本文選題：氨氯地平 + 慶大霉素��； 參考：《武漢大學(xué)》2014年博士論文

【摘要】：第一部分氨氯地平通過調(diào)節(jié)Smad2,7的表達(dá)防止慶大霉素所致大鼠腎小管間質(zhì)纖維化 目的：在慶大霉素所致大鼠腎小管間質(zhì)纖維化損傷整體模型上,觀察氨氯地平通過調(diào)節(jié)Smad2,7的表達(dá)對慶大霉素所致大鼠腎小管損傷的影響。 方法：取SD大鼠28只,分正常對照組,慶大霉素模型組,氨氯地平2.5mg/kg+慶大霉素組,氨氯地平5mg/kg+慶大霉素組,每組7只。氨氯地平2.5mg/kg/d和5mg/kg/d給藥組每日灌胃(i.g.)給藥一次,給藥體積為10mL/kg/d,連續(xù)給藥8天。給藥第2天,模型組及氨氯地平兩個(gè)給藥組腹腔注射(i.p.)慶大霉素100mg/kg/d(體積4mL/kg/d),連續(xù)7天,對照組給予相應(yīng)體積的生理鹽水。給藥第8天置代謝籠中收集24h尿,檢測尿蛋白含量及N-乙酰-β-D-氨基葡萄糖苷酶(NAG)、丫-谷氨酸轉(zhuǎn)換酶(GGT)、堿性磷酸酶(ALP)活性。大鼠稱重后處死,收集血液標(biāo)本檢測血清尿素氮(BUN)和肌酐(SCr)含量。大鼠腎臟用10%福爾馬林固定,石蠟包埋切片后,HE染色,光鏡觀察腎組織形態(tài)學(xué)變化；原位缺口末端標(biāo)記法(d-UTP nick and labeling,TUNEL)檢測腎小管細(xì)胞凋亡情況；免疫組化SP法檢測腎小管Smad2,Smad7表達(dá)水平；其余腎組織制備腎皮質(zhì)勻漿,測定其丙二醛(MDA)、一氧化氮(NO)含量及超氧化物歧化酶(SOD)、一氧化氮合酶(NOS)活性。 結(jié)果：慶大霉素模型組大鼠的24h尿蛋白、血清BUN、SCr的含量明顯升高,與正常對照組比較具有顯著性差異(P0.05,P0.01)；腎臟病理組織學(xué)檢查,慶大霉素模型組近曲小管上皮細(xì)胞濁腫,管腔內(nèi)可見明顯紅細(xì)胞管型和蛋白管型,間質(zhì)呈炎癥反應(yīng),遠(yuǎn)曲小管腔內(nèi)可見充血和紅細(xì)胞管型,說明腎小管間質(zhì)纖維化造模成功。氨氯地平呈劑量依賴性降低慶大霉素模型大鼠的24h尿蛋白含量,血清BUN、SCr含量及尿NAG、GGT、ALP活性的升高趨勢,并且可明顯降低腎組織MDA、 NO含量及NOS活力,提高SOD活性。氨氯地平給藥組對慶大霉素引起的腎小管病理性損傷有明顯的改善及恢復(fù)作用,僅見少量的蛋白管型和紅細(xì)胞管型。同時(shí),氨氯地平也明顯抑制慶大霉素引起的腎小管細(xì)胞凋亡；氨氯地平給藥組顯著降低腎小管Smad2的表達(dá)而增加Smad7的表達(dá)。 結(jié)論：氨氯地平通過調(diào)節(jié)Smad2,7的表達(dá)能防止慶大霉素所致大鼠腎小管間質(zhì)纖維化的進(jìn)程。 第二部分氨氯地平通過調(diào)節(jié)Smad6,7的表達(dá)防止阿霉素所致大鼠腎小球系膜細(xì)胞的損傷 目的：在阿霉素所致大鼠腎小球系膜細(xì)胞纖維化的模型上,觀察氨氯地平通過調(diào)節(jié)Smad6,7的表達(dá)對阿霉素所致大鼠腎小球系膜細(xì)胞損傷的影響。 方法：體外培養(yǎng)腎小球細(xì)膜細(xì)胞(MCs),并按照實(shí)驗(yàn)設(shè)計(jì)對細(xì)胞進(jìn)行分組。分光光度法測定大鼠腎小球系膜細(xì)胞上清液LDH活性；Real-time PCR分析系膜細(xì)胞Smad6,7基因mRNA表達(dá)；Western blot分析系膜細(xì)胞Smad6,7蛋白表達(dá)；細(xì)胞免疫化學(xué)SP法檢測系膜細(xì)胞Smad7的表達(dá)水平。 結(jié)果：氨氯地平呈濃度依賴性降低阿霉素模型組細(xì)胞培養(yǎng)上清液中LDH的活性(P0.01)：對阿霉素+TGF-β1組所致LDH活性的增加也能呈濃度依賴性降低,與阿霉素組比較具有顯著差異(P0.01)。Real-time PCR和Western blot分析顯示,氨氯地平(10-7,10-6mol/L)顯著上調(diào)阿霉素+TGF-β1組系膜細(xì)胞Smad6,7基因和蛋白的表達(dá)(P0.01)；細(xì)胞免疫化學(xué)分析表明,氨氯地平能顯著上調(diào)阿霉素+TGF-β1組所致系膜細(xì)胞Smad7的表達(dá)。 結(jié)論：氨氯地平通過調(diào)節(jié)Smad6,7的表達(dá)防止阿霉素所致大鼠腎小球系膜細(xì)胞的損傷。
[Abstract]:Part 1 amlodipine prevents gentamicin induced tubulointerstitial fibrosis in rats by regulating the expression of Smad2,7.
Objective: To observe the effect of amlodipine on renal tubule injury induced by gentamicin in rats induced by gentamicin induced renal tubulointerstitial fibrosis in rats. The effect of amlodipine on the expression of Smad2,7 was observed.
Methods: 28 SD rats were divided into normal control group, gentamicin model group, amlodipine 2.5mg/kg+ gentamicin group, amlodipine 5mg/kg+ gentamicin group, 7 rats in each group. Amlodipine 2.5mg/kg/d and 5mg/kg/d administration group were administered daily (i.g.), the dosage was 10mL/kg/d, and the drug was given for 8 days. The drug was given for second days, model group and amamlol chloride. The two administration groups were injected intraperitoneally (i.p.) gentamicin 100mg/kg/d (volume 4mL/kg/d) for 7 days, and the control group was given the corresponding volume of normal saline. 24h urine was collected in the metabolic cage for eighth days, and the content of urine protein and N- acetyl beta -D- aminoglucosidase (NAG), GGT, and alkaline phosphatase (ALP) activity in rats were detected. After weighing, the blood samples were collected to detect the content of serum urea nitrogen (BUN) and creatinine (SCr). Rats' kidneys were fixed with 10% formalin and paraffin embedded sections, HE staining, and light microscopy were used to observe the morphological changes of renal tissue; in situ nick end labeling (d-UTP Nick and labeling, TUNEL) was used to detect the apoptosis of renal tubule cells; immunohistochemical SP method was used. The expression of Smad2 and Smad7 in renal tubules was detected, and renal cortex homogenate was prepared in the rest of the renal tissue, and the content of malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD) and nitric oxide synthase (NOS) activity were measured.
Results: the content of 24h urine protein, serum BUN and SCr in the gentamicin group was significantly higher than that in the normal control group (P0.05, P0.01); the renal histopathology examination, the cloudy swelling of the proximal tubular epithelial cells in the gentamicin model group, the obvious tube type and the protein tube type in the lumen, and the interstitial inflammation in the lumen. It should be seen that hyperemia and erythrocyte tube can be seen in the canalicular cavity of the far curvature, indicating the success of renal tubulointerstitial fibrosis. Amlodipine has a dose-dependent reduction of 24h urine protein content in gentamicin model rats, serum BUN, SCr content and NAG, GGT, ALP activity in urine, and can obviously reduce the MDA, NO content and NOS activity of renal tissue, and improve S. OD activity. Amlodipine administration group has obvious improvement and recovery effect on renal tubular pathological injury caused by gentamicin, only a small amount of protein tube type and erythrocyte tube type. Amlodipine also obviously inhibits the apoptosis of renal tubule cells caused by gentamicin, and amlodipine administration group significantly reduces the expression of Smad2 in renal tubule. Add the expression of Smad7.
Conclusion: Amlodipine can prevent gentamicin induced tubulointerstitial fibrosis in rats by regulating the expression of Smad2,7.
The second part of amlodipine prevents doxorubicin induced glomerular mesangial cell injury by regulating the expression of Smad6,7.
Objective: To observe the effect of amlodipine on the damage of glomerular mesangial cells induced by adriamycin in rat glomerular mesangial cell fibrosis induced by doxorubicin (adriamycin).
Methods: the cultured glomerular mesangial cells (MCs) were cultured in vitro, and the cells were grouped according to the experimental design. The activity of LDH in the supernatant of glomerular mesangial cells was measured by spectrophotometric method; the Smad6,7 gene mRNA expression of the mesangial cells of the rat mesangial cells was analyzed by Real-time PCR; the Smad6,7 protein expression in the mesangial cells of the Western blot analysis; and the detection of the cell immunochemistry SP method. The expression level of Smad7 in mesangial cells.
Results: Amlodipine was a concentration dependent decrease in the activity of LDH in the supernatant of cell culture of adriamycin model group (P0.01): the increase of LDH activity caused by adriamycin +TGF- beta 1 groups decreased in a concentration dependent manner, and there was significant difference (P0.01).Real-time PCR and Western blot analysis, amlodipine (10-7,10-6mo, 10-7,10-6mo). L/L) significantly up-regulated the expression of Smad6,7 gene and protein in the mesangial cells of adriamycin +TGF- beta 1 group (P0.01). Cell immuno chemical analysis showed that amlodipine could significantly increase the expression of Smad7 in the mesangial cells induced by adriamycin +TGF- beta 1 group.
Conclusion: Amlodipine can prevent the injury of mesangial cells induced by adriamycin by regulating the expression of Smad6,7.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R965

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