本文選題：抑郁 + 動脈粥樣硬化　； 參考：《廣州中醫(yī)藥大學》2014年碩士論文

【摘要】：目的 通過慢性不可預知溫和應激(UCMS)加高脂飼料(High-fat Diet,HFD)喂養(yǎng)建立實驗性抑郁型動脈粥樣硬化大鼠模型,采用Real-time PCR和Western blot方法,觀察所建立的大鼠模型中與膽固醇逆轉運(RCT)相關基因和蛋白的表達情況。采用液相色譜-質譜聯用技術(HPLC-MS)測定各組大鼠血漿中同型半胱氨酸(Hcy)的含量。初步探討抑郁與動脈粥樣硬化相關的分子機制。 方法 1.抑郁型動脈粥樣硬化大鼠模型的建立 SD大鼠40只,隨機分為對照組、應激組、高脂組和模型組,對照組和應激組給予基礎飼料喂養(yǎng),高脂組和模型組給予高脂飼料,并于實驗前3天按70萬IU· kg-1的總劑量灌胃給予維生素D3,應激組和模型組每日隨機給予下列8種慢性應激：禁水24h、禁食24h、45℃溫水游泳、4℃冰水游泳、提尾旋轉1min、懸尾5mmin、夾尾30min、束縛8h。造模持續(xù)8周,整個過程中無動物死亡。 2.行為學的觀察 造模第8周,取材前對各組大鼠進行行為學的檢測,包括食物消耗量實驗、糖水偏嗜實驗和曠場實驗。 3.取材及處理 第8周末腹腔注射10%水合氯醛(3.5ml· kg-1)麻醉禁食大鼠,仰臥式固定大鼠后開腹,采用10ml注射器從腹主動脈取血,每只大鼠可取到10ml全血。大鼠采血后快速取肝組織,液氮凍存。剖取腹主動脈及肝臟右葉距離肝邊緣0.5cm處相同部位約2cm X2cm X0.3cm的組織小塊,以4%多聚甲醛固定,常規(guī)石蠟包埋。 4.各組大鼠血清指標的檢測 采用全自動生化分析儀,檢測總膽固醇、總甘油三酯、高密度脂蛋白、低密度脂蛋白、丙氨酸轉氨酶、谷草轉氨酶、堿性磷酸酶、白蛋白及球蛋白的含量。按照試劑盒說明書步驟,檢測超氧化物歧化酶和丙二醛的含量。 5.腹主動脈及肝組織的病理改變 取包埋好的腹主動脈和肝組織的石蠟塊,行5μm連續(xù)切片,HE染色,在光鏡下觀察主動脈內膜的厚度、內皮細胞排列情況以及肝臟病理改變。 6.肝組織中LXRα的蛋白表達 參照二步法免疫組化試劑盒說明書,對肝臟免疫切片進行脫蠟至水、抗原修復、血清封閉、抗體結合、細胞核復染及脫水封片,顯微鏡下隨機測定5個視野,用Image-Pro Plus (Media Cybernetics Inc. USA)圖像分析系統(tǒng)測定其平均光密度(IOD)值。 7.膽固醇逆轉運相關基因和蛋白表達的測定 分別采用Real-time PCR和Western blot實驗技術,檢測大鼠肝臟中LXRα、SR-B I、ABCG5、ABCG8、LCAT、CPY7A1mRNA和蛋白的表達。 8.血漿中同型半胱氨酸的含量測定 采用液相色譜-質譜聯用技術(HPLC-MS)測定血漿中同型半胱氨酸(Hcy)的含量。同型半胱氨酸的色譜條件采用等度洗脫,質譜條件采用電噴霧離子源(ESI),正離子掃描,3,4-二羥基苯甲胺氫溴酸鹽(DHBA)作為內標。 9.統(tǒng)計方法 所有數據以均數±標準差表示,應用SPSS13.0統(tǒng)計軟件進行統(tǒng)計分析。多個樣本均數比較采用單因素方差分析,以P0.05為差異有顯著意義。 結果 1.行為學指標觀察結果 與對照組相比較,應激組、高脂組、模型組的食物消耗量明顯減少；應激組和模型組大鼠的糖水消耗量明顯減少,高脂組的糖水消耗量雖有所下降,但結果沒有統(tǒng)計學意義；應激組與模型組大鼠水平得分、垂直得分及總得分均顯著性降低,模型組比應激組大鼠的運動量更為減少。 2.血清指標的檢測結果 與對照組相比較,高脂組和模型組大鼠血清中TC、TTG、LDL含量均明顯上升,HDL含量則明顯下降,應激組只有HDL顯著下降,其余兩個指標差異沒有顯著性意義。與對照組相比較,模型組大鼠ALT和AST均顯著升高,應激組與高脂組的ALT與AST雖有所上升,但與對照組比較,差異沒有顯著性；與對照組相比較,各組大鼠血清中的ALP均明顯上升,高脂組與模型組的ALB和GLB顯著升高。與對照組相比較,高脂組和模型組大鼠血清中SOD活性下降,應激組雖有所下降,但差異不具統(tǒng)計學意義；與對照組相比,應激組和模型組大鼠血清中MDA含量顯著升高,高脂組變化不明顯。 3.腹主動脈和肝臟組織形態(tài)學改變 慢性應激聯合高脂飲食促進不穩(wěn)定的主動脈斑塊的形成和肝臟脂肪沉積。 4.肝組織中LXRα的蛋白表達 與對照組相比較,高脂組、應激組及模型組LXRα的蛋白表達均顯著下降,其中模型組下降更加明顯。 5.膽固醇逆轉運相關基因和蛋白表達的測定結果 與對照組相比較,應激組肝臟中ABCG8、ABCG5、SR-B I和LXRa的表達有所增強,而CYP7A1和LCAT的變化則不明顯。高脂組肝臟中ABCG8、ABCG5、SR-B I、CYP7A1、LXRα及LCAT的表達均出現顯著性的降低。模型組則中和應激和高脂的雙重作用,顯著地上調了SR-B I和LXRa mRNA的表達,同時下調ABCG8、 ABCG5、CYP7A1和LCATmRNA的表達。上述結果與Western blot實驗結果相一致。 6.血漿中同型半胱氨酸的含量測定 與對照組相比較,應激組大鼠血漿中同型半胱氨酸有所升高,但升高程度不明顯；高脂組大鼠血漿中同型半胱氨酸顯著下降,而模型組則顯著升高。 結論 1.模型的建立 本研究利用慢性不可預知溫和應激疊加高脂飼料喂養(yǎng)造模后,大鼠體重、飲食及糖水消耗量明顯比正常對照組少,且曠場活動顯示大鼠的活動性減少,以及對外界的好奇心減弱,大鼠動脈出現了早期動脈硬化的癥狀,內皮細胞突起,內膜出現不同程度的增厚,彈性纖維結構不清,表明成功建立了實驗性抑郁型動脈粥樣硬化大鼠模型。 2.膽固醇逆轉運相關基因的表達 進食高脂飼料模型大鼠肝臟中與膽固醇逆轉運相關的基因和蛋白的表達,相對于單純給予慢性應激的大鼠顯著降低,慢性應激只有聯合高脂飲食后才會顯著影響大鼠肝臟的脂質代謝。 3.血漿中Hcy含量 慢性應激聯合高脂飲食可使模型大鼠血漿中同型半胱氨酸含量顯著升高,單純的慢性應激大鼠血漿中同型半胱氨酸含量卻有所下降。
[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 偽 , 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 鹵 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 - 偽 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 偽 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 偽 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 .

【學位授予單位】：廣州中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R285

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