血管内皮生长因子过表达对肾小管上皮间充质转化的抑制作用及机制探讨

发布时间：2018-05-18 04:19

本文选题：VEGF121 + VEGF165　；参考：《北京协和医学院》2012年博士论文

【摘要】：第一部分血管内皮生长因子(VEGF)稳定过表达肾小管上皮细胞系(HKC细胞系)的建立背景与目的血管内皮生长因子(VEGF)在维持血管内皮细胞生存、增殖及调节血管新生和重构方面有非常重要的作用。正常情况下,胎儿与成人肾组织的VEGF主要来源于足突细胞和肾小管上皮细胞,肾小管上皮细胞可分泌VEGF121和VEGF165,是肾小管-间质中VEGF的主要来源。我们以前的多次实验证实,VEGF能抑制TGF-β1诱导的肾小管上皮-间充质转化(Epithelial-Mesenchymal Transition, EMT)。但是实验都是采用外源性的VEGF刺激体外培养的肾小管上皮细胞(HKC),具体的作用机制还不明确。因此,我们通过建立VEGF稳定过表达HKC细胞系,模仿体内环境,使HKC细胞能同时分泌VEGF121和VEGF165,进一步探讨VEGF对TGF-β1诱导的EMT的抑制作用及其作用机制。方法全基因合成目的序列VEGF121和VEGF165,构建目的基因VEGF121到pBuDCE4.1载体的CMV启动子下,并构建目的基因VEGF165到EF-1α启动子下,同时插入抗性zeocin。测序验证含有目的序列的正确质粒编号为Z7842-2,抗性为Zeocin。采用电转仪转化细胞的方法,将Z7842-2质粒导入HKC细胞,并通过逐步用zeocin加压的方法进行筛选,最后用ELISA法检测培养液上清中VEGF浓度。结果正常HKC细胞培养液上清中VEGF浓度为112.918pg/ml,而被转入Z7842-2质粒的HKC细胞培养液上清中VEGF的浓度为1210.061pg/ml,后者的浓度是前者的10.72倍(p0.05)。因此认为VEGF稳定过表达HKC细胞系(简称为VEGF过表达HKC细胞系)已经建立。结论成功构建VEGF稳定过表达HKC细胞系。第二部分血管内皮生长因子(VEGF)过表达对肾小管上皮间充质转化(EMT)和Smad3表达的抑制背景与目的 TGF-β1主要通过TGF-β1/Smad途径发挥生物学效应,Smad蛋白是TGF-β1受体后主要的信使分子,介导TGF-β1信号从细胞膜传入细胞核。肾小管上皮间充质转化(EMT)是肾间质纤维化的重要途径。Smad3在TGF-β1诱导的EMT过程中发挥重要作用。我们多次的实验证明,VEGF能抑制TGF-β1诱导的肾小管EMT。近来,我们已经成功建立了VEGF稳定过表达HKC细胞系。因此本实验的目的是探讨VEGF过表达抑制EMT和对Smad3的影响。方法将体外培养的细胞分为以下几组：A.正常HKC组,B.正常HKC加入TGF-β1(5μ/l)刺激24小时和48小时组,C.VEGF过表达HKC组,D.VEGF过表达HKC加入TGF-β1(5μg/l)刺激24小时和48小时组。Western-blot方法检测TGF-β1信号通路中的重要分子Smad2及Smad3的表达,同时用RT及Real Time PCR方法检测Smad3基因的表达情况。Western-blot方法及激光共聚焦方法检测上皮细胞的标志蛋白E-钙黏素(E-cadherin)的表达及上皮间充质转化的标志蛋白α-平滑肌动蛋白(a-SMA)表达。ELISA方法检测培养液上清中VEGF的浓度变化。40倍倒置光学显微镜下观察各组细胞在加入TGF-β1刺激不同时间后细胞形态的变化。结果 B组的α-SMA的表达较A组明显增加(p0.05)；B组的E-钙黏素明显低于A组(p0.05)。D组的α-SMA的表达较B组下降(p0.05),E-钙黏素较B组明显增加(p0.05)。B组P-Smad3的表达明显高于A组(p0.05)；D组P-Smad3的表达明显低于B组。C组Smad3的表达较A组明显减少(p0.05), D组Smad3的表达较B组明显减少(p0.05)。B组P-Smad3/Smad3的比值较A组明显增加(p0.05), D组P-Smad3/Smad3的比值较B组下降(p0.05)。 40倍倒置光学显微镜下观察,正常HKC细胞呈鹅卵石样,在加入TGF-β1刺激24小时后,细胞变成梭形；在加入TGF-β1刺激48小时后,这种变化更为明显。而VEGF过表达HKC细胞也是呈鹅卵石样外观,在加入TGF-β1刺激24及48小时后,细胞形态没有明显变化。结论 HKC细胞VEGF过表达能明显减弱TGF-β1诱导的EMT; VEGF的这一作用与TGF-β1信号途径中的Smad3的表达下调及磷酸化抑制有关。第三部分血管内皮生长因子(VEGF)过表达下调Smad3表达通过PI3K/AKT信号通路背景与目的肾小管上皮-间充质细胞转化(EMT)在肾间质纤维化的发生、发展中起重要作用,是肾间质纤维化的重要机制之一。TGF-β1/Smad信号通路在EMT过程中发挥关键作用,而Smad3是TGF-β1/Smad信号通路的关键调节因子。我们的多次研究表明,VEGF能抑制TGFβ1诱导的肾小管EMT。 PI3K/Akt信号通路是VEGF发挥作用的主要通路之一。LY294002是PI3K活性的特异性抑制剂。我们已经成功建立了VEGF过表达HKC细胞系。本部分实验的目的是,探讨VEGF过表达可否通过PI3K/Akt信号通路抑制Smad3的表达,从而抑制TGF-β1诱导的肾小管EMT。方法将体外培养的细胞分为以下几组：.A组：正常HKC组；B组：正常HKC加入TGF-β1(5μg/l)刺激刺激24小时及48小时组；C组：VEGF过表达HKC组；D组：VEGF过表达HKC加入TGF-β1(5μg/l)刺激24小时及48小时组；E组：VEGF过表达HKC加入TGF-β1(5μg/l)及LY294002(20μmol/l)刺激24小时及48小时组。 Western Blot法检测各组细胞磷酸化的PI3K (P-PI3K)、PI3K、磷酸化的Akt、Akt、磷酸化的Smad3、Smad3、平滑肌肌动蛋白(α-SMA)以及E钙黏素(E-cadherin)的表达。荧光实时定量PCR (real time PCR)方法检测Smad3基因的表达情况。激光共聚焦检测α-SMA及E-cadherin的表达情况。倒置光学显微镜下观察细胞形态学的变化。结果 C组PI3K的表达较A组增加(p0.05)；C组的PI3K在P55位点的磷酸化水平较A组增加(p0.05)；VEGF过表达HKC加入TGF-β1刺激48小时的P-PI3K较正常HKC加入TGF-β1刺激48小时明显增加(p0.05)；E组磷酸化的PI3K水平,较其他组明显下降(p0.05)(除A组外)。 AKT的表达除了正常HKC细胞加入TGF-β1刺激48小时较低之外,其他组的表达量没有明显不同。D组磷酸化的AKT表达水平较B组明显升高(p0.05),E组磷酸化的AKT表达水平较D组明显下降(p0.05)。 C组的Smad3基因及蛋白表达较A组减少(p0.05)；D组Smad3基因及蛋白表达较B组减少(p0.05)；但是,E组的Smad3基因及蛋白表达较D组升高(p0.05)。B组的P-Smad3的表达及P-Smad3/Smad3值较A组明显增加(p0.05)；D组的P-Smad3的表达及P-Smad3/Smad3值较B组减少(p0.05)；E组的P-Smad3的表达及P-Smad3/Smad3值较D组增加(p0.05)。 B组的a-SMA较A组和D组明显增加(p0.05),但VEGF过表达HKC加入TGF-β1刺激48小时组的α-SMA较正常HKC加入TGF-β1刺激48小时组明显增加(p0.05)。 A组的E-cadherin明显高于B组(p0.05),但低于C组(p0.05)；VEGF过表达HKC加入TGF-β1刺激24小时组的E-cadherin明显低于VEGF过表达HKC细胞加入TGF-β1及LY294002刺激24小时组(p0.05)。正常HKC细胞及VEGF过表达HKC细胞呈鹅卵石样,在加入TGF-β1刺激24小时后,细胞变成梭形；在加入TGF-β1刺激48小时后,这种变化更为明显。VEGF过表达HKC细胞在加入TGF-β1刺激后,细胞开始有轻度变成梭形细胞的趋势,但变化不甚明显。VEGF过表达HKC细胞加入TGF-β1及LY294002刺激后细胞变为梭形。结论 VEGF过表达能抑制TGF-β1诱导的体外培养的HKC细胞发生EMT,其作用机制可能与VEGF通过PI3K/Akt信号通路抑制TGF-β1信号通路中Smad3表达及磷酸化有关。第四部分血管内皮生长因子(VEGF)过表达抑制肾小管上皮间充质转化(EMT)与miRNA192表达变化的关系背景与目的 TGF-β1诱导的肾小管上皮间充质转化(]EMT)在肾间质纤维化的发生、发展中起重要作用。据前述报道,VEGF能抑制TGF-β1诱导的肾小管EMT,并且VEGF主要通过PI3K/Akt信号通路抑制Smad3的表达与磷酸化,从而抑制EMT,但其机制不明确。近来报道,miR-192与肾脏纤维化密切相关。本部分研究目的是探讨miR192在VEGF抑制TGF-β1诱导的肾小管EMT过程中的调控作用。方法将体外培养的细胞分为以下几组：A.正常HKC组,B.正常HKC加入TGF-β1(5μg/l)刺激24小时及48小时组,C.VEGF过表达HKC组,D. VEGF过表达HKC加入TGF-β1(5μg/l)刺激组刺激24小时及48小时组,E. VEGF过表达HKC加入TGF-β (5μg/l)及LY294002(20μmol/l)刺激24小时及48小时组；荧光实时定量PCR (real time PCR)方法检测miR192及Smad3基因的表达情况；采用SPSS11.5软件对miR192和Smad3基因的表达进行相关分析。结果正常HKC细胞在加入TGF-β1刺激48小时后,miR192的表达较A组明显增加(p0.05)；VEGF过表达HKC细胞在加入TGF-β1刺激48小时后,miR192的表达较正常HKC细胞加入TGF-β1刺激48小时后明显降低(p0.05)；VEGF过表达HKC细胞在加入TGF-β1及LY294002刺激24小时后,miR192的表达较VEGF过表达HKC细胞加入TGF-β1刺激24小时后明显增加(p0.05)。 B组Smad3基因的表达较A组明显升高(p0.05)；C组Smad3基因的表达较A组降低(p0.05)。D组的Smad3基因的表达较B组明显降低(p0.05)。但是,E组Smad3基因的表达较D组明显升高(p0.05)。Smad3基因与1niR192的表达呈中高度正相关。结论在人肾小管上皮细胞中,TGF-β1能明显促进miR192表达；而VEGF能抑制miR192和Smad3的表达。Smad3基因表达与miR192勺表达呈中高度正相关。
[Abstract]:Part 1 Establishment of vascular endothelial growth factor (VEGF) over stable expression of renal tubular epithelial cell line (HKC cell line)
Background and purpose
Vascular endothelial growth factor (VEGF) plays a very important role in maintaining vascular endothelial cell survival, proliferation and regulating angiogenesis and remodeling. In normal cases, the VEGF of fetal and adult renal tissue is mainly derived from the podocyte and renal tubular epithelial cells. Renal tubular epithelial cells can secrete VEGF121 and VEGF165, which are renal tubulointerstitium. The main source of VEGF. Our previous experiments have confirmed that VEGF can inhibit the renal tubular epithelial mesenchymal transition (Epithelial-Mesenchymal Transition, EMT) induced by TGF- beta 1. But the experiment is the use of exogenous VEGF to stimulate the renal tubular epithelial cells (HKC) cultured in vitro. The specific mechanism of action is not clear. Therefore, we pass through To establish VEGF stable overexpression HKC cell line, mimic the body environment, make HKC cells secrete VEGF121 and VEGF165 at the same time, and further explore the inhibitory effect of VEGF on TGF- beta 1 induced EMT and its mechanism.
Method
The whole gene syntheses the target sequence VEGF121 and VEGF165, constructs the target gene VEGF121 to the CMV promoter of the pBuDCE4.1 carrier, and constructs the target gene VEGF165 to the EF-1 alpha promoter, and inserts the resistant zeocin. sequencing to verify the correct plasmid number containing the target sequence as Z7842-2 and the anti sex Zeocin. using the electroporer to transform the cell. The Z7842-2 plasmid was introduced into HKC cells, and then gradually screened by zeocin pressurization. Finally, the VEGF concentration in the supernatant of the culture medium was detected by ELISA.
Result
The concentration of VEGF in the supernatant of normal HKC cell culture medium was 112.918pg/ml, and the concentration of VEGF in the supernatant of HKC cell culture medium of Z7842-2 plasmid was 1210.061pg/ml, the concentration of the latter was 10.72 times of the former (P0.05). Therefore, it was established that VEGF stable overexpression HKC cell line (for short, VEGF overexpression HKC cell line) was established.
conclusion
The VEGF stable overexpression of HKC cell line was successfully constructed.
The second part is the overexpression of vascular endothelial growth factor (VEGF) on the inhibition of renal tubular epithelial mesenchymal transition (EMT) and Smad3 expression.
Background and purpose
TGF- beta 1 plays a biological role mainly through the TGF- beta 1/Smad pathway. Smad protein is the main messenger after TGF- beta 1 receptor, mediating TGF- beta 1 signal from the cell membrane to the nucleus. EMT is an important pathway for renal interstitial fibrosis..Smad3 plays an important role in the EMT process of TGF- beta 1 induced EMT. It has been proved that VEGF can inhibit the renal tubule EMT. induced by TGF- beta 1 recently. We have successfully established the VEGF stable overexpression of HKC cell lines. The aim of this experiment is to explore the effect of VEGF overexpression on the inhibition of EMT and Smad3.
Method
The cultured cells were divided into the following groups: A. normal HKC group, B. normal HKC adding TGF- beta 1 (5 /l) stimulation for 24 hours and 48 hours, C.VEGF overexpression HKC group, D.VEGF overexpression HKC adding TGF- beta 1 (5 mu g/l) stimulation 24 hours and 48 hours RT and Real Time PCR were used to detect the expression of Smad3 gene by.Western-blot method and laser confocal method to detect the expression of the marker protein E- calcain (E-cadherin) in epithelial cells and the marker protein alpha smooth actin (a-SMA) expression.ELISA method for the detection of VEGF concentration in the supernatant of the culture medium. 40 times inverted optical microscope was used to observe the morphological changes of cells in different groups after TGF- beta 1 stimulation.
Result
The expression of alpha -SMA in group B was significantly higher than that in group A (P0.05), and the expression of E- calcain in group B was significantly lower than that in group A (P0.05), and the expression of alpha -SMA in group.D was significantly higher than that in B group (P0.05). 5) the expression of Smad3 in group D was significantly lower than that in group B (P0.05). The ratio of P-Smad3/Smad3 in.B group was significantly higher than that in A group (P0.05), and the ratio of P-Smad3/Smad3 in D group was lower than that in group A.
Under the 40 fold inverted optical microscope, the normal HKC cells were cobblestone like, and the cells became fusiform after adding TGF- beta 1 for 24 hours. After adding TGF- beta 1 for 48 hours, this change was more obvious. The VEGF overexpressed HKC cells were also cobblestone like appearance, and the cell morphology was not obvious after the addition of TGF- beta 1 for 24 and 48 hours. Change.
conclusion
VEGF overexpression in HKC cells can significantly attenuated the EMT induced by TGF- beta 1, which is related to downregulation of Smad3 expression and phosphorylation inhibition in TGF- beta 1 signaling pathway.
The third part of the overexpression of vascular endothelial growth factor (VEGF) downregulated Smad3 expression through PI3K/AKT signaling pathway.
Background and purpose
Renal tubular epithelial mesenchymal cell transformation (EMT) plays an important role in the development of renal interstitial fibrosis. It is one of the important mechanisms of renal interstitial fibrosis..TGF- beta 1/Smad signaling pathway plays a key role in the EMT process. Smad3 is a key regulator of TGF- beta 1/Smad signaling pathway. Many of our studies have shown that VEGF can inhibit TG. F beta 1 induced renal tubule EMT. PI3K/Akt signaling pathway is one of the main pathways that VEGF plays,.LY294002 is a specific inhibitor of PI3K activity. We have successfully established the VEGF overexpression HKC cell line. The purpose of this experiment is to investigate whether VEGF overexpression can inhibit Smad3 expression through PI3K/Akt signaling and thus inhibit TGF. - beta 1 induced renal tubule EMT.
Method
The cells cultured in vitro were divided into the following groups: group.A: normal HKC group; group B: normal HKC added TGF- beta 1 (5 g/l) to stimulate 24 hours and 48 hours; C group: VEGF overexpressed HKC group; D group: VEGF overexpression HKC joined 1 (5 mu) stimulation for 24 hours and 48 hours; 02 (20 mol/l) was stimulated for 24 hours and 48 hours.
Western Blot method was used to detect the expression of phosphorylated PI3K (P-PI3K), PI3K, phosphorylated Akt, Akt, phosphorylated Smad3, Smad3, smooth muscle actin (alpha -SMA) and the expression of E calcalin (E-cadherin). Morphological changes were observed under inverted light microscope.
Result
The expression of PI3K in group C was higher than that in group A (P0.05); the phosphorylation level of PI3K at P55 site in C group was higher than that in A group (P0.05); VEGF overexpressed HKC TGF- beta 1 stimulation for 48 hours was significantly higher than that of normal beta 1 stimulation for 48 hours.
The expression of AKT was lower than that of normal HKC cells added to TGF- beta 1 for 48 hours. There was no significant difference in the expression of AKT in other groups from.D group (P0.05). The AKT expression level of E group was significantly lower than that of the D group (P0.05).
The expression of Smad3 gene and protein in group C decreased (P0.05), and the expression of Smad3 gene and protein in D group decreased (P0.05) than that in B group, but the expression and value of Smad3 gene and protein expression in E group were higher than that in D group (P0.05). .05); the expression of P-Smad3 and P-Smad3/Smad3 in group E increased compared with that in group D (P0.05).
The a-SMA in group B was significantly higher than that in group A and D (P0.05), but VEGF overexpressed HKC added to TGF- beta 1 to stimulate a 48 hour group of alpha -SMA more than normal HKC added to TGF- beta 1 stimulation for 48 hours (P0.05).
The E-cadherin in group A was significantly higher than that in group B (P0.05), but lower than that in group C (P0.05); E-cadherin for VEGF overexpression and TGF- beta 1 stimulation for 24 hours was significantly lower than VEGF overexpression of HKC cells added to the beta 1 and the 24 hour stimulus group.
The normal HKC cells and VEGF overexpressed HKC cells were cobblestone like. After adding TGF- beta 1 for 24 hours, the cells turned into spindle shape. After adding TGF- beta 1 for 48 hours, this change was more obvious in.VEGF overexpressed HKC cells after the addition of TGF- beta 1, the cells began to slightly turn into spindle cells, but the changes were not very obvious in.VEGF over the table. When HKC cells were added TGF- beta 1 and LY294002, the cells became fusiform.
conclusion
VEGF overexpression can inhibit the occurrence of EMT in the cultured HKC cells induced by TGF- beta 1, and its mechanism may be related to the inhibition of Smad3 expression and phosphorylation in TGF- beta 1 signaling pathway through PI3K/Akt signaling pathway.
The fourth part is the relationship between overexpression of vascular endothelial growth factor (VEGF) and the expression of miRNA192 in renal tubular epithelial mesenchymal transition (EMT).
Background and purpose
TGF- beta 1 induced tubuloepithelial mesenchymal transition (]EMT) plays an important role in the development of renal interstitial fibrosis. According to the previous reports, VEGF can inhibit the renal tubule EMT induced by TGF- beta 1, and VEGF mainly inhibits the expression and phosphorylation of Smad3 through the PI3K/Akt signaling pathway, and thus inhibits EMT, but its mechanism is not clear. Recently, the mechanism is reported, miR-192. The aim of this study is to investigate the role of miR192 in regulating VEGF induced TGF- EMT 1 induced tubule EMT.
Method
The cells cultured in vitro were divided into the following groups: A. normal HKC group, B. normal HKC adding TGF- beta 1 (5 g/l) stimulation for 24 hours and 48 hours, C.VEGF overexpression HKC group, D. VEGF overexpression HKC addition to TGF- beta 1 (5 mu) stimulation group stimulated for 24 hours and 48 hours. The expression of miR192 and Smad3 genes was detected by real time fluorescence quantitative PCR (real time PCR) method, and the expression of miR192 and Smad3 genes was analyzed by SPSS11.5 software.
Result
After adding TGF- beta 1 to normal HKC cells for 48 hours, the expression of miR192 was significantly higher than that in the A group (P0.05). VEGF overexpressed HKC cells decreased significantly after the addition of TGF- beta 1 for 48 hours. The expression of miR192 was significantly lower than that of TGF- beta 1 stimulation for 48 hours (P0.05). The expression of miR192 increased significantly after 24 hours of stimulation with VEGF overexpressing HKC cells and TGF- beta 1 (P0.05).
The expression of Smad3 gene in group B was significantly higher than that in group A (P0.05), and the expression of Smad3 gene in group C was lower than that in A group (P0.05), and the expression of Smad3 gene in group.D was significantly lower than that in B group (P0.05).
conclusion
In human renal tubular epithelial cells, TGF- beta 1 can obviously promote the expression of miR192, while VEGF can inhibit the expression of.Smad3 gene expression of miR192 and Smad3 and the positive correlation between the expression of miR192 spoon and the expression of miR192 spoon.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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