【摘要】：第一部分抗氧化應(yīng)激轉(zhuǎn)錄因子Nrf2在VDN誘導(dǎo)的大鼠血管鈣化模型中的作用目的氧化應(yīng)激是多種疾病合并的血管鈣化發(fā)生的共同病理因素,但是抗氧化應(yīng)激機(jī)制對(duì)血管鈣化的研究尚待深入。本研究擬初步探討機(jī)體內(nèi)源性抗氧化應(yīng)激轉(zhuǎn)錄因子Nrf2及其調(diào)控的下游抗氧化應(yīng)激分子在維生素D3 (Vitamin D3)和尼古丁(Nicotine)誘導(dǎo)的大鼠血管鈣化模型(VDN)中的變化,通過在體研究證實(shí)其在血管鈣化發(fā)生中的作用。方法選取20只兩月齡的Wistar雄性大鼠,隨機(jī)分為對(duì)照組和VDN鈣化組,鈣化組大鼠早上9點(diǎn)鐘分別給予尼古丁(25mg/kg)灌胃和維生素D3(30萬單位/kg)肌肉注射,晚上6點(diǎn)再給予尼古丁25mg/kg灌胃一次,對(duì)照組給予生理鹽水注射。兩個(gè)月后取大鼠血管組織：硝酸銀染色測定血管鈣化的發(fā)生程度;試劑盒測定組織鈣含量和ALP活性;Westernblot檢測成骨標(biāo)記蛋白R(shí)unx2、OPN、βcatenin的表達(dá),同時(shí)檢測Nrf2及其下游抗氧化酶NQO1的表達(dá)。結(jié)果與對(duì)照組相比,VDN鈣化組大鼠血管中層出現(xiàn)明顯鈣化,血管組織鈣含量和ALP活性均顯著增加(P0.05),成骨標(biāo)記蛋白R(shí)unx2、OPN、β catenin的表達(dá)顯著增加：同時(shí),與對(duì)照組相比,VDN鈣化組大鼠血管組織Nrf2及其誘導(dǎo)的抗氧化酶NQO1的表達(dá)也均增加。結(jié)論在VDN誘導(dǎo)的大鼠血管鈣化中,Nrf2及其下游抗氧化酶NQO1被激活,參與了血管鈣化的發(fā)生。第二部分抗氧化應(yīng)激轉(zhuǎn)錄因子Nrf2對(duì)高磷誘導(dǎo)的血管平滑肌細(xì)胞鈣化的影響及其分子機(jī)制研究目的氧化應(yīng)激、凋亡和成骨樣表型轉(zhuǎn)變是血管平滑肌細(xì)胞(vascular smooth muscle cells, VSMCs)參與和調(diào)控血管鈣化的主要機(jī)制,作為機(jī)體最重要的內(nèi)源性抗氧化應(yīng)激轉(zhuǎn)錄因子,Nrf2在氧化應(yīng)激防御中發(fā)揮關(guān)鍵作用,具有顯著的抗氧化應(yīng)激和抗凋亡作用,同時(shí)最新的研究也發(fā)現(xiàn),Nrf2是調(diào)控骨代謝和穩(wěn)態(tài)的新型作用因子。本實(shí)驗(yàn)旨在深入探討Nrf2對(duì)高磷誘導(dǎo)的血管平滑肌細(xì)胞鈣化的作用及其分子機(jī)制。方法酶解法分離Wistar大鼠的原代血管平滑肌細(xì)胞,分為對(duì)照組,鈣化組,Nrf2沉默組和Nrf2激活組。對(duì)照組用正常培養(yǎng)基培養(yǎng),鈣化組的細(xì)胞培養(yǎng)基中給予10mMβ磷酸甘油(β glycerophosphate, PGP)培養(yǎng),Nrf2激活組細(xì)胞同時(shí)選用特異性的Nrf2激活劑DMF和tBHQ干預(yù),Nrf2沉默組細(xì)胞應(yīng)用RNA干擾技術(shù)使Nrf2表達(dá)沉默。應(yīng)用茜素紅染色檢測各組細(xì)胞鈣化程度；應(yīng)用試劑盒檢測各組細(xì)胞ALP活性和鈣含量;應(yīng)用Westernblot和RT-PCR技術(shù)檢測各組細(xì)胞Nrf2及其下游NQO1和HO1的表達(dá),以及各組細(xì)胞成骨標(biāo)記BMP2、Runx2、OPN、β catenin的表達(dá),應(yīng)用流式細(xì)胞術(shù)和Westernblot分別檢測細(xì)胞凋亡比例及cleaved caspase3的表達(dá)；應(yīng)用流式細(xì)胞儀及試劑盒分別檢測細(xì)胞ROS活性和MDA含量。結(jié)果①與對(duì)照組相比,鈣化組細(xì)胞培養(yǎng)10天后茜素紅染色出現(xiàn)明顯的鈣化,細(xì)胞內(nèi)鈣含量和ALP活性均顯著增加,成骨標(biāo)記蛋白BMP2、Runx2、OPN、β catenin的表達(dá)均顯著增高,細(xì)胞ROS活性及MDA含量顯著上升,cleaved caspase3表達(dá)及細(xì)胞凋亡率也顯著增加。②與對(duì)照組相比,Nrf2沉默組Nrf2的mRNA水平降低約70%,Nrf2下游抗氧化酶NQO1和HO1表達(dá)也顯著下降,同時(shí)成骨標(biāo)記蛋白BMP2、Runx2、OPN、β catenin的表達(dá)均顯著增高,細(xì)胞鈣含量顯著上升。③與鈣化組相比,Nrf2激活劑DMF和tBHQ干預(yù)之后,Nrf2下游抗氧化酶NQO1被顯著激活,同時(shí)成骨標(biāo)記蛋白BMP2、Runx2、OPN、βcatenin的表達(dá)均顯著下降,細(xì)胞ROS活性及MDA含量顯著下降,cleaved caspase3表達(dá)及細(xì)胞凋亡率也顯著下降。結(jié)論Nrf2通過抑制高磷誘導(dǎo)的血管平滑肌細(xì)胞的成骨樣表型轉(zhuǎn)變、減輕血管平滑肌細(xì)胞的氧化應(yīng)激和凋亡,進(jìn)而抑制高磷誘導(dǎo)的血管平滑肌細(xì)胞鈣化的發(fā)生。
[Abstract]:The oxidative stress of the first part of the anti-oxidative stress transcription factor Nrf2 in the vascular calcification model of the rat induced by VDNN is a common pathological factor of the blood vessel calcification combined with various diseases, but the research of the anti-oxidative stress mechanism to the vascular calcification is still to be studied. This study is to explore the changes of endogenous anti-oxidative stress transcription factor Nrf2 and its regulated downstream anti-oxidative stress molecule in the vascular calcification model (VDN) induced by vitamin D3 (Vitamine D3) and nicotine (Nicotine), The role of this in the occurrence of vascular calcification was demonstrated by the in vivo study. Methods Twenty two-month-old Wistar male rats were randomly divided into the control group and the VDN calcified group. The rats were given nicotine (25mg/ kg) and vitamin D3 (30 000 units/ kg) intramuscularly at 9 o 'clock in the calcified group, and the nicotine was given at 6: 00 at night for one time. The control group was given saline injection. After two months, rat blood vessel tissue: silver nitrate staining was used to determine the degree of blood vessel calcification; the content of calcium and ALP activity of the tissue were determined by the kit; Western blot was used to detect the expression of bone marker protein Runx2, OPN, and Catenin, and the expression of Nrf2 and its downstream anti-oxidation enzyme NQO1 was also detected. Results Compared with the control group, there was a significant increase in the levels of calcium and ALP in the vascular middle layer of the VDN-calcified group (P0.05), and the expression of the bone marker protein Runx2, OPN, and Catenin increased significantly (P0.05). The expression of Nrf2 and its induced antioxidant NQO1 in the vascular tissue of the VDN-calcified group was also increased. Conclusion Nrf2 and its downstream anti-oxidation enzyme NQO1 were activated in the vascular calcification induced by VDNN, and were involved in the occurrence of vascular calcification. The effect of the second part of the anti-oxidative stress transcription factor Nrf2 on the calcification of high-phosphorus-induced vascular smooth muscle cells and its molecular mechanism are the main mechanism of vascular smooth muscle cells (VSMCs) to participate in and control the vascular calcification. As the most important endogenous anti-oxidative stress transcription factor, Nrf2 plays a key role in the prevention of oxidative stress, and has significant anti-oxidative stress and anti-apoptotic effects. The purpose of this study was to investigate the role of Nrf2 on the calcification of vascular smooth muscle cells induced by high phosphorus and its molecular mechanism. Methods The primary vascular smooth muscle cells of Wistar rats were divided into control group, calcification group, Nrf2 silencing group and Nrf2 activation group. The control group was cultured with normal medium, and 10 mM triphosphoglycerate (PGP) was given to the cell culture medium of the calcified group, and the specific Nrf2 activator DMF and tBHQ were selected for the Nrf2 activated group cells, and the Nrf2 expression was silenced by the RNA interference technique in the Nrf2 silencing group cells. The expression of Nrf2 and its downstream NQO1 and HO1 in each group was detected by Western blot and RT-PCR, and the expression of BMP2, Runx2, OPN and Catenin in each group was detected by Western blot and RT-PCR. The cell apoptosis and the expression of clear caspase3 were detected by flow cytometry and Western blot. The activity of ROS and the content of MDA were detected by flow cytometry and kit. Results Compared with the control group, the content of the intracellular calcium and the activity of ALP increased significantly after 10 days after the cell culture of the calcified group, and the expression of the bone marker protein BMP2, Runx2, OPN and Catenin increased significantly, and the activity of ROS and the content of MDA increased significantly. The expression of clear caspase3 and the cell apoptosis rate were also increased. Compared with the control group, the mRNA level of Nrf2 in the Nrf2 group was reduced by about 70%, and the expression of NQO1 and HO1 downstream of Nrf2 was also significantly decreased, while the expression of the bone marker protein BMP2, Runx2, OPN, and Catenin increased significantly, and the content of calcium in the cells increased significantly. Compared with the calcified group, the Nrf2 downstream anti-oxidation enzyme NQO1 was significantly activated after the intervention of the Nrf2 activator DMF and tBHQ, while the expression of the bone marker protein BMP2, Runx2, OPN, and Catenin decreased significantly, and the ROS activity and MDA content of the cells decreased significantly, and the clear caspase3 expression and the cell apoptosis rate were also significantly reduced. Conclusion Nrf2 can reduce the oxidative stress and apoptosis of vascular smooth muscle cells by inhibiting the transformation of bone-like phenotype of high-phosphorus-induced vascular smooth muscle cells, and further inhibit the occurrence of high-phosphorus-induced vascular smooth muscle cell calcification.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R54

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