CREB-1载体的构建及其对HSC中内源性TGF-β3分泌的影响
发布时间：2018-03-15 22:08
本文选题：CREB-1 切入点：干扰载体 出处：《华中科技大学》2012年博士论文 论文类型：学位论文
【摘要】：目的：分别构建及获取有效的CREB-1干扰与过表达质粒载体，为实验提供相关的干预材料及基础。 方法：利用碱基互补配对原理，通过生物信息学软件及CREB-1基因mRNA序列，设计3条理论上能靶向抑制CREB-1表达的茎环状shRNA，分别命名为sh-CREB1-1、sh-CREB1-2与sh-CREB1-3。以pGenesil-1.1为载体，含增强绿色荧光蛋白（EGFP），通过基因工程技术合成含有上述shRNA的质粒，另合成针对内参GADPH的sh-GADPH与不针对任何基因的空白质粒sh-BLANK。CREB-1表达载体pRSV-CREB-1由MichaelE.Greenberg教授赠与，常规从纸片中提取及扩增，并行DNA测序以初步验证其序列。将以上6组质粒分别用脂质体法转染至HSC-T6，转染后12h、24h、36h、48h、60h、72h在荧光显微镜下观察GFP初步判断转染效果及效率，然后分别提取细胞总蛋白，WesternBlot检测CREB-1及内参GAPDH蛋白的表达变化。 结果：pRSV-CREB-1表达载体经DNA测序后与GenBank中CREB-1碱基序列相比对，其编码CREB-1蛋白氨基酸的碱基一致率达93.4%，并且CREB-1磷酸化关键位点（S133）的氨基酸区域完全一致。含有EGFP的质粒组细胞从转染后24小时到72小时均能观察到绿色荧光，转染后48h荧光最强，其转染效率约为15%~20%。转染后48h，WesternBlot结果显示：与转染sh-BLANK的空白组相比，sh-CREB1-1组、sh-CREB1-2组、sh-CREB1-3组、pRSV-CREB-1组HSC中CREB-1蛋白表达分别为0.61±0.17（P0.05）、0.55±0.07（P0.01）、0.94±0.27（P0.05）、1.60±0.11（P0.05）；同时，与空白组相比，sh-GAPDH组的内参GAPDH表达明显下降0.20±0.08（P0.01）。 结论：成功构建及获取CREB-1干扰及过表达质粒载体sh-CREB1与pRSV-CREB-1。3条干扰载体中sh-CREB1-2的抑制效果最佳，可作为后续实验的首选干扰载体。sh-GAPDH组的结果进一步证实本转染体系有效可行。 背景与目的：在大鼠肠上皮细胞中，存在CREB-1与TGF-β3反馈分泌环，即两者可相互促进对方的表达。前期研究显示外源性TGF-β3能够诱导HSC中CREB-1蛋白的表达及促进该蛋白的磷酸化，而HSC中CREB-1是否也存在促进TGF-β3的通路目前尚不明确，本部分实验为探讨CREB-1及其磷酸化状态对HSC中内源性TGF-β3分泌的影响。 方法：利用上一部分工作所建立的转染体系及筛选的CREB-1干扰载体sh-CREB1-2、表达质粒pRSV-CREB1及空白质粒转染HSC，转染后46小时换不含血清的培养基液并加（+）或不加（-）入外源性TGF-β3处理，2小时后留取细胞外液及提取细胞胞浆和胞核蛋白，WesternBlot法检测HSC胞浆蛋白中CREB-1及胞核蛋白中磷酸化CREB-1（p-CREB-1）蛋白的表达，ELISA法检测细胞外液中内源性TGF-β3的分泌。另外，采用抑制CREB-1磷酸化试剂H89与促进CREB-1磷酸化试剂Forskolin（毛喉素）分别处理HSC细胞，检测处理后细胞核蛋白中p-CREB-1表达及细胞外液中TGF-β3的分泌。 结果：与空白转染BLANK（-）组相比，sh-CREB1-2（-）组胞浆蛋白中的CREB-1及胞核蛋白中的p-CREB-1表达降低，分别为0.63±0.11（P0.05）与0.93±0.10（P0.05），细胞外TGF-β3分泌减少为0.81±0.18（P0.05）；pRSV-C（-）组中CREB-1及p-CREB-1蛋白表达上升为1.66±0.14（P0.05）与1.23±0.14（P0.05），细胞外TGF-β3分泌为1.13±0.07（P0.05）。与BLANK（+）组相比，sh-CREB1-2（+）组胞浆蛋白中的CREB-1及胞核蛋白中的p-CREB-1表达亦降低，分别为0.45±0.08（P0.05）与0.32±0.08（P0.05），细胞外TGF-β3分泌减少为0.35±0.05（P0.05）；pRSV-C（+）组胞浆蛋白中的CREB-1及胞核蛋白中的p-CREB-1表达升高，分别为2.19±0.08（P0.05）与2.75±0.14（P0.05），细胞外TGF-β3分泌增多为2.56±0.19（P0.05）。与空白对照（Control）组相比，H89组p-CREB-1蛋白表达降低为0.53±0.07（P0.05），细胞外TGF-β3分泌减少为0.44±0.05（P0.05）；Forskolin组p-CREB-1蛋白表达升高为2.13±0.09（P0.05），细胞外TGF-β3分泌增多达3.76±0.11（P0.05）。 结论：增加HSC中CREB-1蛋白的表达及其磷酸化，可以增加HSC中内源性TGF-β3的分泌；反之，减少HSC中CREB-1蛋白的表达及其磷酸化，则降低HSC中内源性TGF-β3的分泌。提示p-CREB-1是介导HSC内源性TGF-β3分泌的关键因子，为完善TGF-β3抑制肝纤维化机制提供了新的实验依据。
[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.
缁撴灉锛氫笌绌虹櫧杞煋BLANK(-)缁勭浉姣，
本文编号：1617028
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