本文選題：糖原合酶激酶-3β + GSK3β��； 參考：《浙江大學》2007年博士論文

【摘要】： 內皮素-1(endothelin-1,ET-1)作為強效的血管收縮因子和有絲分裂原,在高血壓、冠心病、心肌肥大等疾病的發(fā)病中起重要作用。內皮細胞ET-1表達調節(jié)主要發(fā)生于轉錄水平。大量文獻報道胰島素能刺激內皮細胞ET-1表達;而胰島素抵抗時的高胰島素血癥可激活機體內皮素系統(tǒng),引起的ET-1表達升高,進而導致心血管細胞的增生或肥大以及血管舒縮異常。 胰島素激活PI3激酶(P13K)和絲裂原活化蛋白激酶(MAPK)兩條主要信號通路,現(xiàn)已知PI3K/Akt通路介導胰島素刺激內皮細胞一氧化氮(NO)產生;但不清楚PI3K/Akt通路是否參與胰島素刺激內皮細胞ET-1基因表達。胰島素激活PI3K/Akt,進而磷酸化9位絲氨酸殘基(Ser~9)導致糖原合酶激酶-3β(GSKβ)滅活。GSK3β是一種多功能的絲氨酸/蘇氨酸激酶,可磷酸化多種轉錄因子并調節(jié)其活性,例如:改變轉錄因子與DNA的結合能力。作為胰島素信號下游的重要激酶,GSK3β是否參與調節(jié)內皮細胞ET-1基因表達,尚未見文獻報道。 人ET-1基因啟動子含TATA框,其活性受多種轉錄因子調節(jié),如:AP-1、含鋅指結構轉錄因子GATA-2、缺氧誘導因子-1(HIF-1);但這些轉錄因子的表達并不局限于血管內皮細胞;因而,這些因子單獨作用不大可能引起胰島素誘導的內皮細胞特異性基因ET-1表達。血管內皮鋅指-1(Vezf1)與ET-1核心啟動子結合,是調節(jié)細胞特異性基因ET-1在內皮細胞表達的潛在靶點。目前尚不清楚Vezf1是否參與胰島素對內皮細胞ET-1表達的調節(jié)。 本課題研究:(1)胰島素對GSK3β滅活作用是否引起內皮細胞ET-1基因表達增加;(2)胰島素上調ET-1啟動子活性是否由PI3K介導;(3)Vezf1是否參與胰島素信號對內皮細胞ET-1基因表達調節(jié)。研究目的在于闡明胰島素調節(jié)內皮細胞ET-1表達的分子機制。 材料和方法 1.細胞培養(yǎng) 人肺動脈內皮細胞(PAEC)生長于含2%胎牛血清(FBS)的EBM-2培養(yǎng)液中。人臍靜脈內皮細胞(HUVEC),用含4%FBS的RPMI 1640培養(yǎng)液培養(yǎng)。 2.胰島素和氯化鋰(LiCl)處理 單層培養(yǎng)的人肺動脈內皮細胞(PAEC)血清饑餓6h后,加入含0-100nM重組豬胰島素或20mM LiCl(GSK3β抑制劑)條件培養(yǎng)液,37℃孵育1h。用酶聯(lián)免疫吸附法(ELISA)檢測培養(yǎng)上清液ET-1多肽含量或用Real time定量RT-PCR(qRT-PCR)分析細胞ET-1 mRNA水平。部分細胞經(jīng)胰島素或LiCl處理后,用4%甲醛固定,ELISA法檢測細胞的Ser~9磷酸化GSK3β和總GSK3β蛋白水平(GSK3p活性)。 3.GSK3βsiRNA和Vezf1 siRNA轉染內皮細胞 單層PAEC分別轉染GSK3βNezf1 siRNA和control siRNA(不針對某種特殊序列)。37℃孵育72h后。用ELISA法測定培養(yǎng)上清液ET-1含量;采用qRT-PCR分析細胞ET-1和GSK3βNezf1基因表達;siRNA對目標蛋白的抑制作用經(jīng)Western blot分析證實。 4.重組腺病毒Ad5-GSK3β感染內皮細胞 人PAEC分別感染染重組共表達GSK3β基因和GFP基因的腺病毒表達載體(Ad5-GSK3β)和僅表達GFP基因的空白腺病毒(Ad5-GFP);細胞感染后在含或不含18nM胰島素條件培養(yǎng)液中孵育1h。ELISA法檢測培養(yǎng)上清液ET-1含量;qRT-PCR分析細胞ET-1基因表達。 5.報告基因質粒構建和熒光素酶檢測 從人基因組DNA調取ET-1基因啟動子(-832/+171bp)片斷;將其插入報告基因質粒pGL3-Basic,形成重組質粒pGL3-ET1。以構建好的pGL3-ET1為基礎,將其中Vezf1結合位點(-47bp處)“TTACCCCCACTC”突變成“TTACATCCACTC”,形成一個突變質粒pGL3-ET1-m。 HUVEC用Lipofectin Reagent轉染pGL3-ET1/pGL3-ET1-m;共轉染pRL-SV40(含SV40啟動子的海腎熒光素酶報告基因質粒)作為內參照。轉染48h后,用100nM胰島素刺激1、3和6h;部分細胞在胰島素刺激前30min至刺激結束,培養(yǎng)液中含濃度為100nM的Wortmannin(P13K抑制劑)或10μM的PD-98059(MAPK抑制劑);雙熒光素酶報告檢測系統(tǒng)(Dual-LuciferseReporter Assay System)檢測熒光素酶活性(表示ET-1啟動子活性)。 結果 1.胰島素刺激人內皮細胞分泌ET-1 人PAEC用10、100nM胰島素刺激1h;培養(yǎng)上清液中ET-1含量比對照組明顯增加(P＜0.05或P＜0.01),并呈劑量依賴效應。 2.GSK3β抑制劑LiCl模仿胰島素刺激內皮細胞ET-1基因表達 人PAEC用100nM胰島素或20mM LiCl條件培養(yǎng)液處理1h后,GSK3β的Ser~9磷酸化水平比對照組明顯增高(P＜0.01),提示LiCl模仿胰島素抑制GSK3β活性。PAEC用10、100nM胰島素或20mM LiCl刺激1h,細胞ET-1mRNA水平明顯高于相應對照組(P＜0.05或P＜0.01)。結果表明GSK3β活性抑制與培養(yǎng)內皮細胞ET-1基因表達上調有關。 3.特異性抑制GSK3β表達上調內皮細胞ET-1 mRNA表達和ET-1多肽釋放 為進一步證實GSK3β對ET-1基因表達的影響,人PAEC轉染GSK3βsiRNA/control siRNA后,細胞和培養(yǎng)上清液分別用于qRT-PCR和ELISA分析。GSK3βsiRNA轉染的人內皮細胞GSK3βmRNA表達僅為control siRNA轉染細胞的20%,證實siRNA成功抑制GSK3β基因表達。GSK3p沉默分別上調ET-1mRNA表達50%(P＜0.01)和ET-1多肽釋放100%(P＜0.01)。結果證實GSK3β活性抑制可上調內皮細胞ET-1 mRNA轉錄和ET-1多肽釋放。 4.過表達GSK3β基因減弱胰島素對ET-1表達的刺激作用 為闡明GSK3β在胰島素調節(jié)ET-1產生中的作用,人PAEC感染共表達GSK3β和GFP重組腺病毒后,qRT-PCR和ELISA分別檢測細胞ET-1基因轉錄與ET-1多肽分泌。與空白病毒(無插入片斷)感染細胞相比,過表達GSK3β表達明顯減弱胰島素對ET-1 mRNA表達(P＜0.01)和ET-1多肽釋放的刺激作用(P＜0.05);結果支持GSK3β負調節(jié)內皮細胞ET-1轉錄與ET-1分泌。 5.Vezf1調節(jié)內皮細胞ET-1表達 為確定Vezf1是否調節(jié)內皮細胞ET-1基因表達,人PAEC轉染VeZf1 siRNA后,細胞和培養(yǎng)上清液用于qRT-PCR和ELISA分析。Vezf1 siRNA轉染細胞Vezf1 mRNA水平僅為control siRNA轉染細胞的9%,而相應ET-1 mRNA表達和ET-1多肽釋放僅分別為control siRNA轉染細胞的20%和40%(P＜0.01);結果表明Vezf1參與調節(jié)內皮細胞ET-1基因轉錄和ET-1多肽釋放。 6.胰島素上調ET-1基因啟動子活性由PI3K介導 現(xiàn)有研究已經(jīng)證明,細胞內胰島素滅活GSK3β主要由PI3K介導。為確定內皮細胞中胰島素信號刺激ET-1基因表達是否由PI3K介導,HUVEC共轉染報告基因質粒pGL3-ET1和pRL-SV40,37℃孵育48h后,加入含100nM胰島素培養(yǎng)液37℃,孵育1、3和6h;部分細胞在胰島素刺激之前30min和刺激期間,培養(yǎng)液中含100nM的Wortmannin或10μM的PD-98059;用雙熒光素酶檢測試劑盒檢測ET-1啟動子活性。Wortmannin和PD-98059在無胰島素刺激條件下對ET-1啟動子活性無明顯影響(P＞0.05);胰島素刺激后各時間點ET-1啟動子活性比對照組(無胰島素刺激)明顯增強(P＜0.05或P＜0.01);胰島素+Wortmannin組ET-1啟動子活性明顯低于胰島素單獨處理組(P＜0.05或P＜0.01),而與對照組相比,無顯著性差異(P＞0.05);胰島素+PD-98059組ET-1啟動子活性與胰島素單獨處理組比較,無顯著性差異(P＞0.05),且明顯高于對照組(P＜0.05或P＜0.01)。結果說明胰島素上調ET-1基因啟動子活性由PI3K介導。 7.Vezf1參與胰島素對ET-1基因表達的調節(jié) 為證實胰島素調節(jié)ET-1表達是否通過轉錄因子Vezf1發(fā)揮作用,HUVEC分別轉染pGL3-ET1和Vezf1結合序列突變質粒pGL3-ET1-m;轉染48h后,加入含100nM胰島素培養(yǎng)液,37℃孵育6h;雙熒光素酶檢測試劑盒檢測ET-1啟動子活性。胰島素明顯上調轉染pGL3-ET1內皮細胞的ET-1啟動子活性(P＜0.05);而轉染pGL3-ET1-m內皮細胞的ET-1啟動子活性在胰島素刺激后與對照組無明顯差異。結果說明Vezf1參與胰島素對ET-1基因表達調節(jié)。 結論 1.本實驗首次發(fā)現(xiàn)PI3K-GSK3β信號通路在胰島素調節(jié)內皮細胞ET-1基因表達中起關鍵作用。PI3K-GSK3β信號通路可能是胰島素抵抗時高胰島素血癥引起內皮細胞ET-1表達增加的分子基礎。 2.內皮細胞特異性轉錄因子Vezf1參與胰島素對內皮細胞ET-1基因表達調節(jié)。
[Abstract]:Endothelin -1 (endothelin-1, ET-1), as a powerful vasoconstrictor and mitogen, plays an important role in the pathogenesis of hypertension, coronary heart disease, and myocardial hypertrophy. The regulation of ET-1 expression in endothelial cells mainly occurs at the transcriptional level. A large number of literature reports that insulin can stimulate ET-1 expression in endothelial cells, and the high pancreas in insulin resistance. Islet emia can activate the endothelin system and increase the expression of ET-1, which may lead to the proliferation or hypertrophy of cardiovascular cells and abnormal vasomotion.
Insulin activates two main signaling pathways of PI3 kinase (P13K) and mitogen activated protein kinase (MAPK). It is known that the PI3K/Akt pathway mediates insulin stimulation of nitric oxide (NO) production in endothelial cells, but it is not clear whether the PI3K/Akt pathway participates in the insulin stimulation of the ET-1 gene expression in endothelial cells. Insulin activates PI3K/Akt and then phosphorylates 9 bits of silk. Amino acid residue (Ser~9) causes glycogen synthase kinase -3 beta (GSK beta) inactivation.GSK3 beta as a multifunctional serine / threonine kinase, which can phosphorate a variety of transcription factors and regulate its activity, for example, to change the binding capacity of transcription factors and DNA. As an important kinase downstream of insulin signals, GSK3 beta is involved in regulating the ET-1 gene of endothelial cells. The expression has not been reported in the literature.
The human ET-1 gene promoter contains the TATA frame and its activity is regulated by a variety of transcription factors, such as AP-1, zinc finger structure transcription factor GATA-2, and hypoxia inducible factor -1 (HIF-1), but these transcription factors are not limited to vascular endothelial cells; therefore, these factors are not likely to cause insulin induced endothelial specific genes alone. ET-1 expression. The binding of -1 (Vezf1) to ET-1 core promoter is a potential target for regulating the expression of cell specific gene ET-1 in endothelial cells. It is not clear whether Vezf1 is involved in the regulation of insulin on the expression of ET-1 in endothelial cells.
The study: (1) whether the effect of insulin on GSK3 beta inactivation induces the increase of ET-1 gene expression in endothelial cells; (2) whether insulin up ET-1 promoter activity is mediated by PI3K; (3) whether Vezf1 participates in the regulation of ET-1 gene expression in endothelial cells by insulin signal. The aim of the study is to clarify the molecular mechanism of insulin regulating the ET-1 expression of endothelial cells. System.
Materials and methods
1. cell culture
Human pulmonary artery endothelial cells (PAEC) were grown in EBM-2 culture medium containing 2% fetal bovine serum (FBS). Human umbilical vein endothelial cells (HUVEC) were cultured in RPMI 1640 medium containing 4%FBS.
2. insulin and lithium chloride (LiCl) treatment
After 6h was starved in human pulmonary artery endothelial cells (PAEC) in single cultured human pulmonary artery, 0-100nM recombinant pig insulin or 20mM LiCl (GSK3 beta inhibitor) conditioned medium was added. The content of ET-1 polypeptide in cultured supernatant was detected by enzyme linked immunosorbent assay (ELISA) at 37 degrees or ELISA, or Real time quantitative RT-PCR (Real time). After treatment with insulin or LiCl, the cells were fixed with 4% formaldehyde, and the levels of Ser~9 phosphorylated GSK3 beta and total GSK3 beta protein (GSK3p activity) were detected by ELISA.
Transfection of 3.GSK3 beta siRNA and Vezf1 siRNA to endothelial cells
The monolayer PAEC was incubated with GSK3 beta Nezf1 siRNA and control siRNA respectively (no specific sequence) was incubated at.37 C at.37 C. The ET-1 content of the culture supernatant was determined by ELISA method, and the cell ET-1 and the expression of the gene were analyzed by qRT-PCR, and the inhibitory effect on the target protein was confirmed by the analysis.
4. recombinant adenovirus Ad5-GSK3 beta infection of endothelial cells
Human PAEC infected respectively the recombinant adenovirus expression vector (Ad5-GSK3 beta) and the GFP gene expression vector (Ad5-GSK3 beta) and the blank adenovirus (Ad5-GFP) that only expressed the GFP gene, respectively, and incubated the ET-1 content of the supernatant in the culture medium containing or without 18nM insulin condition culture, and qRT-PCR analyzed the expression of the ET-1 gene of the cells after the infection.
5. report gene plasmid construction and luciferase detection
The ET-1 gene promoter (-832/+171bp) fragment was extracted from the human genome DNA, and was inserted into the reporter gene plasmid pGL3-Basic to form a recombinant plasmid pGL3-ET1., based on the constructed pGL3-ET1, and the Vezf1 binding site (-47bp) "TTACCCCCACTC" was mutated into a "TTACATCCACTC", forming a mutant plasmid pGL3-ET1-m..
HUVEC transfected pGL3-ET1/pGL3-ET1-m with Lipofectin Reagent, CO transfected pRL-SV40 (the sea kidney luciferase reporter gene plasmid containing SV40 promoter) as internal reference. After transfection of 48h, 100nM insulin was used to stimulate 1,3 and 6h. Or 10 M PD-98059 (MAPK inhibitor); double Luciferase Report detection system (Dual-LuciferseReporter Assay System) detected luciferase activity (indicating the activity of ET-1 promoter).
Result
1. insulin stimulates human endothelial cells to secrete ET-1
Human PAEC was stimulated by 10100nM insulin, and ET-1 content in culture supernatant increased significantly (P < 0.05 or P < 0.01), and showed a dose-dependent effect in the culture supernatant (P < 0 or P < 0.01).
2.GSK3 beta inhibitor LiCl mimics insulin stimulated ET-1 gene expression in endothelial cells
After 1h was treated with 100nM insulin or 20mM LiCl conditioned medium, the level of Ser~9 phosphorylation of GSK3 beta was significantly higher than that of the control group (P < 0.01), suggesting that the LiCl mimic insulin inhibition GSK3 beta activity was stimulated by 10100nM insulin or 20mM. The level of the cells was significantly higher than that in the corresponding control group (< 0.05 or 0.01). The inhibition of K3 beta activity was related to the up regulation of ET-1 gene expression in cultured endothelial cells.
3. specific inhibition of GSK3 beta expression upregulated ET-1 mRNA expression and ET-1 peptide release in endothelial cells.
In order to further confirm the effect of GSK3 beta on the expression of ET-1 gene, after transfection of human PAEC to GSK3 beta siRNA/control siRNA, the cells and culture supernatant were used for qRT-PCR and ELISA to analyze.GSK3 beta siRNA transfected respectively. The GSK3 beta mRNA expression was only 20% of the transfected cells. Do not increase ET-1mRNA expression 50% (P < 0.01) and ET-1 polypeptide release 100% (P < 0.01). The results showed that the inhibition of GSK3 beta activity could up regulate the ET-1 mRNA transcription of endothelial cells and the release of ET-1 polypeptide.
4. overexpression of GSK3 beta gene weakens insulin's stimulatory effect on ET-1 expression.
In order to elucidate the role of GSK3 beta in the insulin regulation of ET-1 production, after human PAEC infection co expressed GSK3 beta and GFP recombinant adenovirus, qRT-PCR and ELISA detected the transcription of ET-1 gene and the secretion of ET-1 polypeptide respectively. Compared with the blank virus (no inserted fragment) infected cells, overexpression of GSK3 beta reduced the expression of insulin to ET-1 mRNA (< 0.01). The stimulatory effect of ET-1 peptide release (P < 0.05) results in GSK3 beta negative regulation of ET-1 transcription and ET-1 secretion in endothelial cells.
5.Vezf1 regulates the expression of ET-1 in endothelial cells
In order to determine whether Vezf1 regulates the expression of ET-1 gene in endothelial cells, after transfection of human PAEC to VeZf1 siRNA, cells and culture supernatant are used for qRT-PCR and ELISA to analyze.Vezf1 siRNA transfected cells with Vezf1 mRNA level only 9%, while the corresponding expression and polypeptide release are only 20% of the transfected cells respectively. And 40% (P < 0.01); the results showed that Vezf1 was involved in regulating ET-1 gene transcription and ET-1 peptide release in endothelial cells.
6. insulin upregulates ET-1 gene promoter activity mediated by PI3K.
The current research has shown that intracellular insulin inactivation of GSK3 beta is mainly mediated by PI3K. To determine whether the ET-1 gene expression of insulin signal in endothelial cells is mediated by PI3K, HUVEC co transfection reporter gene plasmid pGL3-ET1 and pRL-SV40,37 C incubate 48h, adding 100nM insulin culture medium at 37 C to incubate 1,3 and 6h; some of the cells are in the pancreas. Before the stimulation of 30min and stimulation, the culture medium contained 100nM Wortmannin or 10 M PD-98059, and the double luciferase detection kit was used to detect the activity of ET-1 promoter.Wortmannin and PD-98059 on the ET-1 promoter activity without insulin stimulation (P > 0.05), and the activity of ET-1 promoter at each time point after insulin stimulation. Compared with the control group (P < 0.05 or P < 0.01), the activity of ET-1 promoter in insulin +Wortmannin group was significantly lower than that in the insulin treatment group (P < 0.05 or P < 0.01), but there was no significant difference compared with the control group (P > 0.05), and the activity of ET-1 promoter in the insulin + PD-98059 group was not significantly higher than that of the individual insulin treatment group. Sexual difference (P > 0.05) was significantly higher than that in the control group (P < 0.05 or P < 0.01). The results showed that the up regulation of ET-1 gene promoter activity was mediated by PI3K.
7.Vezf1 participates in the regulation of ET-1 gene expression by insulin
To verify whether insulin regulated ET-1 expression through the transcription factor Vezf1, HUVEC transfected pGL3-ET1 and Vezf1 binding sequence mutation plasmid pGL3-ET1-m respectively. After transfecting 48h, adding 100nM insulin culture solution and incubating 6h at 37 C, double luciferase detection kit was used to detect ET-1 promoter activity. Insulin obviously up-regulated transfection pGL3-ET1 inside The ET-1 promoter activity of the skin cells (P < 0.05), and the activity of ET-1 promoter transfected with pGL3-ET1-m endothelial cells was not significantly different from that of the control group after insulin stimulation. The results showed that Vezf1 was involved in the regulation of the expression of the ET-1 gene in the insulin.
conclusion
1. for the first time, we found that PI3K-GSK3 beta signaling pathway plays a key role in the expression of ET-1 gene in insulin regulated endothelial cells. The.PI3K-GSK3 beta signaling pathway may be the molecular basis for the increase of ET-1 expression in endothelial cells induced by hyperinsulinemia in insulin resistance.
2. endothelial cell specific transcription factor Vezf1 is involved in the regulation of insulin on ET-1 gene expression in endothelial cells.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R341

【共引文獻】

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