甲基苯丙胺對大鼠腎臟損傷研究
發(fā)布時間：2018-08-11 15:22
【摘要】：《2015中國禁毒報告》顯示[1],全國共破獲毒品犯罪案件14.59萬起,繳獲各類毒品合計(jì)68.96噸,其中冰毒類毒品25.9噸,氯胺酮11.2噸,海洛因9.3噸、大麻4噸。全國累計(jì)登記吸毒人數(shù)295.5萬名,其中濫用新型毒品人員達(dá)145.9萬名,占吸毒人數(shù)的49.4%,首次超過海洛因?yàn)E用群體比例49.3%,且在新型毒品濫用者中,濫用甲基苯丙胺人員達(dá)119萬,占新型毒品吸毒人數(shù)的81.6%,說明中國毒品濫用結(jié)構(gòu)發(fā)生了深刻的變化;以甲基苯丙胺為主體的苯丙胺類興奮劑(AST)濫用與依賴在我國和世界范圍內(nèi)快速蔓延,大有取代海洛因、可卡因等傳統(tǒng)毒品的趨勢,我國面臨著繼海洛因之后的甲基苯丙胺濫用的威脅。云南省地處我國西南,其邊境地區(qū)環(huán)境優(yōu)美,氣候宜人,民族眾多,邊境貿(mào)易繁榮,自古就是歷史文化風(fēng)景名勝。自改革開放以來,從“金三角”毒源地經(jīng)我國云南邊境地區(qū)的毒品運(yùn)輸量愈來愈大,毒品運(yùn)輸愈加頻繁,嚴(yán)重危害邊境人民的正常生活和正常的經(jīng)濟(jì)秩序,也使得我國從過去毒品的過境國逐步變成制造、生產(chǎn)、銷售和消費(fèi)“一條龍”式的受害國。目前由于新型毒品尤其以甲基苯丙胺(冰毒)濫用人群較多,故本研究就甲基苯丙胺對腎臟損傷予以探討。[目的]探討濫用新型毒品甲基苯丙胺(methamphetamine,MA)對腎臟損傷機(jī)制的研究,本研究分為兩部分,第一部分為研究甲基苯丙胺損傷人腎近曲小管上皮細(xì)胞(HK-2cells,HK-2細(xì)胞),采用MTS法進(jìn)行細(xì)胞毒試驗(yàn)及細(xì)胞凋亡情況,以及急性損傷標(biāo)志物中性粒細(xì)胞明膠酶相關(guān)脂質(zhì)運(yùn)載蛋白(Ngal)、腎損傷因子(Kim-1)的表達(dá)、分泌特點(diǎn),并對其在甲基苯丙胺腎毒性損傷中的意義進(jìn)行探討。第二部分建立甲基苯丙胺對大鼠模型腎臟損傷模型,研究甲基苯丙胺對大鼠腎損傷機(jī)制。[方法]觀察甲基苯丙胺對人腎近曲小管上皮細(xì)胞損傷作用。實(shí)驗(yàn)分組如下:正常對照組(僅加入培養(yǎng)液),對照組采用濃度分別為0.75mmol/L、1mmol/L、1.5mmol/L、2mmol/L、3mmol/L、4mmol/L、6mmol/L、8mmol/L、12mmol/L、16mmol/L甲基苯丙胺作用于體外培養(yǎng)人腎近曲小管上皮細(xì)胞,通過MTS法觀察甲基苯丙胺對HK-2的影響,流式細(xì)胞術(shù)觀察細(xì)胞周期改變和凋亡情況,ELISA法檢測培養(yǎng)上清中Ngal與Kim-1的分泌情況。通過建立甲基苯丙胺急性、亞急性、慢性大鼠模型,取大鼠腎組織進(jìn)行切片、HE染色及電鏡掃描。[結(jié)果](1)甲基苯丙胺作用人腎小管上皮細(xì)胞24h,顯微觀察細(xì)胞形態(tài)病使用MTS法測定細(xì)胞生存率。甲基苯丙胺在0.75mmo1～16mmol/L濃度范圍內(nèi),隨著甲基苯丙胺濃度的增加,HK-2細(xì)胞的生存率相應(yīng)降低。增殖實(shí)驗(yàn)結(jié)果顯示:與相同時間內(nèi)的對照組相比,濃度達(dá)6mmol/L～16mmol/L時甲基苯丙胺作用HK-2細(xì)胞24h差異有統(tǒng)計(jì)學(xué)意義(P0.05)。(2)甲基苯丙胺誘導(dǎo)HK-2細(xì)胞凋亡結(jié)果:0.75mmol/L 組:20.51%;1mmol/L 組:24%;1.5mmol/L 組:31.96%;2mmol/L 組:30.15%;3mmol/L 組:31.8%;4mmol/L 組:26.9%;6mmol/L 組:99%;8mmol/L 組:98%;12mmol/L 組:99.16%;16mmol/L 組:99.8%。(3)NGAL結(jié)果:8mmol/L組與對照組在相比,在12h內(nèi),隨著作用時間延長,腎小管上皮細(xì)胞分泌NGAL量增加。12h后腎小管上皮細(xì)胞分泌NGAL逐漸減少。(4)KIM-1結(jié)果:8mmol/L組與對照組在相比,在48h內(nèi),隨著作用時間延長,腎小管上皮細(xì)胞分泌KIM-1量增加。[結(jié)論](1)甲基苯丙胺對HK-2細(xì)胞的有抑制增殖作用,濃度達(dá)6mmol/L～16mmol/L時甲基苯丙胺作用HK-2細(xì)胞24h抑制作用越強(qiáng)。(2)甲基苯丙胺對HK-2細(xì)胞的促進(jìn)凋亡。(3)在12h內(nèi),隨著作用時間延長,腎小管上皮細(xì)胞分泌NGAL量增加。12h后腎小管上皮細(xì)胞分泌NGAL逐漸減少。(4)隨著作用時間延長,腎小管上皮細(xì)胞分泌KIM-1量增加。(5)MA對腎臟有明顯毒性作用。
[Abstract]:According to China's Drug Control Report in 2015, a total of 145,900 drug-related crimes were cracked and 68.96 tons of all kinds of drugs were seized nationwide, including 25.9 tons of methamphetamines, 11.2 tons of ketamine, 9.3 tons of heroin and 4 tons of cannabis. Among the new drug abusers, 1.19 million people abused methamphetamine, accounting for 81.6% of the new drug abusers, indicating that the structure of drug abuse in China has undergone profound changes; the abuse and dependence of amphetamine-type stimulants (AST), mainly methamphetamine, in China and around the world Yunnan Province is located in the southwest of China, with beautiful environment, pleasant climate, numerous nationalities and prosperous border trade. Since the reform and opening up, it has been a historical and cultural scenic spot. Drug traffic in the "Golden Triangle" is increasing in the border areas of Yunnan, China. Drug traffic is becoming more frequent, seriously endangering the normal life and normal economic order of the border people. It has also gradually transformed China from a drug transit country into a "one-stop" victim country of manufacturing, production, sales and consumption. Methamphetamine (MA) is a new drug, especially methamphetamine (methamphetamine) is abused in a large number of people, so this study on methamphetamine-induced renal damage to explore. [Objective] To explore the abuse of methamphetamine (MA) on the mechanism of renal damage, this study is divided into two parts, the first part is to study methamphetamine-induced renal damage in human proximal meander. Tubular epithelial cells (HK-2 cells, HK-2 cells) were used for cytotoxicity test and apoptosis by MTS method. The expression and secretion characteristics of neutrophil gelatinase-associated lipid carrier protein (Ngal) and kidney injury factor (Kim-1) in acute injury markers were studied. The significance of MTS in methamphetamine nephrotoxic injury was also discussed. [Methods] The effects of methamphetamine on renal proximal convoluted tubular epithelial cells in rats were observed. The experimental groups were as follows: normal control group (only adding culture medium), control group with concentrations of 0.75 mmol/L, 1.5 mmol/L, 2 mmol/L, respectively. L, 3mmol/L, 4mmol/L, 6mmol/L, 8mmol/L, 12mmol/L, 16mmol/L methamphetamine acted on cultured human renal proximal tubular epithelial cells in vitro. MTS method was used to observe the effect of methamphetamine on HK-2, flow cytometry was used to observe cell cycle changes and apoptosis. ELISA was used to detect the secretion of Ngal and Kim-1 in culture supernatant. [Results] (1) Methamphetamine treated human renal tubular epithelial cells for 24 hours. Cell survival rate was determined by MTS method. Methamphetamine concentration ranged from 0.75 mmo1 to 16 mmol/L, with the concentration of methamphetamine. The proliferative test showed that there was a significant difference in the effect of methamphetamine on HK-2 cells at the concentration of 6 mmol/L to 16 mmol/L for 24 hours (P 0.05). (2) Methamphetamine-induced apoptosis of HK-2 cells: 0.75 mmol/L group: 20.51%; 1 mmol/L group: 24%; 1.5 mmol/L group: 1.5 mmol/L; 2. L group: 31.96%; 2 mmol/L group: 30.15%; 3 mmol/L group: 31.8%; 4 mmol/L group: 26.9%; 6 mmol/L group: 99%; 8 mmol/L group: 98%; 12 mmol/L group: 99.16%; 16 mmol/L group: 99.8%. (3) NGAL results: 8 mmol/L group compared with the control group, in 12 hours, the amount of NGAL secreted by renal tubular epithelial cells increased. (4) KIM-1 gradually decreased. (4) KIM-1 secretion of renal tubular epithelial cells increased with the prolongation of the treatment time in the 8mmol/L group compared with the control group. [Conclusion] (1) Methamphetamine inhibited the proliferation of HK-2 cells. When the concentration reached 6mmol/L to 16mmol/L, the inhibition of methamphetamine on HK-2 cells became stronger in 24h. (2) Methamphetamine inhibited the proliferation of HK-2 cells. Amine promoted apoptosis of HK-2 cells. (3) Within 12 hours, NGAL secreted by renal tubular epithelial cells increased with the prolongation of treatment time. NGAL secreted by renal tubular epithelial cells decreased gradually after 12 hours. (4) KIM-1 secreted by renal tubular epithelial cells increased with the prolongation of treatment time. (5) MA had a significant toxic effect on kidney.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R749.64
本文編號：2177392
[Abstract]:According to China's Drug Control Report in 2015, a total of 145,900 drug-related crimes were cracked and 68.96 tons of all kinds of drugs were seized nationwide, including 25.9 tons of methamphetamines, 11.2 tons of ketamine, 9.3 tons of heroin and 4 tons of cannabis. Among the new drug abusers, 1.19 million people abused methamphetamine, accounting for 81.6% of the new drug abusers, indicating that the structure of drug abuse in China has undergone profound changes; the abuse and dependence of amphetamine-type stimulants (AST), mainly methamphetamine, in China and around the world Yunnan Province is located in the southwest of China, with beautiful environment, pleasant climate, numerous nationalities and prosperous border trade. Since the reform and opening up, it has been a historical and cultural scenic spot. Drug traffic in the "Golden Triangle" is increasing in the border areas of Yunnan, China. Drug traffic is becoming more frequent, seriously endangering the normal life and normal economic order of the border people. It has also gradually transformed China from a drug transit country into a "one-stop" victim country of manufacturing, production, sales and consumption. Methamphetamine (MA) is a new drug, especially methamphetamine (methamphetamine) is abused in a large number of people, so this study on methamphetamine-induced renal damage to explore. [Objective] To explore the abuse of methamphetamine (MA) on the mechanism of renal damage, this study is divided into two parts, the first part is to study methamphetamine-induced renal damage in human proximal meander. Tubular epithelial cells (HK-2 cells, HK-2 cells) were used for cytotoxicity test and apoptosis by MTS method. The expression and secretion characteristics of neutrophil gelatinase-associated lipid carrier protein (Ngal) and kidney injury factor (Kim-1) in acute injury markers were studied. The significance of MTS in methamphetamine nephrotoxic injury was also discussed. [Methods] The effects of methamphetamine on renal proximal convoluted tubular epithelial cells in rats were observed. The experimental groups were as follows: normal control group (only adding culture medium), control group with concentrations of 0.75 mmol/L, 1.5 mmol/L, 2 mmol/L, respectively. L, 3mmol/L, 4mmol/L, 6mmol/L, 8mmol/L, 12mmol/L, 16mmol/L methamphetamine acted on cultured human renal proximal tubular epithelial cells in vitro. MTS method was used to observe the effect of methamphetamine on HK-2, flow cytometry was used to observe cell cycle changes and apoptosis. ELISA was used to detect the secretion of Ngal and Kim-1 in culture supernatant. [Results] (1) Methamphetamine treated human renal tubular epithelial cells for 24 hours. Cell survival rate was determined by MTS method. Methamphetamine concentration ranged from 0.75 mmo1 to 16 mmol/L, with the concentration of methamphetamine. The proliferative test showed that there was a significant difference in the effect of methamphetamine on HK-2 cells at the concentration of 6 mmol/L to 16 mmol/L for 24 hours (P 0.05). (2) Methamphetamine-induced apoptosis of HK-2 cells: 0.75 mmol/L group: 20.51%; 1 mmol/L group: 24%; 1.5 mmol/L group: 1.5 mmol/L; 2. L group: 31.96%; 2 mmol/L group: 30.15%; 3 mmol/L group: 31.8%; 4 mmol/L group: 26.9%; 6 mmol/L group: 99%; 8 mmol/L group: 98%; 12 mmol/L group: 99.16%; 16 mmol/L group: 99.8%. (3) NGAL results: 8 mmol/L group compared with the control group, in 12 hours, the amount of NGAL secreted by renal tubular epithelial cells increased. (4) KIM-1 gradually decreased. (4) KIM-1 secretion of renal tubular epithelial cells increased with the prolongation of the treatment time in the 8mmol/L group compared with the control group. [Conclusion] (1) Methamphetamine inhibited the proliferation of HK-2 cells. When the concentration reached 6mmol/L to 16mmol/L, the inhibition of methamphetamine on HK-2 cells became stronger in 24h. (2) Methamphetamine inhibited the proliferation of HK-2 cells. Amine promoted apoptosis of HK-2 cells. (3) Within 12 hours, NGAL secreted by renal tubular epithelial cells increased with the prolongation of treatment time. NGAL secreted by renal tubular epithelial cells decreased gradually after 12 hours. (4) KIM-1 secreted by renal tubular epithelial cells increased with the prolongation of treatment time. (5) MA had a significant toxic effect on kidney.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R749.64
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