抑郁对胆固醇逆转运的影响及相关的分子机制

发布时间：2018-04-12 23:07

本文选题：抑郁 + 动脉粥样硬化　；参考：《广州中医药大学》2014年硕士论文

【摘要】：目的通过慢性不可预知温和应激(UCMS)加高脂饲料(High-fat Diet,HFD)喂养建立实验性抑郁型动脉粥样硬化大鼠模型,采用Real-time PCR和Western blot方法,观察所建立的大鼠模型中与胆固醇逆转运(RCT)相关基因和蛋白的表达情况。采用液相色谱-质谱联用技术(HPLC-MS)测定各组大鼠血浆中同型半胱氨酸(Hcy)的含量。初步探讨抑郁与动脉粥样硬化相关的分子机制。方法 1.抑郁型动脉粥样硬化大鼠模型的建立 SD大鼠40只,随机分为对照组、应激组、高脂组和模型组,对照组和应激组给予基础饲料喂养,高脂组和模型组给予高脂饲料,并于实验前3天按70万IU· kg-1的总剂量灌胃给予维生素D3,应激组和模型组每日随机给予下列8种慢性应激：禁水24h、禁食24h、45℃温水游泳、4℃冰水游泳、提尾旋转1min、悬尾5mmin、夹尾30min、束缚8h。造模持续8周,整个过程中无动物死亡。 2.行为学的观察造模第8周,取材前对各组大鼠进行行为学的检测,包括食物消耗量实验、糖水偏嗜实验和旷场实验。 3.取材及处理第8周末腹腔注射10%水合氯醛(3.5ml· kg-1)麻醉禁食大鼠,仰卧式固定大鼠后开腹,采用10ml注射器从腹主动脉取血,每只大鼠可取到10ml全血。大鼠采血后快速取肝组织,液氮冻存。剖取腹主动脉及肝脏右叶距离肝边缘0.5cm处相同部位约2cm X2cm X0.3cm的组织小块,以4%多聚甲醛固定,常规石蜡包埋。 4.各组大鼠血清指标的检测采用全自动生化分析仪,检测总胆固醇、总甘油三酯、高密度脂蛋白、低密度脂蛋白、丙氨酸转氨酶、谷草转氨酶、碱性磷酸酶、白蛋白及球蛋白的含量。按照试剂盒说明书步骤,检测超氧化物歧化酶和丙二醛的含量。 5.腹主动脉及肝组织的病理改变取包埋好的腹主动脉和肝组织的石蜡块,行5μm连续切片,HE染色,在光镜下观察主动脉内膜的厚度、内皮细胞排列情况以及肝脏病理改变。 6.肝组织中LXRα的蛋白表达参照二步法免疫组化试剂盒说明书,对肝脏免疫切片进行脱蜡至水、抗原修复、血清封闭、抗体结合、细胞核复染及脱水封片,显微镜下随机测定5个视野,用Image-Pro Plus (Media Cybernetics Inc. USA)图像分析系统测定其平均光密度(IOD)值。 7.胆固醇逆转运相关基因和蛋白表达的测定分别采用Real-time PCR和Western blot实验技术,检测大鼠肝脏中LXRα、SR-B I、ABCG5、ABCG8、LCAT、CPY7A1mRNA和蛋白的表达。 8.血浆中同型半胱氨酸的含量测定采用液相色谱-质谱联用技术(HPLC-MS)测定血浆中同型半胱氨酸(Hcy)的含量。同型半胱氨酸的色谱条件采用等度洗脱,质谱条件采用电喷雾离子源(ESI),正离子扫描,3,4-二羟基苯甲胺氢溴酸盐(DHBA)作为内标。 9.统计方法所有数据以均数±标准差表示,应用SPSS13.0统计软件进行统计分析。多个样本均数比较采用单因素方差分析,以P0.05为差异有显著意义。结果 1.行为学指标观察结果与对照组相比较,应激组、高脂组、模型组的食物消耗量明显减少；应激组和模型组大鼠的糖水消耗量明显减少,高脂组的糖水消耗量虽有所下降,但结果没有统计学意义；应激组与模型组大鼠水平得分、垂直得分及总得分均显著性降低,模型组比应激组大鼠的运动量更为减少。 2.血清指标的检测结果与对照组相比较,高脂组和模型组大鼠血清中TC、TTG、LDL含量均明显上升,HDL含量则明显下降,应激组只有HDL显著下降,其余两个指标差异没有显著性意义。与对照组相比较,模型组大鼠ALT和AST均显著升高,应激组与高脂组的ALT与AST虽有所上升,但与对照组比较,差异没有显著性；与对照组相比较,各组大鼠血清中的ALP均明显上升,高脂组与模型组的ALB和GLB显著升高。与对照组相比较,高脂组和模型组大鼠血清中SOD活性下降,应激组虽有所下降,但差异不具统计学意义；与对照组相比,应激组和模型组大鼠血清中MDA含量显著升高,高脂组变化不明显。 3.腹主动脉和肝脏组织形态学改变慢性应激联合高脂饮食促进不稳定的主动脉斑块的形成和肝脏脂肪沉积。 4.肝组织中LXRα的蛋白表达与对照组相比较,高脂组、应激组及模型组LXRα的蛋白表达均显著下降,其中模型组下降更加明显。 5.胆固醇逆转运相关基因和蛋白表达的测定结果与对照组相比较,应激组肝脏中ABCG8、ABCG5、SR-B I和LXRa的表达有所增强,而CYP7A1和LCAT的变化则不明显。高脂组肝脏中ABCG8、ABCG5、SR-B I、CYP7A1、LXRα及LCAT的表达均出现显著性的降低。模型组则中和应激和高脂的双重作用,显著地上调了SR-B I和LXRa mRNA的表达,同时下调ABCG8、 ABCG5、CYP7A1和LCATmRNA的表达。上述结果与Western blot实验结果相一致。 6.血浆中同型半胱氨酸的含量测定与对照组相比较,应激组大鼠血浆中同型半胱氨酸有所升高,但升高程度不明显；高脂组大鼠血浆中同型半胱氨酸显著下降,而模型组则显著升高。结论 1.模型的建立本研究利用慢性不可预知温和应激叠加高脂饲料喂养造模后,大鼠体重、饮食及糖水消耗量明显比正常对照组少,且旷场活动显示大鼠的活动性减少,以及对外界的好奇心减弱,大鼠动脉出现了早期动脉硬化的症状,内皮细胞突起,内膜出现不同程度的增厚,弹性纤维结构不清,表明成功建立了实验性抑郁型动脉粥样硬化大鼠模型。 2.胆固醇逆转运相关基因的表达进食高脂饲料模型大鼠肝脏中与胆固醇逆转运相关的基因和蛋白的表达,相对于单纯给予慢性应激的大鼠显著降低,慢性应激只有联合高脂饮食后才会显著影响大鼠肝脏的脂质代谢。 3.血浆中Hcy含量慢性应激联合高脂饮食可使模型大鼠血浆中同型半胱氨酸含量显著升高,单纯的慢性应激大鼠血浆中同型半胱氨酸含量却有所下降。
[Abstract]:Purpose

To establish a model of experimental depression - type atherosclerosis in rats with chronic unpredictable mild stress ( UCMS ) and high - fat Diet ( HFD ) . Real - time PCR and Western blot were used to observe the expression of related genes and proteins in plasma of rats .

method

1 . Establishment of model of depression - type atherosclerosis rats

Forty - four SD rats were randomly divided into control group , stress group , high fat group and model group , control group and stress group were given basal diet feeding , high fat group and model group were given high fat feed .

2 . Observation of ethology

The rats in each group were tested before the 8th week of the molding , including the food consumption experiment , the sugar water offset experiment and the open field experiment .

3 . Materials and treatment

The rats were anesthetized with 10 % chloral hydrate ( 3.5 ml 路 kg - 1 ) at the end of the 8th week , and the abdominal aorta was taken from the abdominal aorta with 10 ml syringe . After blood sampling , 10 ml of whole blood was taken from the abdominal aorta .

4 . Detection of serum markers in each group

Total cholesterol , total triglyceride , high density lipoprotein , low density lipoprotein , alanine aminotransferase , aspartate aminotransferase , alkaline phosphatase , albumin and globulin in total cholesterol , total triglyceride , high density lipoprotein , low density lipoprotein , alanine aminotransferase , aspartate aminotransferase , alkaline phosphatase , albumin and globulin were detected by a full - automatic biochemical analyzer .

5 . Pathological changes of abdominal aorta and liver tissue

The thickness of aortic intima was observed under light microscope , the arrangement of endothelial cells and the pathological changes of liver were observed under light microscope .

6 . Protein expression of LXR.alpha . in liver tissue

The average optical density ( IOD ) values were determined by using Image - Pro Plus ( Media Cybertron Inc . USA ) image analysis system .

7 . Determination of the gene and protein expression related to the reversal of cholesterol

The expression of LXR 伪 , SR - B I , ABCG5 , ABCG8 , LCAT , CPY7A1 mRNA and protein in rat liver was detected by Real - time PCR and Western blot .

8 . Determination of homocysteine in plasma

The content of homocysteine in plasma was determined by liquid chromatography - mass spectrometry ( HPLC - MS ) . The chromatographic conditions of homocysteine were isocratic elution , electrospray ion source ( ESI ) , positive ion scanning , 3,4 - dihydroxybenzene methylamine hydrobromide ( DHBA ) were used as internal standard .

9 . Statistical methods

All the data were expressed by mean 卤 standard deviation , and the statistical analysis was carried out with SPSS 13.0 software . The mean number of multiple samples was analyzed by single factor analysis , and the difference was significant in P0.05 .

Results

1 . Result of observation of ethology index

Compared with the control group , the consumption of food in the stress group , the high fat group and the model group was obviously reduced ;
The consumption of sugar and water in the stress group and the model group decreased obviously , but the consumption of the sugar and water in the high fat group decreased , but the results were not statistically significant ;
The level scores , vertical scores and total scores of the stress group and the model group were significantly lower than those in the stress group .

2 . Test results of serum indicators

Compared with the control group , the levels of TC , TTG and LDL in the high - fat group and the model group were significantly higher than those in the control group , but the HDL content decreased significantly . Compared with the control group , the ALT and AST in the model group increased significantly , while the ALT and AST in the stress group and the high - fat group increased , but the difference was not significant compared with the control group .
Compared with the control group , the levels of ALP in serum of rats with high fat group and model group were significantly higher than those in control group . Compared with control group , the activity of SOD in serum of high fat group and model group decreased and the stress group decreased , but the difference was not statistically significant .
Compared with the control group , the content of MDA in the serum of the stress group and the model group increased significantly , and the changes of the high fat group were not obvious .

3 . Morphological changes of abdominal aorta and liver

Chronic stress combined with high fat diet promotes the formation of unstable aortic plaque and liver fat deposition .

4 . Protein expression of LXR.alpha . in liver tissue

Compared with the control group , the protein expression of LXR - 伪 in the high - fat group , the stress group and the model group decreased significantly , and the model group was more obvious .

5 . Measurement results of the expression of genes and proteins associated with the reversal of cholesterol

The expression of ABCG8 , ABCG5 , SR - B I , CYP7A1 , LXR 伪 and LCAT in the liver of the stress group was significantly decreased compared with the control group . The expression of ABCG8 , ABCG5 , SR - B I , CYP7A1 , LXR 伪 and LCAT in the liver of the high fat group was significantly decreased .

6 . Determination of homocysteine in plasma

Compared with the control group , the homocysteine in the plasma of the stress group increased , but the degree of elevation was not obvious ;
Homocysteine in plasma of high - fat rats decreased significantly , while the model group increased significantly .

Conclusion

1 . Establishment of the Model

In this study , the rats ' weight , diet and water consumption were significantly lower than those of the normal control group . The rats ' body weight , diet and sugar consumption were significantly lower than those in the normal control group .

2 . Expression of genes related to the reversal of cholesterol

The expression of genes and proteins associated with the reversal of cholesterol in the liver of rats fed with high - fat feed model significantly reduced the lipid metabolism in the liver of the rats only after a combination of high - fat diets with respect to rats treated with chronic stress alone .

3 . Plasma homocysteine content

Chronic stress combined with high fat diet can significantly increase homocysteine content in plasma of model rats , but the content of homocysteine in plasma of simple chronic stress rats decreases .

【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R285

【参考文献】