【摘要】：目的： 1、分析ERC(N~5)與人臍靜脈內(nèi)皮細(xì)胞HUVEC結(jié)合的情況 2、觀察ERC(N~5)對(duì)HUVEC細(xì)胞凋亡及侵襲作用的影響 方法： 1、ERC(N~5)小肽的設(shè)計(jì)與合成 將蛇毒鋸鱗蝰素（Echistatin，Ech）氨基酸序列中的RGD模體及其周邊序列直接與C末端序列結(jié)合，突變第五個(gè)氨基酸（D→N）并適當(dāng)減少氨基酸的數(shù)目，設(shè)計(jì)成含16個(gè)氨基酸的小肽，其序列為ARGDNMPRNPHKGPAT，命名為ERC(N~5)，，由西安聯(lián)美生物科技有限公司合成。 2、ERC(N~5)與人臍靜脈內(nèi)皮細(xì)胞HUVEC的結(jié)合情況分析 利用流式細(xì)胞術(shù)（flowcytometry，F(xiàn)CM）檢測(cè)人臍靜脈內(nèi)皮細(xì)胞表面αvβ3的表達(dá)情況；分別將ERC(N~5)、Ech、FITC-LM609（整合素αvβ3的單克隆抗體）與HUVEC細(xì)胞共同孵育后，流式細(xì)胞術(shù)檢測(cè)FITC標(biāo)記的陽(yáng)性細(xì)胞率，分析ERC(N~5)/Ech與抗體競(jìng)爭(zhēng)結(jié)合αvβ3的情況。 3、ERC(N~5)對(duì)HUVEC細(xì)胞凋亡、侵襲作用的研究 倒置顯微鏡觀察ERC(N~5)處理后的HUVEC形態(tài)變化；通過(guò)AO/EB對(duì)細(xì)胞核染色觀察細(xì)胞形態(tài)的變化；AnnexinV-FITC/PI雙染法結(jié)合流式細(xì)胞儀檢測(cè)ERC(N~5)對(duì)HUVEC凋亡率的影響；用RT-PCR檢測(cè)細(xì)胞凋亡基因Caspase-3表達(dá)。利用細(xì)胞劃痕愈合實(shí)驗(yàn)、Transwell遷移實(shí)驗(yàn)和Matrigel侵襲實(shí)驗(yàn)觀察ERC(N~5)對(duì)HUVEC遷移、侵襲能力的影響。 結(jié)果： 1、人臍靜脈內(nèi)皮細(xì)胞HUVEC細(xì)胞表面整合素αvβ3表達(dá)量為68.3%。2、ERC(N~5)、FITC-LM609與HUVEC細(xì)胞共孵育后，F(xiàn)ITC標(biāo)記的陽(yáng)性細(xì)胞率隨著ERC(N~5)濃度的增大而逐漸減小。終濃度為16μM的ERC(N~5)與Ech分別與HUVEC孵育后，其αvβ3陽(yáng)性細(xì)胞率分別為1.2%和26.8%。 3、HUVEC經(jīng)ERC(N~5)作用24h后，細(xì)胞變圓、間隙增大、有細(xì)胞碎片出現(xiàn)。經(jīng)AO/EB染色后，出現(xiàn)核染色質(zhì)著綠色呈固縮狀的早期凋亡細(xì)胞和核染色質(zhì)為橘紅色的晚期凋亡細(xì)胞。細(xì)胞凋亡率檢測(cè)結(jié)果顯示，ERC(N~5)作用HUVEC細(xì)胞后，凋亡率增加5倍。 4、RT-PCR結(jié)果顯示在經(jīng)ERC(N~5)處理后Caspase-3基因表達(dá)較對(duì)照組明顯增加，且有劑量依賴(lài)關(guān)系。 5、ERC(N~5)作用下，HUVEC細(xì)胞的遷移和侵襲能力分別降低至78.5%和87%；細(xì)胞劃痕愈合實(shí)驗(yàn)表明，細(xì)胞培養(yǎng)24小時(shí)后，陰性對(duì)照組細(xì)胞劃痕已經(jīng)基本愈合，ERC(N~5)處理組劃痕依然清晰。 結(jié)論： 1、人臍靜脈內(nèi)皮細(xì)胞HUVEC細(xì)胞表面高表達(dá)整合素αvβ3，ERC(N~5)能夠通過(guò)與αvβ3識(shí)別從而與HUVEC結(jié)合。 2、ERC(N~5)能夠促進(jìn)HUVEC凋亡，從而抑制HUVEC的增殖，也能抑制HUVEC的侵襲和遷移能力。
[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 偽 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 偽 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 偽 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 偽 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 偽 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 偽 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 偽 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 偽 v 尾 3 on the surface of human umbilical vein endothelial cells, and ERC (N 尾 5) can bind to HUVEC through recognition with 偽 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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R363

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