mmLDL損傷小鼠腸系膜動(dòng)脈內(nèi)皮依賴性舒張功能作用及機(jī)制
發(fā)布時(shí)間：2018-11-05 07:57
【摘要】：研究背景與目的弱氧化型低密度脂蛋白（minimally modified low densitylipoprotein，mmLDL）是指僅脂質(zhì)部分被氧化，載脂蛋白B100結(jié)構(gòu)中賴氨酸殘基未被破壞的LDL。離體器官和細(xì)胞培養(yǎng)研究發(fā)現(xiàn)，mmLDL可以顯著上調(diào)心腦血管平滑肌內(nèi)皮素B受體，增強(qiáng)血管的收縮功能；mmLDL還能與巨噬細(xì)胞上的TLR4結(jié)合，啟動(dòng)免疫反應(yīng)誘發(fā)大量炎癥因子的產(chǎn)生，還誘導(dǎo)細(xì)胞吞噬功能改變，，加速泡沫細(xì)胞的形成。但是，mmLDL是否對(duì)整體動(dòng)物血管舒張功能產(chǎn)生影響還不清楚。本實(shí)驗(yàn)將探討mmLDL對(duì)阻力血管腸系膜動(dòng)脈內(nèi)皮依賴性舒張功能的影響及其作用機(jī)制。為抗心腦血管疾病的研究提供一定的理論指導(dǎo)和實(shí)驗(yàn)基礎(chǔ)，并為心腦血管疾病的防治提供新的思路和新的藥物治療靶點(diǎn)。 方法（1）小鼠腸系膜動(dòng)脈mmLDL損傷模型建立：1mg/kg的劑量尾靜脈注射mmLDL，從第1次注射后開(kāi)始計(jì)時(shí)，之后每隔12h再注射1次，第六次注射后（即在0、12、24、36、48及60h各注射1次），將小鼠脫臼處死，取出含腸系膜動(dòng)脈組織，顯微鏡下剝離血管。（2）運(yùn)用微血管張力描記儀測(cè)定mmLDL對(duì)小鼠腸系膜動(dòng)脈收縮功能與舒張功能的影響。（3）運(yùn)用ELISA技術(shù)檢測(cè)血漿中炎癥因子IL-1β、TNF-α的濃度水平。（4）透射電鏡觀察血管內(nèi)皮超微結(jié)構(gòu)。（5）RT-PCR技術(shù)檢測(cè)腸系膜動(dòng)脈中IL-1β、TNF-α、KCa2.3、KCa3.1、 maxi KCa的mRNA表達(dá)水平。（6）采用WesternBlot技術(shù)檢測(cè)腸系膜動(dòng)脈中TLR4、TNF-α、KCa2.3、KCa3.1、 maxi KCa的蛋白表達(dá)量。 結(jié)果（1）尾靜脈注射mmLDL能時(shí)間和劑量依賴性的損傷小鼠腸系膜動(dòng)脈內(nèi)皮依賴性舒張功能，并且在1mg/kg的注射劑量和72小時(shí)作用達(dá)到最大。（2）與生理鹽水組pIC506.42±0.03和Rmax(63±5)%相比，mmLDL顯著損傷EDHF-通路介導(dǎo)的血管內(nèi)皮依賴性舒張功能[pIC505.67±0.07和Rmax(31±3)%，P0.001，P0.001]；與生理鹽水組pIC505.87±0.10和Rmax(47±4)%相比，mmLDL顯著損傷NO-通路介導(dǎo)的血管內(nèi)皮依賴性舒張功能[pIC505.44±0.12和Rmax(31±4)%，P0.01，P0.01]；而對(duì)PGI2-通路的損傷無(wú)顯著影響。（3）與生理鹽水組pIC506.44±0.11和Rmax(40±4)%相比，mmLDL顯著損傷KCa2.3-通路介導(dǎo)的血管內(nèi)皮依賴性舒張功能[pIC505.30±0.13和Rmax(18±3)%，P0.001，P0.01]；與生理鹽水組pIC505.44±0.18和Rmax(21±4)%相比，mmLDL顯著損傷KCa3.1-通路介導(dǎo)的血管內(nèi)皮依賴性舒張功能[pIC504.72±0.13和Rmax(8±2)%，P0.01，P0.01]；而對(duì)maxi KCa-通路的損傷基本無(wú)顯著影響。（4）mmLDL在局部區(qū)域損傷內(nèi)彈力膜，并且誘發(fā)腸系膜動(dòng)脈內(nèi)皮細(xì)胞剝落和水腫，導(dǎo)致了其對(duì)血管內(nèi)皮依賴性舒張功能的損傷。（5）mmLDL還誘導(dǎo)血管組織中TLR4蛋白表達(dá)明顯上調(diào)（P0.01）。（6）與生理鹽水組140.15±22.68pg/mL相比，mmLDL顯著上調(diào)血清中TNF-α的血清濃度水平（328.95±28.85，P0.001）；與生理鹽水組60.73±9.73pg/mL相比，mmLDL顯著上調(diào)血清中IL-1β的血清濃度水平（132.75±13.37，P0.01）；IL-1β和TNF-α的mRNA水平與血清濃度水平趨勢(shì)基本一致。 結(jié)論mmLDL顯著降低血管內(nèi)皮依賴性舒張功能，其機(jī)制與下列因素有關(guān)：（1）通過(guò)上調(diào)TLR4的蛋白表達(dá)量，誘發(fā)炎癥反應(yīng)，損傷內(nèi)皮細(xì)胞超微結(jié)構(gòu)。（2）抑制NO和EDHF信號(hào)通路。（3）下調(diào)KCa2.3-通道和KCa3.1-通道的蛋白表達(dá)。
[Abstract]:Background and objective weakly oxidized low density lipoprotein (mLDL) refers to LDL whose lipid fraction is oxidized and the lysine residue in apolipoprotein B100 structure is not destroyed. From the study of body organ and cell culture, mLDL can significantly upregulate the endothelin B receptor of cardiovascular and cerebrovascular smooth muscle and enhance the contraction function of blood vessel; mmLDL can also bind TLR4 on macrophages, initiate immune response to induce the generation of a great amount of inflammatory factors, and also induce the change of phagocyte function. the formation of foam cells is accelerated. However, it is not clear whether mmLDL has an impact on the whole animal's vasodilation function. This study will investigate the effect of mmLDL on endothelium-dependent vasodilation function of vascular mesenteric artery and its mechanism of action. To provide some theoretical guidance and experimental basis for the study of cardiovascular and cerebrovascular diseases, and to provide new thought and new drug therapy target for the prevention and treatment of cardiovascular and cerebrovascular diseases. Method (1) Mouse mesenteric artery mLDL injury model was established: 1 mg/ kg dose tail vein injection of mmLDL, the timing after the first injection, followed by injection once every 12h, and after the sixth injection (i.e. 1 times at 0, 12, 24, 36, 48, and 60h), the mice were dislocated. Euthanasia, removal of mesenteric artery tissue, dissection under microscope blood vessel. (2) Measurement of the contractile function and diastolic function of mLDL on mesenteric artery in mice by means of microangiogram (3) To detect the concentration of IL-1, TNF-and IL-1 in plasma by ELISA. Observation of vascular endothelial ultrastructure by transmission electron microscope (TEM) Structure. (5) RT-PCR technique was used to detect the mRNA expression of IL-1, TNF-, KCa2 +, KCa3. 1 and maxi KCa in mesenteric artery. Levels of TLR4, TNF-CoV, KCa2 + 3, KCa3. 1 and maxi KCa in mesenteric artery were detected by using Blot technique. Results (1) After intravenous injection of mLDL energy and dose-dependent injury to the mesenteric artery endothelium-dependent relaxation function, and in an injection amount of 1 mg/ kg and 72 hours Compared with normal saline group, pIC504.42 and Rmax (63/ 5)%, mmLDL was significantly injured by EDHF-pathway mediated vasodilation function[pIC505. 67, 0. 07 and Rmax (31% 3)%, P0.001, P0. 001]; compared with saline group, pIC505. 87, 0. 10 and Rmax (47%). Compared with 4%, mLDL was significantly injured by NO-pathway-mediated vasodilation function[pIC505. 44, 0. 12 and Rmax (31/ 4)%, P0.05). There was no significant effect. (3) Compared with saline group, pIC504.44, 0.011 and Rmax (40% 4)%, mmLDL significantly injured KCa2 +-pathway-mediated vasodilation function[pIC505. 30, 0. 13 and Rmax (18, 3)%, P0. 001, P0.01]; and pIC505. 44, 0. 18 and Rmax (21) in physiological saline group. Compared with 4%, mmLDL significantly damaged the endothelium-dependent vasodilation function of KCa3. 1-pathway[pIC504. 72, 0. 13 and Rmax (8, 2)%, P0. 01, P0.01], and the damage to maxi KCa-pathway was basic. There is no significant effect. (4) mLDL is an elastic membrane in local area injury and induces exfoliation and edema of mesenteric artery endothelial cells, leading to its endothelium-dependent relaxation. Functional damage. (5) mmLDL also induces significant upregulation of TLR4 protein expression in vascular tissues (P mLDL significantly upregulated the level of serum concentration (328,95, 28,85, P0.001) in serum as compared to the saline group of 140. 15 vs 22. 68pg/ mL; mLDL significantly upregulated the serum concentration level of IL-1 in serum (132. 75, 13.37, P 0. 01) Levels of mRNA and serum concentration of IL-1 and TNF-and IL-1 in serum Conclusion mmLDL significantly reduces the endothelium-dependent vasodilation function, and its mechanism is related to the following factors: (1) by upregulating the expression of TLR4 protein, inducing inflammatory response, injury, Ultrastructure of endothelial cells. (2) Inhibition of NO and EDHF signaling pathway. (3) down-regulation of KCa2 + 3-channel and KCa3. 1
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R54;R743.3
本文編號(hào)：2311411
[Abstract]:Background and objective weakly oxidized low density lipoprotein (mLDL) refers to LDL whose lipid fraction is oxidized and the lysine residue in apolipoprotein B100 structure is not destroyed. From the study of body organ and cell culture, mLDL can significantly upregulate the endothelin B receptor of cardiovascular and cerebrovascular smooth muscle and enhance the contraction function of blood vessel; mmLDL can also bind TLR4 on macrophages, initiate immune response to induce the generation of a great amount of inflammatory factors, and also induce the change of phagocyte function. the formation of foam cells is accelerated. However, it is not clear whether mmLDL has an impact on the whole animal's vasodilation function. This study will investigate the effect of mmLDL on endothelium-dependent vasodilation function of vascular mesenteric artery and its mechanism of action. To provide some theoretical guidance and experimental basis for the study of cardiovascular and cerebrovascular diseases, and to provide new thought and new drug therapy target for the prevention and treatment of cardiovascular and cerebrovascular diseases. Method (1) Mouse mesenteric artery mLDL injury model was established: 1 mg/ kg dose tail vein injection of mmLDL, the timing after the first injection, followed by injection once every 12h, and after the sixth injection (i.e. 1 times at 0, 12, 24, 36, 48, and 60h), the mice were dislocated. Euthanasia, removal of mesenteric artery tissue, dissection under microscope blood vessel. (2) Measurement of the contractile function and diastolic function of mLDL on mesenteric artery in mice by means of microangiogram (3) To detect the concentration of IL-1, TNF-and IL-1 in plasma by ELISA. Observation of vascular endothelial ultrastructure by transmission electron microscope (TEM) Structure. (5) RT-PCR technique was used to detect the mRNA expression of IL-1, TNF-, KCa2 +, KCa3. 1 and maxi KCa in mesenteric artery. Levels of TLR4, TNF-CoV, KCa2 + 3, KCa3. 1 and maxi KCa in mesenteric artery were detected by using Blot technique. Results (1) After intravenous injection of mLDL energy and dose-dependent injury to the mesenteric artery endothelium-dependent relaxation function, and in an injection amount of 1 mg/ kg and 72 hours Compared with normal saline group, pIC504.42 and Rmax (63/ 5)%, mmLDL was significantly injured by EDHF-pathway mediated vasodilation function[pIC505. 67, 0. 07 and Rmax (31% 3)%, P0.001, P0. 001]; compared with saline group, pIC505. 87, 0. 10 and Rmax (47%). Compared with 4%, mLDL was significantly injured by NO-pathway-mediated vasodilation function[pIC505. 44, 0. 12 and Rmax (31/ 4)%, P0.05). There was no significant effect. (3) Compared with saline group, pIC504.44, 0.011 and Rmax (40% 4)%, mmLDL significantly injured KCa2 +-pathway-mediated vasodilation function[pIC505. 30, 0. 13 and Rmax (18, 3)%, P0. 001, P0.01]; and pIC505. 44, 0. 18 and Rmax (21) in physiological saline group. Compared with 4%, mmLDL significantly damaged the endothelium-dependent vasodilation function of KCa3. 1-pathway[pIC504. 72, 0. 13 and Rmax (8, 2)%, P0. 01, P0.01], and the damage to maxi KCa-pathway was basic. There is no significant effect. (4) mLDL is an elastic membrane in local area injury and induces exfoliation and edema of mesenteric artery endothelial cells, leading to its endothelium-dependent relaxation. Functional damage. (5) mmLDL also induces significant upregulation of TLR4 protein expression in vascular tissues (P mLDL significantly upregulated the level of serum concentration (328,95, 28,85, P0.001) in serum as compared to the saline group of 140. 15 vs 22. 68pg/ mL; mLDL significantly upregulated the serum concentration level of IL-1 in serum (132. 75, 13.37, P 0. 01) Levels of mRNA and serum concentration of IL-1 and TNF-and IL-1 in serum Conclusion mmLDL significantly reduces the endothelium-dependent vasodilation function, and its mechanism is related to the following factors: (1) by upregulating the expression of TLR4 protein, inducing inflammatory response, injury, Ultrastructure of endothelial cells. (2) Inhibition of NO and EDHF signaling pathway. (3) down-regulation of KCa2 + 3-channel and KCa3. 1
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R54;R743.3
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