IGFBPrP1致肝纤维化信号转导通路的筛选与验证

发布时间：2018-06-21 20:05

  本文选题：胰岛素样生长因子结合蛋白相关蛋白1 + 肝纤维化　； 参考：《山西医科大学》2014年博士论文

【摘要】：胰岛素样生长因子结合蛋白相关蛋白1（insulin-like growth factor bindingprotein related protein1，IGFBPrP1），又称为IGFBP7，是一种分泌蛋白，属于胰岛素样生长因子结合蛋白（insulin-like growth factor binding proteins，IGFBPs）超家族成员，与胰岛素样生长因子(insulin-like growth factor，IGF)亲和力低，而与胰岛素呈高结合力，具有调节细胞增殖、分化、黏附、衰老、凋亡及血管形成等生物学活性。 导师前期研究发现IGFBPrP1是肝纤维化新的致病因子，但其致肝纤维化作用的机制尚不完全清楚。 目前已明确的致肝纤维化的信号转导通路有TGFβ通路、Jak-Stat通路、Rho-ROCK通路、NF-κB通路、Wnt通路、Hedgehog（Hh）通路、瘦素信号通路、PPAR介导的信号通路、血管紧张素Ⅱ受体介导的信号通路等。TGFβ通路包括TGFβ/Smad信号通路和非Smad信号通路。TGFβ/Smad信号通路是经典的肝纤维化信号转导通路；非Smad信号通路包括MAPK通路和PI3K/AKT通路等。 导师在前期研究中对TGFβ相关通路进行了探索，发现IGFBPrP1可通过TGFβ/Smad信号通路发挥致肝纤维化作用，且可能与非Smad信号通路中的ERK/MAPK信号通路有关。但IGFBPrP1是否能通过其他信号通路发挥致肝纤维化作用尚不清楚。研究表明，众多信号通路在肝纤维化发生发展的不同环节发挥着重要作用，，不仅TGFβ/Smad信号通路与非Smad信号通路之间，而且TGFβ通路与其他信号通路之间都存在着交互作用（crosstalk）。因此推测，IGFBPrP1也可能通过MAPK、PI3K/AKT等非Smad通路及其他信号通路发挥致肝纤维化作用。 PCR芯片是荧光定量PCR与芯片技术的结合，仅关注那些与研究对象相关的某一信号通路或多个相关基因的表达，所研究的基因范围相对集中，通常只有几百个或更少的基因。与基因芯片相比，获得的信息具有更强的针对性和准确性。 因此本实验拟通过RT2ProfilerTMPCR芯片筛选IGFBPrP1致肝纤维化作用的信号转导通路及差异表达基因，并对主要差异表达基因进行验证，以阐明IGFBPrP1致肝纤维化作用的部分机制，为抗纤维化治疗提供新思路。 第一部分腺病毒介导IGFBPrP1基因转染大鼠肝组织 目的：观察腺病毒载体能否通过尾静脉注射将IGFBPrP1基因成功转染大鼠肝组织及IGFBPrP1在肝组织的表达。 方法：SD雄性大鼠98只，体重120-140g，随机分为3组：腺病毒基因组（Ad-IGFBPrP1，n=43）：通过大鼠尾静脉注射Ad-IGFBPrP14×109pfu/只；腺病毒空载组（Ad-EGFP，n=43）：通过大鼠尾静脉注射Ad-EGFP4×109pfu/只；正常对照组（N，n=12）：通过大鼠尾静脉注射同等剂量的生理盐水。各组分别于注射腺病毒后2/7w（n=3）、1w（n=8）、2w（n=8）、4w（n=8）、6w（n=8）、9w（n=8）末处死大鼠，留取血清和肝组织待测。荧光显微镜观察肝组织中增强型绿色荧光蛋白（EGFP）的表达；流式细胞仪检测肝组织EGFP阳性细胞的百分比；实时荧光定量PCR（qRT-PCR）和Western blot方法分别测定肝组织IGFBPrP1的mRNA和蛋白表达水平。 结果：1.荧光显微镜观察和流式细胞仪检测结果均显示，一次性给予Ad-EGFP4×109pfu和重复给予（0w，2w）Ad-EGFP2×109pfu两种方式转染，大鼠肝组织均有EGFP表达，但一次性给予Ad-EGFP4×109pfu的方式转染效率更高（60.4%vs46.3%）。故后续实验中采用一次性给予Ad-EGFP4×109pfu进行转染。 2.荧光显微镜下观察，腺病毒转染1w时，肝组织可见明亮的绿色荧光，EGFP表达达到高峰，转染2w后绿色荧光逐渐减弱，4w后基本消失。 3.流式细胞仪检测结果显示，腺病毒转染1w时，肝组织EGFP阳性细胞最多，达到60.4%，腺病毒转染效率达高峰；2w后EGFP阳性细胞逐渐减少。 4. qRT-PCR检测结果显示，腺病毒转染1w时，肝组织IGFBPrP1基因mRNA表达水平最高（5.57±1.52）。 5. Western blot检测结果显示，腺病毒转染2w时，肝组织IGFBPrP1蛋白表达达高峰（1.07±0.11），随后逐渐减低。结论：腺病毒载体通过尾静脉注射成功将IGFBPrP1基因导入大鼠肝组织。 第二部分腺病毒介导的IGFBPrP1基因转染致大鼠肝纤维化 目的：观察腺病毒介导的IGFBPrP1是否导致大鼠发生肝纤维化。 方法：实验分组同第一部分。HE和Sirius red染色观察肝组织病理学改变和胶原纤维分布；Western blot方法检测HSC活化标志物α-SMA蛋白的表达；羟脯氨酸（Hyp）含量测定观察肝组织胶原纤维的形成；全自动生化仪检测肝功能观察肝组织损伤程度。 结果：1. HE和Sirius red染色结果显示，IGFBPrP1基因转染大鼠肝组织后，随着病变进展，肝细胞出现水样变性和脂肪变性，点状坏死，甚至灶状坏死，伴有炎细胞浸润，胆管增生。转染4w时汇管区出现增生的纤维组织，胶原纤维逐渐增多，由血管壁向肝小叶内延伸，在汇管区与汇管区之间以及中央静脉与汇管区之间相互连接，病变可达到纤维化S3期。 2. Western blot检测结果显示，随着纤维化病变进展，Ad-IGFBPrP1组肝组织α-SMA蛋白表达逐渐增强。转染9w时，α-SMA蛋白表达量增至Ad-EGFP组的20倍。 3.肝组织Hyp含量检测结果显示，Ad-IGFBPrP1组肝组织Hyp含量随纤维化进展逐渐升高。 4.血清学检测结果显示，Ad-IGFBPrP1组大鼠血清ALT和TBIL水平显著升高，与正常对照组和Ad-EGFP组相比差异有统计学意义（P＜0.05）。各组大鼠血清TP和ALB水平在正常范围内，组间差异无统计学意义（P＞0.05）。 结论：腺病毒介导的IGFBPrP1基因转染导致大鼠发生肝纤维化。 第三部分PCRArray筛选参与IGFBPrP1致肝纤维化作用的信号转导通路的差异表达基因 目的：通过PCRArray筛选出IGFBPrP1致肝纤维化的信号转导通路的差异表达基因。 方法：SD大鼠随机分为3组（n=3）：Ad-IGFBPrP1组、Ad-EGFP组和正常对照组（N）。各组腺病毒处理同第一部分。各组分别于腺病毒转染2w末处死大鼠，留取肝组织待测。提取肝组织mRNA，逆转录为cDNA，然后qRT-PCR方法检测信号转导通路的差异表达基因。 结果：检测Signal Transduction PathwayFinder PCR Array上84个相关基因的mRNA表达水平变化。结果显示，18条信号转导通路中有33个基因mRNA表达有差异，占检测基因的39.29%。其中17个基因mRNA表达上调，16个基因mRNA表达下调。这些差异表达基因涉及16条信号转导通路。其中核转录因子Egr1表达上调，Hedgehog通路的Hhip基因和PI3K/AKT通路的PTEN基因表达均下调。 结论：IGFBPrP1可能通过多条信号转导通路促进肝纤维化发生发展，差异表达基因Egr1、Hhip和PTEN可能是IGFBPrP1致肝纤维化作用信号转导的关键基因。 第四部分在IGFBPrP1诱导的肝纤维化大鼠肝组织中MAPK信号通路的差异表达基因 目的：通过PCR Array检测在IGFBPrP1诱导的肝纤维化大鼠肝组织中MAPK信号通路的差异表达基因。 方法：实验分组同第二部分。提取肝组织mRNA，逆转录为cDNA，然后qRT-PCR方法检测IGFBPrP1诱导的肝纤维化大鼠肝组织中MAPK信号通路的差异表达基因。 结果：MAPK信号通路中有24个基因mRNA表达有差异，占检测基因的28.57%。其中19个基因mRNA表达上调，5个基因mRNA表达下调。这些基因按功能可分为转录因子基因、Raf调控蛋白基因、细胞周期蛋白基因等。其中Map2k2（MEK2）和Mapk3（ERK1）表达均上调。 结论：IGFBPrP1可能通过激活MAPK信号通路的不同环节发挥致肝纤维化作用，通路中差异表达基因Map2k2（MEK2）和Mapk3（ERK1）可能是IGFBPrP1致肝纤维化作用信号转导的关键基因。 第五部分在IGFBPrP1诱导的肝纤维化大鼠肝组织中部分差异表达基因的验证 目的：验证部分差异表达基因在IGFBPrP1诱导的肝纤维化大鼠肝组织的表达。 方法：实验分组同第一部分。应用qRT-PCR和Western blot方法分别检测PCRArray筛选出的差异表达基因的mRNA和蛋白在IGFBPrP1诱导的肝纤维化大鼠肝组织的表达。 结果：1. qRT-PCR检测结果显示，①Ad-IGFBPrP1组Map2k2（MEK2）和Mapk3（ERK1）基因的mRNA水平均升高，与正常对照组和Ad-EGFP组相比，差异有统计学意义（P＜0.05）；②Hhip和PTEN基因的mRNA水平在Ad-IGFBPrP1组显著降低，明显低于正常对照组和Ad-EGFP组，差异有统计学意义（P＜0.05）； ③与正常对照组和Ad-EGFP组相比，Ad-IGFBPrP1组Egr1基因的mRNA水平升高，差异有统计学意义（P＜0.05）。 2. Western blot检测结果显示，①PTEN蛋白表达随肝纤维化病变进展逐渐减弱，各时相的PTEN蛋白表达与正常对照组和Ad-EGFP组相比，均有统计学差异（P＜0.05）。②腺病毒转染后，Egr1蛋白表达增强，转染2w时达到高峰，然后逐渐减弱。转染2w的Egr1蛋白水平与其他各组相比，差异有统计学意义（P＜0.05）。 结论：Map2k2（MEK2）、Mapk3（ERK1）、Hhip、PTEN和Egr1等差异表达基因共同调控IGFBPrP1发挥致肝纤维化作用，可能部分阐明了IGFBPrP1致肝纤维化的作用机制。 第六部分IGFBPrP1和TGFβ1在致肝纤维化中的关系初探 目的：初步探讨IGFBPrP1和TGFβ1在致肝纤维化中的关系。 方法：实验分组同第一部分。Western blot方法检测IGFBPrP1、α-SMA和TGFβ1蛋白在IGFBPrP1诱导的肝纤维化大鼠肝组织的表达，并观察它们在肝纤维化发展过程中的变化趋势。 结果：Western blot检测结果显示，腺病毒转染后，IGFBPrP1蛋白水平先升高，在转染2w时达到高峰，随后逐渐减低。α-SMA和TGFβ1蛋白表达均随着肝纤维化程度的增加而增加，在转染9w时达到高峰。肝组织Hyp含量在腺病毒转染4w后随时间延长逐渐升高，在转染9w时达到高峰。 结论： IGFBPrP1刺激肝组织产生TGFβ1可能是IGFBPrP1致肝纤维化作用的主要途径。
[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.
【学位授予单位】：山西医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R575

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