高碘对大鼠下腔静脉内皮细胞活化的影响
发布时间：2018-08-25 08:06
【摘要】：[目的] 本实验通过以高碘水和偏食饲料饲养Wistar大鼠,建立偏食高碘大鼠模型,通过HE染色、免疫组织化学染色和血浆中相关因子的测定,观察大鼠下腔静脉肝段内皮细胞的活化情况,并探讨高碘引起血管内皮细胞活化的作用机制。 [方法] 1、实验动物与分组：选择断乳后约1个月,体重(110±10)g远交封闭群SPF/VAF级Wistar大鼠,由山东大学动物实验中心提供。适应性喂养一周后根据性别和体重随机分为8组,分别为：①正常对照组(NC)；②正常饲料+10倍碘水组(10HI)；③正常饲料+50倍碘水组(50HI)；④正常饲料+100倍碘水组(100HI)；⑤偏食对照组(MC)；⑥偏食饲料+10倍碘水组(M10HI)；⑦偏食饲料+50倍碘水组(M50HI)；⑧偏食饲料+100倍碘水组(M100HI),每组8只。 2、大鼠体重记录：每周称量各组大鼠体重并记录,喂养6个月。 3、大鼠下腔静脉肝段HE染色：用10%福尔马林固定,常规石蜡包埋,切片,以二甲苯脱蜡,各级乙醇水化,苏木素染色2min,盐酸酒精分化15s,酒精伊红染色2-5分钟,常规脱水透明,封片,观察下腔静脉内皮细胞的形态改变。 4、测定各组大鼠血浆中ET-1、vWF、vWFpp及HCY的含量。 5、大鼠下腔静脉肝段免疫组织化学染色：组织切片经过脱蜡水化,抗原修复,PBS冲洗,加入一抗,4℃过夜,加入二抗,用DAB进行显色,观察E-选择素和ICAM-1在下腔静脉内皮细胞的表达情况。 [结果] 1.大鼠体重的变化： 从开始喂养到6个月结束,各正常饲料+高碘水组与NC组相比无明显差别；偏食对照组(MC)到第24周才与NC组出现统计学差异；第2周开始,各偏食饲料+高碘水组均低于或明显低于NC组。 2、内皮细胞形态的改变： 随着碘浓度的逐渐升高,大鼠下腔静脉肝段内皮细胞出现活化和增生的改变,表现为内皮细胞体积增大,呈立方形向管腔突起,内皮细胞数目增多,内皮下ECM增多,甚至局部内膜增厚,其中以偏食组更为明显。 3、血浆ET-1、vWF、vWFpp及HCY含量测定： 3.1大鼠血浆ET-1 内皮细胞功能受到了一定的影响,与正常对照组相比,50HI组ET-1含量升高,差异具有统计学意义(P0.05)：100HI组、M50HI组和MlOOHI组ET-1含量升高,差异显著(P0.01)。相邻组间比较结果显示,随着碘浓度的逐渐升高,ET-1的含量呈升高趋势(P0.05或P0.01) 3.2大鼠血浆vWF 内皮细胞功能受到了影响,与正常对照组相比,M10HI组和M50HI组vWF含量升高,差异具有统计学意义(P＜0.05)；100HI组和M100HI组vWF含量升高,差异显著(P＜0．01)。相邻组间比较结果显示,随着碘浓度的逐渐升高,vWF的含量逐渐升高。其中,M10HI组与MC组比较有统计学差异(P＜0.05),100HI组与50HI组比较、M100HI组与M50HI组比较差异显著(P0.01)。 3.3大鼠血浆vWFpp 与正常对照组相比,M50HI组vWFpp含量升高(p0.05),其余各组未见升高。 3.4大鼠血浆HCY 与正常对照组相比,50HI组HCY含量升高,差异具有统计学意义(p0.05),100HI组、M10HI组、M50HI组和M100HI组HCY含量升高,差异显著(p0.01)。相邻组间比较结果显示,随着碘浓度的逐渐升高,血浆HCY的含量呈升高趋势(P0.05或P＜0.01) 4、免疫组织化学结果： 4.1ICAM-1的表达 随着碘浓度逐渐升高,内皮细胞ICAM-1表达呈增加趋势。其中,M100HI组ICAM-1表达与正常对照组相比有统计学差异(P0.05)；组间比较均无统计学差异(P0.05) 4.2E-选择素的表达 随着碘浓度逐渐升高,内皮细胞E-选择素表达逐渐增加。其中,与正常对照组相比,100HI组、M50HI组、M100HI组E-选择素表达有统计学差异(P0.05或P0.01)；组间比较显示,M100HI组与M10HI组相比有统计学差异(P0.05) [结论] 1.内皮细胞形态学的改变表明高碘可导致内皮细胞活化、进而损伤和增生,内皮下ECM增多,进而促进内膜增厚,并呈剂量一效应关系,其中,在偏食组更为明显,提示在该动物模型中高碘通过促进内皮细胞活化、损伤和增生,可引起下腔静脉管壁增厚、管腔狭窄,可能对隔膜型布加综合征的发病机制起一定的提示作用。 2.高碘可引起大鼠血浆多种活性因子ET-1、vWF及HCY的含量发生改变：随碘浓度的逐渐升高,血浆ET-1、vWF、HCY的含量逐渐增加；同时,高碘使内皮细胞E-选择素和ICAM-1表达量增加,导致内皮细胞活化。 3.在该动物模型中,偏食所引起的营养不良可加重高碘对大鼠下腔静脉内皮细胞活化的影响。
[Abstract]:[Objective]
In this study, Wistar rats were fed with Iodine-Excess water and diet to establish a partial Iodine-Excess rat model. The activation of endothelial cells in the hepatic segment of the inferior vena cava was observed by HE staining, immunohistochemical staining and determination of related factors in plasma. The mechanism of activation of endothelial cells in the hepatic segment of the inferior vena cava was also discussed.
[method]
1. Laboratory animals and grouping: SPF/VAF Wistar rats, weighing (110 _normal diet + 100 times iodine water group (100HI); _partial diet control group (MC); _partial diet + 10 times iodine water group (M10HI); _partial diet + 50 times iodine water group (M50HI); _partial diet + 100 times iodine water group (M100HI), 8 rats in each group.
2, weight records of rats: weighing the rats in each group and recording them for 6 months each week.
3. HE staining of hepatic segment of inferior vena cava in rats: fixed with 10% formalin, embedded in conventional paraffin, sectioned, dewaxed with xylene, hydrated with ethanol at all levels, stained with hematoxylin for 2 minutes, differentiated with hydrochloric acid for 15 seconds, stained with eosin for 2-5 minutes, routinely dehydrated and transparent, sealed, observed the morphological changes of endothelial cells of inferior vena cava.
4, the contents of ET-1, vWF, vWFpp and HCY in plasma of each group were measured.
5. Immunohistochemical staining of hepatic segment of inferior vena cava in rats: After dewaxing and hydration, antigen repairing, PBS washing, adding first antibody, overnight at 4 C, adding second antibody, using DAB for coloration, the expression of E-selectin and ICAM-1 in endothelial cells of inferior vena cava was observed.
[results]
1. body weight changes in rats:
From the beginning of feeding to the end of 6 months, there was no significant difference between the normal diet + high iodine water group and NC group; there was no significant difference between the control group (MC) and NC group until 24 weeks; from the second week, the diet + high iodine water group was lower or significantly lower than NC group.
2, changes in endothelial cell morphology:
With the increase of iodine concentration, the endothelial cells in the hepatic segment of the inferior vena cava increased in size, cubic protrusion, the number of endothelial cells increased, the number of subendothelial ECM increased, and even the local intima thickened, especially in the partial feeding group.
3, plasma ET-1, vWF, vWFpp and HCY content determination:
3.1 rat plasma ET-1
Endothelial cell function was affected. Compared with the normal control group, the content of ET-1 in 50HI group increased, and the difference was statistically significant (P 0.05):The content of ET-1 in 100HI group, M50HI group and MlOHI group increased significantly (P 0.01).
3.2 rat plasma vWF
Compared with the normal control group, the content of vWF in M10HI group and M50HI group increased significantly (P < 0.05), while that in 100HI group and M100HI group increased significantly (P < 0.01). Compared with MC group, there was significant difference (P < 0.05). Compared with 100HI group and 50HI group, there was significant difference between M100HI group and M50HI group (P 0.01).
3.3 rat plasma vWFpp
Compared with the normal control group, the vWFpp content in the M50HI group increased (P0.05), but no increase was observed in the other groups.
3.4 rat plasma HCY
Compared with the normal control group, the content of HCY in 50HI group increased significantly (p0.05). The content of HCY in 100HI group, M10HI group, M50HI group and M100HI group increased significantly (p0.01).
4, immunohistochemistry results:
Expression of 4.1ICAM-1
With the increase of iodine concentration, the expression of ICAM-1 in endothelial cells increased. The expression of ICAM-1 in M100HI group was significantly different from that in normal control group (P 0.05), and there was no significant difference between groups (P 0.05).
Expression of 4.2E- selectin
The expression of E-selectin in endothelial cells increased with the increase of iodine concentration. Compared with normal control group, the expression of E-selectin in 100HI group, M50HI group and M100HI group was significantly different (P 0.05 or P 0.01); the comparison between groups showed that there was significant difference between M100HI group and M10HI group (P 0.05).
[Conclusion]
1. The morphological changes of endothelial cells showed that iodine excess could induce the activation of endothelial cells, and then damage and proliferation of endothelial cells, and increase the number of ECM in endothelial cells, and then promote the intimal thickening in a dose-dependent manner, especially in the prey group, suggesting that iodine excess could induce the inferior vena cava by promoting the activation, injury and proliferation of endothelial cells in this animal model. Thickening of the wall and narrowing of the lumen may play a role in the pathogenesis of diaphragmatic Budd Chiari syndrome.
2. Iodine excess can change the contents of ET-1, vWF and HCY in plasma of rats: with the increase of iodine concentration, the contents of ET-1, vWF and HCY in plasma increase gradually; at the same time, iodine excess can increase the expression of E-selectin and ICAM-1 in endothelial cells, leading to the activation of endothelial cells.
3. In this animal model, malnutrition caused by partial diet aggravated the effect of excessive iodine on endothelial cell activation of inferior vena cava in rats.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
本文编号：2202280
[Abstract]:[Objective]
In this study, Wistar rats were fed with Iodine-Excess water and diet to establish a partial Iodine-Excess rat model. The activation of endothelial cells in the hepatic segment of the inferior vena cava was observed by HE staining, immunohistochemical staining and determination of related factors in plasma. The mechanism of activation of endothelial cells in the hepatic segment of the inferior vena cava was also discussed.
[method]
1. Laboratory animals and grouping: SPF/VAF Wistar rats, weighing (110 _normal diet + 100 times iodine water group (100HI); _partial diet control group (MC); _partial diet + 10 times iodine water group (M10HI); _partial diet + 50 times iodine water group (M50HI); _partial diet + 100 times iodine water group (M100HI), 8 rats in each group.
2, weight records of rats: weighing the rats in each group and recording them for 6 months each week.
3. HE staining of hepatic segment of inferior vena cava in rats: fixed with 10% formalin, embedded in conventional paraffin, sectioned, dewaxed with xylene, hydrated with ethanol at all levels, stained with hematoxylin for 2 minutes, differentiated with hydrochloric acid for 15 seconds, stained with eosin for 2-5 minutes, routinely dehydrated and transparent, sealed, observed the morphological changes of endothelial cells of inferior vena cava.
4, the contents of ET-1, vWF, vWFpp and HCY in plasma of each group were measured.
5. Immunohistochemical staining of hepatic segment of inferior vena cava in rats: After dewaxing and hydration, antigen repairing, PBS washing, adding first antibody, overnight at 4 C, adding second antibody, using DAB for coloration, the expression of E-selectin and ICAM-1 in endothelial cells of inferior vena cava was observed.
[results]
1. body weight changes in rats:
From the beginning of feeding to the end of 6 months, there was no significant difference between the normal diet + high iodine water group and NC group; there was no significant difference between the control group (MC) and NC group until 24 weeks; from the second week, the diet + high iodine water group was lower or significantly lower than NC group.
2, changes in endothelial cell morphology:
With the increase of iodine concentration, the endothelial cells in the hepatic segment of the inferior vena cava increased in size, cubic protrusion, the number of endothelial cells increased, the number of subendothelial ECM increased, and even the local intima thickened, especially in the partial feeding group.
3, plasma ET-1, vWF, vWFpp and HCY content determination:
3.1 rat plasma ET-1
Endothelial cell function was affected. Compared with the normal control group, the content of ET-1 in 50HI group increased, and the difference was statistically significant (P 0.05):The content of ET-1 in 100HI group, M50HI group and MlOHI group increased significantly (P 0.01).
3.2 rat plasma vWF
Compared with the normal control group, the content of vWF in M10HI group and M50HI group increased significantly (P < 0.05), while that in 100HI group and M100HI group increased significantly (P < 0.01). Compared with MC group, there was significant difference (P < 0.05). Compared with 100HI group and 50HI group, there was significant difference between M100HI group and M50HI group (P 0.01).
3.3 rat plasma vWFpp
Compared with the normal control group, the vWFpp content in the M50HI group increased (P0.05), but no increase was observed in the other groups.
3.4 rat plasma HCY
Compared with the normal control group, the content of HCY in 50HI group increased significantly (p0.05). The content of HCY in 100HI group, M10HI group, M50HI group and M100HI group increased significantly (p0.01).
4, immunohistochemistry results:
Expression of 4.1ICAM-1
With the increase of iodine concentration, the expression of ICAM-1 in endothelial cells increased. The expression of ICAM-1 in M100HI group was significantly different from that in normal control group (P 0.05), and there was no significant difference between groups (P 0.05).
Expression of 4.2E- selectin
The expression of E-selectin in endothelial cells increased with the increase of iodine concentration. Compared with normal control group, the expression of E-selectin in 100HI group, M50HI group and M100HI group was significantly different (P 0.05 or P 0.01); the comparison between groups showed that there was significant difference between M100HI group and M10HI group (P 0.05).
[Conclusion]
1. The morphological changes of endothelial cells showed that iodine excess could induce the activation of endothelial cells, and then damage and proliferation of endothelial cells, and increase the number of ECM in endothelial cells, and then promote the intimal thickening in a dose-dependent manner, especially in the prey group, suggesting that iodine excess could induce the inferior vena cava by promoting the activation, injury and proliferation of endothelial cells in this animal model. Thickening of the wall and narrowing of the lumen may play a role in the pathogenesis of diaphragmatic Budd Chiari syndrome.
2. Iodine excess can change the contents of ET-1, vWF and HCY in plasma of rats: with the increase of iodine concentration, the contents of ET-1, vWF and HCY in plasma increase gradually; at the same time, iodine excess can increase the expression of E-selectin and ICAM-1 in endothelial cells, leading to the activation of endothelial cells.
3. In this animal model, malnutrition caused by partial diet aggravated the effect of excessive iodine on endothelial cell activation of inferior vena cava in rats.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
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