本文選題：卵泡抑素相關蛋白 + 血管內皮細胞��； 參考：《延邊大學》2010年碩士論文

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

【學位授予單位】：延邊大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R363

【參考文獻】

相關期刊論文 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：1797411