卵泡抑素相关蛋白抑制血管内皮细胞凋亡的机制研究

发布时间：2018-04-24 16:31

本文选题：卵泡抑素相关蛋白 + 血管内皮细胞　；参考：《延边大学》2010年硕士论文

【摘要】： 一、研究背景与目的血管内皮细胞(vascular endothelial cell, VEC)是循环血液与血管平滑肌间的机械屏障。VEC的功能相当广泛,除起转运屏障作用外,还具有分泌功能,可以合成分泌多种物质,对调节血管紧张度、血液流通、细胞生成、炎症反应、免疫等功能具有重要作用。多种因素可通过不同机制与途径损伤血管内皮细胞,使内皮屏障作用减弱,血中脂蛋白过多地进入动脉壁沉积下来,被清道夫细胞吞噬形成泡沫细胞；内皮细胞受损后,炎细胞、血小板的粘附与内容物的释放又加重了内皮细胞损伤,从而逐渐形成粥样硬化斑块,因此寻找一种应用方便、专一性强、来源丰富、效果良好的VEC保护剂也越来越引起人们的重视。本课题以血管内皮细胞为靶细胞,以卵泡抑素相关蛋白(Follistatin Related Protein, FRP)为主要研究对象,以Western Blot、Si-RNA、Rt-PCR、ChIP assay等实验技术为研究手段,重点探讨FRP对VEC的保护作用,力求阐明该蛋白对VEC的抗凋亡机制及其在动脉粥样硬化中的病理意义。二、材料与方法 1、根据验证样性的基因编码序列获得FRP编码区cDNA连接于表达载体pET32a,构建表达重组子,转化入Origami (DE3), 1mM IPTG诱导蛋白表达,使用Ni-NTA His Band Resins, Sephacryl S-100纯化蛋白,肠激酶酶切融合蛋白并鉴定。 2、分离脐带静脉血管内皮细胞,并用含有胎牛血清的1640完全培养基进行细胞培养,每2-3天换液一次,定期进行消化传代。 3、将C段带有FLAG标签的FRP基因片段连接到pcDNA 4／TO上,先后将pcDNA 6／TR和pcDNA4/T0/FRP-FLAG转染至内皮细胞系EAHY926中,用blasticidin和zeocin筛选稳定转染的单克隆细胞。 4、制备ApoE基因敲除小鼠为背景的FRP转基因动物,ApoE基因缺陷小鼠与ApoE基因缺陷FRP转基因小鼠,构建动脉粥样硬化动物模型。 5、通过PI-Annexin-V双染后流式细胞仪分析,研究FRP重组蛋白FRP抗ox-LDL诱导的细胞凋亡维持HUVEC的细胞活性。 6、通过rt-PCR WesternBlot等方法检测FRP下游抗凋亡蛋白。三、结果 1.给予ApoE缺失小鼠及ApoE基因缺陷-FRP转基因小鼠喂养12周。TUNEL提示ApoE缺失小鼠动脉粥样硬化斑块形成后血管内皮细胞凋亡增加；ApoE基因缺陷-FRP转基因小鼠TUNEL染色显示凋亡的内皮细胞显著少于ApoE组。用oxLDL的终浓度依次为0ug／ml、10ug／ml、20ug／ml、30ug／ml、50ug／ml(0ug／ml oxLDL作为对照组)处理HUVECs直接影响FRP的表达,结果发现处理后HUVECs的FRP表达量与oxLDL的浓度呈反比。 2.细菌可溶性的蛋白经Ni-His band及Sephadex Sephacryl S-100纯化酶切蛋白后,得到目的蛋白。 3.用浓度为50μg／ml的ox-LDL处理HUVECs,与对照组相比细胞变圆,贴壁细胞减少。100ng／ml的FRP重组蛋白可以抗ox-LDL诱导的细胞凋亡。MTT检测提示,随着ox-LDL浓度的增加,内皮细胞的存活能力逐渐降低；不同浓度的FRP和ox-LDL对内皮细胞进行处理时,发现FRP可以抵抗ox-LDL诱导的内皮细胞凋亡,当FRP浓度为100ng／ml时有显著性差异。FRP浓度大于80ng／ml时可显著提高细胞活性。FACS提示FRP可显著降低凋亡早期annexin V阳性细胞数。 4.按如下方法处理细胞24小时：1、空白对照组+50μg／ml LDL.2、+50μg／mloxLDL.3、+50μg／ml oxLDL+50ng／ml FRP.4、+50μg／ml oxLDL+75ng／ml FRP.5、+50μg／ml oxLDL+100ng／ml FRP。实验发现,FRP可以上调磷酸化Akt的表达,从而激活磷酸化Akt的下游基因——磷酸化Bad的表达。然而,Akt的表达量并未随着FRP的表达量而改变, Bcl2的表达量随着FRP浓度的增高而显著性增加。 5.利用转染Bcl2 siRNA的方法抑制Bcl2的蛋白表达,可使Bcl2蛋白表达量降低约50%(Scramble RNA组为对照组)。用FRP诱导Bcl2表达发现Bcl2 siRNA组与对照组相比Bcl2的表达量无明显增加。在Scramble RNA组中可以观察到FRP对ox-LDL诱导的HUVEC凋亡仍有保护作用,而在Bcl2 siRNA组,FRP的保护作用丧失。四、结论本次课题体内和体外实验都证实了ox-LDL通过抑制内皮细胞中FRP的蛋白表达从而诱导内皮细胞凋亡；同时发现,FRP是通过转录水平上调Bcl2的蛋白表达从而抑制ox-LDL诱导内皮细胞凋亡,发挥其抗内皮细胞凋亡、延缓动脉粥样硬化发展的功能。
[Abstract]:I . Background and purpose of research

vascular endothelial cell ( vec ) is a mechanical barrier between circulating blood and vascular smooth muscle .
After the endothelial cells are damaged , the adhesion of the inflammatory cells , platelets and the release of the contents further aggravate the endothelial cell injury , thereby gradually forming the atheromatous plaque , and therefore , it is becoming more and more important to find a vec protective agent which has the advantages of convenient application , strong specificity , rich source and good effect .

In this study , vascular endothelial cells were used as target cells . Follistatin Related Protein ( FRP ) was used as the main research object . Western Blot , Si - RNA , Rt - PCR , ChIP assay and other experimental techniques were used as the research methods .

II . MATERIALS AND METHODS

1 . The cDNA of the coding region of the FRP was ligated to the expression vector pET32a according to the verification - like gene coding sequence to construct the expression vector pET32a . Then , the expressed recombinant was transformed into Origami ( DE3 ) and 1 mM IPTG - induced protein expression .

2 . The umbilical vein endothelial cells were isolated and the cells were cultured with 1640 complete medium containing fetal bovine serum , once every 2 - 3 days , the culture was performed on a regular basis .

3 . The fragment of the FRP gene with the tag in C segment was ligated to pcDNA 4 / TO , pcDNA 6 / TR was transfected into the endothelial cell line EAHY926 , and the stably transfected monoclonal cell was screened by using zeocin .

and 4 , preparing an E gene knockout mouse as a background FRP transgenic animal , an apolipoprotein E gene deficient mouse and an apolipoprotein E gene defective FRP transgenic mouse , and constructing an atherosclerosis animal model .

5 . Apoptosis of HUVEC induced by anti - ox - LDL induced by FRP recombinant protein FRP was studied by flow cytometry after PI - annexin - V double staining .

6 . The downstream anti - apoptotic proteins of FRP were detected by rt - PCR Western Blot .

III . Results

1 . Induction of apoptosis of vascular endothelial cells after the formation of atherosclerotic plaques in E - deficient mice was demonstrated by the administration of E - deficient mice and the deficient - FRP transgenic mice for 12 weeks .
The results showed that the expression of FRP was inversely proportional to the concentration of oxLDL after treatment with oxLDL . The results showed that the expression of FRP was inversely proportional to oxLDL .

2 . Bacterial soluble proteins were purified by Ni - His band and Sephadex Sephacryl S - 100 to obtain the desired protein .

3 . The cells were treated with ox - LDL with concentration of 50 渭g / ml . Compared with the control group , the cells became round , adherent cells decreased . 100ng / ml of FRP recombinant protein could resist ox - LDL - induced apoptosis . MTT assay suggested that the survival ability of endothelial cells decreased with the increase of ox - LDL .
At different concentrations of FRP and ox - LDL , it was found that FRP can resist the apoptosis of endothelial cells induced by ox - LDL . When the concentration of FRP is 100 ng / ml , the cell activity can be significantly improved when the concentration of FRP is greater than 80ng / ml . FACS suggests that FRP can significantly reduce the number of annexin V positive cells in early apoptotic cells .

4 . The cells were treated 24 hours : 1 , blank control group + 50 渭g / ml LDL . 2 , + 50 渭g / ml oxLDL + 50 ng / ml FRP . 4 , + 50 渭g / ml oxLDL + 75 ng / ml FRP . 5 , + 50 渭g / ml oxLDL + 100 ng / ml FRP .

5 . The expression of Bcl - 2 protein was inhibited by the transfection of Bcl - 2 siRNA , and the expression of Bcl - 2 protein was reduced by about 50 % ( Scramble RNA group was the control group ) . Compared with the control group , the expression of Bcl - 2 was not significantly increased compared with the control group . In the Scramble RNA group , the protective effect of FRP on the apoptosis of HUVEC induced by ox - LDL was observed .

IV . Conclusions

In vivo and in vitro experiments , it was confirmed that ox - LDL could induce apoptosis of endothelial cells by inhibiting the expression of FRP in endothelial cells .
At the same time , it was found that the expression of Bcl 2 was up - regulated by the level of transcription , which inhibited the apoptosis of endothelial cells induced by ox - LDL , and played a role in preventing the apoptosis of endothelial cells and delaying the development of atherosclerosis .

【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

【参考文献】