【作者】 李疆；

【导师】 王国斌；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【摘要】 第一部分体外研究次级淋巴组织趋化因子/趋化因子受体7对稳定转染SW480细胞株的基质金属蛋白酶9表达调控目的在稳定转染CCR7-shRNA质粒细胞株SW480/CCR7ˉ和阴性对照质粒细胞株SW480/control中检测次级淋巴组织趋化因子(secondary lymphoid tissue chemokine, SLC/CCL21)对基质金属蛋白酶9(matrix metalloproteinases 9, MMP-9)蛋白水平和酶活性的调控,分析次级淋巴组织趋化因子/趋化因子受体7(CC chemokine receptor 7,CCR7)对MMP-9的影响。方法0ng/ml和100ng/ml CCL21处理SW480/CCR7"和SW480/control细胞24小时后,酶联免疫吸附试验测定细胞上清中的MMP-9蛋白水平；明胶酶谱法检测其酶活性。结果100ng/ml CCL21能够明显刺激SW480/control细胞中MMP-9蛋白水平和酶活性升高,差异有统计学意义(P<0.05)；在抑制CCR7表达的SW480/CCR7ˉ细胞中,CCL21的刺激作用则不明显,CCR7基因沉默能够阻断CCL21诱导的MMP-9上调。结论CCL21通过作用于肿瘤细胞表面的CCR7能够诱导人结肠癌细胞株SW480MMP-9表达上调。第二部分体内研究CCL21/CCR7对结肠癌MMP-9表达调节和淋巴结转移的影响目的在裸鼠移植瘤模型中研究CCR7的表达沉默对肿瘤组织中MMP-9的蛋白水平和酶活性的影响；对肿瘤生长和淋巴结转移的影响以及对裸鼠生存期的影响。方法按照种植SW480/CCR7-和SW480/control细胞将裸鼠分为SW480/CCR7-和SW480/control组。采用活体荧光成像技术观察裸鼠移植瘤的生长和荧光强度；使用酶联免疫吸附试验检测移植瘤组织中的MMP-9蛋白水平；明胶酶谱检测其活性；使用免疫组织化学方法检测淋巴结转移；使用Kaplan-Meier进行生存分析。结果SW480/control组肿瘤体积大于SW480/CCR7-组(P<0.05)；SW480/control组肿瘤组织MMP-9蛋白水平和酶活性明显高于SW480/CCR7-组(P＜0.05)；SW480/CCR7-组肿瘤淋巴结转移少于SW480/control组(P<0.05)；SW480/CCR7-组动物生存期长于SW480/control组(P<0.05)。结论CCR7表达沉默能够抑制结肠癌的MMP-9产生和淋巴结转移。第三部分原代培养人类骨髓间充质干细胞对SW480细胞增殖和侵袭的影响目的原代培养人骨髓间充质干细胞(human bone marrow mesenchymal stem cells, hBMSCs)并鉴定；检测间充质干细胞中CCR7的表达；初步探讨人类骨髓间充质于细胞对结肠癌细胞SW480生长和侵袭的影响。方法结合密度梯度离心法和贴壁法原代培养人骨髓间充质干细胞；使用流式细胞分析检测细胞表面分子标志；使用诱导分化试验来鉴定细胞的分化潜能；使用免疫荧光染色和免疫印迹法分析检测细胞的CCR7表达；使用MTT法和Transwell小室检测人类骨髓间充质干细胞对结肠癌细胞SW480生长和侵袭的影响。结果成功培养人骨髓间充质干细胞；其表面分子标志为CD34ˉ、CD45ˉ、CD44+、CD90+；骨髓间充质干细胞能够在条件培养基的作用下向脂肪细胞和成骨细胞分化；骨髓间充质干细胞有CCR7的表达；骨髓间充质干细胞的条件培养基能够刺激SW480的生长(P<0.05)；共培养骨髓间充质干细胞和SW480细胞能够刺激肿瘤细胞的侵袭增强(P<0.05)。结论骨髓间充质干细胞表达CCR7蛋白并能够刺激结肠癌SW480细胞增殖和侵袭。第四部分体内研究CCR7介导的人类骨髓间充质干细胞对结肠癌淋巴结转移的影响目的在裸鼠移植瘤模型上研究人类骨髓间充质干细胞对结肠癌淋巴结转移的影响；并探讨CCR7在其中的作用机制。方法按照种植SW480/CCR7ˉ,SW480/control, SW480/CCR7-+hBMSCs和SW480/control+hBMSCs细胞将裸鼠分为SW480/CCR7-, SW480/control, SW480/CCR7-+hBMSCs和SW480/control+hBMSCs组；采用活体荧光成像技术观察裸鼠移植瘤的生长和荧光强度；使用免疫组织化学方法检测淋巴结转移；使用Kaplan-Meier生存曲线进行生存分析。结果SW480/control+hBMSCs组肿瘤大小和淋巴结转移高于SW480/control组(P<0.05)；SW480/CCR7-+hBMSCs组肿瘤大小和淋巴结转移高于SW480/CCR7组,生存期则较短(P<0.05)。结论人类骨髓间充质干细胞可以通过CCR7介导刺激结肠癌淋巴结转移。更多还原

【Abstract】 Part I Effect of CCL21/CCR7 on MMP-9 expression in the stable transfected SW480 cell line with the CCR7 shRNA vector in vitroObjective To investigate effect of CCL21/CCR7 on MMP-9 expression in the stable transfected SW480 cell line with CCR7 shRNA vector (SW480/CCR7-) and negative control vector (SW480/control)Methods SW480/CCR7-and SW480/control cells were treated with Ong/ml and 100ng/ml CCL21 for 24 hours. Levels of MMP-9 protein and MMP-9 activity were analyzed by enzyme linked immunosorbent assay and gelatin zymography.Results 100ng/ml CCL21 could significantly regulate MMP-9 protein levels and activity in SW480/control (P<0.05).The effect of CCL21 was not obvious in SW480/CCR7- cells (P>0.05).CCR7 gene silencing blocked MMP-9 production induced by CCL21.Conclusion CCL21/CCR7 could induce production of MMP-9 in human colon cancer cell line SW480. Part II Effect of CCR7 gene silencing on MMP-9 expression and lymph node metastasis of colon cancer in vivoObjective To investigate effect of CCR7 gene silencing on MMP-9 expression in cancerous tissues, the growth and lymph node metastasis of colon cancer and the survival of nude mice in a xenografted mouse model.Methods The mice were divided into SW480/CCR7" group treated with SW480/CCR7-cells and SW480/control group treated with SW480/control cells. The growth of colon cancer was observed by using whole body fluorescence imaging. Levels of MMP-9 protein and activity in xenografted tissues were analyzed by enzyme linked immunosorbent assay and gelatin zymography. Lymph node metastasis was detected by immunohistochemistry analysis. Kaplan-Meier survival analysis evaluated the survival of nude mice.Results The MMP-9 protein levels and activity in SW480/control group cancerous tissues were significantly higher than SW480/CCR7- group (P<0.05).The xenografts in SW480/control group had greater tumor size and more lymph node metastases than SW480/CCR7- group (P<0.05).The mice in SW480/control group had shorter survival as compared with SW480/CCR7" group (P<0.05).Conclusion Silencing expression of CCR7 could inhibit MMP-9 production and lymph node metastasis of colon cancer. PartⅢEffect of human bone marrow mesenchmal stem cells on the proliferation and invasion of colon cancer cell line SW480Objective To culture and identify human bone marrow mesenchymal stem cells (hBMSCs); to detect expression of CCR7 in hBMSCs; to evaluat effect of hBMSCs on the proliferation and invasion of colon cancer cell line SW480.Methods hBMSCs were primary cultured by density gradient centrifugation and adherent from human bone marrow. The cell surface molecular markers were identified by flow cytometry. Differentiation culture of hBMSCs for mesenchymal lineage was achieved when cells were treated with adipogenic differentiation medium and osteogenic differentiation medium for two weeks. Expression of CCR7 was analyzed by immunofluorescence and western blotting in hBMSCs. Effect of hBMSCs on the proliferation and invasion of colon cancer cell line SW480 was evaluated by MTT and invasion assayResults hBMSCs were sucessfully cultured. Cells surface molecular markers were CD34", CD45-, CD44+and CD90+. They had the ability of adipogenic differentiation and osteogenic differentiation. CCR7 expressed in hBMSCs. The conditioned medium of hBMSCs could stimulate the growth of SW480 cells (P<0.05). Co-culture of hBMSCs and SW480 could stimulate tumor cells invasion (P<0.05).Conclusion Human bone marrow mesenchymal stem cells had expression of CCR7 protein. They could stimulate the proliferation and invasion of SW480 cells. Part IV Effect of human bone marrow mesenchymal stem cells on the lymph node metastasis of colon cancer by CCR7-mediated mechanism in vivoObjective To evaluate effect of human bone marrow mesenchymal stem cells on the lymph node metastasis of colon cancer; to discuss the role of CCR7 in this process of lymph node metastasis which hBMSCs interacted with colon cancer cells.Method The mice were divided into SW480/CCR7-, SW480/control, SW480/CCR7-+hBMSCs and SW480/control+hBMSCs group. The growth of colon cancer was observed by using whole body fluorescence imaging. Lymph node metastasis was detected by immunohistochemistry analysis. Kaplan-Meier survival analysis evaluated the survival of nude mice.Results The mice in SW480/control+hBMSCs group had greater tumor size, more lymph node metastases than SW480/control group (P<0.05). The mice in SW480/CCR7-+hBMSCs group had greater tumor size, more lymph node metastases and shorter survival than SW480/CCR7- group (P<0.05).Conclusion The human bone marrow mesenchymal stem cells could stimulate cancer cells growth and lymph node metastasis by CCR7-mediated molecular mechanism.更多还原