抗人乙酰肝素酶單克隆抗體聯(lián)合紫杉醇對(duì)肺癌的抑制作用
發(fā)布時(shí)間：2019-06-14 06:46
【摘要】：目的探討抗乙酰肝素酶(heparanase,HPA)單抗對(duì)人肺癌A549細(xì)胞HPA m RNA、HPA蛋白表達(dá)水平以及HPA活性的影響。同時(shí)評(píng)價(jià)抗HPA單抗聯(lián)合化療藥物紫杉醇對(duì)人肺癌A549細(xì)胞增殖、遷移和侵襲能力的影響。在抗HPA單抗聯(lián)合紫杉醇體外抑制人肺癌A549細(xì)胞增殖,遷移和侵襲的基礎(chǔ)上,建立荷Lewis肺癌小鼠皮下移植瘤模型,探討抗HPA單抗與紫杉醇單獨(dú)或聯(lián)用對(duì)移植瘤生長的影響。方法采用RT-PCR和Western blot檢測抗HPA單抗對(duì)人肺癌A549細(xì)胞HPA m RNA和HPA蛋白表達(dá)水平的影響;采用四唑藍(lán)比色法和多糖電泳法檢測抗HPA單抗對(duì)人肺癌A549細(xì)胞HPA活性的影響;HPA單抗與紫杉醇單獨(dú)或聯(lián)合干預(yù)人肺癌A549細(xì)胞,采用MTT法檢測對(duì)人肺癌A549細(xì)胞增殖的影響;利用流式細(xì)胞術(shù)分析人肺癌A549細(xì)胞周期和細(xì)胞凋亡的改變,通過細(xì)胞劃痕和Transwell小室實(shí)驗(yàn)觀察人肺癌A549細(xì)胞遷移和侵襲能力的變化。建立荷Lewis肺癌C57BL/6小鼠皮下移植瘤模型,隨機(jī)分為生理鹽水組、鼠Ig G組、抗HPA單抗組、紫杉醇組、抗HPA單抗與紫杉醇聯(lián)用組。稱量小鼠體重,繪制體重變化曲線;測量皮下移植瘤體積,繪制腫瘤生長曲線;稱量裸瘤重并計(jì)算抑瘤率,評(píng)價(jià)藥物對(duì)移植瘤生長的抑制作用。HE染色觀察腫瘤組織病理學(xué)變化,免疫組織化學(xué)檢測腫瘤組織微血管密度。結(jié)果抗HPA單抗干預(yù)后,A549細(xì)胞的HPA m RNA和HPA蛋白相對(duì)表達(dá)水平與溶劑對(duì)照組和鼠Ig G組相比降低(P0.05),A549細(xì)胞的HPA的活性與陰性對(duì)照組相比降低�？笻PA單抗和紫杉醇單獨(dú)應(yīng)用呈濃度依賴性地抑制人肺癌A549細(xì)胞增殖,抗HPA單抗(100μg/ml)和紫杉醇(10nmol/L)聯(lián)用對(duì)細(xì)胞增殖的抑制作用與任意單藥作用組相比增強(qiáng)(P0.05),呈協(xié)同作用。與紫杉醇和抗HPA單抗單獨(dú)干預(yù)相比,抗HPA單抗(100μg/ml)和紫杉醇(10nmol/L)聯(lián)合干預(yù)進(jìn)一步阻滯人肺癌細(xì)胞周期于G2/M期(P0.01),細(xì)胞凋亡比例增加(P0.01)�？笻PA單抗可以部分增強(qiáng)紫杉醇對(duì)人肺癌A549細(xì)胞遷移和侵襲能力的抑制作用。抗HPA單抗和紫杉醇單獨(dú)干預(yù)荷Lewis肺癌小鼠后,與生理鹽水組和鼠Ig G組相比,腫瘤生長速度減慢,體積減小和瘤重量減輕�？笻PA單抗和紫杉醇聯(lián)合干預(yù)組與生理鹽水組和鼠Ig G組相比,對(duì)荷Lewis肺癌小鼠皮下移植瘤的生長有明顯抑制作用,與抗HPA單抗和紫杉醇單獨(dú)干預(yù)組相比瘤體積和瘤重量均減小。HE染色檢查腫瘤形態(tài)學(xué)變化發(fā)現(xiàn),治療各組與生理鹽水組和鼠Ig G組相比腫瘤細(xì)胞生長受到抑制,壞死更為明顯。免疫組化發(fā)現(xiàn),抗HPA單抗與生理鹽水組相比瘤組織MVD減少,抗HPA單抗和PTX聯(lián)合干預(yù)組與生理鹽水組相比瘤組織MVD減少。結(jié)論抗HPA單抗部分增強(qiáng)紫杉醇對(duì)人肺癌A549細(xì)胞增殖、遷移和侵襲能力的抑制作用。其作用機(jī)制可能與抗HPA單抗下調(diào)A549細(xì)胞HPA m RNA和蛋白表達(dá)水平,抑制HPA活性有關(guān)。也可能與抗HPA單抗與紫杉醇聯(lián)用進(jìn)一步阻滯細(xì)胞于G2/M期同時(shí)誘導(dǎo)細(xì)胞的凋亡有關(guān)�？笻PA單抗與紫杉醇聯(lián)用對(duì)荷Lewis肺癌小鼠皮下移植瘤生長的抑制率高于單一藥組,抗HPA單抗可能成為潛在的化療增敏藥物。
[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.
【學(xué)位授予單位】：廣東藥學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：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.
【學(xué)位授予單位】：廣東藥學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R734.2
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