乳腺钠/碘转运体表达与功能的调控研究
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摘要
目的
1．研究正常和良／恶性乳腺肿瘤组织中钠碘转运体(NIS)基因和蛋白的表达情况，探讨NIS与乳腺肿瘤疾病之间的关系。
2．研究甾体激素、促甲状腺激素(TSH)、三碘甲腺原氨酸(T3)、碘化钠和细胞因子对乳腺癌细胞钠碘转运体(NIS)基因表达的影响，探讨乳腺癌NIS基因表达和调控的因素。
3．研究维甲酸(RA)、泌乳素(PRL)和瘦素(leptin)对乳腺癌细胞钠碘转运体(NIS)基因、蛋白表达和摄碘功能的影响，探讨其与乳腺生理功能调控和病理变化之间的内在联系。
方法
1．乳腺组织取自乳腺纤维瘤、乳腺小叶增生以及乳腺癌患者，另取癌旁正常乳腺组织作为对照，分别运用半定量逆转录聚合酶链反应(RT-PCR)、Western Blot方法和免疫组织化学技术检测NIS的基因和蛋白表达情况。
2．两株乳腺癌细胞(MCF-7和MDA-MB-453)进行培养，分别给予不同浓度的甾体激素(包括雌二醇、孕酮、睾酮、双氢睾酮、脱氢表雄酮或地塞米松等)、TSH、T3及碘化钠或细胞因子(包括肿瘤坏死因子-α(TNF-α)，干扰素-γ(IFN-γ)，白细胞介素-6(IL-6))刺激72小时。采用RT-PCR方法检测细胞中NIS的mRNA表达情况。
3．采用乳腺癌细胞MCF-7进行培养，分别给予不同浓度的RA、PRL和瘦素进行刺激72小时或同浓度刺激不同时间。分别采用RT-PCR、Western blot和细胞免疫化学方法检测细胞中NIS的mRNA和蛋白表达，并用~(125)I测定细胞对碘摄取的能力。
结果
1．癌旁正常乳腺组织和良性乳腺肿瘤组织未检出NIS基因的蛋
南京医科大学博士学位论文
白表达，而乳腺癌组织均见MS基因和蛋白的表达，但免疫组织化学
结果显示MS蛋白主要表达在乳腺癌细胞胞浆中。
2.两株乳腺癌细胞在经过雌二醇、孕酮及肇酮或TSH、T3刺激
后，细胞中NIS的mRNA表达比对照组有明显增加，并且与刺激物
浓度成正相关。双氢辜酮或脱氢表雄酮对乳腺癌细胞中MS的mRNA
表达没有明显影响。而经过地塞米松或碘化钠以及细胞因子TNF一a，
IFN一Y，lL一6刺激后，细胞中Nls的mRNA表达比对照组有明显降
低，并与刺激物浓度成反比。
3.乳腺癌细胞MCF一7在经过RA、PRL和瘦素刺激后，Nls mRNA
和蛋白的表达比对照组明显增加，并与刺激物浓度成正比;且MS
mRNA表达与刺激时间也成正比。免疫细胞化学染色的结果显示，经
过RA和瘦素刺激的Nls蛋白染色在细胞内主要呈颗粒状浓集分布，
而经过PRL刺激的NIS蛋白染色在细胞内主要呈均匀分布。而且，
RA和瘦素使乳腺癌细胞的摄碘功能明显增强，PRL培养的细胞摄碘
功能并无明显变化。
结论
乳腺癌NIS基因和蛋白表达受苗体激素、TSH、T3、碘化钠、细
胞因子、RA、PRL和瘦素等多种因素调控，但摄碘功能的恢复需要
Nis蛋白功能正常化，本研究中只发现RA和瘦素有此调节作用。MS
蛋白翻译和翻译后水平的缺陷可能是乳腺癌组织摄碘能力下降的原
因之一。
OBJECTIVES
1. To study the expression of sodium-iodide symporter (NIS) mRNA and protein in normal and benign or malignant breast tissue, and investigate the association between NIS and breast disease.
2. To investigate the effect of steroids, thyrotropin (TSH), triiodothyronine (T3), iodide, and cytokines on the expression of NIS mRNA in breast cancer cells.
3. To investigate the effect of retinoic acid (RA), prolactin and leptin on the expression of gene and protein of NIS and the relationship between NIS expression and the ability of iodide uptake in breast cancer cell.
METNODS
1. Breast tissues were taken at surgery from patients with breast adenoma, nodule of lobuli glandulae mammariae and breast cancer. Normal samples adjacent to breast cancer were used as control. NIS mRNA and protein were assessed in these samples by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry methods.
2. Breast cancer cell lines (MCF-7 and MDA-MB-453) were cultured in the absence or presence of different concentrations of estradiol, progesterone, testosterone, dehydrotestosterone, dehydroepiandrosterone , dexamethasone, TSH, T3, Nal, TNF- a, IFN- r, or IL-6 for 72h. The level of NIS gene expression was assessed by RT-PCR.
3. The breast cancer cell line (MCF-7) was cultured with or without different concentrations of RA, PRL or leptin for various times. NIS mRNA and protein in cultured breast cancer cells was determined by RT-PCR, Western blot and immunocytochemistry. The activity of iodide uptake was determined by 125I uptake rate.
RESULTS
1. NIS mRNA and protein were undetectable in benign breast nodules and normal breast tissues, while they could be found by RT-PCR and Western blot methods in all breast cancer samples, though this protein was found mainly in cytoplasm as detected by immunohistochemistry.
2. Estradiol, progesterone, testosterone, TSH and T3 promoted NIS mRNA expression in breast cancer cells in a dose-dependent manner, while dexamethasone, Nal, TNF- a , IFN-r and IL-6 inhibited NIS mRNA in breast cancer cells dose-dependently. Dehydrotestosterone and dehydroepiandrosterone had no effect on the expression of NIS mRNA in these cells.
3. NIS gene expression was enhanced by RA, PRL or leptin in a dose- and time-dependent manner. RA, PRL or leptin also dose-dependently up-regulated NIS protein expression. The activity of iodide uptake had been found to be increased by RA and leptin, whereas there was no effect of prolactin.
CONCLUSION
Expression of NIS gene and protein in breast cancer could be regulated by many kinds of factors including steroids, thyrotropin, triiodothyronine, sodium iodide, TNF- a, IFN- y , IL-6, retinoic acid, prolactin, and leptin. But only RA and leptin could increase iodide-uptake in breast cancer cell because the resumption of the ability of iodide-uptake needs NIS protein normal function. The decreased ability of iodide uptake by breast cancer might result from the defect of NIS mRNA on translational or post-translational pathways.
1．研究正常和良／恶性乳腺肿瘤组织中钠碘转运体(NIS)基因和蛋白的表达情况，探讨NIS与乳腺肿瘤疾病之间的关系。
2．研究甾体激素、促甲状腺激素(TSH)、三碘甲腺原氨酸(T3)、碘化钠和细胞因子对乳腺癌细胞钠碘转运体(NIS)基因表达的影响，探讨乳腺癌NIS基因表达和调控的因素。
3．研究维甲酸(RA)、泌乳素(PRL)和瘦素(leptin)对乳腺癌细胞钠碘转运体(NIS)基因、蛋白表达和摄碘功能的影响，探讨其与乳腺生理功能调控和病理变化之间的内在联系。
方法
1．乳腺组织取自乳腺纤维瘤、乳腺小叶增生以及乳腺癌患者，另取癌旁正常乳腺组织作为对照，分别运用半定量逆转录聚合酶链反应(RT-PCR)、Western Blot方法和免疫组织化学技术检测NIS的基因和蛋白表达情况。
2．两株乳腺癌细胞(MCF-7和MDA-MB-453)进行培养，分别给予不同浓度的甾体激素(包括雌二醇、孕酮、睾酮、双氢睾酮、脱氢表雄酮或地塞米松等)、TSH、T3及碘化钠或细胞因子(包括肿瘤坏死因子-α(TNF-α)，干扰素-γ(IFN-γ)，白细胞介素-6(IL-6))刺激72小时。采用RT-PCR方法检测细胞中NIS的mRNA表达情况。
3．采用乳腺癌细胞MCF-7进行培养，分别给予不同浓度的RA、PRL和瘦素进行刺激72小时或同浓度刺激不同时间。分别采用RT-PCR、Western blot和细胞免疫化学方法检测细胞中NIS的mRNA和蛋白表达，并用~(125)I测定细胞对碘摄取的能力。
结果
1．癌旁正常乳腺组织和良性乳腺肿瘤组织未检出NIS基因的蛋
南京医科大学博士学位论文
白表达，而乳腺癌组织均见MS基因和蛋白的表达，但免疫组织化学
结果显示MS蛋白主要表达在乳腺癌细胞胞浆中。
2.两株乳腺癌细胞在经过雌二醇、孕酮及肇酮或TSH、T3刺激
后，细胞中NIS的mRNA表达比对照组有明显增加，并且与刺激物
浓度成正相关。双氢辜酮或脱氢表雄酮对乳腺癌细胞中MS的mRNA
表达没有明显影响。而经过地塞米松或碘化钠以及细胞因子TNF一a，
IFN一Y，lL一6刺激后，细胞中Nls的mRNA表达比对照组有明显降
低，并与刺激物浓度成反比。
3.乳腺癌细胞MCF一7在经过RA、PRL和瘦素刺激后，Nls mRNA
和蛋白的表达比对照组明显增加，并与刺激物浓度成正比;且MS
mRNA表达与刺激时间也成正比。免疫细胞化学染色的结果显示，经
过RA和瘦素刺激的Nls蛋白染色在细胞内主要呈颗粒状浓集分布，
而经过PRL刺激的NIS蛋白染色在细胞内主要呈均匀分布。而且，
RA和瘦素使乳腺癌细胞的摄碘功能明显增强，PRL培养的细胞摄碘
功能并无明显变化。
结论
乳腺癌NIS基因和蛋白表达受苗体激素、TSH、T3、碘化钠、细
胞因子、RA、PRL和瘦素等多种因素调控，但摄碘功能的恢复需要
Nis蛋白功能正常化，本研究中只发现RA和瘦素有此调节作用。MS
蛋白翻译和翻译后水平的缺陷可能是乳腺癌组织摄碘能力下降的原
因之一。
OBJECTIVES
1. To study the expression of sodium-iodide symporter (NIS) mRNA and protein in normal and benign or malignant breast tissue, and investigate the association between NIS and breast disease.
2. To investigate the effect of steroids, thyrotropin (TSH), triiodothyronine (T3), iodide, and cytokines on the expression of NIS mRNA in breast cancer cells.
3. To investigate the effect of retinoic acid (RA), prolactin and leptin on the expression of gene and protein of NIS and the relationship between NIS expression and the ability of iodide uptake in breast cancer cell.
METNODS
1. Breast tissues were taken at surgery from patients with breast adenoma, nodule of lobuli glandulae mammariae and breast cancer. Normal samples adjacent to breast cancer were used as control. NIS mRNA and protein were assessed in these samples by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry methods.
2. Breast cancer cell lines (MCF-7 and MDA-MB-453) were cultured in the absence or presence of different concentrations of estradiol, progesterone, testosterone, dehydrotestosterone, dehydroepiandrosterone , dexamethasone, TSH, T3, Nal, TNF- a, IFN- r, or IL-6 for 72h. The level of NIS gene expression was assessed by RT-PCR.
3. The breast cancer cell line (MCF-7) was cultured with or without different concentrations of RA, PRL or leptin for various times. NIS mRNA and protein in cultured breast cancer cells was determined by RT-PCR, Western blot and immunocytochemistry. The activity of iodide uptake was determined by 125I uptake rate.
RESULTS
1. NIS mRNA and protein were undetectable in benign breast nodules and normal breast tissues, while they could be found by RT-PCR and Western blot methods in all breast cancer samples, though this protein was found mainly in cytoplasm as detected by immunohistochemistry.
2. Estradiol, progesterone, testosterone, TSH and T3 promoted NIS mRNA expression in breast cancer cells in a dose-dependent manner, while dexamethasone, Nal, TNF- a , IFN-r and IL-6 inhibited NIS mRNA in breast cancer cells dose-dependently. Dehydrotestosterone and dehydroepiandrosterone had no effect on the expression of NIS mRNA in these cells.
3. NIS gene expression was enhanced by RA, PRL or leptin in a dose- and time-dependent manner. RA, PRL or leptin also dose-dependently up-regulated NIS protein expression. The activity of iodide uptake had been found to be increased by RA and leptin, whereas there was no effect of prolactin.
CONCLUSION
Expression of NIS gene and protein in breast cancer could be regulated by many kinds of factors including steroids, thyrotropin, triiodothyronine, sodium iodide, TNF- a, IFN- y , IL-6, retinoic acid, prolactin, and leptin. But only RA and leptin could increase iodide-uptake in breast cancer cell because the resumption of the ability of iodide-uptake needs NIS protein normal function. The decreased ability of iodide uptake by breast cancer might result from the defect of NIS mRNA on translational or post-translational pathways.
引文
[1] 刘超，武晓泓．钠／碘转运体．国外医学内分泌分册，2000，20(2)：
59-62．
[2] 贾悦，唐伟，刘超．钠／碘转运体在治疗甲状腺恶性肿瘤治疗中的作用．国外医学内分泌分册，2003，23(1)：37-9．
[3] 贾悦，唐伟，刘超．钠／碘转运体转基因介导放射性碘治疗非甲状腺恶性肿瘤的研究进展．国外医学肿瘤学分册，2003，30(3)：197-9．
[4] Cho JY, Leveille R, Kao R, et al. Hormonal regplation of radioiodide uptake activity and Na+/Ⅰ-symporter expression in mammary glands. J Clin Endocrinol Metab. 2000, 85(8): 2936-43.
[5] Tazebay UH, Wapnir IL, Levy O, et al. The mammary gland iodide transporter is expressed during lactation and in breast cancer. Nat Med. 2000, 6(8): 871-8.
[6] Perron B, Rodriguez AM, Leblanc G, et al. Cloning of the mouse sodium iodide symporter and its expression in the mammary gland and other tissues. J Endocrinol. 2001, 170(1): 185-96.
59-62．
[2] 贾悦，唐伟，刘超．钠／碘转运体在治疗甲状腺恶性肿瘤治疗中的作用．国外医学内分泌分册，2003，23(1)：37-9．
[3] 贾悦，唐伟，刘超．钠／碘转运体转基因介导放射性碘治疗非甲状腺恶性肿瘤的研究进展．国外医学肿瘤学分册，2003，30(3)：197-9．
[4] Cho JY, Leveille R, Kao R, et al. Hormonal regplation of radioiodide uptake activity and Na+/Ⅰ-symporter expression in mammary glands. J Clin Endocrinol Metab. 2000, 85(8): 2936-43.
[5] Tazebay UH, Wapnir IL, Levy O, et al. The mammary gland iodide transporter is expressed during lactation and in breast cancer. Nat Med. 2000, 6(8): 871-8.
[6] Perron B, Rodriguez AM, Leblanc G, et al. Cloning of the mouse sodium iodide symporter and its expression in the mammary gland and other tissues. J Endocrinol. 2001, 170(1): 185-96.