發(fā)布時(shí)間：2018-04-15 05:36

  本文選題：鉛 + 男(雄)性　； 參考：《武漢大學(xué)》2013年博士論文

【摘要】：研究背景 鉛存在于人們生活與工作的環(huán)境中,由于它廣泛分布,并能產(chǎn)生男(雄)性生殖毒性,在現(xiàn)代優(yōu)生優(yōu)育與男性生殖健康問題中極為重視。鉛嚴(yán)重地影響了男性的生殖健康,它能通過血-睪屏障儲(chǔ)留在睪丸組織中,并對睪丸組織產(chǎn)生直接的毒性損害。Nrf2是轉(zhuǎn)錄因子中CNC家族的成員,被認(rèn)為是細(xì)胞內(nèi)重要的氧化應(yīng)激反應(yīng)調(diào)節(jié)子,與抗氧化反應(yīng)元件密切相關(guān)。Nrf2可通過激活Keapl-Nrf2-ARE信號(hào)通路對其下游靶基因進(jìn)行調(diào)控,這包括一些抗氧化酶、Ⅱ相解毒酶、藥物外排泵、清道夫受體及其轉(zhuǎn)錄調(diào)節(jié)子等的調(diào)節(jié)。Mrp1是一個(gè)ATP依賴的外排泵,被視為是有代表性的谷胱甘肽-S-共軛外排泵,能夠向細(xì)胞外輸送多種物質(zhì)�，F(xiàn)代研究表明,每一個(gè)有機(jī)體都有其自身的防御系統(tǒng),某些重金屬,諸如汞、鎘等可以通過Mrpl實(shí)現(xiàn)自身機(jī)體的自我解毒防御作用,與Nrf2的激活有一定關(guān)系。Long等人研究也顯示包括鉛在內(nèi)的一些有毒重金屬能夠誘導(dǎo)斑馬魚MRP1基因的轉(zhuǎn)錄表達(dá)。 關(guān)于鉛對男性生殖系統(tǒng)睪丸毒性作用機(jī)制至今仍不完全明了,目前在鉛中毒防治過程中常規(guī)使用藥物又因其副作用而受到不少質(zhì)疑,進(jìn)一步搞清鉛的毒性作用機(jī)制并積極尋找有效的排鉛等有效治療方法意義重大。本研究通過開展體內(nèi)實(shí)驗(yàn)和體外實(shí)驗(yàn),探討低鉛環(huán)境中鉛對雄性大鼠睪丸和小鼠睪丸支持細(xì)胞(TM4細(xì)胞)的Nrf2和Mrp1的影響,尋求可能的自身防御性排鉛解毒機(jī)制；同時(shí)運(yùn)用Meta分析方法對鉛致我國男性生殖健康影響進(jìn)行定量綜合分析,為探尋新的、更有效的重金屬鉛的解毒途徑提供理論依據(jù)。 第一部分鉛對我國男性生殖健康影響Meta分析 目的研究鉛對男性生殖健康的影響。材料與方法采用Meta分析方法對已在正式刊物上發(fā)表的有關(guān)鉛對男性生殖健康影響的文獻(xiàn)進(jìn)行綜合定量分析。結(jié)果鉛接觸男性陽痿、早泄的合并相對危險(xiǎn)度(Relative risk,RR)分別為2.55(95%C1=1.39-4.70)、2.23(95%CI=1.38-3.61),而精子總數(shù)、精子畸形率和精子活動(dòng)率的合并加權(quán)均數(shù)差(weighted mean difference,WMD)則分別為-13.39×109/L(95%CI=-21.49×109/L～5.30×109/L)、8.49%(95%CI=6.0l%～10.97%)和-8.29%(95%CI=-12.94%～-3.64%),差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論鉛可對男性的生殖健康造成損傷,引起陽痿率和早泄率的升高,精子總數(shù)的減少,精子畸形率的上升和精于活動(dòng)率的下降。 第二部分長期低鉛作用對大鼠睪丸Nrf2與Mrpl表達(dá)的影響及其相關(guān)指標(biāo)研究 目的本研究旨在探討低濃度鉛長期慢性作用對大鼠睪丸Nrf2和MRP1表達(dá)的影響。材料與方法適應(yīng)性喂養(yǎng)后,雌性SD大鼠隨機(jī)分為3組,通過自由飲水染毒,從懷孕前10天至斷乳各組分別用0,0.8和1.5g/L的醋酸鉛喂養(yǎng)；然后取各組15只雄性子代也通過自由飲水染毒,從斷奶到6月齡各組分別用0,0.3和0.9g/L的醋酸鉛喂養(yǎng)。應(yīng)用RT-PCR,蛋白質(zhì)印跡法,免疫組化和組織免疫熒光法檢測大鼠睪丸Nrf2與MRP1(?)勺表達(dá)水平及Nrf2的分布定位。采用原子吸收光譜法,分光光度法、酶標(biāo)法分別測定睪丸組織鉛含量、GST活力及GSH含量等。結(jié)果隨著鉛染毒劑量的增加,大鼠睪丸組織鉛含量增高,GST活力與GSH含量下降,Nrf2和MRP1(?)勺表達(dá)均呈現(xiàn)上升趨勢。Nrf2與MRP1在蛋白水平的表達(dá)與其在mRNA水平的表達(dá)基本一致。MRP1的相對表達(dá)量在各組間的差異都有顯著性(P0.05),Nrf2的顯著性差異存在于兩低鉛劑量組與對照組間(P0.05)。免疫熒光結(jié)果顯示,染鉛劑量越高,Nrf2核移位發(fā)生明顯。結(jié)論MRP1在低鉛染毒環(huán)境中可能通過激活Nrf2而促進(jìn)了細(xì)胞內(nèi)鉛的外排來實(shí)現(xiàn)機(jī)體的去毒防御功能。 第三部分低鉛暴露下TM4細(xì)胞Nrf2對Mrp1表達(dá)的調(diào)節(jié)作用 目的本部分旨在通過體外實(shí)驗(yàn)(細(xì)胞實(shí)驗(yàn))對Nrf2分別應(yīng)用激活劑tBHQ(?)口抑制劑ATRA處理,進(jìn)一步探討鉛染毒生殖細(xì)胞(TM4細(xì)胞)可能存在的自身解毒防御機(jī)制。材料與方法利用CCK-8比色法檢測不同濃度tBHQ和ATRA處理TM4細(xì)胞的存活率。建立空白對照組、20μM醋酸鉛處理組、20μM醋酸鉛tBHQ處理組及20pM醋酸鉛+ATRA處理組4個(gè)細(xì)胞實(shí)驗(yàn)?zāi)Ｐ徒M,利用原子吸收光譜法測定各組細(xì)胞內(nèi)的鉛含量,qRT-PCR檢測各組Nrf2與Mrp1的表達(dá)水平,同時(shí)應(yīng)用細(xì)胞免疫熒光法觀察Nrf2在細(xì)胞中TM4細(xì)胞內(nèi)Nrf2的定位及變化。結(jié)果加入40μM tBHQ組的TM4細(xì)胞存活率與正常對照組比較無差異,不存在統(tǒng)計(jì)學(xué)差別(P0.05)；加入2μM ATRA組的TM4細(xì)胞存活率與對照組存活率比,稍有增加,但也無統(tǒng)計(jì)學(xué)意義(P0.05)。Nrf2與Mrp1兩者的相對表達(dá)量在總的升降趨勢上是基本一致的；與空白對照比,其它染鉛毒組的Nrf2與Mrpl(?)分量均有上調(diào),且都有統(tǒng)計(jì)學(xué)差異(P0.05)；與單純低鉛處理組比,同時(shí)加入tBHQ的組Nrf2與Mrpl表達(dá)都增加,其中Nrf2的表達(dá)在兩者間有顯著性差異(P0.05),而同時(shí)加入ATRA的組Nrf2與Mrpl表達(dá)都下降,其中Mrpl的表達(dá)在兩者間有顯著性差異(P0.05)。細(xì)胞免疫熒光檢測中未見明顯核轉(zhuǎn)位,但同時(shí)加了tBHQ的組中Nrf2熒光強(qiáng)度感觀上較其它3組有所增加。空白對照組細(xì)胞的鉛含量很低；與對照組比,其它3組的細(xì)胞鉛含量均有顯著性的增高(P0.05)；與單純低鉛處理組比,同時(shí)加了tBHQ組的細(xì)胞鉛含量減少,同時(shí)加了ATRA組的細(xì)胞鉛含量增高,且有顯著性變化(P0.05)。結(jié)論鉛染毒TM4細(xì)胞可以通過Nrf2的激活上調(diào)Mrpl的表達(dá),增強(qiáng)Mrpl的外排泵功能,從而起到細(xì)胞解毒作用。
[Abstract]:Background of the study

Nrf2 is a member of the CNC family in the transcription factor . It is considered to be a representative glutathione - S - conjugated efflux pump , which is closely related to the anti - oxidation reaction element . Nrf2 is a representative glutathione - S - conjugated efflux pump , which is closely related to the activation of the Nrf2 . Long et al studies also show that some toxic heavy metals including lead can induce the transcriptional expression of the MRP1 gene of zebrafish .

In this study , the effects of lead on the testis and mouse testis supporting cells ( TM4 cells ) of male rat testis and mouse testis supporting cells ( TM4 cells ) were investigated by in vivo and in vitro experiments .
At the same time , Meta - analysis is applied to the quantitative analysis of the effect of lead - induced male reproductive health , which provides the theoretical basis for exploring new and more effective ways of detoxification of heavy metal lead .

Meta - analysis on the Effect of Lead on Reproductive Health in China

Objective To study the effects of lead on male reproductive health . Meta - analysis was used to quantify the effects of lead on reproductive health of male . Results Lead exposure to male impotence , premature ejaculation and relative risk ( RR ) were 2.55 ( 95 % CI = 1.39 - 4.70 ) and - 8.29 % ( 95 % CI = - 12.94 % ~ - 3.64 % ) , respectively .

The Effect of Long - term and Low - Lead on the Expression of Nrf2 in Rat Testis and Its Related Indexes

Objective To investigate the effects of low concentration lead on the expression of Nrf2 and MRP1 in rat testis . After adaptive feeding , female SD rats were randomly divided into 3 groups .
The expression level of Nrf2 and MRP1 ( ? ) and the distribution of Nrf2 were determined by AAS , Western blotting , immunohistochemistry and immunofluorescence staining . The results showed that the higher the dose of lead exposed to lead , the higher the content of GST and GSH in the testis , the higher the expression of Nrf2 and MRP1 , the higher the expression of Nrf2 and MRP1 ( P0.05 ) . Conclusion The higher the dose of lead exposure , the higher the expression of Nrf2 and MRP1 ( ? ) , the higher the level of Nrf2 and MRP1 , the higher the Nrf2 nuclear translocation . Conclusion MRP1 may promote the external discharge of lead in the cells by activating Nrf2 .

Regulatory Effects of TM4 Cells Nrf2 on the Expression of Mr p1 in the Third Part of Low Lead Exposure

Objective To investigate the effects of different concentrations of tBHQ and ATRA on the survival rate of TM4 cells ( TM4 cells ) .
The survival rate of TM4 cells in 2渭M ATRA group was significantly higher than that in control group , but there was no statistical significance ( P0.05 ) .
Compared with the control of blank control , the components of Nrf2 in other lead - exposed groups were up - regulated and that of the other groups were statistically different ( P0.05 ) .
Compared with the simple low - lead treatment group , the expression of Nrf2 in the group of tBHQ was significantly different from that in the other three groups ( P0.05 ) , but the expression of Nrf2 was significantly different between the two groups ( P0.05 ) . However , there was no significant nuclear translocation in the expression of Nrf2 in the cell immunofluorescence assay , but the fluorescence intensity of Nrf2 in the group with tBHQ was higher than that in the other 3 groups .
Compared with the control group , the lead content of other three groups increased significantly ( P0.05 ) .
Compared with the simple low - lead treatment group , the lead content of the cells in the tBHQ group was decreased , and the lead content of the cells in the ATRA group was increased , and there was a significant change ( P0.05 ) .

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R114

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