非肌细胞肌球蛋白Ⅱ重链在乳腺癌中作用的研究
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摘要
目的:确定乳腺癌中存在的非肌细胞肌球蛋白Ⅱ重链亚型;观察非肌细胞肌球蛋白Ⅱ重链亚型在乳腺癌中的表达情况;探讨非肌细胞肌球蛋白Ⅱ重链亚型与乳腺癌恶性程度及浸润转移的关系。
方法:应用Elston与Ellis的改良的Bloom-Richardson系统进行乳腺癌标本分级;应用免疫组化SP法检测非肌细胞肌球蛋白Ⅱ重链亚型A、B在乳腺癌中的定位及表达特点;应用Image-Pro Plus 6.0软件测定NMHCⅡA、NMHCⅡB在乳腺癌中表达强度;应用glycerol SDS-PAGE结合Western blot,观察NMHCⅡA、NMHCⅡB在乳腺癌细胞和正常乳腺细胞中的表达情况;应用INSTAT软件进行统计学分析。
结果:免疫组织化学染色结果显示,①NMHCⅡA和NMHCⅡB均在乳腺癌中表达。②NMHCⅡA主要表达于癌细胞胞浆中;随恶性程度增高,NMHCⅡA阳性表达增强,差别有统计学意义(P<0.05)。③NMHCⅡB主要表达于癌细胞胞膜和胞浆中;NMHCⅡB在乳腺癌Ⅲ级中的表达明显高于Ⅰ级、Ⅱ级和癌旁组织,其差异有统计学意义(P<0.001)。④NMHCⅡA在有淋巴结转移组中的表达强度高于无淋巴结转移组,但无统计学意义(P>0.05);NMHCⅡB在有淋巴结转移组中的表达强度显著高于无淋巴结转移组,差别有统计学意义(P <0.05)。⑤在NMHCⅡA和NMHCⅡB阳性表达的乳腺癌中,癌巢周边部比内部表达强,部分散在的、黏附性差的癌细胞呈强阳性表达。⑥NMHCⅡA和NMHCⅡB在乳腺癌间质中呈强阳性表达,在癌旁组织中不表达,差别有统计学意义(P<0.05)。Western blot结果显示,①NMHCⅡA和NMHCⅡB在HB细胞和MCF-7细胞中均有表达。②NMHCⅡB在乳腺癌细胞中表达明显高于正常乳腺细胞,且有统计学意义(P <0.0001)。
结论:1、乳腺癌与正常乳腺中存在两种重链亚型NMHCⅡA和NMHCⅡB,且这两种亚型在乳腺癌中表达均高于正常乳腺。2、NMHCⅡA与乳腺癌恶性程度密切相关。3、NMHCⅡB可以作为乳腺癌淋巴结转移的评估指标。4、NMHCⅡA、NMHCⅡB在乳腺癌间质中呈阳性表达,考虑与肿瘤转移有关。
As one of the most common malignant cancer of women in the world, breast cancer is from lobular epithelial cell of the breast terminal ductal, with the first incidence of cancer among women.The causes are not yet clear.Hormones, genetic, and some related proteins involve in the evolution process of breast cancer.Malignant tumor metastasis is the most common cause of death.In some literature, the initial stages of tumor metastasis involve in the epithelial and stromal migration: loss of polarized epithelial cell layer, cell adhesion and cytoskeleton remodeling.Tumor metastasis is associated with cell movement.The study also shows roles of nonmuscle myosinⅡ( NMⅡ) in cell motility:cell polarization, cell migration and cell adhesion.Meanwhile, the literature shows NMⅡenhances tumor cell invasion and metastasis by regulating cell movement.
As the hexamer, NMⅡincludes a pair of heavy chain (NMHCⅡ,230kDa) and two pairs of light chain (NMLCⅡ,20 and 17 kDa).NMLCⅡmainly exercises regulatory functions.In the myosin molecule, the light chain(MLC) combines with the heavy chain(MHC) by non-covalently, which works in stabilizing and regulating the structure and activation of myosin.Each heavy chain consists of a long fibrous tails ofα-helix and a complete spherical head.A lot of literature suggests that the spherical head of nonmuscle myosin heavy chain(N-terminal) is an important functional areas, including decomposing ATP, producing energy and affecting actin.In vitro, the ATP enzyme sites of the single heavy chain head area (S1) can be combined with the ATP enzyme and activate enzymes, in order to generate power and to drive actin filament movement.Thus, in NMⅡmolecules, NMLCⅡmainly exercises regulatory functions.NMHCⅡis the important functional areas, which cause cell movement and make biological activities.So there are important significances in studying the relationship between NMHCⅡand breast cancer by studying the relationship between NMHCⅡand cell movement.
Objective: To determine NMHCⅡsubtype of breast cancer and normal breast; To observe the expressions of NMHCⅡA, B in breast cancer cases; To compare the expressions of NMHCⅡA, B in breast cancer cells to the expressions in normal breast cells; To investigate the relationship among NMHCⅡA, B and malignancy, invasion and metastasis of breast cancer.
Methods:Application of HE staining and the modified Bloom-Richardson system by Elston and Ellis, combined with the comprehensive evaluation analysis of the structure and morphology atypical nuclei, classificated breast specimens. Immunohistochemistry staining (SP) was performed to detecte the location and expression of NMHCⅡA, B in breast cancer.Software Image-Pro Plus 6.0 was to study the cumulative optical density (IOD) of expression of NMHCⅡA, B inⅠ,Ⅱ,Ⅲgrade breast cancer and to do in breast cancer with lymph node metastasis and without lymph node metastasis.Glycerol SDS-PAGE combined with Western blot was to observe the expression of NMHCⅡA, B in the breast cancer cells (MCF-7) and the normal breast cells (HB).Application of gel imaging analysis system was to determine optical density of Western blot.INSTAT software was used for statistical analysis.
Results: According to the degree of malignancy, 47 invasive ductal breast cancer were dividedⅠ,Ⅱ,Ⅲgrade, with gradeⅠ8 cases, gradeⅡ32 cases and gradeⅢ7 cases.Immunohistochemical staining showed that,①NMHCⅡA and NMHCⅡB were expressed both in breast cancer and in adjacent tissues, and expression in breast cancer was higher than that in adjacent tissue, with statistical significance(P <0.05).②N MHCⅡA expressed mainly in the cytoplasm of cancer nests, followed in the interstitial and vascular wall.InⅠ,Ⅱ,Ⅲgrade breast cancer and in adjacent tissues, the differences of positive IOD values were statistically significant(P <0.05), with the level higher, IOD values greater.③NMHCⅡB expressed in the major parts of the membrane, cytoplasm, followed by localization in interstitial and vascular wall.The result that histological gradeⅢof breast cancer respectively comparied with gradeⅠ,Ⅱbreast cancer and adjacent tissue showed that the differences of positive IOD values were statistically significant(P <0.001), but the differences of positive IOD values among gradeⅠ,Ⅱbreast cancer and adjacent tissue were not statistically significant (P >0.05), with the degree of malignancy increasing, IOD values greater, and expressions of NMHCⅡB increasing.④T he positive expressions of NMHCⅡA in breast cancer had no relations with lymphatic metastasis, with no significant differences (P >0.05), but in the tissue of lymph node metastasis, with histological grade level increasing, IOD values increasing.The positive expressions of NMHCⅡB in breast cancer had relationship with lymphatic metastasis, with significant differences(P <0.05).⑤The results of observating NMHCⅡA and NMHCⅡB were nests lordosis position.There were more positive expressions in the cells of the cancer nest periphery than in cancer nests, with a part of the scattered or the poor adhesion cancer expressing stronger.⑥In breast cancer tissues with NMHCⅡA positive, 77.27% in the fibrous stroma of breast cancer was positive, with the fibrous stroma of adjacent tissue negative.The difference was statistically significant (P <0.05).In breast cancer tissues with NMHCⅡB positive, 81.08% in the fibrous stroma of breast cancer was positive, with the fibrous stroma of adjacent tissue negative.The difference was statistically significant (P <0.05).⑦Comparative study found that in gradeⅠ,Ⅱ,Ⅲbreast cancer, IOD values of NMHCⅡA expression were significantly higher than IOD values of NMHCⅡB expression, and there were significant differences among three groups(P <0.01).Western blot showed that NMHCⅡA and NMHCⅡB expressed both in HB and in MCF-7.Under the same protein sample, optical densities of Western blot were measured and analyzed statistically.①The optical density of NMHCⅡA in breast cancer cells(MCF-7) was higher than in normal breast cells (HB), but no statistical significance (P >0.05).②The optical density of NMHCⅡB in breast cancer cells(MCF-7) was higher than in normal breast cells (HB), with statistical significance(P <0.0001).
Conclusion: 1. There are two expression NMHCⅡA and NMHCⅡB in breast cancer and in the normal breast, with both more expressed in breast cancer and less expressed in the normal breast. 2. NMHCⅡA are closely related with the malignancy of breast cancer. 3. NMHCⅡB can be used as evaluation indicators of breast cancer metastasis. 4. NMHCⅡA and NMHCⅡB in breast cancer stroma were positive, perhaps involving in tumor metastasis.
方法:应用Elston与Ellis的改良的Bloom-Richardson系统进行乳腺癌标本分级;应用免疫组化SP法检测非肌细胞肌球蛋白Ⅱ重链亚型A、B在乳腺癌中的定位及表达特点;应用Image-Pro Plus 6.0软件测定NMHCⅡA、NMHCⅡB在乳腺癌中表达强度;应用glycerol SDS-PAGE结合Western blot,观察NMHCⅡA、NMHCⅡB在乳腺癌细胞和正常乳腺细胞中的表达情况;应用INSTAT软件进行统计学分析。
结果:免疫组织化学染色结果显示,①NMHCⅡA和NMHCⅡB均在乳腺癌中表达。②NMHCⅡA主要表达于癌细胞胞浆中;随恶性程度增高,NMHCⅡA阳性表达增强,差别有统计学意义(P<0.05)。③NMHCⅡB主要表达于癌细胞胞膜和胞浆中;NMHCⅡB在乳腺癌Ⅲ级中的表达明显高于Ⅰ级、Ⅱ级和癌旁组织,其差异有统计学意义(P<0.001)。④NMHCⅡA在有淋巴结转移组中的表达强度高于无淋巴结转移组,但无统计学意义(P>0.05);NMHCⅡB在有淋巴结转移组中的表达强度显著高于无淋巴结转移组,差别有统计学意义(P <0.05)。⑤在NMHCⅡA和NMHCⅡB阳性表达的乳腺癌中,癌巢周边部比内部表达强,部分散在的、黏附性差的癌细胞呈强阳性表达。⑥NMHCⅡA和NMHCⅡB在乳腺癌间质中呈强阳性表达,在癌旁组织中不表达,差别有统计学意义(P<0.05)。Western blot结果显示,①NMHCⅡA和NMHCⅡB在HB细胞和MCF-7细胞中均有表达。②NMHCⅡB在乳腺癌细胞中表达明显高于正常乳腺细胞,且有统计学意义(P <0.0001)。
结论:1、乳腺癌与正常乳腺中存在两种重链亚型NMHCⅡA和NMHCⅡB,且这两种亚型在乳腺癌中表达均高于正常乳腺。2、NMHCⅡA与乳腺癌恶性程度密切相关。3、NMHCⅡB可以作为乳腺癌淋巴结转移的评估指标。4、NMHCⅡA、NMHCⅡB在乳腺癌间质中呈阳性表达,考虑与肿瘤转移有关。
As one of the most common malignant cancer of women in the world, breast cancer is from lobular epithelial cell of the breast terminal ductal, with the first incidence of cancer among women.The causes are not yet clear.Hormones, genetic, and some related proteins involve in the evolution process of breast cancer.Malignant tumor metastasis is the most common cause of death.In some literature, the initial stages of tumor metastasis involve in the epithelial and stromal migration: loss of polarized epithelial cell layer, cell adhesion and cytoskeleton remodeling.Tumor metastasis is associated with cell movement.The study also shows roles of nonmuscle myosinⅡ( NMⅡ) in cell motility:cell polarization, cell migration and cell adhesion.Meanwhile, the literature shows NMⅡenhances tumor cell invasion and metastasis by regulating cell movement.
As the hexamer, NMⅡincludes a pair of heavy chain (NMHCⅡ,230kDa) and two pairs of light chain (NMLCⅡ,20 and 17 kDa).NMLCⅡmainly exercises regulatory functions.In the myosin molecule, the light chain(MLC) combines with the heavy chain(MHC) by non-covalently, which works in stabilizing and regulating the structure and activation of myosin.Each heavy chain consists of a long fibrous tails ofα-helix and a complete spherical head.A lot of literature suggests that the spherical head of nonmuscle myosin heavy chain(N-terminal) is an important functional areas, including decomposing ATP, producing energy and affecting actin.In vitro, the ATP enzyme sites of the single heavy chain head area (S1) can be combined with the ATP enzyme and activate enzymes, in order to generate power and to drive actin filament movement.Thus, in NMⅡmolecules, NMLCⅡmainly exercises regulatory functions.NMHCⅡis the important functional areas, which cause cell movement and make biological activities.So there are important significances in studying the relationship between NMHCⅡand breast cancer by studying the relationship between NMHCⅡand cell movement.
Objective: To determine NMHCⅡsubtype of breast cancer and normal breast; To observe the expressions of NMHCⅡA, B in breast cancer cases; To compare the expressions of NMHCⅡA, B in breast cancer cells to the expressions in normal breast cells; To investigate the relationship among NMHCⅡA, B and malignancy, invasion and metastasis of breast cancer.
Methods:Application of HE staining and the modified Bloom-Richardson system by Elston and Ellis, combined with the comprehensive evaluation analysis of the structure and morphology atypical nuclei, classificated breast specimens. Immunohistochemistry staining (SP) was performed to detecte the location and expression of NMHCⅡA, B in breast cancer.Software Image-Pro Plus 6.0 was to study the cumulative optical density (IOD) of expression of NMHCⅡA, B inⅠ,Ⅱ,Ⅲgrade breast cancer and to do in breast cancer with lymph node metastasis and without lymph node metastasis.Glycerol SDS-PAGE combined with Western blot was to observe the expression of NMHCⅡA, B in the breast cancer cells (MCF-7) and the normal breast cells (HB).Application of gel imaging analysis system was to determine optical density of Western blot.INSTAT software was used for statistical analysis.
Results: According to the degree of malignancy, 47 invasive ductal breast cancer were dividedⅠ,Ⅱ,Ⅲgrade, with gradeⅠ8 cases, gradeⅡ32 cases and gradeⅢ7 cases.Immunohistochemical staining showed that,①NMHCⅡA and NMHCⅡB were expressed both in breast cancer and in adjacent tissues, and expression in breast cancer was higher than that in adjacent tissue, with statistical significance(P <0.05).②N MHCⅡA expressed mainly in the cytoplasm of cancer nests, followed in the interstitial and vascular wall.InⅠ,Ⅱ,Ⅲgrade breast cancer and in adjacent tissues, the differences of positive IOD values were statistically significant(P <0.05), with the level higher, IOD values greater.③NMHCⅡB expressed in the major parts of the membrane, cytoplasm, followed by localization in interstitial and vascular wall.The result that histological gradeⅢof breast cancer respectively comparied with gradeⅠ,Ⅱbreast cancer and adjacent tissue showed that the differences of positive IOD values were statistically significant(P <0.001), but the differences of positive IOD values among gradeⅠ,Ⅱbreast cancer and adjacent tissue were not statistically significant (P >0.05), with the degree of malignancy increasing, IOD values greater, and expressions of NMHCⅡB increasing.④T he positive expressions of NMHCⅡA in breast cancer had no relations with lymphatic metastasis, with no significant differences (P >0.05), but in the tissue of lymph node metastasis, with histological grade level increasing, IOD values increasing.The positive expressions of NMHCⅡB in breast cancer had relationship with lymphatic metastasis, with significant differences(P <0.05).⑤The results of observating NMHCⅡA and NMHCⅡB were nests lordosis position.There were more positive expressions in the cells of the cancer nest periphery than in cancer nests, with a part of the scattered or the poor adhesion cancer expressing stronger.⑥In breast cancer tissues with NMHCⅡA positive, 77.27% in the fibrous stroma of breast cancer was positive, with the fibrous stroma of adjacent tissue negative.The difference was statistically significant (P <0.05).In breast cancer tissues with NMHCⅡB positive, 81.08% in the fibrous stroma of breast cancer was positive, with the fibrous stroma of adjacent tissue negative.The difference was statistically significant (P <0.05).⑦Comparative study found that in gradeⅠ,Ⅱ,Ⅲbreast cancer, IOD values of NMHCⅡA expression were significantly higher than IOD values of NMHCⅡB expression, and there were significant differences among three groups(P <0.01).Western blot showed that NMHCⅡA and NMHCⅡB expressed both in HB and in MCF-7.Under the same protein sample, optical densities of Western blot were measured and analyzed statistically.①The optical density of NMHCⅡA in breast cancer cells(MCF-7) was higher than in normal breast cells (HB), but no statistical significance (P >0.05).②The optical density of NMHCⅡB in breast cancer cells(MCF-7) was higher than in normal breast cells (HB), with statistical significance(P <0.0001).
Conclusion: 1. There are two expression NMHCⅡA and NMHCⅡB in breast cancer and in the normal breast, with both more expressed in breast cancer and less expressed in the normal breast. 2. NMHCⅡA are closely related with the malignancy of breast cancer. 3. NMHCⅡB can be used as evaluation indicators of breast cancer metastasis. 4. NMHCⅡA and NMHCⅡB in breast cancer stroma were positive, perhaps involving in tumor metastasis.
引文
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[2]吴在德,吴肇汉.外科学(第七版).人民卫生出版社,2008:308-312.
[3]吴功侃,赵荣宇.试论我国乳腺癌的诊治现状与改革对策[J].医师进修杂志,2000,23(11):59-62.
[4]Elston CW,Ellis IO. Pathological prognostic factors in breast cancer.Ⅰ.The value of histological grade in breast cancer:experience from a large study with long-term follow-up[J]. Histopathology,1991,19 (5):403-410.
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