本文選題：雷公藤甲素 + 缺氧誘導(dǎo)因子1�。� 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:探討缺氧環(huán)境下雷公藤甲素(Triptolide,TP)對(duì)人臍靜脈內(nèi)皮細(xì)胞(Human umbilical vein endothelial cells,HUVECs)中缺氧誘導(dǎo)因子1α(HIF1α)和血管內(nèi)皮生長(zhǎng)因子(VEGF)表達(dá)的影響。在人近曲腎小管上皮細(xì)胞(Human kidney proximal tubular epithelial cell,HK-2)中,研究TP對(duì)TGF-β1誘導(dǎo)的上皮-間充質(zhì)轉(zhuǎn)化(Epithelial-mesenchymal transition,EMT)的影響,從而探討TP對(duì)腎臟纖維化過(guò)程的影響及其可能的分子機(jī)制。方法:為了研究TP在缺氧情況下對(duì)HIF1α和VEGF表達(dá)的影響,(1)我們選取原代人臍靜脈內(nèi)皮細(xì)胞培養(yǎng)于專(zhuān)門(mén)的缺氧培養(yǎng)小室中。實(shí)驗(yàn)所做的分組及處理如下:HUVECs加入0、40、80、160、320 nmol/L的TP(分別設(shè)為缺氧組,TP40組、TP80組、TP160組、TP320組)在缺氧條件下(37℃、5%CO2、1%O2、94%N2)培養(yǎng)12 h,同時(shí)設(shè)常氧組(不加TP)作為對(duì)照,Western blot檢測(cè)各組HIF1α蛋白表達(dá)。(2)細(xì)胞設(shè)TP80組、缺氧組和常氧組,免疫熒光法檢測(cè)HIF1α在細(xì)胞中的表達(dá)定位。(3)設(shè)TP80組和缺氧組,處理結(jié)束后Western blot檢測(cè)VEGF蛋白表達(dá)水平。(4)加入HIF1α的抑制劑KC7F2,設(shè)缺氧組、TP80組,TP80+KF20組(80 nmol/L TP和20μmol/L KC7F2)、TP80+KF30組(80 nmol/L TP和30μmol/L KC7F2),處理12 h后Western blot檢測(cè)各組HIF1α、VEGF蛋白表達(dá)。為了研究TP在TGF-β1誘導(dǎo)的HK-2細(xì)胞EMT過(guò)程中的作用,(1)HK-2細(xì)胞的實(shí)驗(yàn)分組如下:1正常對(duì)照組,常規(guī)下培養(yǎng)細(xì)胞48h;2TGF-β1刺激組,10ng/ml的TGF-β1刺激細(xì)胞培養(yǎng)48h;3TP組,20μmol/L的TP加上10ng/ml的TGF-β1共同刺激培養(yǎng)細(xì)胞48h。每組實(shí)驗(yàn)重復(fù)一次,提取細(xì)胞的總RNA,純化后以Illumina Hi Seq平臺(tái)進(jìn)行RNA測(cè)序。(2)分析處理轉(zhuǎn)錄組的測(cè)序信息,并與指定參考基因組進(jìn)行序列比對(duì),采用FPKM方法計(jì)算出單個(gè)基因在各個(gè)處理組中的表達(dá)量。(3)分析各處理組之間基因表達(dá)量的差異,找出上調(diào)基因和下調(diào)基因,并對(duì)差異表達(dá)基因的數(shù)目進(jìn)行統(tǒng)計(jì)。(4)對(duì)差異表達(dá)基因進(jìn)行KEGG的通路富集分析。(5)根據(jù)KEGG通路富集分析的結(jié)果,對(duì)TGF-β-smad信號(hào)通路進(jìn)行蛋白表達(dá)水平的驗(yàn)證,實(shí)驗(yàn)的分組及處理?xiàng)l件如前述(1),相差顯微鏡來(lái)觀察細(xì)胞形態(tài)的改變。(6)Western blot檢測(cè)各種TGF-β-smad信號(hào)通路的相關(guān)指標(biāo)(如smad2/3、TGF-βR?和TGF-βR??)的表達(dá),同時(shí)也檢測(cè)了EMT的相關(guān)指標(biāo)(如E-cadherin、Vimentin和N-cadherin)的表達(dá)。結(jié)果:(1)常氧條件下HUVECs不表達(dá)HIF1α;缺氧條件下,TP可以誘導(dǎo)HIF1α的高表達(dá),且TP濃度為80 nmol/L時(shí),HIF1α的表達(dá)達(dá)到峰值,TP80組HIF1α表達(dá)水平較缺氧組明顯升高(P0.05),而TP160組和TP320組HIF1α表達(dá)與缺氧組相比無(wú)明顯變化。(2)細(xì)胞免疫熒光結(jié)果顯示,HIF1α主要表達(dá)于細(xì)胞核。(3)TP80組VEGF表達(dá)水平較缺氧組升高(P0.05)。(4)TP作用后,TP80組較缺氧組HIF1α、VEGF表達(dá)水平都升高;但經(jīng)過(guò)KC7F2干預(yù)后,TP80+KF20組和TP80+KF30組HIF1α、VEGF表達(dá)水平較TP80組明顯降低(P0.05)。(5)在NC、TGF-β1和TGF-β1+TP三個(gè)處理組中,計(jì)算出了14903個(gè)基因在不同處理組中的表達(dá)量(即FPKM值)。(6)通過(guò)對(duì)差異基因的兩兩比較,NC組與TGF-β1組比較,有161個(gè)上調(diào)基因和48個(gè)下調(diào)基因,NC組與TGF-β1+TP組比較中是2997個(gè)上調(diào)和1954個(gè)下調(diào)基因,而TGF-β1組與TGF-β1+TP組中,分析出的上調(diào)基因數(shù)為2982,下調(diào)基因數(shù)為1827個(gè)。TGF-β1+TP組與TGF-β1組比較中,火山圖顯示TP較TGF-β1處理引起更多基因的上調(diào)和下調(diào)。(7)差異表達(dá)基因在KEGG通路富集分析了兩兩比較中顯著性Q值最小的前20個(gè)通路。其中Pathway in cancer富集最為顯著,說(shuō)明TP影響了腫瘤相關(guān)的通路,如TGF-β,Hippo等,多與細(xì)胞極性、EMT等過(guò)程有關(guān)。(8)在TGF-β1組與TP+TGF-β1組差異基因的KEGG富集通路TP影響了與許多與細(xì)胞粘附有關(guān)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的表達(dá)下調(diào),伴隨BAMB1和Smad5/8的上調(diào)。在Hippo signaling pathway通路中分析得到TP也下調(diào)Smad2/3的表達(dá),但對(duì)E-cadherin的表達(dá)無(wú)影響。TP也影響了Pancreatic cancer通路中的許多基因的表達(dá)。(9)10ng/ml的TGF-β1刺激HK-2細(xì)胞,細(xì)胞形態(tài)從上皮鋪路石樣變成梭型,伴隨TGF-β-smad信號(hào)通路蛋白Smad2/3、TGF-βR?、TGF-βR??和間充質(zhì)的相關(guān)指標(biāo)如N-cadherin、Vimentin蛋白表達(dá)上調(diào),上皮特性的相關(guān)指標(biāo)E-cadherin的下調(diào)。當(dāng)TP的加入,TGF-β1+TP組與TGF-β1相比較,這些通路蛋白和間充質(zhì)的指標(biāo)都下調(diào),但E-cadherin的表達(dá)卻沒(méi)有顯著上調(diào),細(xì)胞形態(tài)也未變成鋪路石樣,而是有些細(xì)胞開(kāi)始變成圓形。結(jié)論:缺氧環(huán)境下TP可通過(guò)提高HIF1α來(lái)影響內(nèi)皮細(xì)胞VEGF的表達(dá)。同時(shí)TP可能通過(guò)影響TGF-β-smad信號(hào)通路的相關(guān)基因的表達(dá),對(duì)TGF-β1誘導(dǎo)的EMT有一定的抑制作用,TP與腎臟纖維化的治療有關(guān)。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R692

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