本文選題：代謝綜合征 + 早期腎損害��； 參考：《廣州醫(yī)學(xué)院》2011年碩士論文

【摘要】：研究目的: 1.構(gòu)建代謝綜合征大鼠模型。 2.探討代謝綜合征大鼠模型早期腎臟α-SMA的表達。 3.RAAS拮抗藥物及降脂藥物分別干預(yù)代謝綜合征大鼠模型后早期腎臟α-SMA的表達變化。 研究方法: 1.動物模型的構(gòu)建 選擇3周齡SPF級雄性Wistar大鼠36只,隨機分為正常飼料(含0.5%NaCl)喂養(yǎng)組(normal sodium, NS,n=12)、高鹽高脂飼料(4%NaCl+49%Fat)喂養(yǎng)組(high sodium and fat, HSF,n=24)。飼養(yǎng)12周時,每組隨機取6只處死(為NS-1組和HSF-1組),稱體重,測量頸總動脈有創(chuàng)血壓、血脂、血糖及血胰島素濃度;根據(jù)穩(wěn)態(tài)胰島素抵抗指數(shù)(HOMA-IR index:fasting insulin X fasting glucose/22.5 [1])評價是否存在胰島素抵抗,根據(jù)代謝綜合征的診斷標(biāo)準(zhǔn)及統(tǒng)計學(xué)方法進行組間比較評價模型是否構(gòu)建成功。 2.藥物干預(yù) NS組(n=6)繼續(xù)以正常飼料喂養(yǎng),為NS-2組。HSF組隨機分為3組:一組繼續(xù)單以高鹽高脂飼料喂養(yǎng)(HSF-2組,n=6),其余動物在喂養(yǎng)高鹽高脂飼料同時分別加入RAAS拮抗藥替米沙坦(HSF+T,n=6,10mg/kg/d)及降脂藥物辛伐他汀(HSF+S,n=6,10mg/kg/d)干預(yù)。至30周時結(jié)束實驗,觀察藥物干預(yù)情況下體重、頸總動脈有創(chuàng)血壓、血脂、血糖、血胰島素濃度、計算穩(wěn)態(tài)胰島素抵抗指數(shù)。 3.代謝綜合征大鼠腎組織形態(tài)學(xué)觀察及免疫組化a-SMA表達建立以高鹽高脂誘導(dǎo)的代謝綜合征動物模型,飼養(yǎng)12周處死的大鼠及繼續(xù)飼養(yǎng)18周到30周后的大鼠分離出腎臟組織,HE染色、Masson染色(20×10倍)使用Leica DM4000顯微鏡自帶的顯微成像系統(tǒng)在相同的條件下采集數(shù)碼圖片。免疫組化切片在Leica DM4000顯微鏡下按統(tǒng)一條件采集,每個組取3個連續(xù)切片,每個切片在10×20倍視野下隨機取5個非重疊視野,避開切片邊緣和組織破損區(qū),拍照,用Image Pro Plus 6.0軟件分析每張照片中陽性染色面積,選擇常用的參數(shù)陽性面積(Area)的百分比代表a-SMA表達量。5個比值的平均值作為每個切片腎組織區(qū)域α-SMA的陽性率(%)。 研究結(jié)果: 1.體重變化 1.1高鹽高脂致體重明顯增加:實驗12周,HSF-1組比NS-1組體重明顯增加(326.45±6.84g比303.3±9.13g ,P0.01);但實驗30周時,NS-2組(434.73±10.19g),HSF-2組(458.83±12.85g),兩組比較,P0.05。結(jié)果提示高鹽高脂飲食可在短期內(nèi)增加體重。 1.2 RAAS拮抗藥物對體重改變不明顯:使用替米沙坦干預(yù)18周后測量體重,HSF+T組與NS-2及HSF-2組差異無統(tǒng)計學(xué)意義(P0.05),結(jié)果提示:使用RAAS拮抗藥物對體重?zé)o影響。 1.3辛伐他汀對體重改變不明顯:使用辛伐他汀治療18周后,HSF+S體重與NS-2及HSF-2組無差異(P0.05)。結(jié)果提示:使用辛伐他汀干預(yù)對體重?zé)o影響。 2.頸總動脈有創(chuàng)血壓 2.1高鹽高脂導(dǎo)致收縮壓升高:實驗12周,HSF-1組(189.43±14.65mmHg)明顯高于NS-1組(153.65±20.53mmHg ,P0.01),同樣,實驗30周時HSF-2組(190.03±14.48mmHg)收縮壓明顯高于NS-2組(161.00±19.49mmHg,P0.05)。結(jié)果提示長期高鹽高脂飼料喂養(yǎng)正常Wistar大鼠可誘導(dǎo)形成高血壓模型。 2.2替米沙坦有效降低收縮壓:給予替米沙坦干預(yù)18周后檢測收縮壓發(fā)現(xiàn),HSF+T組收縮壓(161.95±15.62mmHg)較HSF-2組低(P0.05);HSF+T組與NS-2組間無差異(P0.05)。結(jié)果提示RAAS拮抗藥能有效地降低由高鹽高脂誘導(dǎo)的高血壓。 2.3辛伐他汀對血壓影響不明顯:給予辛伐他汀干預(yù)18周后檢測收縮壓發(fā)現(xiàn),HSF+S組收縮壓(180.36±9.94mmHg)與HSF-2組比較無明顯變化(P0.05),均高于NS-2組(P0.05)。 3.血脂改變 3.1實驗12周,TG、TC及LDL在HSF-1組分別為0.97±0.54mmol/L、1.51±0.24mmol/L、0.90±0.23mmol/L,明顯高于NS-1組(TG、TC、LDL分別為0.31±0.17mmol/L、0.88±0.12mmol/L、0.53±0.23mmol/L,P0.05);兩組HDL相近(0.83±0.17mmol/L對0.89±0.13mmol/L,P0.05)。 3.2實驗30周: 3.2.1 HSF-2組TG及LDL高于NS-2組(TG 2.96±0.56mmol/L對1.10±0.24mmol/L,LDL 2.60±0.51mmol/L對0.81±0.10mmol/L,P0.05)。 3.2.2替米沙坦干預(yù)后血脂改變不明顯:HSF+T組TG及LDL與HSF-2組比較無差異,P0.05;HSF+T組高于NS-2組,P0.05。 3.2.3辛伐他汀干預(yù)后TG及LDL降低:HSF+S組(TG 1.51±0.26mmol/L,LDL 1.17±0.26mmol/L)低于HSF-2組,P0.05;兩組TC及HDL差異無統(tǒng)計學(xué)意義,P0.05。 4.血糖(FBG)、胰島素(INS)及穩(wěn)態(tài)胰島素抵抗指數(shù)(HOMA-IR)的改變:實驗12周,HSF-1組FBG和HOMA-IR明顯高于NS-1組(8.01±1.52mmol/L對6.86±1.18mmol/L, 2.39±0.36對1.84±0.41,P0.01)。INS水平兩組比較差異無統(tǒng)計學(xué)意義,P0.05。實驗30周,HSF-2組HOMA-IR高于NS-2組,HSF-2組、HSF-T組及HSF-S組兩兩比較結(jié)果無統(tǒng)計學(xué)意義。結(jié)果表明:以高鹽高脂飼料喂養(yǎng)正常Wistar大鼠可以導(dǎo)致胰島素抵抗。 綜上,高鹽高脂能夠誘導(dǎo)大鼠在12周后出現(xiàn)典型的代謝綜合征改變:①體重增加:HSF-1組較NS-1組大鼠體重明顯增加(BW:326.45±6.84g vs.303.3±9.13g,P0.01); ②高血壓:HSF-1組較NS-1組大鼠血壓明顯增高(BP:189.43±14.65 mmHg vs 153.65±20.53 mmHg,P0.01); ③血脂異常:HSF-1組較NS-1組大鼠血脂明顯增高(TC:1.51±0.24mmol/L vs. 0.88±0.12mmol/L, TG:0.97±0.54mmol/L vs. 0.31±0.17mmol/L, LDL-C: 0.90±0.23mmol/L vs. 0.53±0.23mmol/L,三者均P0.05,但HDL-C:0.83±0.17mmol/L vs. 0.89±0.13mmol/L,P0.05); ④胰島素抵抗:HSF-1組大鼠胰島素抵抗指數(shù)較NS-1組明顯升高(HOMA-IR index:2.39±0.36 vs. 1.84±0.41,P0.01)。 5.腎組織形態(tài)學(xué)檢測 5.1 HE染色切片光鏡下觀察:NS-1、NS-2組無明顯病理改變,表現(xiàn)為腎小球結(jié)構(gòu)無改變,腎小管上皮細胞胞質(zhì)豐富、小管管腔小、間質(zhì)面積少。HSF-1組可見腎小球系膜輕度節(jié)段性增生、基底膜稍增厚,部分腎小管上皮細胞空泡變性、部分管腔擴張。HSF-2組病變較HSF-1組增加。HSF-T組、HSF-S組組織形態(tài)學(xué)類似HSF-2組。提示HSF組腎臟有輕度病理改變。 5.2 Masson染色光鏡下觀察:所見病理改變同HE染色,膠原纖維被染成蘭色,細胞成紅色,NS組Masson染色未見明顯改變,腎小管基膜處見線狀分布的蘭染的膠原組織,HSF組膠原纖維較NS組稍增多。 6.腎臟組織免疫組化檢測a-SMA表達: 6.1實驗12周,HSF-1組腎臟a-SMA表達面積陽性百分比(0.03±0.005)稍高于NS-1組(0.015±0.008),但兩組比較,P0.05;提示12周時HSF組腎臟a-SMA表達增高。 6.2實驗30周,HSF-2組(0.101±0.003)、HSF+T組(0.038±0.006)、HSF+S組(0.082±0.003)、NS-2組(0.02±0.004),四組比較,腎臟a-SMA表達面積陽性百分比存在差異(P0.01),組間比較發(fā)現(xiàn)HSF-2組、HSF+S組腎臟a-SMA表達面積陽性百分比明顯高于HSF+T組、NS-2組,而HSF-2組與HSF+S組,HSF+T組與NS-2組之間未發(fā)現(xiàn)明顯差異,HSF+T組與HSF-2組比較(0.038±0.006 vs 0.101±0.003),P0.05。提示30周時,HSF組腎臟a-SMA表達增高;替米沙坦治療能有效減少a-SMA的表達;辛伐他汀對a-SMA表達影響不大。 結(jié)論: 1.高鹽高脂飲食喂養(yǎng)12周大鼠出現(xiàn)代謝綜合征,此時腎組織呈輕度腎損害,表現(xiàn)為腎小球系膜輕度節(jié)段性增生、基底膜稍增厚,部分腎小管上皮細胞空泡變性、部分管腔擴張;α-SMA在大鼠腎臟的表達增加,并隨著飼養(yǎng)時間增加a-SMA的表達增高。 2.RAAS拮抗藥物能有效降低代謝綜合征大鼠腎臟α-SMA的表達,而降脂藥物降低腎臟α-SMA的表達不明顯。
[Abstract]:The purpose of the study is:
1. the rat model of metabolic syndrome was constructed.
2. to investigate the expression of renal -SMA in the early stage of metabolic syndrome in rats.
3.RAAS antagonist and lipid-lowering drugs interfere the expression of alpha -SMA in the early stage of metabolic syndrome in rats.
Research methods:
The construction of 1. animal models
36 SPF grade male Wistar rats of 3 weeks old were randomly divided into normal feed (0.5%NaCl) feeding group (normal sodium, NS, n=12), high salt and high fat feed (4%NaCl+49%Fat) feeding group (high sodium and fat). After 12 weeks, each group was randomly selected and weighed to measure the blood pressure and blood lipid of the common carotid artery. The blood glucose and blood insulin concentration; the evaluation of insulin resistance based on the HOMA-IR index:fasting insulin X fasting glucose/22.5 [1], and the success of a comparative evaluation model based on the diagnostic criteria and statistical methods of metabolic syndrome.
2. drug intervention
Group NS (n=6) continued to feed in normal diet for group NS-2.HSF randomly divided into 3 groups: one group continued to feed with high salt and high fat diet (HSF-2 group, n=6), and the other animals were fed with RAAS antagonist telmisartan (HSF+T, n=6,10mg/kg/d) and lipid-lowering drug simvastatin (HSF+S, n=6,10mg/kg/d) at the same time. To 30 weeks, the other animals were fed with high salt and high fat diet. At the end of the experiment, the body weight, blood pressure, blood fat, blood sugar and blood insulin concentration of the common carotid artery were observed, and the steady-state insulin resistance index was calculated.
The renal histomorphology of 3. metabolic syndrome rats and the expression of immunohistochemical a-SMA were established with high salt and high fat induced metabolic syndrome animal model. The rats were fed for 12 weeks and the rats were kept for 18 weeks to 30 weeks. The rats were separated from the kidney tissue, HE staining, and Masson staining (20 x 10 times) using the Leica DM4000 microscope self-contained microscopic imaging. The system collected digital pictures under the same conditions. The immunohistochemical slices were collected under the unified conditions under the Leica DM4000 microscope. Each group was taken 3 consecutive slices. Each slice was randomly selected from 10 x 20 times to take 5 non overlapping fields of vision, avoiding the edge of the slice and the damaged area of the tissue, taking photos and analyzing each photo with Image Pro Plus 6 software. The percentage of the positive area (Area) of the commonly used parameters represented the average value of the.5 ratio of the a-SMA expression as the positive rate (%) of the alpha -SMA in each section of the renal tissue.
The results of the study:
1. body weight change
1.1 high salt and high fat induced weight increased significantly: the body weight of group HSF-1 was significantly increased (326.45 + 6.84g than 303.3 + 9.13g, P0.01) at 12 weeks, but at 30 weeks, group NS-2 (434.73 + 10.19g), HSF-2 group (458.83 + 12.85g), two groups compared, P0.05. results suggested that high salt and high fat diet could increase weight in the short term.
1.2 RAAS antagonists did not have significant weight change: the use of telmisartan after 18 weeks of intervention to measure weight, HSF+T group and NS-2 and HSF-2 group had no statistically significant difference (P0.05), the results showed that the use of RAAS antagonists had no effect on weight.
1.3 simvastatin did not have significant weight change: there was no difference between the HSF+S body weight and the NS-2 and HSF-2 groups after 18 weeks of simvastatin treatment (P0.05). The results showed that the use of simvastatin did not affect the weight.
2. common carotid artery to create blood pressure
2.1 high salt and high fat led to the increase of systolic pressure: 12 weeks in the experiment, group HSF-1 (189.43 + 14.65mmHg) was significantly higher than that in group NS-1 (153.65 + 20.53mmHg, P0.01). Also, the systolic pressure of group HSF-2 (190.03 + 14.48mmHg) was significantly higher than that of NS-2 group (161 + 19.49mmHg, P0.05) at 30 weeks. Hypertension model.
2.2 telmisartan reduced the systolic pressure effectively: after the intervention of telmisartan for 18 weeks, the detection of systolic blood pressure showed that the systolic pressure of HSF+T group (161.95 + 15.62mmHg) was lower than that of the HSF-2 group (P0.05), and there was no difference between group HSF+T and NS-2 (P0.05). The results suggested that the RAAS antagonist could effectively reduce the high blood pressure induced by high salt and high fat.
2.3 the effect of simvastatin on blood pressure was not obvious: after the intervention of simvastatin for 18 weeks, the detection of systolic blood pressure showed that the systolic pressure (180.36 + 9.94mmHg) in group HSF+S was not significantly different from that in the HSF-2 group (P0.05), which was higher than that in the NS-2 group (P0.05).
3. blood lipid changes
3.1 after 12 weeks of experiment, TG, TC and LDL were 0.97 + 0.54mmol/L, 1.51 + 0.24mmol/L and 0.90 + 0.23mmol/L respectively in group HSF-1 (TG, TC, LDL were 0.31 + 0.17mmol/L, 0.88 and 0.53, respectively), and the two groups were close (0.83 + 0.89 +).
3.2 the experiment was 30 weeks.
TG and LDL in group 3.2.1 HSF-2 were higher than those in group NS-2 (TG 2.96 + 0.56mmol/L to 1.10 + 0.24mmol/L, LDL 2.60 + 0.51mmol/L to 0.81 + 0.10mmol/L, P0.05).
After 3.2.2 telmisartan intervention, blood lipid did not change significantly: HSF+T group TG and LDL had no difference compared with HSF-2 group, P0.05 and HSF+T group were higher than NS-2 group, P0.05.
TG and LDL decreased after 3.2.3 simvastatin: group HSF+S (TG 1.51 + 0.26mmol/L, LDL 1.17 + 0.26mmol/L) were lower than group HSF-2, P0.05, and there was no statistical difference between two groups of TC and HDL.
4. blood glucose (FBG), insulin (INS) and homeostasis insulin resistance index (HOMA-IR) changes: 12 weeks of experiment, FBG and HOMA-IR in group HSF-1 were significantly higher than those in group NS-1 (8.01 + 1.52mmol/L against 6.86 + 1.18mmol/L, 2.39 + 0.36 against 1.84 + 0.41, P0.01).INS level two group had no statistical difference, 30 weeks of P0.05. experiment. There was no significant difference between group HSF-T and group HSF-S in 22. The results showed that normal Wistar rats fed with high salt and high fat diet could lead to insulin resistance.
To sum up, high salt and high fat could induce a typical change of metabolic syndrome in rats after 12 weeks: (1) weight gain: the body weight of group HSF-1 was significantly higher than that in group NS-1 (BW:326.45 + 6.84g vs.303.3 + 9.13g, P0.01).
Hypertension: the blood pressure of HSF-1 group was significantly higher than that of NS-1 group (BP:189.43 + 14.65 mmHg vs 153.65 + 20.53 mmHg, P0.01).
Dyslipidemia: the blood lipid of group HSF-1 was significantly higher than that in group NS-1 (TC:1.51 + 0.24mmol/L vs. 0.88 + 0.12mmol/L, TG:0.97 + 0.54mmol/L vs. 0.31 + 0.17mmol/L, LDL-C: 0.90 + 0.23mmol/L 0.53).
Insulin resistance: the insulin resistance index of HSF-1 group was significantly higher than that of NS-1 group (HOMA-IR index:2.39 + 0.36 vs. 1.84 + 0.41, P0.01).
5. renal histomorphological detection
5.1 HE staining section under light microscope: NS-1, group NS-2 had no obvious pathological changes, showing no changes in the glomerular structure, abundant cytoplasm of renal tubular epithelial cells, small tubules and small tubules, low interstitial area in group.HSF-1, which showed mild segmental hyperplasia of mesangial membrane, slightly thickening of basal membrane, vacuolation of some renal tubular epithelial cells, and partial lumen dilatation of.HS In group F-2, the lesions in group.HSF-T were increased compared with those in group HSF-1, while those in group HSF-S were similar to those in group HSF-2, suggesting that there were mild pathological changes in the HSF group.
5.2 Masson staining under light microscope: the pathological changes were stained with HE, the collagen fibers were dyed blue, the cells were red, and the Masson staining in the NS group did not change obviously. The collagenous tissue in the basement membrane of the renal tubule was linearly distributed, and the collagen fibers in the HSF group were slightly more than those in the NS group.
6. the expression of a-SMA in renal tissue was detected by immunohistochemistry.
6.1 after 12 weeks of experiment, the positive percentage of a-SMA expression area in group HSF-1 (0.03 + 0.005) was slightly higher than that in group NS-1 (0.015 + 0.008), but the two groups were compared, P0.05. It was suggested that the expression of a-SMA in the kidney of HSF group was increased at 12 weeks.
6.2 and 30 weeks, group HSF-2 (0.101 + 0.003), group HSF+T (0.038 + 0.006), group HSF+S (0.082 + 0.003), group NS-2 (0.02 + 0.004), compared with group four, the positive percentage of a-SMA expression area in kidney was different (P0.01). The percentage of HSF-2 in HSF-2 group was found in group HSF+S, and the percentage of a-SMA expression area in HSF+S group was significantly higher than that in HSF+T group and NS-2 group. There was no significant difference between group HSF+T and group NS-2. Group HSF+T was compared with group HSF-2 (0.038 + 0.006 vs 0.101 + 0.003), P0.05. suggested that the expression of a-SMA in group HSF increased at 30 weeks, and telmisartan could effectively reduce the expression of a-SMA; simvastatin had little effect on the expression of a-SMA.
Conclusion:
1. the rats fed with high salt and high fat diet for 12 weeks had metabolic syndrome. At this time, the renal tissue showed mild renal damage, characterized by mild segmental hyperplasia of mesangial membrane, slightly thickening of the basement membrane, vacuolation of some tubular epithelial cells and partial dilatation of the lumen. The expression of alpha -SMA in the kidney of rats increased and increased with the increase of the expression of a-SMA in the feeding time. High.
2.RAAS antagonist can effectively reduce the expression of alpha -SMA in the kidney of rats with metabolic syndrome, while the decrease of renal -SMA expression by lipid-lowering drugs is not obvious.

【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R589;R-332

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