SDF-1和MMP-7在人乳腺癌中的表达及其相关性研究
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摘要
第一部分:SDF-1、MMP-7、Alpha-SMA在人乳腺癌中表达
目的:研究乳腺癌组织中基质细胞衍生因子-1(SDF-1)、a-平滑肌肌动蛋白(alpha-SMA)和MMP-7的表达情况。
方法:从有腋窝淋巴结转移的33例乳腺癌患者取癌组织和癌旁组织的标本,另取乳腺正常组织33例,每一蜡块连续切片3张,免疫组化方法检测SDF-1、MMP-7和alpha-SMA分别在乳腺良性病变的正常组织、乳腺癌及癌旁组织中的表达情况。
结果:在乳腺癌及癌旁组织中SDF-1、MMP-7均高表达,与正常乳腺组织相比,差异有显著性意义,SDF-1与alpha-SMA在乳腺癌中的表达部位相似。SDF-1和MMP-7的表达具有相关性。
结论:
1、SDF-1、MMP-7、alpha-SMA在乳腺癌及癌旁组织中高表达,与肿瘤关系密切。
2、SDF-1和MMP-7在乳腺癌中的表达具有相关性。
第二部分DECM对人乳腺癌细胞株MCF-7增殖和侵袭的影响
目的:观察Ⅳ型胶原酶降解基质后产物(DECM)对人乳腺癌细胞株MCF-7增殖和侵袭的影响,对MMP-7蛋白和SDF-1蛋白表达的影响。探讨胶原酶Ⅳ及DECM在肿瘤降解中的作用,及MMP-7与SDF-1间相互关系。
方法:采用胰酶消化法,分别分离来自人正常乳腺组织、乳腺癌旁组织及乳腺癌组织切取物中细胞和基质,提取基质成分,再以Ⅳ型胶原酶降解,分成7组分别是1.空白对照组,2.正常乳腺(无胶原酶组)3.正常乳腺组(胶原酶组),4.癌旁(无胶原酶组),5.癌旁(胶原酶组),6.乳腺癌(无胶原酶组),7.乳腺癌(胶原酶组),得到DECM;分别作用于人乳腺癌细胞株MCF-7,通过软琼脂克隆形成实验观察瘤细胞增殖的变化,用Transwell实验观察瘤细胞侵袭能力的变化,通过Western Blot检测人乳腺癌细胞株MCF-7细胞中MMP-7和SDF-1蛋白的表达变化。
结果:Ⅳ型胶原酶作用人乳腺癌基质后产物DECM可以使MCF-7人乳腺癌细胞株增殖和转移能力的增加,同一组织来源的胶原酶组与无胶原酶组比较差异有显著性意义;Ⅳ型胶原酶作用人乳腺癌基质后产物DECM可同时上调MMP-7和SDF-1蛋白的表达。
结论:
1、Ⅳ型胶原酶作用人乳腺癌基质后产物DECM具有促进肿瘤增殖及其侵袭活性的生物学活性。
2、肿瘤降解基质的生物行为可能与MMP-7和SDF-1蛋白表达的增加有关。
第三部分人乳腺癌细胞株MCF-7中MMP-7与SDF-1的相互作用影响的研究
目的:研究SDF-1对人乳腺癌细胞株MCF-7生物学行为的影响,并研究SDF-1和MMP-7之间的相互作用关系。
方法:不同浓度的SDF-1作用于人乳腺癌细胞株MCF-7后,观察MCF-7细胞增殖和侵袭能力变化,Western Blot法检测SDF-1在不同浓度和时间引起MMP-7蛋白表达的变化;Western Blot法检测MMP-7在不同浓度和和不同时间下作用于MCF-7后,SDF-1蛋白表达的变化。使用CXCR4抑制剂T140抑制了SDF-1功能后,Westernblot检测MMP-7蛋白表达变化;siRNA抑制MMP-7表达后,Westernblot检测SDF-1蛋白表达变化。
结果:SDF-1呈浓度梯度促进MCF-7细胞的增殖和侵袭。SDF-1能促进MCF-7细胞中MMP-7蛋白表达的增加;MMP-7也可促进MCF-7细胞中SDF-1蛋白的表达增加。T140抑制SDF-1表达后,MMP-7蛋白表达下调;siRNA抑制MMP-7蛋白表达后,SDF-1蛋白表达下调。
结论:1、SDF-1可促进乳腺癌细胞株MCF-7细胞的增殖和侵袭,其侵袭能力增加可能与上调MMP-7表达有关。2、在人乳腺癌细胞株MCF-7中MMP-7与SDF-1呈相互促进和相互抑制的关系,他们之间可能存在一种正反馈影响。
Part one:Expression of SDF-1,MMP-7 and Alpha-SMA in breast cancer
Objective:To Study expression of Stromal cell-derived factor-1(SDF-1),MMP-7 and Alpha-Smooth Muscle Actin(Alpha-SMA) in breast cancer by Immunohistochemical.
Methods:To observe the expression of SDF-1,MMP-7 and Alpha-sma in breast cancer tissue,cancer nearby tissue and normal breast tissue,and contrast the expression of SDF-1 and Alpha-SMA in vicinity paraffin section.
Results:The expression of SDF-1,MMP-7 and Alpha-SMA were higher in breast cancer tissue and cancer nearby tissue;there are similar position and intensity expression of between SDF-1 and Alpha-SMA. And there was a relationship between the expression of SDF-1 and MMP-7.
Conclusion:1.SDF-1,MMP-7 and alpha-SMA were high expression in breast cancer tissue and cancer nearby tissue,they were all correlated with tumor.2.The expression of SDF-1 was correlation with the expression of MMP-7.
Part two:Study the growth and invasion influence of degrdn extracellular matrix byⅣcollagenase in humam breast cancer line MCF-7
Objective:To observe the growth and invasion influence of degrdn extracellular matrix byⅣcollagenase from breast cancer stroma on humam breast cancer line MCF-7.To study the activity of DECM andⅣcollagenase in the tumor degradation and the relationship between SDF-1 and MMP-7 protein influenced by the DECM.
Methods:Segregating stroma and cell through digestion by pamcreatin from normal breast tissue,cancer side tissue and cancer tissue,then extracted stroma,observingⅣcollagenase degradate stroma in different groups,there were 7 groups:1.blank group,2.normal breast tissue group(NoⅣcollagenase),3.normal breast tissue group(Ⅳcollagenase),4.brast cancer side group(NoⅣcollagenase),5.brast cancer side group(Ⅳcollagenase),6.breast cancer group(NoⅣcollagenase),7 breast cancer group(Ⅳcollagenase),the same quantity DECM were added in human breast cancer line MCF-7.The tumor cell growth was observed by soft agar clone formation method,the tumor cell metastasis was studed by Transwells method,the expressive of MMP-7 and SDF-1 protein were detected in MCF-7 by Western blot method.
Results:The DECM from breast cancer stroma could promote human breast cancer line MCF-7 proliferate and invasive,there was significantly distinction betweenⅣcollagenase group and No collagenase group in the same tissue,the expression of SDF-1 and MMP-7 were elevated by DECM.
Conclusion:1 The DECM fromⅣcollagenase degrdn breast cancer stroma possessed tumor biologic activity which promoted hu man breast cancer line MCF-7 proliferate and invasive.2 Degrdnin g stroma in tumor could simultaneously elevate the expression of SDF-1 and MMP-7.
Part three:The interaction of SDF-1 and MMP-7 in humam breast cancer line MCF-7
Objective:To Study the biological influence of SDF-1 in MCF-7 and the interaction of SDF-1 and MMP-7.
Methods:The tumor cell proliferation and invasion were observed in MCF-7 by different concentration SDF-1,the expression of MMP-7 was detected by Western blotting with different density SDF-1 and at different time.In the same way,the expression of SDF-1 was detected with different density MMP-7 and at different time.The expression of MMP-7 was detected by Western blotting after using CXCR4 inhibitor T140 in MCF-7,the expression of SDF-1 was detected after using small interfere RNA anti-MMP-7 in MCF-7 by Western blotting.
Results:SDF-1 enhanced tumor cell proliferation and invasion of MCF-7 in a concentration-dependent way.SDF-1 could upregulate the expression of MMP-7 in concentration- and time-dependent way.And vice versa.The expression of MMP-7 was down regulated after inhibited the expression of SDF-1 by T140,and SDF-1 also was down regulated after inhibited MMP-7 by siRNA.
Conclusion:1.SDF-1 could enhance tumor cell proliferation and invasion in the breast cancer line MCF-7 in a concentration- and time-dependent way.2.There was interaction between SDF-1 and MMP-7,it seems like a regenerative feedback between them.
目的:研究乳腺癌组织中基质细胞衍生因子-1(SDF-1)、a-平滑肌肌动蛋白(alpha-SMA)和MMP-7的表达情况。
方法:从有腋窝淋巴结转移的33例乳腺癌患者取癌组织和癌旁组织的标本,另取乳腺正常组织33例,每一蜡块连续切片3张,免疫组化方法检测SDF-1、MMP-7和alpha-SMA分别在乳腺良性病变的正常组织、乳腺癌及癌旁组织中的表达情况。
结果:在乳腺癌及癌旁组织中SDF-1、MMP-7均高表达,与正常乳腺组织相比,差异有显著性意义,SDF-1与alpha-SMA在乳腺癌中的表达部位相似。SDF-1和MMP-7的表达具有相关性。
结论:
1、SDF-1、MMP-7、alpha-SMA在乳腺癌及癌旁组织中高表达,与肿瘤关系密切。
2、SDF-1和MMP-7在乳腺癌中的表达具有相关性。
第二部分DECM对人乳腺癌细胞株MCF-7增殖和侵袭的影响
目的:观察Ⅳ型胶原酶降解基质后产物(DECM)对人乳腺癌细胞株MCF-7增殖和侵袭的影响,对MMP-7蛋白和SDF-1蛋白表达的影响。探讨胶原酶Ⅳ及DECM在肿瘤降解中的作用,及MMP-7与SDF-1间相互关系。
方法:采用胰酶消化法,分别分离来自人正常乳腺组织、乳腺癌旁组织及乳腺癌组织切取物中细胞和基质,提取基质成分,再以Ⅳ型胶原酶降解,分成7组分别是1.空白对照组,2.正常乳腺(无胶原酶组)3.正常乳腺组(胶原酶组),4.癌旁(无胶原酶组),5.癌旁(胶原酶组),6.乳腺癌(无胶原酶组),7.乳腺癌(胶原酶组),得到DECM;分别作用于人乳腺癌细胞株MCF-7,通过软琼脂克隆形成实验观察瘤细胞增殖的变化,用Transwell实验观察瘤细胞侵袭能力的变化,通过Western Blot检测人乳腺癌细胞株MCF-7细胞中MMP-7和SDF-1蛋白的表达变化。
结果:Ⅳ型胶原酶作用人乳腺癌基质后产物DECM可以使MCF-7人乳腺癌细胞株增殖和转移能力的增加,同一组织来源的胶原酶组与无胶原酶组比较差异有显著性意义;Ⅳ型胶原酶作用人乳腺癌基质后产物DECM可同时上调MMP-7和SDF-1蛋白的表达。
结论:
1、Ⅳ型胶原酶作用人乳腺癌基质后产物DECM具有促进肿瘤增殖及其侵袭活性的生物学活性。
2、肿瘤降解基质的生物行为可能与MMP-7和SDF-1蛋白表达的增加有关。
第三部分人乳腺癌细胞株MCF-7中MMP-7与SDF-1的相互作用影响的研究
目的:研究SDF-1对人乳腺癌细胞株MCF-7生物学行为的影响,并研究SDF-1和MMP-7之间的相互作用关系。
方法:不同浓度的SDF-1作用于人乳腺癌细胞株MCF-7后,观察MCF-7细胞增殖和侵袭能力变化,Western Blot法检测SDF-1在不同浓度和时间引起MMP-7蛋白表达的变化;Western Blot法检测MMP-7在不同浓度和和不同时间下作用于MCF-7后,SDF-1蛋白表达的变化。使用CXCR4抑制剂T140抑制了SDF-1功能后,Westernblot检测MMP-7蛋白表达变化;siRNA抑制MMP-7表达后,Westernblot检测SDF-1蛋白表达变化。
结果:SDF-1呈浓度梯度促进MCF-7细胞的增殖和侵袭。SDF-1能促进MCF-7细胞中MMP-7蛋白表达的增加;MMP-7也可促进MCF-7细胞中SDF-1蛋白的表达增加。T140抑制SDF-1表达后,MMP-7蛋白表达下调;siRNA抑制MMP-7蛋白表达后,SDF-1蛋白表达下调。
结论:1、SDF-1可促进乳腺癌细胞株MCF-7细胞的增殖和侵袭,其侵袭能力增加可能与上调MMP-7表达有关。2、在人乳腺癌细胞株MCF-7中MMP-7与SDF-1呈相互促进和相互抑制的关系,他们之间可能存在一种正反馈影响。
Part one:Expression of SDF-1,MMP-7 and Alpha-SMA in breast cancer
Objective:To Study expression of Stromal cell-derived factor-1(SDF-1),MMP-7 and Alpha-Smooth Muscle Actin(Alpha-SMA) in breast cancer by Immunohistochemical.
Methods:To observe the expression of SDF-1,MMP-7 and Alpha-sma in breast cancer tissue,cancer nearby tissue and normal breast tissue,and contrast the expression of SDF-1 and Alpha-SMA in vicinity paraffin section.
Results:The expression of SDF-1,MMP-7 and Alpha-SMA were higher in breast cancer tissue and cancer nearby tissue;there are similar position and intensity expression of between SDF-1 and Alpha-SMA. And there was a relationship between the expression of SDF-1 and MMP-7.
Conclusion:1.SDF-1,MMP-7 and alpha-SMA were high expression in breast cancer tissue and cancer nearby tissue,they were all correlated with tumor.2.The expression of SDF-1 was correlation with the expression of MMP-7.
Part two:Study the growth and invasion influence of degrdn extracellular matrix byⅣcollagenase in humam breast cancer line MCF-7
Objective:To observe the growth and invasion influence of degrdn extracellular matrix byⅣcollagenase from breast cancer stroma on humam breast cancer line MCF-7.To study the activity of DECM andⅣcollagenase in the tumor degradation and the relationship between SDF-1 and MMP-7 protein influenced by the DECM.
Methods:Segregating stroma and cell through digestion by pamcreatin from normal breast tissue,cancer side tissue and cancer tissue,then extracted stroma,observingⅣcollagenase degradate stroma in different groups,there were 7 groups:1.blank group,2.normal breast tissue group(NoⅣcollagenase),3.normal breast tissue group(Ⅳcollagenase),4.brast cancer side group(NoⅣcollagenase),5.brast cancer side group(Ⅳcollagenase),6.breast cancer group(NoⅣcollagenase),7 breast cancer group(Ⅳcollagenase),the same quantity DECM were added in human breast cancer line MCF-7.The tumor cell growth was observed by soft agar clone formation method,the tumor cell metastasis was studed by Transwells method,the expressive of MMP-7 and SDF-1 protein were detected in MCF-7 by Western blot method.
Results:The DECM from breast cancer stroma could promote human breast cancer line MCF-7 proliferate and invasive,there was significantly distinction betweenⅣcollagenase group and No collagenase group in the same tissue,the expression of SDF-1 and MMP-7 were elevated by DECM.
Conclusion:1 The DECM fromⅣcollagenase degrdn breast cancer stroma possessed tumor biologic activity which promoted hu man breast cancer line MCF-7 proliferate and invasive.2 Degrdnin g stroma in tumor could simultaneously elevate the expression of SDF-1 and MMP-7.
Part three:The interaction of SDF-1 and MMP-7 in humam breast cancer line MCF-7
Objective:To Study the biological influence of SDF-1 in MCF-7 and the interaction of SDF-1 and MMP-7.
Methods:The tumor cell proliferation and invasion were observed in MCF-7 by different concentration SDF-1,the expression of MMP-7 was detected by Western blotting with different density SDF-1 and at different time.In the same way,the expression of SDF-1 was detected with different density MMP-7 and at different time.The expression of MMP-7 was detected by Western blotting after using CXCR4 inhibitor T140 in MCF-7,the expression of SDF-1 was detected after using small interfere RNA anti-MMP-7 in MCF-7 by Western blotting.
Results:SDF-1 enhanced tumor cell proliferation and invasion of MCF-7 in a concentration-dependent way.SDF-1 could upregulate the expression of MMP-7 in concentration- and time-dependent way.And vice versa.The expression of MMP-7 was down regulated after inhibited the expression of SDF-1 by T140,and SDF-1 also was down regulated after inhibited MMP-7 by siRNA.
Conclusion:1.SDF-1 could enhance tumor cell proliferation and invasion in the breast cancer line MCF-7 in a concentration- and time-dependent way.2.There was interaction between SDF-1 and MMP-7,it seems like a regenerative feedback between them.
引文
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