【摘要】：目的建立正己烷(n-hexane)靜式染毒的SD大鼠實(shí)驗(yàn)?zāi)Ｐ�，并�?duì)實(shí)驗(yàn)?zāi)Ｐ偷姆�(wěn)定性、可重復(fù)性及其改良應(yīng)用進(jìn)行探討。在確定正己烷導(dǎo)致SD大鼠睪丸和卵巢損傷的基礎(chǔ)上，展開(kāi)關(guān)于損傷過(guò)程可能毒性機(jī)制的初步觀察研究，為流行病學(xué)和職業(yè)病有關(guān)正己烷的毒理研究和制定防護(hù)措施提供參考依據(jù)。方法SD大鼠48只，隨機(jī)分為4組(每組12只，雌雄各半)，1個(gè)對(duì)照組和3個(gè)染毒組(1d、3d和7d組)。置于特制染毒箱中，采用靜式染毒法，染毒時(shí)間為8h／d共7d，正己烷初始濃度設(shè)為35.2g／m~3。各染毒組大鼠于染毒結(jié)束后30min內(nèi)取動(dòng)脈血，氣相色譜-質(zhì)譜儀檢測(cè)全血正己烷濃度；取睪丸或卵巢稱量并計(jì)算臟器系數(shù)。睪丸和卵巢組織，一側(cè)制組織勻漿測(cè)定谷胱甘肽過(guò)氧化物酶(GSH-Px)、谷胱甘肽(GSH)、超氧化物歧化酶(SOD)活性和丙二醛(MDA)的含量，另一側(cè)4％多聚甲醛固定后常規(guī)石蠟切片觀察病理變化。免疫組織化學(xué)兩步法，檢測(cè)正己烷靜式染毒致?lián)p害的SD大鼠睪丸組織和卵巢組織中Fas、FasL、bax、bcl-2等凋亡相關(guān)蛋白的的表達(dá)。結(jié)果染毒1d、3d和7d組大鼠，經(jīng)檢出血液正己烷平均濃度分別為：636.92μg／L、913.49μg／L和1090.69μg／L，均明顯高于正常，表明已達(dá)到染毒要求。顯微鏡觀察發(fā)現(xiàn)，實(shí)驗(yàn)SD大鼠睪丸和卵巢結(jié)構(gòu)均出現(xiàn)明顯病理變化。染毒實(shí)驗(yàn)組的SD大鼠，隨染毒時(shí)間延長(zhǎng)，睪丸和卵巢顯微結(jié)構(gòu)的破壞逐漸明顯和加重。睪丸生精小管中支持細(xì)胞形態(tài)模糊不清，各級(jí)生精細(xì)胞排列疏松，部分小管中生精細(xì)胞嚴(yán)重脫落，有的管腔出現(xiàn)多核巨細(xì)胞。SD大鼠卵巢中生長(zhǎng)卵泡數(shù)量減少，一些生長(zhǎng)卵泡中卵母細(xì)胞的核膜及核仁不清晰，透明帶模糊甚至已溶解，黃體中淤血致血細(xì)胞浸潤(rùn)。染毒鼠的體重和生殖腺重量均有所降低，染毒7d組最為明顯(p＜0.05)，器官體重比也呈現(xiàn)明顯下降趨勢(shì)，3d組和7d組具有統(tǒng)計(jì)學(xué)意義(p＜0.05)。隨染毒時(shí)間延長(zhǎng)，睪丸或卵巢組織中SOD、GSH、GSH-Px均有降低，MDA含量升高，尤以7d組最為顯著(p＜0.01)，顯示正己烷染毒所致的不同程度的脂質(zhì)過(guò)氧化損傷。睪丸中Fas、FasL、bax蛋白在7d組均有增強(qiáng)，位于生精小管和生精細(xì)胞；卵巢中各凋亡相關(guān)蛋白，隨著染毒時(shí)間的延長(zhǎng)表達(dá)強(qiáng)度有所增加，主要表現(xiàn)在黃體細(xì)胞和顆粒細(xì)胞。結(jié)論1．本研究采用“正己烷急性吸入靜式染毒法”，建立的正己烷吸入致SD大鼠睪丸和卵巢損傷的實(shí)驗(yàn)?zāi)Ｐ�，具有操作�?jiǎn)單、效果穩(wěn)定和可重復(fù)性強(qiáng)等優(yōu)點(diǎn)，可為正己烷和其他毒物致代謝器官損害的實(shí)驗(yàn)研究提供方法學(xué)依據(jù)。2．正己烷靜式染毒導(dǎo)致?lián)p傷的SD大鼠，體重下降，睪丸或卵巢的濕重和臟器系數(shù)降低、睪丸和卵巢的結(jié)構(gòu)出現(xiàn)病理變化，證實(shí)正己烷毒性作用導(dǎo)致睪丸和卵巢損傷。染毒時(shí)間越長(zhǎng)，睪丸和卵巢的結(jié)構(gòu)組織損傷越嚴(yán)重。3．SD大鼠經(jīng)正己烷染毒后，所測(cè)出睪丸和卵巢勻漿中的SOD、GSH-Px和GSH和MDA含量變化表明，，隨著染毒時(shí)間的延長(zhǎng)，睪丸或卵巢中SOD、GSH-Px活力均呈明顯下降，而MDA含量呈明顯升高，反映正己烷的脂質(zhì)過(guò)氧化毒性作用已導(dǎo)致睪丸和卵巢清除自由基能力明顯下降，脂質(zhì)過(guò)氧化產(chǎn)物大量堆積。脂質(zhì)過(guò)氧化損傷是正己烷導(dǎo)致生殖腺損傷的機(jī)制之一。4．本研究檢測(cè)與細(xì)胞凋亡相關(guān)的Fas、FasL、bax和bcl-2等的免疫組化表達(dá)特征，結(jié)果表明，正己烷中毒對(duì)睪丸和卵巢的影響，最終導(dǎo)致生殖細(xì)胞凋亡的過(guò)程。由于雄性和雌性個(gè)體的生殖細(xì)胞發(fā)育過(guò)程具有不同的特點(diǎn)，正己烷中毒對(duì)睪丸和卵巢的干擾和影響表現(xiàn)出不同的狀況。但預(yù)示正己烷的毒性作用將促使和導(dǎo)致生殖細(xì)胞凋亡，必然會(huì)影響卵巢生殖功能。
[Abstract]:Objective To establish an experimental model of n-hexane-based SD rat model, and to study the stability, repeatability and application of n-hexane. Based on the determination of the testicular and ovarian damage of SD rats caused by n-hexane, a preliminary study on the possible toxicity mechanism of the injury process was conducted to provide a reference for the toxicological study and protective measures of n-hexane related to epidemiology and occupational disease. Methods Forty-eight SD rats were randomly divided into 4 groups (12 in each group, half of male and female), 1 control group and 3 groups of exposure (1d, 3d and 7d). The initial concentration of n-hexane was 35. 2g/ m ~ 3, and the initial concentration of n-hexane was 35. 2g/ m ~ 3. Blood and gas chromatography-mass spectrometer (GC-MS) were used to measure the concentration of n-hexane in the whole blood for 30min after the end of the exposure, and the testis or the ovary was weighed and the coefficient of the organ was calculated. The contents of glutathione peroxidase (GSH-Px), glutathione (GSH), superoxide dismutase (SOD) and malondialdehyde (MDA) were measured on one side of the testis and the ovary tissue, and the pathological changes were observed in the normal paraffin section after the fixation of 4% paraformaldehyde on the other side. The expression of Fas, FasL, bax, bcl-2 and other apoptosis-related proteins in the testis and ovarian tissues of SD rats was detected by two-step immunohistochemical method. Results The average concentration of n-hexane in the rats was 636. 92. mu. g/ L, 913. 49. m u.g/ L and 1090. 69. m u.g/ L, respectively. The results showed that both the testis and the ovary structure of SD rats had obvious pathological changes. The SD rats of the experimental group were prolonged with the time of exposure, and the destruction of the microstructure of the testis and the ovary became more and more serious. In the testis spermatogenic small tube, the cell morphology is blurred, and the spermatogenic cells at all levels are arranged to be loose, and the spermatogenic cells in some small tubes are seriously detached, so that the tube cavity has a plurality of giant cells. The number of follicles in the ovary of SD rats was decreased, and the nuclear membrane and the nucleolus of the oocytes in some of the growing follicles were not clear, the clear band was blurry and even dissolved, and the blood cells in the corpus luteum were infiltrates. The weight of the rats and the weight of the gonads decreased, and the group was the most obvious (p <0.05), and the weight ratio of the organs decreased significantly, and the 3d and 7d groups were of statistical significance (p <0.05). The contents of SOD, GSH and GSH-Px in the testis or the ovary were decreased with the time of exposure, and the content of MDA increased, especially in the 7dgroup (p <0.01). The expression of Fas, FasL and bax in the testis was enhanced in the 7dgroup, and the expression of Fas, FasL and bax in the ovary was increased, and the expression of Fas, FasL and bax in the ovary increased with the time of exposure, mainly in the yellow cells and the granulosa cells. Conclusion 1. The experimental model of the rat's testis and the ovary injury induced by n-hexane inhalation by using the 鈥渘-hexane acute inhalation and static exposure method鈥

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