本文選題：VEGF121 + VEGF165��； 參考：《北京協(xié)和醫(yī)學院》2012年博士論文

【摘要】：第一部分血管內(nèi)皮生長因子(VEGF)穩(wěn)定過表達腎小管上皮細胞系(HKC細胞系)的建立 背景與目的 血管內(nèi)皮生長因子(VEGF)在維持血管內(nèi)皮細胞生存、增殖及調(diào)節(jié)血管新生和重構(gòu)方面有非常重要的作用。正常情況下,胎兒與成人腎組織的VEGF主要來源于足突細胞和腎小管上皮細胞,腎小管上皮細胞可分泌VEGF121和VEGF165,是腎小管-間質(zhì)中VEGF的主要來源。我們以前的多次實驗證實,VEGF能抑制TGF-β1誘導的腎小管上皮-間充質(zhì)轉(zhuǎn)化(Epithelial-Mesenchymal Transition, EMT)。但是實驗都是采用外源性的VEGF刺激體外培養(yǎng)的腎小管上皮細胞(HKC),具體的作用機制還不明確。因此,我們通過建立VEGF穩(wěn)定過表達HKC細胞系,模仿體內(nèi)環(huán)境,使HKC細胞能同時分泌VEGF121和VEGF165,進一步探討VEGF對TGF-β1誘導的EMT的抑制作用及其作用機制。 方法 全基因合成目的序列VEGF121和VEGF165,構(gòu)建目的基因VEGF121到pBuDCE4.1載體的CMV啟動子下,并構(gòu)建目的基因VEGF165到EF-1α啟動子下,同時插入抗性zeocin。測序驗證含有目的序列的正確質(zhì)粒編號為Z7842-2,抗性為Zeocin。采用電轉(zhuǎn)儀轉(zhuǎn)化細胞的方法,將Z7842-2質(zhì)粒導入HKC細胞,并通過逐步用zeocin加壓的方法進行篩選,最后用ELISA法檢測培養(yǎng)液上清中VEGF濃度。 結(jié)果 正常HKC細胞培養(yǎng)液上清中VEGF濃度為112.918pg/ml,而被轉(zhuǎn)入Z7842-2質(zhì)粒的HKC細胞培養(yǎng)液上清中VEGF的濃度為1210.061pg/ml,后者的濃度是前者的10.72倍(p0.05)。因此認為VEGF穩(wěn)定過表達HKC細胞系(簡稱為VEGF過表達HKC細胞系)已經(jīng)建立。 結(jié)論 成功構(gòu)建VEGF穩(wěn)定過表達HKC細胞系。 第二部分血管內(nèi)皮生長因子(VEGF)過表達對腎小管上皮間充質(zhì)轉(zhuǎn)化(EMT)和Smad3表達的抑制 背景與目的 TGF-β1主要通過TGF-β1/Smad途徑發(fā)揮生物學效應,Smad蛋白是TGF-β1受體后主要的信使分子,介導TGF-β1信號從細胞膜傳入細胞核。腎小管上皮間充質(zhì)轉(zhuǎn)化(EMT)是腎間質(zhì)纖維化的重要途徑。Smad3在TGF-β1誘導的EMT過程中發(fā)揮重要作用。我們多次的實驗證明,VEGF能抑制TGF-β1誘導的腎小管EMT。近來,我們已經(jīng)成功建立了VEGF穩(wěn)定過表達HKC細胞系。因此本實驗的目的是探討VEGF過表達抑制EMT和對Smad3的影響。 方法 將體外培養(yǎng)的細胞分為以下幾組：A.正常HKC組,B.正常HKC加入TGF-β1(5μ/l)刺激24小時和48小時組,C.VEGF過表達HKC組,D.VEGF過表達HKC加入TGF-β1(5μg/l)刺激24小時和48小時組。Western-blot方法檢測TGF-β1信號通路中的重要分子Smad2及Smad3的表達,同時用RT及Real Time PCR方法檢測Smad3基因的表達情況。Western-blot方法及激光共聚焦方法檢測上皮細胞的標志蛋白E-鈣黏素(E-cadherin)的表達及上皮間充質(zhì)轉(zhuǎn)化的標志蛋白α-平滑肌動蛋白(a-SMA)表達。ELISA方法檢測培養(yǎng)液上清中VEGF的濃度變化。40倍倒置光學顯微鏡下觀察各組細胞在加入TGF-β1刺激不同時間后細胞形態(tài)的變化。 結(jié)果 B組的α-SMA的表達較A組明顯增加(p0.05)；B組的E-鈣黏素明顯低于A組(p0.05)。D組的α-SMA的表達較B組下降(p0.05),E-鈣黏素較B組明顯增加(p0.05)。B組P-Smad3的表達明顯高于A組(p0.05)；D組P-Smad3的表達明顯低于B組。C組Smad3的表達較A組明顯減少(p0.05), D組Smad3的表達較B組明顯減少(p0.05)。B組P-Smad3/Smad3的比值較A組明顯增加(p0.05), D組P-Smad3/Smad3的比值較B組下降(p0.05)。 40倍倒置光學顯微鏡下觀察,正常HKC細胞呈鵝卵石樣,在加入TGF-β1刺激24小時后,細胞變成梭形；在加入TGF-β1刺激48小時后,這種變化更為明顯。而VEGF過表達HKC細胞也是呈鵝卵石樣外觀,在加入TGF-β1刺激24及48小時后,細胞形態(tài)沒有明顯變化。 結(jié)論 HKC細胞VEGF過表達能明顯減弱TGF-β1誘導的EMT; VEGF的這一作用與TGF-β1信號途徑中的Smad3的表達下調(diào)及磷酸化抑制有關(guān)。 第三部分血管內(nèi)皮生長因子(VEGF)過表達下調(diào)Smad3表達通過PI3K/AKT信號通路 背景與目的 腎小管上皮-間充質(zhì)細胞轉(zhuǎn)化(EMT)在腎間質(zhì)纖維化的發(fā)生、發(fā)展中起重要作用,是腎間質(zhì)纖維化的重要機制之一。TGF-β1/Smad信號通路在EMT過程中發(fā)揮關(guān)鍵作用,而Smad3是TGF-β1/Smad信號通路的關(guān)鍵調(diào)節(jié)因子。我們的多次研究表明,VEGF能抑制TGFβ1誘導的腎小管EMT。 PI3K/Akt信號通路是VEGF發(fā)揮作用的主要通路之一。LY294002是PI3K活性的特異性抑制劑。我們已經(jīng)成功建立了VEGF過表達HKC細胞系。本部分實驗的目的是,探討VEGF過表達可否通過PI3K/Akt信號通路抑制Smad3的表達,從而抑制TGF-β1誘導的腎小管EMT。 方法 將體外培養(yǎng)的細胞分為以下幾組：.A組：正常HKC組；B組：正常HKC加入TGF-β1(5μg/l)刺激刺激24小時及48小時組；C組：VEGF過表達HKC組；D組：VEGF過表達HKC加入TGF-β1(5μg/l)刺激24小時及48小時組；E組：VEGF過表達HKC加入TGF-β1(5μg/l)及LY294002(20μmol/l)刺激24小時及48小時組。 Western Blot法檢測各組細胞磷酸化的PI3K (P-PI3K)、PI3K、磷酸化的Akt、Akt、磷酸化的Smad3、Smad3、平滑肌肌動蛋白(α-SMA)以及E鈣黏素(E-cadherin)的表達。熒光實時定量PCR (real time PCR)方法檢測Smad3基因的表達情況。激光共聚焦檢測α-SMA及E-cadherin的表達情況。倒置光學顯微鏡下觀察細胞形態(tài)學的變化。 結(jié)果 C組PI3K的表達較A組增加(p0.05)；C組的PI3K在P55位點的磷酸化水平較A組增加(p0.05)；VEGF過表達HKC加入TGF-β1刺激48小時的P-PI3K較正常HKC加入TGF-β1刺激48小時明顯增加(p0.05)；E組磷酸化的PI3K水平,較其他組明顯下降(p0.05)(除A組外)。 AKT的表達除了正常HKC細胞加入TGF-β1刺激48小時較低之外,其他組的表達量沒有明顯不同。D組磷酸化的AKT表達水平較B組明顯升高(p0.05),E組磷酸化的AKT表達水平較D組明顯下降(p0.05)。 C組的Smad3基因及蛋白表達較A組減少(p0.05)；D組Smad3基因及蛋白表達較B組減少(p0.05)；但是,E組的Smad3基因及蛋白表達較D組升高(p0.05)。B組的P-Smad3的表達及P-Smad3/Smad3值較A組明顯增加(p0.05)；D組的P-Smad3的表達及P-Smad3/Smad3值較B組減少(p0.05)；E組的P-Smad3的表達及P-Smad3/Smad3值較D組增加(p0.05)。 B組的a-SMA較A組和D組明顯增加(p0.05),但VEGF過表達HKC加入TGF-β1刺激48小時組的α-SMA較正常HKC加入TGF-β1刺激48小時組明顯增加(p0.05)。 A組的E-cadherin明顯高于B組(p0.05),但低于C組(p0.05)；VEGF過表達HKC加入TGF-β1刺激24小時組的E-cadherin明顯低于VEGF過表達HKC細胞加入TGF-β1及LY294002刺激24小時組(p0.05)。 正常HKC細胞及VEGF過表達HKC細胞呈鵝卵石樣,在加入TGF-β1刺激24小時后,細胞變成梭形；在加入TGF-β1刺激48小時后,這種變化更為明顯。VEGF過表達HKC細胞在加入TGF-β1刺激后,細胞開始有輕度變成梭形細胞的趨勢,但變化不甚明顯。VEGF過表達HKC細胞加入TGF-β1及LY294002刺激后細胞變?yōu)樗笮巍?結(jié)論 VEGF過表達能抑制TGF-β1誘導的體外培養(yǎng)的HKC細胞發(fā)生EMT,其作用機制可能與VEGF通過PI3K/Akt信號通路抑制TGF-β1信號通路中Smad3表達及磷酸化有關(guān)。 第四部分血管內(nèi)皮生長因子(VEGF)過表達抑制腎小管上皮間充質(zhì)轉(zhuǎn)化(EMT)與miRNA192表達變化的關(guān)系 背景與目的 TGF-β1誘導的腎小管上皮間充質(zhì)轉(zhuǎn)化(]EMT)在腎間質(zhì)纖維化的發(fā)生、發(fā)展中起重要作用。據(jù)前述報道,VEGF能抑制TGF-β1誘導的腎小管EMT,并且VEGF主要通過PI3K/Akt信號通路抑制Smad3的表達與磷酸化,從而抑制EMT,但其機制不明確。近來報道,miR-192與腎臟纖維化密切相關(guān)。本部分研究目的是探討miR192在VEGF抑制TGF-β1誘導的腎小管EMT過程中的調(diào)控作用。 方法 將體外培養(yǎng)的細胞分為以下幾組：A.正常HKC組,B.正常HKC加入TGF-β1(5μg/l)刺激24小時及48小時組,C.VEGF過表達HKC組,D. VEGF過表達HKC加入TGF-β1(5μg/l)刺激組刺激24小時及48小時組,E. VEGF過表達HKC加入TGF-β (5μg/l)及LY294002(20μmol/l)刺激24小時及48小時組；熒光實時定量PCR (real time PCR)方法檢測miR192及Smad3基因的表達情況；采用SPSS11.5軟件對miR192和Smad3基因的表達進行相關(guān)分析。 結(jié)果 正常HKC細胞在加入TGF-β1刺激48小時后,miR192的表達較A組明顯增加(p0.05)；VEGF過表達HKC細胞在加入TGF-β1刺激48小時后,miR192的表達較正常HKC細胞加入TGF-β1刺激48小時后明顯降低(p0.05)；VEGF過表達HKC細胞在加入TGF-β1及LY294002刺激24小時后,miR192的表達較VEGF過表達HKC細胞加入TGF-β1刺激24小時后明顯增加(p0.05)。 B組Smad3基因的表達較A組明顯升高(p0.05)；C組Smad3基因的表達較A組降低(p0.05)。D組的Smad3基因的表達較B組明顯降低(p0.05)。但是,E組Smad3基因的表達較D組明顯升高(p0.05)。Smad3基因與1niR192的表達呈中高度正相關(guān)。 結(jié)論 在人腎小管上皮細胞中,TGF-β1能明顯促進miR192表達；而VEGF能抑制miR192和Smad3的表達。Smad3基因表達與miR192勺表達呈中高度正相關(guān)。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R363

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