本文選題：草酸鈣 + 乙二醇　； 參考：《蘭州大學(xué)》2017年博士論文

【摘要】：目的泌尿系統(tǒng)中結(jié)石的形成主要包括了結(jié)石結(jié)晶的成核、生長、聚集及滯留。其全球發(fā)病率為1%-20%,平均約為10%。伴隨微創(chuàng)外科新技術(shù)的不斷進(jìn)步,結(jié)石患者絕大多數(shù)得到了滿意的療效,但結(jié)石的高復(fù)發(fā)率,抗結(jié)石藥物的不良反應(yīng),仍是一個困擾人類的大難題。雖然研究表明,噻嗪類利尿劑與檸檬酸鹽等能在一定程度上預(yù)防泌尿系統(tǒng)結(jié)石的形成與生長,但發(fā)現(xiàn)長期使用該類藥物會有較為明顯的副作用及不良反應(yīng)。因此,人們目前試圖解決的一大難題便是重新開發(fā)研究一些較為安全有效、價格低廉、能夠長期服用并且并發(fā)癥相對較少的抗結(jié)石藥物。近幾年,以銀杏葉、紫杉醇、青蒿素與喜樹堿等一批植物為研究基礎(chǔ)研發(fā)了一些新的藥物,其抗結(jié)石作用得到了肯定,更多植物制劑的研究為人們的研究提供了新的思路。祖國醫(yī)學(xué)中,連錢草常用來治療腎結(jié)石,但其作用并不肯定。我們擬將中藥多作用靶點的特點運用起來,通過蛋白組學(xué)的方法,探討其預(yù)防泌尿系結(jié)石的可能作用機(jī)制。方法細(xì)胞實驗中,通過草酸鈣結(jié)晶(67μg/cm2)持續(xù)24小時來刺激人腎皮質(zhì)近曲小管上皮細(xì)胞(HK-2)來建立草酸鈣結(jié)石的細(xì)胞模型。試驗時,細(xì)胞培養(yǎng)液中給予不同濃度的連錢草(0.5,1.0,2.0,or 4.0mg/mL)or 1.0mg/mL檸檬酸鉀,當(dāng)CaOx結(jié)晶作用HK-2細(xì)胞1天后,收集細(xì)胞及培養(yǎng)液,并檢測氧化應(yīng)激的指標(biāo):丙二醛(MDA)、超氧化物歧化酶(SOD)以及過氧化氫酶(CAT)。而后用免疫印跡(Western blotting)檢測泌尿結(jié)石相關(guān)蛋白骨橋蛋白(OPN)和腎損傷分子-1(KIM-1)的表達(dá)情況。動物實驗中,60只成年的雄性SD大鼠被隨機(jī)分入6組(分別為正常組、實驗組、陽性對照組、低中高濃度連錢草組)。當(dāng)大鼠適應(yīng)環(huán)境一周以后,予以實驗組大鼠1%的乙二醇飼料喂養(yǎng)4周,同時以不同濃度水平的連錢草灌胃。于第28天將其置入代謝籠中,留取24h尿液標(biāo)本。10%的水合氯醛行腹腔注射麻醉大鼠后,采集血標(biāo)本和腎標(biāo)本。而后檢測尿液中的各項生化指標(biāo),在顯微鏡下觀察結(jié)晶的沉淀情況及病理變化情況,同時測定OPN以及KIM-1的免疫組化、免疫印跡和氧化應(yīng)激相關(guān)指標(biāo)。結(jié)果體外和體內(nèi)實驗均證明,與陽性對照組相比,連錢草顯著降低了草酸鈣引起的OPN、KIM-1表達(dá)及氧化應(yīng)激水平(p0.05)。和結(jié)石組大鼠相比,連錢草組的大鼠表現(xiàn)出顯著減少的草酸鈣沉積和病理改變(p0.05)。此外,尿液標(biāo)本中草酸、肌酐和尿素的含量亦明顯低于結(jié)石組的大鼠,而檸檬酸的含量則明顯增高。結(jié)論連錢草提取物預(yù)防結(jié)石的作用比檸檬酸鉀更好。
[Abstract]:Objective the formation of stone in urinary system mainly includes nucleation, growth, aggregation and retention of stone crystallization. Its global incidence is 1-20, with an average of about 10. With the development of the new technique of minimally invasive surgery, the majority of calculi patients get satisfactory curative effect. However, the high recurrence rate of stones and the adverse reaction of anti-stone drugs are still a big problem for human being. Although studies have shown that thiazide diuretics and citrate can prevent the formation and growth of urinary calculi to some extent, it is found that long-term use of these drugs has obvious side effects and adverse reactions. Therefore, one of the most difficult problems that people are trying to solve is to re-develop and study anti-stone drugs that are safe, effective, inexpensive, can be taken for a long time and have relatively few complications. In recent years, some new drugs were developed on the basis of ginkgo leaf, paclitaxel, artemisinin and camptothecin. In traditional Chinese medicine, even herbs are often used to treat kidney stones, but their role is not certain. We intend to apply the characteristics of multi-action targets of traditional Chinese medicine to explore the possible mechanism of preventing urinary calculi by proteomics. Methods the cell model of calcium oxalate calculi was established by stimulating the epithelial cells of proximal tubules of human renal cortex with calcium oxalate crystal 67 渭 g / cm 2 for 24 hours. In the experiment, the cells were treated with different concentrations of 4.0mg/mL)or 1.0mg/mL citric acid or 4.0mg/mL)or 1.0mg/mL in different concentrations. After CaOx crystallized for 1 day, the cells and the culture medium were collected, and the cells and the culture medium were collected. The indexes of oxidative stress were: malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT). Then Western blotting was used to detect the expression of osteopontin (OPN) and renal injury molecule-1 (KIM-1) in urolithiasis. In animal experiment, 60 adult male SD rats were randomly divided into 6 groups (normal group, experimental group, positive control group, low, medium and high concentration group). After one week of adaptation, rats in the experimental group were fed with 1% ethylene glycol forage for 4 weeks. On the 28th day, the rats were placed in a metabolic cage and 24 h urine samples were collected. 10% chloral hydrate was injected intraperitoneally into anesthetized rats. Blood and kidney samples were collected. Then the biochemical indexes in urine were detected, the precipitation and pathological changes of crystals were observed under microscope, and the immunohistochemical, immunoblotting and oxidative stress related indexes of OPN and KIM-1 were also determined. Results both in vitro and in vivo experiments showed that the expression of OPNN KIM-1 and the level of oxidative stress (P 0.05) were significantly decreased by Lianqiangcao compared with the positive control group. Compared with the lithiasis group, the rats in Lianchencao group showed significantly decreased calcium oxalate deposition and pathological changes (p 0.05). In addition, the contents of oxalic acid, creatinine and urea in urine samples were significantly lower than those in rats with calculi, while the content of citric acid was significantly increased. Conclusion the extract of Lianqiangcao is more effective than potassium citrate in preventing stone.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R691.4

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