乳腺微浸潤性癌臨床病理特征和導(dǎo)管內(nèi)癌浸潤機(jī)制的研究
發(fā)布時間：2018-08-06 09:35
【摘要】：目的探討乳腺微浸潤性癌和導(dǎo)管內(nèi)癌(ductal carcinoma in situ,DCIS)的臨床病理特征、分子分型及預(yù)后情況。研究上皮間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)和肌上皮表型改變是否參與導(dǎo)管內(nèi)癌進(jìn)展到浸潤性乳腺癌的進(jìn)程。方法收集131例乳腺微浸潤性癌和451例導(dǎo)管內(nèi)癌。通過免疫組化檢測ER、PR、HER2和Ki67。對微浸潤性癌進(jìn)行臨床病理特征的評估,如臨床表現(xiàn),病理特征,分子亞型和臨床預(yù)后結(jié)果,并與導(dǎo)管內(nèi)癌作比較。以導(dǎo)管內(nèi)癌和浸潤性癌為研究對象,通過免疫組化檢測EMT標(biāo)記物及肌上皮細(xì)胞標(biāo)記物的表達(dá)情況。使用TGF-β1誘導(dǎo)乳腺腺上皮表型細(xì)胞MCF-7發(fā)生EMT,并建立肌上皮表型細(xì)胞MDA-MB-231與MCF-7的共培養(yǎng)體系。觀察細(xì)胞形態(tài)學(xué)變化,通過QRT-PCR和Western blot檢測EMT相關(guān)標(biāo)記物的表達(dá)水平,MTT法檢測細(xì)胞增殖能力的改變,劃痕實(shí)驗(yàn)和Transwell實(shí)驗(yàn)檢測細(xì)胞遷移及侵襲能力的變化,通過ELISA法檢測TGF-β1對共培養(yǎng)上清液MMP-9和IL-6分泌量的影響。結(jié)果1、比較乳腺微浸潤性癌和導(dǎo)管內(nèi)癌的臨床病理特征,在腫瘤直徑、淋巴結(jié)轉(zhuǎn)移方面微浸潤性癌高于導(dǎo)管內(nèi)癌;而在年齡、絕經(jīng)狀況、家族史、分子分型方面,兩者差異無統(tǒng)計(jì)學(xué)意義。比較不同分子分型乳腺微浸潤性癌與臨床病理特征,Her-2過表達(dá)型和TNBCs在組織學(xué)級別方面較高;而在年齡、腫瘤直徑、淋巴結(jié)轉(zhuǎn)移、絕經(jīng)狀況、家族史方面,不同分子分型之間差異無統(tǒng)計(jì)學(xué)意義。比較不同分子分型導(dǎo)管內(nèi)癌與臨床病理特征,Her-2過表達(dá)型和TNBCs在年齡、腫瘤直徑、核級別方面較高;而在淋巴結(jié)轉(zhuǎn)移、家族史方面,不同分子分型之間差異無統(tǒng)計(jì)學(xué)意義。2、隨訪時間自治療時間起,中位隨訪時間分別為69和62個月。乳腺微浸潤性癌和導(dǎo)管內(nèi)癌的5年總生存率分別為99.0%和99.2%,微浸潤性癌與導(dǎo)管內(nèi)癌的OS差異無統(tǒng)計(jì)學(xué)意義。乳腺微浸潤性癌和導(dǎo)管內(nèi)癌的5年無病生存率分別為95.2%和95.9%,微浸潤性癌和導(dǎo)管內(nèi)癌的DFS差異無統(tǒng)計(jì)學(xué)意義。3、以正常乳腺組織、導(dǎo)管內(nèi)癌和浸潤性癌為研究對象,通過免疫組化檢測EMT標(biāo)記物的表達(dá)情況。E-cadherin在正常組織表達(dá)較高,在導(dǎo)管內(nèi)癌組及IDC組表達(dá)降低。N-cadherin、Snail、Vimentin、Twist和Zeb1在正常組織中不表達(dá),在導(dǎo)管內(nèi)癌組及IDC組表達(dá)明顯增高,差異有統(tǒng)計(jì)學(xué)意義。4、以正常乳腺組織、導(dǎo)管內(nèi)癌和浸潤性癌為研究對象,通過免疫組化檢測肌上皮細(xì)胞標(biāo)記物的表達(dá)情況。SMA、p63、Calponin蛋白表達(dá)在正常組織和導(dǎo)管內(nèi)癌中呈陽性表達(dá),在浸潤性癌幾乎不著色。5、TGF-β1刺激MCF-7細(xì)胞誘導(dǎo)發(fā)生EMT,部分處理組細(xì)胞較對照組發(fā)生形態(tài)學(xué)變化。通過QRT-PCR檢測TGF-β1刺激MCF-7后EMT相關(guān)標(biāo)記物mRNA的表達(dá)水平,處理組較對照組E-cadherin下調(diào),Vimentin、N-cadherin上調(diào)。通過Western blot檢測TGF-β1刺激MCF-7后EMT相關(guān)標(biāo)記物蛋白的表達(dá)水平,處理組較對照組E-cadherin下調(diào),Vimentin、N-cadherin上調(diào)。MTT法發(fā)現(xiàn)處理組較對照組細(xì)胞增殖沒有差異。劃痕實(shí)驗(yàn)檢測TGF-β1刺激MCF-7后,處理組較對照組細(xì)胞遷移明顯增加。6、TGF-βl影響共培養(yǎng)體系下MDA-MB-231的形態(tài)學(xué)發(fā)生變化。TGF-βl對共培養(yǎng)體系下MDA-MB-231的增殖能力沒有影響。TGF-βl增強(qiáng)共培養(yǎng)條件下MDA-MB-231的遷移和侵襲能力。TGF-βl提高共培養(yǎng)條件下MDA-MB-231分泌MMP-9和IL-6的含量。結(jié)論總之,乳腺微浸潤性癌與導(dǎo)管內(nèi)癌在臨床病理特征及預(yù)后方面相似。相比于導(dǎo)管內(nèi)癌,浸潤性癌中間質(zhì)標(biāo)記物表達(dá)增多、E-cadherin表達(dá)缺失和肌上皮表型標(biāo)記物表達(dá)下降。通過TGF-β1的刺激,可以有效促進(jìn)乳腺癌腺上皮表型細(xì)胞的侵襲和遷移能力,也促進(jìn)乳腺癌肌上皮表型細(xì)胞的侵襲和遷移能力。表明EMT和肌上皮表型改變可能參與導(dǎo)管內(nèi)癌進(jìn)展到浸潤性乳腺癌的進(jìn)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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R737.9
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