微囊蛋白在小鼠膽囊膽固醇結(jié)石癥形成中的作用
發(fā)布時間：2018-07-24 22:02
【摘要】：目的：研究微囊蛋白在致石飼料誘導(dǎo)的小鼠膽囊膽固醇結(jié)石癥形成中的作用,為臨床膽囊膽固醇結(jié)石癥的診療提供新的思路。 方法：以結(jié)石易感小鼠C57BL/6小鼠為研究對象,分別給予普通飼料和含高脂高膽固醇致石飼料飼養(yǎng)4周。4%水合氯醛腹腔注射,待小鼠麻醉后,進(jìn)行小鼠外科實驗。依次剪開皮膚、皮下筋膜、腹膜,充分暴露肝膽組織,肉眼觀察小鼠肝臟、膽囊外觀及膽囊結(jié)石形成情況。結(jié)扎膽囊管及膽總管下端,在膽總管插入PE10號管1h留取膽汁-20℃保存。下腔靜脈采血于含EDTA的生化管中,3000rpm4℃離心15min收集上層血清4℃保存。最后切取肝臟和膽囊組織,肝臟稱重,肝膽組織保存于-80℃冰箱。全自動生化分析儀檢測血脂及膽汁成分。RT-PCR及Westren blot分別檢測小鼠肝臟及膽囊CAV1、CAV3、SR-B1、CCK-AR的基因和蛋白水平。 結(jié)果：致石飼料飼養(yǎng)4周后,實驗組小鼠膽囊增大,膽囊內(nèi)可見大量泥沙狀膽固醇結(jié)石沉積,成石率為100%(6/6),而普通飼料飼養(yǎng)的對照組小鼠膽囊略小,均未發(fā)現(xiàn)有結(jié)石。實驗組小鼠肝體重比顯著增加(0.08±0.01vs0.05+0.01,P0.01),肝臟外觀較灰暗、腫大,而普通飼料飲食的對照組小鼠肝臟色澤紅潤,體積較小實驗組小鼠膽汁渾濁,膽固醇和磷脂含量較對照組明顯升高(1.33±0.33vs0.21±0.11,P0.01；3.55±1.40vs1.55±0.63,P0.05),而總膽汁酸含量卻顯著下降(726.48±51.83vs839.83+23.75,P0.01)。實驗組小鼠血清總膽固醇、高密度脂蛋白和低密度脂蛋白含量明顯高于對照組(TC：4.22±0.46vs2.21±0.11,P0.01；HDL：1.86±0.10vs1.35±0.11,P0.01,LDL：2.18±0.44vs0.58±0.12,P0.01),甘油三酯、極低密度脂蛋白(水平未有明顯改變(TG：0.75+0.04vs0.83+0.15,VLDL：0.18±0.12vs0.28±0.09)。與對照組小鼠相比,實驗組小鼠肝臟和膽囊CAV1mRNA和蛋白表達(dá)均顯著下降(肝臟CAV1基因和蛋白分別為0.53±±0.13vs1±±0.32,P0.01；0.39±0.07vs0.9±0.06,P0.01；膽囊CAV1基因和蛋白分別為0.44-0.22vs1±0.22,P0.01；1.04±0.07vs1.34±0.04,P0.01),膽囊CCKAR基因和蛋白水平亦明顯降低(0.32±0.20vs1±0.25,P0.01；0.07±0.02vs0.35±0.04,P0.01),兩組小鼠肝臟均未能檢測到CCK-AR的表達(dá)。肝臟CAV3mRNA和蛋白表達(dá)增加(肝臟CAV3基因和蛋白分別為3.38±1.47vs1±0.17,P0.01；0.93±0.04vs0.5±±0.06,P0.01),膽囊CAV3mRNA水平改變沒有統(tǒng)計學(xué)意義(0.87±0.27vs1±0.40),蛋白水平顯著升高(0.25±0.03vs0.17±0.02,P0.05)；SR-B1基因和蛋白均未有明顯改變(肝臟基因和蛋白分別為0.25±0.03vs0.17±0.02,0.9±0.04vs0.92±0.04；膽囊基因和蛋白分別為0.86±0.27vs1±0.35,0.89±0.03vs1.044±0.07)。 結(jié)論：含高脂高膽固醇高膽酸的致石餐作為一外界干預(yù)因素,能誘導(dǎo)小鼠血脂含量升高,膽道膽固醇高分泌,總膽汁酸合成減少,促使小鼠膽囊膽固醇結(jié)石形成。小鼠膽囊膽固醇結(jié)石產(chǎn)生伴隨CAV1和CAV3在肝膽組織基因和蛋白表達(dá)水平發(fā)生變化,提示CAV1及CAV3可能與膽囊膽固醇結(jié)石的形成有關(guān)。SR-B1可能對C57BL/6小鼠膽囊膽固醇結(jié)石的形成無顯著影響。CAV1在肝膽組織表達(dá)下降,可能造成膽固醇轉(zhuǎn)運(yùn)功能受損,脂質(zhì)代謝紊亂,膽固醇在肝細(xì)胞內(nèi)大量堆積。CAV3可能通過抑制膽囊CCKAR的表達(dá),減弱膽囊平滑肌的收縮,膽囊動力低下,膽汁淤滯,促進(jìn)膽固醇結(jié)晶析出,聚集以及結(jié)石形成。CAV1、CAV3有望成為臨床膽囊膽固醇結(jié)石癥治療的藥物靶標(biāo)分子或/和診斷標(biāo)志物。
[Abstract]:Objective: To study the role of microcystin in the formation of gallbladder cholesterol gallstone induced by stone feed in mice, and to provide a new idea for the diagnosis and treatment of cholelithiasis in clinical gallbladder.
Methods: the C57BL/6 mice were treated with common fodder and high cholesterol gallstone feed for 4 weeks. The mice were treated with chloral hydrate and chloral hydrate for 4 weeks. After the mice were anesthetized, the mice were treated with the skin, the subcutaneous fascia, the peritoneum, the liver and the hepatobiliary body, the liver and the outside of the gallbladder. The formation of cholecystolithiasis. Ligation of the cystic duct and the lower end of the common bile duct, the bile duct was inserted into the PE10 tube 1h to retain the bile -20 centigrade. The inferior vena cava was collected in a biochemical tube containing EDTA, 3000rpm4 centigrade centrifugation 15min to collect the upper serum at 4 C. Finally, the liver and gallbladder tissues were removed, the liver was weighed, and the hepatobiliary tissue was preserved at -80 centigrade refrigerator. .RT-PCR and Westren blot were detected by automatic biochemical analyzer to detect the gene and protein levels of CAV1, CAV3, SR-B1 and CCK-AR in liver and gallbladder of mice.
Results: after 4 weeks of feeding, the gallbladder in the experimental group was enlarged and a large number of silt like cholesterol stones were deposited in the gallbladder. The rate of stone formation was 100% (6/6), while the gallbladder in the control group of the control group of the normal feed was slightly smaller and no stones were found. The liver weight of the mice in the experimental group was increased (0.08 + 0.01vs0.05+0.01, P0.01), and the appearance of the liver was more gray. The liver color of the control group of the control group of the normal diet diet was red, and the bile was cloudy in the small experimental group and the content of cholesterol and phospholipid was significantly higher than that of the control group (1.33 + 0.33vs0.21 + 0.11, P0.01; 3.55 + 1.40vs1.55 + 0.63, P0.05), but the total bile acid content decreased significantly (726.48 + 51.83vs839.83+23.75, P0.01). The content of serum total cholesterol, high density lipoprotein and low density lipoprotein in the group of mice was significantly higher than that in the control group (TC:4.22 + 0.46vs2.21 + 0.11, P0.01; HDL:1.86 + 0.10vs1.35 + 0.11, P0.01, LDL:2.18 + 0.44vs0.58 + 0.12, P0.01), triglyceride and extremely low density lipoprotein (TG:0.75+0.04vs0.83+0.15, VLDL:0.18) 0.12vs0.28 + 0.09). Compared with the control group, the expression of CAV1mRNA and protein in the liver and gallbladder of the experimental group decreased significantly (the liver CAV1 gene and protein were 0.53 + + + 0.13vs1 + 0.32, P0.01, 0.39 + 0.07vs0.9 + 0.06, P0.01, and CAV1 gene and protein of the gallbladder were 0.44-0.22vs1 + 0.22, P0.01; 1.04 + 0.07vs1.34 + 0.04, P0.01). The CCKAR gene and protein level of gallbladder also decreased significantly (0.32 + 0.20vs1 + 0.25, P0.01, 0.07 + 0.02vs0.35 + 0.04, P0.01). The expression of CCK-AR was not detected in the two groups of mice, and the expression of CAV3mRNA and protein in the liver was increased (the liver CAV3 gene and protein were 3.38 + 1.47vs1 + 0.17, P0.01; 0.93 + 0.04vs0.5 + + 0.06, P0.01). The level of mRNA was not statistically significant (0.87 + 0.27vs1 + 0.40), the protein level increased significantly (0.25 + 0.03vs0.17 + 0.02, P0.05), and there was no significant change in the SR-B1 gene and protein (the liver gene and protein were 0.25 + 0.03vs0.17 + 0.02,0.9 + 0.04vs0.92 + 0.04, respectively, and the gene and protein of the gallbladder were 0.86 + 0.27vs1 + 0.35,0.89 + 0.03vs1., respectively. 044 + 0.07).
Conclusion: the stony meal containing high cholesterol and hypercholesterolic hypercholic acid can induce the increase of blood lipid content, the hypersecretion of cholesterol in the bile duct and the decrease of total bile acid synthesis in mice. The cholesterol gallstones of gallbladder in mice are associated with the gene and protein expression levels of CAV1 and CAV3 in the liver and gallbladder tissues. The results suggest that CAV1 and CAV3 may be related to the formation of gallbladder cholesterol stones..SR-B1 may have no significant effect on the formation of cholesterol gallstones in the gallbladder of C57BL/6 mice..CAV1 may decrease in the expression of.CAV1 in the liver and gallbladder tissue, which may cause impaired cholesterol transport function, lipid metabolism disorder, and a large accumulation of.CAV3 in the liver cells. The expression of CCKAR can weaken the contraction of the smooth muscle of the gallbladder, low gallbladder motility, cholestasis, promote the crystallization of cholesterol, accumulate and form.CAV1. CAV3 is expected to be a drug target molecule or / and diagnostic marker for the treatment of cholelithiasis in the gallbladder.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R575.6
本文編號：2142788
[Abstract]:Objective: To study the role of microcystin in the formation of gallbladder cholesterol gallstone induced by stone feed in mice, and to provide a new idea for the diagnosis and treatment of cholelithiasis in clinical gallbladder.
Methods: the C57BL/6 mice were treated with common fodder and high cholesterol gallstone feed for 4 weeks. The mice were treated with chloral hydrate and chloral hydrate for 4 weeks. After the mice were anesthetized, the mice were treated with the skin, the subcutaneous fascia, the peritoneum, the liver and the hepatobiliary body, the liver and the outside of the gallbladder. The formation of cholecystolithiasis. Ligation of the cystic duct and the lower end of the common bile duct, the bile duct was inserted into the PE10 tube 1h to retain the bile -20 centigrade. The inferior vena cava was collected in a biochemical tube containing EDTA, 3000rpm4 centigrade centrifugation 15min to collect the upper serum at 4 C. Finally, the liver and gallbladder tissues were removed, the liver was weighed, and the hepatobiliary tissue was preserved at -80 centigrade refrigerator. .RT-PCR and Westren blot were detected by automatic biochemical analyzer to detect the gene and protein levels of CAV1, CAV3, SR-B1 and CCK-AR in liver and gallbladder of mice.
Results: after 4 weeks of feeding, the gallbladder in the experimental group was enlarged and a large number of silt like cholesterol stones were deposited in the gallbladder. The rate of stone formation was 100% (6/6), while the gallbladder in the control group of the control group of the normal feed was slightly smaller and no stones were found. The liver weight of the mice in the experimental group was increased (0.08 + 0.01vs0.05+0.01, P0.01), and the appearance of the liver was more gray. The liver color of the control group of the control group of the normal diet diet was red, and the bile was cloudy in the small experimental group and the content of cholesterol and phospholipid was significantly higher than that of the control group (1.33 + 0.33vs0.21 + 0.11, P0.01; 3.55 + 1.40vs1.55 + 0.63, P0.05), but the total bile acid content decreased significantly (726.48 + 51.83vs839.83+23.75, P0.01). The content of serum total cholesterol, high density lipoprotein and low density lipoprotein in the group of mice was significantly higher than that in the control group (TC:4.22 + 0.46vs2.21 + 0.11, P0.01; HDL:1.86 + 0.10vs1.35 + 0.11, P0.01, LDL:2.18 + 0.44vs0.58 + 0.12, P0.01), triglyceride and extremely low density lipoprotein (TG:0.75+0.04vs0.83+0.15, VLDL:0.18) 0.12vs0.28 + 0.09). Compared with the control group, the expression of CAV1mRNA and protein in the liver and gallbladder of the experimental group decreased significantly (the liver CAV1 gene and protein were 0.53 + + + 0.13vs1 + 0.32, P0.01, 0.39 + 0.07vs0.9 + 0.06, P0.01, and CAV1 gene and protein of the gallbladder were 0.44-0.22vs1 + 0.22, P0.01; 1.04 + 0.07vs1.34 + 0.04, P0.01). The CCKAR gene and protein level of gallbladder also decreased significantly (0.32 + 0.20vs1 + 0.25, P0.01, 0.07 + 0.02vs0.35 + 0.04, P0.01). The expression of CCK-AR was not detected in the two groups of mice, and the expression of CAV3mRNA and protein in the liver was increased (the liver CAV3 gene and protein were 3.38 + 1.47vs1 + 0.17, P0.01; 0.93 + 0.04vs0.5 + + 0.06, P0.01). The level of mRNA was not statistically significant (0.87 + 0.27vs1 + 0.40), the protein level increased significantly (0.25 + 0.03vs0.17 + 0.02, P0.05), and there was no significant change in the SR-B1 gene and protein (the liver gene and protein were 0.25 + 0.03vs0.17 + 0.02,0.9 + 0.04vs0.92 + 0.04, respectively, and the gene and protein of the gallbladder were 0.86 + 0.27vs1 + 0.35,0.89 + 0.03vs1., respectively. 044 + 0.07).
Conclusion: the stony meal containing high cholesterol and hypercholesterolic hypercholic acid can induce the increase of blood lipid content, the hypersecretion of cholesterol in the bile duct and the decrease of total bile acid synthesis in mice. The cholesterol gallstones of gallbladder in mice are associated with the gene and protein expression levels of CAV1 and CAV3 in the liver and gallbladder tissues. The results suggest that CAV1 and CAV3 may be related to the formation of gallbladder cholesterol stones..SR-B1 may have no significant effect on the formation of cholesterol gallstones in the gallbladder of C57BL/6 mice..CAV1 may decrease in the expression of.CAV1 in the liver and gallbladder tissue, which may cause impaired cholesterol transport function, lipid metabolism disorder, and a large accumulation of.CAV3 in the liver cells. The expression of CCKAR can weaken the contraction of the smooth muscle of the gallbladder, low gallbladder motility, cholestasis, promote the crystallization of cholesterol, accumulate and form.CAV1. CAV3 is expected to be a drug target molecule or / and diagnostic marker for the treatment of cholelithiasis in the gallbladder.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R575.6
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