本文關(guān)鍵詞： 氯胺酮 腎損傷 褪黑素 氧化應(yīng)激 凋亡　出處：《廣西醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景目前研究發(fā)現(xiàn),氯胺酮對(duì)膀胱的損傷機(jī)制可能與氯胺酮的直接毒性作用、炎癥損傷、血管損傷以及氧化應(yīng)激的有關(guān)。但其對(duì)腎臟的損害作用機(jī)制尚不明確。本研究通過(guò)建立氯胺酮致SD大鼠腎臟損傷動(dòng)物模型,觀察SD大鼠行為學(xué)及體重變化、取腎臟行HE染色、馬松染色觀察病理學(xué)變化；測(cè)定腎組織勻漿中丙二醛(MDA)的含量、超氧化物氣化酶(SOD)和谷胱甘肽過(guò)氧化物酶(GSH-P x)的活力,以及抗氧化劑褪黑素(Melatonni,MT)干預(yù)后是否對(duì)腎臟有保護(hù)作用。研究目的：探討氯胺酮所致腎臟損傷的可能機(jī)制以及MT對(duì)氯胺酮染毒大鼠腎臟損傷的保護(hù)作用。研究方法：選擇成年雄性SD大鼠30只,隨機(jī)分為三組：(1)生理鹽水組(對(duì)照組)：每日注射生理鹽水lml；(2)氯胺酮組(K實(shí)驗(yàn)組)：氯胺酮按100mg/kg用生理鹽水稀釋至1m1每天腹腔注射。(3)MT+氯胺酮聯(lián)合給藥組(MT保護(hù)組)：氯胺酮按10Omg/kg+褪黑素10mg/kg用生理鹽水稀釋至1m1每天腹腔注射。三組大鼠飼養(yǎng)時(shí)間為12周。12周后經(jīng)腹主動(dòng)脈采血,測(cè)量血清中肌酐、尿素氮評(píng)估大鼠的腎臟功能。然后處死大鼠,取腎臟制作組織勻漿,測(cè)量丙二醛(MDA)的含量、超氧化物氣化酶(SOD)和微量還原型谷胱甘肽(GSH)的活力。另取腎臟組織行HE、Masson染色和TUNEL凋亡檢測(cè)。研究結(jié)果：1.大鼠行為學(xué)和體重變化對(duì)照組大鼠在注射等量生理鹽水后,大鼠行為未見(jiàn)明顯異常。而氯胺酮組和MT保護(hù)組大鼠在腹腔注射藥物后,大鼠先出現(xiàn)興奮增加,走路不穩(wěn)、后肢癱瘓和共濟(jì)失調(diào)等表現(xiàn)。其后均在lmin內(nèi)出現(xiàn)深度麻醉,表現(xiàn)為嗜睡、對(duì)刺激無(wú)反應(yīng)等。從第2周至12周,對(duì)照組大鼠的平均體重均高于K實(shí)驗(yàn)組和MT保護(hù)組,三組大鼠的體重變化差異有統(tǒng)計(jì)學(xué)意義(P0.05)。并且MT保護(hù)組大鼠體重平均值也高于K實(shí)驗(yàn)組,二者比較差異有統(tǒng)計(jì)學(xué)意義(P0.05)。2.大鼠腎功能檢測(cè)結(jié)果大鼠血清Scr、BUN在各組之間差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。但MT保護(hù)組和K實(shí)驗(yàn)組的Scr和BUN值仍然分別有14只(14/20)、12只(12/20)高于對(duì)照組的均值,提示氯胺酮可能對(duì)大鼠的腎臟濾過(guò)功能有一定的損害作用。3.腎組織勻漿氧化指標(biāo)測(cè)定結(jié)果三組腎組織勻漿MDA含量差異有統(tǒng)計(jì)學(xué)意義(P0.001),K實(shí)驗(yàn)組MDA含量明顯高于對(duì)照組(P0.001)；而MT保護(hù)組MT明顯抑制了氯胺酮引起的大鼠腎臟組織MDA的升高趨勢(shì),與K組比較明顯降低(K0.001)。三組大鼠腎組織勻漿SOD活力差異有統(tǒng)計(jì)學(xué)意義(P0.001),K實(shí)驗(yàn)組明顯低于對(duì)照組(P0.001),MT組增加了腎組織勻漿SOD活力,與K實(shí)驗(yàn)組比較差異有統(tǒng)計(jì)學(xué)意義(P0.05)。三組大鼠腎組織勻漿GSH-Px活力差異有統(tǒng)計(jì)學(xué)意義(P0.001),K實(shí)驗(yàn)組明顯低于對(duì)照組(P0.001),MT組增加了腎組織勻漿GSH-Px活力,與K實(shí)驗(yàn)組比較差異有統(tǒng)計(jì)學(xué)意義(P0.05)。4.大鼠腎臟HE染色結(jié)果對(duì)照組大鼠的腎臟結(jié)構(gòu)組織形態(tài)學(xué)正常,無(wú)腎小管水腫、淤血或管型形成。K實(shí)驗(yàn)組腎小管、腎小球周圍能夠見(jiàn)到大量炎癥細(xì)胞浸潤(rùn),腎間質(zhì)有纖維化條索形成。腎小管大量水腫,正常結(jié)構(gòu)消失,出現(xiàn)大量空泡樣改變；腎小球變性萎縮,甚至出現(xiàn)閉鎖；MT保護(hù)組腎小管、腎小球炎癥細(xì)胞浸潤(rùn)、腎間質(zhì)纖維化較K實(shí)驗(yàn)組減輕,部分腎小球出現(xiàn)萎縮。5.大鼠腎臟Masson染色結(jié)果與對(duì)照組比較,K實(shí)驗(yàn)組、MT保護(hù)組腎間質(zhì)膠原纖維染色明顯加深,可見(jiàn)纖維化條索形成,提示在長(zhǎng)期腹腔注射氯胺酮后,腎間質(zhì)膠原沉積增加、纖維化加重。但MT保護(hù)組較K實(shí)驗(yàn)組腎間質(zhì)膠原沉積減少,纖維化程度減輕。半定量分析發(fā)現(xiàn),三組間比較差異有統(tǒng)計(jì)學(xué)意義(F=178.83,P0.001)。對(duì)照組腎間質(zhì)平均膠原面積比是(8.97%±0.02)；K實(shí)驗(yàn)組平均膠原面積比是(41.49%±0.03)；MT保護(hù)組平均膠原面積比是(28.54%±0.04)。K實(shí)驗(yàn)組和對(duì)照組比較,差異有統(tǒng)計(jì)學(xué)意義(P0.001),提示氯胺酮導(dǎo)致腎間質(zhì)發(fā)生纖維化;MT保護(hù)組與K實(shí)驗(yàn)組比較,差異有統(tǒng)計(jì)學(xué)意義(P0.001),提示褪黑素在一定程度上能夠減輕腎間質(zhì)的纖維化程度。6.大鼠腎臟腎小球TUNE凋亡細(xì)胞檢測(cè)對(duì)照組偶見(jiàn)凋亡陽(yáng)性細(xì)胞。K實(shí)驗(yàn)組和MT保護(hù)組,凋亡細(xì)胞呈現(xiàn)明顯增多。半定量分析發(fā)現(xiàn),腎小管平均每高倍鏡下陽(yáng)性細(xì)胞K實(shí)驗(yàn)組為171.76±14.67個(gè),MT保護(hù)組為149.92±27.87個(gè),NS對(duì)照組為64.56±17.90個(gè)。三者比較,差異有統(tǒng)計(jì)學(xué)意義(F=73.33,P0.001)。K實(shí)驗(yàn)組和對(duì)照組比較,差異有統(tǒng)計(jì)學(xué)意義(P0.001),MT保護(hù)組和對(duì)照組比較,差異有統(tǒng)計(jì)學(xué)意義(P0.001)。MT保護(hù)組比K實(shí)驗(yàn)組凋亡細(xì)胞計(jì)數(shù)明顯減少,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。研究結(jié)論：1.長(zhǎng)期腹腔注射氯胺酮是制造大鼠氯胺酮性腎臟損傷模型的可靠方法。2.氯胺酮導(dǎo)致大鼠腎組織勻漿中MDA的水平升高,SOD和GSH-Px的活力下降,并且在應(yīng)用MT治療后,可明顯降低大鼠腎組織勻漿中MDA的含量,增加SOD和GSH-Px的活力。提示大鼠體內(nèi)存在氧化損傷和氧化-抗氧化失衡,可能為氯胺酮致腎臟損傷的機(jī)制之一,褪黑素對(duì)腎臟具有一定的保護(hù)作用。3.氯胺酮導(dǎo)致腎臟炎癥細(xì)胞浸潤(rùn)和腎臟細(xì)胞凋亡增加,而MT能夠減輕腎臟的細(xì)胞凋亡,MT的保護(hù)機(jī)制可能和增強(qiáng)體內(nèi)抗氧化應(yīng)激能力有關(guān)系。
[Abstract]:On the background of the present study found a direct toxic effect, the possible mechanism of ketamine and ketamine on injury of bladder inflammation injury, vascular injury and oxidative stress damage to the kidneys. But its mechanism is still not clear. In this study we established a model of renal injury induced by ketamine animal SD rats, and to observe the change of body weight SD the behavior in the kidneys, HE staining was performed to observe the pathological changes of Masson staining; Determination of malondialdehyde in kidney homogenates (MDA) content, superoxide enzyme gasification (SOD) and glutathione peroxidase (GSH-P x) activity and melatonin (Melatonni, MT) after the intervention have protective effects on kidney research. Objective: To investigate the possible mechanism of renal injury induced by ketamine and the protective effect of MT on renal injury in rats exposed to ketamine. Methods: 30 male SD rats were randomly. Divided into three groups: (1) normal saline group (control group): daily injection of saline LML; (2) ketamine group (K group): Ketamine 100mg/kg diluted to 1m1 with normal saline by intraperitoneal injection every day. (3) MT+ ketamine combined drug group (MT group): according to the 10Omg/kg+ of melatonin 10mg/kg diluted with saline to 1m1 daily intraperitoneal injection of ketamine. Three rats were fed for 12 weeks.12 weeks after abdominal aortic blood, serum creatinine measurements, evaluation of rat kidney function of urea nitrogen. Then the rats were sacrificed and kidneys tissue homogenate, measurement of malondialdehyde (MDA) content, superoxide gasification enzyme (SOD) and trace glutathione (GSH) activity. The other kidney tissues were collected for detection of HE, Masson staining and TUNEL apoptosis. Results: 1. the behavior of rats and the change of body weight of rats in control group after the injection of normal saline, rats had no obvious differences Often. And ketamine group and MT group rats after intraperitoneal injection of drugs, rats first appeared excited increase, ataxia, ataxia and hind limb paralysis. Subsequently occurred within lmin depth of anesthesia, as lethargy, no response to stimulation. From second to 12 weeks, the average weight of control rats were higher than that of K group and MT group, there were statistically significant differences in body weight changes of the three groups of rats (P0.05) and MT protection group weight average value is higher than that of K group, there was significant difference between the two groups (P0.05) in serum of Scr rats renal function in rats with.2. detection results there was no significant difference of BUN between the groups (P0.05). But the MT protection group and K experimental group Scr and BUN were still 14 (14/20), 12 (12/20) higher than the control group, suggesting that ketamine may on rat glomerular filtration function have a certain role in.3. damage kidney group Oxidative index determination results between homogenate homogenate MDA content of renal tissue of three groups was statistically significant (P0.001), K MDA in the experimental group were significantly higher than the control group (P0.001); while the MT protection group MT obviously inhibited by ketamine in renal tissue of MDA rats increased, compared with the K group decreased significantly (K0.001). The three groups had statistical significance in kidney homogenates of rat SOD activity (P0.001), K group was significantly lower than the control group (P0.001), MT group increased SOD activity in renal tissue homogenate, and the difference had statistical significance K experimental group (P0.05). The three groups had statistical significance in kidney homogenates of rat GSH-Px activity the difference of K (P0.001), the experimental group was significantly lower than the control group (P0.001), MT group increased GSH-Px activity in renal tissue homogenate, and the difference had statistical significance in experimental group K (P0.05) HE in the kidney of.4. rat kidney tissue staining structure of rats in control group were morphological Often, no renal tubular edema, congestion or tube type.K experimental group of renal tubule formation can be seen around the glomerular infiltration of inflammatory cells, renal interstitial fibrosis cord formation. Renal tubular edema, normal structure disappeared, the emergence of a large number of vacuolar changes; glomerular atrophy and degeneration, even atresia; MT group of renal tubules glomerular, inflammatory cell infiltration, renal interstitial fibrosis than K experimental group, partial glomerular atrophy of renal Masson in rats with.5. staining results compared with the control group, K group, MT group of renal interstitial collagen fiber staining was deepened, fibrosis cord formation, suggesting that in the long term after intraperitoneal injection of ketamine, kidney collagen deposition and fibrosis. But the MT protection group compared with the K experimental group of renal interstitial collagen deposition decreased, reduce the degree of fibrosis. Semi quantitative analysis showed that there were significant differences between the three groups (F=178 .83, P0.001). The control group of renal interstitial collagen area ratio is the average (8.97% + 0.02); K group average collagen area ratio is (41.49% + 0.03); MT group average collagen area ratio is (28.54% + 0.04).K experimental group and the control group, the difference was statistically significant (P0.001). It indicates that ketamine lead to renal interstitial fibrosis; MT group and K experimental group, the difference was statistically significant (P0.001), suggesting that the degree of fibrosis in.6. rat kidney TUNE cell apoptosis of melatonin can reduce the renal interstitial in a certain extent, the control group occasionally positive apoptotic cells in experimental group.K and MT group. Apoptotic cells showed significantly increased. Semi quantitative analysis showed that the renal tubular average high magnification of K positive cells in experimental group was 171.76 + 14.67, 149.92 + 27.87 MT protection group, NS control group is 64.56 + 17.90. Three comparison, the difference was statistically significant (F=73. 33, P0.001).K experimental group and the control group, the difference was statistically significant (P0.001), MT group and control group, the difference was statistically significant (P0.001) in.MT group was obviously lower than that of K group the number of apoptotic cells, the difference was statistically significant (P0.05). Conclusions: 1. long term ketamine intraperitoneal injection ketamine is a reliable method for manufacturing renal injury in rats.2. ketamine resulted in elevated rat renal tissue homogenate levels of MDA, SOD and GSH-Px activity decreased, and the application of MT after treatment, can significantly reduce the content of rat kidney tissue homogenate in MDA, SOD and GSH-Px increased vitality. Suggesting the presence of oxidation damage and oxidant / antioxidant imbalance in rats, one of the possible mechanisms of renal damage induced by ketamine, melatonin has a protective effect on renal.3. ketamine must lead to renal inflammatory cell infiltration and apoptosis of kidney cells, MT can reduce the apoptosis of the kidneys, and the protective mechanism of MT may be associated with the enhancement of anti oxidative stress in the body.

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R692

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