雷公藤甲素干预内皮细胞和肾脏纤维化相关基因的研究

发布时间：2018-06-19 00:22

本文选题：雷公藤甲素 + 缺氧诱导因子1　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的:探讨缺氧环境下雷公藤甲素(Triptolide,TP)对人脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVECs)中缺氧诱导因子1α(HIF1α)和血管内皮生长因子(VEGF)表达的影响。在人近曲肾小管上皮细胞(Human kidney proximal tubular epithelial cell,HK-2)中,研究TP对TGF-β1诱导的上皮-间充质转化(Epithelial-mesenchymal transition,EMT)的影响,从而探讨TP对肾脏纤维化过程的影响及其可能的分子机制。方法:为了研究TP在缺氧情况下对HIF1α和VEGF表达的影响,(1)我们选取原代人脐静脉内皮细胞培养于专门的缺氧培养小室中。实验所做的分组及处理如下:HUVECs加入0、40、80、160、320 nmol/L的TP(分别设为缺氧组,TP40组、TP80组、TP160组、TP320组)在缺氧条件下(37℃、5%CO2、1%O2、94%N2)培养12 h,同时设常氧组(不加TP)作为对照,Western blot检测各组HIF1α蛋白表达。(2)细胞设TP80组、缺氧组和常氧组,免疫荧光法检测HIF1α在细胞中的表达定位。(3)设TP80组和缺氧组,处理结束后Western blot检测VEGF蛋白表达水平。(4)加入HIF1α的抑制剂KC7F2,设缺氧组、TP80组,TP80+KF20组(80 nmol/L TP和20μmol/L KC7F2)、TP80+KF30组(80 nmol/L TP和30μmol/L KC7F2),处理12 h后Western blot检测各组HIF1α、VEGF蛋白表达。为了研究TP在TGF-β1诱导的HK-2细胞EMT过程中的作用,(1)HK-2细胞的实验分组如下:1正常对照组,常规下培养细胞48h;2TGF-β1刺激组,10ng/ml的TGF-β1刺激细胞培养48h;3TP组,20μmol/L的TP加上10ng/ml的TGF-β1共同刺激培养细胞48h。每组实验重复一次,提取细胞的总RNA,纯化后以Illumina Hi Seq平台进行RNA测序。(2)分析处理转录组的测序信息,并与指定参考基因组进行序列比对,采用FPKM方法计算出单个基因在各个处理组中的表达量。(3)分析各处理组之间基因表达量的差异,找出上调基因和下调基因,并对差异表达基因的数目进行统计。(4)对差异表达基因进行KEGG的通路富集分析。(5)根据KEGG通路富集分析的结果,对TGF-β-smad信号通路进行蛋白表达水平的验证,实验的分组及处理条件如前述(1),相差显微镜来观察细胞形态的改变。(6)Western blot检测各种TGF-β-smad信号通路的相关指标(如smad2/3、TGF-βR?和TGF-βR??)的表达,同时也检测了EMT的相关指标(如E-cadherin、Vimentin和N-cadherin)的表达。结果:(1)常氧条件下HUVECs不表达HIF1α;缺氧条件下,TP可以诱导HIF1α的高表达,且TP浓度为80 nmol/L时,HIF1α的表达达到峰值,TP80组HIF1α表达水平较缺氧组明显升高(P0.05),而TP160组和TP320组HIF1α表达与缺氧组相比无明显变化。(2)细胞免疫荧光结果显示,HIF1α主要表达于细胞核。(3)TP80组VEGF表达水平较缺氧组升高(P0.05)。(4)TP作用后,TP80组较缺氧组HIF1α、VEGF表达水平都升高;但经过KC7F2干预后,TP80+KF20组和TP80+KF30组HIF1α、VEGF表达水平较TP80组明显降低(P0.05)。(5)在NC、TGF-β1和TGF-β1+TP三个处理组中,计算出了14903个基因在不同处理组中的表达量(即FPKM值)。(6)通过对差异基因的两两比较,NC组与TGF-β1组比较,有161个上调基因和48个下调基因,NC组与TGF-β1+TP组比较中是2997个上调和1954个下调基因,而TGF-β1组与TGF-β1+TP组中,分析出的上调基因数为2982,下调基因数为1827个。TGF-β1+TP组与TGF-β1组比较中,火山图显示TP较TGF-β1处理引起更多基因的上调和下调。(7)差异表达基因在KEGG通路富集分析了两两比较中显著性Q值最小的前20个通路。其中Pathway in cancer富集最为显著,说明TP影响了肿瘤相关的通路,如TGF-β,Hippo等,多与细胞极性、EMT等过程有关。(8)在TGF-β1组与TP+TGF-β1组差异基因的KEGG富集通路TP影响了与许多与细胞粘附有关的通路,如TGF-beta signaling pathway、Hippo signaling pathway。在TGF-beta signaling pathway中Smad2/3、THBS1、Noggin、Rbx1、SARA、ROCK1和c-Myc的表达下调,伴随BAMB1和Smad5/8的上调。在Hippo signaling pathway通路中分析得到TP也下调Smad2/3的表达,但对E-cadherin的表达无影响。TP也影响了Pancreatic cancer通路中的许多基因的表达。(9)10ng/ml的TGF-β1刺激HK-2细胞,细胞形态从上皮铺路石样变成梭型,伴随TGF-β-smad信号通路蛋白Smad2/3、TGF-βR?、TGF-βR??和间充质的相关指标如N-cadherin、Vimentin蛋白表达上调,上皮特性的相关指标E-cadherin的下调。当TP的加入,TGF-β1+TP组与TGF-β1相比较,这些通路蛋白和间充质的指标都下调,但E-cadherin的表达却没有显著上调,细胞形态也未变成铺路石样,而是有些细胞开始变成圆形。结论:缺氧环境下TP可通过提高HIF1α来影响内皮细胞VEGF的表达。同时TP可能通过影响TGF-β-smad信号通路的相关基因的表达,对TGF-β1诱导的EMT有一定的抑制作用,TP与肾脏纤维化的治疗有关。
[Abstract]:Objective: To investigate the effect of Triptolide (TP) on the expression of hypoxia inducible factor 1 alpha (HIF1 alpha) and vascular endothelial growth factor (VEGF) in human umbilical vein endothelial cells (Human umbilical vein endothelial cells, HUVECs) under anoxic environment. The effect of TP on TGF- beta 1 induced epithelial mesenchymal transition (Epithelial-mesenchymal transition, EMT) and to explore the effect of TP on the process of renal fibrosis and its possible molecular mechanism. Methods: To study the effect of TP on the expression of HIF1 A and VEGF under the condition of hypoxia. (1) we selected the culture of human umbilical vein endothelial cells. In the special hypoxia culture compartment, the group and treatment of the experiment were as follows: HUVECs added to 0,40,80160320 nmol/L TP (set anoxia group, TP40 group, TP80 group, TP160 group, TP320 group) under hypoxia (37 degrees, 5%CO2,1%O2,94%N2) to cultivate 12 h, and set the normal oxygen group (without TP) as the control. (2) the cells were set up in TP80 group, hypoxia group and oxygen group, and the expression of HIF1 alpha in cells was detected by immunofluorescence. (3) group TP80 and hypoxia group, VEGF protein expression level was detected by Western blot after treatment. (4) HIF1 alpha inhibitor KC7F2, anoxic group, TP80 group, TP80+KF20 group (80 nmol/L TP and 20 mu), 8 0 nmol/L TP and 30 mol/L KC7F2) and Western blot after 12 h to detect HIF1 alpha and VEGF protein expression in each group. In order to study the role of TP in HK-2 cells induced by TGF- beta 1, (1) the experimental groups of the cells were as follows: 1 normal control group, conventional cultured cell culture, beta 1 stimulation cell culture, 2 group, 2 0 mu mol/L TP plus 10ng/ml TGF- beta 1 co stimulated the culture cells to repeat each experiment for one time and extract the total RNA of the cells. After purification, the Illumina Hi Seq platform was used to sequence the RNA. (2) the sequencing information of the transcriptional group was analyzed, and the sequence was compared with the specified reference genome, and the individual gene was calculated by FPKM method. (3) analysis of the difference in gene expression between the treatment groups, find the up-regulated and down regulated genes, and count the number of differentially expressed genes. (4) KEGG pathway enrichment analysis of the differentially expressed genes. (5) the protein expression level of the TGF- beta -smad signaling pathway is carried out according to the results of the enrichment analysis of the KEGG pathway. Validation, experimental grouping and processing conditions such as the foregoing (1), phase contrast microscope to observe the changes in cell morphology. (6) Western blot detection of various TGF- beta -smad signaling pathways (such as smad2/3, TGF- beta R? And TGF- beta R?) expression, and also the expression of EMT related indicators (E-cadherin, Vimentin, and -smad). Results: (1 Under the condition of normal oxygen, HUVECs did not express HIF1 alpha, and TP could induce the high expression of HIF1 alpha, and the expression of HIF1 a was peak when the concentration of TP was 80 nmol/L, and the expression level of HIF1 alpha in TP80 group was significantly higher than that in the hypoxia group (P0.05), while the expression of TP160 group and TP320 group was not significantly different from that of the hypoxia group. (2) the results of cell immunofluorescence were obvious. The expression of HIF1 alpha was mainly expressed in the nucleus. (3) the expression level of VEGF in the group TP80 was higher than that in the hypoxia group (P0.05). (4) the expression level of HIF1 alpha and VEGF in the TP80 group was higher than that in the anoxic group after TP, but the expression level of HIF1 alpha in TP80+KF20 and TP80+KF30 groups was significantly lower than that in the KC7F2 group. (5) three treatment In the group, the expression of 14903 genes in different treatment groups (FPKM value) was calculated. (6) by comparing 22 of the difference genes, there were 161 up-regulated genes and 48 down-regulation genes in group NC and TGF- beta 1, and 2997 up regulation and 1954 down regulated genes in group NC and TGF- beta 1+TP group, while the TGF- beta 1 group and TGF- beta 1+TP group were analyzed. The number of up-regulated genes was 2982, and the number of down regulated genes was 1827.TGF- beta 1+TP groups and TGF- beta 1 groups. The volcano map showed that TP was up regulation and down regulation of more genes than TGF- beta 1. (7) the differential expression genes were enriched and analyzed in the KEGG pathway for the first 20 pathways with the smallest Q value in the 22 comparison. Among them, Pathway in cancer enrichment was the most significant. It is suggested that TP affects the tumor related pathways, such as TGF- beta, Hippo and so on, which are related to the process of cell polarity, EMT and so on. (8) the KEGG enrichment pathway of the TGF- beta 1 group and the TP+TGF- beta 1 group is affected by a number of pathways associated with cell adhesion, such as TGF-beta signaling pathway. The expression of Smad2/3, THBS1, Noggin, Rbx1, SARA, ROCK1 and c-Myc is down regulated, accompanied by the up regulation of BAMB1 and Smad5/8. Cell morphology changed from the epithelia paving stone to the spindle type, with the TGF- beta -smad signaling pathway protein Smad2/3, TGF- beta R?, TGF- beta R? And the correlation indices of the mesenchyme, such as N-cadherin, the up regulation of the Vimentin protein expression, the down regulation of the epithelial properties associated with E-cadherin. The expression of mesenchyme was down, but the expression of E-cadherin was not significantly up-regulated, and the cell morphology did not become pave like, but some cells began to become round. Conclusion: TP can affect the expression of VEGF in endothelial cells by increasing HIF1 alpha in anoxic environment. Meanwhile, TP may be expressed through the expression of related genes affecting the TGF- beta -smad signaling pathway. It has a certain inhibitory effect on EMT induced by TGF- beta 1, and TP is related to the treatment of renal fibrosis.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692

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