本文選題：卵泡抑素相關(guān)蛋白 + 血管內(nèi)皮細(xì)胞�。� 參考：《延邊大學(xué)》2010年碩士論文

【摘要】： 一、研究背景與目的 血管內(nèi)皮細(xì)胞(vascular endothelial cell, VEC)是循環(huán)血液與血管平滑肌間的機(jī)械屏障。VEC的功能相當(dāng)廣泛,除起轉(zhuǎn)運(yùn)屏障作用外,還具有分泌功能,可以合成分泌多種物質(zhì),對(duì)調(diào)節(jié)血管緊張度、血液流通、細(xì)胞生成、炎癥反應(yīng)、免疫等功能具有重要作用。多種因素可通過不同機(jī)制與途徑損傷血管內(nèi)皮細(xì)胞,使內(nèi)皮屏障作用減弱,血中脂蛋白過多地進(jìn)入動(dòng)脈壁沉積下來,被清道夫細(xì)胞吞噬形成泡沫細(xì)胞；內(nèi)皮細(xì)胞受損后,炎細(xì)胞、血小板的粘附與內(nèi)容物的釋放又加重了內(nèi)皮細(xì)胞損傷,從而逐漸形成粥樣硬化斑塊,因此尋找一種應(yīng)用方便、專一性強(qiáng)、來源豐富、效果良好的VEC保護(hù)劑也越來越引起人們的重視。 本課題以血管內(nèi)皮細(xì)胞為靶細(xì)胞,以卵泡抑素相關(guān)蛋白(Follistatin Related Protein, FRP)為主要研究對(duì)象,以Western Blot、Si-RNA、Rt-PCR、ChIP assay等實(shí)驗(yàn)技術(shù)為研究手段,重點(diǎn)探討FRP對(duì)VEC的保護(hù)作用,力求闡明該蛋白對(duì)VEC的抗凋亡機(jī)制及其在動(dòng)脈粥樣硬化中的病理意義。 二、材料與方法 1、根據(jù)驗(yàn)證樣性的基因編碼序列獲得FRP編碼區(qū)cDNA連接于表達(dá)載體pET32a,構(gòu)建表達(dá)重組子,轉(zhuǎn)化入Origami (DE3), 1mM IPTG誘導(dǎo)蛋白表達(dá),使用Ni-NTA His Band Resins, Sephacryl S-100純化蛋白,腸激酶酶切融合蛋白并鑒定。 2、分離臍帶靜脈血管內(nèi)皮細(xì)胞,并用含有胎牛血清的1640完全培養(yǎng)基進(jìn)行細(xì)胞培養(yǎng),每2-3天換液一次,定期進(jìn)行消化傳代。 3、將C段帶有FLAG標(biāo)簽的FRP基因片段連接到pcDNA 4／TO上,先后將pcDNA 6／TR和pcDNA4/T0/FRP-FLAG轉(zhuǎn)染至內(nèi)皮細(xì)胞系EAHY926中,用blasticidin和zeocin篩選穩(wěn)定轉(zhuǎn)染的單克隆細(xì)胞。 4、制備ApoE基因敲除小鼠為背景的FRP轉(zhuǎn)基因動(dòng)物,ApoE基因缺陷小鼠與ApoE基因缺陷FRP轉(zhuǎn)基因小鼠,構(gòu)建動(dòng)脈粥樣硬化動(dòng)物模型。 5、通過PI-Annexin-V雙染后流式細(xì)胞儀分析,研究FRP重組蛋白FRP抗ox-LDL誘導(dǎo)的細(xì)胞凋亡維持HUVEC的細(xì)胞活性。 6、通過rt-PCR WesternBlot等方法檢測(cè)FRP下游抗凋亡蛋白。 三、結(jié)果 1.給予ApoE缺失小鼠及ApoE基因缺陷-FRP轉(zhuǎn)基因小鼠喂養(yǎng)12周。TUNEL提示ApoE缺失小鼠動(dòng)脈粥樣硬化斑塊形成后血管內(nèi)皮細(xì)胞凋亡增加；ApoE基因缺陷-FRP轉(zhuǎn)基因小鼠TUNEL染色顯示凋亡的內(nèi)皮細(xì)胞顯著少于ApoE組。用oxLDL的終濃度依次為0ug／ml、10ug／ml、20ug／ml、30ug／ml、50ug／ml(0ug／ml oxLDL作為對(duì)照組)處理HUVECs直接影響FRP的表達(dá),結(jié)果發(fā)現(xiàn)處理后HUVECs的FRP表達(dá)量與oxLDL的濃度呈反比。 2.細(xì)菌可溶性的蛋白經(jīng)Ni-His band及Sephadex Sephacryl S-100純化酶切蛋白后,得到目的蛋白。 3.用濃度為50μg／ml的ox-LDL處理HUVECs,與對(duì)照組相比細(xì)胞變圓,貼壁細(xì)胞減少。100ng／ml的FRP重組蛋白可以抗ox-LDL誘導(dǎo)的細(xì)胞凋亡。MTT檢測(cè)提示,隨著ox-LDL濃度的增加,內(nèi)皮細(xì)胞的存活能力逐漸降低；不同濃度的FRP和ox-LDL對(duì)內(nèi)皮細(xì)胞進(jìn)行處理時(shí),發(fā)現(xiàn)FRP可以抵抗ox-LDL誘導(dǎo)的內(nèi)皮細(xì)胞凋亡,當(dāng)FRP濃度為100ng／ml時(shí)有顯著性差異。FRP濃度大于80ng／ml時(shí)可顯著提高細(xì)胞活性。FACS提示FRP可顯著降低凋亡早期annexin V陽性細(xì)胞數(shù)。 4.按如下方法處理細(xì)胞24小時(shí)：1、空白對(duì)照組+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。實(shí)驗(yàn)發(fā)現(xiàn),FRP可以上調(diào)磷酸化Akt的表達(dá),從而激活磷酸化Akt的下游基因——磷酸化Bad的表達(dá)。然而,Akt的表達(dá)量并未隨著FRP的表達(dá)量而改變, Bcl2的表達(dá)量隨著FRP濃度的增高而顯著性增加。 5.利用轉(zhuǎn)染Bcl2 siRNA的方法抑制Bcl2的蛋白表達(dá),可使Bcl2蛋白表達(dá)量降低約50%(Scramble RNA組為對(duì)照組)。用FRP誘導(dǎo)Bcl2表達(dá)發(fā)現(xiàn)Bcl2 siRNA組與對(duì)照組相比Bcl2的表達(dá)量無明顯增加。在Scramble RNA組中可以觀察到FRP對(duì)ox-LDL誘導(dǎo)的HUVEC凋亡仍有保護(hù)作用,而在Bcl2 siRNA組,FRP的保護(hù)作用喪失。 四、結(jié)論 本次課題體內(nèi)和體外實(shí)驗(yàn)都證實(shí)了ox-LDL通過抑制內(nèi)皮細(xì)胞中FRP的蛋白表達(dá)從而誘導(dǎo)內(nèi)皮細(xì)胞凋亡；同時(shí)發(fā)現(xiàn),FRP是通過轉(zhuǎn)錄水平上調(diào)Bcl2的蛋白表達(dá)從而抑制ox-LDL誘導(dǎo)內(nèi)皮細(xì)胞凋亡,發(fā)揮其抗內(nèi)皮細(xì)胞凋亡、延緩動(dòng)脈粥樣硬化發(fā)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 .

【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Molecular signal transduction in vascular cell apoptosis[J];Cell Research;2001年04期

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本文編號(hào)：1797411