抗人乙酰肝素酶单克隆抗体联合紫杉醇对肺癌的抑制作用
发布时间：2019-06-14 06:46
【摘要】：目的探讨抗乙酰肝素酶(heparanase,HPA)单抗对人肺癌A549细胞HPA m RNA、HPA蛋白表达水平以及HPA活性的影响。同时评价抗HPA单抗联合化疗药物紫杉醇对人肺癌A549细胞增殖、迁移和侵袭能力的影响。在抗HPA单抗联合紫杉醇体外抑制人肺癌A549细胞增殖,迁移和侵袭的基础上,建立荷Lewis肺癌小鼠皮下移植瘤模型,探讨抗HPA单抗与紫杉醇单独或联用对移植瘤生长的影响。方法采用RT-PCR和Western blot检测抗HPA单抗对人肺癌A549细胞HPA m RNA和HPA蛋白表达水平的影响;采用四唑蓝比色法和多糖电泳法检测抗HPA单抗对人肺癌A549细胞HPA活性的影响;HPA单抗与紫杉醇单独或联合干预人肺癌A549细胞,采用MTT法检测对人肺癌A549细胞增殖的影响;利用流式细胞术分析人肺癌A549细胞周期和细胞凋亡的改变,通过细胞划痕和Transwell小室实验观察人肺癌A549细胞迁移和侵袭能力的变化。建立荷Lewis肺癌C57BL/6小鼠皮下移植瘤模型,随机分为生理盐水组、鼠Ig G组、抗HPA单抗组、紫杉醇组、抗HPA单抗与紫杉醇联用组。称量小鼠体重,绘制体重变化曲线;测量皮下移植瘤体积,绘制肿瘤生长曲线;称量裸瘤重并计算抑瘤率,评价药物对移植瘤生长的抑制作用。HE染色观察肿瘤组织病理学变化,免疫组织化学检测肿瘤组织微血管密度。结果抗HPA单抗干预后,A549细胞的HPA m RNA和HPA蛋白相对表达水平与溶剂对照组和鼠Ig G组相比降低(P0.05),A549细胞的HPA的活性与阴性对照组相比降低。抗HPA单抗和紫杉醇单独应用呈浓度依赖性地抑制人肺癌A549细胞增殖,抗HPA单抗(100μg/ml)和紫杉醇(10nmol/L)联用对细胞增殖的抑制作用与任意单药作用组相比增强(P0.05),呈协同作用。与紫杉醇和抗HPA单抗单独干预相比,抗HPA单抗(100μg/ml)和紫杉醇(10nmol/L)联合干预进一步阻滞人肺癌细胞周期于G2/M期(P0.01),细胞凋亡比例增加(P0.01)。抗HPA单抗可以部分增强紫杉醇对人肺癌A549细胞迁移和侵袭能力的抑制作用。抗HPA单抗和紫杉醇单独干预荷Lewis肺癌小鼠后,与生理盐水组和鼠Ig G组相比,肿瘤生长速度减慢,体积减小和瘤重量减轻。抗HPA单抗和紫杉醇联合干预组与生理盐水组和鼠Ig G组相比,对荷Lewis肺癌小鼠皮下移植瘤的生长有明显抑制作用,与抗HPA单抗和紫杉醇单独干预组相比瘤体积和瘤重量均减小。HE染色检查肿瘤形态学变化发现,治疗各组与生理盐水组和鼠Ig G组相比肿瘤细胞生长受到抑制,坏死更为明显。免疫组化发现,抗HPA单抗与生理盐水组相比瘤组织MVD减少,抗HPA单抗和PTX联合干预组与生理盐水组相比瘤组织MVD减少。结论抗HPA单抗部分增强紫杉醇对人肺癌A549细胞增殖、迁移和侵袭能力的抑制作用。其作用机制可能与抗HPA单抗下调A549细胞HPA m RNA和蛋白表达水平,抑制HPA活性有关。也可能与抗HPA单抗与紫杉醇联用进一步阻滞细胞于G2/M期同时诱导细胞的凋亡有关。抗HPA单抗与紫杉醇联用对荷Lewis肺癌小鼠皮下移植瘤生长的抑制率高于单一药组,抗HPA单抗可能成为潜在的化疗增敏药物。
[Abstract]:Objective To study the effect of anti-B-heparanase (HPA) on the expression of HPA mRNA, HPA protein and HPA in human lung cancer A549 cells. The effect of anti-HPA plus chemotherapy on the proliferation, migration and invasion of human lung cancer A549 cells was also evaluated. On the basis of anti-HPA and paclitaxel in vitro inhibition of the proliferation, migration and invasion of human lung cancer A549 cells, a model of subcutaneous transplantation of Lewis lung cancer mice was established, and the effect of anti-HPA and paclitaxel on the growth of transplanted tumor was discussed. Methods The effect of anti-HPA (HPA) on the expression of HPA mRNA and HPA protein in human lung cancer A549 cells was detected by RT-PCR and Western blot. the effect of hpa monoclonal antibody and paclitaxel on the proliferation of human lung cancer A549 cells is detected by using the MTT method alone or in combination, and the cell cycle and the cell apoptosis change of the human lung cancer A549 are analyzed by flow cytometry, The changes of cell migration and invasion ability of human lung cancer A549 were observed by cell scratch and Transwell cell test. The model of the subcutaneous transplantation of Lewis lung cancer C57BL/6 mice was established. The mice were randomly divided into the normal saline group, the mouse Ig G group, the anti-HPA monoclonal antibody group, the paclitaxel group, the anti-HPA monoclonal antibody and the paclitaxel combined group. Weighing the weight of the mouse, and drawing a weight change curve; measuring the volume of the subcutaneous transplanted tumor, and drawing a tumor growth curve; and weighing the bare tumor and calculating the tumor inhibition rate, and evaluating the inhibition effect of the drug on the growth of the transplanted tumor. The pathological changes of the tumor were observed by HE staining, and the microvessel density of the tumor was detected by immunohistochemistry. Results The relative expression of HPA mRNA and HPA protein in A549 cells was lower than that of the control group and the control group (P0.05), and the activity of HPA in A549 cells was lower than that of the negative control group. The inhibition of human lung cancer A549 cell proliferation, anti-HPA (100. mu.g/ ml) and paclitaxel (10 nmol/ L) on the proliferation of human lung cancer A549 cells was enhanced by the combination of anti-HPA (100. mu.g/ ml) and paclitaxel (10 nmol/ L) compared with any single-agent group (P0.05). Compared with paclitaxel and anti-HPA, anti-HPA (100. mu.g/ ml) and paclitaxel (10 nmol/ L) combined to further block the cell cycle of human lung cancer in the G2/ M phase (P0.01), and the proportion of apoptosis increased (P0.01). The anti-HPA monoclonal antibody can partially enhance the inhibitory effect of the paclitaxel on the migration and the invasion ability of the human lung cancer A549 cells. The tumor growth rate, volume reduction and tumor weight were reduced in the mice with Lewis lung cancer treated with anti-HPA and paclitaxel alone. Compared with the normal saline group and the mouse Ig G group, the anti-HPA monoclonal antibody and the paclitaxel combined intervention group have obvious inhibition effect on the growth of the subcutaneous transplanted tumor of the Lewis lung cancer mouse, and the tumor volume and the tumor weight are reduced compared with the anti-HPA monoclonal antibody and the paclitaxel-alone intervention group. It was found that the growth of the tumor cells was inhibited and the necrosis was more obvious in the treatment group than in the normal saline group and the mouse Ig G group. It was found that the MVD of the anti-HPA and the anti-HPA was lower than that of the normal saline group, and the MVD in the anti-HPA and PTX group was lower than that of the normal saline group. Conclusion The anti-HPA monoclonal antibody can inhibit the proliferation, migration and invasion of human lung cancer A549 cells. The mechanism of action may be related to the down-regulation of the expression of HPA m RNA and protein of A549 cells and the inhibition of the activity of HPA. It may also be related to the combination of anti-HPA and paclitaxel to further arrest the apoptosis of cells at the same time as the G2/ M phase. The inhibition rate of the anti-HPA and the combination of the anti-HPA and the paclitaxel on the growth of the subcutaneous transplanted tumor of the Lewis lung cancer mice is higher than that of the single treatment group, and the anti-HPA monoclonal antibody can be a potential chemosensitizing drug.
【学位授予单位】：广东药学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2
[Abstract]:Objective To study the effect of anti-B-heparanase (HPA) on the expression of HPA mRNA, HPA protein and HPA in human lung cancer A549 cells. The effect of anti-HPA plus chemotherapy on the proliferation, migration and invasion of human lung cancer A549 cells was also evaluated. On the basis of anti-HPA and paclitaxel in vitro inhibition of the proliferation, migration and invasion of human lung cancer A549 cells, a model of subcutaneous transplantation of Lewis lung cancer mice was established, and the effect of anti-HPA and paclitaxel on the growth of transplanted tumor was discussed. Methods The effect of anti-HPA (HPA) on the expression of HPA mRNA and HPA protein in human lung cancer A549 cells was detected by RT-PCR and Western blot. the effect of hpa monoclonal antibody and paclitaxel on the proliferation of human lung cancer A549 cells is detected by using the MTT method alone or in combination, and the cell cycle and the cell apoptosis change of the human lung cancer A549 are analyzed by flow cytometry, The changes of cell migration and invasion ability of human lung cancer A549 were observed by cell scratch and Transwell cell test. The model of the subcutaneous transplantation of Lewis lung cancer C57BL/6 mice was established. The mice were randomly divided into the normal saline group, the mouse Ig G group, the anti-HPA monoclonal antibody group, the paclitaxel group, the anti-HPA monoclonal antibody and the paclitaxel combined group. Weighing the weight of the mouse, and drawing a weight change curve; measuring the volume of the subcutaneous transplanted tumor, and drawing a tumor growth curve; and weighing the bare tumor and calculating the tumor inhibition rate, and evaluating the inhibition effect of the drug on the growth of the transplanted tumor. The pathological changes of the tumor were observed by HE staining, and the microvessel density of the tumor was detected by immunohistochemistry. Results The relative expression of HPA mRNA and HPA protein in A549 cells was lower than that of the control group and the control group (P0.05), and the activity of HPA in A549 cells was lower than that of the negative control group. The inhibition of human lung cancer A549 cell proliferation, anti-HPA (100. mu.g/ ml) and paclitaxel (10 nmol/ L) on the proliferation of human lung cancer A549 cells was enhanced by the combination of anti-HPA (100. mu.g/ ml) and paclitaxel (10 nmol/ L) compared with any single-agent group (P0.05). Compared with paclitaxel and anti-HPA, anti-HPA (100. mu.g/ ml) and paclitaxel (10 nmol/ L) combined to further block the cell cycle of human lung cancer in the G2/ M phase (P0.01), and the proportion of apoptosis increased (P0.01). The anti-HPA monoclonal antibody can partially enhance the inhibitory effect of the paclitaxel on the migration and the invasion ability of the human lung cancer A549 cells. The tumor growth rate, volume reduction and tumor weight were reduced in the mice with Lewis lung cancer treated with anti-HPA and paclitaxel alone. Compared with the normal saline group and the mouse Ig G group, the anti-HPA monoclonal antibody and the paclitaxel combined intervention group have obvious inhibition effect on the growth of the subcutaneous transplanted tumor of the Lewis lung cancer mouse, and the tumor volume and the tumor weight are reduced compared with the anti-HPA monoclonal antibody and the paclitaxel-alone intervention group. It was found that the growth of the tumor cells was inhibited and the necrosis was more obvious in the treatment group than in the normal saline group and the mouse Ig G group. It was found that the MVD of the anti-HPA and the anti-HPA was lower than that of the normal saline group, and the MVD in the anti-HPA and PTX group was lower than that of the normal saline group. Conclusion The anti-HPA monoclonal antibody can inhibit the proliferation, migration and invasion of human lung cancer A549 cells. The mechanism of action may be related to the down-regulation of the expression of HPA m RNA and protein of A549 cells and the inhibition of the activity of HPA. It may also be related to the combination of anti-HPA and paclitaxel to further arrest the apoptosis of cells at the same time as the G2/ M phase. The inhibition rate of the anti-HPA and the combination of the anti-HPA and the paclitaxel on the growth of the subcutaneous transplanted tumor of the Lewis lung cancer mice is higher than that of the single treatment group, and the anti-HPA monoclonal antibody can be a potential chemosensitizing drug.
【学位授予单位】：广东药学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2
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