PEDF-Fc融合蛋白的制備及其對(duì)肺腺癌A549細(xì)胞增殖和凋亡的影響
發(fā)布時(shí)間：2018-10-08 06:19
【摘要】：目的構(gòu)建真核重組表達(dá)質(zhì)粒pc DNA3.4-PEDF-Fc,在HEK293F細(xì)胞中表達(dá)并純化,以期獲得高表達(dá)量、純度高、具有生物學(xué)活性的PEDF-Fc融合蛋白,并體外實(shí)驗(yàn)探究PEDF-Fc融合蛋白單用及其與順鉑聯(lián)合應(yīng)用對(duì)肺腺癌A549細(xì)胞增殖和凋亡的影響。方法采用PCR法擴(kuò)增PEDF c DNA編碼序列,將擴(kuò)增的PEDF c DNA編碼序列與編碼人免疫球蛋白Ig G1Fc段恒定區(qū)基因的質(zhì)粒pc DNA3.4-Fc融合,構(gòu)建pc DNA3.4-PEDF-Fc重組質(zhì)粒,脂質(zhì)體法將重組質(zhì)粒轉(zhuǎn)染到HEK293F細(xì)胞中;293F細(xì)胞培養(yǎng)上清液過柱,親和介質(zhì)Mabselect Su Re純化Fc融合蛋白,超濾離心管濃縮融合蛋白;BCA法測定融合蛋白的濃度;SDS-PAGE、Western Blot對(duì)表達(dá)的蛋白進(jìn)行檢測和鑒定;采用MTT法分別檢測不同濃度的PEDF-Fc融合蛋白(2.5、5、10、20、40μg/m L)干預(yù)48h后對(duì)HUVECs和肺癌A549細(xì)胞的增殖抑制作用;MTT法檢測不同濃度順鉑(0.625、1.25、2.5、5、10μg/m L)聯(lián)合PEDF-Fc融合蛋白(10μg/m L)對(duì)肺腺癌A549細(xì)胞的增殖抑制作用;采用流式細(xì)胞術(shù)檢測不同濃度PEDF-Fc融合蛋白(10、40μg/m L)對(duì)HUVECs凋亡的影響以及不同濃度PEDF-Fc融合蛋白(10、40μg/m L)單用及與順鉑(2μg/m L)聯(lián)合應(yīng)用對(duì)A549細(xì)胞凋亡的影響。結(jié)果雙酶切鑒定及基因測序結(jié)果共同證實(shí)成功構(gòu)建了pc DNA3.4-PEDF-Fc重組質(zhì)粒,真核表達(dá)載體構(gòu)建成功;BCA法測得PEDF-Fc融合蛋白的濃度約為3.65mg/m L,PEDF-Fc融合蛋白的表達(dá)量約為90mg/L;SDS-PAGE法和Western Blot法證實(shí)獲得了純度較高的PEDF-Fc融合蛋白,還原性PEDF-Fc融合蛋白的分子量為75KDa左右;MTT結(jié)果表明,不同濃度PEDF-Fc融合蛋白(2.5、5、10、20、40μg/m L)對(duì)HUVECs具有增殖抑制作用,并且抑制作用隨著濃度的增加而增強(qiáng),具有一定的濃度依賴性(P0.05);當(dāng)PEDF-Fc融合蛋白濃度≥5μg/m L時(shí),PEDF-Fc融合蛋白對(duì)A549細(xì)胞的增殖具有明顯的抑制作用,并且具有濃度依賴性(P0.05);以PEDF起效較明顯的濃度干預(yù)組(10μg/m L)與不同濃度的DDP聯(lián)合作用于A549細(xì)胞結(jié)果顯示,聯(lián)合用藥組對(duì)A549細(xì)胞的增殖抑制作用比單獨(dú)使用DDP組更明顯(P0.05);流式結(jié)果表明,PEDF-Fc干預(yù)組(10、40μg/m L)對(duì)HUVECs的凋亡率均高于對(duì)照組(P0.05),且高濃度干預(yù)組細(xì)胞凋亡率明顯高于低濃度干預(yù)組(P0.05);相對(duì)于PEDF-Fc或者順鉑單用組,PEDF-Fc與順鉑聯(lián)用對(duì)A549細(xì)胞具有更強(qiáng)的凋亡促進(jìn)作用(P0.05)。結(jié)論成功構(gòu)建了PEDF-Fc融合蛋白真核表達(dá)載體,且獲得了純度高、具有生物學(xué)活性的PEDF-Fc融合蛋白,為研究PEDF蛋白在肺癌治療中的作用奠定了基礎(chǔ)。結(jié)果表明,PEDF-Fc融合蛋白和低劑量順鉑聯(lián)用對(duì)肺腺癌A549細(xì)胞的增殖抑制和凋亡促進(jìn)作用較PEDF-Fc融合蛋白或者順鉑單用強(qiáng),提示PEDF-Fc融合蛋白和順鉑聯(lián)用對(duì)于肺癌治療的潛在價(jià)值值得進(jìn)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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R734.2
本文編號(hào)：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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R734.2
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