本文選題：子癇前期 + 左卡尼汀�。� 參考：《天津醫(yī)科大學》2017年博士論文

【摘要】：研究目的:子癇前期(Preeclampsia,PE)不僅是導(dǎo)致孕產(chǎn)婦死亡和圍產(chǎn)兒死亡的主要原因,而且患者與其子代遠期心血管疾病(Cardiovascular Disease,CVD)發(fā)病率明顯增加。近年來研究顯示循環(huán)單核細胞亞群及蛻膜巨噬細胞(Decidual Macrophage,DMs)M2表型偏移參與妊娠期間免疫抑制和螺旋動脈重塑,是成功妊娠的關(guān)鍵。左卡尼汀(L-Carnitine,LC)抗炎、抗氧化,應(yīng)用于許多CVD中,同時還可以促進體外胚胎發(fā)育并提高體外受精成功率、上調(diào)組織PPAR-γ的表達。因此本研究通過觀察LC對PE樣小鼠妊娠結(jié)局及M2型DMs的影響,探索LC在PE中的應(yīng)用前景及潛在機制,并探討pStat6/Stat6-PPAR-γ信號通路在LC中介的干預(yù)效應(yīng)中的作用。內(nèi)容與方法:(1)7-8周齡雌性和雄性C57BL/6J小鼠適應(yīng)性喂養(yǎng)一周后,無創(chuàng)監(jiān)測鼠尾血壓,隨機將血壓穩(wěn)定的雌鼠分為8組,分別為對照組(A組)、PE組[妊娠第7天(Gestation Day 7,GD7)皮下注射L-NAME,B組]、PE+LC高劑量干預(yù)組(800mg/kg/d、C至E組)和PE+LC低劑量干預(yù)組(400mg/kg/d、F至H組)。C/F組、D/G、E/H組分別于孕前1周、GD0、GD9開始給予LC灌胃直至GD18結(jié)束妊娠。監(jiān)測各組血壓、24小時尿蛋白;高頻小動物超聲評價各組GD18血管功能;觀察子代宮內(nèi)生長發(fā)育狀況以及胎盤/腎臟結(jié)構(gòu)改變;分析A至E組外周血單核細胞Ly6Chi亞群、蛻膜巨噬細胞表型偏移的變化;檢測A至E組血漿氧化三甲胺(Trimethylamine Oxide,TMAO)濃度;監(jiān)測B組和C組動脈粥樣硬化程度;檢測A至E組糞便16S rDNA分析菌群分布情況。(2)L-NAME、LC以及GW9662(PPAR-γ阻滯劑)共同干預(yù)RAW 264.7細胞,觀察iNOS、IL-1β、TNF-α、CD206、ARG1、IL-10和PPAR-γm RNA表達水平以及PPAR-γ和Stat6信號通路相關(guān)蛋白pStat6/Stat6表達水平,探索LC對小鼠巨噬細胞表型偏移的影響及其機制。結(jié)果:(1)L-NAME可以引起C57BL/6J小鼠PE樣表現(xiàn),如高血壓、蛋白尿、宮內(nèi)生長受限和血管功能受損等,同時還可以誘導(dǎo)外周血Ly6Chi單核細胞亞群增加,蛻膜巨噬細胞M2表型偏移減少。Ly6Chi亞群與血壓、蛋白尿呈正相關(guān);與胎鼠重、頂臀長和胎盤直徑呈負相關(guān)(r=0.6363,r=0.6752,r=-0.6694,r=-0.6623,r=-0.6623,all P0.001);蛻膜組織巨噬細胞M2表型與血壓、蛋白尿呈負相關(guān),與胎鼠重、頂臀長和胎盤重呈正相關(guān)(r=-0.5214,r=-0.6562,r=0.4882,r=0.5701,r=0.5701,all P0.05)。(2)不同時間點給予不同劑量LC可以不同程度降低PE樣小鼠血壓、24小時尿蛋白,改善血管內(nèi)皮功能,改善子代宮內(nèi)生長受限,降低臍動脈和子宮-胎盤動脈的搏動指數(shù)(Plusatility Index,PI)和阻力指數(shù)(Resistance Index,RI);LC改善圍產(chǎn)期結(jié)局的程度隨著LC劑量的增加而增加,差異有統(tǒng)計學意義(all P0.05)。(3)流式細胞術(shù)分析結(jié)果顯示與A組相比,B組外周血單核細胞Ly6Chi亞群升高(61.7±8.66%vs.72.2±6.02%),蛻膜組織巨噬細胞M2表型表達減少(65.4±9.66 vs.53.7±4.08),差異有統(tǒng)計學意義(all P0.01)。孕前1周給予高劑量LC后Ly6Chi亞群比例下調(diào)(72.2±6.02%vs.61.25±3.21%),蛻膜巨噬細胞M2表型表達增加(53.7±4.08 vs.65.8±6.14),差異有統(tǒng)計學意義(all P0.01)。B組蛻膜組織M1表型標志物(TNF-α、IL-1β、iNOS)基因相對表達量較對照組增加,差異有統(tǒng)計學意義(1.00 vs.1.41±0.15;1.00 vs.5.35±1.14;1.00 vs.81.3±16.3;all P0.001);孕前1周、GD0給予高劑量LC干預(yù)后TNF-α、IL-1β、iNOS基因表達量均下降,差異有統(tǒng)計學意義(1.42±0.15 vs.0.34±0.14;5.35±1.14 vs.3.64±1.13;81.3±16.3 vs.8.84±1.41;all P0.001;1.42±0.15 vs.0.41±0.11;5.35±1.14vs.4.39±0.89;81.3±16.3 vs.12.32±1.04;all P0.05);GD9給予高劑量LC干預(yù)后iNOS基因表達量下降,差異有統(tǒng)計學意義(81.3±16.3 vs.20.2±3.37;P0.001)。B組蛻膜組織M2表型標志物CD206基因相對表達量較A組下調(diào),差異有統(tǒng)計學意義(1.00 vs.0.60±0.17;P0.001);IL-10、ARG1基因表達量與對照組差異無統(tǒng)計學意義(1.00 vs.1.46±0.31;1.00 vs.1.04±0.15;P0.05);孕前1周給予高劑量LC干預(yù)后CD206、IL-10基因表達量上升,差異有統(tǒng)計學意義(0.60±0.17vs.1.00±0.20;1.46±0.31 vs.2.57±0.66;all P0.001);GD0給予高劑量LC干預(yù)后,IL-10基因表達量上升,差異有統(tǒng)計學意義(1.46±0.31 vs.1.77±0.48;P0.05)。(4)蛻膜組織M2型巨噬細胞與孕鼠GD18血壓、24小時蛋白尿呈負相關(guān)(r=-0.5196,P0.001;r=-0.5132,all P0.001);與胎鼠重、頂臀長呈正相關(guān)(r=0.5408,r=0.5779,all P0.001)。(5)圍產(chǎn)期LC總量與蛻膜M2型巨噬細胞呈正相關(guān)(r=0.59,P0.001);與蛻膜組織TNF-α、IL-1β、iNOS基因表達量呈負相關(guān)(r=-0.80,r=-0.50,r=-0.96,all P0.001);與蛻膜組織CD206、IL-10基因表達量呈正相關(guān)(r=0.47,r=0.53,all P0.001)。(6)L-NAME可刺激RAW264.7細胞系M1巨噬細胞表型標志物(TNF-α、IL-1β、iNOS)基因表達量增加,差異有統(tǒng)計學意義(1.00 vs.1.56±0.19;1.00 vs.1.82±0.42;1.00 vs.3.90±0.80;all P0.05);聯(lián)合LC干預(yù)后TNF-α表達量下降,差異有統(tǒng)計學意義(1.56±0.19 vs.0.81±0.16;P0.01),IL-1β、iNOS表達量有下降趨勢,但差異無統(tǒng)計學意義(1.82±0.42 vs.1.58±0.42;2.67±0.80 vs.3.90±0.80;all P0.05),M2巨噬細胞表型標志物CD206、IL-10和ARG1基因表達量以及PPAR-γ基因表達量增加,差異有統(tǒng)計學意義(1.50±0.70 vs.6.43±1.36;3.45±0.65 vs.5.51±1.84;1.11±0.20 vs.2.21±0.53;1.02±0.27 vs.1.47±0.28;all P0.05);同時給予L-NAME、LC和GW9662(PPAR-γ阻滯劑)干預(yù)后M1、M2巨噬細胞表型標志物和PPAR-γ基因表達量與單純L-NAME刺激組相似,差異無統(tǒng)計學意義(all P0.05);L-NAME+LC組PPAR-γ和Stat6信號通路相關(guān)蛋白pStat6/Stat6表達水平較對照組增加,差異有統(tǒng)計學意義(all P0.05);加用GW9662后上述蛋白表達量下降,與對照組差異無統(tǒng)計學意義(all P0.05)。(7)A至E組血漿TMAO濃度差異無統(tǒng)計學意義;B組與C組相比主動脈瓣、主動脈及其主要分支動脈粥樣硬化差異無統(tǒng)計學意義(all P0.05)。A至E組小鼠糞便16S rDNA高通量測序結(jié)果顯示各組厚壁菌門和擬桿菌門占主要門類分布,組間差異無統(tǒng)計學意義,與血漿TMAO、圍產(chǎn)期服用LC總量無相關(guān)性;屬水平組間梭狀芽胞桿菌、甲型變形菌綱、梭菌屬XlVa、厭氧細桿菌屬、棍狀厭氧菌、歐陸森氏菌屬和八疊球菌屬差異有統(tǒng)計學意義,其中歐陸森氏菌屬與圍產(chǎn)期口服LC總量呈負相關(guān)(r=-0.7989,P0.01)。結(jié)論:本研究結(jié)果顯示孕鼠GD7皮下注射L-NAME可以引起PE樣表現(xiàn),是比較合理的PE樣模型。LC可能通過pStat6/Stat6-PPAR-γ信號通路介導(dǎo)蛻膜巨噬細胞M2表型偏移,改善PE樣小鼠圍產(chǎn)期妊娠結(jié)局;PE樣小鼠孕前1周給予口服LC直至GD18結(jié)束妊娠(800 mg/kg/d)對動脈粥樣硬化的發(fā)生發(fā)展無明顯影響,提示LC可能是防治PE的一個新的方法。
[Abstract]:Objective: Preeclampsia (PE) is not only the main cause of maternal mortality and perinatal death, but also the incidence of Cardiovascular Disease (CVD) in the patients and their progeny is significantly increased. In recent years, the study showed the circulating mononuclear subgroup and the M2 form of the decidual macrophage (Decidual Macrophage, DMs). Type migration involved in immunosuppression and spiral artery remodeling during pregnancy is the key to successful pregnancy. L-Carnitine (LC), anti-inflammatory and antioxidant, is used in many CVD, and can also promote the development of in vitro embryos and improve the success rate of in vitro fertilization and up regulation of the expression of tissue PPAR- gamma. Therefore, this study was conducted by observing LC to PE like mice. The effect of pregnancy outcome and M2 type DMs, explore the potential application and potential mechanism of LC in PE, and explore the role of pStat6/Stat6-PPAR- gamma signaling pathway in the intervention effect of LC mediator. (1) 7-8 weeks old female and male C57BL/6J mice are fed for one week after adaptive feeding, and the blood pressure of the rat tail is not monitored, and the female rats with stable blood pressure are randomly divided. In the 8 groups, the control group (group A), group PE [seventh days of pregnancy (Gestation Day 7, GD7) subcutaneous injection of L-NAME, B group], PE+LC high dose group (800mg/kg/d, C to E group) and PE+LC low dose intervention group, respectively at 1 weeks before pregnancy. Hourly urine protein; high frequency small animals to evaluate GD18 vascular function in each group; observe the development of intrauterine and placental / renal structural changes in the offspring; analyze the Ly6Chi subgroup in the peripheral blood mononuclear cells from A to E group, the change of the phenotypic migration of decidual macrophages; detect the concentration of Trimethylamine Oxide (TMAO) in the plasma of A to E, and monitor B. The degree of atherosclerosis in group and C group and the distribution of 16S rDNA in the stool of A to E. (2) L-NAME, LC and GW9662 (PPAR- gamma blocker) were used to interfere with RAW 264.7 cells. The effect of cable LC on the phenotypic migration of mouse macrophages and its mechanism. Results: (1) L-NAME can cause PE like performance in C57BL/6J mice, such as hypertension, proteinuria, intrauterine growth restriction and vascular dysfunction, and can also induce the increase of Ly6Chi monocyte subgroup in peripheral blood, and the M2 phenotypic migration of decidual macrophages to decrease the.Ly6Chi subgroup and blood. Positive correlation between pressure and proteinuria; negative correlation with fetal weight, top hip length and placental diameter (r=0.6363, r=0.6752, r=-0.6694, r=-0.6623, r=-0.6623, all P0.001). The M2 phenotype of decidual macrophages was negatively correlated with blood pressure and proteinuria, and was positively correlated with fetal weight, top hip length and placental weight (r=-0.5214, r=-0.6562, r=0.4882, r=0.5701, r=0.5701). 0.05) (2) different doses of LC at different time points can reduce blood pressure in PE like mice to varying degrees, 24 hours of urine protein, improve vascular endothelial function, improve intrauterine growth restriction, and reduce the pulsation index (Plusatility Index, PI) and resistance index (Resistance Index, RI) of the umbilical artery and uterine placental artery (Resistance Index, RI); LC improves the perinatal outcome. With the increase of LC dose, the difference was statistically significant (all P0.05). (3) flow cytometry analysis showed that compared with the A group, the Ly6Chi subgroup of peripheral blood mononuclear cells in the B group increased (61.7 + 8.66%vs.72.2 + 6.02%), and the M2 phenotype expression in decidual macrophages decreased (65.4 + 9.66 vs.53.7 + 4.08), and the difference was statistically significant (all P0.. 01) the proportion of Ly6Chi subgroup decreased (72.2 + 6.02%vs.61.25 + 3.21%) and M2 phenotype expression of decidual macrophages increased (53.7 + 4.08 vs.65.8 + 6.14) after 1 weeks of pregnancy. The difference was statistically significant (all P0.01) and the relative expression of M1 phenotypic markers (TNF- a, IL-1 beta, iNOS) was increased in.B group Decidua Tissue (TNF- a, IL-1 beta, iNOS), and the difference was statistically significant Significance (1 vs.1.41 + 0.15; 1 vs.5.35 + 1.14; 1 vs.81.3 + 16.3; all P0.001); TNF- a, IL-1 beta, iNOS gene expression decreased in 1 weeks before pregnancy, and the difference was statistically significant (1.42 + 0.15 vs.0.34 + 0.14, 5.35 + 1.14 vs.3.64 + 1.13, 5.35 + 1.14) 1.14vs.4.39 + 0.89, 81.3 + 16.3 vs.12.32 + 1.04, all P0.05), iNOS gene expression decreased after GD9 was given to high dose LC, and the difference was statistically significant (81.3 + 16.3 vs.20.2 + 3.37; P0.001).B group M2 phenotype marker. 0, there was no significant difference between the ARG1 gene expression and the control group (1 vs.1.46 + 0.31; 1 vs.1.04 + 0.15; P0.05); the prognosis of IL-10 gene expression was higher in the 1 weeks before pregnancy, and the expression of IL-10 gene increased, and the difference was statistically significant (0.60 + 0.17vs.1.00 + 0.20; 1.46 + 0.31 vs.2.57 + 0.66; all P0.001). The difference was statistically significant (1.46 + 0.31 vs.1.77 + 0.48; P0.05). (4) the M2 type macrophages in the decidua tissue were negatively correlated with the blood pressure of GD18 in pregnant rats and 24 hours of proteinuria (r=-0.5196, P0.001; r=-0.5132, all P0.001); and the weight of the fetal rat, the top hip length Cheng Zhengxiang (r=0.5408, r=0.5779, all). (5) the perinatal total and decidua giant macrophages Positive correlation (r=0.59, P0.001), and negative correlation with TNF- alpha, IL-1 beta and iNOS gene expression in decidua (r=-0.80, r=-0.50, r=-0.96, all P0.001), and a positive correlation with the CD206 of decidual tissue. (6) it stimulates the phenotype marker of macrophage The difference was statistically significant (1 vs.1.56 + 0.19; 1 vs.1.82 + 0.42; 1 vs.3.90 + 0.80; all P0.05); TNF- alpha expression decreased after combined LC intervention, and the difference was statistically significant (1.56 + 0.19 vs.0.81 + 0.16; P0.01), IL-1 beta and iNOS expression decreased, but the difference was not statistically significant (1.82 + 0.42 vs.1.58 + 0.42; 2.; 2.) 67 + 0.80 vs.3.90 + 0.80; all P0.05), M2 macrophage phenotypic markers CD206, IL-10 and ARG1 gene expression and PPAR- gamma gene expression increased, the difference was statistically significant (1.50 + 0.70 vs.6.43 + 1.36; 3.45 + 0.65 vs.5.51 + 1.84; 1.11 + 0.20 vs.2.21 + 0.53; 1.02 + 0.27 vs.1.47) PAR- gamma blocker) after M1, the phenotype markers of M2 macrophages and the expression of PPAR- gamma gene were similar to those of the L-NAME stimulus group, and there was no statistical difference (all P0.05). The pStat6/Stat6 expression level of the PPAR- gamma and Stat6 signaling pathway related proteins in L-NAME+LC group was higher than that in the control group, and the difference was statistically significant (all). There was no significant difference between the expression of the protein and the control group (all P0.05). (7) there was no significant difference in plasma TMAO concentration in group A to E; there was no significant difference between the aortic valve and the aorta and the main branches of the aorta and the main branches of the B group (all P0.05), and the high throughput sequencing of the.A to E group of the.A to E group showed that the high throughput sequencing of the fecal 16S There was no significant difference in the distribution of the main phylum and bacteriobacteria. There was no significant difference between the group and the plasma TMAO and the total amount of LC in perinatal period. The difference between the group of Clostridium Clostridium, a Proteus, Clostridium, XlVa, anaerobes, anaerobes, oontococcus and eight The total amount of LC was negatively correlated with the total perinatal oral administration (r=-0.7989, P0.01). Conclusion: the results of this study showed that GD7 subcutaneous injection of L-NAME in pregnant rats can cause PE like performance. It is a more reasonable PE like model.LC may mediate the migration of the M2 phenotype of the decidua macrophage by pStat6/Stat6-PPAR- gamma pathway and improve the perinatal pregnancy induced pregnancy in PE like mice. The outcome of pregnancy; PE like mice given LC 1 weeks before pregnancy until the end of GD18 (800 mg/kg/d) has no significant influence on the development of atherosclerosis, suggesting that LC may be a new method for the prevention and control of PE.

【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R714.244

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