ERC（N~5）小肽对人脐静脉内皮细胞系HUVEC增殖、侵袭作用的研究
发布时间：2019-04-17 19:36
【摘要】：目的： 1、分析ERC(N~5)与人脐静脉内皮细胞HUVEC结合的情况 2、观察ERC(N~5)对HUVEC细胞凋亡及侵袭作用的影响 方法： 1、ERC(N~5)小肽的设计与合成 将蛇毒锯鳞蝰素（Echistatin，Ech）氨基酸序列中的RGD模体及其周边序列直接与C末端序列结合，突变第五个氨基酸（D→N）并适当减少氨基酸的数目，设计成含16个氨基酸的小肽，其序列为ARGDNMPRNPHKGPAT，命名为ERC(N~5)，，由西安联美生物科技有限公司合成。 2、ERC(N~5)与人脐静脉内皮细胞HUVEC的结合情况分析 利用流式细胞术（flowcytometry，FCM）检测人脐静脉内皮细胞表面αvβ3的表达情况；分别将ERC(N~5)、Ech、FITC-LM609（整合素αvβ3的单克隆抗体）与HUVEC细胞共同孵育后，流式细胞术检测FITC标记的阳性细胞率，分析ERC(N~5)/Ech与抗体竞争结合αvβ3的情况。 3、ERC(N~5)对HUVEC细胞凋亡、侵袭作用的研究 倒置显微镜观察ERC(N~5)处理后的HUVEC形态变化；通过AO/EB对细胞核染色观察细胞形态的变化；AnnexinV-FITC/PI双染法结合流式细胞仪检测ERC(N~5)对HUVEC凋亡率的影响；用RT-PCR检测细胞凋亡基因Caspase-3表达。利用细胞划痕愈合实验、Transwell迁移实验和Matrigel侵袭实验观察ERC(N~5)对HUVEC迁移、侵袭能力的影响。 结果： 1、人脐静脉内皮细胞HUVEC细胞表面整合素αvβ3表达量为68.3%。2、ERC(N~5)、FITC-LM609与HUVEC细胞共孵育后，FITC标记的阳性细胞率随着ERC(N~5)浓度的增大而逐渐减小。终浓度为16μM的ERC(N~5)与Ech分别与HUVEC孵育后，其αvβ3阳性细胞率分别为1.2%和26.8%。 3、HUVEC经ERC(N~5)作用24h后，细胞变圆、间隙增大、有细胞碎片出现。经AO/EB染色后，出现核染色质着绿色呈固缩状的早期凋亡细胞和核染色质为橘红色的晚期凋亡细胞。细胞凋亡率检测结果显示，ERC(N~5)作用HUVEC细胞后，凋亡率增加5倍。 4、RT-PCR结果显示在经ERC(N~5)处理后Caspase-3基因表达较对照组明显增加，且有剂量依赖关系。 5、ERC(N~5)作用下，HUVEC细胞的迁移和侵袭能力分别降低至78.5%和87%；细胞划痕愈合实验表明，细胞培养24小时后，阴性对照组细胞划痕已经基本愈合，ERC(N~5)处理组划痕依然清晰。 结论： 1、人脐静脉内皮细胞HUVEC细胞表面高表达整合素αvβ3，ERC(N~5)能够通过与αvβ3识别从而与HUVEC结合。 2、ERC(N~5)能够促进HUVEC凋亡，从而抑制HUVEC的增殖，也能抑制HUVEC的侵袭和迁移能力。
[Abstract]:Objective: 1. To analyze the binding of ERC (N) to HUVEC of human umbilical vein endothelial cells (HUVEC) 2, and to observe the effect of ERC (N) on apoptosis and invasion of HUVEC cells. Design and Synthesis of a small Peptide of ERC (N) the RGD motif and its peripheral sequence in the amino acid sequence of viperin (Echistatin,Ech) were directly combined with the C-terminal sequence. A peptide containing 16 amino acids was designed by mutating the fifth amino acid (D N) and reducing the number of amino acids. The sequence of the peptide was named ERC (ARGDNMPRNPHKGPAT, 5), which was synthesized by Xi'an Lianmei Biotechnology Co., Ltd. 2. Binding analysis of ERC (N) with human umbilical vein endothelial cells (HUVEC). Flow cytometry (flowcytometry,FCM) was used to detect the expression of 伪 v 尾 3 on the surface of human umbilical vein endothelial cells (HUVECs). After HUVEC cells were incubated with ERC (N5) and Ech,FITC-LM609 (monoclonal antibody against integrin 伪 v 尾 3) respectively, the positive rate of FITC labeled cells was detected by flow cytometry, and the competitive binding of ERC (Nm 5) / Ech with antibody 伪 v 尾 3 was analyzed. 3. Study on apoptosis and invasion of HUVEC cells by ERC (N). The morphological changes of HUVEC were observed by inverted microscope, the morphological changes of HUVEC cells were observed by nuclear staining with AO/EB, and the morphological changes of ERC cells were observed by nuclear staining with AO/EB, and the morphological changes of ERC cells were observed by inverted microscope. AnnexinV-FITC/PI double staining and flow cytometry were used to detect the effect of ERC on the apoptosis rate of HUVEC, and the expression of apoptosis gene Caspase-3 was detected by RT-PCR. Cell scratch healing test, Transwell migration test and Matrigel invasion test were used to observe the effect of ERC on the migration and invasiveness of HUVEC. Results: 1. The expression of integrin 伪 v 尾 3 on the surface of human umbilical vein endothelial cells (HUVEC) was 68.3%. ERC (N + 5), FITC-LM609 co-incubated with HUVEC cells, the expression of integrin 伪 v 尾 3 on the surface of human umbilical vein endothelial cells was 68.3%. The positive rate of FITC-labeled cells decreased with the increase of ERC (N _ (n) _ (5) concentration. The percentage of 伪 v 尾 3 positive cells was 1.2% and 26.8% after ERC (Nm 5) and Ech were incubated with HUVEC at the final concentration of 16 渭 M, respectively, and the positive rate of 伪 v 尾 3 positive cells was 1.2% and 26.8%, respectively. 3. HUVECs were treated with ERC (N + 5) for 24 h, the cells became round and the gap increased, and some cell fragments appeared in HUVECs. After AO/EB staining, early apoptotic cells with green chromatin pyknosis and terminal apoptotic cells with orange chromatin in nuclear chromatin were observed. The apoptosis rate of HUVEC cells was increased by 5-fold after treated with, ERC (- N-5 (P < 0.05). 4. The results of RT-PCR showed that the expression of Caspase-3 gene was significantly increased in ERC (N) treated group than that in control group, and there was a dose-dependent relationship between the expression of RT-PCR and that of control group. 5. The migration and invasion ability of HUVEC cells decreased to 78.5% and 87%, respectively. The cell scratch healing experiment showed that after 24 hours of cell culture, the scratch in the negative control group was basically healed by, ERC (N\ -\ {5}\ {5\}, and the scratch was still clear in the treatment group. Conclusion: 1. HUVEC cells express integrin 伪 v 尾 3 on the surface of human umbilical vein endothelial cells, and ERC (N 尾 5) can bind to HUVEC through recognition with 伪 v 尾 3. 2. ERC (N + 5) can promote the apoptosis of HUVEC, inhibit the proliferation of HUVEC, and also inhibit the invasion and migration of HUVEC.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
[Abstract]:Objective: 1. To analyze the binding of ERC (N) to HUVEC of human umbilical vein endothelial cells (HUVEC) 2, and to observe the effect of ERC (N) on apoptosis and invasion of HUVEC cells. Design and Synthesis of a small Peptide of ERC (N) the RGD motif and its peripheral sequence in the amino acid sequence of viperin (Echistatin,Ech) were directly combined with the C-terminal sequence. A peptide containing 16 amino acids was designed by mutating the fifth amino acid (D N) and reducing the number of amino acids. The sequence of the peptide was named ERC (ARGDNMPRNPHKGPAT, 5), which was synthesized by Xi'an Lianmei Biotechnology Co., Ltd. 2. Binding analysis of ERC (N) with human umbilical vein endothelial cells (HUVEC). Flow cytometry (flowcytometry,FCM) was used to detect the expression of 伪 v 尾 3 on the surface of human umbilical vein endothelial cells (HUVECs). After HUVEC cells were incubated with ERC (N5) and Ech,FITC-LM609 (monoclonal antibody against integrin 伪 v 尾 3) respectively, the positive rate of FITC labeled cells was detected by flow cytometry, and the competitive binding of ERC (Nm 5) / Ech with antibody 伪 v 尾 3 was analyzed. 3. Study on apoptosis and invasion of HUVEC cells by ERC (N). The morphological changes of HUVEC were observed by inverted microscope, the morphological changes of HUVEC cells were observed by nuclear staining with AO/EB, and the morphological changes of ERC cells were observed by nuclear staining with AO/EB, and the morphological changes of ERC cells were observed by inverted microscope. AnnexinV-FITC/PI double staining and flow cytometry were used to detect the effect of ERC on the apoptosis rate of HUVEC, and the expression of apoptosis gene Caspase-3 was detected by RT-PCR. Cell scratch healing test, Transwell migration test and Matrigel invasion test were used to observe the effect of ERC on the migration and invasiveness of HUVEC. Results: 1. The expression of integrin 伪 v 尾 3 on the surface of human umbilical vein endothelial cells (HUVEC) was 68.3%. ERC (N + 5), FITC-LM609 co-incubated with HUVEC cells, the expression of integrin 伪 v 尾 3 on the surface of human umbilical vein endothelial cells was 68.3%. The positive rate of FITC-labeled cells decreased with the increase of ERC (N _ (n) _ (5) concentration. The percentage of 伪 v 尾 3 positive cells was 1.2% and 26.8% after ERC (Nm 5) and Ech were incubated with HUVEC at the final concentration of 16 渭 M, respectively, and the positive rate of 伪 v 尾 3 positive cells was 1.2% and 26.8%, respectively. 3. HUVECs were treated with ERC (N + 5) for 24 h, the cells became round and the gap increased, and some cell fragments appeared in HUVECs. After AO/EB staining, early apoptotic cells with green chromatin pyknosis and terminal apoptotic cells with orange chromatin in nuclear chromatin were observed. The apoptosis rate of HUVEC cells was increased by 5-fold after treated with, ERC (- N-5 (P < 0.05). 4. The results of RT-PCR showed that the expression of Caspase-3 gene was significantly increased in ERC (N) treated group than that in control group, and there was a dose-dependent relationship between the expression of RT-PCR and that of control group. 5. The migration and invasion ability of HUVEC cells decreased to 78.5% and 87%, respectively. The cell scratch healing experiment showed that after 24 hours of cell culture, the scratch in the negative control group was basically healed by, ERC (N\ -\ {5}\ {5\}, and the scratch was still clear in the treatment group. Conclusion: 1. HUVEC cells express integrin 伪 v 尾 3 on the surface of human umbilical vein endothelial cells, and ERC (N 尾 5) can bind to HUVEC through recognition with 伪 v 尾 3. 2. ERC (N + 5) can promote the apoptosis of HUVEC, inhibit the proliferation of HUVEC, and also inhibit the invasion and migration of HUVEC.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
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