【作者】 彭强；

【导师】 苏海；

【作者基本信息】 南昌大学医学院 ， 内科学， 2012， 博士

【摘要】 第1部分TFA异构体对人脐静脉内皮细胞生存及凋亡的影响目的：流行病学研究证实反式脂肪酸(Trans Fatty Acids, TFA)摄入可促进动脉粥样硬化性疾病的发生,而且不同来源TFA的致冠心病(Coronary Heart Disease,CHD)风险不同,工业产TFA (Industrially Produced TFA, IP-TFA)的危害明显高于反刍动物源TFA (Ruminant TFA, R-TFA).由于不同来源TFA所含的异构体不同,提示不同的TFA异构体心血管生物学效应可能不同,但其机理目前不清。血管内皮的损害是动脉粥样硬化的始动机制,本部分研究重点观察五种TFA异构体在体外培养条件下对人脐静脉内皮细胞(Human Umbilical Vein Endothelial Cell, HUVEC)生存及凋亡的影响是否存在差异,以解释临床和流行病学研究结果的机理。方法：原代HUVEC细胞分为6组：正常对照组、T-C16：1组、Δ6T-C18：1组、△9T-C18：1组、△11T-C18：1组和T-C18：2组。正常对照组为普通培养基培养,其余五组分别用含相应TFA异构体培养基培养。分别以50umol/1、100umol/1、200umol/1、500umol/1浓度,观测第0、6、12、24小时HUVEC存活率和死亡率,以观察不同TFA异构体对HUVEC存活影响的时效和量效关系。在上述实验结果的基础上,选择采用200umol/1浓度作为最佳实验刺激条件,测定第0、6、12、24小时HUVEC存活率及凋亡率,并比较T-C16：1和其他四种18碳TFA异构体对HUVEC存活率及凋亡率的差异。结果：1、五种TFA异构体包括反式软油酸(T-C16：1)、反式油酸(△6T-C18:1、△9T-C18:1、△11T-C18:1)、反式亚油酸(T-C18：2)在50umol/1浓度时,HUVEC的存活率与正常对照组相比无差别,500umol/1浓度时15小时左右HUVEC几乎全部死亡。而200umol/1浓度导致的细胞损害比100umol/1浓度出现更早、且随时间变化的趋势更为明显。结果表明,五种TFA在一定浓度均降低HUVEC的存活率,并呈现时间和浓度依赖性规律。2.200umol/1浓度干预条件下,五种TFA异构体均明显降低HUVEC存活率。在12h时点,T-C18：2组存活率即明显低于T-C16：1组；在24h时点,Δ6T-C18：1、△9T-C18:1组存活率也明显低于T-C16：1组,然而△11T-C18：1组的存活率与T-C16：1组相似。与其它浓度比较,200umol/1浓度组存活率下降出现较早、且呈明显的时效关系,利于观察不同TFA异构体间的差异；而且200umol/1浓度更接近普通人群血浆中TFA的含量,因此定位为最佳的实验干预浓度。3.200umol/1浓度干预条件下,五种TFA异构体均明显增加HUVEC凋亡率,但各自的效应不同,由强到弱依次为T-C18:2.△6T-C18:1.△9T-C18:1、△11T-C18:1、T-C16:1。且T-C18:2、△6T-C18:1和Δ9T-C18：1组HUVEC凋亡率明显高于T-C16：1组,但Δ11T-C18：1与T-C16：1组间无明显差异性。结论：1、在体外培养条件下,T-C16：1、△6T-C18:1、△9T-C18:1、△11T-C18:1和T-C18：2均可通过增加凋亡的途径降低HUVEC的存活率,并且其危害呈现时效和量效关系。2、在200umol/1浓度干预下,以T-C16：1作为对照组,T-C18：2致HUVEC凋亡的效应最强、Δ6T-C18：1和△9T-C18:1次之、而△11T-C18：1最弱。证实同为18碳链的四种TFA异构体致HUVEC的凋亡效应存在明显差异性。3、四种18碳链的TFA异构体致HUVEC凋亡的程度存在显著差异,可以认为反式双键的数量和位置与其生物学效应有关。4、鉴于△9T-C18:1与T-C16：1的反式双键的位置相同,但它们的致细胞凋亡作用不同,提示碳链长度也是决定不同TFA诱导HUVEC凋亡差异的一个影响因素。第2部分TFA异构体对人脐静脉内皮细胞NOS-NO的影响目的：一氧化氮(Nitric Oxide,NO)不仅是体内强有力的舒血管分子,也是重要的抗动脉粥样硬化分子,适度的NO对维持血管系统的正常功能甚为重要。NOS-NO是体内重要的分泌NO的系统。TFA异构体对于HUVEC的NOS-NO系统的影响目前并不清楚。本部分研究主要观察：在体外培养条件下TFA异构体对血管内皮NOS-NO系统的影响是否存在差异性。方法：实验分组与第一部分相同,以200umol/1为TFA实验浓度,检测0、6、12、24小时HUVEC培养上清液中NO含量和总NOS活力变化；检测24小时HUVEC eNOS mRNA以及eNOS蛋白表达的变化。并以T-C16：1组作为对照,比较分析四种18碳链TFA异构体对上述参数的影响是否存在差异。同时采用两变量相关分析细胞培养上清液NO浓度与HUVEC凋亡率的关联。结果：1、与正常对照组和基础状态相比,五种TFA异构体均能降低HUVEC上清液NO浓度。在12h时点,T-C18：2组上清液NO浓度明显低于T-C16：1组；在24h时点,△6T-C18:1、△9T-C18:1和T-C18：2组的NO浓度均明显低于T-C16：1组,尤其是T-C18：2组；然而△11T-C18：1组的NO浓度一直与T-C16：1组相似。2、与正常对照组和基础状态相比,五种TFA异构体均能降低上清液总NOS活性。但组间出现显著差异的时间点比NO浓度变化的时间点更早,在6h时点,△6T-C18:1、△9T-C18:1和T-C18：2组总NOS活性明显低于T-C16：1组,而△11T-C18：1的总NOS活性也一直与T-C16：1组相似。3、细胞上清液NO浓度与HUVEC凋亡率呈负相关(r=-0.949,P<0.05)。4、与正常对照组比较,五种TFA异构体均能抑制eNOS的mRNA表达,T-C16：1组的NOS mRNA表达为正常对照组的32%、△11T-C18：1为23%、△6T-C18：1为13%、A9T-C18：1为8%、T-C18：2仅为3%。其中△6T-C18:1、△9T-C18:1和T-C18：2组的eNOS mRNA表达明显T-C16：1组,而△11T-C18：1组与T-C16：1组之间无统计学差别(18.55±0.05比19.06±0.07,P>0.05)。5、五种TFA异构体组eNOS蛋白表达量均明显低于正常对照组。与T-C16：1比较,T-C18:2、△6T-C18:1和A9T-C18：1组的eNOS蛋白表达量明显更低；而△11T-C18：1组与T-C16：1组eNOS蛋白表达量无统计学差异。结论：1、在体外培养状态下,T-C18：2、△6T-C18：1、△9T-C18:1、△11T-C18:1和T-C16：1均可降低HUVEC培养上清液NO浓度和总NOS活性,下调HUVEC的eNOS mRNA和蛋白表达。2、不同TFA异构体对于HUVEC的NOS-NO系统的抑制效应(降低NO浓度、NOS活性、下调eNOS mRNA和蛋白表达)具有差异性,T-C18：2的抑制作用最强、△6T-C18:1和△9T-C18:1次之,而△11T-C18:1与T-C16：1最弱。3、NO浓度和HUVEC凋亡率升高之间呈显著负相关,提示不同TFA异构体对于NOS-NO系统抑制程度的差异是它们致细胞凋亡差异的基础原因。第3部分TFA异构体对人脐静脉内皮细胞PRMT/ADMA/DDAH的影响目的：非对称性二甲基精氨酸(Asymmetric Dimethylarginine, ADM A)是机体内调节NOS-NO系统重要的物质,因为具有竞争性抑制NOS的作用,而被称为“内源性的NOS抑制剂”。甲基化蛋白转移酶Ⅰ (Protein Arginine Methyl Transferase Ⅰ, PRMT Ⅰ)是ADMA生成的主要限速酶,其活性高低与体内ADMA的生成密切相关。而体内90%以上的ADMA是经二甲基精氨酸二甲胺水解酶(Dimethylarginine Dimethylaminohydrolase, DDAH)分解代谢的。因为三者之间存在密不可分的关联,故被称为PRMT-ADMA-DDAH系统。我们第一、二部分的研究已经发现,TFA异构体可明显抑制NOS-NO系统,然而深层的机制不明。因此我们提出假设：不同TFA异构体对PRMT-ADMA-DDAH系统具有不同的影响,进而造成对NOS-NO系统影响不同。本研究部分主要观察体外培养条件下,不同TFA异构体对血管内皮细胞的PRMT-ADMA-DDAH系统的影响是否存在差异性。方法：实验分组与第一部分相同,以200umol/l为TFA实验浓度,检测0、6、12、24小时HUVEC培养上清液中ADMA含量和DDAH活性；检测24小时HUVEC的PRMTⅠ、DDAH Ⅰ和DDAH Ⅱ mRNA以及蛋白表达的变化。并以T-C16：1组作为对照,比较分析四种18碳链TFA异构体对上述参数的影响是否存在差异。同时采用两变量相关分析细胞培养上清液DDAH活性与ADMA浓度及ADMA浓度与总NOS活性的关联。结果：1、与正常对照组和基础状态相比,五种TFA异构体均能升高HUVEC上清液ADMA浓度。在12h和24h时点,△6T-C18:1、△9T-C18:1和T-C18：2组上清液ADMA浓度明显高于T-C16：1组；但五种TFA异构体升高ADMA幅度不同,T-C18：2升高幅度最大；其次为△6T-C18:1和A9T-C18：1；而△11T-C18：1与T-C16：1作用最弱。2、与正常对照组和基础状态相比,五种TFA异构体均不同程度地降低上清液DDAH活性。细胞上清液DDAH活性与ADMA浓度呈负相关(r=-0.975,P<0.05)。ADMA浓度与总NOS活性也呈负相关(r=-0.918,P<0.05)。3、在五种TFA异构体干预各组,HUVEC的PRMTⅠmRNA及蛋白表达与正常对照组无明显差别。4、五种TFA异构体干预组,HUVEC的DDAH Ⅰ和DDAH Ⅱ mRNA和蛋白表达均明显低于正常对照组。五个TFA干预组间比较发现：△6T-C18:1、△9T-C18:1和T-C18：2组的DDAH Ⅰ和DDAH Ⅱ mRNA和蛋白表达明显低于T-C16：1组,而△11T-C18：1组与T-C16：1组无统计学差别。结论：1、在体外培养状态下,T-C18:2、△6T-C18:1、△9T-C18:1、△11T-C18:1和T-C16：1均可升高HUVEC培养上清液ADMA浓度,同时导致DDAH活性下降。ADMA浓度和DDAH活性下降之间呈负相关。2、由于五种TFA异构体均不影响ADMA的合成酶PRMTⅠ mRNA和蛋白表达。因此可以认为,培养上清液ADMA浓度增加不是源于ADMA合成增加。3、在体外培养状态下,五种TFA异构体均可下调DDAH Ⅰ和DDAH II mRNA和蛋白表达。因此可以认为,培养上清液ADMA浓度增加是源于ADMA分解减少。4、在体外培养的HUVEC,虽然不同TFA干预均能抑制PRMT-ADMA-DDAH系统中DDAH合成环节而使ADMA降解减少,但其程度有明显不同。T-C18：2的作用最强、Δ6T-C18：1和A9T-C18：1次之,而△11T-C18：1与T-C16：1最弱。更多还原

【Abstract】 Part I The influence of trans fatty acids isomers on survival and apoptosis of human umbilical vein endothelial cells in vitroObjective:Many epidemiological studies indicated that coronary heart disease (CHD) is associated with high intake of trans fatty acids (TFA), but this correlation is dependent on the sources of TFA. Industrially produced TFA (IP-TFA) has been found to correspond to CHD, but ruminant TFA (R-TFA) does not. At present, the mechanisms for the different cardiovascular effects of various TFAs were still unclear. Because the damage of vascular endothelium is the initiating process of CHD, and TFA isomers of IP-TFA are different from those of R-TFA, we give a hypothesis that the TFA isomers contained in IP-TFA may have stronger damage effects on HUVEC than those from R-TFA. The aim of the first part of this study was to investigate whether there were different impacts of five kinds of TFA isomers on the survival and apoptosis of human umbilical vein endothelial cell (HUVEC) in vitro.Method:Five TFA isomers, including trans palmitoleic acid (T-C16:1), trans oleic acid (△6T-C18:1;△9T-C18:1;△11T-C18:1), trans linoleic acid (T-C18:2) were investigated in this study. Six groups were divided according to the TFA isomers used as:control group HUVECs were cultured in general media; T-C16:1group;△6T-C18:1group;△9T-C18:1group;△11T-C18:1group and T-C18:2groups. The latter five groups were treated with corresponding TFA isomer respectively. For evaluating the impacts of TFA isomers on the survival rate of HUVEC, four concentrations of TFA isomers (50umol/1,100umol/1,200umol/1and500umol/1) were used and the HUVEC counts were detected at before (0) and6,12,24hours after TFA intervention.Based on the results of above experiment, TFA of200umol/1was selected as the most proper intervention concentration. The survival and apoptosis rates of HUVEC were detected at0,4,8,12hours.Results:1. Compared with the control group, the survival rates of HUVEC in five TFA treated groups in50umol/1were similar. When TFA of500umol/1concentration was used, the HUVECs in all five treatment groups were almost death at15hours. Significant time and dose-dependent relation was seen in the100umol/l and200umol/l TFA treated groups, and as the TFA of200umol/1is similar to the level in human, TFA of200umol/1was selected as the intervention concentration.2. In200umol/l concentration, five TFA isomers significantly reduced HUVEC survival rate of HUVECs. The survival rate of HUVECs was significantly lower in T-C18.2group than in T-C16:1group at12hours. At24hours, similar changes were observed between△6T-C18:1or A9T-C18:1and T-C16:1groups, but the survival rates between△11T-C18:1group and T-C16:1group were similar.3. In200umol/1concentration, the HUVEC apoptosis rate significantly increased in five TFA isomers groups, but obvious group variations were found. On the decline sequence of apoptosis rate, the order was T-C18:2,△6T-C18:1,△9T-C18:1,△11T-C18:1and T-C16:1. Compared with the T-C16:1group, the apoptosis rates of T-C18:2group,△6T-C18:1and△9T-C18:1group were obviously higher, but no significant difference was seen between△11T-C18:1group and T-C16:1group.Conclusions:1. In100umol/1and200umol/1concentration,T-C16:1,△6T-C18:1,△9T-C18:1,△11T-C18:1and T-C18:2could reduce the survival rate of HUVECs in vitro through increasing apoptosis rate, with time and dose-dependent way.2. In200umol/1concentration, compared with the T-C16:1group, four18-carbon TFA isomers induced significantly higher apoptosis rates of HUVEC, with the order from strong to weak: T-C18:2,△6T-C18:1,△9T-C18:1and△11T-C18:1. It was clear showed that the apoptogenic effects on HUVEC of the four18-carbon TFA isomers were significantly different. 3. Based on above-mentioned data, in it reasonable to suggested that the difference of biological effects of TFA isomers was related to the number and position of the trans double bond.4. Although the trans double bond of△9T-C18:1and T-C16:1molecules located in the same position, their different effects on HUVEC apoptosis rate suggested that the carbon chain length might be a influencing factor of the apoptogenic effect.Part II The influence of trans fatty acids isomers on NOS-NO system of human umbilical vein endothelial cells in vitroObjective:Nitric Oxide (NO) is not only a strong vasodilator molecule, but also an important anti-atherosclerotic molecule. In body, proper NO concentration is needed to maintain the normal function of the cardiovascular system. Nitric oxide synthase (NOS) is key link for NO secretion. So NOS-NO system play an important role in keeping cardiovascular system health. As the apoptogenic effects on HUVEC of the different TFA isomers varied, we give a hypothesis the difference of their apoptogenic effects is associated with the influence of TFA isomers on the NOS-NO system. The aim of the second part of this study was to investigate whether there were different influences of five TFA isomers on NOS-NO in HUVEC in vitro.Method:The experimental groups were the same as in the first part of this study, and the200umol/1concentration of TFA isomers was used also. The NO concentration and total NOS activity in HUVEC culture medium were detected before (0) and6,12,24hours after intervention. The expression levels of endothelial nitric oxide synthase (eNOS) mRNA and protein were detected at24hours by real-time PCR and western blotting respectively. The relation between NO concentration and the apoptosis rate of HUVEC were analyzed by linear correlation. Results:1. Compared with the control group and the basic state, the five TFA isomers significantly reduced NO concentration of HUVEC culture medium. The NO concentration was significantly lower in T-C18:2group than in T-C16:1group at12hours. At24hours, similar changes were also observed in△6T-C18:1,△9T-C18:1and T-C18:2groups, but the NO concentration between△11T-C18:1group and T-C16:1group was similar.2. Compared with the control group and the basic state, the five TFA isomers could reduce total NOS activity of HUVEC. But in time point, the change of total NOS activity was earlier than the time of NO. We could find total NOS activity was significantly lower in△6T-C18:1、△9T-C18:1and T-C18:2groups than in T-C16:1group at6hours, while this change was similar between△11T-C18:1and T-C16:1group.3. There was a negative correlation between the NO concentration and the apoptosis rate of HUVEC (r=-0.949,P<0.05).4. Compared with the control group, five TFA isomers significantly down-regulated the expression of eNOS mRNA. If the expression level of eNOS mRNA in the control group was taken as the reference of100%, the expression level of eNOS mRNA was32%in T-C16:1group;23%in△11T-C18:1group;13%in△6T-C18:1group;8%in△9T-C18:1group;3%only in T-C18:2group. Meanwhile, the expression level of eNOS mRNA was significantly lower in△6T-C18:1、△9T-C18:1and T-C18:2groups than in T-C16:1group. However, the expression level of eNOS mRNA was similar between△11T-C18:1and T-C16:1group.5. Five TFA isomers used in this study significantly down-regulated the expression of eNOS protein. Compared with T-C16:1group, the expression level of eNOS protein were significantly lower in△6T-C18:1,△9T-C18:1and T-C18:2groups. But expression levels of eNOS protein of both△11T-C18:1and T-C16:1groups were similar.Conclusions: 1. In vitro culture conditions, T-C16:1,△6T-C18:1,△9T-C18:1,△11T-C18:1and T-C18:2significantly reduced NO concentration and total NOS activity in HUVEC culture medium, as well as down-regulate the expression of eNOS mRNA and protein.2. Different TFA isomers had different inhibitory effect on NOS-NO system in HUVEC. The inhibitory effect of T-C18:2was the strongest, then the△6T-C18:1and△9T-C18:1, while△11T-C18:1and T-C16:1was the weakest.3. Because there was a negative relation between NO concentration and HUVEC apoptosis rate, it suggested that the various apoptogenic effects of different TFA isomers was based on the different inhibitory effects on NOS-NO system.Part III The influence of trans fatty acids isomers on PRMT/ADMA/DDAH of human umbilical vein endothelial cells in vitroObjective:Asymmetric dimethylarginine (ADMA) plays a very important role in regulating NOS-NO system and is called "the endogenous NOS inhibitor". Protein-arginine methyl-transferase I (PRMT I) is a rate-limiting enzyme of ADMA generation. More than90%of ADMA is metabolized by Dimethylarginine-dimethylamino-hydrolase (DDAH). In the second part of our research, we found that five TFA isomers had different effects on NOS-NO system in HUVEC. So we give a hypothesis that the different effects of five TFA isomers on NOS-NO system is associated with the influences of them on the PRMT-ADMA-DDAH system. The aim of the third part of this study was to investigate whether there were different influences of five TFA isomers on PRMT-ADMA-DDAH in HUVEC.Method:The experimental groups were the same as in the second part of this study, and the200umol/1concentration of TFA isomers was used also. The ADMA concentration and DDAH activity in HUVEC culture medium were detected before (0) and6,12,24hours after intervention. The mRNA and proteins’expression levels of PRMT I, DDAH I and DDAH II were detected at24hours by real-time PCR and western blotting respectively. And the relations between DDAH activity and ADMA concentration, as well as ADMA concentration and NOS activity were analyzed by linear correlation.Results:1. Compared with the control group and the basic state, ADMA concentration of HUVEC culture medium was significantly increased in five TFA groups. At12and24hours, ADMA concentration was significantly higher in△6T-C18:1,△9T-C18:1and T-C18：2groups than in T-C16:1group. But there were difference in the levels of ADMA concentration among five TFA groups, with the order from high to low:T-C18:2,△6T-C18:1,△9T-C18:1,△11T-C18:1and T-C16:1.2. Compared with the control group and the basic state, the DDAH activity of HUVEC culture medium was reduced in five TFA groups. And a negative relation was found between DDAH activity and ADMA concentration (r=-0.975, P<0.05), as well as a negative relation was also found between ADMA concentration and total NOS activity (r=-0.918, P<0.05).3. The mRNA and protein expression levels of PRMT I were similar in five TFA groups.4. Compared with the control group, the mRNA and protein expression levels of DDAH I and DDAH II were down-regulated in five TFA groups. The mRNA and protein expression levels of DDAH I and DDAH II in△6T-C18:1,△9T-C18:1and T-C18:2groups were significantly lower than in T-C16:1group, but the mRNA and protein expression levels of DDAH I and DDAH II were similar between△11T-C18:1and T-C16:1groups.Conclusions:1. T-C16:1,△6T-C18:1,△9T-C18:1,△11T-C18:1and T-C18:2could significantly increase ADMA concentration and decrease DDAH activity of HUVEC.2. The mRNA and protein expression levels of PRMT I were similar in five TFA groups, it suggested that the change of ADMA concentration of HUVEC was not derived from the ADMA synthase.3. The mRNA and protein expression levels of DDAH I and DDAH ⅡI were down-regulated in T-C16:1,△6T-C18:1,△9T-C18:1,△11T-C18:1and T-C18:2groups. So it suggested that the change of ADMA concentration of HUVEC was derived from the decline of ADM A degradation.4. Five TFA isomers had different effects on PRMT-ADMA-DDAH system by increasing ADMA concentration and decreasing DDAH activity. But obvious group variations were found, T-C18:2was the strongest, then the△6T-C18:1and△9T-C18:1followed, while△11T-C18:1and T-C16:1were the weakest.更多还原