本文選題：慢性氟中毒 + 神經(jīng)細(xì)胞�。� 參考：《貴陽(yáng)醫(yī)學(xué)院》2015年碩士論文

【摘要】：目的:通過(guò)檢測(cè)慢性氟中毒大鼠腦組織中神經(jīng)細(xì)胞凋亡率、凋亡誘導(dǎo)因子(apoptosis-inducing factor,AIF)和相關(guān)凋亡因子caspase在蛋白表達(dá)的水平,了解AIF介導(dǎo)的非caspase依賴性細(xì)胞凋亡途徑是否參與慢性氟中毒大鼠大腦神經(jīng)細(xì)胞凋亡過(guò)程,并探討其發(fā)生機(jī)制。方法:實(shí)驗(yàn)用Sprague Dawley(SD)大鼠60只,雌雄各半,隨機(jī)分為兩組,即正常對(duì)照組:自由飲用自來(lái)水,飲水中含氟量0.5 mg/L;染氟組:飲水中加入氟化鈉,使氟含量為50 mg/L。兩組均飼以標(biāo)準(zhǔn)飼料(氟含量小于0.5mg/kg),實(shí)驗(yàn)期為10個(gè)月。實(shí)驗(yàn)結(jié)束時(shí),觀察大鼠氟斑牙發(fā)生情況;用氟離子選擇電極法測(cè)定大鼠尿氟、骨氟含量;HE染色觀察大鼠大腦神經(jīng)組織學(xué)改變,尼氏染色觀察大鼠腦組織神經(jīng)細(xì)胞內(nèi)尼氏小體形態(tài)學(xué)改變;免疫組化檢測(cè)大腦皮質(zhì)及海馬AIF分布;蛋白印跡(Western blotting)檢測(cè)大腦皮質(zhì)及海馬AIF、caspase-3及caspase-9的蛋白表達(dá)水平;流式細(xì)胞術(shù)(flow cytometr,FCM)檢測(cè)大鼠大腦皮質(zhì)及海馬細(xì)胞凋亡率。結(jié)果:染氟成年大鼠出現(xiàn)不同程度的氟斑牙,尿氟及骨氟含量明顯高于對(duì)照組(P0.05);HE染色病理形態(tài)學(xué)檢查顯示,各組大鼠腦組織未見(jiàn)明顯異常改變,尼氏染色發(fā)現(xiàn)染氟組大鼠腦組織神經(jīng)細(xì)胞中尼氏體數(shù)量較對(duì)照組顏色變淺,數(shù)量減少;免疫組化結(jié)果顯示,氟中毒組海馬及皮質(zhì)頂葉神經(jīng)細(xì)胞AIF陽(yáng)性表達(dá)率與染色程度明顯高于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P0.05),但在額葉和枕葉差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);蛋白印跡結(jié)果顯示,染氟組大鼠腦組織神經(jīng)細(xì)胞線粒體內(nèi)AIF(57-k Da)的蛋白表達(dá)水平明顯低于對(duì)照組(P0.01),而細(xì)胞核中AIF的蛋白表達(dá)水平明顯高于對(duì)照組(P0.01);染氟組大鼠腦組織神經(jīng)細(xì)胞含半胱氨酸的天冬氨酸蛋白水解酶(cysteinyl aspartate specific proteinase,Caspase)-3的蛋白表達(dá)水平明顯高于對(duì)照組(P0.01),caspase-9的蛋白表達(dá)水平高于對(duì)照組(P0.05);流式細(xì)胞術(shù)結(jié)果顯示,染氟組大鼠海馬和皮質(zhì)神經(jīng)細(xì)胞凋亡率與對(duì)照組相比顯著增高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:以AIF為主的非caspase依賴性凋亡途徑與通過(guò)激活caspase介導(dǎo)的經(jīng)典途徑共同參與慢性氟中毒大鼠腦組織神經(jīng)細(xì)胞凋亡,是慢性氟中毒引起的腦損傷發(fā)生機(jī)制之一。
[Abstract]:Objective: to investigate the expression of apoptosis-inducing factor (AIFF) and apoptosis-inducing factor (caspase) in the brain of rats with chronic fluorosis. Objective: to investigate whether AIF mediated caspase dependent cell apoptosis is involved in the process of neurocyte apoptosis in rats with chronic fluorosis and to explore its mechanism. Methods: sixty male and female Sprague Dawley SD rats were randomly divided into two groups: normal control group: drinking tap water freely, fluorine content in drinking water 0.5 mg / L, fluoride group: adding sodium fluoride into drinking water, making fluorine content 50 mg / L. Both groups were fed with standard diet (fluoride content less than 0.5 mg / kg 路kg ~ (-1) for 10 months. At the end of the experiment, the occurrence of dental fluorosis in rats was observed, the urine fluoride was determined by fluorine ion selective electrode method, and the histological changes of brain nerve were observed by bone fluorine content and HE staining. Nissl's staining was used to observe the morphological changes of neuronal Nissl corpuscles in rat brain, the distribution of AIF in cerebral cortex and hippocampus was detected by immunohistochemistry, and the expression of AIF-caspase-3 and caspase-9 in cerebral cortex and hippocampus were detected by Western blotting (Western blotting). Apoptosis rate of cerebral cortex and hippocampal cells in rats was detected by flow cytometry (FCM). Results: dental fluorosis of different degrees was found in adult rats exposed to fluoride. The contents of urine fluoride and bone fluorine in adult rats were significantly higher than those in control group (P 0.05) and HE staining showed that there were no obvious abnormal changes in brain tissue of rats in each group. Nissl staining showed that the number of Nissl bodies in brain tissue of fluoride group was lighter than that of control group. The positive expression rate and staining degree of AIF in hippocampus and parietal cortex in fluorosis group were significantly higher than those in control group (P 0.05), but there was no significant difference in frontal lobe and occipital lobe (P 0.05). The expression level of AIF(57-k Daa in brain mitochondria of rats exposed to fluoride was significantly lower than that of control group (P 0.01), while the expression of AIF protein in nucleus was significantly higher than that in control group, and cysteine was found in nerve cells of rats exposed to fluoride. The protein expression level of cysteinyl aspartate specific proteinase Caspase- 3 was significantly higher than that of control group (P 0.01), and the expression of caspase-9 was significantly higher than that of control group (P 0.05), and the results of flow cytometry showed that the expression of caspase-9 was significantly higher than that of control group (P < 0.05), and the expression of caspase-9 was significantly higher than that of control group. The apoptosis rate of hippocampal and cortical neurons in fluoride group was significantly higher than that in control group (P 0.05). Conclusion: the AIF independent caspase dependent apoptosis pathway and the classical pathway mediated by caspase are involved in the neuronal apoptosis in the brain tissue of rats with chronic fluorosis, which is one of the mechanisms of brain injury induced by chronic fluorosis.
【學(xué)位授予單位】：貴陽(yáng)醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R599.1

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