Transfection of the Human Sodium/Iodide Symporter(NIS) Gene with Liposomes and the Expression of the NIS Protein in Human Lung A549 Cancer Cells

OBJECTIVE To examine the possibility of human sodium iodide symporter （hNIS） protein expression in lung cancer cells. METHODS Human lung A549 cancer cells were thawed and cultured in vitro. The cells were divided into an experimental group transfected with a recombinant pcDNA3-hNIS plasmid and a control group transfected only with a pcDNA3 plasmid. The recombinant plasmid vector encoding the hNIS gene （pcDNA3-hNIS） was amplified, purified and identified. The hNIS gene was followed by DNA sequencing. A Western blot and an immunohistochemical assay were applied to detect the hNIS protein expression in the transfected human lung A549 cancer cells. RESULTS Restriction enzyme digestion and DNA sequencing results showed the size and direction of the inserted gene in the recombinant pcD- NA3-hNIS plasmid was correct. The Western blot method and immunohistochemical analysis showed a positive NIS protein expression in the experimental group. The NIS protein was detected mainly in the cell membranes showing a positive rate up to 70.6% with no expression of the NIS protein in the control group. There was a significant difference between two groups （P=0.000）. CONCLUSION The hNIS gene was transfected effectively into human lung A549 cancer cells mediated by Lipofectamine 2000, and was expressed with its protein in vitro.

Radioiodine therapy for castration-resistant prostate cancer following prostate-specific membrane antigen promoter-mediated transfer of the human sodium iodide symporter

Radioiodine therapy, the most effective form of systemic radiotherapy available, is currently useful only for thyroid cancer because of the thyroid-specific expression of the human sodium iodide symporter （hNIS）. Here, we explore the efficacy of a novel form of gene therapy using prostate-specific membrane antigen （PSMA） promoter-mediated hNIS gene transfer followed by radioiodine administration for the treatment of castration-resistant prostate cancer （CRPC）. The androgen-dependent C33 LNCaP cell line and the androgen-independent C81 LNCaP cell line were transfected by adenovirus. PSMA promoter-hNIS （Ad.PSMApro-hNIS） or adenovirus.cytomegalovirus-hNIS containing the cytomegalovirus promoter （Ad.CMM-hNIS） or a control virus. The iodide uptake was measured in vitro. The in vivo iodide uptake by C81 cell xenografts in nude mice injected with an adenovirus carrying the hNIS gene linked to PSMA and the corresponding tumor volume fluctuation were assessed. Iodide accumulation was shown in different LNCaP cell lines after Ad.PSMApro-hNIS and Ad.CMV-hNIS infection, but not in different LNCaP cell lines after adenovirus.cytomegalovirus （Ad.CMV） infection. At each time point, higher iodide uptake was shown in the C81 cells infected with Ad.PSMApro-hNIS than in the C33 cells （P 〈 0.05）. An in vivo animal model showed a significant difference in 1311 radioiodine uptake in the tumors infected with Ad.PSMApro-hNIS, Ad.CMV-hNIS and control virus （P 〈 0.05） and a maximum reduction of tumor volume in mice infected with Ad.PSMApro-hNIS. These results show prostate-specific expression of the hNIS gene delivered by the PSMA promoter and effective radioiodine therapy of CRPC by the PSMA promoter-driven hNIS transfection.

Correlation between sodium-iodide symporter expression and circulating tumor cell positivity in differentiated thyroid carcinoma

Objective We investigated the correlation between the expression of the sodium-iodide symporter(NIS) and the detection of circulating tumor cells(CTCs) in differentiated thyroid carcinoma(DTC).Methods NIS expression in differentiated thyroid and the positive rate of CTCs in the peripheral blood were determined by immunohistochemistry S-P and flow cytometry from the records of 172 cases of differentiated thyroid carcinoma.Results Seventy-six cases(44.2%) expressed NIS in the differentiated thyroid and 63 cases(36.6%) were positive for CTCs in the peripheral blood. There was a significant difference between N0 and N1 in the expression of NIS(χ~2 = 6.015, P = 0.014) and the positive rate of CTCs(χ~2 = 14.035, P = 0.001). N0 and N1 also differed significantly in the expression of NIS(r =-0.383,-0.610, P = 0.002, < 0.001). The differences in the NIS expression, but not in the positive rate of CTCs, were significant among the different pathological subtypes(χ~2 = 7.897, P = 0.005; χ~2 = 1.455, P = 0.228, respectively). There was a significant negative correlation between the highly differentiated type and intermediate differentiation type both in the expression of NIS and positive rate of CTCs(r =-0.591,-0.443, P < 0.001, P = 0.002). Conclusion There was a significant negative correlation between the expression of tissue NIS and positive rate of CTCs in the peripheral blood in DTC. The malignancy level and lymph node metastasis in differentiated thyroid carcinoma were negatively correlated with NIS expression and positively correlated with the positive rate of CTC.

Effect of Cytokine on the Expression of Sodium Iodide Symporter Gene in Breast Cancer Cell

Objective: To investigate the effect of cytokines (TNF-α,IFN-γ and IL-6) on the expression of sodium-iodide symporter(NIS)gene in breast cancer cell(MCF-7). Methods:The breast cancer cell was cultureds with negative control culture or culture with different concentrations of cytokines for 72 h.NIS gene mRNA in breast cancer cells cultured was determined by reverse transcriptase-polymerase chain reaction(RT-PCR). Results:Expression of sodium-iodide symporter mRNA can be found decreasing along with increasing the concentration of cytokine dose-dependently. Conclusion: Cytokine may play a role in iodide-uptake modulating in breast cancer cells by their effect on NIS gene expression.

Correlation analysis between serum β2-MG and sodium/iodide symporter in patients with thyroid carcinoma

Objective： The aim of our study was to investigate the correlation between the expression of sodium/iodide symporter, serum levels of β2-MG and prognosis of thyroid carcinoma patients. Methods： Ninty-five cases with thyroid carcinoma, using enzyme-linked immunosorbent assay with double-antibody sandwich to detect the serum β2-MG levels and immunehistochemistry to detect NIS expression of thyroid cancer tissue. Results： Thirty-seven cases showed positive expression of sodium/iodide symporter （38.9%） and 30 cases showed positive expression of β2-MG （31.57%）. There were significant differences of NIS expression （X2 = 8.207, P = 0.017） and β2-MG expression （X2 = 10.121, P = 0.006） between different pathological types of thyroid carcinoma, but there was no correlation between the positive rate of the two research groups （r = -0.546, P = 0.633）. The significant differences was observed in expression of sodium/iodide symporter （X2 = 9.272, P=0.002） and expression of β2-MG （X2 = 4.441, P = 0.035） between the group with neck lymph node metastasis and the group without neck lymph node metastasis and both positive rate was significantly negatively correlated （r = -1.000, P = 0.000）. The significant differences was observed in expression of sodium/iodide symporter （X2 = 9.272, P = 0.002） and expression of β2-MG （X2 = 3.867, P = 0.043） between the group with distant organ metastasis and the group without distant organ metastasis （X2 = 11.985, P = 0.001） and both positive rate was significantly negatively correlated （r = -1.000, P = 0.000）. Conclusion： There are significantly negatively correlated between neck lymph node metastasis, distant organ metastasis and expression of sodium/iodide symporter and expression of β2-MG. Thyroid cancer lymph node and distant organ metastasis, the tumor tissue NIS expression and serum levels of β2-MG is significantly negatively correlated. The detection of serum β2-MG provides clinical reference value for the effects on radionuclide therapy and prognosis assessment of thyroid carcinoma. Serum β2-MG levels is negatively correlated with prognosis in thyroid cancer patients.

Radiofrequency induction on sodium/iodide symporter expression of thyroid cancer

Objective; The aim of this study was to investigate the effects of radiofrequency treatment on sodium/iodide symporter expression of thyroid cancer ceils. Methods： In 29 thyroid cancer patients with low or no expression of soda ／ iodide symporter, the radio frequency combined 1311 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 radiofrequenc_y 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 effective, 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 efficacy of 131I therapy in dedifferentiated thyroid cancer.

Effects of BRAF^(V600E) Mutation on Na^+/I^- Symporter Expression in Papillary Thyroid Carcinoma

Radioiodine ablation（RIA） therapy is one of the most important treatments for papillary thyroid carcinoma（PTC）, but some patients who received 131 I have radioiodine-refractory disease caused by the decreased expression of the Na^＋/I^- symporter（NIS）. BRAF^V600E mutation is one possible risk factor that can disturb the NIS expression, but the roles are unclear in clinical practice. This research discussed the association of BRAF^V600E mutation and NIS expression in PTC tissue and the clinical implications in RIA therapy. 134 PTC samples were collected between June 2013 and June 2014 from Tongji Hospital affiliated to Tongji Medical College, and their clinical characteristics were analyzed. RT-PCR was used to detect the BRAF^V600E mutation from formalin-fixed paraffin-embedded samples, and immunohistochemistry was applied to detect the NIS expression. IPP software was used to calculate the relative expression quantity of NIS. We found that there was no significant correlation between the absorbance（A） values of NIS and clinicopathologic features in these cases, even thyroid stimulating hormone. BRAF^V600E mutation showed inhibitory effect on the NIS expression without statistically significant difference in all PTC cases（β=–0.0195, P=0.085）, but in the subgroup without hashimoto＇s thyroiditis（HT）, BRAF^V600E mutation could significantly inhibit the NIS expression（β=–0.0257, P=0.046）. The results indicate that BRAF^V600E mutation is correlated with a lower expression of NIS in PTCs without HT, suggesting the radioiodine-refractory effects during RIA therapy in these patients.

Radioiodide uptake in melanoma cells after transfer of human NaI symporter gene

To obtain human sodium/iodide symporter gene cDNA for studying its potential ability as a radioiodide treatment for melanoma, the hNIS gene cDNA was amplified with total RNA from human thyroid tissue by RT-PCR. The hNIS cDNA was inserted into cloning vector pUCm-T and subcloned into eukaryotic expression vector pc-DNA3. The pc-DNA3-hNIS and pc-DNA3 were transduced into melanoma cells (B16) by electroporation, and two cell lines termed B16-A and B16-B respectively were established. The uptake and efflux of iodide was examined in vitro. The three cell lines (B16-A, B16-B, B16) were injected subcutaneously into the right flank of C57 mice. Biodistribution study and tumor imaging were performed when the tumor reached approximately 10mm in diameter. The cloned hNIS cDNA sequence was identical with the published sequence. Two novel cell lines named 16-A containing pc-DNA3-hNIS and B16-B containing pc-DNA3 only were established. The resultant cell line B16-A accumulated 17 and 19 times more radioiodide in vitro than B16 and B16-B respectively. The iodide uptake reached the half-maximal level within 10 min, and reached a plateau at 30 min. The efflux of iodide was also rapid (T1/2eff=10min). The imaging shows in vivo uptake in expected sites including the salivary glands, thyroid, stomach, and hNIS-transduced tumor, whereas the nontransduced tumor was not visualized. The %ID/g of B16-A tumors at 1, 2, 4, 12, and 24h after injec- tion of 125I were 12.22±0.71, 10.91±0.72, 8.73±0.99, 1.24±0.29, and 0.19±0.03, respectively, which were signifi- cantly higher percentages than those for controlling tumors, p<0.01. However, biologic T1/2 was about 6 h. Our pre- liminary data indicate that the transduction of the hNIS gene per se is sufficient to induce iodide transport in mela- noma cells both in vitro and in vivo, but T1/2eff is short.

Human sodium/iodide symporter gene induced iodine uptake in human lung adenocarcinoma via baculovirus

To investigate human sodium/iodide symporter (hNIS) induced iodine uptake in human lung adenocarcinoma via baculovirus, a recombinant baculovirus encoding hNIS gene was constructed under the control of CMV promoter (Bac-CMV-hNIS). In vitro, baculovirus infected A549 cells accumulated about 27 times more 125I than that of noninfected cells. The 125I uptake was maximal after 30-min incubation of the cells, and efflux of the radioactivity was rapid, with 50% lost during the first 2 min after 125I-containing medium had been replaced by nonradioactive medium. Competition experiments in the presence of sodium perchlorate revealed a dose-dependent decrease of 125I uptake. Bac-CMV-hNIS infected tumor cells were selectively killed by exposure to 131I, as revealed by clonogenic assays. In nude mice, Bac-CMV-hNIS infected A549 cells accumulated more 131I than that of the control monitored by 1-h scintigraphy after 131I administration. The transduction of hNIS gene through baculovirus is sufficient to induce iodine transporting in A549 cells in vitro and in vivo, outlining the potential of this novel tumor gene imaging approach. But a rapid efflux of radioactivity from the tumor was shown in vivo and the in vivo therapy test showed no sign of effect.

Molecular rotaxane shuttle-relay accelerates K^(+)/Cl^(-) symport across a lipid membrane

Synthetic molecules that can mediate the coupled transport of Cl^(-) with K^(+) and/or Na+across the lipid bilayers have aroused great interest due to their potential as a novel therapeutic strategy by disrupting cellular ion homeostasis.Based on the structural advantages of molecular rotaxanes,we herein show that two rotaxane-based transporters[2]R and[3]R induce coupled K^(+)/Cl^(-) channel transport by introducing Cl^(-) recognition sites in the thread and K^(+) binding group in the wheel,respectively.The welldesigned molecular structures allow the insertion of unimolecular rotaxanes into the lipid bilayer,thus achieving effective ion transport by means of thermodynamically controlled movement and driven by the difference in ion concentration inside and outside the vesicles.In addition,the use of a three-component rotaxane can accelerate ion transport through a cooperative shuttlerelay mechanism in which two wheels move along the thread in the lipid membrane,thereby enabling[3]R to have higher ion transport capacity.This work represents a major advance in the use of rotaxane molecules to accomplish more complex and effective tasks.

电针对紫杉醇诱导大鼠神经病理性疼痛的影响

目的观察电针(EA)对紫杉醇诱导神经病理性疼痛大鼠脊髓背角NKCC1、KCC2表达和小胶质细胞活化的影响及其可能机制。方法将48只雄性SD大鼠随机分为溶媒组(Vehicle组)、紫杉醇组(PTX组)、紫杉醇+电针组(PTX+EA组)、紫杉醇+假电针组(PTX+Sham EA组),每组12只。采用腹腔注射PTX的方法建立PTX诱导的神经病理性疼痛大鼠动物模型,建模完成后,PTX+EA组给予“足三里”、“阳陵泉”电针刺激,连续7 d。于紫杉醇注射前2 d和注射后第1、3、5、7、14、21天进行机械撤足阈值和热缩足潜伏期痛行为学测试。利用免疫荧光染色技术和Western blot技术检测脊髓背角组织中钠钾氯联合转运蛋白1(NKCC1)、钾氯联合转运蛋白2(KCC2)和小胶质细胞标志物离子钙结合衔接分子1(Iba1)表达的变化。结果与Vehicle组比较,PTX组大鼠出现双后足机械和热痛觉过敏,脊髓背角组织中NKCC1表达升高和活化的小胶质细胞数增加。与PTX组比较,PTX+EA组大鼠在第14、21天的机械和热痛觉过敏得到显著改善,脊髓背角组织中NKCC1和Iba1表达量降低,4组间KCC2表达差异无统计学意义。结论电针可有效缓解紫杉醇诱导的神经病理性疼痛,其机制可能与抑制大鼠脊髓背角组织中NKCC1表达和小胶质细胞活化有关。

钠碘同向转运体表达与功能调控的研究进展

钠碘同向转运体(sodium iodine symporter)广泛分布于人体各个器官,是器官转运I-的结构和功能基础。钠碘同向转运体主要表达在细胞的基底侧膜上,为各种生命活动提供所需的I-。目前已知,钠碘同向转运体的调控机制涉及激素、细胞因子、转录因子及信号分子的作用。此外,钠碘同向转运体作为甲状腺癌的重要治疗靶点逐渐受到关注,然而在治疗过程中唾液腺等其他器官吸收放射性碘而产生的并发症也值得重视。本文重点综述了钠碘同向转运体的表达、作用及其调控机制的研究进展,旨在为临床干预钠碘同向转运体异常相关疾病进程提供新思路。

甲状腺癌碘-131治疗抵抗发生的分子机制研究进展

甲状腺癌是内分泌系统最常见的恶性肿瘤,其中分化型甲状腺癌(differentiated thyroid carcinoma,DTC)占90%以上。多数DTC患者经过系统治疗后预后良好,但少数患者肿瘤原发灶或转移灶出现失分化现象,进展为放射性碘难治性DTC(radioiodine-refractory DTC,RAIR-DTC),预后明显变差,是甲状腺癌致死的主要原因。钠碘转运体(sodium iodide symporter,NIS)的表达和功能异常,是导致甲状腺癌碘-131治疗抵抗的主要原因,其发生受遗传学改变、表观遗传学改变、肿瘤微环境作用、自噬作用等多因素影响。遗传学改变如BRAF基因的V600E位点突变、RET/PTC基因重排等导致致癌信号通路的激活,直接或间接地影响NIS的表达及其在细胞膜上的正常定位。表观遗传学调控特定基因的表达模式,调节NIS的表达水平,进而影响甲状腺细胞的碘摄取功能。肿瘤微环境中的免疫细胞、细胞因子和细胞外基质等成分也可能通过降低NIS的表达水平和/或干扰其在细胞膜上的正常功能导致细胞碘摄取障碍。此外,自噬作为一种细胞内部的代谢调节机制,也可以调节NIS的表达及其在细胞内的分布,从而影响碘的摄取和碘-131治疗的敏感性。通过综述以上因素在甲状腺癌失分化中的作用机制,可以更全面地理解RAIR-DTC的发生和发展过程,有助于探寻新的治疗靶点,改善预后,并为患者提供更有效的个体化治疗策略。

Adenovirus-mediated and tumor-specific transgene expression of the sodium-iodide symporter from the human telomerase reverse transcriptase promoter enhances killing of lung cancer cell line in vitro

Background The sodium-iodide symporter （NIS） protein can mediate the active radioiodine uptake.The human telomerase reverse transcriptase （hTERT） promoter is known to be selectively reactivated in majority of tumors and hence could be used for tumor targeting.We constructed a recombinant adenovirus containing the human sodium iodide symporter （hNIS） gene directed by the hTERT promoter, characterized the ability of infected cells in uptaking iodide, and explored the therapeutic efficacy of 131I in a lung cancer cell line in vitro.Methods The hTERT promoter was amplified by PCR from DNA isolated from log-phase HepG2 cells, subcloned into lineralized FL＊-hNIS/pcDNA3, and then the hTERT-hNIS sequence was subcloned into the shuttle plasmid pAdTrack.The recombinant adenovirus Ad-hTERT-hNIS was constructed by AdEasy system.A positive control adenovirusAd-CMV-hNIS and a negative control adenovirus Ad-CMV were created similarly.A549 cells were transduced with recombinant adenoviruses.125I uptake studies and sodium perchlorate suppression studies were used to confirm hNIS expression and function.Toxic effects of 131I on tumor cells were studied by in vitro clonogenic assay.Results We first successfully constructed an adenovirus mediated transgene expression system of the hNIS under the control of hTERT promoter.When infected with recombinant adenovirus constructs expressing hNIS directed by hTERTand CMV-promoters （Ad-hTERT-hNIS and Ad-CMV-hNIS, respectively）, the lung cancer cell line A549 had increased ability to uptake radioiodide up to 23- and 30- fold compared to the control parental cells, respectively.The radioiodide uptake ability of both the Ad-CMV-hNIS and Ad-hTERT-hNIS transduced cell lines were repressed 11-fold by sodium perchlorate （NaCIO4）.The subsequent in vitro clonogenic assay of the infected A549 cell line was further repressed to 23% （Ad-CMV-hNIS） and 30% （Ad-hTERT-hNIS） of the control group after receiving radioiodide for 7 hours （P 〈0.001）.Conclusion Our preliminary study indicates that an adenovirus mediated transgene expression system of the hNIS under the control of hTERT promoter has the potential to become an effective wide-spectrum yet highly specific anti-cancer strategy.

SGLT-2抑制剂对糖尿病小鼠血管内皮细胞的影响

【目的】探讨钠-葡萄糖协同转运蛋白2(SGLT-2)抑制剂对糖尿病小白鼠血管内皮细胞的影响。【方法】将24只SPF级8周龄雄性ICR小白鼠腹腔注射低剂量链脲佐菌素(STZ)(50 mg/kg),建立糖尿病模型,造模成功,随机分为模型组和治疗组(达格列净治疗12周),6只小白鼠作为对照组,皮下注射生理盐水。三组均普通饲料喂养,动态观察每周小鼠体重、血糖的变化,比较三组小白鼠血浆肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)、单核细胞趋化因子-1(MCP-1)、超氧化物歧化酶(SOD)、丙二醛(MDA)水平。【结果】第2周末至第12周末,模型组、治疗组体重均低于对照组,治疗组体重均高于模型组,差异有统计学意义(P<0.05)。与对照组比较,模型组、治疗组血糖显著升高,差异有统计学意义(P<0.05);与模型组比较,3周末至12周末,治疗组小白鼠血糖水平显著下降,差异有统计学意义(P<0.05)。模型组血浆TNF-α、IL-1β、MCP-1水平显著高于对照组,治疗组血浆TNF-α、IL-1β、MCP-1水平显著低于模型组,差异有统计学意义(P<0.05)。模型组血浆SOD水平低于对照组,治疗组血浆SOD水平高于模型组,差异有统计学意义(P<0.05);与对照组比较,模型组血浆MAD水平高于对照组,治疗组血浆MAD水平低于模型组,差异有统计学意义(P<0.05)。【结论】SGLT-2抑制剂能有效抑制糖尿病小鼠血管内皮细胞炎症因子的释放,改善血管内皮细胞功能。

Capsaicin restores sodium iodine symportermediated radioiodine uptake through bypassing canonical TSH–TSHR pathway in anaplastic thyroid carcinoma cells

Anaplastic thyroid cancer(ATC)is a rare but highly lethal disease.ATCs are resistant to standard therapies and are extremely difficult to manage.The stepwise cell dedifferentiation results in the impairment of the iodine-metabolizing machinery and the infeasibility of radioiodine treatment in ATC.Hence,reinducing iodine-metabolizing gene expression to restore radioiodine avidity is considered as a promising strategy to fight against ATC.In the present study,capsaicin(CAP),a natural potent transient receptor potential vanilloid type 1(TRPV1)agonist,was discovered to reinduce ATC cell differentiation and to increase the expression of thyroid transcription factors(TTFs including TTF-1,TTF-2,and PAX8)and iodine-metabolizing proteins,including thyroidstimulating hormone receptor(TSHR),thyroid peroxidase,and sodium iodine symporter(NIS),in two ATC cell lines,8505C and FRO.Strikingly,CAP treatment promoted NIS glycosylation and its membrane trafficking,resulting in a significant enhancement of radioiodine uptake of ATC cells in vitro.Mechanistically,CAP-activated TRPV1 channel and subsequently triggered Ca2þinflux,cyclic adenosine monophosphate(cAMP)generation,and cAMP-responsive element-binding protein(CREB)signal activation.Next,CREB recognized and bound to the promoter of SLC5A5 to facilitate its transcription.Moreover,the TRPV1 antagonist CPZ,the calcium chelator BAPTA,and the PKA inhibitor H-89 effectively alleviated the redifferentiation exerted by CAP,demonstrating that CAP might improve radioiodine avidity through the activation of the TRPV1–Ca2þ/cAMP/PKA/CREB signaling pathway.In addition,our study indicated that CAP might trigger a novel cascade to redifferentiate ATC cells and provide unprecedented opportunities for radioiodine therapy in ATC,bypassing canonical TSH–TSHR pathway.

The effects of thyroid-stimulating hormone,estradiol and prolactin on sodium/iodide symporter mRNA expression in mouse lactating mammary gland cells under different iodine levels

Objective The present study investigated the sodium/iodide symporter mRNA expression in mouse lactating mammary gland cells under different iodine levels and the effects of thyroid-stimulating hormone(TSH),estradiol(E2)and prolactin(PRL)on NIS mRNA expression in mouse lactating mammary gland cells.

溴结构域蛋白4经PTEN/PI3K/AKT通路对甲状腺乳头状癌细胞摄碘相关指标影响的研究

目的研究溴结构域蛋白4(BRD4)在甲状腺乳头状癌(PCT)中的表达水平及其抑制剂JQ1对PCT细胞增殖、迁移能力的影响,进一步探索影响摄碘能力的调控机制。方法采用实时荧光定量聚合酶链反应(qRT-PCR)技术及蛋白质印迹法分别检测BRD4在正常甲状腺细胞Nthy-ori 3-1及人PCT细胞TPC-1、K1的表达水平。采用CCK-8及划痕实验分别检测JQ1对TPC-1、K1细胞增殖及迁移能力的影响。采用高内涵细胞成像与分析系统观察TPC-1、K1细胞在JQ1作用下的形态及数目变化。采用qRT-PCR技术检测影响PCT摄碘能力相关基因的表达。结果BRD4在PCT细胞及癌组织中高表达。JQ1对TPC-1、K1细胞增殖能力呈时间及浓度依赖性抑制。TPC-1、K1细胞的48 h半抑制浓度分别为(9.045±0.772)、(14.169±1.406)μmol/L。JQ1可以抑制TPC-1、K1细胞的迁移,且具有统计学意义(P<0.05)。JQ1分别作用于TPC-1、K1细胞48 h后,实验组BRD4、磷脂酰肌醇3激酶(PI3K)和蛋白激酶B(AKT)的mRNA表达水平降低。第10号染色体同源缺失性磷酸酶-张力蛋白基因(PTEN)、钠-碘转运蛋白的mRNA表达升高。结论BRD4在PCT中呈现高表达状态。JQ1可有效抑制BRD4的表达,并影响PTEN/PI3K/AKT通路从而抑制PCT细胞的增殖及迁移,使得其摄碘能力增加。

Mechanism of intervention of medicine combined with mild moxibustion on hypothyroidism rat model

Objective:To explore the intervention effect of medicine combined with mild moxibustion on immune factor and Na/I symporter(NIS)in hypothyroidism rat model.Methods:The model was successfully made by intragastric administration of propylthiouracil(PTU)solution medicine and medicine combined with mild moxibustion groups were given levothyroxine sodium suspension 60μg/kg body weight by gavage,once a day.In the medicine combined with mild moxibustion group,mild moxibustion was applied in“Dazhui”,“Mingmen”,“Pishu”,“Shenshu”,and 10 minutes per point,once a day,and one day off every six days;four weeks in a row.The model group and medicine was fixed in the same way as the medicine combined with mild moxibustion group.The blank group received no treatment.The contents of thyrotropin-releasing hormone(TSH),tatalthyroxine(TT4),thyroid peroxidase antibody(TPOAb),thyroglobulin antibodies(TGAb),Interleukin-4(IL-4)and Interleukin-23(IL-23)in serum were determined by Enzyme Linked Immunosorbent Assay(ELISA).The content of NIS and the expression level of NISmRNA in thyroid tissues of each group were detected by immunohistochemistry and real-time polymerase chain reaction(R-T PCR).Results:Compared with the blank group,the contents of TSH,TPOAb,TGAb and IL-23 in the serum of rats in the model group were increased,the contents of IL-4 and TT4 were decreased,and the contents of NIS and NISmRNA in thyroid tissue were decreased,with statistical significance(P<0.01).Compared with model group,the contents of TSH,TPOAb,TGAb and IL-23 in serum of medicine group and medicine combined with mild moxibustion group were decreased,while the contents of IL-4 and TT4 were increased;the NIS content and NISmRNA expression in thyroid tissues were increased,and the differences were statistically significant(P<0.01).Compared with the medicine group,NISmRNA expression in thyroid tissues of medicine combined with mild moxibustion groups was increased,and the difference was statistically significant(P<0.05).Conclusion:Medicine combined with mild moxibustion can decrease the contents of TPOAb,TGAb and IL-23,and increase the content of IL-4,increasing the content and expression of NIS to interfere with the hypothyroidism rat model.

西药联合温和灸对甲状腺功能减退大鼠模型干预作用的机制研究

目的:探究西药联合温和灸对甲减模型大鼠免疫因子及钠/碘共转运体(NIS)干预作用的机制。方法:丙硫氧嘧啶(PTU)溶液灌胃造模成功后,西药及西药加温和灸组以左甲状腺素钠混悬液60μg/kg体重灌胃,每日一次;西药加温和灸组在大椎、命门、脾俞、肾俞施温和灸,每穴10 min,每日1次,每6日休息1日;连续4周。模型组与西药组同方式、同频率固定;空白组不做处理。ELISA法检测血清中促甲状腺激素(TSH)、总甲状腺激素(TT4)、甲状腺过氧化物酶抗体(TPOAb)、甲状腺球蛋白抗体(TGAb)、白细胞介素4(IL-4)、白细胞介素23(IL-23)的含量;免疫组化及R-T PCR法检测各组大鼠甲状腺组织中钠/碘共转运体(NIS)的含量及NISmRNA的表达量。结果:(1)与空白组比较,模型组大鼠血清中TSH、TPOAb、TGAb、IL-23含量上升,IL-4、TT4含量下降,甲状腺组织中NIS含量及NISmRNA表达量下降,差异有统计学意义(P<0.01);(2)与模型组比较西药组及西药加温和灸组大鼠血清中TSH、TPOAb、TGAb、IL-23含量下降,IL-4、TT4含量上升;甲状腺组织中NIS含量及NISmRNA表达量上升,差异有统计学意义(P<0.01);(3)与西药组相比西药加温和灸组大鼠甲状腺组织中NISmRNA表达量上升,差异有统计学意义(P<0.05);结论:西药加温和灸可以通过降低TPOAb、TGAb、及IL-23的含量,升高IL-4的含量;增加NIS的含量及NISmRNA的表达量,干预甲减模型大鼠。