高碘對大鼠下腔靜脈內(nèi)皮細(xì)胞活化的影響
發(fā)布時間：2018-08-25 08:06
【摘要】：[目的] 本實(shí)驗(yàn)通過以高碘水和偏食飼料飼養(yǎng)Wistar大鼠,建立偏食高碘大鼠模型,通過HE染色、免疫組織化學(xué)染色和血漿中相關(guān)因子的測定,觀察大鼠下腔靜脈肝段內(nèi)皮細(xì)胞的活化情況,并探討高碘引起血管內(nèi)皮細(xì)胞活化的作用機(jī)制。 [方法] 1、實(shí)驗(yàn)動物與分組：選擇斷乳后約1個月,體重(110±10)g遠(yuǎn)交封閉群SPF/VAF級Wistar大鼠,由山東大學(xué)動物實(shí)驗(yàn)中心提供。適應(yīng)性喂養(yǎng)一周后根據(jù)性別和體重隨機(jī)分為8組,分別為：①正常對照組(NC)；②正常飼料+10倍碘水組(10HI)；③正常飼料+50倍碘水組(50HI)；④正常飼料+100倍碘水組(100HI)；⑤偏食對照組(MC)；⑥偏食飼料+10倍碘水組(M10HI)；⑦偏食飼料+50倍碘水組(M50HI)；⑧偏食飼料+100倍碘水組(M100HI),每組8只。 2、大鼠體重記錄：每周稱量各組大鼠體重并記錄,喂養(yǎng)6個月。 3、大鼠下腔靜脈肝段HE染色：用10%福爾馬林固定,常規(guī)石蠟包埋,切片,以二甲苯脫蠟,各級乙醇水化,蘇木素染色2min,鹽酸酒精分化15s,酒精伊紅染色2-5分鐘,常規(guī)脫水透明,封片,觀察下腔靜脈內(nèi)皮細(xì)胞的形態(tài)改變。 4、測定各組大鼠血漿中ET-1、vWF、vWFpp及HCY的含量。 5、大鼠下腔靜脈肝段免疫組織化學(xué)染色：組織切片經(jīng)過脫蠟水化,抗原修復(fù),PBS沖洗,加入一抗,4℃過夜,加入二抗,用DAB進(jìn)行顯色,觀察E-選擇素和ICAM-1在下腔靜脈內(nèi)皮細(xì)胞的表達(dá)情況。 [結(jié)果] 1.大鼠體重的變化： 從開始喂養(yǎng)到6個月結(jié)束,各正常飼料+高碘水組與NC組相比無明顯差別；偏食對照組(MC)到第24周才與NC組出現(xiàn)統(tǒng)計(jì)學(xué)差異；第2周開始,各偏食飼料+高碘水組均低于或明顯低于NC組。 2、內(nèi)皮細(xì)胞形態(tài)的改變： 隨著碘濃度的逐漸升高,大鼠下腔靜脈肝段內(nèi)皮細(xì)胞出現(xiàn)活化和增生的改變,表現(xiàn)為內(nèi)皮細(xì)胞體積增大,呈立方形向管腔突起,內(nèi)皮細(xì)胞數(shù)目增多,內(nèi)皮下ECM增多,甚至局部內(nèi)膜增厚,其中以偏食組更為明顯。 3、血漿ET-1、vWF、vWFpp及HCY含量測定： 3.1大鼠血漿ET-1 內(nèi)皮細(xì)胞功能受到了一定的影響,與正常對照組相比,50HI組ET-1含量升高,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)：100HI組、M50HI組和MlOOHI組ET-1含量升高,差異顯著(P0.01)。相鄰組間比較結(jié)果顯示,隨著碘濃度的逐漸升高,ET-1的含量呈升高趨勢(P0.05或P0.01) 3.2大鼠血漿vWF 內(nèi)皮細(xì)胞功能受到了影響,與正常對照組相比,M10HI組和M50HI組vWF含量升高,差異具有統(tǒng)計(jì)學(xué)意義(P＜0.05)；100HI組和M100HI組vWF含量升高,差異顯著(P＜0．01)。相鄰組間比較結(jié)果顯示,隨著碘濃度的逐漸升高,vWF的含量逐漸升高。其中,M10HI組與MC組比較有統(tǒng)計(jì)學(xué)差異(P＜0.05),100HI組與50HI組比較、M100HI組與M50HI組比較差異顯著(P0.01)。 3.3大鼠血漿vWFpp 與正常對照組相比,M50HI組vWFpp含量升高(p0.05),其余各組未見升高。 3.4大鼠血漿HCY 與正常對照組相比,50HI組HCY含量升高,差異具有統(tǒng)計(jì)學(xué)意義(p0.05),100HI組、M10HI組、M50HI組和M100HI組HCY含量升高,差異顯著(p0.01)。相鄰組間比較結(jié)果顯示,隨著碘濃度的逐漸升高,血漿HCY的含量呈升高趨勢(P0.05或P＜0.01) 4、免疫組織化學(xué)結(jié)果： 4.1ICAM-1的表達(dá) 隨著碘濃度逐漸升高,內(nèi)皮細(xì)胞ICAM-1表達(dá)呈增加趨勢。其中,M100HI組ICAM-1表達(dá)與正常對照組相比有統(tǒng)計(jì)學(xué)差異(P0.05)；組間比較均無統(tǒng)計(jì)學(xué)差異(P0.05) 4.2E-選擇素的表達(dá) 隨著碘濃度逐漸升高,內(nèi)皮細(xì)胞E-選擇素表達(dá)逐漸增加。其中,與正常對照組相比,100HI組、M50HI組、M100HI組E-選擇素表達(dá)有統(tǒng)計(jì)學(xué)差異(P0.05或P0.01)；組間比較顯示,M100HI組與M10HI組相比有統(tǒng)計(jì)學(xué)差異(P0.05) [結(jié)論] 1.內(nèi)皮細(xì)胞形態(tài)學(xué)的改變表明高碘可導(dǎo)致內(nèi)皮細(xì)胞活化、進(jìn)而損傷和增生,內(nèi)皮下ECM增多,進(jìn)而促進(jìn)內(nèi)膜增厚,并呈劑量一效應(yīng)關(guān)系,其中,在偏食組更為明顯,提示在該動物模型中高碘通過促進(jìn)內(nèi)皮細(xì)胞活化、損傷和增生,可引起下腔靜脈管壁增厚、管腔狹窄,可能對隔膜型布加綜合征的發(fā)病機(jī)制起一定的提示作用。 2.高碘可引起大鼠血漿多種活性因子ET-1、vWF及HCY的含量發(fā)生改變：隨碘濃度的逐漸升高,血漿ET-1、vWF、HCY的含量逐漸增加；同時,高碘使內(nèi)皮細(xì)胞E-選擇素和ICAM-1表達(dá)量增加,導(dǎo)致內(nèi)皮細(xì)胞活化。 3.在該動物模型中,偏食所引起的營養(yǎng)不良可加重高碘對大鼠下腔靜脈內(nèi)皮細(xì)胞活化的影響。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363
本文編號：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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363
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，本文編號：2202280
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