【摘要】：目的:高同型半胱氨酸血癥(HHcy)是心血管疾病的獨立危險因素(CVD)。我們實驗團隊先前證明,同型半胱氨酸(Hcy)抑制血管內皮細胞(EC)細胞增殖、遷移和損傷后修復,但Hcy誘導的EC損傷分子機制尚不清楚。在這項研究中,我們發現了一種新的F-BAR蛋白Carom,可介導的Hcy誘導的EC遷移和血管生成抑制。方法:1、實時熒光定量聚合酶鏈反應(RT-PCR)和蛋白免疫印跡實驗(WB)檢測人動脈內皮細胞(HAEC),24h 50μm DL-Hcy刺激后,Carom m RNA和蛋白在內皮細胞的表達。2、高通量液相質譜法檢測CBS KO小鼠同型半胱氨酸(Hcy)水平。WB檢測CBS KO小鼠主動脈Carom的表達,WB及流式細胞儀技術(FACS)檢測肺組織內皮細胞中Carom的表達。3、高通量液相質譜法檢測HAEC Hcy、S-腺蛋氨酸(SAM)和S-腺苷高半胱氨酸(SAH)水平,并計算SAM/SAH比例。用Actinnomyocin D及AZC處理內皮細胞,RT-PCR檢測內皮細胞Carom。4、Adv-Carom病毒或Carom Sh-RNA病毒轉染HAEC 72h,提取完整RNA,進行Carom相關的microarray及cytokine array。5、Adv-Carom病毒或Carom Sh-RNA病毒轉染HAEC 72h,RT-PCR檢測Carom及CXCL10 m RNA的表達水平。6、Hcy干預24h,Adv-Carom或Carom Sh-RNA病毒轉染HAEC后72h,進行細胞劃痕實驗,觀察Hcy和Carom對內皮細胞遷移功能的影響。同時加入CXCL10中和抗體,細胞劃痕實驗檢測內皮細胞Carom和CXCLl0對細胞遷移功能的影響。7、Hcy干預細胞后,提取不同細胞成分的蛋白,WB鑒定Carom在細胞質,膜,細胞核及細胞骨架上表達變化。設計并合成Carom相關的NLS肽,內皮細胞同時給予Hcy和NLS肽處理后,WB檢測Carom在染色質結合細胞核(CB)中的表達。內皮細胞Adv-Carom轉染和NLS肽處理后,RT-PCR檢測CXCLl0m RNA的表達。8、給予內皮細胞Hcy或Carom相關病毒轉染后,體外進行血管再生實驗,檢測Hcy和Carom對內皮細胞血管再生的抑制作用。9、內皮細胞給予Hcy或Carom相關病毒轉染后,分別給予兩種細胞內吞抑制劑Mitmab和Chroquine,流式細胞儀檢測Hcy和Carom對人血管生長因子受體2(VEGFR2)表達的影響。10、PIP array觀察GST-Carom融合蛋白對磷酸肌醇的結合能力。用生物素標記膜蛋白,Hcy干預后觀察細胞膜蛋白內吞的情況。11、內皮細胞過表達Carom,免疫共沉淀聯合質譜篩選,Carom可能的相互作用蛋白。結果:1、RT-PCR發現Hcy可誘導HAEC Carom m RNA的表達,分別為對照組1.87倍(12h),2.72倍(24h)和2.45倍(48h)P0.05。同時DL-Hcy可誘導內皮細胞Carom蛋白表達增加,并呈時間依賴性,48h Carom蛋白表達最高為對照組的2.2倍(P0.05)。2、發現12-16周Tg-h CBS Cbs-/-小鼠血漿Hcy水平為50-100μm,Tg-h CBS Cbs+/-和Tg-h CBS Cbs+/+小鼠血漿Hcy水平小于10μm。血漿Hcy水平高的Tg-h CBS Cbs-/-小鼠主動脈及肺組織內皮細胞Carom的表達高于對照組小鼠,是對照組小鼠的1.4倍和1.6倍(P0.05)。3、Hcy干預可增加內皮細胞SAH水平約為對照組的1.8倍(P0.05),降低了內皮細胞SAM/SAH的比例由原來2.2降為0.5左右(P0.05),導致內皮細胞的低甲基化水平。Actinomyosin D可阻斷Hcy誘導內皮細胞Carom m RNA的表達。不同濃度甲基化抑制劑AZC干預內皮細胞后,Carom蛋白的表達增加分別為對照組的1.9和2.2倍(P0.05)。4、Carom microarray篩選發現,與Adv-CT轉染組相比,Adv-Carom轉染組可使459個基因表達上調和460個基因表達下降(P0.05,FC1.2),Sh-RNA Carom可使653個基因表達下降,537個基金表達升高(P0.05,FC1.2)。其中有42個基因同時受Adv-Carom升高和sh-RNA Carom下降的調節。13個基因同時受Adv-Carom下降和sh-RANA Carom升高的調節。其中受Carom病毒轉染變化最大的基因是CXCL10。5、DL-Hcy 50μm分別干預HAEC 12h、24h、48h小時,RT-PCR發現同型半胱氨酸增加內皮細胞Carom m RNA表達的同時,也增加CXCL10 m RNA的表達,并且在24h表達最高,升高為對照組22.1倍(P0.05)。用內皮細胞過表達Carom后發現,Adv-Carom不僅能升高Carom m RNA的表達,同時CXCL10 m RNA也升高為對照組的27.5倍(P0.05)。Carom Sh-RNA轉染后,可抑制同型半胱氨酸誘導Carom的表達0.32倍(P0.05),同時抑制Hcy誘導CXCL10 m RNA的表達1.1倍(P0.05)。6、細胞劃痕實驗發現,Hcy刺激或Adv Carom病毒轉染可抑制內皮細胞的遷移,Carom sh-RNA可改善Hcy對內皮細胞遷移的抑制作用(P0.05),CXCLl0中和抗體(a-CXCL10)可減輕Carom對內皮細胞遷移的抑制作用(P0.05)。7、WB結果發現Carom除了在細胞膜(ME)表達最多外,Carom還可以在細胞質(CE),可溶性細胞核(NE),染色質結合細胞核(CB)和細胞骨架(PE)中表達。同型半胱氨酸干預內皮細胞(HAEC)24h,Carom蛋白主要在CB和PE中表達增加,分別為對照組2.2倍和1.75倍(P0.05)。Carom NLS肽可減少同型半胱氨酸誘導Carom在染色質結合細胞核(CB)中的表達,同時可抑制Carom誘導的CXCLl0 m RNA的表達,提示Carom調節CXCLl0的表達可能與細胞核內Carom的表達和Carom的入核運動相關。8、內皮細胞小管形成實驗發現,內皮細胞降低Carom表達后,可改善同型半胱氨酸對內皮細胞血管生成的抑制作用,由原來的35%升高為80%(P0.05),新生血管數量由原來的20上升至42(P0.05);內皮細胞過表達Carom可明顯抑制內皮細胞血管再生過程(P0.05)。Carom對內皮細胞的增值作用無影響。9、流式細胞儀檢測發現,與Adv-CT相比,過表達Carom組內皮細胞膜VEGFR2明顯減少為28.6%(P0.05),同型半胱氨酸也可抑制VEGFR2在內皮細胞的表達為41%(P0.05),同時給予Sh-RNA Carom病毒轉染后VEGFR2的表達升高為49%(P0.05,無統計學意義)。內吞抑制劑Mitmab可改善同型半胱氨酸和Adv-Carom對內皮細胞膜VEGFR2表達的抑制,分別由原來39%升高至58%,39%升高至51%(P0.05),Chroquine對VEGFR2表達無影響。10、PIP array表明GST-Carom融合蛋白易與各種磷酸肌醇結合,對Ptdlns(3)P及Ptdlns(3,5)P親和力最大。細胞膜蛋白生物素標記細胞內吞實驗表明,Hcy可誘導內皮細胞Carom及VEGFR2的內吞作用。11、免疫共沉淀蛋白印跡實驗(Co-IP)發現在Hcy刺激下,Carom不能與VEGFR2,CASK和MAGI1連接發生相互作用。過表達Carom免疫共沉淀聯合蛋白質譜分析發現,Carom可能在內皮細胞有13個相互作用蛋白,其中TRIM21可能通過與Carom連接,參與和介導Carom在內皮細胞的內吞。結論:1、Carom可在Hcy誘導的內皮細胞和高Hcy血癥的小鼠肺內皮細胞和主動脈中高表達,其機制可能與內皮細胞低甲基化水平相關。2、過表達Carom可增加CXCLl0 m RNA的表達,Carom可能通過CXCLl0介導Hcy對內皮細胞遷移功能的抑制,其機制可能與Carom NLS介導的染色質結合細胞核(CB)Carom的表達增加及Carom的入核運動相關。3、Carom可介導Hcy對內皮細胞血管生成的抑制,抑制內皮細胞膜VEGFR2的表達,其機制可能與Carom介導的細胞內吞作用相關。
[Abstract]:AIM: Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease (CVD). Our team has previously demonstrated that homocysteine inhibits the proliferation, migration and repair of vascular endothelial cells (EC) cells, but the molecular mechanism of Hcy-induced EC injury remains unclear. F-BAR protein Carom can mediate Hcy-induced EC migration and angiogenesis inhibition. Methods: 1. Real-time fluorescence quantitative polymerase chain reaction (RT-PCR) and protein immunoblotting assay (WB) were used to detect the expression of Carom m RNA and protein in human arterial endothelial cells (HAEC), 24 h 50 micron DL-Hcy stimulation, and high-throughput liquid chromatography-mass spectrometry was used to detect CBS KO. The levels of homocysteine (Hcy) in mice were measured by WB. The expression of Carom in the aorta of CBS KO mice was detected by WB. The expression of Carom in lung endothelial cells was detected by WB and FACS. The levels of HAEC Hcy, S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) were detected by high-throughput liquid chromatography-mass spectrometry. The ratio of SAM to SAH was calculated by Actinnomyocin D and FACS. Endothelial cells were treated with AZC. Carom 4, Adv-Carom virus or Carom Sh-RNA virus were transfected into HAEC by RT-PCR for 72 hours. Complete RNA was extracted and transfected with Carom-related microarray and cytokine array. 72 hours after transfection of HAEC with rom-Sh-RNA virus, the effects of Hcy and Carom on the migration of endothelial cells were observed. At the same time, CXCL-10 neutralizing antibody was added. The effects of Carom and CXCLl-0 on the migration of endothelial cells were detected by cell scratch test. 7. After Hcy interfered with cells, the proteins of different cell components were extracted and the Carom was identified by WB. Carom-related NLS peptides were designed and synthesized. After endothelial cells were treated with Hcy and NLS peptides, the expression of Carom in chromatin-binding nucleus (CB) was detected by WB. After endothelial cells were transfected with Adv-Carom and NLS peptides, the expression of CXCLl0m RNA was detected by RT-PCR. Hcy or Caro peptides were given to endothelial cells. Vascular regeneration test was carried out in vitro after transfection of m-associated virus to detect the inhibitory effect of Hcy and Carom on vascular regeneration of endothelial cells. 9. After transfection of Hcy or Carom-related virus, endothelial cells were given two kinds of endocytosis inhibitors Mitmab and Chroquine respectively. The expression of human vascular growth factor receptor 2 (VEGFR2) was detected by flow cytometry. PIP array was used to observe the binding ability of GST-Carom fusion protein to inositol phosphate.Biotin-labeled membrane protein was used to observe the endocytosis of membrane protein after Hcy intervention.11.Overexpression of Carom in endothelial cells was observed.Immunocoprecipitation combined with mass spectrometry was used to screen the possible interaction protein of Carom.Results:1.RT-PCR showed that Hcy could induce HAEC Caro. The expression of M RNA was 1.87 times (12h), 2.72 times (24h) and 2.45 times (48h) of the control group, respectively, P 0.05. DL-Hcy could induce the expression of Carom protein in endothelial cells in a time-dependent manner. The highest expression of Carom protein was 2.2 times (P 0.05). It was found that the plasma Hcy level of Tg-h CBS Cbs-/-mice at 12-16 weeks was 50-100um, Tg-h CBS Cbs+/-and Tg-h CBS Cbs-/-2, respectively. The levels of plasma Hcy in Tg-h CBS Cbs+/+ mice were less than 10 microns. The expression of Carom in endothelial cells of aorta and lung tissue of Tg-h CBS Cbs-/- mice with high plasma Hcy level was 1.4 and 1.6 times higher than that of control mice (P 0.05). Hcy intervention could increase the level of SAH in endothelial cells by 1.8 times (P 0.05), and decrease the level of SAH in endothelial cells. Actinomyosin D blocked Hcy-induced expression of Carom m RNA in endothelial cells. The expression of Carom protein increased by 1.9 and 2.2 times (P 0.05). Carom microarray sieve and AZC at different concentrations increased the expression of Carom protein in endothelial cells, respectively. Compared with the Adv-CT transfection group, the Adv-Carom transfection group could up-regulate the expression of 459 genes and down-regulate the expression of 460 genes (P 0.05, FC1.2), Sh-RNA Carom could down-regulate the expression of 653 genes and up-regulate the expression of 537 funds (P 0.05, FC1.2). Among them, CXCL10.5 was the most variable gene transfected by Carom virus, and DL-Hcy 50 micron interfered with HAEC 12 h, 24 h and 48 h respectively. RT-PCR showed that homocysteine increased the expression of Carom RNA and CXCL10 m RNA in endothelial cells, and the expression was the highest at 24 h, up to 0. Overexpression of Carom in endothelial cells showed that Adv-Carom not only increased the expression of Carom m RNA, but also increased the expression of CXCL10 m RNA by 27.5 times (P 0.05). Carom Sh-RNA transfection inhibited homocysteine-induced expression of Carom by 0.32 times (P 0.05) and inhibited Hcy-induced expression of CXCL10 m RNA by 1.1 times (P 0.0). 5) 6. Cell scratch test showed that Hcy stimulation or Adv Carom virus transfection could inhibit endothelial cell migration, Carom sh-RNA could improve the inhibition of Hcy on endothelial cell migration (P 0.05), CXCLl0 neutralizing antibody (a-CXCL10) could reduce the inhibition of Carom on endothelial cell migration (P 0.05). 7. WB results showed that Carom was expressed in the cell membrane (ME) except for Hcy. At most, Carom could also be expressed in cytoplasm (CE), soluble nucleus (NE), chromatin-binding nucleus (CB) and cytoskeleton (PE). Homocysteine interfered with endothelial cells (HAEC) for 24 hours, and the expression of Carom protein increased mainly in CB and PE, which were 2.2 and 1.75 times higher than that in control group (P 0.05). Carom NLS peptide decreased the induction of Ca by homocysteine. The expression of ROM in chromatin-binding cell nucleus (CB) and the expression of CXCLl0 m RNA induced by Carom were inhibited, suggesting that the expression of CXCLl0 regulated by Carom may be related to the expression of Carom in the nucleus and the movement of Carom into the nucleus. 8. Tubular formation experiment of endothelial cells showed that endothelial cells could improve homocysteine pairs by reducing the expression of Carom. The inhibition of endothelial cell angiogenesis increased from 35% to 80% (P 0.05), and the number of neovascularization increased from 20% to 42 (P 0.05). Overexpression of Carom in endothelial cells significantly inhibited the process of endothelial cell angiogenesis (P 0.05). Carom had no effect on endothelial cell proliferation. The expression of vascular endothelial growth factor R2 was significantly decreased by 28.6% (P Inhibition of the expression of VEGF R2 on the cell membrane increased from 39% to 58% and 39% to 51% (P 0.05), respectively. Chroquine had no effect on the expression of VEGF R2. 10. PIP array showed that GST-Carom fusion protein was easy to bind to various inositol phosphate and had the highest affinity for Ptdlns (3) P and Ptdlns (3,5) P. Carom could not interact with the binding of VEGFR2, CASK and MAGI1 under the stimulation of Hcy. Overexpression of Carom immunoprecipitation combined with protein profiling revealed that Carom may have 13 interacting proteins in endothelial cells, including TRIM21. Conclusion: 1. Carom can be highly expressed in Hcy-induced endothelial cells and in the pulmonary endothelial cells and aortas of mice with hypercythemia. The mechanism may be related to the hypomethylation level of endothelial cells. 2. Overexpression of Carom can increase the expression of CXCLl0 m RNA. Carom may increase the expression of CXCLl0 m RNA through CXCLl0. The mechanism of Hcy-mediated inhibition of endothelial cell migration may be related to the increased expression of Carom in the chromatin-binding nucleus (CB) mediated by Carom NLS and the movement of Carom into the nucleus. 3. Carom can mediate the inhibition of Hcy on endothelial cell angiogenesis and inhibit the expression of VEGF R2 in endothelial cell membrane. The mechanism may be Carom-mediated endocytosis. Function related.
【學位授予單位】：南昌大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R54

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8 苑t，

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