本文選題：胰島素樣生長(zhǎng)因子結(jié)合蛋白相關(guān)蛋白1 + 肝纖維化��； 參考：《山西醫(yī)科大學(xué)》2014年博士論文

【摘要】：胰島素樣生長(zhǎng)因子結(jié)合蛋白相關(guān)蛋白1（insulin-like growth factor bindingprotein related protein1，IGFBPrP1），又稱(chēng)為IGFBP7，是一種分泌蛋白，屬于胰島素樣生長(zhǎng)因子結(jié)合蛋白（insulin-like growth factor binding proteins，IGFBPs）超家族成員，與胰島素樣生長(zhǎng)因子(insulin-like growth factor，IGF)親和力低，而與胰島素呈高結(jié)合力，具有調(diào)節(jié)細(xì)胞增殖、分化、黏附、衰老、凋亡及血管形成等生物學(xué)活性。 導(dǎo)師前期研究發(fā)現(xiàn)IGFBPrP1是肝纖維化新的致病因子，但其致肝纖維化作用的機(jī)制尚不完全清楚。 目前已明確的致肝纖維化的信號(hào)轉(zhuǎn)導(dǎo)通路有TGFβ通路、Jak-Stat通路、Rho-ROCK通路、NF-κB通路、Wnt通路、Hedgehog（Hh）通路、瘦素信號(hào)通路、PPAR介導(dǎo)的信號(hào)通路、血管緊張素Ⅱ受體介導(dǎo)的信號(hào)通路等。TGFβ通路包括TGFβ/Smad信號(hào)通路和非Smad信號(hào)通路。TGFβ/Smad信號(hào)通路是經(jīng)典的肝纖維化信號(hào)轉(zhuǎn)導(dǎo)通路；非Smad信號(hào)通路包括MAPK通路和PI3K/AKT通路等。 導(dǎo)師在前期研究中對(duì)TGFβ相關(guān)通路進(jìn)行了探索，發(fā)現(xiàn)IGFBPrP1可通過(guò)TGFβ/Smad信號(hào)通路發(fā)揮致肝纖維化作用，且可能與非Smad信號(hào)通路中的ERK/MAPK信號(hào)通路有關(guān)。但I(xiàn)GFBPrP1是否能通過(guò)其他信號(hào)通路發(fā)揮致肝纖維化作用尚不清楚。研究表明，眾多信號(hào)通路在肝纖維化發(fā)生發(fā)展的不同環(huán)節(jié)發(fā)揮著重要作用，，不僅TGFβ/Smad信號(hào)通路與非Smad信號(hào)通路之間，而且TGFβ通路與其他信號(hào)通路之間都存在著交互作用（crosstalk）。因此推測(cè)，IGFBPrP1也可能通過(guò)MAPK、PI3K/AKT等非Smad通路及其他信號(hào)通路發(fā)揮致肝纖維化作用。 PCR芯片是熒光定量PCR與芯片技術(shù)的結(jié)合，僅關(guān)注那些與研究對(duì)象相關(guān)的某一信號(hào)通路或多個(gè)相關(guān)基因的表達(dá)，所研究的基因范圍相對(duì)集中，通常只有幾百個(gè)或更少的基因。與基因芯片相比，獲得的信息具有更強(qiáng)的針對(duì)性和準(zhǔn)確性。 因此本實(shí)驗(yàn)擬通過(guò)RT2ProfilerTMPCR芯片篩選IGFBPrP1致肝纖維化作用的信號(hào)轉(zhuǎn)導(dǎo)通路及差異表達(dá)基因，并對(duì)主要差異表達(dá)基因進(jìn)行驗(yàn)證，以闡明IGFBPrP1致肝纖維化作用的部分機(jī)制，為抗纖維化治療提供新思路。 第一部分腺病毒介導(dǎo)IGFBPrP1基因轉(zhuǎn)染大鼠肝組織 目的：觀察腺病毒載體能否通過(guò)尾靜脈注射將IGFBPrP1基因成功轉(zhuǎn)染大鼠肝組織及IGFBPrP1在肝組織的表達(dá)。 方法：SD雄性大鼠98只，體重120-140g，隨機(jī)分為3組：腺病毒基因組（Ad-IGFBPrP1，n=43）：通過(guò)大鼠尾靜脈注射Ad-IGFBPrP14×109pfu/只；腺病毒空載組（Ad-EGFP，n=43）：通過(guò)大鼠尾靜脈注射Ad-EGFP4×109pfu/只；正常對(duì)照組（N，n=12）：通過(guò)大鼠尾靜脈注射同等劑量的生理鹽水。各組分別于注射腺病毒后2/7w（n=3）、1w（n=8）、2w（n=8）、4w（n=8）、6w（n=8）、9w（n=8）末處死大鼠，留取血清和肝組織待測(cè)。熒光顯微鏡觀察肝組織中增強(qiáng)型綠色熒光蛋白（EGFP）的表達(dá)；流式細(xì)胞儀檢測(cè)肝組織EGFP陽(yáng)性細(xì)胞的百分比；實(shí)時(shí)熒光定量PCR（qRT-PCR）和Western blot方法分別測(cè)定肝組織IGFBPrP1的mRNA和蛋白表達(dá)水平。 結(jié)果：1.熒光顯微鏡觀察和流式細(xì)胞儀檢測(cè)結(jié)果均顯示，一次性給予Ad-EGFP4×109pfu和重復(fù)給予（0w，2w）Ad-EGFP2×109pfu兩種方式轉(zhuǎn)染，大鼠肝組織均有EGFP表達(dá)，但一次性給予Ad-EGFP4×109pfu的方式轉(zhuǎn)染效率更高（60.4%vs46.3%）。故后續(xù)實(shí)驗(yàn)中采用一次性給予Ad-EGFP4×109pfu進(jìn)行轉(zhuǎn)染。 2.熒光顯微鏡下觀察，腺病毒轉(zhuǎn)染1w時(shí)，肝組織可見(jiàn)明亮的綠色熒光，EGFP表達(dá)達(dá)到高峰，轉(zhuǎn)染2w后綠色熒光逐漸減弱，4w后基本消失。 3.流式細(xì)胞儀檢測(cè)結(jié)果顯示，腺病毒轉(zhuǎn)染1w時(shí)，肝組織EGFP陽(yáng)性細(xì)胞最多，達(dá)到60.4%，腺病毒轉(zhuǎn)染效率達(dá)高峰；2w后EGFP陽(yáng)性細(xì)胞逐漸減少。 4. qRT-PCR檢測(cè)結(jié)果顯示，腺病毒轉(zhuǎn)染1w時(shí)，肝組織IGFBPrP1基因mRNA表達(dá)水平最高（5.57±1.52）。 5. Western blot檢測(cè)結(jié)果顯示，腺病毒轉(zhuǎn)染2w時(shí)，肝組織IGFBPrP1蛋白表達(dá)達(dá)高峰（1.07±0.11），隨后逐漸減低。結(jié)論：腺病毒載體通過(guò)尾靜脈注射成功將IGFBPrP1基因?qū)氪笫蟾谓M織。 第二部分腺病毒介導(dǎo)的IGFBPrP1基因轉(zhuǎn)染致大鼠肝纖維化 目的：觀察腺病毒介導(dǎo)的IGFBPrP1是否導(dǎo)致大鼠發(fā)生肝纖維化。 方法：實(shí)驗(yàn)分組同第一部分。HE和Sirius red染色觀察肝組織病理學(xué)改變和膠原纖維分布；Western blot方法檢測(cè)HSC活化標(biāo)志物α-SMA蛋白的表達(dá)；羥脯氨酸（Hyp）含量測(cè)定觀察肝組織膠原纖維的形成；全自動(dòng)生化儀檢測(cè)肝功能觀察肝組織損傷程度。 結(jié)果：1. HE和Sirius red染色結(jié)果顯示，IGFBPrP1基因轉(zhuǎn)染大鼠肝組織后，隨著病變進(jìn)展，肝細(xì)胞出現(xiàn)水樣變性和脂肪變性，點(diǎn)狀壞死，甚至灶狀壞死，伴有炎細(xì)胞浸潤(rùn)，膽管增生。轉(zhuǎn)染4w時(shí)匯管區(qū)出現(xiàn)增生的纖維組織，膠原纖維逐漸增多，由血管壁向肝小葉內(nèi)延伸，在匯管區(qū)與匯管區(qū)之間以及中央靜脈與匯管區(qū)之間相互連接，病變可達(dá)到纖維化S3期。 2. Western blot檢測(cè)結(jié)果顯示，隨著纖維化病變進(jìn)展，Ad-IGFBPrP1組肝組織α-SMA蛋白表達(dá)逐漸增強(qiáng)。轉(zhuǎn)染9w時(shí)，α-SMA蛋白表達(dá)量增至Ad-EGFP組的20倍。 3.肝組織Hyp含量檢測(cè)結(jié)果顯示，Ad-IGFBPrP1組肝組織Hyp含量隨纖維化進(jìn)展逐漸升高。 4.血清學(xué)檢測(cè)結(jié)果顯示，Ad-IGFBPrP1組大鼠血清ALT和TBIL水平顯著升高，與正常對(duì)照組和Ad-EGFP組相比差異有統(tǒng)計(jì)學(xué)意義（P＜0.05）。各組大鼠血清TP和ALB水平在正常范圍內(nèi)，組間差異無(wú)統(tǒng)計(jì)學(xué)意義（P＞0.05）。 結(jié)論：腺病毒介導(dǎo)的IGFBPrP1基因轉(zhuǎn)染導(dǎo)致大鼠發(fā)生肝纖維化。 第三部分PCRArray篩選參與IGFBPrP1致肝纖維化作用的信號(hào)轉(zhuǎn)導(dǎo)通路的差異表達(dá)基因 目的：通過(guò)PCRArray篩選出IGFBPrP1致肝纖維化的信號(hào)轉(zhuǎn)導(dǎo)通路的差異表達(dá)基因。 方法：SD大鼠隨機(jī)分為3組（n=3）：Ad-IGFBPrP1組、Ad-EGFP組和正常對(duì)照組（N）。各組腺病毒處理同第一部分。各組分別于腺病毒轉(zhuǎn)染2w末處死大鼠，留取肝組織待測(cè)。提取肝組織mRNA，逆轉(zhuǎn)錄為cDNA，然后qRT-PCR方法檢測(cè)信號(hào)轉(zhuǎn)導(dǎo)通路的差異表達(dá)基因。 結(jié)果：檢測(cè)Signal Transduction PathwayFinder PCR Array上84個(gè)相關(guān)基因的mRNA表達(dá)水平變化。結(jié)果顯示，18條信號(hào)轉(zhuǎn)導(dǎo)通路中有33個(gè)基因mRNA表達(dá)有差異，占檢測(cè)基因的39.29%。其中17個(gè)基因mRNA表達(dá)上調(diào)，16個(gè)基因mRNA表達(dá)下調(diào)。這些差異表達(dá)基因涉及16條信號(hào)轉(zhuǎn)導(dǎo)通路。其中核轉(zhuǎn)錄因子Egr1表達(dá)上調(diào)，Hedgehog通路的Hhip基因和PI3K/AKT通路的PTEN基因表達(dá)均下調(diào)。 結(jié)論：IGFBPrP1可能通過(guò)多條信號(hào)轉(zhuǎn)導(dǎo)通路促進(jìn)肝纖維化發(fā)生發(fā)展，差異表達(dá)基因Egr1、Hhip和PTEN可能是IGFBPrP1致肝纖維化作用信號(hào)轉(zhuǎn)導(dǎo)的關(guān)鍵基因。 第四部分在IGFBPrP1誘導(dǎo)的肝纖維化大鼠肝組織中MAPK信號(hào)通路的差異表達(dá)基因 目的：通過(guò)PCR Array檢測(cè)在IGFBPrP1誘導(dǎo)的肝纖維化大鼠肝組織中MAPK信號(hào)通路的差異表達(dá)基因。 方法：實(shí)驗(yàn)分組同第二部分。提取肝組織mRNA，逆轉(zhuǎn)錄為cDNA，然后qRT-PCR方法檢測(cè)IGFBPrP1誘導(dǎo)的肝纖維化大鼠肝組織中MAPK信號(hào)通路的差異表達(dá)基因。 結(jié)果：MAPK信號(hào)通路中有24個(gè)基因mRNA表達(dá)有差異，占檢測(cè)基因的28.57%。其中19個(gè)基因mRNA表達(dá)上調(diào)，5個(gè)基因mRNA表達(dá)下調(diào)。這些基因按功能可分為轉(zhuǎn)錄因子基因、Raf調(diào)控蛋白基因、細(xì)胞周期蛋白基因等。其中Map2k2（MEK2）和Mapk3（ERK1）表達(dá)均上調(diào)。 結(jié)論：IGFBPrP1可能通過(guò)激活MAPK信號(hào)通路的不同環(huán)節(jié)發(fā)揮致肝纖維化作用，通路中差異表達(dá)基因Map2k2（MEK2）和Mapk3（ERK1）可能是IGFBPrP1致肝纖維化作用信號(hào)轉(zhuǎn)導(dǎo)的關(guān)鍵基因。 第五部分在IGFBPrP1誘導(dǎo)的肝纖維化大鼠肝組織中部分差異表達(dá)基因的驗(yàn)證 目的：驗(yàn)證部分差異表達(dá)基因在IGFBPrP1誘導(dǎo)的肝纖維化大鼠肝組織的表達(dá)。 方法：實(shí)驗(yàn)分組同第一部分。應(yīng)用qRT-PCR和Western blot方法分別檢測(cè)PCRArray篩選出的差異表達(dá)基因的mRNA和蛋白在IGFBPrP1誘導(dǎo)的肝纖維化大鼠肝組織的表達(dá)。 結(jié)果：1. qRT-PCR檢測(cè)結(jié)果顯示，①Ad-IGFBPrP1組Map2k2（MEK2）和Mapk3（ERK1）基因的mRNA水平均升高，與正常對(duì)照組和Ad-EGFP組相比，差異有統(tǒng)計(jì)學(xué)意義（P＜0.05）；②Hhip和PTEN基因的mRNA水平在Ad-IGFBPrP1組顯著降低，明顯低于正常對(duì)照組和Ad-EGFP組，差異有統(tǒng)計(jì)學(xué)意義（P＜0.05）； ③與正常對(duì)照組和Ad-EGFP組相比，Ad-IGFBPrP1組Egr1基因的mRNA水平升高，差異有統(tǒng)計(jì)學(xué)意義（P＜0.05）。 2. Western blot檢測(cè)結(jié)果顯示，①PTEN蛋白表達(dá)隨肝纖維化病變進(jìn)展逐漸減弱，各時(shí)相的PTEN蛋白表達(dá)與正常對(duì)照組和Ad-EGFP組相比，均有統(tǒng)計(jì)學(xué)差異（P＜0.05）。②腺病毒轉(zhuǎn)染后，Egr1蛋白表達(dá)增強(qiáng)，轉(zhuǎn)染2w時(shí)達(dá)到高峰，然后逐漸減弱。轉(zhuǎn)染2w的Egr1蛋白水平與其他各組相比，差異有統(tǒng)計(jì)學(xué)意義（P＜0.05）。 結(jié)論：Map2k2（MEK2）、Mapk3（ERK1）、Hhip、PTEN和Egr1等差異表達(dá)基因共同調(diào)控IGFBPrP1發(fā)揮致肝纖維化作用，可能部分闡明了IGFBPrP1致肝纖維化的作用機(jī)制。 第六部分IGFBPrP1和TGFβ1在致肝纖維化中的關(guān)系初探 目的：初步探討IGFBPrP1和TGFβ1在致肝纖維化中的關(guān)系。 方法：實(shí)驗(yàn)分組同第一部分。Western blot方法檢測(cè)IGFBPrP1、α-SMA和TGFβ1蛋白在IGFBPrP1誘導(dǎo)的肝纖維化大鼠肝組織的表達(dá)，并觀察它們?cè)诟卫w維化發(fā)展過(guò)程中的變化趨勢(shì)。 結(jié)果：Western blot檢測(cè)結(jié)果顯示，腺病毒轉(zhuǎn)染后，IGFBPrP1蛋白水平先升高，在轉(zhuǎn)染2w時(shí)達(dá)到高峰，隨后逐漸減低。α-SMA和TGFβ1蛋白表達(dá)均隨著肝纖維化程度的增加而增加，在轉(zhuǎn)染9w時(shí)達(dá)到高峰。肝組織Hyp含量在腺病毒轉(zhuǎn)染4w后隨時(shí)間延長(zhǎng)逐漸升高，在轉(zhuǎn)染9w時(shí)達(dá)到高峰。 結(jié)論： IGFBPrP1刺激肝組織產(chǎn)生TGFβ1可能是IGFBPrP1致肝纖維化作用的主要途徑。
[Abstract]:Insulin like growth factor binding protein related protein 1 (insulin-like growth factor bindingprotein related protein1, IGFBPrP1), also known as IGFBP7, is a secretory protein, belonging to the insulin like growth factor binding protein (insulin-like growth factor binding) superfamily members, and insulin-like growth factors. Lin-like growth factor, IGF) with low affinity and high junction with insulin, has biological activity to regulate cell proliferation, differentiation, adhesion, senescence, apoptosis and angiogenesis.
Previous studies showed that IGFBPrP1 is a new pathogenic factor of liver fibrosis, but the mechanism of its effect on liver fibrosis is not yet clear.
TGF beta pathway, Jak-Stat pathway, Rho-ROCK pathway, NF- kappa B pathway, Wnt pathway, Hedgehog (Hh) pathway, leptin signaling pathway, PPAR mediated signaling pathway, and angiotensin II receptor mediated signaling pathway, including TGF beta /Smad signal pathways and non signaling pathways, have been identified. F beta /Smad signaling pathway is a classic signal transduction pathway in liver fibrosis, and non Smad signaling pathway includes MAPK pathway and PI3K/AKT pathway.
In the previous study, the tutor explored the TGF beta related pathway, and found that IGFBPrP1 could play a role in liver fibrosis through the TGF beta /Smad signaling pathway, and may be related to the ERK/MAPK signaling pathway in the non Smad signaling pathway. However, it is not clear whether IGFBPrP1 can play the role of liver fibrosis through other signaling pathways. The multi signal pathway plays an important role in the development of liver fibrosis, not only between the TGF beta /Smad signaling pathway and the non Smad signaling pathway, but also the interaction between the TGF beta pathway and the other signaling pathways (crosstalk). Therefore, it is speculated that IGFBPrP1 can also pass through the MAPK, PI3K/AKT and other non Smad pathways and other signals. The road plays a role in liver fibrosis.
The PCR chip is a combination of fluorescence quantitative PCR and chip technology. It is concerned only with the expression of a signal pathway or a number of related genes related to the research object. The gene range is relatively concentrated, usually only a few hundred or less genes. Compared with the gene chip, the information obtained is more pertinent and more accurate.
Therefore, this experiment is designed to screen the signal transduction pathway and differentially expressed genes of liver fibrosis induced by IGFBPrP1 by RT2ProfilerTMPCR chip, and to verify the main differentially expressed genes, in order to elucidate the mechanism of IGFBPrP1 induced liver fibrosis and provide new ideas for anti fibrosis treatment.
Part I adenovirus mediated IGFBPrP1 gene transfection into rat liver tissue
Objective: To observe whether adenovirus vector can successfully transfect IGFBPrP1 gene into rat liver tissue and express IGFBPrP1 in liver tissue through tail vein injection.
Methods: 98 male SD rats, weight 120-140g, were randomly divided into 3 groups: Ad-IGFBPrP1 (n=43): Ad-IGFBPrP14 x 109pfu/ injection in rat tail vein; adenovirus free load group (Ad-EGFP, n=43): intravenous Ad-EGFP4 x 109pfu/ by rat tail vein; normal control group (N, n=12): rat tail vein injection same 2/7w (n=3), 1W (n=8), 2W (n=8), 4W (n=8), 6W (n=8), 9W (n=8) were killed in rats after the injection of adenovirus, and the serum and liver tissues were left to be measured. The expression of enhanced green fluorescent protein in liver tissue was observed by fluorescence microscope; the percentage of liver tissue positive cells was detected by flow cytometry; real time Fluorescence quantitative PCR (qRT-PCR) and Western blot methods were used to detect the expression of mRNA and protein in liver tissue IGFBPrP1.
Results: the results of 1. fluorescence microscopy and flow cytometry showed that the transfection of Ad-EGFP4 x 109pfu and repeated giving (0W, 2W) Ad-EGFP2 * 109pfu were all EGFP expression in rat liver tissues, but the transfection efficiency of Ad-EGFP4 * 109pfu was higher (60.4%vs46.3%). The sex was given to Ad-EGFP4 x 109pfu for transfection.
Under 2. fluorescence microscope, when adenovirus transfected to 1W, the liver tissue showed bright green fluorescence, and the expression of EGFP reached the peak. After transfection of 2W, the green fluorescence decreased gradually, and the 4W disappeared after 4W.
The results of 3. flow cytometry showed that when adenovirus transfected to 1W, the EGFP positive cells in liver tissue were most, up to 60.4%, the transfection efficiency of adenovirus reached the peak, and the EGFP positive cells gradually decreased after 2W.
4. qRT-PCR detection showed that the expression level of IGFBPrP1 gene mRNA was highest in liver tissues (5.57 + 1.52) when adenovirus was transfected into 1W.
The results of 5. Western blot showed that the expression of IGFBPrP1 protein in the liver tissues reached the peak (1.07 + 0.11) and then decreased gradually when adenovirus transfected to 2W. Conclusion: the adenovirus vector was successfully injected into the rat liver tissue through the injection of the tail vein.
The second part of adenovirus mediated IGFBPrP1 gene transfection induces liver fibrosis in rats.
Objective: To observe whether adenovirus mediated IGFBPrP1 can induce liver fibrosis in rats.
Methods: the pathological changes of liver tissue and the distribution of collagen fibers were observed in the experimental group with the first part.HE and Sirius red staining, and the expression of the HSC activation marker alpha -SMA protein was detected by Western blot; the formation of collagen fibrils in the liver tissue was observed by the determination of the hydroxyproline (Hyp) content; the liver function was detected by the automatic biochemical analyzer to observe the liver tissue damage. The degree of injury.
Results: the results of 1. HE and Sirius red staining showed that after the IGFBPrP1 gene transfected to the rat liver tissue, with the progression of the lesion, the hepatocyte appeared water like degeneration and fatty degeneration, punctate necrosis, even focal necrosis, accompanied by inflammatory cell infiltration, and bile duct hyperplasia. The proliferation of fibrous tissue in the manifold area was gradually increased when transfected to 4W, and the collagen fibers gradually increased from the vascular wall. It extends to the lobules of liver, and connects between the portal area and the portal area, and between the central vein and the portal area. The lesion can reach the stage of fibrosis S3.
The results of 2. Western blot showed that the expression of alpha -SMA protein in the liver tissue of group Ad-IGFBPrP1 increased gradually with the progression of fibrosis, and the expression of alpha -SMA protein increased to 20 times that of the Ad-EGFP group when 9W transfected.
3. the detection of Hyp content in liver tissue showed that the level of Hyp in liver tissue gradually increased with the progression of fibrosis in group Ad-IGFBPrP1.
4. serological test results showed that the serum level of ALT and TBIL in the Ad-IGFBPrP1 group was significantly higher than that in the normal control group and the Ad-EGFP group (P < 0.05). The serum TP and ALB levels were in the normal range, and there was no significant difference between the groups (P > 0.05).
Conclusion: adenovirus mediated IGFBPrP1 gene transfection can induce liver fibrosis in rats.
The third part is to screen differentially expressed genes involved in signal transduction pathways involved in IGFBPrP1 induced liver fibrosis by PCRArray.
Objective: to screen differentially expressed genes of signal transduction pathways induced by IGFBPrP1 in liver fibrosis by PCRArray.
Methods: SD rats were randomly divided into 3 groups (n=3):Ad-IGFBPrP1 group, Ad-EGFP group and normal control group (N). The adenovirus in each group was treated with the first part. Each group was killed by adenovirus transfection 2W at the end of 2W, and the liver tissue was left to be measured. The liver tissue mRNA was extracted, reverse transcriptase cDNA, and then qRT-PCR method was used to detect the differential expression genes in the signal transduction pathway.
Results: the change of mRNA expression level of 84 related genes on Signal Transduction PathwayFinder PCR Array was detected. The results showed that there were 33 gene mRNA expressions in 18 signal transduction pathways, which accounted for the increase of mRNA expression in the 39.29%. of the detected gene, and the expression of mRNA expression of 16 genes down. These differentially expressed genes involved 16 Signal transduction pathway, in which the expression of nuclear transcription factor Egr1 is up-regulated, and the PTEN gene expression of Hedgehog pathway Hhip and PI3K/AKT pathway is down regulated.
Conclusion: IGFBPrP1 may promote the development of liver fibrosis through multiple signal transduction pathways. The differentially expressed genes, Egr1, Hhip and PTEN, may be the key genes in the signal transduction of liver fibrosis induced by IGFBPrP1.
The fourth part is the differentially expressed genes of MAPK signaling pathway in liver tissue of IGFBPrP1 induced liver fibrosis rats.
Objective: to detect the differentially expressed genes of MAPK signaling pathway in liver tissues of rats with liver fibrosis induced by IGFBPrP1 by PCR Array.
Methods: the experimental group was divided into second parts. The liver tissue mRNA was extracted and reverse transcriptase was cDNA. Then qRT-PCR method was used to detect the differential expression gene of MAPK signaling pathway in liver tissue of rat liver fibrosis induced by IGFBPrP1.
Results: there were 24 gene mRNA expressions in the MAPK signaling pathway, which accounted for 19 of the 28.57%. gene expression up-regulated and 5 genes down regulated. These genes could be divided into transcription factor gene, Raf regulation protein gene, cyclin gene and so on, among which Map2k2 (MEK2) and Mapk3 (ERK1) were up regulated.
Conclusion: IGFBPrP1 may play a role in liver fibrosis by activating the different links of the MAPK signaling pathway. The differential expression gene Map2k2 (MEK2) and Mapk3 (ERK1) in the pathway may be the key genes in the signal transduction of liver fibrosis induced by IGFBPrP1.
The fifth part is the validation of some differentially expressed genes in liver tissues of IGFBPrP1 induced liver fibrosis rats.
Objective: to verify the expression of some differentially expressed genes in liver tissue of IGFBPrP1 induced liver fibrosis in rats.
Methods: the experimental group was divided into the first part. The expression of mRNA and protein of the differentially expressed genes screened by PCRArray was detected by qRT-PCR and Western blot, respectively, in the liver tissue of rat liver fibrosis induced by IGFBPrP1.
Results: the results of 1. qRT-PCR detection showed that the mRNA level of Map2k2 (MEK2) and Mapk3 (ERK1) genes in group Ad-IGFBPrP1 increased, and the difference was statistically significant (P < 0.05) compared with the normal control group and Ad-EGFP group (P < 0.05), and the mRNA level of Hhip and PTEN genes in the group was significantly lower than that of the normal control group and the normal control group, and the difference was significantly lower than that of the normal control group and the Ad-EGFP group. Statistical significance (P < 0.05);
(3) compared with the normal control group and the Ad-EGFP group, the mRNA level of Egr1 gene in Ad-IGFBPrP1 group increased, the difference was statistically significant (P < 0.05).
The results of 2. Western blot showed that the expression of PTEN protein decreased gradually with the progression of hepatic fibrosis, and the expression of PTEN protein in each phase was significantly different from that in the normal control group and the Ad-EGFP group (P < 0.05). (2) after adenovirus transfection, the expression of Egr1 protein was enhanced and the transfection of 2W reached its peak and then gradually weakened. Egr1 eggs transfected with 2W were found. White level was significantly different from other groups (P < 0.05).
Conclusion: Map2k2 (MEK2), Mapk3 (ERK1), Hhip, PTEN, Egr1 and other differentially expressed genes regulate the role of IGFBPrP1 to induce liver fibrosis, which may partly clarify the mechanism of the action of IGFBPrP1 induced liver fibrosis.
The sixth part is the relationship between IGFBPrP1 and TGF beta 1 in liver fibrosis.
Objective: To investigate the relationship between IGFBPrP1 and TGF beta 1 in liver fibrosis.
Methods: the experimental group and the first part of the.Western blot method were used to detect the expression of IGFBPrP1, alpha -SMA and TGF beta 1 protein in the liver tissue of rats with hepatic fibrosis induced by IGFBPrP1, and to observe their changes in the development of liver fibrosis.
Results: the results of Western blot detection showed that after transfection of adenovirus, the level of IGFBPrP1 protein increased first, reached the peak at the time of transfection of 2W, and then decreased gradually. The expression of alpha -SMA and TGF beta 1 protein increased with the increase of liver fibrosis, and reached the peak when transfected to 9W. The Hyp content of liver tissue was gradually prolonged after adenovirus transfection of 4W. Up to the peak when transfected with 9W.
Conclusion: IGFBPrP1 stimulates liver tissue to produce TGF beta 1, which may be the main pathway of liver fibrosis induced by IGFBPrP1.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R575

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