏度为1CFU/10~3ml。这两种检测方法的建立,为逆转录病毒载体用于临床基因治疗的安全性提供一定的保证。
1996, 3(3):226-229.
DOI: 10.3872/j.issn.1007-385X.1996.3.017
Abstract:
将表达人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)的重组真核表达质粒(pCG1),以30μg和60μg的剂量分别直接注射小鼠骨骼肌,观察不同剂量的质粒(pCG1)在小鼠体内分泌表达rhGM-CSF的情况。ELISA检测表明,注射60μg质粒DNA的小鼠血液中测得的rhGM-CSF含量与对照组相比有显著差别,而注射30μg质粒DNA的小鼠血液中rhGM-CSF含量与对照组相比无显著差别。以60μg质粒DNA直接注射骨骼肌后第15～20天左右表达量最高,平均约91ng/ml。生物学活性检测结果显示,注射60μg质粒DNA的小鼠血液能维持GM-CSF依赖的TF-1细胞的生长。这提示用质粒DNA直接注射小鼠骨骼肌时,质粒DNA必须达到足够的量。
将表达人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)的重组真核表达质粒(pCG1),以30μg和60μg的剂量分别直接注射小鼠骨骼肌,观察不同剂量的质粒(pCG1)在小鼠体内分泌表达rhGM-CSF的情况。ELISA检测表明,注射60μg质粒DNA的小鼠血液中测得的rhGM-CSF含量与对照组相比有显著差别,而注射30μg质粒DNA的小鼠血液中rhGM-CSF含量与对照组相比无显著差别。以60μg质粒DNA直接注射骨骼肌后第15～20天左右表达量最高,平均约91ng/ml。生物学活性检测结果显示,注射60μg质粒DNA的小鼠血液能维持GM-CSF依赖的TF-1细胞的生长。这提示用质粒DNA直接注射小鼠骨骼肌时,质粒DNA必须达到足够的量。
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- 《中国肿瘤生物治疗杂志》
- 1994年创刊
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Supported by:Beijing E-Tiller Technology Development Co., Ltd.