hSSTR2基因转染肺癌细胞的肿瘤核素显像与杀伤研究
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摘要
目的将人生长抑素受体2亚型(hSSTR2)基因转染至该受体表达阴性的肿瘤细胞,研究125I-伐普肽(RC-160)与其结合的规律,以及131I-RC-160对转染肿瘤细胞的杀伤作用,并进行该受体介导的肿瘤显像研究。
方法1.应用反转录PCR(RT-PCR)方法获得hSSTR2基因并构建真核表达载体;2.将hSSTR2基因转染至hSSTR2表达阴性的肺腺癌A549细胞系,RT-PCR及流式细胞术检测受体的表达,并筛选hSSTR2的高表达的细胞株;3.采用放射性配基结合分析法,以125I-RC-160为放射性配基,对高表达hSSTR2基因的细胞株进行该受体体外结合特性研究;4.采用四甲基偶氮唑蓝(MTT)法测不同浓度131I-RC-160、Na131I、RC-160对转染细胞作用24、48、72和96h的杀伤效应;5.建立裸鼠移植瘤模型,以99mTc-RC-160为受体显像剂,探索该受体介导的肿瘤显像方法。
结果获得经测序完全正确的hSSTR2基因,RT-PCR及流式细胞术鉴定选取高表达hSSTR2基因的细胞株,体外结合实验测得平衡解离常数KD=5.65×10-10mol/L,最大结合容量Bmax=2.01×104结合位点/细胞,半数抑制浓度IC50=1.88×10-9mol/L; 131I-RC-160对A549-hSSTR2细胞的杀伤作用较其对A549-pcDNA3细胞杀伤作用明显增强,并呈一定的剂量-效应和时间-效应关系,在96h时,3.7MBq/ml的131I-RC-160对A549-hSSTR2的抑制率达78.8±5.9%。裸鼠肿瘤模型显像显示静脉注射显像剂99mTc-RC-160后0.5-1h肿瘤显影明显,之后肿瘤显影渐消退,于注射后24h肿瘤部位仍见显影。
结论将hSSTR2转染至该受体表达阴性的肿瘤细胞并进行受体介导的肿瘤显像是可行的,适宜显像时间为注射99mTc-RC-160后0.5-1h;131I-RC-160对A549-hSSTR2细胞的杀伤作用较131I或RC-160对A549-hSSTR2细胞的杀伤作用明显增强,说明131I-RC-160对转染hSSTR2后的肿瘤杀伤具有核素照射与生物抑制的协同效应。不表达SSTR2的肿瘤转染hSSTR2后,生长抑素类似物与hSSTR2结合,可以介导放射性核素对肿瘤进行靶向杀伤效应,从而提高对这类肿瘤的治疗效果,为外源性受体基因介导核素靶向性内照射治疗肿瘤提供了实验依据。
Objective To introduce human somatostatin receptors subtype-2(hSSTR2) gene to A549 lung carcinoma cell which does not express the gene, investigate the binding characters between the radioiodinated somatostatin analog 125I-RC-160 and hSSTR2 expressed on the transfected A549 lung carcinoma cells (A549-hSSTR2) ,and to observe the lethal effect of 131I-RC-160 on A549-hSSTR2 cells and tumor scintigraphy mediated by the receptor .
Methods An enkaryotic expression vector including hSSTR2 was constructed. In vitro human lung adenocarcinoma A549 cells were transfected with the vector. The expression of SSTR2 in the resulting cell clone was investigated by RT-PCR and flow cytometry and the clones overexpressing SSTR2 were selected for in vitro radioligand binding assay with 125I-RC-160. Used 3-(4,5-dimethylthiazol-2.y1)-2,5-diphenyltetrazolium bromide(MTT) assay to observe the lethal effect of various dosage of 131I-RC-160、Na131I、RC-160 on A549-hSSTR2 after different incubation periods. Established the xenograft tumor models in nude mice with A549- SSTR2 by incubation subcutaneously. SPECT was performed in nude mice bearing A549 tumors overexpressing SSTR2.
Results A549 cell clones stably transfected with hSSTR2 was generated, and the expression of SSTR2 in those clones was confirmed by RT-PCR and flow cytometry. SSTR2 expressed on membrane of the above cells showed high affinity and specificity. 125I-RC-160 rapidly combined with the membrane receptor of A549-hSSTR2. Scatchard analysis of 125I-RC-160 and tumor cell revealed that KD=5.65×10-10mol/L,Bmax =2.01×104/cell. In competitive binding assay, IC50=1.88×10-9mol/L. The inhibition effect of 131I-RC-160 on the growth of A549-hSSTR2 is stronger than that on A549- pcDNA3 cells, and is time-,and concentration-dependent. The inhibition ratio of A549-hSSTR2 cells is 78.8±5.9% after incubated 96h with 7.4MBq/ml of 131I-RC-160. Tumor lesions in nude mice was detected with 99mTc-RC-160 and clear images were achieved at 0.5-1h postinjection. At 24h postinjection images were showed again.
Conclusion 125I-RC-160 can bind with hSSTR2 expressed on the cells transfected with hSSTR2 gene; and the lethal effect of 131I-RC-160 on A549-hSSTR2 cells is enhanced obviously than that on control cells. This study demonstrates that it is available to transfer hsstr2 to tumor cell line which does not express the gene and detect the tumor by the receptor-mediated SPECT.These can provide experimental evidence for the concept of targeted exogenous receptor gene-mediated internalradiation therapy of human cancer.
方法1.应用反转录PCR(RT-PCR)方法获得hSSTR2基因并构建真核表达载体;2.将hSSTR2基因转染至hSSTR2表达阴性的肺腺癌A549细胞系,RT-PCR及流式细胞术检测受体的表达,并筛选hSSTR2的高表达的细胞株;3.采用放射性配基结合分析法,以125I-RC-160为放射性配基,对高表达hSSTR2基因的细胞株进行该受体体外结合特性研究;4.采用四甲基偶氮唑蓝(MTT)法测不同浓度131I-RC-160、Na131I、RC-160对转染细胞作用24、48、72和96h的杀伤效应;5.建立裸鼠移植瘤模型,以99mTc-RC-160为受体显像剂,探索该受体介导的肿瘤显像方法。
结果获得经测序完全正确的hSSTR2基因,RT-PCR及流式细胞术鉴定选取高表达hSSTR2基因的细胞株,体外结合实验测得平衡解离常数KD=5.65×10-10mol/L,最大结合容量Bmax=2.01×104结合位点/细胞,半数抑制浓度IC50=1.88×10-9mol/L; 131I-RC-160对A549-hSSTR2细胞的杀伤作用较其对A549-pcDNA3细胞杀伤作用明显增强,并呈一定的剂量-效应和时间-效应关系,在96h时,3.7MBq/ml的131I-RC-160对A549-hSSTR2的抑制率达78.8±5.9%。裸鼠肿瘤模型显像显示静脉注射显像剂99mTc-RC-160后0.5-1h肿瘤显影明显,之后肿瘤显影渐消退,于注射后24h肿瘤部位仍见显影。
结论将hSSTR2转染至该受体表达阴性的肿瘤细胞并进行受体介导的肿瘤显像是可行的,适宜显像时间为注射99mTc-RC-160后0.5-1h;131I-RC-160对A549-hSSTR2细胞的杀伤作用较131I或RC-160对A549-hSSTR2细胞的杀伤作用明显增强,说明131I-RC-160对转染hSSTR2后的肿瘤杀伤具有核素照射与生物抑制的协同效应。不表达SSTR2的肿瘤转染hSSTR2后,生长抑素类似物与hSSTR2结合,可以介导放射性核素对肿瘤进行靶向杀伤效应,从而提高对这类肿瘤的治疗效果,为外源性受体基因介导核素靶向性内照射治疗肿瘤提供了实验依据。
Objective To introduce human somatostatin receptors subtype-2(hSSTR2) gene to A549 lung carcinoma cell which does not express the gene, investigate the binding characters between the radioiodinated somatostatin analog 125I-RC-160 and hSSTR2 expressed on the transfected A549 lung carcinoma cells (A549-hSSTR2) ,and to observe the lethal effect of 131I-RC-160 on A549-hSSTR2 cells and tumor scintigraphy mediated by the receptor .
Methods An enkaryotic expression vector including hSSTR2 was constructed. In vitro human lung adenocarcinoma A549 cells were transfected with the vector. The expression of SSTR2 in the resulting cell clone was investigated by RT-PCR and flow cytometry and the clones overexpressing SSTR2 were selected for in vitro radioligand binding assay with 125I-RC-160. Used 3-(4,5-dimethylthiazol-2.y1)-2,5-diphenyltetrazolium bromide(MTT) assay to observe the lethal effect of various dosage of 131I-RC-160、Na131I、RC-160 on A549-hSSTR2 after different incubation periods. Established the xenograft tumor models in nude mice with A549- SSTR2 by incubation subcutaneously. SPECT was performed in nude mice bearing A549 tumors overexpressing SSTR2.
Results A549 cell clones stably transfected with hSSTR2 was generated, and the expression of SSTR2 in those clones was confirmed by RT-PCR and flow cytometry. SSTR2 expressed on membrane of the above cells showed high affinity and specificity. 125I-RC-160 rapidly combined with the membrane receptor of A549-hSSTR2. Scatchard analysis of 125I-RC-160 and tumor cell revealed that KD=5.65×10-10mol/L,Bmax =2.01×104/cell. In competitive binding assay, IC50=1.88×10-9mol/L. The inhibition effect of 131I-RC-160 on the growth of A549-hSSTR2 is stronger than that on A549- pcDNA3 cells, and is time-,and concentration-dependent. The inhibition ratio of A549-hSSTR2 cells is 78.8±5.9% after incubated 96h with 7.4MBq/ml of 131I-RC-160. Tumor lesions in nude mice was detected with 99mTc-RC-160 and clear images were achieved at 0.5-1h postinjection. At 24h postinjection images were showed again.
Conclusion 125I-RC-160 can bind with hSSTR2 expressed on the cells transfected with hSSTR2 gene; and the lethal effect of 131I-RC-160 on A549-hSSTR2 cells is enhanced obviously than that on control cells. This study demonstrates that it is available to transfer hsstr2 to tumor cell line which does not express the gene and detect the tumor by the receptor-mediated SPECT.These can provide experimental evidence for the concept of targeted exogenous receptor gene-mediated internalradiation therapy of human cancer.
引文
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3. Schally AV, Comaru-Schally AM, Nagy A, et al. Hypothalamic hormones and cancer. Front Neuroendocrinol, 2001,22(4):248–291.
4. Yamada Y, Post S, Wang K, et al. Cloning and functional characterization of a family of human and mouse somatostatin receptors expressed in brain, gastrointestinal tract, and kidney. Proc Natl Acad Sci U S A,1992, 89(1):251-255.
5. Yamada Y, Reisine T, Law SF, et al. Somatostatin receptors, an expanding gene family: cloning and functional characterization of human SSTR3, a protein coupled to adenylyl cyclase. Mol Endocrinol, 1992, 6(12):2136-2142
6. Yamada Y, Kagimoto S, Kubota A, et al. Cloning, functional expression and pharmacological characterization of a fourth (hSSTR4) and a fifth (hSSTR5) human somatostatin receptor subtype. Biochem Biophys Res Commun,1993,195(2): 844-852.
7. Patel YC, Greenwood M, Kent G, et al. Multiple gene transcripts of the somatostatin receptor SSTR2: tissue selective distribution and cAMP regulation. Biochem Biophys Res Commun, 1993, 192(1):288-294.
8. Reisine T, Bell GJ. Molecular biology of somatostatin receptors.Endocrinol. Rev, 1995,16:427-442.
9. O'Carroll AM, Raynor K, Lolait SJ, et al. Characterization of cloned human somatostatin receptor SSTR5. Mol Pharmacol, 1994,48:291-298.
10. Liebow C, Reilly C, Serrano M, et al. SRIF analogues inhibit the growth of pancreatic cancer by stimulating tyrosine phosphatase. Proc. Natl Acad Sci USA , 1989,86:2003-2007.
11. Lamberts S, Krenning E, Reubi J. The role of SRIF and its analogs in the diagnosis and treatment of tumors.Endocrinol. Rev, 1991,12: 450-482.
12. Hofand LJ, Visser-Wisselaar HA, Lamberts SW. Somatostatin analogs:clinical application in relation to human somatostatin receptor subtypes. Biochem.Pharmacol, 1995,50: 287-297.
13. Reubi JC, Waser B, Schaer JC, Laissue JA. Somatostatin receptor sst1–sst5 expression in normal and neoplastic human tissues using receptor autoradiography with subtype-selective ligands. Eur J Nucl Med, 2001;28:836–46.
14. Breeman WAP, de Jong M, Kwekkeboom DJ, et al. Somatostatin receptor mediated imaging and therapy: basic science, current knowledge, limitations and future perspectives. Eur J Nucl Med, 2001;28: 1421–9.
15. Mishima M, Yano T, Jimbo H, et al. Inhibition of human endometrial cancer cell growth in vitro and in vivo by somatostatin analog RC-160. Am J Obstel Gynecol, 1999,181:583-599.
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