本文關(guān)鍵詞：內(nèi)毒素對大鼠附睪特異基因Bin1b表達(dá)的影響、機(jī)制及生物學(xué)意義　出處：《第二軍醫(yī)大學(xué)》2008年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 附睪 β防御素 Bin1b 脂多糖 精子成熟

【摘要】： 附睪不僅是精子貯存和運(yùn)輸?shù)膱鏊?還參與了精子成熟的過程。由于B淋巴細(xì)胞的缺失,天然免疫在附睪防御系統(tǒng)中的地位顯得尤為重要。附睪中有這樣一類組織特異性表達(dá)的分子,它們具有防御素樣的三葉草結(jié)構(gòu)基序,又能夠與精子結(jié)合,執(zhí)行抗微生物入侵和促精子成熟的雙重功能。Bin1b(Spag11e)是中科院張永蓮院士實驗室首先克隆并報道的附睪特異表達(dá)蛋白。該實驗室的前期研究發(fā)現(xiàn),Bin1b基因編碼一條68個氨基酸的多肽,結(jié)構(gòu)上屬β防御素超家族成員。Bin1b的表達(dá)具有嚴(yán)格的空間和時間特異性,它只表達(dá)于大鼠附睪頭部中段的上皮細(xì)胞中,性成熟時表達(dá)豐度最高。Bin1b能有效抑制附睪上皮原代培養(yǎng)上清液中E.coli的生長,還可在附睪不同區(qū)域以不同的方式與精子頭部結(jié)合,通過上調(diào)Ca~(2+)誘導(dǎo)未成熟精子的前向運(yùn)動。盡管目前對Bin1b生理功能的研究已有較大進(jìn)展,但是對病理狀態(tài)下的變化卻了解的不多。本文首次觀察了大腸桿菌脂多糖(lipopolysaccharide,LPS)對大鼠附睪頭部Bin1b和其它12個附睪特異β防御素基因的表達(dá)的影響。在發(fā)現(xiàn)LPS能夠下調(diào)附睪中Bin1b和另一些β防御素表達(dá)的基礎(chǔ)上,進(jìn)一步探討了炎癥時Bin1b的表達(dá)減少與精子活力改變之間的關(guān)系,并在原代培養(yǎng)的大鼠附睪頭部上皮細(xì)胞中研究了參與介導(dǎo)Bin1b下調(diào)表達(dá)的可能因素,以闡明附睪炎癥條件下Bin1b基因的改變、機(jī)制和可能的病理意義。 一、LPS下調(diào)大鼠附睪頭部Bin1b和其它附睪頭部特異性β防御素的表達(dá) 我們采用單側(cè)附睪頭部直接注射LPS的方法復(fù)制了大鼠附睪炎動物模型,用注射等量生理鹽水的對側(cè)附睪作自身對照。HE染色證實,200μg LPS處理2天可以誘發(fā)單側(cè)附睪明顯的炎癥性改變,包括附睪管間隙增寬,間質(zhì)內(nèi)大量白細(xì)胞浸潤,以及毛細(xì)血管充血。Northern blot方法證實病變側(cè)附睪促炎細(xì)胞因子IL-1βmRNA的表達(dá)也呈LPS劑量和時間依賴性增加,表明附睪炎模型的復(fù)制是成功的。在此基礎(chǔ)上,我們采用Northern blot和Western blot的方法觀察了LPS處理對附睪頭部組織中Bin1b表達(dá)的影響。結(jié)果發(fā)現(xiàn),200μg LPS處理2天,可使Bin1b mRNA和蛋白的表達(dá)水平減少90%以上(p＜0.01)。 為明確LPS導(dǎo)致的附睪Bin1b下調(diào)是否也發(fā)生在其它附睪特異性β防御素家族成員中,我們又觀察了大鼠附睪頭部特異性表達(dá)的其它12個β防御素基因在LPS誘導(dǎo)的炎癥附睪中的表達(dá)。結(jié)果發(fā)現(xiàn)LPS可以導(dǎo)致附睪頭部11個β防御素基因(Defb12,17,21,25,27,39,41,42,44,51和52)的表達(dá)明顯下調(diào);只有1個成員(Defb29)的表達(dá)不受LPS調(diào)節(jié)。附睪特異性β防御素樣分子雖然是β防御素超家族中的成員,但是它們無論在功能(身兼抗微生物和促精子成熟的雙職)或是表達(dá)調(diào)控方面都具有一定的特殊性。與文獻(xiàn)所報道的在其它組織中β防御素往往受炎癥誘導(dǎo)性表達(dá)的模式不同,在附睪中LPS不是上調(diào),而是下調(diào)附睪中β防御素基因的表達(dá),提示它們在促精子成熟方面的功能可能更為重要。 二、LPS減少大鼠附睪頭部Bin1b表達(dá)是導(dǎo)致附睪尾部精子活力下降的重要原因 Bin1b能與附睪精子的頭部結(jié)合,幫助未成熟精子獲得前向運(yùn)動能力。因此,我們推測附睪炎時Bin1b的表達(dá)減少可能會引起精子運(yùn)動能力的改變,從而導(dǎo)致附睪源性男性不育的發(fā)生。實驗采用計算機(jī)輔助精子分析(computer assisted sperm analysis,CASA)的方法,觀察LPS處理后附睪不同區(qū)域精子活力的改變,結(jié)果發(fā)現(xiàn)200μg LPS處理2天可明顯降低附睪體、尾部精子的總活力和前向運(yùn)動率。其中,附睪體部精子的總活力和前向運(yùn)動率分別減少48.1%和55.8%(p＜0.05);尾部精子總活力和前向運(yùn)動率分別減少49.6%和57.1%(p＜0.01)。為證明LPS引起的附睪精子活力下降和Bin1b表達(dá)減少之間的關(guān)系,我們進(jìn)而將大鼠Bin1b表達(dá)載體瞬時轉(zhuǎn)染附睪頭部上皮PCI細(xì)胞株獲得了含重組Bin1b蛋白的細(xì)胞培養(yǎng)上清,用其孵育LPS處理后的附睪尾部精子,結(jié)果顯示體外補(bǔ)充重組Bin1b蛋白可使精子的總活力和前向運(yùn)動率較LPS處理后分別增加1.06倍和1.31倍(p＜0.01)。免疫熒光化學(xué)和流式細(xì)胞儀檢測的結(jié)果與CASA結(jié)果相一致,LPS處理后精子頭部結(jié)合的Bin1b量明顯減少,補(bǔ)充Bin1b蛋白后,Bin1b蛋白結(jié)合量又恢復(fù)至生理鹽水對照側(cè)水平,這進(jìn)一步證明在LPS引起的大鼠附睪炎中,Bin1b的表達(dá)減少是導(dǎo)致精子活力下降的重要原因之一。 三、附睪頭部上皮細(xì)胞存在LPS受體,但是LPS不能直接下調(diào)上皮中Bin1b的表達(dá) 以上我們證實了在體LPS處理可導(dǎo)致大鼠附睪頭部組織中Bin1b的表達(dá)下調(diào)。但是下調(diào)的機(jī)制還不清楚,既可能是LPS直接作用于附睪上皮細(xì)胞,通過在該細(xì)胞中誘發(fā)的信號轉(zhuǎn)導(dǎo)通路下調(diào)Bin1b的表達(dá);也可能是在LPS導(dǎo)致的附睪炎癥反應(yīng)中,其他因素的改變對上皮細(xì)胞的間接作用。由于體內(nèi)參與炎癥的因素很多,內(nèi)環(huán)境復(fù)雜,為探討LPS導(dǎo)致Bin1b下調(diào)的機(jī)制,我們原代分離并培養(yǎng)了大鼠附睪頭部上皮細(xì)胞,首先檢測了該細(xì)胞的LPS受體和信號轉(zhuǎn)導(dǎo)通路的反應(yīng)性,結(jié)果顯示,原代培養(yǎng)的附睪頭部上皮細(xì)胞表達(dá)Toll樣受體4(Toll-like receptor 4,TLR4),且LPS可使原代細(xì)胞中IL-1βmRNA的表達(dá)呈時間依賴性上調(diào),其中200μg/ml LPS處理24小時,IL-1βmRNA的表達(dá)增加至對照的8.27倍(p＜0.01),表明LPS可通過上皮細(xì)胞中的自身受體和激活的信號轉(zhuǎn)導(dǎo)通路誘導(dǎo)下游靶基因的表達(dá)。我們繼而觀察了LPS對該細(xì)胞中Bin1b表達(dá)的影響,結(jié)果發(fā)現(xiàn)與生理鹽水處理的對照細(xì)胞相比,用不同濃度的LPS處理0—2天,原代細(xì)胞中Bin1b的表達(dá)并沒有明顯改變,說明LPS在體內(nèi)下調(diào)Bin1b的表達(dá),并不是通過與附睪上皮細(xì)胞的直接作用,而是一種間接的作用的結(jié)果。如LPS通過增加對Bin1b表達(dá)具有抑制作用的因子,或者減少能促進(jìn)Bin1b表達(dá)的因子導(dǎo)致該基因的下調(diào),這方面的研究有待于進(jìn)一步深入。 四、雄激素能上調(diào)原代培養(yǎng)的附睪頭部上皮細(xì)胞中Bin1b的表達(dá),而雌激素和糖皮質(zhì)激素沒有調(diào)節(jié)作用 在原代培養(yǎng)的附睪頭部上皮細(xì)胞中,我們還意外地發(fā)現(xiàn)Bin1b mRNA的表達(dá)隨培養(yǎng)時間延長而逐步減少,培養(yǎng)5天后的附睪上皮細(xì)胞中幾乎測不到其表達(dá)。這提示可能在體內(nèi)某些未知的Bin1b表達(dá)誘導(dǎo)因子在體外培養(yǎng)液中缺少或者濃度降低,從而不足以維持Bin1b的表達(dá)。已知一些附睪蛋白的表達(dá)受甾體激素的調(diào)節(jié),因此我們觀察了不同濃度的雄激素—二氫睪酮(dihydrotestosterone,DHT),雌激素—雌二醇(estradiol,E_2)和糖皮質(zhì)激素—地塞米松(dexamethasone,Dex)單獨(dú)作用對Bin1b表達(dá)的影響,結(jié)果發(fā)現(xiàn)不同濃度的DHT處理后,Bin1b mRNA表達(dá)量較對照組均明顯增加(p＜0.05),其中10~(-9)M的DHT可使Bin1b表達(dá)比對照細(xì)胞增加約30%,而E_2或Dex處理的細(xì)胞Bin1b mRNA的表達(dá)與對照相比沒有明顯的差別,這表明雄激素能上調(diào)Bin1b的表達(dá),而雌激素或糖皮質(zhì)激素則無此作用。盡管雄激素有上調(diào)Bin1b的作用,但是在體外雄激素單獨(dú)作用并不足以維持Bin1b在體內(nèi)的表達(dá)水平,提示體內(nèi)除了雄激素外,還存在其他能夠上調(diào)Bin1b表達(dá)的因子,尋找這些調(diào)節(jié)因子是今后要開展的工作。 同樣的體外培養(yǎng)衰減表達(dá)模式也存在于附睪頭部特異β防御素Defb21中。巧合的是,Defb21在LPS處理后的附睪組織中的表達(dá)也呈時間和劑量依賴性下調(diào),而另一個附睪頭部特異β防御素Defb29在附睪組織的表達(dá)不受LPS調(diào)節(jié),在原代培養(yǎng)過程中其mRNA的表達(dá)量在所觀察的時間內(nèi)也不發(fā)生衰減。LPS導(dǎo)致的Bin1b表達(dá)下調(diào),是否有可能與維持其生理性表達(dá)的因子在炎癥時的水平下降有關(guān),值得進(jìn)一步研究。 綜上所述,我們發(fā)現(xiàn)LPS可以抑制大鼠附睪頭部Bin1b基因的表達(dá),使附睪精子與Bin1b蛋白的結(jié)合減少,從而影響精子成熟過程,使附睪尾部精子運(yùn)動能力降低。并且,LPS誘導(dǎo)的下調(diào)表達(dá)可能還是附睪特異表達(dá)的β防御素樣分子在炎癥時普遍存在的一種調(diào)節(jié)模式。附睪頭部上皮細(xì)胞表達(dá)TLR4受體,LPS刺激能誘導(dǎo)上皮細(xì)胞中IL-1βmRNA的表達(dá),但LPS下調(diào)組織中Bin1b表達(dá)的主要機(jī)制并非通過與上皮細(xì)胞的直接作用。在原代培養(yǎng)的附睪頭部上皮細(xì)胞中,Bin1b mRNA的表達(dá)呈時間依賴性減少,DHT對該細(xì)胞中Bin1b的表達(dá)有一定上調(diào)作用,但并不能完全逆轉(zhuǎn)這種時間依賴性表達(dá)減少的趨勢,E_2或Dex單獨(dú)處理對該細(xì)胞中Bin1b的表達(dá)沒有影響。以上發(fā)現(xiàn)將有助于更深入地了解β防御素樣附睪特異蛋白在炎癥和精子成熟過程中的病理生理作用,幫助闡明附睪炎與男性不育之間的關(guān)系,并可能為附睪源性男性不育和性傳播疾病的防治提供藥靶位點。
[Abstract]:Not only is the epididymal sperm storage and transport sites, is also involved in sperm maturation. Because of the absence of B lymphocytes, natural immune status in epididymal defense system is particularly important. This kind of expression has tissue specificity in the epididymis, they have the defense trefoil motif in kind, but also can with sperm anti microbial invasion and promote the dual functions of.Bin1b sperm maturation (Spag11e) is specifically expressed in the CAS academician Zhang Yonglian laboratory first cloned and reported by previous study. The epididymis protein laboratory found that Bin1b gene encoding a polypeptide of 68 amino acids, the structure is the expression of beta defensin superfamily.Bin1b has the time and space specificity strictly, it only expressed in the middle part of the caput epididymis epithelial cells, mature.Bin1b can effectively inhibit the expression of the highest abundance of epididymis Epithelial primary culture supernatant of E.coli growth, but also in the different regions of the epididymis in different ways and the sperm head through the up regulation of Ca~ binding (2+) induced by immature sperm prior to the movement. Although the physiological function of Bin1b research has made great progress, but the changes of pathological condition but don't know much about it. The first observation of Escherichia coli lipopolysaccharide (lipopolysaccharide, LPS) expression in the rat epididymis head Bin1b and the other 12 epididymis specific beta defensin gene. In that LPS can cut foundation in the epididymis of Bin1b and some other beta defensin expression, to further explore the relationship between inflammation and decrease the expression of Bin1b sperm motility change, and study mediated by down regulated expression of Bin1b may be the factors in the epididymal epithelial cells of primary cultured rat Bin1b gene, to clarify the condition of inflammation of epididymis The change, mechanism, and possible pathological significance.
1. LPS down regulated the expression of Bin1b and other epididymal specific beta defensins in the epididymis of rats
We use the method of unilateral epididymis head direct injection of LPS replication in the rat animal model with epididymitis, injection of saline on the side of the epididymis as control.HE staining confirmed that 200 g LPS treatment for 2 days can be induced by unilateral epididymis inflammatory change, including epididymis tube gap widened, interstitial infiltration of a large number of white blood cells in.Northern and blot confirmed the expression of capillary congestion in ipsilateral epididymis proinflammatory cytokines of IL-1 beta mRNA also showed LPS dose and time dependent increase, show that the model is successful replication of epididymitis. On this basis, we use the method of Northern blot and Western blot to observe the effect of LPS treatment on the expression of Bin1b of brain tissue of epididymis the results showed that 200 g LPS treatment for 2 days, the expression level of Bin1b mRNA and protein decreased more than 90% (P < 0.01).
As a result of LPS Bin1b down is also occurred in the epididymis of other epididymis specific beta defensin family members, we also observed the expression of rat epididymal head specific other 12 beta defense apoptin gene expression in LPS induced inflammation in the epididymis. The results showed that LPS can cause epididymis head beta defensin 11 (Defb12,17,21,25,27,39,41,42,44,51 and 52) gene expression was significantly reduced; only 1 members (Defb29) expression is not regulated by LPS. The epididymis specific beta defensin beta defensin like molecules is a member of the family of, but they both in function (as anti microbial and promote sperm maturation or have dual roles) is a special expression in other tissues. And the beta defense reported in inflammation induced expression is often affected by the different modes in the epididymis, LPS is not increased, but decreased in epididymal beta The expression of the defensin gene suggests that they may be more important in the function of spermatogenesis.
Two, LPS reduces the expression of Bin1b in the epididymal head of rats as an important cause of the decrease of sperm motility in the tail of epididymis
Bin1b and the sperm head with help of immature motility. Therefore, we speculate that when epididymitis reduced expression of Bin1b may cause sperm motility changes, resulting in the epididymis derived male infertility. The experiment by computer assisted sperm analysis (computer assisted sperm analysis, CASA) method after LPS treatment, observation of different regions of epididymis sperm motility changes, results showed that 200 g 2 day LPS treatment can significantly reduce the total activity of the epididymis, sperm tail and the forward moving rate. Among them, the total activity of the epididymis and sperm forward motility rate reduced by 48.1% and 55.8% respectively (P < 0.05); the total sperm activity and forward movement rate reduced by 49.6% and 57.1% respectively (P < 0.01). To prove that LPS induced epididymal sperm activity decreased Bin1b expression and the relationship between the decrease, then we will Bin1b rats As the carrier of transient transfection of epididymal epithelial PCI cell line was obtained with the recombinant Bin1b protein of the cell culture supernatant, with the incubation tail epididymis sperm after LPS treatment, the result shows that the total activity in vitro of recombinant Bin1b protein can make the sperm and the forward moving rate is LPS after treatment respectively increased 1.06 times and 1.31 times (P < 0.01). Consistent with immunofluorescence and flow cytometry results with the results of CASA, Bin1b combined with the amount of sperm head decreased significantly after treatment with LPS, Bin1b protein, and to restore the saline control side Bin1b protein binding capacity, which further proved in LPS rats caused by epididymitis. The decrease of Bin1b expression is one of the main causes of decreased sperm motility.
Three, the epididymal epithelial cells of the epididymis have LPS receptor, but LPS can not directly down the expression of Bin1b in the epithelium.
We confirmed that the LPS treatment can lead to decrease the expression of Bin1b in epididymal tissue in rats. But the mechanism of down-regulation is unclear, it may be a direct effect of LPS on epididymal epithelial cells through signal transduction pathway in the cells induced by the downregulation of Bin1b expression; may also be epididymis inflammation in LPS lead in the indirect effects of other factors on the epithelial cells. Because of the many factors involved in inflammation in vivo, in complex environment, in order to explore the mechanism of the LPS induced down-regulation of Bin1b, we isolated and cultured primary rat epididymal epithelial cells, the results showed first examined the reactivity, the cells of the LPS receptor and signal transduction pathway, primary cultured epididymal epithelial cells of the head of the expression of Toll like receptor 4 (Toll-like receptor 4, TLR4), and LPS can increase the expression of IL-1 beta mRNA cells in a time dependent increase, which 200 g/ml LPS treatment for 24 hours, the expression of IL-1 beta mRNA increased to 8.27 times than the control (P < 0.01), showed that the expression of LPS in epithelial cells through its receptor and signal transduction pathway induced activation of downstream target genes. Then we observed the effects of LPS on the expression of Bin1b in the cell, the results found compared with the saline treated control cells and treated with different concentrations of LPS 0 - 2 days, the expression of Bin1b in primary cells did not change significantly, indicating that LPS down-regulation of Bin1b expression in vivo, and not by direct interaction with epididymal epithelial cells, which is an indirect effect. Such as factor has the inhibitory effect of LPS by increasing the expression of Bin1b, or the reduction factor can promote the expression of Bin1b leads to down-regulation of the gene, research in this area is to be further studied.
Four, androgen can increase the expression of Bin1b in the epithelia of primary epididymis, while estrogen and glucocorticoid have no regulatory effect.
In the epididymal epithelial cells of primary culture, we also unexpectedly found that the expression of Bin1b mRNA decreased gradually with the prolongation of the culture time, after 5 days of culture of epididymal epithelial cells almost undetectable expression. This may induce the expression of cytokines in lower concentration in the culture medium or lack of in vitro and in vivo of some unknown Bin1b thus, the expression is not sufficient to maintain the Bin1b. The expression of some known epididymal protein is regulated by steroid hormones, we observed different concentrations of androgen - two dihydrotestosterone (dihydrotestosterone, DHT), estrogen - estradiol (estradiol, E_2) and glucocorticoid dexamethasone (dexamethasone, Dex) to separate the expression of Bin1b. The results showed that different concentrations of DHT after treatment, Bin1b mRNA expression was significantly increased compared with the control group (P < 0.05), 10~ (-9) M DHT the expression of Bin1b than in control cells An increase of about 30%, while the expression of E_2 or Dex cells treated with Bin1b mRNA compared with the control group no significant difference, indicating that the male hormone can increase the expression of Bin1b and estrogen or glucocorticoids had no such effect. Although androgen is to upregulate Bin1b in vitro, but androgen alone is not sufficient to maintain in Bin1b the expression level of the body, suggesting that in addition to androgen, there are other factors can upregulate the expression of Bin1b, looking for the regulator is work to be done in the future.
The expression pattern of culture attenuation also exists in the epididymis head specific beta defensins in Defb21 in vitro. Coincidentally, the expression of Defb21 in LPS after the treatment of epididymal tissues also showed time and dose dependent downregulation of expression, while another specific epididymis head beta defensin Defb29 in the epididymis is not regulated by LPS, in the process of primary culture in the expression of mRNA in the observed time nor attenuation of.LPS induced Bin1b expression, and the factor whether it is possible to maintain its physiological level of expression in inflammation associated with decreased, it is worthy of further study.
In summary, we found that LPS can inhibit the expression of Bin1b gene in the rat caput epididymal sperm, combine with Bin1b protein reduced, thus affecting the sperm maturation process, reduce the epididymal sperm motility and LPS induced down-regulation of a regulated expression pattern might be epididymis specific expression of beta defensin like molecules exist in inflammation. The expression of TLR4 receptor expression on epithelial cells of epididymis, LPS stimulation can induce IL-1 beta mRNA in epithelial cells, but the expression of Bin1b LPS reduction is not the main mechanism of organization by direct interaction with epithelial cells. In epididymal epithelial cells of primary culture, the expression of Bin1b mRNA in a time dependent manner. Reduce the expression of DHT on Bin1b in the cells is upregulated, but did not completely reverse this time dependent expression decreased, E_2 or Dex alone in the No effect on the expression of Bin1b in cells. These findings will contribute to a deeper understanding of the pathophysiological role of beta defensin like epididymis specific protein in inflammation and sperm maturation, help to elucidate the relationship between epididymitis and male infertility, and may provide drug target sites for prevention and treatment of epididymis derived male infertility and sexual transmission disease.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R363

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