生长抑素2型受体的核素报告基因显像和对胰腺癌抑制作用的研究
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摘要
背景与目的：生长抑素是一种在人体中广泛分布的激素，通过靶细胞膜上的生长抑素受体(somatostatin receptor，SSTR)介导，负性调节多种生理功能。SSTR共有5种亚型，其中2型受体(somatostatin receptor 2，SSTR2)被认为与肿瘤的关系最为密切。生长抑素或生长抑素类似物可通过SSTR2抑制肿瘤细胞的增生。胰腺癌中SSTR2表达缺失或是低表达，导致SSA治疗胰腺癌的效果不佳。通过基因转移的方法，将SSTR2基因导入肿瘤细胞内，通过SSTR2在细胞表面的再表达可以进行基因治疗。为评价基因治疗，需要随时对治疗基因的定位和表达进行监测，随着分子影像学的发展，可以利用放射性核素报告基因显像技术探测基因治疗中治疗基因表达情况。报告基因是监测治疗基因表达的外源基因，SSTR2可作为报告基因，利用放射性核素标记的生长抑素类似物进行显像，通过SSTR2基因的表达情况来间接评价治疗基因的表达情况。在SSTR2基因治疗胰腺癌的过程中，SSTR2既是报告基因，又是治疗基因。为此，本研究以SSTR2为报告基因，建立放射性核素锝标记奥曲肽(~(99m)Tc-奥曲肽)在胰腺癌基因治疗中的报告基因表达显像的方法，为胰腺癌基因治疗的疗效监测提供新的途径，观察SSTR2基因对裸鼠人胰腺癌移植瘤生长的影响，并以调控肿瘤生长的关键基因为靶点，对其影响机制作初步探讨。
方法：①构建重组腺病毒穿梭质粒pDC316-SSTR2和pDC316-LacZ，分别通过脂质体与腺病毒骨架质粒pBHGloxdeltaE1，3Cre共转染293细胞，经位点特异性重组获得重组腺病毒Ad-SSTR2和Ad-LacZ，并通过PCR鉴定Ad-SSTR2和Ad-LacZ，再经293细胞扩增，纯化制备高滴度病毒感染液，用X-gal染色法测定重组腺病毒感染能力。②以病毒感染液感染人胰腺癌细胞capan-2，应用RT-PCR、Western blot和免疫细胞化学方法检测SSTR2 mRNA和蛋白的表达。③MTT法检测Ad-SSTR2对capan-2细胞生长的影响。④SD大鼠经静脉注射~(99m)Tc-奥曲肽后，在不同时间内显像并测定心血池放射性计数，计算奥曲肽血药
Background & Objective: Somatostatin is a widely distributed peptide
that negatively regulates multiple physiological functions. Somatostatin inhibits cell proliferation and growth hormone secretion through interaction with somatostatin receptors (SSTRs). Five SSTRs have been cloned in the human, among those SSTRs, the SSTR2 gene is found to be the most important SSTR subtype for tumors. Many studies have demonstrated tumor cell growth inhibition by somatostatin and somatostatin analogs in SSTR2-positive tumors. However, functional SSTR2 are lost in most pancreatic cancers. Loss of SSTR2 expression may lead to a deficiency in negative regulation of cell growth by somatostatin analogs. Accordingly, gene therapy may be a promising strategy. Treating pancreatic cancer by reintroducing SSTR2 gene back to the cancer cells may be a good way. To evaluate the efficiency of gene therapy, it is requisite to monitor localization and expression of the therapeutic gene in vivo. Radionuclide reporter gene imaging techniques are currently being developed to map the topography and level of gene expression following gene therapy. Reporter gene can be used to monitor therapeutic gene and the efficacy of transgene targeting and transduction. SSTR2 is under evaluation as a reporter gene. The reporter SSTR2 protein is expressed on the plasma membrane of target cells and detected by imaging the specific accumulation of radiolabeled somatostatin analogs. The expression of therapeutic gene was indirectly evaluated through the expression of SSTR2 gene. In the course of gene therapy using SSTR2 in pancreatic cancer, SSTR2, the therapeutic gene also function as a reporter gene. Thus in the present study we established a radionuclide reporter gene imaging technology for monitoring adenoviral-mediated SSTR2 gene transfer to pancreatic cancer xenografts and wanted to supply a new way to evaluate the effects of gene therapy in pancreatic cancer. Furthermore, we also
方法：①构建重组腺病毒穿梭质粒pDC316-SSTR2和pDC316-LacZ，分别通过脂质体与腺病毒骨架质粒pBHGloxdeltaE1，3Cre共转染293细胞，经位点特异性重组获得重组腺病毒Ad-SSTR2和Ad-LacZ，并通过PCR鉴定Ad-SSTR2和Ad-LacZ，再经293细胞扩增，纯化制备高滴度病毒感染液，用X-gal染色法测定重组腺病毒感染能力。②以病毒感染液感染人胰腺癌细胞capan-2，应用RT-PCR、Western blot和免疫细胞化学方法检测SSTR2 mRNA和蛋白的表达。③MTT法检测Ad-SSTR2对capan-2细胞生长的影响。④SD大鼠经静脉注射~(99m)Tc-奥曲肽后，在不同时间内显像并测定心血池放射性计数，计算奥曲肽血药
Background & Objective: Somatostatin is a widely distributed peptide
that negatively regulates multiple physiological functions. Somatostatin inhibits cell proliferation and growth hormone secretion through interaction with somatostatin receptors (SSTRs). Five SSTRs have been cloned in the human, among those SSTRs, the SSTR2 gene is found to be the most important SSTR subtype for tumors. Many studies have demonstrated tumor cell growth inhibition by somatostatin and somatostatin analogs in SSTR2-positive tumors. However, functional SSTR2 are lost in most pancreatic cancers. Loss of SSTR2 expression may lead to a deficiency in negative regulation of cell growth by somatostatin analogs. Accordingly, gene therapy may be a promising strategy. Treating pancreatic cancer by reintroducing SSTR2 gene back to the cancer cells may be a good way. To evaluate the efficiency of gene therapy, it is requisite to monitor localization and expression of the therapeutic gene in vivo. Radionuclide reporter gene imaging techniques are currently being developed to map the topography and level of gene expression following gene therapy. Reporter gene can be used to monitor therapeutic gene and the efficacy of transgene targeting and transduction. SSTR2 is under evaluation as a reporter gene. The reporter SSTR2 protein is expressed on the plasma membrane of target cells and detected by imaging the specific accumulation of radiolabeled somatostatin analogs. The expression of therapeutic gene was indirectly evaluated through the expression of SSTR2 gene. In the course of gene therapy using SSTR2 in pancreatic cancer, SSTR2, the therapeutic gene also function as a reporter gene. Thus in the present study we established a radionuclide reporter gene imaging technology for monitoring adenoviral-mediated SSTR2 gene transfer to pancreatic cancer xenografts and wanted to supply a new way to evaluate the effects of gene therapy in pancreatic cancer. Furthermore, we also
引文
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