本文選題：血管內(nèi)皮細胞 + 凋亡　； 參考：《山東大學》2008年博士論文

【摘要】： 研究背景和目的 血管內(nèi)皮細胞(VEC)在血管發(fā)育中起重要作用。大量實驗結果表明,血管內(nèi)皮細胞凋亡導致的血管功能異常,將引起多種炎癥和退行性疾病的發(fā)生。這些重大疾病嚴重危害人類健康,如何防治這些疾病是目前亟待解決的問題。 新血管形成(angiogenesis)在生理過程(如胚胎發(fā)育)和病理過程(如腫瘤生長、動脈粥樣硬化)中都起到關鍵作用。新血管形成與內(nèi)皮細胞凋亡有密切關系,抑制血管內(nèi)皮細胞凋亡是促進新血管形成的關鍵因素,但是兩者關聯(lián)的分子機制目前尚未搞清。 從化學遺傳學的角度,利用小分子化合物可以發(fā)現(xiàn)參與血管內(nèi)皮細胞凋亡和血管形成的新關鍵因子,因此可為闡明上述科學問題提供實驗證據(jù)。先前的研究證明去除血清和生長因子可誘導人臍靜脈內(nèi)皮細胞(HUVEC)凋亡,在此條件下,6-氨基-2,3-二氫-3-羥甲基-1,4-苯并VA嗪衍生物(ABO)能提高血管內(nèi)皮細胞的存活率,因此推斷ABO可能是血管內(nèi)皮細胞凋亡抑制劑和血管形成促進劑。 本論文研究目的是從化學遺傳學角度,以小分子化合物ABO為工具,研究血管內(nèi)皮細胞凋亡、血管形成以及兩者關聯(lián)的分子機制。為血管退行性重大疾病的防治提供新線索和靶點。 已知細胞內(nèi)的氧化還原反應(redox)在細胞凋亡和血管形成中起重要作用,參與redox的成分包括:線粒體、活性氧(ROS)、NADPH氧化酶、超氧化物歧化酶(SOD)以及一氧化氮(NO)/內(nèi)皮型一氧化氮合酶(eNOS)等。因此,本論文首先研究了ABO對redox信號分子的調節(jié)機制。隨后研究了在ABO的作用下,其它與凋亡和成血管密切相關的分子(如:磷脂酰膽堿特異性磷脂酶C(PC-PLC)、P53、膜整連蛋白β_4、核因子κB(NF-κB)以及H-ras)的變化情況。 研究內(nèi)容 1.ABO抑制去除血清和生長因子誘導血管內(nèi)皮細胞凋亡的作用 2.ABO促進血管生成的作用 3.ABO抑制血管內(nèi)皮細胞凋亡和促進血管生成的分子機制研究 研究方法 1.血管內(nèi)皮細胞培養(yǎng):人臍靜脈內(nèi)皮細胞的提取和培養(yǎng)參考Jaffe EA et al的方法[Jaffe EA et al,1973] 2.細胞凋亡檢測: 1)MTT檢測細胞存活率 2)倒置相差顯微鏡觀察細胞形態(tài)變化 3)吖啶橙染色結合激光掃描共聚焦顯微鏡,觀察細胞核凝集及片斷化 4)TUNEL方法,檢測細胞凋亡率 3.體外血管形成檢測:Matrigel方法,參考[Kureishi Y et al,2000] 4.體內(nèi)血管形成檢測:雞胚尿囊膜(CAM)方法,參考[Ribatti et al,1997] 5.體外細胞遷移檢測:平面單層細胞損傷愈合實驗,結合倒置相差顯微鏡觀察,參考[Bürk,1973;Vasvari et al,2007] 6.線粒體膜電位(MMP)檢測:利用熒光探針(TMRM)并結合激光掃描共聚焦顯微術檢測,參考[Falchi et al,2005] 7.ROS檢測:利用熒光探針(DCHF)并結合激光掃描共聚焦顯微術檢測 8.NADPH氧化酶活性檢測:參考Li et al的方法[Li et al,2002] 9.SOD活性檢測:利用SOD檢測試劑盒 10.NO含量檢測:利用NO檢測試劑盒 11.eNOS活性檢測:利用eNOS檢測試劑盒 12.PC-PLC活性檢測:參考吳興中等人的方法[Wu et al,1997] 13.細胞內(nèi)蛋白分布及表達水平檢測: 1)免疫細胞化學法結合激光掃描共聚焦顯微術,檢測P53和NF-κB蛋白的表達水平及分布 2)Western blot方法,檢測P53、膜整連蛋白β_4和H-ras蛋白的表達水平 研究結果 1.ABO抑制去除血清和生長因子誘導的血管內(nèi)皮細胞凋亡 在去除血清和FGF-2的條件下,ABO(50-200μM)處理血管內(nèi)皮細胞24 h,HUVEC的存活率明顯升高(圖2,p＜0.05),其最佳濃度為50μM,因此在以下研究中均用該濃度處理血管內(nèi)皮細胞。用ABO處理細胞12和24 h,凋亡小體明顯減少(圖3);吖啶橙染色和TUNEL染色結果表明,ABO明顯抑制血管內(nèi)皮細胞凋亡(圖4和圖5,p＜0.05)。 2.ABO促進血管生成 2.1體外Matrigel毛細血管樣結構形成實驗結果表明,在去除血清和FGF-2的條件下,ABO以時間依賴的方式(24-96 h)明顯促進血管生成(圖6,p＜0.01);存在FGF-2(70ng/mL)的條件下,ABO和FGF-2可協(xié)同促進血管生成(圖7);僅有存在血清(20%,v/v)的條件下,ABO不能促進血管生成(圖8);血清(20%,v/v)和FGF-2(70ng/mL)都存在的條件下,在培養(yǎng)后期,ABO促進血管生成(圖9)。 2.2體內(nèi)雞胚尿囊膜血管形成實驗顯示,ABO(20nmol/100μL)促進血管網(wǎng)形成(圖10,p＜0.05)。 2.3單層細胞損傷愈合實驗結果顯示,ABO以時間依賴的方式(6-24 h)顯著促進細胞遷移(圖11,p＜0.01)。 3.ABO抑制血管內(nèi)皮細胞凋亡和促進血管生成的分子機制 3.1用ABO處理細胞12和24 h,可以明顯降低線粒體膜電位(圖12和圖13,p＜0.05)和細胞內(nèi)ROS水平(圖14和圖15,p＜0.05)。 3.2用ABO處理細胞12 h,可以明顯降低NADPH氧化酶(圖16,p＜0.05)和PC-PLC的活性(圖19,p＜0.05),但對SOD的活性沒有影響(圖17,p＞0.05)。 3.3用ABO處理細胞12 h,明顯增強eNOS的活性(圖18B,p＜0.05)并增加NO含量(圖18A,p＜0.05),但是處理6和24 h,eNOS的活性和NO釋放量沒有變化(圖18)。 3.4免疫細胞化學檢測結果表明,用ABO處理細胞6和12 h,NF-κB在細胞質中均勻分布,未觀察到NF-κB的核移位現(xiàn)象(圖23);用ABO處理細胞24 h,明顯抑制P53蛋白的表達并阻止了P53的核移位(圖20,p＜0.05)。 3.5 Western Blot檢測結果表明,用ABO處理細胞24 h,明顯抑制P53蛋白(圖21B,p＜0.05)和膜整連蛋白β_4的表達(圖22B,p＜0.05),但是用ABO處理12 h,P53和膜整連蛋白β_4的表達均沒有變化(圖21A和圖22A,p＞0.05)。用ABO處理細胞12和24 h,均明顯抑制H-ras蛋白的表達(圖24,p＜0.05)。 結論 1.ABO明顯抑制去除血清和生長因子誘導的血管內(nèi)皮細胞凋亡。 2.ABO有效促進血管生成。 3.在去除血清和生長因子的條件下,ABO通過穩(wěn)定線粒體膜電位,防止超極化,維持線粒體的功能,同時通過降低NADPH氧化酶的活性,抑制了細胞內(nèi)過高的ROS水平而有效抑制了細胞凋亡,并促進了細胞遷移和血管生成。此外,ABO還通過增強eNOS的活性使NO的含量上升,進一步加強血管生成作用。 4.ROS、PC-PLC和p53之間有密切聯(lián)系且均與線粒體相關。ABO很可能通過影響這一條信號轉導途徑中的相關因子,即控制ROS的水平,抑制PC-PLC的活性以及P53、膜整連蛋白β_4和H-ras蛋白的表達,抑制了去除血清和生長因子誘導的HUVEC凋亡并促進了血管形成。說明ABO是研究血管內(nèi)皮細胞凋亡、血管形成和兩者關聯(lián)機制的有效工具。
[Abstract]:Background and purpose of research
Vascular endothelial cells (VEC) play an important role in the development of blood vessels. A large number of experimental results show that abnormal vascular function caused by vascular endothelial cell apoptosis will cause a variety of inflammatory and degenerative diseases. These major diseases seriously harm human health, and how to prevent and control these diseases is a problem to be solved urgently.
New vascular formation (angiogenesis) plays a key role in physiological processes (such as embryonic development) and pathological processes (such as tumor growth, atherosclerosis). New vascular formation is closely related to endothelial cell apoptosis. Inhibition of vascular endothelial cell apoptosis is a key factor in the promotion of new vascular formation. However, the molecular mechanisms associated with them are currently involved. It is not clear yet.
From the perspective of chemical genetics, small molecular compounds can be used to detect new key factors involved in the apoptosis and angiogenesis of vascular endothelial cells, and thus provide experimental evidence to clarify the scientific problems mentioned above. Previous studies have shown that the removal of serum and growth factors can induce apoptosis of human umbilical vein endothelial cells (HUVEC), and in this condition, 6- ammonia Base -2,3- two hydroxy -3- hydroxymethyl -1,4- benzo VA derivatives (ABO) can increase the survival rate of vascular endothelial cells. Therefore, ABO may be an inhibitor of vascular endothelial cell apoptosis and angiogenesis promoter.
The purpose of this study is to study the apoptosis, angiogenesis and the molecular mechanism of vascular endothelial cells from the perspective of chemical genetics, with small molecular compound ABO as a tool for the prevention and treatment of vascular degenerative diseases.
The known redox reaction (redox) plays an important role in cell apoptosis and angiogenesis, and the components involved in redox include mitochondria, reactive oxygen species (ROS), NADPH oxidase, superoxide dismutase (SOD) and nitric oxide (NO) / endothelial nitric oxide synthase (eNOS). Therefore, this paper first studied the redox signal molecule of ABO. The changes in other molecules that are closely related to apoptosis and angiogenesis, such as phosphatidylcholine specific phospholipase C (PC-PLC), P53, membrane protein beta _4, nuclear factor kappa B (NF- kappa B) and H-ras, are subsequently studied under the action of ABO.
research contents
1.ABO inhibits vascular endothelial cell apoptosis induced by serum and growth factors
The role of 2.ABO in promoting angiogenesis
Molecular mechanism of 3.ABO inhibiting apoptosis and promoting angiogenesis in vascular endothelial cells
research method
1. endothelial cell culture: extraction and culture of human umbilical vein endothelial cells by reference to Jaffe EA et al [Jaffe EA et al 1973]
2. detection of cell apoptosis:
1) MTT detection of cell viability
2) observation of cell morphological changes by inverted phase contrast microscope
3) acridine orange staining combined with laser scanning confocal microscopy to observe nuclear agglutination and fragmentation.
4) TUNEL method, detection of apoptosis rate
3. in vitro angiogenesis detection: Matrigel method, reference [Kureishi Y et al, 2000]
4. detection of blood vessel formation in vivo: chicken embryo allantoic membrane (CAM) method, refer to [Ribatti et al, 1997]
5. in vitro cell migration assay: planar monolayer cell injury healing experiment, combined with inverted phase contrast microscope, with reference to [B u rk, 1973; Vasvari et al, 2007]
6. mitochondrial membrane potential (MMP) detection: fluorescence probe (TMRM) combined with laser scanning confocal microscopy, reference [Falchi et al, 2005]
7.ROS detection: fluorescence probe (DCHF) combined with laser scanning confocal microscopy.
8.NADPH oxidase activity detection: reference Li et al method [Li et al, 2002]
9.SOD activity detection: using SOD detection kit
10.NO content detection: using NO detection kit
11.eNOS activity detection: using eNOS detection kit
12.PC-PLC activity detection: reference to Wu Xingzhong et al. Method [Wu et al, 1997]
The distribution and expression level of protein in 13. cells were detected.
1) immunocytochemistry and laser scanning confocal microscopy were used to detect the expression level and distribution of P53 and NF- kappa B protein.
2) Western blot method was used to detect the expression level of P53, _4 and H-ras protein.
Research results
1.ABO inhibits serum and growth factor induced apoptosis of vascular endothelial cells
Under the conditions of removing serum and FGF-2, ABO (50-200 mu M) treated vascular endothelial cells 24 h, and the survival rate of HUVEC increased significantly (Figure 2, P < 0.05). The optimum concentration was 50 mu M. Therefore, the concentration of vascular endothelial cells was treated with this concentration in the following studies. The apoptotic bodies were significantly reduced by ABO treatment of 12 and 24 h (Fig. 3); acridine orange staining and TUNEL staining. The results showed that ABO significantly inhibited the apoptosis of vascular endothelial cells (FIGS. 4 and 5, P < 0.05).
2.ABO promotes angiogenesis
2.1 in vitro Matrigel capillary like structure formation experimental results showed that, under the condition of removing serum and FGF-2, the time dependent manner (24-96 h) significantly promoted angiogenesis (Figure 6, P < 0.01); under the condition of FGF-2 (70ng/mL), ABO and FGF-2 could promote angiogenesis (Fig. 7); only under the presence of serum (20%, v/v), ABO was not Promoting angiogenesis (Fig. 8); in serum (20%, v/v) and FGF-2 (70ng/mL), ABO promoted angiogenesis at the later stage of culture (Fig. 9).
2.2 angiogenesis experiments in chicken chorioallantoic membrane showed that ABO (20nmol/100 L) promoted the formation of vascular network (FIG. 10, P < 0.05).
2.3 the single cell injury healing experiment showed that ABO significantly promoted cell migration in a time-dependent manner (6-24 h) (FIG. 11, P < 0.01).
Molecular mechanism of 3.ABO inhibiting apoptosis and promoting angiogenesis in vascular endothelial cells
3.1 treatment of cells 12 and 24 h with ABO significantly reduced mitochondrial membrane potential (Figs 12 and 13, P < 0.05) and intracellular ROS level (Figs 14 and 15, P < 0.05).
3.2 cell 12 h treated with ABO could significantly reduce the activity of NADPH oxidase (Figure 16, P < 0.05) and PC-PLC (Fig. 19, P < 0.05), but did not affect the activity of SOD (FIG. 17, P > 0.05).
3.3 the cell 12 h treated with ABO significantly enhanced the activity of eNOS (Figure 18B, P < 0.05) and increased the content of NO (Fig. 18A, P < 0.05), but the activity of 6 and 24 h, eNOS activity and NO release did not change (Fig. 18).
3.4 the results of immunocytochemical detection showed that the cell 6 and 12 h were treated with ABO, NF- kappa B was evenly distributed in the cytoplasm, and the nuclear shift of NF- kappa B was not observed (Fig. 23); the expression of 24 h by ABO was used to inhibit the expression of P53 protein and prevent the nuclear shift of P53 (FIG. 20, P < 0.05).
The results of 3.5 Western Blot test showed that the expression of P53 protein (21B, P < 0.05) and the expression of membrane protein beta _4 were obviously inhibited by ABO treatment of 24 h (Fig. 22B, P < 0.05), but there was no change in the expression of 12 h. Expression (Figure 24, P < 0.05).
conclusion
1.ABO significantly inhibited the apoptosis of vascular endothelial cells induced by serum and growth factor.
2.ABO effectively promotes angiogenesis.
3. under the conditions of removing serum and growth factors, ABO can prevent hyperpolarization and maintain mitochondrial function by stabilizing mitochondrial membrane potential, and reducing the high ROS level by reducing the activity of NADPH oxidase, which effectively inhibits apoptosis and promotes cell migration and angiogenesis. In addition, ABO is also enhanced by eNOS. The activity increased the content of NO and further enhanced angiogenesis.
The close links between 4.ROS, PC-PLC and p53 and mitochondrial related.ABO are likely to affect the level of ROS, the inhibition of the activity of PC-PLC, and the expression of P53, the expression of the membrane protein beta _4 and H-ras protein, which inhibit the removal of HUVEC apoptosis induced by the removal of serum and growth factors and promote the promotion of the apoptosis of HUVEC. Angiogenesis indicates that ABO is an effective tool for studying endothelial cell apoptosis, angiogenesis and their correlation mechanisms.

【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R363

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