PEDF-Fc融合蛋白的制备及其对肺腺癌A549细胞增殖和凋亡的影响
发布时间：2018-10-08 06:19
【摘要】：目的构建真核重组表达质粒pc DNA3.4-PEDF-Fc,在HEK293F细胞中表达并纯化,以期获得高表达量、纯度高、具有生物学活性的PEDF-Fc融合蛋白,并体外实验探究PEDF-Fc融合蛋白单用及其与顺铂联合应用对肺腺癌A549细胞增殖和凋亡的影响。方法采用PCR法扩增PEDF c DNA编码序列,将扩增的PEDF c DNA编码序列与编码人免疫球蛋白Ig G1Fc段恒定区基因的质粒pc DNA3.4-Fc融合,构建pc DNA3.4-PEDF-Fc重组质粒,脂质体法将重组质粒转染到HEK293F细胞中;293F细胞培养上清液过柱,亲和介质Mabselect Su Re纯化Fc融合蛋白,超滤离心管浓缩融合蛋白;BCA法测定融合蛋白的浓度;SDS-PAGE、Western Blot对表达的蛋白进行检测和鉴定;采用MTT法分别检测不同浓度的PEDF-Fc融合蛋白(2.5、5、10、20、40μg/m L)干预48h后对HUVECs和肺癌A549细胞的增殖抑制作用;MTT法检测不同浓度顺铂(0.625、1.25、2.5、5、10μg/m L)联合PEDF-Fc融合蛋白(10μg/m L)对肺腺癌A549细胞的增殖抑制作用;采用流式细胞术检测不同浓度PEDF-Fc融合蛋白(10、40μg/m L)对HUVECs凋亡的影响以及不同浓度PEDF-Fc融合蛋白(10、40μg/m L)单用及与顺铂(2μg/m L)联合应用对A549细胞凋亡的影响。结果双酶切鉴定及基因测序结果共同证实成功构建了pc DNA3.4-PEDF-Fc重组质粒,真核表达载体构建成功;BCA法测得PEDF-Fc融合蛋白的浓度约为3.65mg/m L,PEDF-Fc融合蛋白的表达量约为90mg/L;SDS-PAGE法和Western Blot法证实获得了纯度较高的PEDF-Fc融合蛋白,还原性PEDF-Fc融合蛋白的分子量为75KDa左右;MTT结果表明,不同浓度PEDF-Fc融合蛋白(2.5、5、10、20、40μg/m L)对HUVECs具有增殖抑制作用,并且抑制作用随着浓度的增加而增强,具有一定的浓度依赖性(P0.05);当PEDF-Fc融合蛋白浓度≥5μg/m L时,PEDF-Fc融合蛋白对A549细胞的增殖具有明显的抑制作用,并且具有浓度依赖性(P0.05);以PEDF起效较明显的浓度干预组(10μg/m L)与不同浓度的DDP联合作用于A549细胞结果显示,联合用药组对A549细胞的增殖抑制作用比单独使用DDP组更明显(P0.05);流式结果表明,PEDF-Fc干预组(10、40μg/m L)对HUVECs的凋亡率均高于对照组(P0.05),且高浓度干预组细胞凋亡率明显高于低浓度干预组(P0.05);相对于PEDF-Fc或者顺铂单用组,PEDF-Fc与顺铂联用对A549细胞具有更强的凋亡促进作用(P0.05)。结论成功构建了PEDF-Fc融合蛋白真核表达载体,且获得了纯度高、具有生物学活性的PEDF-Fc融合蛋白,为研究PEDF蛋白在肺癌治疗中的作用奠定了基础。结果表明,PEDF-Fc融合蛋白和低剂量顺铂联用对肺腺癌A549细胞的增殖抑制和凋亡促进作用较PEDF-Fc融合蛋白或者顺铂单用强,提示PEDF-Fc融合蛋白和顺铂联用对于肺癌治疗的潜在价值值得进一步研究。
[Abstract]:Objective to construct and purify eukaryotic recombinant expression plasmid pc DNA3.4-PEDF-Fc, in HEK293F cells in order to obtain PEDF-Fc fusion protein with high expression quantity, high purity and biological activity. The effects of PEDF-Fc fusion protein alone and its combination with cisplatin on proliferation and apoptosis of lung adenocarcinoma cell line A549 were investigated in vitro. Methods PCR method was used to amplify the PEDF c DNA coding sequence. The amplified PEDF c DNA coding sequence was fused with the plasmid pc DNA3.4-Fc encoding the constant region gene of human immunoglobulin Ig G1Fc segment, and the pc DNA3.4-PEDF-Fc recombinant plasmid was constructed. The recombinant plasmid was transfected into HEK293F cells by liposome method. The supernatant of the cell culture supernatant was supernatant. The affinity medium Mabselect Su Re was used to purify the Fc fusion protein. The concentration of fusion protein was determined by BCA method in ultrafiltration centrifuge tube. SDS-PAGEG Western Blot was used to detect and identify the expressed protein. MTT assay was used to detect the proliferation inhibitory effect of different concentrations of PEDF-Fc fusion protein (2.5mL) on HUVECs and lung cancer A549 cells after 48 h intervention. The proliferation inhibitory effect of different concentrations of cisplatin (0.625 ~ 1.252.5 渭 g / mL) combined with PEDF-Fc fusion protein (10 渭 g / mL) on the proliferation of lung adenocarcinoma A549 cells was detected by MTT assay. The effects of different concentrations of PEDF-Fc fusion protein (10 ~ 40 渭 g / mL) on apoptosis of A549 cells were detected by flow cytometry. The effects of different concentrations of PEDF-Fc fusion protein (10 ~ 40 渭 g / mL) alone and combined with cisplatin (2 渭 g / mL) on apoptosis of A549 cells were investigated. Results double restriction endonuclease digestion and gene sequencing confirmed the successful construction of pc DNA3.4-PEDF-Fc recombinant plasmid. The eukaryotic expression vector was successfully constructed. The concentration of PEDF-Fc fusion protein was about 90 mg 路L ~ (-1) 3.65mg/m PEDF-Fc, which was confirmed by Western Blot method and 90 mg / L SDS-PAGE, and a high purity PEDF-Fc fusion protein was obtained. The molecular weight of the reductive PEDF-Fc fusion protein was about 75KDa. The results showed that PEDF-Fc fusion protein (2.5G / mL) could inhibit the proliferation of HUVECs, and the inhibitory effect increased with the increase of the concentration. When the concentration of PEDF-Fc fusion protein was more than 5 渭 g / mL, PEDF-Fc fusion protein could significantly inhibit the proliferation of A549 cells. The results showed that the A549 cells were treated with 10 渭 g / mL of DDP and 10 渭 g / mL of PEDF in a dose-dependent manner (P0.05). The proliferation inhibition of A549 cells in combination group was more obvious than that in DDP group alone (P0.05), and the flow rate showed that the apoptosis rate of HUVECs in PEDF-Fc intervention group (1040 渭 g / mL) was higher than that in control group (P0.05), and the apoptosis rate in high concentration intervention group was significantly higher than that in low concentration group (P0.05). Degree intervention group (P0.05); compared with PEDF-Fc or cisplatin alone group PEDF-Fc combined with cisplatin has a stronger apoptosis promotion effect on A549 cells (P0.05). Conclusion the eukaryotic expression vector of PEDF-Fc fusion protein was successfully constructed, and the PEDF-Fc fusion protein with high purity and biological activity was obtained, which laid a foundation for the study of the role of PEDF protein in the treatment of lung cancer. The results showed that PEDF-Fc fusion protein combined with low dose cisplatin could inhibit the proliferation and apoptosis of lung adenocarcinoma A549 cells more effectively than PEDF-Fc fusion protein or cisplatin alone. It suggests that the potential value of PEDF-Fc fusion protein combined with cisplatin in the treatment of lung cancer deserves further study.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
本文编号：2255737
[Abstract]:Objective to construct and purify eukaryotic recombinant expression plasmid pc DNA3.4-PEDF-Fc, in HEK293F cells in order to obtain PEDF-Fc fusion protein with high expression quantity, high purity and biological activity. The effects of PEDF-Fc fusion protein alone and its combination with cisplatin on proliferation and apoptosis of lung adenocarcinoma cell line A549 were investigated in vitro. Methods PCR method was used to amplify the PEDF c DNA coding sequence. The amplified PEDF c DNA coding sequence was fused with the plasmid pc DNA3.4-Fc encoding the constant region gene of human immunoglobulin Ig G1Fc segment, and the pc DNA3.4-PEDF-Fc recombinant plasmid was constructed. The recombinant plasmid was transfected into HEK293F cells by liposome method. The supernatant of the cell culture supernatant was supernatant. The affinity medium Mabselect Su Re was used to purify the Fc fusion protein. The concentration of fusion protein was determined by BCA method in ultrafiltration centrifuge tube. SDS-PAGEG Western Blot was used to detect and identify the expressed protein. MTT assay was used to detect the proliferation inhibitory effect of different concentrations of PEDF-Fc fusion protein (2.5mL) on HUVECs and lung cancer A549 cells after 48 h intervention. The proliferation inhibitory effect of different concentrations of cisplatin (0.625 ~ 1.252.5 渭 g / mL) combined with PEDF-Fc fusion protein (10 渭 g / mL) on the proliferation of lung adenocarcinoma A549 cells was detected by MTT assay. The effects of different concentrations of PEDF-Fc fusion protein (10 ~ 40 渭 g / mL) on apoptosis of A549 cells were detected by flow cytometry. The effects of different concentrations of PEDF-Fc fusion protein (10 ~ 40 渭 g / mL) alone and combined with cisplatin (2 渭 g / mL) on apoptosis of A549 cells were investigated. Results double restriction endonuclease digestion and gene sequencing confirmed the successful construction of pc DNA3.4-PEDF-Fc recombinant plasmid. The eukaryotic expression vector was successfully constructed. The concentration of PEDF-Fc fusion protein was about 90 mg 路L ~ (-1) 3.65mg/m PEDF-Fc, which was confirmed by Western Blot method and 90 mg / L SDS-PAGE, and a high purity PEDF-Fc fusion protein was obtained. The molecular weight of the reductive PEDF-Fc fusion protein was about 75KDa. The results showed that PEDF-Fc fusion protein (2.5G / mL) could inhibit the proliferation of HUVECs, and the inhibitory effect increased with the increase of the concentration. When the concentration of PEDF-Fc fusion protein was more than 5 渭 g / mL, PEDF-Fc fusion protein could significantly inhibit the proliferation of A549 cells. The results showed that the A549 cells were treated with 10 渭 g / mL of DDP and 10 渭 g / mL of PEDF in a dose-dependent manner (P0.05). The proliferation inhibition of A549 cells in combination group was more obvious than that in DDP group alone (P0.05), and the flow rate showed that the apoptosis rate of HUVECs in PEDF-Fc intervention group (1040 渭 g / mL) was higher than that in control group (P0.05), and the apoptosis rate in high concentration intervention group was significantly higher than that in low concentration group (P0.05). Degree intervention group (P0.05); compared with PEDF-Fc or cisplatin alone group PEDF-Fc combined with cisplatin has a stronger apoptosis promotion effect on A549 cells (P0.05). Conclusion the eukaryotic expression vector of PEDF-Fc fusion protein was successfully constructed, and the PEDF-Fc fusion protein with high purity and biological activity was obtained, which laid a foundation for the study of the role of PEDF protein in the treatment of lung cancer. The results showed that PEDF-Fc fusion protein combined with low dose cisplatin could inhibit the proliferation and apoptosis of lung adenocarcinoma A549 cells more effectively than PEDF-Fc fusion protein or cisplatin alone. It suggests that the potential value of PEDF-Fc fusion protein combined with cisplatin in the treatment of lung cancer deserves further study.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
【参考文献】
相关期刊论文 前7条
1 Zhan-Wu Yu;Ying-Hua Ju;Cheng-Liang Yang;Han-Bing Yu;Quan Luo;Ye-Gang Ma;Yong-Yu Liu;;Antitumor effect of recombinant human endostatin combined with cisplatin on rats with transplanted Lewis lung cancer[J];Asian Pacific Journal of Tropical Medicine;2015年08期
2 娄志霞;束军;金程;沈继龙;;PEDF对人肺腺癌A549细胞增殖与凋亡的影响[J];安徽医科大学学报;2014年10期
3 吴桢珍;查王健;陈菲菲;杨光宇;黄茂;;PEDF对低氧状态下人支气管上皮细胞表达VEGF的影响[J];南京医科大学学报(自然科学版);2014年04期
4 刘正兵;束军;;色素上皮衍生因子对非小细胞肺癌NCI-H460细胞增殖和凋亡的影响[J];安徽医科大学学报;2013年06期
5 郑金旭;许清;管淑红;刘继柱;宋萍;汤燕;吴立艳;;柴胡皂甙d干预肺纤维化VEGF/PEDF表达的实验研究[J];天津医药;2012年08期
6 李瑞霞;陈海冰;;色素上皮衍生因子与氧化应激[J];医学综述;2012年13期
7 郑民;梁岳培;王洋;;非小细胞肺癌组织中VEGF、PEDF的表达变化及意义[J];山东医药;2011年18期
相关硕士学位论文 前1条
1 赵俊;胸腔积液中VEGF-A、VEGF-C、PEDF和CEA水平及其与中医辨证分型的关系研究[D];南京中医药大学;2013年
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