hNIS基因转导黑色素瘤细胞介导放射性碘摄取的实验研究

目的获取人钠/碘同向转运体(hNIS)基因cDNA,研究其转导黑色素瘤细胞在体外和体内能否介导放射性碘摄取,从而探索该策略用于黑色素瘤放射性碘治疗的可能性.方法运用逆转录聚合酶链反应从人甲状腺组织总RNA中扩增出hNIS基因cDNA,将其克隆至真核载体pc-DNA3中,电转化法分别将重组质粒pc-DNA3-hNIS及空质粒pc-DNA3转导黑色素瘤细胞(B16),分别建立了细胞系B16-A和B16-B.在体外培养条件下检测其对放射性碘的摄取及外流情况.继而将三种细胞系接种C57小鼠行131I显像和肿瘤摄取125I动态定量测定.结果成功克隆到hNIS基因,并建立了能稳定表达hNIS的新型细胞系B16-A.B16-A细胞的摄碘能力较B16细胞高17倍,较B16-B细胞高19倍.碘的外流过程迅速,T1/2eff仅10 min.体内实验发现B16-A细胞所形成肿瘤能摄取放射性碘,腹腔注射125I后1、2、4、12、24 h肿瘤组织的%ID/g平均为12.22.10.91、8.73、1.24、0.19,125I在肿瘤组织内的生物半衰期约为6 h.B16-A细胞系所成肿瘤摄碘量与对照组相比较,P＜0.01,差别具有高度统计意义.结论hNIS基因转导黑色素瘤细胞足以介导放射性碘摄取,但有效半衰期较短,难以产生足够的治疗剂量,有必要进一步研究如何增加放射性碘的摄取量及延长碘在细胞内的滞留时间以提高其放射生物学效应.     

The rat sodium iodide symporter gene permits more effective radioisotope concentration than the human sodium iodide symporter gene in human and rodent cancer cells

Expression of the sodium iodide symporter (NIS) gene in tumor cells may provide a novel mechanism for treating cancer. The NIS mediates the normal physiological transport of iodide across the thyroid cell membrane. This mechanism of iodide uptake has been used to both diagnose and treat thyroid cancer. Tissue expression of the NIS is largely limited to the thyroid; therefore, expression of the NIS gene in cancer cells would allow for specific iodine uptake, radioisotope accumulation, and treatment. In this study, we directly compared the human and rat NIS (rNIS) for their ability to concentrate radioisotope into human and rodent cancer cells. Perchlorate-sensitive (125)I uptake in multiple cell lines was demonstrated following transduction with retroviral vectors expressing either the human or rNIS gene. Surprisingly, iodine uptake was consistently higher with the rNIS gene, up to 5-fold greater, when compared to the human gene, even within a variety of human tumor cell lines. This iodine uptake allowed for cell killing following (131)I treatment in NIS-transduced cells when assayed by in vitro clonogenic assays. These results demonstrate that the rNIS gene provides superior iodine uptake ability, and may be preferable for use in designing anticancer gene therapy approaches.     

Sodium/iodide symporter gene transfection increases radionuclide uptake in human cisplatin-resistant lung cancer cells

The sodium/iodide symporter (NIS) is involved in iodide uptake and has been used for the diagnosis and treatment of thyroid cancer. Transfection of the NIS gene in A549 human lung cancer cells can induce radioactive iodine (¹³¹I) and radioactive technetium (^99mTc) uptake. The aim of the present study was to assess the role of NIS in ^99mTc and ¹³¹I uptake by the A549/DDP human cisplatin-resistant lung cancer cell line. To do so, recombinant adenovirus, adenovirus-enhanced green fluorescent protein-human NIS (Ad-eGFP-hNIS) and Ad-eGFP-rat NIS (Ad-eGFP-rNIS) vectors were established. These vectors were transfected into A549/DDP cells and xenograft tumors in nude mice. Assessment of ^99mTc and ¹³¹I uptake was performed. Results showed that the transfection efficiency of Ad-eGFP-hNIS and Ad-eGFP-rNIS in A549/DDP cells was at least 90 % in all experiments, and that the uptake ability of ^99mTc and ¹³¹I was highly enhanced (14–18 folds for ^99mTc, and 12–16 folds for ¹³¹I). However, the radionuclide concentration in transfected NIS genes’ A549/DDP cells reached a plateau within 30–60 min, indicating that NIS transport led rapidly to ^99mTc and ¹³¹I saturation in cells. In xenograft tumor models, uptake of ^99mTcO₄ ^? was obviously higher in the hNIS and rNIS groups compared with controls. In conclusion, these results support the hypothesis that A549/DDP cells can effectively uptake ^99mTc and ¹³¹I when transfected with the hNIS and rNIS gene. The rNIS or hNIS gene could be used as an effective method for the effective delivery of radioactive products to specific tissues for imagery and/or treatment.     

全反式维甲酸对甲状腺癌细胞NIS基因表达及吸碘能力影响的实验研究

目的：探讨全反式维甲酸（ATRA）对甲状腺癌细胞株钠/碘同向转运体（NIS）基因表达、吸碘能力的影响,为ATRA用于放射性碘治疗甲状腺癌提供理论依据。方法：分别以不同浓度（10^-7mol/L、10^-6mol/L、10^-5mol/L、10^-4mol/L）的ATRA处理体外培养的甲状腺癌细胞株（FTC-133）,48h后利用半定量RT-PCR检测细胞NISmRNA表达,γ-计数仪检测细胞吸碘能力。结果：ARTA浓度在（0～10%-5）mol/L范围内,细胞NIS基因表达及吸碘能力随ARTA剂量的增加而增加（P〈0.05）。当ARTA浓度达10%-4mol/L时,增加与前一浓度相比无统计学意义（P〉0.05）。结论：ATRA可上调甲状腺癌FTC-133细胞NIS基因表达,增强其吸碘能力,而且这种作用在一定浓度范围内具有剂量依赖性。     

重组腺病毒携带钠碘转运体基因介导放射性碘治疗小鼠胶质瘤

目的 验证转染人钠碘转运体基因(hNIS)介导放射性碘治疗肿瘤的有效性.方法 利用重组腺病毒将hNIS基因及人甲状腺过氧化物酶(hTPO)基因转染入人胶质瘤细胞系U251中,使肿瘤细胞获得hNIS和 hTPO基因,然后进行体外摄125I实验、过氯酸盐抑制实验、体外125I反流及内流实验.应用转染hNIS和hTPO基因及未转染hNIS和hTPO基因的细胞株,分别建立荷瘤裸鼠模型,并检测131I对肿瘤的抑制作用.结果 利用腺病毒可以将hNIS和hTPO基因成功转染到U251细胞系中,转染上述基因的肿瘤细胞系可以摄取碘,而且这种摄碘的功能是由hNIS基因所介导的,转染后的hNIS-U251细胞系摄碘能力是U251细胞的121.2倍,hNIS-hTPO-U251细胞系摄碘能力是U251细胞的172.0倍.131I对裸鼠移植瘤的治疗结果表明,在131I作用下,对照组肿瘤均继续迅速生长,而hNIS转染组及hNIS和hTPO共转染组移植瘤体积均有所减小.结论 在肿瘤细胞中转染hNIS和hTPO基因后,可以提高其摄取12I的活性.131I 可以有效杀伤荷瘤裸鼠体内的中瘤细胞.

Abstract：

Objective To explore the possibility of tranfecting hNIS and hTPO genes into gliomas cells by recombinant adenovirus for radioactive iodide treatment. Methods To tranfect hNIS gene into human glioma cell line U251 by recombinant adenovirus.The biological functions of the cells stably expressing hNIS and hTPO genes were assessed by 1251 uptake assay,125I influx-course and 12I-effluxcourse.A glioma model was established with inoculation of the U251 cells in nude mice,and the inhibiting effect of 131 I on the tumor growth was tested in the mouse models. Results The hNIS and hTPO genes were successfully transfected into human gliomas cell line U251 cells by recombinant adenovirus.The radioactive iodide could be intaken by the tumor cells mediated by hNIS gene.The uptake of 125I was higher in cell lines hNIS-U251 and hNIS-hTPO-U251 cells than in cell line U251 cells.The tumor volume of the mice after 131I treatment was significantly decreased in comparison with that before treatment.Conclusion Radioactive 131I treatment after HNIS-based gene transfer can be enhanced and effectively inhibite the tumor growth in nude mice.     

甲状腺乳头状癌BRAFV600E基因突变与钠碘同向转运体表达的研究

目的:探讨甲状腺乳头状癌(PTC)患者中BRAFV600E基因突变及钠碘同向转运体(NIS)蛋白的表达及两者间的相关性.方法:收集2008-01-01—2011-01-01青岛大学医学院附属医院病理科PTC石蜡包埋组织30例,其中癌旁组织为正常组织(G正常组)15例,癌旁组织为结节性甲状腺肿组织(G结甲组)15例.对PTC组织行DNA提取、PCR扩增、基因测序检测BRAFV600E基因突变,同时采用免疫组化的方法分析NIS蛋白表达,结果以免疫组化评分(IHS)表示.结果:30例PTC均无BRAFV600E基因突变;30例PTC组织中NIS蛋白表达,从0～12分,G正常组IHS=(7.92±3.01)分,G结甲组IHS=(6.58±2.71)分,两组NIS差异无统计学1意义,t=1.11,P=0.95;BRAFV600E突变与NIS蛋白表达无相关性.结论:尚未发现癌旁不同的PTCBRAFV600E基因有突变及NIS蛋白表达的差异.BRAFV600E基因突变与NIS表达在PTC中的关系有待进一步研究.     

Expression of sodium iodide symporter in metastatic and follicular human thyroid tissues

Active iodide uptake across the basal membrane mediated by human sodiumiodide symporter (hNIS) has been shown to be a process coupled with the flowof sodium. There is still controversy as to the amount of hNIS expressionpresent in different kinds of human thyroid cancer tissues. In this study, wepresent a 58-year-old women with follicular thyroid carcinoma with vertebraand skull metastases. ²⁰¹Tl and 5 mCi ¹³¹I scans clearlydemonstrated the metastatic lesions in the brain of this patient. Thyroid andmetastatic tissues were then obtained for this study, which is aimed atcomparing the iodide trapping ability in vivo and in vitro of hNIS, and then comparing their expression in both thyroid tissue andmetastatic tissues. Polyclonal antibodies to hNIS and competitive RT–PCRwere used to analyze the symporter protein and mRNA expressed in follicularhuman thyroid and metastatic tissues. Positive staining of the symporterprotein was performed in the follicular thyroid carcinomas, otherwise, themetastatic tissues could not have demonstrated the protein in the staining.Follicular thyroid carcinoma tissues from thyroid were revealed around 5 pghNIS expressed in follicular thyroid carcinoma tissues from the thyroid.Otherwise, there was almost an absence of hNIS expression in the metastatictissue. These discrepancies of the expression in hNIS in vivo and in vitrostudies need further investigation.     

Radiofrequency induction on sodium/iodide symporter expression of thyroid cancer

Objective:The aim of this study was to investigate the ef ects of radiofrequency treatment on sodium/iodide symporter expression of thyroid cancer cells. Methods:In 29 thyroid cancer patients with low or no expression of sodaiodide symporter, the radio frequency combined 131I therapy was used, the whole-body scintigraphy and serum Ig were detected before and after the radiofrequency treatment. Results:The whole-body scintigraphy showed that 4 cases (4/29) before radiofrequency treatment had positive iodine uptake, 19 cases (19/29) two weeks after radiofrequency treatment had the positive iodine uptake, 12 cases (12/29) four weeks after radiofrequency treatment had the positive iodine uptake. Four weeks after radiofrequency treatment, 5 cases had increased serum Ig levels, 17 cases had decreased serum Ig levels, 7 cases showed no change. 25 cases (25/29) were ef ective, 15 cases (15/29) were cured. Conclusion:The radiofrequency induced the non-expressed the sodium/iodide symporter of thyroid cancer cells regain the iodine intake ability, it improved the clinical ef icacy of 131I therapy in dedif erentiated thyroid cancer.     

钠/碘转运体在甲状腺癌中的表达

目的:探讨钠/碘转运体(sodium iodide symporter,NIS)在甲状腺癌组织中的表达.方法:应用实时荧光定量PCR(real-time fluorescence quantitative-PCR, RFQ-PCR)和免疫组织化学法检测NIS mRNA和蛋白在18例正常甲状腺组织、27例结节性甲状腺肿组织和23例甲状腺癌组织中的表达.结果: 甲状腺癌组织中NIS mRNA的表达显著低于正常甲状腺组织和结节性甲状腺肿组织(P=0.002).免疫组织化学检测结果表明,甲状腺癌组织中NIS蛋白主要定位于细胞质,强阳性表达13例(56.6%)、阳性表达3例(13.0%),强阳性表达率显著高于正常甲状腺组织(P=0.010).与正常甲状腺组织相比,甲状腺癌组织中NIS mRNA表达下降并且NIS蛋白表达升高者17例(74%),显著高于结节性甲状腺肿组织(χ2=4.428,P=0.035).结论:甲状腺癌组织中NIS mRNA的表达下降而NIS蛋白的表达增强,推测NIS蛋白主要位于细胞质可能是NIS不能发挥正常的运输功能,导致甲状腺癌患者不能聚碘的重要原因之一.     

人类DBC2基因重组腺病毒的构建及其在膀胱癌中的表达(英文)

Objective: The aim of the study was to construct recombinant type 5 adenovirus expressing the human DBC2(deleted in breast cancer 2) gene for in vitro and in vivo assay in human bladder cancer research. Methods: The human DBC2gene was first subcloned into a shuttle plasmid pAdTrack-CMV. After recombining with pAdEasy-1 vector in BJ5183 cells, thenew recombinant vector pAdEasy-DBC2-CMV was transfected into HEK-293 cells to produce adenovirus. The human bladdercancer cell line T24 was infected with DBC2-containing adenovirus particles. Both RNA and protein were collected from cellsharvested at 72 h after infection. Real time quantitative PCR (qPCR) and Western blot were used to examine mRNA and proteinlevels. Fluorescence microscopy was utilized to observe the expression of reporter green fluorescence protein. Results:Electrophoresis showed there was a 2.2 kb size band produced from high fidelity PCR. Pac I digest of the final producedrecombinant vector yielded band sizes of approximately 30 kb and 4.5 kb. After virus infection with the pAdEasy-DBC2-CMVvector, the T24 cell line was observed to highly express green fluorescence protein under a fluorescence microscope. qPCRand Western blot assay identified that the DBC2 gene was overexpressed at both the mRNA and protein levels in virustransfected cells. Conclusion: By using the pAdEasy adenovirus system, we successfully constructed an adenovirus thatcould highly overexpress the tumor suppressor DBC2 gene in a bladder cancer cell line. This viral construct would be widelyused for our further research in gene functional assays and gene therapy in bladder cancer.     

细胞因子对乳腺癌细胞钠碘转运体基因表达的调控

目的本实验通过三种细胞因子(肿瘤坏死因子-α,干扰素-γ和白细胞介素-6)对乳腺细胞钠碘转运体(NIS)基因表达的影响,探讨乳腺肿瘤组织NIS的表达特点和调控因素.方法采用乳腺癌细胞MCF-7和MB453进行培养,分别给予不同浓度的肿瘤坏死因子-α,干扰素-γ或白细胞介素-6刺激72 h.采用RT-PCR方法检测细胞中NIS mRNA表达情况.结果乳腺癌细胞在经过肿瘤坏死因子-α,干扰素-γ或白细胞介素-6刺激后,细胞中NIS的mRNA表达比对照组明显降低,并且与细胞因子浓度呈负相关.结论肿瘤坏死因子-α,干扰素-γ和白细胞介素-6对乳腺癌细胞MCF-7和MB453的NIS基因mRNA表达有抑制作用,这提示细胞因子可能通过调节乳腺癌NIS基因mRNA的表达,从而影响乳腺癌细胞对放射性碘治疗的敏感性.     

Adenovirus-mediated transfer of the thyroid sodium/iodide symporter gene into tumors for a targeted radiotherapy

The Na+/I- symporter (NIS) present in the membranes of thyroid cells is responsible for the capacity of the thyroid to concentrate iodide. This allows treatment of thyroid cancers with 131I. We propose to enlarge this therapeutic strategy to nonthyroid tumors by using an adenoviral vector to deliver the NIS gene into the tumor cells. We constructed a recombinant adenovirus encoding the rat NIS gene under the control of the cytomegalovirus promoter (AdNIS). Infection of SiHa cells (human cervix tumor cells) with AdNIS resulted in perchlorate-sensitive 125I uptake by these cells to a level 125-225 times higher than that in noninfected cells. Similar results were obtained for other human tumor cell lines, including MCF7 and T-47D (mammary gland), DU 145 and PC-3 (prostate), A549 (lung), and HT-29 (colon), demonstrating that the AdNIS vector can function in tumor cells of various origins. In addition, AdNIS-infected tumor cells were selectively killed by exposure to 131I, as revealed by clonogenic assays. To assess the efficiency of this cancer gene therapy strategy in vivo, we injected the AdNIS vector in human tumors (SiHa or MCF7 cells) established s.c. in nude mice. Immunohistological analysis confirmed the expression of the NIS protein in the tumor. Three days after intratumoral injection, AdNIS-treated tumors could specifically accumulate 125I or 123I, as revealed by kinetics and imaging experiments. A quantitative analysis demonstrated that the uptake in AdNIS-injected tumors was 4-25 times higher than that in nontreated tumors. On average, 11% of the total amount of injected 125I could be recovered per gram of AdNIS-treated tumor tissue. Altogether, these data indicate that AdNIS is very efficient in triggering significant iodide uptake by a tumor, outlining the potential of this novel cancer gene therapy approach for a targeted radiotherapy.     

Ectopic expression of the thyroperoxidase gene augments radioiodide uptake and retention mediated by the sodium iodide symporter in non-small cell lung cancer

Radioiodide is an effective therapy for thyroid cancer. This treatment modality exploits the thyroid-specific expression of the sodium iodide symporter (NIS) gene, which allows rapid internalization of iodide into thyroid cells. To test whether a similar treatment strategy could be exploited in nonthyroid malignancies, we transfected non-small cell lung cancer (NSCLC) cell lines with the NIS gene. Although the expression of NIS allowed significant radioiodide uptake in the transfected NSCLC cell lines, rapid radioiodide efflux limited tumor cell killing. Because thyroperoxidase (TPO) catalyzes iodination of proteins and subsequently causes iodide retention within thyroid cells, we hypothesized that coexpression of both NIS and TPO genes would overcome this deficiency. Our results show that transfection of NSCLC cells with both human NIS and TPO genes resulted in an increase in radioiodide uptake and retention and enhanced tumor cell apoptosis. These findings suggest that single gene therapy with only the NIS gene may have limited efficacy because of rapid efflux of radioiodide. In contrast, the combination of NIS and TPO gene transfer, with resulting TPO-mediated organification and intracellular retention of radioiodide, may lead to more effective tumor cell death. Thus, TPO could be used as a therapeutic strategy to enhance the NIS-based radioiodide concentrator gene therapy for locally advanced lung cancer.     

Dual therapy of ovarian cancer using measles viruses expressing carcinoembryonic antigen and sodium iodide symporter.

PURPOSE: MV-CEA is an oncolytic measles virus currently being tested in patients with ovarian cancer and whose propagation can be monitored by measuring blood carcinoembryonic antigen (CEA) levels. MV-NIS is an oncolytic measles virus coding for the thyroidal sodium iodide symporter (NIS) whose propagation can be mapped by serial radioiodine imaging. Expression of both CEA and NIS genes from a single virus would combine sensitive, quantitative expression monitoring (CEA) with radioisotopic expression mapping (NIS). Because of the unfavorable replication kinetics of measles viruses expressing both CEA and NIS, we explored the feasibility of combining MV-CEA with MV-NIS for comprehensive virotherapy monitoring in ovarian cancer. EXPERIMENTAL DESIGN AND RESULTS: Mice implanted with i.p. SKOV3ip.1 ovarian cancer xenografts received MV-CEA alone, MV-NIS alone, or a combination of MV-CEA plus MV-NIS. Viral gene expression was monitored by measuring blood CEA levels, and the location of virus-infected cells was monitored by gamma camera imaging. Surprisingly, mice receiving the combination of MV-CEA plus MV-NIS showed greatly superior responses to therapy, but this was associated with 10-fold lower plasma levels of CEA compared with mice treated with MV-CEA alone. In vitro studies showed superior replication kinetics of MV-NIS relative to MV-CEA. The gamma camera scans were considerably less sensitive than the plasma CEA marker for monitoring virus infection. CONCLUSIONS: Dual therapy with MV-CEA and MV-NIS is superior to treatment with either virus alone, and it allows noninvasive monitoring of virotherapy via soluble marker peptide and gamma camera imaging. This has important implications for the clinical development of oncolytic measles viruses.     

hTPO和hNIS共转染胶质瘤细胞碘摄取和^131I治疗的实验研究

目的构建含有人甲状腺过氧化物酶基因（hTPO）的重组腺病毒,并与人钠-碘转运体基因（hNIS）共转染至神经胶质瘤细胞中,研究其摄碘能力及对肿瘤细胞的杀伤能力,探讨131I治疗胶质瘤的可能性。方法应用AdEasy系统构建重组腺病毒AdTPO,利用重组质粒将hNIS基因转染入神经胶质瘤细胞系U251中获得hNIS—U251细胞系作为阴性对照组,再利用重组腺病毒AdTPO将TPO基因转染入hNIS—U251细胞系中获得AdTPO—hNIS—U251作为实验组,未转入hTPO和hNIS的细胞U251作为空白对照组。研究三组细胞的摄碘实验、过氯酸盐抑制实验、有机化测定实验检测其摄碘功能,细胞克隆形成实验评价131I对转染肿瘤的杀伤作用。结果成功构建出重组腺病毒AdTPO,并在稳定表达hNIS的U251细胞中实现了hTPO的共转染。hNIS—U251组（每分钟放射性计数55769．96±4353．26）比空白对照组（每分钟放射性计数507．67±57．69）摄碘能力增高约110倍,有效半衰期7分钟,有机化程度约为0．1％,细胞克隆形成率（9．08±2．86）％,较对照组减低约10倍。AdTPO—hNIS—U251组（每分钟放射性计数74647．53±3605．88）比空白对照组摄碘能力增高约147倍,有效半衰期延长至13分钟,有机化程度增高至10％,细胞克隆形成率（6．80±2．09）％,较对照组减低约13倍。结论将hTPO和hNIS共转染至神经胶质瘤细胞后,可有效提高细胞的摄碘能力,hTPO延长了放射性碘在细胞中的停留时间,131I对瘤细胞有较强的杀伤作用。     

Targeting sodium/iodide symporter gene expression for estrogen-regulated imaging and therapy in breast cancer

Expression of the sodium iodide symporter (hNIS) has been detected in breast cancer tissue, but frequently, not at the levels necessary to mediate (131)I accumulation. Transducing the hNIS gene into breast cancer cells with adenovirus could be a tractable strategy to render breast cancer susceptible to radioiodide therapy. We constructed the replication-incompetent virus, AdSERE, in which an estrogen-responsive promoter directs the expression of hNIS. In vitro, we demonstrate that AdSERE mediates hNIS expression and iodide uptake in ER+ breast cancer cells. In vivo, we show that AdSERE-infected ER+ tumors can be imaged due to tracer accumulation; in addition, AdSERE in combination with therapeutic doses of (131)I suppresses tumor growth.