AMPK信号通路在热诱导猪睾丸支持细胞紧密连接蛋白表达中的作用

发布时间：2018-05-20 19:48

  本文选题：AMPK信号通路 + 热处理　； 参考：《西南大学》2016年博士论文

【摘要】：热应激(heat stress,HS)指动物对热环境的舒适区上限温度所产生的一系列生理反应。随着温室效应的加剧,夏季持续高温引起的热应激严重影响了家畜的繁殖性能,对畜牧业造成的损失越来越严重。因此研究热应激对家畜繁殖性能的影响对于减少夏季热应激对动物的不良影响,提高经济效益具有十分重要的意义。猪皮下脂肪较厚,汗腺不发达,其体内热量较难通过皮肤蒸发,因此猪很不耐热。公猪对环境温度尤其敏感,高温环境可对公猪的生殖性能产生明显影响,引起精液量减少、精子活力降低、精子畸形率升高。尽管冷冻精液技术已有相当长的历史,但猪冷冻精液由于繁殖成绩不理想未得到大面积应用,因此夏季热应激引起公猪生精能力降低对整个养猪业产生的影响较难通过使用冷冻精液而改善。全面、深入地研究热应激对公猪精子发生过程的影响,可为预防和缓解热应激对公猪繁殖性能的影响提供重要的理论依据。支持细胞间的紧密连接是构成血睾屏障的重要结构,在精子发生过程中起了关键性的作用。它将生精上皮分为基底室和近腔室。细线前期精母细胞必须穿过紧密连接结构进入近腔室才能完成减数分裂过程进而分化为成熟的精子。此外,其屏障作用为精子发生提供了稳定的微环境。热应激可引起紧密连接相关蛋白表达下调及表达异位,从而破坏紧密连接结构。目前关于热应激对紧密连接影响的报道多为在体实验,体外实验也多聚焦于成熟的体外培养的支持细胞,但未成熟的支持细胞内紧密连接相关蛋白是否表达、表达部位如何以及热应激对未成熟支持细胞内紧密连接蛋白的作用均未见报道。尽管热应激可通过改变成熟支持细胞内紧密连接相关蛋白的表达进而影响精子发生,但由于支持细胞间紧密连接是逐步形成的,热应激对未成熟的支持细胞内紧密连接相关蛋白的影响也可能影响成熟后血睾屏障的形成。鉴于此,本文以仔猪睾丸支持细胞为研究对象,探究热应激对未成熟的支持细胞内紧密连接蛋白表达的影响,并解析其中的分子机制。AMPK是真核细胞内能量代谢调节的关键分子,同时也参与调节上皮细胞间紧密连接的功能:AMPK的激活可促进紧密连接形成,敲除AMPK则可导致紧密连接功能丧失,但AMPK对睾丸内紧密连接的调控作用未见报道。另外,热应激常引起细胞内氧化应激水平升高,使细胞遭受氧化损伤。研究发现ROS可通过调节AMPK上游调控分子Ca MKKβ的表达量进而调节AMPK的活性。基于以上研究背景,本研究提出假设:热应激使支持细胞发生氧化应激反应,进而通过抑制AMPK信号通路调控紧密连接相关蛋白的表达。本文所得研究结果如下:1.支持细胞体外HS模型的建立及HS对紧密连接相关蛋白表达的影响首先比较两种热处理方法(43℃水浴30 min与43℃气浴30 min)对支持细胞活力的影响,发现气浴法可引起支持细胞活力发生可逆性下降(降低34.46%,P0.01),而随着恢复时间的延长,其活力在48 h可恢复至正常;水浴法亦可引起细胞活力降低(降低57.94%,P0.01),但至热处理后第48 h,活力进一步降低为对照组的29.49%,因此选择43℃气浴30 min作为支持细胞体外热处理模型。利用此模型发现,热处理可通过降低线粒体膜电位显著增加支持细胞早期凋亡率(增加367.87%,P0.01),撤除热处理后,随着时间的延长,支持细胞早期凋亡率逐渐降低并恢复至正常。利用western blotting及荧光定量PCR对紧密连接相关蛋白(Claudin-11、JAM-A、Occludin、ZO-1)及m RNA进行检测,发现四种蛋白均能在体外培养的未成熟的公猪支持细胞内表达;热处理后紧密连接相关蛋白的m RNA及蛋白水平显著下调;撤除热处理后随着时间的延长,紧密连接相关蛋白表达量逐渐恢复。通过免疫荧光技术对Claudin-11在支持细胞内的表达部位进行检测,发现尽管体外培养的支持细胞间未形成紧密连接结构,但Claudin-11大量表达于支持细胞胞膜、胞质及囊泡膜上,且表达呈弥散型。热处理后Claudin-11仅少量表达于囊泡膜上,撤除热处理后48 h,Claudin-11在支持细胞内的表达可得到恢复。以上结果表明:热处理可引起支持细胞内紧密连接相关蛋白的m RNA及蛋白水平发生可逆性下调,并使Claudin-11的表达部位发生变化。2.AMPK信号通路在HS影响紧密连接相关蛋白表达中的作用本研究首先发现热处理可抑制AMPK活性,使AMPK磷酸化水平降低60.04%(P0.01),而这一变化可随恢复时间延长逐渐恢复。随后对AMPK上游调控因子(ATP、Ca MKKβ、LKB1)进行检测,发现热处理后支持细胞内ATP水平降低为对照组的48.92%(P0.01),Ca MKKβ表达量下调为对照组的46.18%(P0.01),而LKB1活性未见变化。利用Ca MKKβ的特异性抑制剂STO609(10μmol/L,2 h)对Ca MKKβ进行抑制,发现可在热处理后进一步抑制AMPK活性,相比于单独热处理组下降62.19%(P0.01),而STO609单独作用却对AMPK活性无明显影响,说明热处理通过抑制Ca MKKβ的表达进而抑制AMPK的活性。为了进一步探究AMPK在热处理下调紧密连接相关蛋白表达中是否发挥作用,本研究利用AMPK特异性激活剂AICAR(2 mmol/L,2 h)处理支持细胞,结果发现,先激活AMPK再进行热处理,紧密连接相关蛋白表达量的下降幅度明显减小;随后利用过表达载体pc DNA-AMPK/si RNA对AMPK进行过表达/干涉,结果发现过表达AMPK可部分挽救热处理引起的紧密连接相关蛋白表达下调。免疫荧光结果表明,热处理后Claudin-11在支持细胞中的定位也在AMPK被激活或过表达后发生变化,从仅表达于囊泡膜表面变为弥散于胞质、胞膜及囊泡膜表面。而干涉AMPK后再行热处理,紧密连接相关蛋白表达量较单独热处理组进一步下调,且Claudin-11在支持细胞内几乎检测不到。这说明Ca MKKβ介导的AMPK在热处理调控紧密连接相关蛋白表达中起了关键作用。3.氧化应激在HS调控紧密连接相关蛋白表达中的作用热处理后支持细胞内ROS及MDA水平显著上调,而抗氧化酶SOD、GSH-Px、CAT活性在热处理后显著降低。随着热处理后恢复时间的延长,以上指标均逐渐恢复至正常水平。这表明热处理可使支持细胞内氧化应激水平升高。通过提前添加抗氧化剂NAC(N-acetyl-L-cysteine,N-乙酰半胱氨酸),发现NAC(1 mmol/L,2 h)通过降低热处理后支持细胞内ROS及MDA水平,提高抗氧化酶SOD、GSH-Px、CAT的活性,从而减轻热处理后支持细胞的氧化损伤。同时,NAC的添加亦可降低支持细胞在热处理后的早期凋亡率,进而提高热处理后支持细胞的活性。本研究进一步发现NAC可上调热处理后支持细胞内Ca MKKβ表达量,并提高AMPK的活性,结果说明热处理通过ROS的产生抑制Ca MKKβ的表达,进而抑制AMPK活性。最后本研究还发现NAC的添加可有效缓解热处理引起的紧密连接相关蛋白表达下调和Claudin-11在细胞内异位。综上所述,热处理通过提高支持细胞内氧化应激水平,抑制Ca MKKβ的表达,进而抑制AMPK活性,从而引起紧密连接相关蛋白表达和在细胞中的定位发生可逆性变化。本结论揭示了热处理影响未成熟猪睾丸支持细胞内紧密连接相关蛋白表达的分子机制,这为人工调控热应激对公猪生精的不利影响提供了理论依据。
[Abstract]:Heat stress (HS) refers to a series of physiological responses to the upper limit temperature of the animal's comfort zone in the thermal environment. With the intensification of the greenhouse effect, the heat stress caused by the continuous high temperature in summer seriously affects the reproductive performance of livestock, and the loss of animal husbandry is becoming more and more severe. Therefore, the effect of heat stress on the reproductive performance of domestic animals is studied. It is of great significance to reduce the adverse effects of heat stress on animals in summer and improve the economic benefits. The fat under the pigskin is thicker and the sweat glands are not developed, the heat of the body is difficult to evaporate through the skin, so the pig is very heat-resistant. The pig is especially sensitive to the ambient temperature, and the high temperature environment can have a clear effect on the reproductive performance of the boar and cause the sperm. As the amount of liquid decreased, sperm vitality decreased and sperm abnormality increased. Although cryopreserved semen technology had a long history, the cryopreservation semen had not been applied in large area because of poor reproductive performance. Therefore, the effect of heat stress in summer on the production of pigs in the whole pig industry in summer was difficult to improve by using frozen semen. A thorough study of the effect of heat stress on the process of spermatogenesis in the boar can provide an important theoretical basis for preventing and alleviating the effect of heat stress on the reproductive performance of the boar. The support of the close connections between cells is an important structure of the blood testis barrier and plays a key role in the process of spermatogenesis. It divides the spermatogenic epithelium into a base. The prophase spermatocytes must pass through the tightly connected structure into the proximal chamber to complete the meiosis process and then differentiate into mature sperm. In addition, the barrier effect provides a stable microenvironment for spermatogenesis. Heat stress can cause the downregulation and ectopic expression of the closely linked egg white expression, and thus the damage is tight. Most of the reports about the influence of heat stress on tight connections are in body experiments, and in vitro experiments are mostly focused on mature support cells in vitro. However, the expression of close linked proteins in the immature support cells, how the expression site is and the heat stress on the close connexin in the immature support cells Although heat stress can change the expression of close linked proteins in the mature support cells and then affect spermatogenesis, the effect of heat stress on the tight junction related proteins in immature support cells may also affect the blood testis barrier after maturation. In view of this, this paper studies the effect of heat stress on the expression of tight connexin in immature support cells, and analyzes the molecular mechanism.AMPK is a key molecule in the regulation of energy metabolism in eukaryotic cells, and also participates in the function of regulating the close connection between epithelial cells: AMPK Activation can promote the formation of tight junctions, and knocking out AMPK can lead to the loss of tight junction function, but the regulatory role of AMPK on tight junction in the testis has not been reported. In addition, heat stress often causes increased intracellular oxidative stress and oxidative damage to cells. The study found that ROS can regulate the expression of Ca MKK beta by regulating the upstream of AMPK. Based on the above research background, this study hypothesizes that heat stress induces oxidative stress response in support cells, and then regulates the expression of tightly connected associated proteins by inhibiting the AMPK signaling pathway. The results of this study are as follows: 1. the results of this study are as follows: 1. support the establishment of HS model in vitro and the close linked protein table of HS The influence of the two heat treatment methods (43 centigrade bath 30 min and 43 centigrade gas bath 30 min) on the viability of the support cells was first compared. It was found that the gas bath method could cause the reversibility of the support cell viability to decrease (34.46%, P0.01), and with the prolongation of the recovery time, its vitality could be restored to normal at 48 h, and the water bath method could also cause the cell vitality. Lower (57.94%, P0.01), but to forty-eighth h after heat treatment, the vitality was further reduced to 29.49% in the control group, so 43 C 30 min was selected as the model of the support cells in vitro heat treatment. The model found that heat treatment could reduce the apoptosis rate of the support cells by reducing the mitochondrial membrane potential significantly (367.87%, P0.01), and the heat treatment was removed. After the treatment, the early apoptosis rate of the support cells gradually decreased and returned to normal. The Western blotting and fluorescence quantitative PCR were used to detect the close linked proteins (Claudin-11, JAM-A, Occludin, ZO-1) and m RNA, and the four proteins were found to be expressed in the immature boar support cells in vitro; The level of M RNA and protein in the closely connected protein was down significantly. After the removal of heat treatment, the expression of closely linked protein was gradually restored. The expression of Claudin-11 in the supporting cells was detected by immunofluorescence. It was found that there was no close connection between the support cells in the culture in vitro. Structure, but Claudin-11 was expressed in the cell membrane, cytoplasm and vesicle membrane, and the expression was diffuse. After heat treatment, only a small amount of Claudin-11 was expressed on the vesicle, and 48 h after the heat treatment was removed. The expression of Claudin-11 in the supporting cells could be recovered. The above results showed that heat treatment could cause the close connection in the supporting cells. A reversible downregulation of M RNA and protein levels in the protein and changes in the expression site of Claudin-11 in the expression of.2.AMPK signaling pathway in the effect of HS on the expression of closely linked proteins; the first study found that heat treatment inhibited the activity of AMPK and reduced the level of AMPK phosphorylation by 60.04% (P0.01), and this change could be prolonged with the time of recovery. Then, the AMPK upstream regulator (ATP, Ca MKK beta, LKB1) was detected. It was found that the level of ATP in the support cells decreased to 48.92% (P0.01) in the control group after heat treatment, and the MKK beta expression of Ca was down to 46.18% (P0.01) in the control group, but the LKB1 activity was not changed. Inhibition was found to further inhibit the activity of AMPK after heat treatment, which decreased by 62.19% (P0.01) compared to the single heat treatment group, while STO609 alone had no significant effect on AMPK activity, indicating that heat treatment inhibited the expression of Ca MKK beta and then inhibited the activity of AMPK. In order to further explore AMPK in heat treatment, the closely linked eggs were down regulated. Whether the white expression plays a role, this study uses the AMPK specific activator AICAR (2 mmol/L, 2 h) to treat the support cells. The results showed that the AMPK was activated first and then the heat treatment was activated, and the decrease in the expression of closely linked protein decreased obviously; then the overexpression vector PC DNA-AMPK/si RNA was used to express / interfere with AMPK, and the results were found. Overexpression of AMPK can partly save the expression of close linked protein related proteins caused by heat treatment. Immunofluorescence results show that the location of Claudin-11 in the supporting cells after heat treatment is also changed after AMPK is activated or overexpressed, from the surface of the vesicular membrane to diffuse to the cytoplasm, the membrane and the vesicle surface. And after interfering with AMPK Further heat treatment, the expression of closely linked proteins is further down-regulated than that in the single heat treatment group, and Claudin-11 is almost not detected in the support cells. This indicates that the Ca MKK beta mediated AMPK plays a key role in the expression of closely linked protein related proteins in heat treatment and regulation of.3. oxidative stress in the expression of closely linked protein related proteins regulated by HS After heat treatment, the level of ROS and MDA increased significantly, while the activity of antioxidant enzyme SOD, GSH-Px and CAT decreased significantly after heat treatment. With the extension of recovery time after heat treatment, the above indexes gradually recovered to the normal level. This indicated that the heat treatment could increase the level of oxidative stress in the support cells. NAC (N-acetyl-L-cysteine, N- acetylcysteine) found that NAC (1 mmol/L, 2 h) increased the level of ROS and MDA in cells by reducing heat treatment and increased the activity of antioxidant enzymes SOD, GSH-Px, CAT, thus reducing the oxidative damage of support cells after heat treatment. This study further found that NAC could increase the Ca MKK beta expression in cells and improve the activity of AMPK after heat treatment. The results showed that heat treatment inhibited the expression of Ca MKK beta through ROS production and inhibited AMPK activity. Finally, the addition of NAC could effectively relieve heat treatment induced by heat treatment. In conclusion, heat treatment can inhibit the expression of Ca MKK beta and inhibit the activity of AMPK by increasing the level of oxidative stress in the cells, and thus inhibiting the activity of MKK, thus causing the reversible changes in the expression of closely linked proteins and the localization of the cells in the cells. The molecular mechanism that affects the expression of closely linked proteins in the testis supporting cells of immature pigs provides a theoretical basis for the adverse effects of artificial heat stress on the spermatogenesis of the boars.
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S828
，

本文编号：1915992