逆转录病毒介导的人endostatin基因疗法抗肿瘤作用研究
目的：探讨逆转录病毒介导人血管内皮抑制素基因（endostatin）对人肝癌细胞的生长抑制作用。方法：利用逆转录病毒载体pLocx构建含有人endostatin基因及信号肽的重组逆转录病毒质粒，并将其导入PA317细胞制备重组病毒。用重组病毒感染人肝癌细胞SMMC7721获得稳定转入endostatin基因细胞株(SMM-endo)。同样用空白病毒质粒制备对照细胞株SMMC-pLocx。免疫组化及Western blot检测人endostatin基因在转染细胞中的表达和分泌。应用血管内皮细胞生长抑制性试验证转染细胞株表达的人endostatin的生物学活性。同时对转染细胞株在体内外的生长情况进...
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| Published in | Chinese medical journal Vol. 115; no. 11; pp. 1664 - 1669 |
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| Main Author | |
| Format | Journal Article |
| Language | Chinese English |
| Published |
南京大学医学院,南京军区总院普外研究所,南京,210002%南京大学生命科学系,南京,210002%南京军区军医学院分子生物学研究室,南京,210002
2002
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| Abstract | 目的：探讨逆转录病毒介导人血管内皮抑制素基因（endostatin）对人肝癌细胞的生长抑制作用。方法：利用逆转录病毒载体pLocx构建含有人endostatin基因及信号肽的重组逆转录病毒质粒，并将其导入PA317细胞制备重组病毒。用重组病毒感染人肝癌细胞SMMC7721获得稳定转入endostatin基因细胞株(SMM-endo)。同样用空白病毒质粒制备对照细胞株SMMC-pLocx。免疫组化及Western blot检测人endostatin基因在转染细胞中的表达和分泌。应用血管内皮细胞生长抑制性试验证转染细胞株表达的人endostatin的生物学活性。同时对转染细胞株在体内外的生长情况进行观察。结果：免疫组化及Western blot印迹分析证实转染人endostatin基因的肝癌细胞能表达并分泌人endostatin.与转染空白对照组（SMMC-pLncx）相比，表达人endostatin的SMMC7721浓缩上清可显著抑制人脐静脉血管内皮细胞生长，抑制率达48%,明显高于对照组10.2％（P<0.01）。体外生长实验显示，SMMC-endo细胞与对照细胞SMMC-pLnex体外生长速率无明显差别。而在转染细胞接种裸鼠22d后，转染人endostatin基因的肿瘤细胞生长明显受到抑制，仅在部分裸鼠（3/5只）体内长出肿瘤，并且产生的腹侧肿瘤体积较对照组肿瘤缩小94.5％（P<0.001）。结论：逆转录病毒介导人endostatin基因疗法是治疗肿瘤的一个有效方法。 |
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| AbstractList | 目的：探讨逆转录病毒介导人血管内皮抑制素基因（endostatin）对人肝癌细胞的生长抑制作用。方法：利用逆转录病毒载体pLocx构建含有人endostatin基因及信号肽的重组逆转录病毒质粒，并将其导入PA317细胞制备重组病毒。用重组病毒感染人肝癌细胞SMMC7721获得稳定转入endostatin基因细胞株(SMM-endo)。同样用空白病毒质粒制备对照细胞株SMMC-pLocx。免疫组化及Western blot检测人endostatin基因在转染细胞中的表达和分泌。应用血管内皮细胞生长抑制性试验证转染细胞株表达的人endostatin的生物学活性。同时对转染细胞株在体内外的生长情况进行观察。结果：免疫组化及Western blot印迹分析证实转染人endostatin基因的肝癌细胞能表达并分泌人endostatin.与转染空白对照组（SMMC-pLncx）相比，表达人endostatin的SMMC7721浓缩上清可显著抑制人脐静脉血管内皮细胞生长，抑制率达48%,明显高于对照组10.2％（P<0.01）。体外生长实验显示，SMMC-endo细胞与对照细胞SMMC-pLnex体外生长速率无明显差别。而在转染细胞接种裸鼠22d后，转染人endostatin基因的肿瘤细胞生长明显受到抑制，仅在部分裸鼠（3/5只）体内长出肿瘤，并且产生的腹侧肿瘤体积较对照组肿瘤缩小94.5％（P<0.001）。结论：逆转录病毒介导人endostatin基因疗法是治疗肿瘤的一个有效方法。 R73; 目的探讨逆转录病毒介导人血管内皮抑制素基因(endostatin)对人肝癌细胞的生长抑制作用.方法利用逆转录病毒载体pLncx构建含有人endostatin基因及信号肽的重组逆转录病毒质粒,并将其导入PA317细胞制备重组病毒.用重组病毒感染人肝癌细胞SMMC7721获得稳定转人endostatin基因细胞株(SMMC-endo).同样用空白病毒质粒制备对照细胞株SMMC-pLncx.免疫组化及Western blot检测人endostatin 基因在转染细胞株中的表达和分泌.应用血管内皮细胞生长抑制试验验证转染细胞株表达的人endostatin的生物学活性.同时对转染细胞株在体内外的生长情况进行观察.结果免疫组化及Western blot印迹分析证实转染人endostatin基因的肝癌细胞能表达并分泌人endostatin.与转染空白对照组(SMMC-pLncx)相比,表达人endostatin的SMMC7721浓缩上清可显著抑制人脐静脉血管内皮细胞生长,抑制率达48%,明显高于对照组10.2%(P<0.01).体外生长实验显示,SMMC-endo细胞与对照细胞SMMC-pLncx体外生长速率无明显差别.而在转染细胞接种裸鼠22d后,转染人endostatin基因的肿瘤细胞生长明显受到抑制,仅在部分裸鼠(3/5只)体内长出肿瘤,并且产生的腹侧肿瘤体积较对照组肿瘤缩小94.5%(P<0.001).结论逆转录病毒介导的人endostatin基因疗法是治疗肿瘤的一个有效方法. |
| Abstract_FL | Objective To explore the inhibitory effect of human endostatin gene mediated by retroviral vector on the growth of human liver carcinoma.Methods A recombinant retroviral plasmid containing human endostatin gene and signal peptide was engineered and transferred into PA317 cells to produce retrovirus. Human liver carcinoma cells (SMMC7721) were infected with the above retrovirus to build a stable endostatin-transfected liver carcinoma cell line (SMMC-endo). The control liver carcinoma cell line (SMMC-pLncx) was developed in a similar way except that the plasmid was replaced by an empty retroviral vector. Immunohistochemistry and Western blot were used to test the expression and secretion of human endostatin. The biological activity of the expressed human endostatin was assessed by endothelial cell proliferation assay. The growth rates of SMMC-endo and control SMMC-pLncx cells in vivo and in vitro were also observed.Results The expression and secretion of human endostatin by endostatin-transfected SMMC-endo cells were confirmed by immunohistochemistry and Western blot. Compared with the control group, concentrated supernatant of SMMC-endo cells remarkably inhibited the proliferation of human umbilical vein endothelial cells by 48%, significantly higher than the inhibition by the control (10.2%; P<0.01). The endostatin-transfected SMMC-endo cells had similar in vitro growth rates to SMMC-pLncx cells. The in vivo experiment showed that the growth rate of SMMC-endo cells was slowed. Only in 3 out of 5 mice were tumors formed and flank tumors of SMMC-endo cells were 94.5% smaller than those of control cells 22 days after inoculation into nude mice (P<0.001).Conclusions Gene transfer of human endostatin mediated by retroviral vector is an effective form of cancer therapy. |
| Author | 王轩 李希 等 |
| AuthorAffiliation | 南京大学医学院，南京军区总院普外研究所，南京210002 南京大学生命科学系，南京210002 |
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| Author_FL | XU Genxing LI Jieshou LI Xi Liu Fukun WANG Xuan |
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| SubjectTerms | 人肝癌细胞 内皮抑制素 基因疗法 逆转录病毒 |
| Title | 逆转录病毒介导的人endostatin基因疗法抗肿瘤作用研究 |
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