17β-雌二醇对涎腺粘液表皮样癌高转移细胞Mc3转移相关生物学特性的影响
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摘要
雌激素因其多种作用，作为一线药物广泛应用于临床，并取得明显的效果。近年来，其可能存在的致瘤、促瘤作用引起了人们的关注。目前，已有研究表明雌激素不仅对于乳腺癌，子宫内膜癌、卵巢癌等肿瘤有促进作用，对于非雌激素依赖的靶器官肿瘤组织中，如肺癌、肝脏肿瘤、口腔粘膜白斑和鳞状细胞癌、恶性胃肠肿瘤、胰腺癌、喉癌、前列腺癌等肿瘤也有明显的促增殖作用，但有关雌激素对与肿瘤转移所发挥的作用，一直存在争议。本研究旨在通过对雌激素对其非靶向组织肿瘤涎腺粘液表皮样癌高转移Mc3细胞系转移相关生物学特性的研究，进一步探讨雌激素对与肿瘤转移生物学特性的影响。
本研究应用细胞体外粘附试验、体外移动试验、化学趋化性试验、明胶底物SDS—PAGE凝胶电泳对基质金属蛋白酶(Matrix metalloproteinases，MMPs)活性测定、裸鼠人工肺转移模型等的方法研究17β-雌二醇(17-beta estradiol，E2)对涎腺粘液表皮样癌细胞系粘附、侵袭和移动力可能存在的作用；并应用免疫组化的方法对Mc3细胞的雌激素受体进行检测以及E2对Mc3细胞VEGF表达的影响。
主要实验结果如下：
1．10~(-9)、10~(-8)、10~(-7)、10~(-6)mol/L的E2处理Mc3细胞72h后，
第四军医大学硕士学位论文
其粘附率分别为38.3%、50.4%、69.2%、91.1%;而对照组为
25.0%。作用4sh后，10一9、10一8、10一7、10一6 mol/L的EZ对
Mc3细胞在纤维粘连蛋白上的移动促进率分别为16.9%、40.9
%、36.4%、38.8%。在10一mo比的EZ的作用下，其对FN
的化学趋化性增加60.3%。
2.10一9、10一8、10一7、10一6 moFL的EZ均可不同程度的促进 Mc3
细胞基质金属蛋白酶2(MMpZ)的活性，在10一moUL时尤
为显著。
3.应用临床药用剂量(5 .2闻d)的EZ处理的裸鼠肺转移瘤结节
数显著高于对照组。EZ组和对照组裸鼠肺表面转移瘤结节数
分别为119.2士69.6个和142士11.8个
4.10一7mof几的EZ在体外可促进Mc3细胞血管内皮生长因子
(vacular endothelial growth factor，泥J)的表达。应用临床
药用剂量(5 .2冈酬d)的 EZ处理的裸鼠肺转移瘤中VEGF的表
达明显高于对照组。
5.在Mc3细胞及裸鼠肺转移瘤中均发现ER的表达。
上述结果表明，生理浓度和药用浓度的EZ可以促进Mc3
细胞对细胞外重要基质成分FN的粘附;促进Mc3细胞在FN
基质上的移动:促进Mc3细胞对FN的趋化性:并可增强Mc3
细胞N伍企2的活性以及VEGF的表达。应用临床药用剂量
(5 .2叫酬d)就E2处理的裸鼠肺转移瘤结节数显著高于对照组。
同时，我们发现ER在Mc3细胞中的表达。结论:生理浓度和
药用浓度的EZ可增强涎腺粘液表皮样癌Mc3细胞的转移力。
Estrogen is effective in the treatment of many diseases and wildely used in clinical. In recent years, studies have shown that it may has the potential to induce or promote some estrogen-dependent tumors as well as many estrogen-independent cancers such as lung cancer, liver cancer, throat cancer and so on. However, the role it plays in tumor cell metastatic process is remained undefined. In this study, the effect of 17beta-estradiol(E2) on the metastatic potential of human salivary gland mucoepidermoid carcinoma Mc3 cells was investigated and the role of E2 in the tumor metastasis was studied.
The effects of E2 on adhesion, invasion and motility potential of salivary mucoepidermoid carcinoma Mc3 cells were investigated with cell attachment assay on fibronectin (FN), wound assay, chemotaxis assay, gelatin -incorporated SDS-PAGE electrophoresis and tumor metastasis experiment in nude mice. Moreover, the effects of E2 on the expression of VEGF in highly metastatic salivary gland mucoepidermoid carcinoma Mc3 cells were investigated with the methods of immunohistochemistry. In Mc3 cells and the samples of
the metastastic foci sectioned from the surfaces of nude mouse lungs, estrogen receptor (ERa) was determined by immunohistochemistry assay.
Following results were obtained:
1. Attachment rates of Mc3 cells treated withE2 at 10-9, 10-8, 10-7, 10-6M were 38.3%, 50.4%, 69.2% and 91.1% respectively, whereas that of the control was 25.0%. When exposed to E2 at 10-9,10-8,10-7 and 10-6 M for 48h, motility of Mc3 cells on FN was increased by 16.9%, 40.9%, 36.4% and 38.8% respectively. When at 10-6M, E2 increased chemotaxis potential of Mc3 cells to FN by 60.3%.
2. The activity of 68kDa matrix metalloproteinase (MMP2) of Mc3 cells was enhanced at different levels by 10-9, 10-8 and 10-7M of E2, the most enhancement was observed at 10-6M.
3. The number of metastatic foci on the lung surfaces in the nude mice treated with E2 at 5.2ug/day and that in the controls were 119.2 + 69.6 and 14.2+11.8 respectively.
4. In vitro, the VEGF expression was significantly higher in Mc3 cells which had been treated with E2 at 10-7M for 24h than in the controls. Consisting with the assay in vitro, the higher VEGF expression was also detected in the samples of foci in the E2-treated nude mice.
5. By immunohistochemistry, we detected nuclear ERa expression in vitro as well as in vivo.
Conclusion: physiological and pharmacological concentration of E2 can promote the adhension of Mc3 cells to fibronectin(FN) membrane, increase mortility on FN and chemotaxis to FN, enhance the activity of matrix metalloproteinase and the expression of vascular endothelial growth factor(VEGF); increase the metastatic foci of Mc3 cells in lungs of nude mice treated with of E2 at clinical dose. At the same time, we detected
Era in Mc3 cells as well as in the metastatic foci. So, we concluded that physiological and pharmacological concentration of E2 can enhance the metastatic protential of human salivary gland mucoepidermoid carcinoma Mc3 cells.
本研究应用细胞体外粘附试验、体外移动试验、化学趋化性试验、明胶底物SDS—PAGE凝胶电泳对基质金属蛋白酶(Matrix metalloproteinases，MMPs)活性测定、裸鼠人工肺转移模型等的方法研究17β-雌二醇(17-beta estradiol，E2)对涎腺粘液表皮样癌细胞系粘附、侵袭和移动力可能存在的作用；并应用免疫组化的方法对Mc3细胞的雌激素受体进行检测以及E2对Mc3细胞VEGF表达的影响。
主要实验结果如下：
1．10~(-9)、10~(-8)、10~(-7)、10~(-6)mol/L的E2处理Mc3细胞72h后，
第四军医大学硕士学位论文
其粘附率分别为38.3%、50.4%、69.2%、91.1%;而对照组为
25.0%。作用4sh后，10一9、10一8、10一7、10一6 mol/L的EZ对
Mc3细胞在纤维粘连蛋白上的移动促进率分别为16.9%、40.9
%、36.4%、38.8%。在10一mo比的EZ的作用下，其对FN
的化学趋化性增加60.3%。
2.10一9、10一8、10一7、10一6 moFL的EZ均可不同程度的促进 Mc3
细胞基质金属蛋白酶2(MMpZ)的活性，在10一moUL时尤
为显著。
3.应用临床药用剂量(5 .2闻d)的EZ处理的裸鼠肺转移瘤结节
数显著高于对照组。EZ组和对照组裸鼠肺表面转移瘤结节数
分别为119.2士69.6个和142士11.8个
4.10一7mof几的EZ在体外可促进Mc3细胞血管内皮生长因子
(vacular endothelial growth factor，泥J)的表达。应用临床
药用剂量(5 .2冈酬d)的 EZ处理的裸鼠肺转移瘤中VEGF的表
达明显高于对照组。
5.在Mc3细胞及裸鼠肺转移瘤中均发现ER的表达。
上述结果表明，生理浓度和药用浓度的EZ可以促进Mc3
细胞对细胞外重要基质成分FN的粘附;促进Mc3细胞在FN
基质上的移动:促进Mc3细胞对FN的趋化性:并可增强Mc3
细胞N伍企2的活性以及VEGF的表达。应用临床药用剂量
(5 .2叫酬d)就E2处理的裸鼠肺转移瘤结节数显著高于对照组。
同时，我们发现ER在Mc3细胞中的表达。结论:生理浓度和
药用浓度的EZ可增强涎腺粘液表皮样癌Mc3细胞的转移力。
Estrogen is effective in the treatment of many diseases and wildely used in clinical. In recent years, studies have shown that it may has the potential to induce or promote some estrogen-dependent tumors as well as many estrogen-independent cancers such as lung cancer, liver cancer, throat cancer and so on. However, the role it plays in tumor cell metastatic process is remained undefined. In this study, the effect of 17beta-estradiol(E2) on the metastatic potential of human salivary gland mucoepidermoid carcinoma Mc3 cells was investigated and the role of E2 in the tumor metastasis was studied.
The effects of E2 on adhesion, invasion and motility potential of salivary mucoepidermoid carcinoma Mc3 cells were investigated with cell attachment assay on fibronectin (FN), wound assay, chemotaxis assay, gelatin -incorporated SDS-PAGE electrophoresis and tumor metastasis experiment in nude mice. Moreover, the effects of E2 on the expression of VEGF in highly metastatic salivary gland mucoepidermoid carcinoma Mc3 cells were investigated with the methods of immunohistochemistry. In Mc3 cells and the samples of
the metastastic foci sectioned from the surfaces of nude mouse lungs, estrogen receptor (ERa) was determined by immunohistochemistry assay.
Following results were obtained:
1. Attachment rates of Mc3 cells treated withE2 at 10-9, 10-8, 10-7, 10-6M were 38.3%, 50.4%, 69.2% and 91.1% respectively, whereas that of the control was 25.0%. When exposed to E2 at 10-9,10-8,10-7 and 10-6 M for 48h, motility of Mc3 cells on FN was increased by 16.9%, 40.9%, 36.4% and 38.8% respectively. When at 10-6M, E2 increased chemotaxis potential of Mc3 cells to FN by 60.3%.
2. The activity of 68kDa matrix metalloproteinase (MMP2) of Mc3 cells was enhanced at different levels by 10-9, 10-8 and 10-7M of E2, the most enhancement was observed at 10-6M.
3. The number of metastatic foci on the lung surfaces in the nude mice treated with E2 at 5.2ug/day and that in the controls were 119.2 + 69.6 and 14.2+11.8 respectively.
4. In vitro, the VEGF expression was significantly higher in Mc3 cells which had been treated with E2 at 10-7M for 24h than in the controls. Consisting with the assay in vitro, the higher VEGF expression was also detected in the samples of foci in the E2-treated nude mice.
5. By immunohistochemistry, we detected nuclear ERa expression in vitro as well as in vivo.
Conclusion: physiological and pharmacological concentration of E2 can promote the adhension of Mc3 cells to fibronectin(FN) membrane, increase mortility on FN and chemotaxis to FN, enhance the activity of matrix metalloproteinase and the expression of vascular endothelial growth factor(VEGF); increase the metastatic foci of Mc3 cells in lungs of nude mice treated with of E2 at clinical dose. At the same time, we detected
Era in Mc3 cells as well as in the metastatic foci. So, we concluded that physiological and pharmacological concentration of E2 can enhance the metastatic protential of human salivary gland mucoepidermoid carcinoma Mc3 cells.
引文
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78．朱启锭吴明章顾文祥主编。《组织胚胎学》第四版