【作者】 张晓清；

【导师】 刘鹏熙；

【作者基本信息】 广州中医药大学 ， 中西医结合临床， 2011， 博士

【摘要】 研究背景与目的乳腺癌是严害威胁妇女心身健康的恶性肿瘤,在女性恶性肿瘤中其发病率和死亡率均居前列。在我国一些大城市中乳腺癌的发病率逐年递增,已跃居女性恶性肿瘤的首位。乳腺与甲状腺同属于垂体激素的靶器官,内分泌功能的变化与腺体疾病的发生发展有着密切的关系,乳腺组织的细胞癌变后仍具有激素受体功能,其原来所依赖的激素仍然调节着肿瘤细胞的生长。甲状腺激素在正常乳腺的发育和分化过程中起着促进作用。甲状腺激素可直接调控机体的生长和细胞的增殖,其含量的不足或过量均可改变垂体前叶和靶器官激素及化学致癌因子的代谢,因此认为乳腺癌的病程演进可能会受到甲状腺激素含量高低不同的影响。甲状腺激素对乳腺癌的作用仍不明,有可能既是肿瘤细胞形成的促进因子,也是对抗肿瘤细胞形成和生长的保护因子。研究发现促甲状腺激素受体和甲状腺激素受体在乳腺组织中有表达,反过来雌激素也影响着甲状腺的发育、生理及病理过程。本研究探讨甲状腺疾病与乳腺癌之间的相关性,了解甲状腺疾病对乳腺癌患者预后的影响。根据国家中医药管理局颁布的《中医病症疗效标准》ZY/T001.2-94将乳腺癌分为3型：肝郁痰凝证、冲任失调证、正虚毒炽证。而甲状腺疾病则属于中医“瘿病”范畴,瘿病的发生主要与情志内伤、饮食及水土失宜有关。在致病因素作用下,肝郁不舒,脾失健运,脏腑失调,经络阻滞,导致气滞、血瘀、痰凝结于颈部而发病。此外,其发病与肝肾不足、六淫外感也有一定关系。常见证候有肝气郁结、肝郁脾虚、肝肾阴虚、肝阳上亢等。甲状腺疾病和乳腺癌的发生均与肝、脾、肾三脏有密切关系,可见两者的病因病机在中医理论的角度也存在相关性。本研究通过调查伴随甲状腺疾病的乳腺癌患者和甲状腺正常的乳腺癌患者的临床症状及症状分级量化值,探讨伴随甲状腺疾病的乳腺癌患者的证型分布情况,了解甲状腺疾病是否影响乳腺癌患者的中医证型分布。钠碘转运体(Na+/I-Symporter, NIS)是一种糖化膜蛋白,即“碘泵”,其功能主要是借助于钠/钾交换而产生的细胞内外钠离子浓度梯度来转运血液内活性碘进入细胞,使胞内碘含量浓聚为血浆的20-100倍。NIS浓聚碘是甲状腺激素生物合成、诊断甲状腺核素显像以及甲亢、甲状腺肿瘤放射性碘治疗的基础。已经证实哺乳期的乳腺导管细胞上表达NIS并能浓聚碘,研究发现约85%的ER阳性乳腺癌及其转移组织细胞表面存在大量NIS,可主动将血液中的碘转运到乳腺癌组织,使其碘浓聚远高于正常组织,并发现87%的浸润性乳腺癌和83%的导管内癌表达NIS,而正常乳腺组织无NIS表达,但乳腺癌组织摄碘能力并没因为NIS表达的增加而增加。用放射性碘治疗恶性肿瘤的是肿瘤综合治疗的方法之一,而放射性碘发挥杀伤肿瘤细胞效应的基础是肿瘤细胞必须具有摄碘能力,但乳腺癌组织摄取碘的能力较低,使放射性碘治疗难以发挥效应。研究发现催乳激素、雌激素、cAMP刺激物、维甲酸类,糖皮质激素通过增加NIS基因表达以增强乳腺癌细胞的碘摄取。昆布(Thallus japonica)是大叶藻科植物大叶藻的全草,俗称“海带”,具有软坚散结、利水泄热等功效,昆布是一种含碘量很高的海藻,碘对维持甲状腺与乳腺组织细胞的正常功能具有重要作用。昆布中含有很多活性成分,它的大部分活性成分与它的多糖成分关系密切,目前对昆布多糖的研究越来越受到重视。昆布多糖是一种β-1,3葡聚糖的聚合物,可以通过激活巨噬细胞,产生细胞毒性作用,抑制肿瘤细胞增殖而杀伤肿瘤细胞；也可以通过抑制肿瘤血管生成而抑制肿瘤生长。本课题主要研究昆布多糖对乳腺癌细胞(MCF-7) NIS基因和蛋白表达的影响,并探讨其与细胞摄碘功能之间的关系。第一部分乳腺癌与甲状腺疾病关系的临床研究目的调查乳腺癌患者中甲状腺疾病的发病率,通过与对照组中甲状腺疾病的发病率比较,初探乳腺癌与甲状腺疾病之间的关系；初步了解甲状腺疾病对乳腺癌患者预后的影响。方法采用临床非随机对照研究的方法,选取我院可手术乳腺癌患者100例作为乳腺癌组,选取体检中心体检者100例作为对照组,分别接受甲状腺彩超、甲状腺抗体2项(TPOAb、TgAb)及甲功5项(TT3、TT4、FT3、FT4、TSH)检查,分别统计两组患者的TT3、TT4、FT3、FT4、TSH值,TPOAb及TgAb阳性率,甲状腺左右叶体积,甲状腺疾病的发病率。分别统计伴随甲状腺疾病的乳腺癌患者及甲状腺正常的乳腺癌患者的病理及生物学特征。分别统计两组患者的年龄、月经状况、体重指数(BMI)等基线特征。结果乳腺癌组和对照组的甲状腺左右叶体积大小比较,差异无统计学意义(P>0.05)；两组患者甲状腺疾病的总发病率分别为62%和38%,差异有统计学意义(P<0.05)；两组患者之间的TT3、TT4、FT3、FT4、TSH值比较,差异均无统计学意义(P>0.05)；两组患者之间的甲状腺单发结节、甲状腺腺瘤、甲状腺囊腺瘤、甲状腺囊肿、桥本氏甲状腺炎、甲亢、甲减、亚临床甲亢、亚临床甲减的发病率比较,差异均无统计学意义(P>0.05)；与对照组相比较,乳腺癌组的甲状腺多发结节、结节性甲状腺肿的发病率高,差异有统计学意义(P<0.05)；与对照组比较,乳腺癌组的TPOAb和TgAb性的发病率高,差异有统计学意义(P<0.05)；与甲状腺正常的乳腺癌患者相比较,伴随甲状腺疾病的乳腺癌患者的Her-2阳性的表达率高,Ki-67>30%的增殖率增高,差异均有统计学意义(P<0.05)；两组的区域淋巴结、组织学分级、病理分期、脉管癌栓、雌激素受体、孕激素受体等病理特征比较,差异均无统计学意义(P>0.05)。两组患者的年龄、月经状况、BMI等基线特征比较,差异均无统计学意义(P>0.05)；结论乳腺癌组的甲状腺疾病总发病率高于对照组；乳腺癌患者的甲状腺多发结节、结节性甲状腺肿的发病率较高,提示乳腺癌患者多伴发甲状腺病理形态方面的改变；乳腺癌组TPOAb及TgAb阝日性率高,提示乳腺癌与自身免疫性甲状腺疾病可能相关；伴随甲状腺疾病的乳腺癌患者Her-2阳性的表达率较高,Ki-67增殖率较高,提示可能预后较差；而两组的区域淋巴结、组织学分级、病理分期、脉管癌栓、雌激素受体、孕激素受体等病理特征比较,差异均无统计学意义。尚需进一步随访明确甲状腺疾病对乳腺癌患者预后的影响。第二部分伴随甲状腺疾病的乳腺癌患者中医证型分布研究目的探讨同时伴随甲状腺疾病的乳腺癌患者中医证型的分布情况,并进一步比较伴发甲状腺疾病组和甲状腺正常组的乳腺癌中医证型的差异,初探甲状腺疾病是否影响乳腺癌中医证型的分布。方法乳腺癌患者入院时填写症状调查表,分别统计伴随甲状腺疾病的乳腺癌患者及甲状腺正常的乳腺癌患者的中医证型分布情况及症状分级量化值。结果伴随甲状腺疾病的乳腺癌组中,肝郁脾虚证占50%,其次是肾阴虚(19.35%)、血瘀证(16.13%)、肾阳虚(14.52%)。甲状腺正常的乳腺癌组中,肝郁脾虚证占57.89%,其次是血瘀证(21.05%)、肾阴虚(10.53%)、肾阳虚(10.53%)。两组之间的各证型分布,差异均无统计学意义(P>0.05)。两组乳腺癌患者各中医证型的症状分级量化值比较,差异均无统计学意义(P>0.05)。结论甲状腺疾病对乳腺癌患者的中医证型分布无明显影响。两组患者的症状分级量化值比较,无明显差异。第三部分昆布多糖对乳腺癌MCF-7细胞钠碘转运体的基因和蛋白表达及摄碘率的影响目的探讨掌状昆布提取物昆布多糖对乳腺癌细胞株MCF-7钠碘转运体(NIS)基因、蛋白表达和摄碘功能的影响,为昆布多糖辅助同位素早期诊断和治疗乳腺癌提供理论依据。方法对乳腺癌细胞株(MCF-7)进行培养,分别给予不同浓度的昆布多糖(0,10ug/mL, 50ug/mL,100ug/mL,200ug/mL),分别孵育24小时、48小时及72小时。采用Real-timePCR法、Western blot法检测细胞中NIS的mRNA和蛋白表达,并用125I测定细胞的摄碘能力。结果不同浓度的昆布多糖刺激乳腺癌细胞(MCF-7) 24h后,NISmRNA和蛋白的表达与空白对照组比较均增加,差异有统计学意义(P<0.05),并与刺激浓度呈正比,各组间比较中,除100ug/ml组和200ug/ml组之间无明显差异(P>0.05),余组间比较,差异均有统计学意义(P<0.05)；不同浓度的昆布多糖刺激乳腺癌细胞(MCF-7) 48h后,NISmRNA和蛋白的表达与空白对照组比较均增加,差异有统计学意义(P<0.05),且与刺激浓度呈正比,各组间比较,差异均有统计学意义(P<0.05)；不同浓度的昆布多糖刺激乳腺癌细胞(MCF-7) 72h后,NISmRNA和蛋白的表达与空白对照组比较均增加,差异有统计学意义(P<0.05),提示与刺激浓度呈正比,各组间比较中,除100ug/ml组和200ug/ml组之间无明显差异(P>0.05),余组间比较,差异均有统计学意义(P<0.05)。不同浓度的昆布多糖刺激MCF-7细胞48h后,与空白对照组比较,10ug/ml组的摄碘率无明显增加,差异无统计学意义(P>0.05),随着浓度增加,摄碘率却显示明显增加,差异均有统计学意义(P<0.05),提示与刺激浓度成正比。余组间比较,差异均有统计学意义(P<0.05)。结论不同浓度的昆布多糖刺激乳腺癌细胞(MCF-7),使NISmRNA和蛋白的表达均增加,且与刺激浓度呈正比。昆布多糖可能通过调节乳腺癌细胞NIS基因和蛋白的表达而改善细胞摄碘能力。更多还原

【Abstract】 Background and objectiveBreast cancer is one of the most common malignant tumors which seriously threaten the health of women. The incidence and mortality of breast cancer are rank the first among the famale malignancy tumors. Especially in some economic flourishing regions and big city in our country, the incidence was resided the first female malignant tumor. Breast and thyroid belong to organs which are sensitive to pituitrin. The change of endocrine can leads to gland diseases. Breast cancer cells which still have normal function of hormone receptor grow depending on original hormones. Thyroid hormones promote the development and atomization of normal breast tissues. Thyroid hormones which can directly control the proliferation of cells change the metabolism of anterior pituitary, germen hormones and carcinogen by the shortage and excessive of its content.Thus, the thyroid hormones may promote the growth of tumor cells and also may suppress the development of tumor cells. Thyroid hormone receptor and Thyroid stimulating hormone receptor expresses in the breast tissues, estrogen effects developments physiology and pathology of thyroid. We discuss the relationship between breast cancer and thyroid diseases and realize whether thyroid diseases effect the progonosis of breast cancer.According to " The standard of efficacy of TCM sympotomes " pubished by State Administration of Traditional Chinese Medicine (ZY/T001.2-94), the sympotomes of breast cancer include three categories:Stagnation of liver-qi and accumulation of phlegm, Incoordination of the thoroughfare and conception vessels, Excessive toxin due to deficient qi. Thyroid diseases which belong to " goiter " in TCM is relevance of emotion, diet and natural enviroment and dimate. Stagnation of liver-qi, disharmony of the spleen, imbalance of zang-fu and retardent of meridians and collaterals which lead to qi stagnation, blood stasis and accumulation of phlegm which coagulate the neck. In addition, it is relevance with deficiency of liver and kidney and extrnal infection.The goiter and breast cancer are both relate to kidney, liver and spleen. Thus, they have the relevance in the pathogenesis of TCM theory. We discuss the distribution of breast cancer patients having thyroid diseases and further study thyroid diseases whether influenced its distribution.The sodium-iodide symporter (NIS) which is an integral membrane protein of thyroid follicular cells transports active iodide from vessels into cells through exchange Na+ and K+. It transports active iodide necessary for thyroid hormone synthesis. Accordingly, radioiodines are used for the diagnosis and treatment of various thyroid diseases. Most differentiated thyroid carcinomas retain NIS protein activity. This provides the basis for the use of radioiodine in the detection and therapy of thyroid cancer. Moreover, iodide also accumulates in the lactating mammary gland and is secreted into milk. Like thyroid cancers, it is possible that breast cancers express the NIS gene and protein. Several investigators have reported the expression of NIS protein and the NIS gene in various cases of human breast cancer. There is a possibility of being able to use NIS and radionuclide for the detection and treatment of breast cancer. Basal NIS gene expression is detected in about 85% breast cancer specimens, but the fraction with functional iodide transport is relatively low. The radioiodine is difficult to treat breast cancer. Retinoic acid has been used to stimulate NIS expression in both thyroid and breast cancer. Agents that promote tumor differentiation, or directly stimulate NIS gene expression, may result in iodine concentration in ’scan-negative’thyroid cancer and some breast cancer. Lactogenic hormones, estrogen, cAMP stimulus, glucocorticoid can stimulate NIS expression and increase radioiodide uptake.Thallus japonica which is Laminarin japonica Aresch has the effect of softening hardness to dissipate stagnation and disinhibiting water to draining heat. Thallus japonica is rich in iodide which maintains normal function of thyroid and breast tissue.It has lots of active ingredients which relate to its polysaccharide ingredients. Laminarin which is aβ-1,3 glucosan polymer can activate macrophagocyte and produce cytotoxicity in order to restrain the proliferati on of tumor cells. In addition, it can suppress tumor angiogenesis to control tumor growth. We research the effect of Laminarin to NIS gene and protein expression of MCF-7 cells and discuss the relationship between iodide uptake and NIS gene and protein expression.Part I The clinic research of the relationship between breast cancer and thyroid pathologyObjectiveThe aim of the study was to comfirm the incidence of the thyroid diseases among breast cancer patients and discuss whether thyroid diseases effect the prognosis of breast cancer patients.MethodsOur prospective open study was conducted on 100 consecutive patients operated for breast carcinoma. As a control group,100 individuals with normal breast examination, who did not have any known malignancy and/or thyroid disease, were included in the study. We aimed to identify the occurrence of thyroid pathologies, thyroid volume, the positive rate of TPOAb and TgAb and the number of TT3、TT4、FT3、FT4、TSH、TPOAb、TgAb in both groups to compare the ultrasound and the laboratory features of thyroid disease (TT3、TT4、FT3、FT4、TSH、TPOAb、TgAb) and breast carcinoma in an attempt to contribute to the studies investigating the relationship between these two entities.In addition,we also identified the pathology and biology features of breast cancer patients with/without thyroid diseases. We definited the baseline features such as age, menstruation and BMI of two groups.ResultsThe thyroid volumes of breast cancer patients and the control group did not differ significantly (P>0.05). When breast carcinoma patients were compared with the control group, thyroid pathology were found to be significa-ntly high in patients with breast cancer(62% vs 38%, P<0.05). There was no statistically significant difference between the two groups with respect to TPOAb、TgAb、TT3、TT4、FT3、FT4、TSH levels (P>0.05). Multiple nodular and nodular goiter thyroid pathologies were significantly higher in the breast cancer group when compared to the control group(P<0.05). There was no statistically significant difference between the two groups with respect to single nodular,adenoma cystadenoma, cystis thyroid pathologies and hyperthyroidism, hypothyroidism, subclinical hypothyroidism, subclinical hyperthyroidism, Hashimoto’s thyroiditis(P>0.05). The incidence of TPOAb and TgAb positive are significantly higher in the breast cancer group when compared to the control group (P>0.05). In breast cancer patients with thyroid pathology, the incidence of HER-2 positive and Ki-67>30% were significantly more compared to the patients without thyroid disease(P<0.05). There was no significant relationship between other pathology features such as lymph nodes, the classification of histology, the stage of pathology, cancer embolus in vessel, progestational hormone receptor and estrogenic hormone receptor of the breast cancer patients and the presence of thyroid pathology (P>0.05). There was no statistically significant difference between the baseline features such as age, menstruation and BMI(P>0.05).ConclusionIn conclusion, the frequency of thyroid pathology is higher in breast cancer patients compared to controls indicating a relationship between breast carcinoma and thyroid pathology. Our study shows that breast cancer patients accompany with thyroid morphology changes such as multiple nodular and nodular goiter. The incidence of TPOAb and TgAb positive are significant-ly higher in the breast cancer group, it is indicated that breast cancer may be relevance to autoimmune thyroid diseases. Although the incidence of HER-2 positive and Ki-67>30% are higher in breast cancer patients with thyroid thyroid pathology, there was no significant relationship between other pathology features such as lymph nodes, the classification of histolog-y, the stage of pathology, cancer embolus in vessel, progestational hormone receptor and estrogenic hormone receptor of the breast cancer patients and the presence of thyroid pathology, we need further studies are needed to investigate the prognosis and survival of breast cancer patients in the presence of thyroid pathology. PartⅡResearch on the d i str i but i on of TCM syndromes i n breast cancer patients with thyroid pathology Object iveTo study the distribution of TCM syndromes in breast cancer patients with thyroid pathology and disscuss thyroid pathology whether influences the the distribution of TCM syndromes in breast cancer. MethodsWhen breast cancer patients were hospitalized, the questionnaire was written in time. Through satatistic analysis, it is confirmed that the distribution of TCM syndromes in breast cancer patients with thyroid pathology and control group. ResultsIn breast cancer patients with thyroid pathology, Stagnation of liver-qi and deficiency of spleen occupied 50%, deficiency of kidney-yin occupied 19.35%, blood stasis occupied 16.13%, deficiency of kidney-yang occupied 14.52%. In the control group, Stagnation of liver-qi and deficiency of spleen occupied 57.89%, deficiency of kidney-yin occupied 10.53%, blood stasis occupied 21.05%, deficiency of kidney-yang occupied 10.53%. There was no statistically significant difference between the two groups with respect to the distribution and the grading numbers of TCM syndromes (P>0.05).ConclusionThyroid pathology did not effect the distribution of TCM syndromes in breast cancer patients. There was no statistically significant difference between the two groups with respect to the grading numbers of TCM syndromes.PartⅢThe effect of Laminar in on the expression of NIS gene and protein expression and rate of iodide uptake in breast cell(MCF-7).ObjectiveTo investigate the effect of Laminarin 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.MethodsThe breast cancer cell line(MCF-7) was cultured with different concentrations of Laminarin(0, 10ug/mL,50ug/mL,100ug/mL,200ug/mL). NTS mRNA and protein in cultured breast cancer cells was determined by Real-time PCR and Western blot after 24h,48h,72h. The activity of iodide uptake was determined by 125I uptake rate. ResultsWhen compared with blank control group, NIS gene expression and prote in expression were enhanced by Laminarin in dose-dependent manner after s timulating 24h (P<0.05). There was no statistically significant differen ce between 100ug/ml and 200ug/ml after stimulating 24h(P>0.05). Compared among other groups, they all have statistically significant diffe rence (P<0.05). When compared with blank control group, NIS gene expressi on and protein expression were enhanced by Laminarin in dose-dependent ma nner after stimulating 48h(P<0.05). Compared among groups, they allhave st atistically significant difference (P<0.05). When compared withblank cont rol group, NIS gene expression and protein expression were enh-anced by La minarin in dose-dependent manner after stimulating 72h(P<0.05). There was no statistically significant difference between 100ug/ml and 200ug/ml af ter stimulating 72h(P>0.05). Compared among other groups, they all have st atistically significant difference(P<0.05). The activity of iodide uptak e had been found to be increased by Laminarin in dose-dependent manner(P <0.05). There was no statistically significant diffe-rence between Oug/ml and 10ug/ml (P>0.05).ConclusionNIS gene expression and protein expression were enhanced by Laminari-n in dose-dependent manner. The activity of iodide uptake had been found to be increased by Laminarin which may be attribute to enhance the NIS gene experessin and protein expression.更多还原