發(fā)布時(shí)間：2018-05-20 19:48

  本文選題：AMPK信號(hào)通路 + 熱處理��； 參考：《西南大學(xué)》2016年博士論文

【摘要】：熱應(yīng)激(heat stress,HS)指動(dòng)物對(duì)熱環(huán)境的舒適區(qū)上限溫度所產(chǎn)生的一系列生理反應(yīng)。隨著溫室效應(yīng)的加劇,夏季持續(xù)高溫引起的熱應(yīng)激嚴(yán)重影響了家畜的繁殖性能,對(duì)畜牧業(yè)造成的損失越來越嚴(yán)重。因此研究熱應(yīng)激對(duì)家畜繁殖性能的影響對(duì)于減少夏季熱應(yīng)激對(duì)動(dòng)物的不良影響,提高經(jīng)濟(jì)效益具有十分重要的意義。豬皮下脂肪較厚,汗腺不發(fā)達(dá),其體內(nèi)熱量較難通過皮膚蒸發(fā),因此豬很不耐熱。公豬對(duì)環(huán)境溫度尤其敏感,高溫環(huán)境可對(duì)公豬的生殖性能產(chǎn)生明顯影響,引起精液量減少、精子活力降低、精子畸形率升高。盡管冷凍精液技術(shù)已有相當(dāng)長(zhǎng)的歷史,但豬冷凍精液由于繁殖成績(jī)不理想未得到大面積應(yīng)用,因此夏季熱應(yīng)激引起公豬生精能力降低對(duì)整個(gè)養(yǎng)豬業(yè)產(chǎn)生的影響較難通過使用冷凍精液而改善。全面、深入地研究熱應(yīng)激對(duì)公豬精子發(fā)生過程的影響,可為預(yù)防和緩解熱應(yīng)激對(duì)公豬繁殖性能的影響提供重要的理論依據(jù)。支持細(xì)胞間的緊密連接是構(gòu)成血睪屏障的重要結(jié)構(gòu),在精子發(fā)生過程中起了關(guān)鍵性的作用。它將生精上皮分為基底室和近腔室。細(xì)線前期精母細(xì)胞必須穿過緊密連接結(jié)構(gòu)進(jìn)入近腔室才能完成減數(shù)分裂過程進(jìn)而分化為成熟的精子。此外,其屏障作用為精子發(fā)生提供了穩(wěn)定的微環(huán)境。熱應(yīng)激可引起緊密連接相關(guān)蛋白表達(dá)下調(diào)及表達(dá)異位,從而破壞緊密連接結(jié)構(gòu)。目前關(guān)于熱應(yīng)激對(duì)緊密連接影響的報(bào)道多為在體實(shí)驗(yàn),體外實(shí)驗(yàn)也多聚焦于成熟的體外培養(yǎng)的支持細(xì)胞,但未成熟的支持細(xì)胞內(nèi)緊密連接相關(guān)蛋白是否表達(dá)、表達(dá)部位如何以及熱應(yīng)激對(duì)未成熟支持細(xì)胞內(nèi)緊密連接蛋白的作用均未見報(bào)道。盡管熱應(yīng)激可通過改變成熟支持細(xì)胞內(nèi)緊密連接相關(guān)蛋白的表達(dá)進(jìn)而影響精子發(fā)生,但由于支持細(xì)胞間緊密連接是逐步形成的,熱應(yīng)激對(duì)未成熟的支持細(xì)胞內(nèi)緊密連接相關(guān)蛋白的影響也可能影響成熟后血睪屏障的形成。鑒于此,本文以仔豬睪丸支持細(xì)胞為研究對(duì)象,探究熱應(yīng)激對(duì)未成熟的支持細(xì)胞內(nèi)緊密連接蛋白表達(dá)的影響,并解析其中的分子機(jī)制。AMPK是真核細(xì)胞內(nèi)能量代謝調(diào)節(jié)的關(guān)鍵分子,同時(shí)也參與調(diào)節(jié)上皮細(xì)胞間緊密連接的功能:AMPK的激活可促進(jìn)緊密連接形成,敲除AMPK則可導(dǎo)致緊密連接功能喪失,但AMPK對(duì)睪丸內(nèi)緊密連接的調(diào)控作用未見報(bào)道。另外,熱應(yīng)激常引起細(xì)胞內(nèi)氧化應(yīng)激水平升高,使細(xì)胞遭受氧化損傷。研究發(fā)現(xiàn)ROS可通過調(diào)節(jié)AMPK上游調(diào)控分子Ca MKKβ的表達(dá)量進(jìn)而調(diào)節(jié)AMPK的活性。基于以上研究背景,本研究提出假設(shè):熱應(yīng)激使支持細(xì)胞發(fā)生氧化應(yīng)激反應(yīng),進(jìn)而通過抑制AMPK信號(hào)通路調(diào)控緊密連接相關(guān)蛋白的表達(dá)。本文所得研究結(jié)果如下:1.支持細(xì)胞體外HS模型的建立及HS對(duì)緊密連接相關(guān)蛋白表達(dá)的影響首先比較兩種熱處理方法(43℃水浴30 min與43℃氣浴30 min)對(duì)支持細(xì)胞活力的影響,發(fā)現(xiàn)氣浴法可引起支持細(xì)胞活力發(fā)生可逆性下降(降低34.46%,P0.01),而隨著恢復(fù)時(shí)間的延長(zhǎng),其活力在48 h可恢復(fù)至正常;水浴法亦可引起細(xì)胞活力降低(降低57.94%,P0.01),但至熱處理后第48 h,活力進(jìn)一步降低為對(duì)照組的29.49%,因此選擇43℃氣浴30 min作為支持細(xì)胞體外熱處理模型。利用此模型發(fā)現(xiàn),熱處理可通過降低線粒體膜電位顯著增加支持細(xì)胞早期凋亡率(增加367.87%,P0.01),撤除熱處理后,隨著時(shí)間的延長(zhǎng),支持細(xì)胞早期凋亡率逐漸降低并恢復(fù)至正常。利用western blotting及熒光定量PCR對(duì)緊密連接相關(guān)蛋白(Claudin-11、JAM-A、Occludin、ZO-1)及m RNA進(jìn)行檢測(cè),發(fā)現(xiàn)四種蛋白均能在體外培養(yǎng)的未成熟的公豬支持細(xì)胞內(nèi)表達(dá);熱處理后緊密連接相關(guān)蛋白的m RNA及蛋白水平顯著下調(diào);撤除熱處理后隨著時(shí)間的延長(zhǎng),緊密連接相關(guān)蛋白表達(dá)量逐漸恢復(fù)。通過免疫熒光技術(shù)對(duì)Claudin-11在支持細(xì)胞內(nèi)的表達(dá)部位進(jìn)行檢測(cè),發(fā)現(xiàn)盡管體外培養(yǎng)的支持細(xì)胞間未形成緊密連接結(jié)構(gòu),但Claudin-11大量表達(dá)于支持細(xì)胞胞膜、胞質(zhì)及囊泡膜上,且表達(dá)呈彌散型。熱處理后Claudin-11僅少量表達(dá)于囊泡膜上,撤除熱處理后48 h,Claudin-11在支持細(xì)胞內(nèi)的表達(dá)可得到恢復(fù)。以上結(jié)果表明:熱處理可引起支持細(xì)胞內(nèi)緊密連接相關(guān)蛋白的m RNA及蛋白水平發(fā)生可逆性下調(diào),并使Claudin-11的表達(dá)部位發(fā)生變化。2.AMPK信號(hào)通路在HS影響緊密連接相關(guān)蛋白表達(dá)中的作用本研究首先發(fā)現(xiàn)熱處理可抑制AMPK活性,使AMPK磷酸化水平降低60.04%(P0.01),而這一變化可隨恢復(fù)時(shí)間延長(zhǎng)逐漸恢復(fù)。隨后對(duì)AMPK上游調(diào)控因子(ATP、Ca MKKβ、LKB1)進(jìn)行檢測(cè),發(fā)現(xiàn)熱處理后支持細(xì)胞內(nèi)ATP水平降低為對(duì)照組的48.92%(P0.01),Ca MKKβ表達(dá)量下調(diào)為對(duì)照組的46.18%(P0.01),而LKB1活性未見變化。利用Ca MKKβ的特異性抑制劑STO609(10μmol/L,2 h)對(duì)Ca MKKβ進(jìn)行抑制,發(fā)現(xiàn)可在熱處理后進(jìn)一步抑制AMPK活性,相比于單獨(dú)熱處理組下降62.19%(P0.01),而STO609單獨(dú)作用卻對(duì)AMPK活性無明顯影響,說明熱處理通過抑制Ca MKKβ的表達(dá)進(jìn)而抑制AMPK的活性。為了進(jìn)一步探究AMPK在熱處理下調(diào)緊密連接相關(guān)蛋白表達(dá)中是否發(fā)揮作用,本研究利用AMPK特異性激活劑AICAR(2 mmol/L,2 h)處理支持細(xì)胞,結(jié)果發(fā)現(xiàn),先激活A(yù)MPK再進(jìn)行熱處理,緊密連接相關(guān)蛋白表達(dá)量的下降幅度明顯減小;隨后利用過表達(dá)載體pc DNA-AMPK/si RNA對(duì)AMPK進(jìn)行過表達(dá)/干涉,結(jié)果發(fā)現(xiàn)過表達(dá)AMPK可部分挽救熱處理引起的緊密連接相關(guān)蛋白表達(dá)下調(diào)。免疫熒光結(jié)果表明,熱處理后Claudin-11在支持細(xì)胞中的定位也在AMPK被激活或過表達(dá)后發(fā)生變化,從僅表達(dá)于囊泡膜表面變?yōu)閺浬⒂诎�質(zhì)、胞膜及囊泡膜表面。而干涉AMPK后再行熱處理,緊密連接相關(guān)蛋白表達(dá)量較單獨(dú)熱處理組進(jìn)一步下調(diào),且Claudin-11在支持細(xì)胞內(nèi)幾乎檢測(cè)不到。這說明Ca MKKβ介導(dǎo)的AMPK在熱處理調(diào)控緊密連接相關(guān)蛋白表達(dá)中起了關(guān)鍵作用。3.氧化應(yīng)激在HS調(diào)控緊密連接相關(guān)蛋白表達(dá)中的作用熱處理后支持細(xì)胞內(nèi)ROS及MDA水平顯著上調(diào),而抗氧化酶SOD、GSH-Px、CAT活性在熱處理后顯著降低。隨著熱處理后恢復(fù)時(shí)間的延長(zhǎng),以上指標(biāo)均逐漸恢復(fù)至正常水平。這表明熱處理可使支持細(xì)胞內(nèi)氧化應(yīng)激水平升高。通過提前添加抗氧化劑NAC(N-acetyl-L-cysteine,N-乙酰半胱氨酸),發(fā)現(xiàn)NAC(1 mmol/L,2 h)通過降低熱處理后支持細(xì)胞內(nèi)ROS及MDA水平,提高抗氧化酶SOD、GSH-Px、CAT的活性,從而減輕熱處理后支持細(xì)胞的氧化損傷。同時(shí),NAC的添加亦可降低支持細(xì)胞在熱處理后的早期凋亡率,進(jìn)而提高熱處理后支持細(xì)胞的活性。本研究進(jìn)一步發(fā)現(xiàn)NAC可上調(diào)熱處理后支持細(xì)胞內(nèi)Ca MKKβ表達(dá)量,并提高AMPK的活性,結(jié)果說明熱處理通過ROS的產(chǎn)生抑制Ca MKKβ的表達(dá),進(jìn)而抑制AMPK活性。最后本研究還發(fā)現(xiàn)NAC的添加可有效緩解熱處理引起的緊密連接相關(guān)蛋白表達(dá)下調(diào)和Claudin-11在細(xì)胞內(nèi)異位。綜上所述,熱處理通過提高支持細(xì)胞內(nèi)氧化應(yīng)激水平,抑制Ca MKKβ的表達(dá),進(jìn)而抑制AMPK活性,從而引起緊密連接相關(guān)蛋白表達(dá)和在細(xì)胞中的定位發(fā)生可逆性變化。本結(jié)論揭示了熱處理影響未成熟豬睪丸支持細(xì)胞內(nèi)緊密連接相關(guān)蛋白表達(dá)的分子機(jī)制,這為人工調(diào)控?zé)釕?yīng)激對(duì)公豬生精的不利影響提供了理論依據(jù)。
[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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S828
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本文編號(hào)：1915992