乳腺微浸润性癌临床病理特征和导管内癌浸润机制的研究
发布时间：2018-08-06 09:35
【摘要】：目的探讨乳腺微浸润性癌和导管内癌(ductal carcinoma in situ,DCIS)的临床病理特征、分子分型及预后情况。研究上皮间质转化(epithelial-mesenchymal transition,EMT)和肌上皮表型改变是否参与导管内癌进展到浸润性乳腺癌的进程。方法收集131例乳腺微浸润性癌和451例导管内癌。通过免疫组化检测ER、PR、HER2和Ki67。对微浸润性癌进行临床病理特征的评估,如临床表现,病理特征,分子亚型和临床预后结果,并与导管内癌作比较。以导管内癌和浸润性癌为研究对象,通过免疫组化检测EMT标记物及肌上皮细胞标记物的表达情况。使用TGF-β1诱导乳腺腺上皮表型细胞MCF-7发生EMT,并建立肌上皮表型细胞MDA-MB-231与MCF-7的共培养体系。观察细胞形态学变化,通过QRT-PCR和Western blot检测EMT相关标记物的表达水平,MTT法检测细胞增殖能力的改变,划痕实验和Transwell实验检测细胞迁移及侵袭能力的变化,通过ELISA法检测TGF-β1对共培养上清液MMP-9和IL-6分泌量的影响。结果1、比较乳腺微浸润性癌和导管内癌的临床病理特征,在肿瘤直径、淋巴结转移方面微浸润性癌高于导管内癌;而在年龄、绝经状况、家族史、分子分型方面,两者差异无统计学意义。比较不同分子分型乳腺微浸润性癌与临床病理特征,Her-2过表达型和TNBCs在组织学级别方面较高;而在年龄、肿瘤直径、淋巴结转移、绝经状况、家族史方面,不同分子分型之间差异无统计学意义。比较不同分子分型导管内癌与临床病理特征,Her-2过表达型和TNBCs在年龄、肿瘤直径、核级别方面较高;而在淋巴结转移、家族史方面,不同分子分型之间差异无统计学意义。2、随访时间自治疗时间起,中位随访时间分别为69和62个月。乳腺微浸润性癌和导管内癌的5年总生存率分别为99.0%和99.2%,微浸润性癌与导管内癌的OS差异无统计学意义。乳腺微浸润性癌和导管内癌的5年无病生存率分别为95.2%和95.9%,微浸润性癌和导管内癌的DFS差异无统计学意义。3、以正常乳腺组织、导管内癌和浸润性癌为研究对象,通过免疫组化检测EMT标记物的表达情况。E-cadherin在正常组织表达较高,在导管内癌组及IDC组表达降低。N-cadherin、Snail、Vimentin、Twist和Zeb1在正常组织中不表达,在导管内癌组及IDC组表达明显增高,差异有统计学意义。4、以正常乳腺组织、导管内癌和浸润性癌为研究对象,通过免疫组化检测肌上皮细胞标记物的表达情况。SMA、p63、Calponin蛋白表达在正常组织和导管内癌中呈阳性表达,在浸润性癌几乎不着色。5、TGF-β1刺激MCF-7细胞诱导发生EMT,部分处理组细胞较对照组发生形态学变化。通过QRT-PCR检测TGF-β1刺激MCF-7后EMT相关标记物mRNA的表达水平,处理组较对照组E-cadherin下调,Vimentin、N-cadherin上调。通过Western blot检测TGF-β1刺激MCF-7后EMT相关标记物蛋白的表达水平,处理组较对照组E-cadherin下调,Vimentin、N-cadherin上调。MTT法发现处理组较对照组细胞增殖没有差异。划痕实验检测TGF-β1刺激MCF-7后,处理组较对照组细胞迁移明显增加。6、TGF-βl影响共培养体系下MDA-MB-231的形态学发生变化。TGF-βl对共培养体系下MDA-MB-231的增殖能力没有影响。TGF-βl增强共培养条件下MDA-MB-231的迁移和侵袭能力。TGF-βl提高共培养条件下MDA-MB-231分泌MMP-9和IL-6的含量。结论总之,乳腺微浸润性癌与导管内癌在临床病理特征及预后方面相似。相比于导管内癌,浸润性癌中间质标记物表达增多、E-cadherin表达缺失和肌上皮表型标记物表达下降。通过TGF-β1的刺激,可以有效促进乳腺癌腺上皮表型细胞的侵袭和迁移能力,也促进乳腺癌肌上皮表型细胞的侵袭和迁移能力。表明EMT和肌上皮表型改变可能参与导管内癌进展到浸润性乳腺癌的进程。
[Abstract]:Objective to investigate the clinicopathological features, molecular typing and prognosis of ductal carcinoma in situ (DCIS) in breast microinvasive carcinoma and intraductal carcinoma (DCIS). To investigate whether epithelial mesenchymal transition (epithelial-mesenchymal transition, EMT) and myoepithelial phenotype changes are involved in the progression of intraductal carcinoma to invasive breast cancer. Methods 131 cases of breast cancer were collected. Adenosine infiltrating carcinoma and 451 cases of intraductal carcinoma. The clinicopathological features of microinvasive carcinoma were assessed by immunohistochemical detection of ER, PR, HER2 and Ki67., such as clinical, pathological, molecular subtypes and clinical prognosis, and compared with intraductal carcinoma. The study of intraductal and infiltrative cancers was conducted by immunohistochemical detection of EMT The expression of markers and myoepithelial cell markers. TGF- beta 1 was used to induce EMT in the phenotype of mammary gland epithelial phenotype, and the co culture system of MDA-MB-231 and MCF-7 in myoepithelial cells was established. The morphological changes were observed and the expression level of EMT related markers was detected by QRT-PCR and Western blot. The MTT method was used to detect the cell growth. Changes in colonization, scratch test and Transwell test were used to detect changes in cell migration and invasion ability. The effect of TGF- beta 1 on the secretion of MMP-9 and IL-6 in co culture supernatant was detected by ELISA. Results 1, the clinicopathological features of breast microinvasive carcinoma and intraductal carcinoma were compared, and the microinvasive cancer was higher in the diameter of the tumor and the lymph node metastasis. There were no statistically significant differences in age, menopause, family history, and molecular typing. Compared with different molecular types of breast microinvasive carcinoma and clinicopathological features, Her-2 overexpression and TNBCs were higher in histological grade, but in age, tumor diameter, lymph node metastasis, menopause, family history, and no family history. There was no statistical difference between the same molecular typing. Compared with the different molecular types of intraductal carcinoma and clinicopathological features, Her-2 overexpression and TNBCs were higher in age, tumor diameter and nuclear grade, while in lymph node metastasis and family history, there was no statistical difference between different molecular types, and the time of follow-up was from the time of treatment from.2. The total 5 year survival rates of microinvasive and intraductal cancers were 99% and 99.2% respectively. There was no significant difference in the OS difference between microinvasive and intraductal carcinoma. The 5 year disease survival rates of microinvasive and intraductal cancers were 95.2% and 95.9% respectively, and the difference in DFS from microinvasive and intraductal carcinoma No statistical significance.3, with normal breast tissue, intraductal carcinoma and invasive carcinoma as the research object, the expression of EMT markers in the normal tissue was higher by immunohistochemistry. The expression of.E-cadherin in the intraductal carcinoma group and the IDC group decreased.N-cadherin, Snail, Vimentin, Twist and Zeb1 were not expressed in the normal tissue, and in the intraductal carcinoma. The expression of the group and IDC group was significantly higher, and the difference was statistically significant.4. The expression of myoepithelial cell markers in normal breast tissue, intraductal carcinoma and invasive carcinoma was detected by immunohistochemical staining.SMA, p63, Calponin protein expression was positive in normal tissue and intraductal carcinoma, and almost no.5, TG was stained in invasive carcinoma. TG F- beta 1 stimulated MCF-7 cells to induce EMT, and some cells in the treatment group had morphological changes compared with the control group. The expression level of mRNA in EMT related markers after TGF- beta 1 stimulated MCF-7, the treatment group was down regulated by the control group, Vimentin, N-cadherin up. The expression level of protein in the treatment group was lower than that of the control group E-cadherin, Vimentin, N-cadherin up regulation.MTT method found that there was no difference in cell proliferation between the treatment group and the control group. After the scratch test, TGF- beta 1 stimulated MCF-7, and the treatment group increased the cell migration significantly more.6 than the control group, and TGF- beta l influenced the morphological change.T of MDA-MB-231 under the co culture system. GF- beta l had no effect on the proliferation of MDA-MB-231 under co culture system..TGF- beta l enhanced the migration and invasion of MDA-MB-231 under co culture conditions,.TGF- beta l increased the MDA-MB-231 secretion MMP-9 and IL-6 content under co culture conditions. Conclusion in conclusion, breast microinvasive carcinoma and intraductal carcinoma are similar to the clinicopathological features and prognosis. In intraductal carcinoma, the expression of intermediate markers of infiltrative carcinoma increased, E-cadherin expression was absent and the expression of myoepithelial phenotypic markers decreased. Through the stimulation of TGF- beta 1, the invasion and migration ability of mammary gland epithelial phenotype cells could be effectively promoted, and the invasion and migration of myoepithelial epithelioid cells in breast cancer were also promoted. It showed that EMT and myoepithelial cells were on the muscle. Alterations in skin phenotype may be involved in the progression of ductal carcinoma to invasive breast cancer.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9
[Abstract]:Objective to investigate the clinicopathological features, molecular typing and prognosis of ductal carcinoma in situ (DCIS) in breast microinvasive carcinoma and intraductal carcinoma (DCIS). To investigate whether epithelial mesenchymal transition (epithelial-mesenchymal transition, EMT) and myoepithelial phenotype changes are involved in the progression of intraductal carcinoma to invasive breast cancer. Methods 131 cases of breast cancer were collected. Adenosine infiltrating carcinoma and 451 cases of intraductal carcinoma. The clinicopathological features of microinvasive carcinoma were assessed by immunohistochemical detection of ER, PR, HER2 and Ki67., such as clinical, pathological, molecular subtypes and clinical prognosis, and compared with intraductal carcinoma. The study of intraductal and infiltrative cancers was conducted by immunohistochemical detection of EMT The expression of markers and myoepithelial cell markers. TGF- beta 1 was used to induce EMT in the phenotype of mammary gland epithelial phenotype, and the co culture system of MDA-MB-231 and MCF-7 in myoepithelial cells was established. The morphological changes were observed and the expression level of EMT related markers was detected by QRT-PCR and Western blot. The MTT method was used to detect the cell growth. Changes in colonization, scratch test and Transwell test were used to detect changes in cell migration and invasion ability. The effect of TGF- beta 1 on the secretion of MMP-9 and IL-6 in co culture supernatant was detected by ELISA. Results 1, the clinicopathological features of breast microinvasive carcinoma and intraductal carcinoma were compared, and the microinvasive cancer was higher in the diameter of the tumor and the lymph node metastasis. There were no statistically significant differences in age, menopause, family history, and molecular typing. Compared with different molecular types of breast microinvasive carcinoma and clinicopathological features, Her-2 overexpression and TNBCs were higher in histological grade, but in age, tumor diameter, lymph node metastasis, menopause, family history, and no family history. There was no statistical difference between the same molecular typing. Compared with the different molecular types of intraductal carcinoma and clinicopathological features, Her-2 overexpression and TNBCs were higher in age, tumor diameter and nuclear grade, while in lymph node metastasis and family history, there was no statistical difference between different molecular types, and the time of follow-up was from the time of treatment from.2. The total 5 year survival rates of microinvasive and intraductal cancers were 99% and 99.2% respectively. There was no significant difference in the OS difference between microinvasive and intraductal carcinoma. The 5 year disease survival rates of microinvasive and intraductal cancers were 95.2% and 95.9% respectively, and the difference in DFS from microinvasive and intraductal carcinoma No statistical significance.3, with normal breast tissue, intraductal carcinoma and invasive carcinoma as the research object, the expression of EMT markers in the normal tissue was higher by immunohistochemistry. The expression of.E-cadherin in the intraductal carcinoma group and the IDC group decreased.N-cadherin, Snail, Vimentin, Twist and Zeb1 were not expressed in the normal tissue, and in the intraductal carcinoma. The expression of the group and IDC group was significantly higher, and the difference was statistically significant.4. The expression of myoepithelial cell markers in normal breast tissue, intraductal carcinoma and invasive carcinoma was detected by immunohistochemical staining.SMA, p63, Calponin protein expression was positive in normal tissue and intraductal carcinoma, and almost no.5, TG was stained in invasive carcinoma. TG F- beta 1 stimulated MCF-7 cells to induce EMT, and some cells in the treatment group had morphological changes compared with the control group. The expression level of mRNA in EMT related markers after TGF- beta 1 stimulated MCF-7, the treatment group was down regulated by the control group, Vimentin, N-cadherin up. The expression level of protein in the treatment group was lower than that of the control group E-cadherin, Vimentin, N-cadherin up regulation.MTT method found that there was no difference in cell proliferation between the treatment group and the control group. After the scratch test, TGF- beta 1 stimulated MCF-7, and the treatment group increased the cell migration significantly more.6 than the control group, and TGF- beta l influenced the morphological change.T of MDA-MB-231 under the co culture system. GF- beta l had no effect on the proliferation of MDA-MB-231 under co culture system..TGF- beta l enhanced the migration and invasion of MDA-MB-231 under co culture conditions,.TGF- beta l increased the MDA-MB-231 secretion MMP-9 and IL-6 content under co culture conditions. Conclusion in conclusion, breast microinvasive carcinoma and intraductal carcinoma are similar to the clinicopathological features and prognosis. In intraductal carcinoma, the expression of intermediate markers of infiltrative carcinoma increased, E-cadherin expression was absent and the expression of myoepithelial phenotypic markers decreased. Through the stimulation of TGF- beta 1, the invasion and migration ability of mammary gland epithelial phenotype cells could be effectively promoted, and the invasion and migration of myoepithelial epithelioid cells in breast cancer were also promoted. It showed that EMT and myoepithelial cells were on the muscle. Alterations in skin phenotype may be involved in the progression of ductal carcinoma to invasive breast cancer.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9
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