本文選題：CREB-1　切入點：干擾載體　出處：《華中科技大學(xué)》2012年博士論文　論文類型：學(xué)位論文

【摘要】：目的：分別構(gòu)建及獲取有效的CREB-1干擾與過表達(dá)質(zhì)粒載體，為實驗提供相關(guān)的干預(yù)材料及基礎(chǔ)。 方法：利用堿基互補配對原理，通過生物信息學(xué)軟件及CREB-1基因mRNA序列，設(shè)計3條理論上能靶向抑制CREB-1表達(dá)的莖環(huán)狀shRNA，分別命名為sh-CREB1-1、sh-CREB1-2與sh-CREB1-3。以pGenesil-1.1為載體，含增強綠色熒光蛋白（EGFP），通過基因工程技術(shù)合成含有上述shRNA的質(zhì)粒，另合成針對內(nèi)參GADPH的sh-GADPH與不針對任何基因的空白質(zhì)粒sh-BLANK。CREB-1表達(dá)載體pRSV-CREB-1由MichaelE.Greenberg教授贈與，常規(guī)從紙片中提取及擴增，并行DNA測序以初步驗證其序列。將以上6組質(zhì)粒分別用脂質(zhì)體法轉(zhuǎn)染至HSC-T6，轉(zhuǎn)染后12h、24h、36h、48h、60h、72h在熒光顯微鏡下觀察GFP初步判斷轉(zhuǎn)染效果及效率，然后分別提取細(xì)胞總蛋白，WesternBlot檢測CREB-1及內(nèi)參GAPDH蛋白的表達(dá)變化。 結(jié)果：pRSV-CREB-1表達(dá)載體經(jīng)DNA測序后與GenBank中CREB-1堿基序列相比對，其編碼CREB-1蛋白氨基酸的堿基一致率達(dá)93.4%，并且CREB-1磷酸化關(guān)鍵位點（S133）的氨基酸區(qū)域完全一致。含有EGFP的質(zhì)粒組細(xì)胞從轉(zhuǎn)染后24小時到72小時均能觀察到綠色熒光，轉(zhuǎn)染后48h熒光最強，其轉(zhuǎn)染效率約為15%~20%。轉(zhuǎn)染后48h，WesternBlot結(jié)果顯示：與轉(zhuǎn)染sh-BLANK的空白組相比，sh-CREB1-1組、sh-CREB1-2組、sh-CREB1-3組、pRSV-CREB-1組HSC中CREB-1蛋白表達(dá)分別為0.61±0.17（P0.05）、0.55±0.07（P0.01）、0.94±0.27（P0.05）、1.60±0.11（P0.05）；同時，與空白組相比，sh-GAPDH組的內(nèi)參GAPDH表達(dá)明顯下降0.20±0.08（P0.01）。 結(jié)論：成功構(gòu)建及獲取CREB-1干擾及過表達(dá)質(zhì)粒載體sh-CREB1與pRSV-CREB-1。3條干擾載體中sh-CREB1-2的抑制效果最佳，可作為后續(xù)實驗的首選干擾載體。sh-GAPDH組的結(jié)果進(jìn)一步證實本轉(zhuǎn)染體系有效可行。 背景與目的：在大鼠腸上皮細(xì)胞中，存在CREB-1與TGF-β3反饋分泌環(huán)，即兩者可相互促進(jìn)對方的表達(dá)。前期研究顯示外源性TGF-β3能夠誘導(dǎo)HSC中CREB-1蛋白的表達(dá)及促進(jìn)該蛋白的磷酸化，而HSC中CREB-1是否也存在促進(jìn)TGF-β3的通路目前尚不明確，本部分實驗為探討CREB-1及其磷酸化狀態(tài)對HSC中內(nèi)源性TGF-β3分泌的影響。 方法：利用上一部分工作所建立的轉(zhuǎn)染體系及篩選的CREB-1干擾載體sh-CREB1-2、表達(dá)質(zhì)粒pRSV-CREB1及空白質(zhì)粒轉(zhuǎn)染HSC，轉(zhuǎn)染后46小時換不含血清的培養(yǎng)基液并加（+）或不加（-）入外源性TGF-β3處理，2小時后留取細(xì)胞外液及提取細(xì)胞胞漿和胞核蛋白，WesternBlot法檢測HSC胞漿蛋白中CREB-1及胞核蛋白中磷酸化CREB-1（p-CREB-1）蛋白的表達(dá)，ELISA法檢測細(xì)胞外液中內(nèi)源性TGF-β3的分泌。另外，采用抑制CREB-1磷酸化試劑H89與促進(jìn)CREB-1磷酸化試劑Forskolin（毛喉素）分別處理HSC細(xì)胞，檢測處理后細(xì)胞核蛋白中p-CREB-1表達(dá)及細(xì)胞外液中TGF-β3的分泌。 結(jié)果：與空白轉(zhuǎn)染BLANK（-）組相比，sh-CREB1-2（-）組胞漿蛋白中的CREB-1及胞核蛋白中的p-CREB-1表達(dá)降低，分別為0.63±0.11（P0.05）與0.93±0.10（P0.05），細(xì)胞外TGF-β3分泌減少為0.81±0.18（P0.05）；pRSV-C（-）組中CREB-1及p-CREB-1蛋白表達(dá)上升為1.66±0.14（P0.05）與1.23±0.14（P0.05），細(xì)胞外TGF-β3分泌為1.13±0.07（P0.05）。與BLANK（+）組相比，sh-CREB1-2（+）組胞漿蛋白中的CREB-1及胞核蛋白中的p-CREB-1表達(dá)亦降低，分別為0.45±0.08（P0.05）與0.32±0.08（P0.05），細(xì)胞外TGF-β3分泌減少為0.35±0.05（P0.05）；pRSV-C（+）組胞漿蛋白中的CREB-1及胞核蛋白中的p-CREB-1表達(dá)升高，分別為2.19±0.08（P0.05）與2.75±0.14（P0.05），細(xì)胞外TGF-β3分泌增多為2.56±0.19（P0.05）。與空白對照（Control）組相比，H89組p-CREB-1蛋白表達(dá)降低為0.53±0.07（P0.05），細(xì)胞外TGF-β3分泌減少為0.44±0.05（P0.05）；Forskolin組p-CREB-1蛋白表達(dá)升高為2.13±0.09（P0.05），細(xì)胞外TGF-β3分泌增多達(dá)3.76±0.11（P0.05）。 結(jié)論：增加HSC中CREB-1蛋白的表達(dá)及其磷酸化，可以增加HSC中內(nèi)源性TGF-β3的分泌；反之，減少HSC中CREB-1蛋白的表達(dá)及其磷酸化，則降低HSC中內(nèi)源性TGF-β3的分泌。提示p-CREB-1是介導(dǎo)HSC內(nèi)源性TGF-β3分泌的關(guān)鍵因子，為完善TGF-β3抑制肝纖維化機制提供了新的實驗依據(jù)。
[Abstract]:Objective: to construct and obtain effective plasmid vectors for CREB-1 interference and overexpression, and to provide relevant intervention materials and basis for the experiment.
Methods: using the principle of complementary base pairing, through bioinformatics software and CREB-1 gene mRNA sequence, 3 design theory can stem loop targeting shRNA inhibits the expression of CREB-1, which were named sh-CREB1-1, sh-CREB1-2 and sh-CREB1-3. in the pGenesil-1.1 vector containing enhanced green fluorescent protein (EGFP), synthetic plasmid by genetic engineering technology with the shRNA, the synthesis of sh-GADPH for internal control GADPH and not for any blank plasmid sh-BLANK.CREB-1 gene expression vector pRSV-CREB-1 given by Professor MichaelE.Greenberg, from the conventional disk extraction and amplification, parallel DNA sequencing to verify its preliminarysequence. The above 6 groups respectively with the plasmid was transfected into HSC-T6 and 12h after transfection. 24h, 36h, 48h, 60H, 72h, GFP to determine the initial transfection efficiency and efficiency was observed under the fluorescence microscope, and then extract the cell total protein respectively, WesternBlot detection C Expression of REB-1 and GAPDH reference protein.
Results: the pRSV-CREB-1 expression vector by DNA sequencing and CREB-1 sequence in GenBank compared to CREB-1, its encoding protein amino acid base consistent rate was 93.4%, and the CREB-1 phosphorylation sites (S133) key amino acid regions are exactly the same. Plasmid group of cells containing EGFP from 24 hours to 72 hours after transfection were observed in green after transfection, 48h fluorescence, fluorescence intensity, the transfection efficiency is about 15%~20%. 48h after transfection, WesternBlot results showed that: compared with the blank group, transfection of sh-BLANK sh-CREB1-1 group, sh-CREB1-2 group, sh-CREB1-3 group, pRSV-CREB-1 group, HSC CREB-1 expression were 0.61 + 0.17, 0.55 + 0.07 (P0.05) (P0.01, 0.94). 0.27 (P0.05), 1.60 + 0.11 (P0.05); at the same time, compared with the control group, sh-GAPDH group, control GAPDH expression was significantly decreased in 0.20 + 0.08 (P0.01).
Conclusion: successful construction and acquisition of CREB-1 interference and over expression plasmid vector sh-CREB1 and pRSV-CREB-1.3 interference vector sh-CREB1-2 inhibit the best effect, which can serve as the preferred carrier of interference in subsequent experiments. The results of.Sh-GAPDH group further confirm that the transfection system is effective and feasible.
Background and purpose: in the intestinal epithelial cells in rats, CREB-1 and TGF- beta 3 secretion feedback loop, which both can promote each other each other. Previous studies have shown that expression of exogenous TGF- beta 3 expression can induce CREB-1 protein in HSC and promote the protein phosphorylation of HSC in CREB-1, and whether there is a pathway to promote TGF- beta 3 is still not clear, this part of the experiment is to explore the effects of CREB-1 and its phosphorylation on HSC in endogenous TGF- beta 3 secretion.
Methods: using CREB-1 interference plasmid sh-CREB1-2 transfection and screening system established on the part of the work, pRSV-CREB1 expression plasmid and blank plasmid HSC, 46 hours after transfection for liquid culture without serum plus (+) or without (-) into the exogenous TGF- beta 3 treatment, 2 hours after leaving the extraction of extracellular fluid and cell nucleus and cytoplasm protein, phosphorylation of CREB-1 CREB-1 and nuclear protein detection in HSC cytoplasmic protein in WesternBlot (p-CREB-1) protein expression and extracellular ELISA assay solution of endogenous TGF- beta 3 secretion. In addition, the inhibition of CREB-1 phosphorylation reagent H89 and promoting CREB-1 phosphorylation reagent Forskolin (Mao Housu) were treated with HSC cells, TGF- p-CREB-1 expression detection of nuclear protein after treatment and extracellular fluid in beta 3 secretion.
緇撴灉錛氫笌絀虹櫧杞煋BLANK(-)緇勭浉姣，

本文編號：1617028