内毒素对大鼠附睾特异基因Bin1b表达的影响、机制及生物学意义

发布时间：2018-01-07 09:19

本文关键词：内毒素对大鼠附睾特异基因Bin1b表达的影响、机制及生物学意义　出处：《第二军医大学》2008年博士论文　论文类型：学位论文

【摘要】： 附睾不仅是精子贮存和运输的场所,还参与了精子成熟的过程。由于B淋巴细胞的缺失,天然免疫在附睾防御系统中的地位显得尤为重要。附睾中有这样一类组织特异性表达的分子,它们具有防御素样的三叶草结构基序,又能够与精子结合,执行抗微生物入侵和促精子成熟的双重功能。Bin1b(Spag11e)是中科院张永莲院士实验室首先克隆并报道的附睾特异表达蛋白。该实验室的前期研究发现,Bin1b基因编码一条68个氨基酸的多肽,结构上属β防御素超家族成员。Bin1b的表达具有严格的空间和时间特异性,它只表达于大鼠附睾头部中段的上皮细胞中,性成熟时表达丰度最高。Bin1b能有效抑制附睾上皮原代培养上清液中E.coli的生长,还可在附睾不同区域以不同的方式与精子头部结合,通过上调Ca~(2+)诱导未成熟精子的前向运动。尽管目前对Bin1b生理功能的研究已有较大进展,但是对病理状态下的变化却了解的不多。本文首次观察了大肠杆菌脂多糖(lipopolysaccharide,LPS)对大鼠附睾头部Bin1b和其它12个附睾特异β防御素基因的表达的影响。在发现LPS能够下调附睾中Bin1b和另一些β防御素表达的基础上,进一步探讨了炎症时Bin1b的表达减少与精子活力改变之间的关系,并在原代培养的大鼠附睾头部上皮细胞中研究了参与介导Bin1b下调表达的可能因素,以阐明附睾炎症条件下Bin1b基因的改变、机制和可能的病理意义。一、LPS下调大鼠附睾头部Bin1b和其它附睾头部特异性β防御素的表达我们采用单侧附睾头部直接注射LPS的方法复制了大鼠附睾炎动物模型,用注射等量生理盐水的对侧附睾作自身对照。HE染色证实,200μg LPS处理2天可以诱发单侧附睾明显的炎症性改变,包括附睾管间隙增宽,间质内大量白细胞浸润,以及毛细血管充血。Northern blot方法证实病变侧附睾促炎细胞因子IL-1βmRNA的表达也呈LPS剂量和时间依赖性增加,表明附睾炎模型的复制是成功的。在此基础上,我们采用Northern blot和Western blot的方法观察了LPS处理对附睾头部组织中Bin1b表达的影响。结果发现,200μg LPS处理2天,可使Bin1b mRNA和蛋白的表达水平减少90%以上(p＜0.01)。为明确LPS导致的附睾Bin1b下调是否也发生在其它附睾特异性β防御素家族成员中,我们又观察了大鼠附睾头部特异性表达的其它12个β防御素基因在LPS诱导的炎症附睾中的表达。结果发现LPS可以导致附睾头部11个β防御素基因(Defb12,17,21,25,27,39,41,42,44,51和52)的表达明显下调;只有1个成员(Defb29)的表达不受LPS调节。附睾特异性β防御素样分子虽然是β防御素超家族中的成员,但是它们无论在功能(身兼抗微生物和促精子成熟的双职)或是表达调控方面都具有一定的特殊性。与文献所报道的在其它组织中β防御素往往受炎症诱导性表达的模式不同,在附睾中LPS不是上调,而是下调附睾中β防御素基因的表达,提示它们在促精子成熟方面的功能可能更为重要。二、LPS减少大鼠附睾头部Bin1b表达是导致附睾尾部精子活力下降的重要原因 Bin1b能与附睾精子的头部结合,帮助未成熟精子获得前向运动能力。因此,我们推测附睾炎时Bin1b的表达减少可能会引起精子运动能力的改变,从而导致附睾源性男性不育的发生。实验采用计算机辅助精子分析(computer assisted sperm analysis,CASA)的方法,观察LPS处理后附睾不同区域精子活力的改变,结果发现200μg LPS处理2天可明显降低附睾体、尾部精子的总活力和前向运动率。其中,附睾体部精子的总活力和前向运动率分别减少48.1%和55.8%(p＜0.05);尾部精子总活力和前向运动率分别减少49.6%和57.1%(p＜0.01)。为证明LPS引起的附睾精子活力下降和Bin1b表达减少之间的关系,我们进而将大鼠Bin1b表达载体瞬时转染附睾头部上皮PCI细胞株获得了含重组Bin1b蛋白的细胞培养上清,用其孵育LPS处理后的附睾尾部精子,结果显示体外补充重组Bin1b蛋白可使精子的总活力和前向运动率较LPS处理后分别增加1.06倍和1.31倍(p＜0.01)。免疫荧光化学和流式细胞仪检测的结果与CASA结果相一致,LPS处理后精子头部结合的Bin1b量明显减少,补充Bin1b蛋白后,Bin1b蛋白结合量又恢复至生理盐水对照侧水平,这进一步证明在LPS引起的大鼠附睾炎中,Bin1b的表达减少是导致精子活力下降的重要原因之一。三、附睾头部上皮细胞存在LPS受体,但是LPS不能直接下调上皮中Bin1b的表达以上我们证实了在体LPS处理可导致大鼠附睾头部组织中Bin1b的表达下调。但是下调的机制还不清楚,既可能是LPS直接作用于附睾上皮细胞,通过在该细胞中诱发的信号转导通路下调Bin1b的表达;也可能是在LPS导致的附睾炎症反应中,其他因素的改变对上皮细胞的间接作用。由于体内参与炎症的因素很多,内环境复杂,为探讨LPS导致Bin1b下调的机制,我们原代分离并培养了大鼠附睾头部上皮细胞,首先检测了该细胞的LPS受体和信号转导通路的反应性,结果显示,原代培养的附睾头部上皮细胞表达Toll样受体4(Toll-like receptor 4,TLR4),且LPS可使原代细胞中IL-1βmRNA的表达呈时间依赖性上调,其中200μg/ml LPS处理24小时,IL-1βmRNA的表达增加至对照的8.27倍(p＜0.01),表明LPS可通过上皮细胞中的自身受体和激活的信号转导通路诱导下游靶基因的表达。我们继而观察了LPS对该细胞中Bin1b表达的影响,结果发现与生理盐水处理的对照细胞相比,用不同浓度的LPS处理0—2天,原代细胞中Bin1b的表达并没有明显改变,说明LPS在体内下调Bin1b的表达,并不是通过与附睾上皮细胞的直接作用,而是一种间接的作用的结果。如LPS通过增加对Bin1b表达具有抑制作用的因子,或者减少能促进Bin1b表达的因子导致该基因的下调,这方面的研究有待于进一步深入。四、雄激素能上调原代培养的附睾头部上皮细胞中Bin1b的表达,而雌激素和糖皮质激素没有调节作用在原代培养的附睾头部上皮细胞中,我们还意外地发现Bin1b mRNA的表达随培养时间延长而逐步减少,培养5天后的附睾上皮细胞中几乎测不到其表达。这提示可能在体内某些未知的Bin1b表达诱导因子在体外培养液中缺少或者浓度降低,从而不足以维持Bin1b的表达。已知一些附睾蛋白的表达受甾体激素的调节,因此我们观察了不同浓度的雄激素—二氢睾酮(dihydrotestosterone,DHT),雌激素—雌二醇(estradiol,E_2)和糖皮质激素—地塞米松(dexamethasone,Dex)单独作用对Bin1b表达的影响,结果发现不同浓度的DHT处理后,Bin1b mRNA表达量较对照组均明显增加(p＜0.05),其中10~(-9)M的DHT可使Bin1b表达比对照细胞增加约30%,而E_2或Dex处理的细胞Bin1b mRNA的表达与对照相比没有明显的差别,这表明雄激素能上调Bin1b的表达,而雌激素或糖皮质激素则无此作用。尽管雄激素有上调Bin1b的作用,但是在体外雄激素单独作用并不足以维持Bin1b在体内的表达水平,提示体内除了雄激素外,还存在其他能够上调Bin1b表达的因子,寻找这些调节因子是今后要开展的工作。同样的体外培养衰减表达模式也存在于附睾头部特异β防御素Defb21中。巧合的是,Defb21在LPS处理后的附睾组织中的表达也呈时间和剂量依赖性下调,而另一个附睾头部特异β防御素Defb29在附睾组织的表达不受LPS调节,在原代培养过程中其mRNA的表达量在所观察的时间内也不发生衰减。LPS导致的Bin1b表达下调,是否有可能与维持其生理性表达的因子在炎症时的水平下降有关,值得进一步研究。综上所述,我们发现LPS可以抑制大鼠附睾头部Bin1b基因的表达,使附睾精子与Bin1b蛋白的结合减少,从而影响精子成熟过程,使附睾尾部精子运动能力降低。并且,LPS诱导的下调表达可能还是附睾特异表达的β防御素样分子在炎症时普遍存在的一种调节模式。附睾头部上皮细胞表达TLR4受体,LPS刺激能诱导上皮细胞中IL-1βmRNA的表达,但LPS下调组织中Bin1b表达的主要机制并非通过与上皮细胞的直接作用。在原代培养的附睾头部上皮细胞中,Bin1b mRNA的表达呈时间依赖性减少,DHT对该细胞中Bin1b的表达有一定上调作用,但并不能完全逆转这种时间依赖性表达减少的趋势,E_2或Dex单独处理对该细胞中Bin1b的表达没有影响。以上发现将有助于更深入地了解β防御素样附睾特异蛋白在炎症和精子成熟过程中的病理生理作用,帮助阐明附睾炎与男性不育之间的关系,并可能为附睾源性男性不育和性传播疾病的防治提供药靶位点。
[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.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R363

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