Immune response to firefly luciferase as a naked DNA

Firefly luciferase (Fluc) has been widely used as a reporter gene. The aim of this study was to investigate immune response to luciferase protein after an intradermal injection of pcDNA3.1-Fluc in immunocompetent BALB/c mice. We observed bioluminescence at injection sites from one to seven days post-injection when pcDNA3.1-Fluc was intradermally injected into ear-pinnae. To observe induced immune response, the percentages of CD8+IFNgamma+ cells in the draining lymphoid cells of immunocompetent BALB/c mice immunized by pcDNA3.1-Fluc were measured. And the tumor growths of CT26/Fluc in pcDNA3.1-Fluc group were monitored by observing bioluminescent signals and measuring tumor mass, and these were compared with those of the pcDNA3.1 group in immunocompetent BALB/c mice and immunodeficient Nu/Nu mice. In the immunocompetent BALB/c mice, percentages of CD8+IFNgamma+ cells in the pcDNA3.1-Fluc group were higher than those in the pcDNA3.1 group. Ten days after tumor inoculation, tumor growth inhibition was found in the pcDNA3.1-Fluc group, but not in the pcDNA3.1 group in the immunocompetent BALB/c mice. No significant difference in tumor growth inhibition was observed when CT26/Fluc was injected into immunodeficient Nu/Nu mice. In terms of cytokine profiles of draining lymphoid cells of immunized mice, IFNgamma protein levels in the pcDNA3.1-Fluc group were higher than in pcDNA3.1 group animals among the immunocompetent BALB/c mice. In conclusion, Fluc induced a Th1 immune response to Fluc protein delivered by injecting pcDNA3.1-Fluc into immunocompetent BALB/c mice. We suggest that immune response to the Fluc gene is cautionary in preclinical or clinical trials involving the Fluc gene, and that the immunologic potential of firefly luciferase as a naked DNA may be useful in cancer immunotherapy.     

Combined E7-dendritic cell-based immunotherapy and human sodium/iodide symporter radioiodine gene therapy with monitoring of antitumor effects by bioluminescent imaging in a mouse model of uterine cervical cancer

Using a uterine cervical cancer cell line expressing human papillomavirus (HPV) 16 E7 antigen and bioluminescent imaging (BLI), we evaluated the therapeutic potential of combined immunotherapy using transfected dendritic cells (DC-E7) and human sodium/iodide symporter (hNIS) radioiodine gene therapy in a xenograft animal cancer model. Dendritic cells expressing either E7 antigen (DC-E7) or no-insert (DC-no insert) were made for immunization materials, and murine uterine cervical cancer cell line coexpressing E7, firefly luciferase, hNIS, and EGFP genes (TC-1/FNG) were prepared for the animal tumor model. C57BL/6 mice were divided into five therapy groups (phosphate-buffered saline [PBS], DC-no insert, DC-E7, I-131, and DC-E7+I-131 groups). Single therapy with either DC-E7 or I-131 induced greater retardation in tumor growth compared with PBS or DC-no insert groups, and it resulted in some tumor-free mice (DC-E7 and I-131 groups, 40% and 20%, respectively). Combination therapy with DC-E7 and I-131 dramatically inhibited tumor growth, thus causing complete disappearance of tumors in all mice, and these effects were further confirmed by BLI in vivo. In conclusion, complete disappearance of the tumor was achieved with combined DC-E7 vaccination and hNIS radioiodine gene therapy in a mouse model with E7-expressing uterine cervical cancer, and serial BLIs successfully demonstrated antitumor effects in vivo.     

Human sodium iodide symporter added to multidrug resistance 1 small hairpin RNA in a single gene construct enhances the therapeutic effects of radioiodine in a nude mouse model of multidrug resistant colon cancer

The objective of this study was to investigate the therapeutic potential of 131I added to doxorubicin therapy in multidrug resistance (MDR) mouse colon cancer coexpressing the MDR1 small hairpin RNA (shRNA) and human sodium iodide symporter (hNIS) gene in a single gene construct and to visualize the antitumor effects using molecular nuclear imaging. HCT-15 coexpressing shRNA for MDR1 gene (MDR1 shRNA) and hNIS gene with a single construct was established (referred to as MN61 cell). Inhibition of P-gp function by MDR1 shRNA and functional activity of hNIS gene was assessed using a ??(m)Tc sestamibi uptake and 12?I uptake, respectively. Cytotoxic effects by a combination of doxorubicin and 131I were determined in parental (HCT-15) or MN61 cells using an in vitro clonogenic assay. Therapeutic effect of either combination therapy (doxorubicin and 131I) or single therapy (doxorubicin or 131I alone) was evaluated by tumor volume measurement. ??(m)Tc-sestamibi, 123I, and ??(m)Tc-pertechnetate images of mice were acquired to evaluate functional assessment in vivo. Cellular uptake of ??(m)Tc-sestamibi and 12?I was approximately 2-fold and 100-fold higher in MN61 cells than in parental cells, respectively. Combination of 131I and doxorubicin resulted in higher cytotoxcity in MN61 cells as compared with parental cells. Scintigraphic imaging showed higher uptake of ??(m)Tc-sestamibi and 123I in MN61 tumor as compared with parental tumor. In mice treated with doxorubicin, there was a slight delay in tumor growth in the MN61 tumor but not in the parental tumor. Cancer treatment with 131I or doxorubicin induced a rapid reduction of tumor volume in the MN61 tumor but not in the parental tumor. Combination therapy further generated a rapid reduction of tumor volume as compared with 131I therapy alone (p?相似文献   

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.     

Exosomes: a new delivery system for tumor antigens in cancer immunotherapy

Exosomes are small membrane vesicles that are released into the extracellular environment during fusion of multivesicular bodies with plasma membrane. Exosomes are secreted by various cell types including hematopoietic cells, normal epithelial cells and even some tumor cells. They are known to carry MHC class I, various costimulatory molecules and some tetraspanins. Recent studies have shown the potential of using native exosomes as immunologic stimulants. Here, we demonstrate a novel means of using exosomes engineered to express a specific tumor antigen to generate an immune response against tumors. We expressed a target tumor antigen, human MUC1 (hMUC1), in 2 MHC type-distinct mouse cell lines, CT26 and TA3HA. Analysis of exosomes purified from these cells revealed that exosomes contained the target MUC1 antigen on their surfaces as well as other well-described exosomal proteins, including Hsc70 and MHC class I molecules. In addition, both autologous and allogenic exosomes were able to stimulate the activation of immune cells and suppress hMUC1-expressing tumor growth in a MUC1-specific and dose-related manner. Therefore, these data suggest that exosomes can be engineered from tumor cell lines to deliver a target immunogen capable of inducing an effective immune response and that they may represent a new cell-free tumor vaccine.     

In vivo long-term imaging and radioiodine therapy by sodium-iodide symporter gene expression using a lentiviral system containing ubiquitin C promoter

To establish stable and long-term gene expression in vitro and in vivo, we developed a lentiviral vector system carrying sodium iodide symporter (hNIS) gene under UbC promoter, and transfected this into a colon cancer cell line. The in vitro and in vivo kinetics of radioiodine and [99mTc]-pertechnetate were then investigated, and the therapeutic effect of 1-131 was evaluated in this system. The hNIS gene was transferred into CT26 cells using lentivirus containing UbC promoter. In vitro iodide uptake and efflux were measured in CT26-hNIS cells at various time points. In addition, scintigraphic images were acquired at 30 min after injecting [99mTc]-pertechnetate i.p. into Balb/C mice for 27 days after CT26-hNIS induction. Biodistribution studies were performed at 10 and 30 min and at 1.5, 6 and 24 h after [99mTc]-pertechnetate injections, and the therapeutic effects of radioiodine were investigated by measuring tumor size using a caliper or by quantifying tumor radioactivity levels in scintigraphic images. The iodide uptakes of CT26-hNIS tumors were 10-fold greater than those of CT26 tumors. In addition, iodide uptake was completely blocked by 100 microM potassium perchlorate. The accumulation of [99mTc]-pertechnetate in hNIS expressing tumor cells was found to be positively related to tumor growth. In biodistribution studies, the %ID/g values of CT26-hNIS were 84.0 +/- 4.5 at 1.5 h and 40.8 +/- 3.9 at 24 h and these were approximately 60 times greater than those of CT26 at these time points. Tumor growth in mice treated with 131I was retarded until 46 days post-tumor challenge. The devised lentiviral vector system carrying hNIS controlled by UbC promoter was found to be suitable for the long-term monitoring and radionuclide therapy of cancer in living organism.     

miR-222通过靶向RB1促进视网膜母细胞瘤细胞生长与侵袭

背景与目的：视网膜母细胞瘤基因1(retinoblastoma 1,RB1)能够抑制多种肿瘤的发生、发展,且与细胞周期、分化、衰老、凋亡及生长抑制等调控密切相关。该研究旨在明确miR-222是否通过靶向RB1表达而促进视网膜母细胞瘤细胞的生长与侵袭,进一步揭示miR-222促瘤作用的分子机制。方法：将miR-222(miR-222模拟物)+RB1-wt(野生型RB1的3’-非翻译区的荧光素酶报告载体)、miR-NC(无关序列对照)+RB1-wt、miR-222+RB1-mut(突变型RB1的3’-非翻译区的荧光素酶报告载体)及miR-NC+RB1-mut共转染人视网膜母细胞瘤细胞株Y79,并采用单光子检测荧光素酶活性。采用蛋白[质]印迹法(Western blot)检测RB1表达水平的改变。将miR-222与miR-NC、RB1(pcDNA3.1-RB1)与vector(pcDNA3.1)、miR-222+RB1及miR-NC+vector转染Y79细胞,MTS检测细胞生长增殖活性,Transwell侵袭实验检测Y79细胞生长与侵袭能力的影响。结果：与miR-NC+RB1-wt组比较,共转染miR-222+RB1-wt组的荧光素酶活性强度降低了约56.67%(P<0.05)。与miR-NC比较,miR-222组RB1蛋白水平显著下调(P<0.05)。转染miR-222组细胞生长速度显著高于miR-NC组(P<0.05)。与pcDNA3.1组比,pcDNA3.1-RB1组可显著抑制Y79细胞的生长(P<0.05),而miR-222+pcDNA3.1-RB1组和miR-NC+pcDNA3.1组比较,细胞生长速度差异无统计学意义(P>0.05)。转染miR-222组穿过基底膜的细胞数分别为(193±10),与对照组(144±11)比较能明显加快Y79细胞的穿膜能力,差异有统计学意义(P<0.05)。而miR-NC+pcDNA3.1组和miR-222+pcDNA3.1-RB1组比较,穿过基底膜的细胞数差异无统计学意义(P>0.05)。结论：miR-222通过靶向调控RB1表达而促进视网膜母细胞瘤细胞的生长与侵袭。     

MIDGE/hNIS vaccination generates antigen-associated CD8+IFN-gamma+ T cells and enhances protective antitumor immunity

Human sodium iodide symporter (hNIS) is a transmembrane protein that actively transports iodide ions into thyroid cells. hNIS is over-expressed in some cases of the thyroid cancers compared with the surrounding normal tissues and has been considered to be an attractive target for immunotherapy. The aim of this study is to determine the feasibility of utilizing the hNIS antigenic protein in enhanced-antigen-associated immunotherapy using image analysis with a gamma counter. To accomplish this, minimalistic immunogenically defined gene expression (MIDGE), either plain or coupled to a nuclear localization signal (NLS) peptide, was used as a vector system. Vaccination with MIDGE/hNIS, MIDGE/hNIS-NLS and pcDNA3.1/hNIS produced a significant increase in the number of hNIS-associated IFN-gamma-secreting CD8(+) T cells, with MIDGE/hNIS having the strongest effect. In addition, immunization with the hNIS encoding vectors induced antigen-mediated antitumor activity against NIS-expressing CT26 tumors in vivo, with the highest tumor free rate (100%) and lowest tumor growth being observed up to 40 days after the CT26/NIS tumor challenge with MIDGE/hNIS than those resulting from other immunization groups. Tumor progression could be followed noninvasively and repetitively by monitoring levels of hNIS gene expression in the tumors using scintigraphic image analysis. Overall, hNIS has a potential use as an antigen for immunization approaches, and vaccination with MIDGE/hNIS vectors is an effective means of generating hNIS-associated immune responses in mice.     

自噬基因Beclin1对宫颈癌HeLa细胞生长影响的体内外研究

目的 研究自噬基因Beclin1在宫颈癌细胞株HeLa中过表达对HeLa细胞体内外生长的影响.方法 构建自噬基因Beclin1的真核表达载体pcDNA3.1(+)-Beclin1,转染HeLa细胞[pcDNA3.1(+)-Beclin1组],同时设空载体转染组[pcDNA3.1(+)组]和空白对照组.采用荧光定量PCR和Western blot法检测3组HeLa细胞中Beclin1 mRNA和蛋白的表达变化,采用四甲基偶氮唑蓝(MTT)法检测3组HeLa细胞生长的变化,采用流式细胞术检测3组HeLa细胞的凋亡情况,采用单丹磺酰尸胺染色检测3组HeLa细胞的自噬情况.将3组HeLa细胞分别接种于裸鼠皮下,观察体内成瘤性和生长情况.结果 pcDNA3.1(+)-Beclin1组、pcDNA3.1(+)组和空白对照组HeLa细胞中Beclin1 mRNA的表达水平分别为994.718 ±468.764、0.570±0.121和0.736±0.251.pcDNA3.1 (+ )-Beclin1组HeLa细胞中Beclin1 mRNA的表达水平较pcDNA3.1(+)组和空白对照组明显增高(P＜0.05),而pcDNA3.1(+)组与空白对照组间的差异无统计学意义(P＞0.05);3组HeLa细胞中Beclin1蛋白的表达情况与mRNA相似.pcDNA3.1(+)-Beclin1组的自噬细胞率为10.9％,明显高于空白对照组和pcDNA3.1(+)组(分别为2.5％和3.1％,P＜0.05).pcDNA3.1(+)-Beclin1组HeLa细胞的凋亡率为(28.2±2.3)％,明显高于pcDNA3.1(+)组和空白对照组[分别为(14.6±4.6)％和(11.2±3.0)％,P＜0.05].Beclin1在HeLa细胞中过表达可抑制肿瘤细胞在体外的生长,抑制率为58.7％;并可使HeLa细胞在裸鼠体内成瘤时间延长,瘤体体积和重量明显小于pcDNA3.1 (+)组和HeLa组(P＜0.05),抑瘤率为52.2％.结论 自噬基因Beclin1过表达可抑制HeLa细胞在体内外的增殖,促进细胞的自噬与凋亡,为宫颈癌基因治疗提供了新的选择途径.     

FOLFOX4联合1-MT对胃癌小鼠皮下移植瘤的抑制作用

目的： 观察FOLFOX4联合1-甲基-色氨酸 (1-methyl tryptophan,1-MT)对荷胃癌小鼠皮下移植瘤生长的抑制作用,以及对胃癌组织吲哚胺-2,3-双加氧酶 (indoleamine-2,3-dioxygenase,IDO)表达的影响。方法：将IDO真核表达质粒 (pcDNA3.1-IDO)转染小鼠胃癌细胞MFC,建立稳定表达IDO的细胞株,RT-PCR与Western blot法检测细胞中IDO的表达。小鼠皮下接种转染pcDNA3.1-IDO的MFC细胞悬液,建立高表达IDO的小鼠胃癌皮下移植瘤模型 (32只),同时设空白对照 (8只,接种未转染的MFC细胞)和阴性对照 (8只,接种转染pcDNA3.1质粒的MFC细胞),观察各组小鼠成瘤情况。将32只模型小鼠随机分为1-MT治疗组、FOLFOX4治疗组、FOLFOX4+1-MT联合治疗组和未治疗组 (阳性对照组),每组8只。治疗组分别注射给予1-MT、 FOLFOX4或FOLFOX4+1-MT,阳性对照组给予生理盐水。观察各组小鼠一般情况及肿瘤质量差异。注射细胞悬液后第12天处死所有小鼠,剥离瘤体并称重,计算各组小鼠平均瘤质量及抑瘤率,另取肿瘤标本采用免疫组织化学染色法检测胃癌组织中IDO的表达。结果：RT-PCR与Western blot均检测到pcDNA3.1-IDO转染细胞中IDO的表达。高表达IDO的胃癌皮下移植瘤模型小鼠肿瘤质量大于空白对照组和阴性对照组小鼠 (P<0.05),且其胃癌组织IDO的表达亦较空白对照组和阴性对照组明显增加 (P<0.05)。给予1-MT、FOLFOX4 和FOLFOX4+1-MT治疗后,模型小鼠进食量均不同程度增加,行动较之前灵活,嗜睡也有不同程度好转,FOLFOX4+1-MT联合治疗组小鼠症状基本缓解。1-MT治疗组、 FOLFOX4治疗组和FOLFOX4+1-MT联合治疗组小鼠肿瘤质量均小于未治疗组 (P<0.05),其抑瘤率分别为 8.91%、80.20%、86.13%,FOLFOX4+1-MT联合治疗组小鼠肿瘤质量小于1-MT治疗组和FOLFOX4治疗组 (P<0.05)。1-MT治疗组、FOLFOX4治疗组和FOLFOX+1-MT联合治疗组小鼠胃癌组织中IDO的表达均低于未治疗组 (P<0.05);FOLFOX4+1-MT联合治疗组胃癌组织中IDO的表达低于1-MT治疗组和FOLFOX4治疗组 (P<0.05)。结论：1-MT对胃癌小鼠皮下移植瘤的生长和胃癌组织IDO的表达具有抑制作用,并能与FOLFOX4发挥协同抗肿瘤作用。     

Probasin promoter (ARR(2)PB)-driven, prostate-specific expression of the human sodium iodide symporter (h-NIS) for targeted radioiodine therapy of prostate cancer

Prostate cancer is one of the most promising candidates for sodium iodide symporter (NIS)-mediated gene therapy. Adenovirus-mediated expression of NIS that is driven by prostate-specific promoters induces generous radioiodine accumulation in prostate cancer cells that may be used for therapy with (131)I. We have recently developed a replication-deficient adenovirus carrying the human NIS cDNA linked to a composite probasin promoter, ARR(2)PB, aiming toward specific expression of the human NIS gene (h-NIS) in prostate tissue for targeted radioactive iodide therapy of prostate cancer (Ad-ARR(2)PB/hNIS). The ability of Ad-ARR(2)PB/hNIS to cause NIS expression in tumor cells was characterized by iodide uptake assay and compared with Ad-CMV/hNIS in which the h-NIS expression is driven by the cytomegalovirus (CMV) promoter. Androgen-dependent prostate cancer cell lines (LNCaP) and non-prostate origin tumor cell lines (SNU449, MCF-7, HCT116, OVCAR-3, and Panc-1) were infected with the viral constructs, and perchlorate-sensitive (125)I uptake and NIS protein expression were measured. Ad-ARR(2)PB/hNIS-infected LNCaP cells showed androgen-dependent and perchlorate-sensitive iodide uptake. Iodide accumulation in LNCaP cells infected with Ad-ARR(2)PB/hNIS, followed by incubation with synthetic androgen, was 5.3-fold increased compared with those coincubated with perchlorate (15,184 +/- 1,173 cpm versus 2,837 +/- 187 cpm). Ad-ARR(2)PB/hNIS-infected LNCaP cells revealed a 3.2-fold increase of iodide accumulation compared with those infected with Ad-CMV/hNIS (multiplicity of infection = 30). Iodide uptake in a panel of non-prostate tumor cell lines infected with Ad-ARR(2)PB/hNIS was no more than 2,500 cpm, demonstrating the tissue specificity of this construct. These results indicate that Ad-ARR(2)PB/hNIS can be used to achieve high-magnitude and tissue-specific expression of h-NIS in prostate tissue and is a promising candidate for cancer gene therapy of prostate cancer.     

Radioimmunotherapy of human colon cancer xenografts by using 131I labeled-CAb1 F(ab')2

PURPOSE: Therapeutic efficacy, suitable dose, and administration times of 131I-CAb1 F(ab')2, a new monoclonal antibody therapeutics specifically directed against a cell surface-associated glycoprotein of colon cancer, were investigated in this article. METHODS AND MATERIALS: In human colon cancer xenografts, 131I-CAb1 F(ab')2 at the dose of 125 muCi, 375 muCi, and 1125 muCi were administrated intraperitoneally on Days 6 and 18 after implantation of HR8348 cells with CAb1 high reactivity. Survival time and tumor growth inhibition rate were used to evaluate the efficacy and safety of 131I-CAb1 F(ab')2 in treatment of colon cancer xenografts. RESULTS: Treatment of 125, 375, and 1125 muCi 131I-CAb1 F(ab')2 did not significantly decrease the mean survival time of nude mice when compared with nontreated groups (p = 0.276, 0.865, 0.582, respectively). Moreover, the mean survival times of nude mice receiving 375 muCi and 1125 muCi 131I-CAb1 F(ab')2 were significantly longer than that of 5-FU-treated groups (p = 0.018 and 0.042). Tumor growth inhibition rates of the first therapy were 35.67% and 41.37%, with corresponding 131I-labeled antibody dosage of 375 muCi and 1125 muCi. After single attack dosage, second reinforcement therapy may rise efficacy significantly. Tumor growth inhibition rates of 125 muCi, 375 muCi, and 1125 muCi 131I-labeled antibody on Day 20 posttherapy were 42.65%, 56.56%, and 84.41%, respectively. Histopathology examination revealed that tissue necrosis of various degrees was found in 131I-CAb1 F(ab')2-treated groups. CONCLUSION: 131I-CAb1 F(ab')2 is safe and effective for colon cancer. It may be a novel and potentially adjuvant therapeutics for colon cancer.     

重组白介素-12治疗小鼠Lewis肺癌的实验研究

背景与目的:白介素-12(interleukin-12,IL-12)是具有抗瘤活性的细胞因子,本研究建立稳定表达小鼠IL-12(murineinterleukin-12,mIL-12)的小鼠Lewis肺癌(Lewislungcarcinoma,LLC)细胞系LLC/mIL-12,评估mIL-12重组质粒及LLC/mIL-12瘤苗治疗小鼠Lewis肺癌移植瘤的疗效。方法:脂质体转染LLC获得LLC/mIL-12瘤苗。于C57BL/6小鼠皮下接种LLC细胞2×106个,成瘤后将小鼠随机分成4组(n=10),第1、4、7天瘤内注射质粒或瘤苗,第14天处死小鼠,观察肿瘤生长曲线、脾细胞中CTL细胞和NK细胞活性以及肿瘤浸润淋巴细胞。结果:LLC/mIL-12瘤苗组和pcDNA3.1( )-mIL-12质粒组肿瘤体积显著缩小,mIL-12能增强NK细胞和CTL细胞活性,两者均以LLC/mIL-12治疗组更显著,LLC/mIL-12和pcDNA3.1( )-mIL-12治疗组有大量CD4 、CD8 淋巴细胞浸润。结论:mIL-12基因能增强NK细胞和CTL活性,LLC/mIL-12及pcDNA3.1( )-mIL-12均能产生抗瘤免疫反应,且前者作用更强。     

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。     

lncRNA MEG3调控NLRP3/caspase-1/GSDMD通路影响三阴性乳腺癌对紫杉醇的敏感性

目的:探究长链非编码RNA（long non-coding RNA,lncRNA）母系表达基因3（maternally expressed gene 3,MEG3）调控含NLR家族Pyrin域蛋白3（NLR family,Pyrin domain containing protein 3,NLRP3）/含半胱氨酸的天冬氨酸蛋白水解酶（cysteinyl aspartate specific proteinase 1,caspase-1）/消皮素D（gasdermin D,GSDMD）通路对三阴性乳腺癌（triple-negative breast cancer,TNBC）对紫杉醇（paclitaxel,PTX）敏感性的影响。方法:通过逐渐增加PTX剂量间歇作用的方法诱导TNBC耐药细胞MDA-MB-231/R,qRT-PCR检测lncRNA MEG3表达;将MDA-MB-231细胞分为对照组（未转染+PTX）、Vector组（空载体+PTX）、pcDNA3.1-MEG3组（pcDNA3.1-MEG3表达载体+PTX）、pcDNA3.1-MEG3+BAY11-7082组（pcDNA3.1-MEG3表达载体+PTX+5 μmol/L NLRP3抑制剂BAY11-7082）,qRT-PCR检测转染后lncRNA MEG3表达;CCK-8法检测MDA-MB-231细胞增殖情况;通过免疫荧光染色和扫描电镜（scanning electron microscope,SEM）观察MDA-MB-231细胞焦亡情况;Western blot检测MDA-MB-231细胞中NLRP3/caspase-1/GSDMD通路蛋白表达;体内成瘤实验检测肿瘤质量。结果:与MDA-MB-231细胞相比,MDA-MB-231/R细胞中lncRNA MEG3表达水平显著降低（P＜0.05）;与对照组相比,pcDNA3.1-MEG3组细胞增殖抑制率、GSDMD-N+细胞数量、细胞焦亡、细胞凋亡率及NLRP3、cleaved-caspase 1/caspase-1、GSDMD-N/GSDMD表达水平显著增加,IC50、肿瘤质量显著降低（P＜0.05）;与pcDNA3.1-MEG3组相比,pcDNA3.1-MEG3+BAY11-7082组细胞增殖抑制率、GSDMD-N+细胞数量、细胞焦亡、细胞凋亡率及NLRP3、cleaved-caspase 1/caspase-1、GSDMD-N/GSDMD表达水平显著降低,IC50、肿瘤质量显著增加（P＜0.05）。结论:上调lncRNA MEG3表达可通过激活NLRP3/caspase-1/GSDMD通路,促进MDA-MB-231细胞焦亡,以此增加TNBC对PTX的敏感性。     

lncRNA MEG3通过调控miR-21表达增加肺癌细胞放射敏感性

目的 探讨长链非编码RNA (lncRNA) MEG3对肺癌细胞H1299的放射敏感性调控机制。方法 运用qRT-PCR法检测具有放射抗性的H1299细胞中MEG3、miR-21-5p的表达。将过表达对照组(转染pcDNA3.1)、过表达MEG3组(转染pcDNA3.1-MEG3)、抑制miR-NC组(转染anti-miR-NC)、抑制miR-21-5p组(转染anti-miR-21-5p)、过表达MEG3+过表达miR-NC组(转染pcDNA3.1-MEG3和miR-NC)、过表达MEG3+过表达miR-21-5p组(转染pcDNA3.1-MEG3和miR-21-5p mimics)均用脂质体法转染。克隆形成实验检测细胞存活分数,流式细胞术检测细胞凋亡,双荧光素酶报告基因检测实验检测细胞中MEG3与miR-21-5p的结合力。结果 与正常肺上皮细胞相比,H1299细胞中MEG3表达明显降低,miR-21-5p表达明显升高;过表达MEG3或抑制miR-21-5p均可促进H1299细胞凋亡,增强放射敏感性;MEG3可靶向调控miR-21-5p的表达。过表达miR-21-5p可逆转MEG3对H1299细胞放射增强作用。结论 lncRNA MEG3可增强H1299细胞放射敏感性,其机制也许可能与靶向miR-21-5p有关。     

lncRNA MEG3 increases the radiosensitivity of lung cancer cells by regulating miR-21

Objective To investigate the regulatory mechanism of long-chain non-coding RNA (lncRNA) MEG3 on the sensitivity of lung cancer cell line H1299 to irradiation. Methods The expression of MEG3 and miR-21-5p in lung cancer cell line H1299 was detected by qRT-PCR. Overexpression control group (transfected with pcDNA3.1), MEG3 overexpression group (transfected with pcDNA3.1-MEG3), miR-NC inhibition group (transfected anti-miR-NC), miR-21-5p inhibition group (transfected with anti-miR-21-5p), MEG3 overexpression+miR-NC overexpression group (co-transfected with pcDNA3.1-MEG3 and miR-NC), MEG3 overexpression+miR-21-5p overexpression group (co-transfected with pcDNA3.1-MEG3 and miR-21-5p mimics) were all transfected into H1299 cells by liposome method treated with 4Gy irradiation. Cell survival fraction was detected by colony formation assay. Cell apoptosis was detected by flow cytometry. The binding of MEG3 to miR-21-5p in cells was assessed by dual luciferase reporter assay. Results Compared with normal lung epithelial cells, the expression of MEG3 was significantly decreased, whereas the expression of miR-21-5p was significantly increased in the radioresistant lung cancer cells H1299. Overexpression of MEG3 or inhibition of miR-21-5p could promote the apoptosis and enhance the radiosensitivity of H1299 cells. MEG3 could targetedly regulate the expression of miR-21-5p. Overexpression of miR-21-5p could reverse the enhanced radiosensitivity of MEG3 to H1299 cells. Conclusion LncRNA MEG3 can enhance the sensitivity of lung cancer cells H1299 to irradiation. The mechanism may be related to targeting miR-21-5p.     

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.     

脂质体介导入钠/碘同向转运体基因转染肺癌A549细胞及其蛋白表达

目的:研究人钠/碘同向转运体(NIS)基因转染肺癌细胞及其蛋白表达。方法:将体外培养的肺癌A549细胞分为实验组和对照两组:以脂质体Lipofectamihe2000为载体,分别介导转染重组质粒pcDNA3-hNIS和空质粒pcDNA3。NIS基因重组质粒pcDNA3-hNIS进行扩增、纯化,并经酶切鉴定和DNA测序。采用Western Blot法和免疫组化法分别检测转染肺癌细胞中NIS蛋白表达。结果:酶切鉴定和DNA测序结果表明重组质粒pcDNA3-hNIS中插入的hNIS基因片段大小、方向正确。WesternBlot法和免疫组化染色结果显示实验组有NIS蛋白表达,其阳性率达70.6%且主要分布于细胞膜而对照组无表达。两组比较有显著性差异(P=0.000)。结论:脂质体Lipofectamine 2000介导转染人钠/碘同向转运体基因pcDAN3-hNIS肺癌细胞能够成功地表达NIS蛋白。