本文關(guān)鍵詞：凋亡誘導(dǎo)因子（AIF）在小鼠毛囊周期中的表達(dá)調(diào)控研究　出處：《吉林大學(xué)》2015年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 毛囊周期 退行期 凋亡誘導(dǎo)因子 Caspase3 凋亡

【摘要】：目的 本研究旨在探究凋亡誘導(dǎo)因子(Apotosisi inducing factor, AIF)是否在小鼠毛囊組織中表達(dá)以及該表達(dá)對(duì)毛囊周期、特別是退行期毛囊細(xì)胞凋亡的影響，為毛囊生物學(xué)研究提供理論依據(jù)，也為毛發(fā)相關(guān)疾病的治療提供潛在的思路。 方法 首先我們通過(guò)酶消化法從C57BL/6小鼠背部皮膚組織中分離得到了純凈的毛囊組織，并以此為原料，從中提取總RNA和蛋白質(zhì)。首先應(yīng)用PCR和Westernblot等技術(shù)，檢測(cè)AIF是否在毛囊組織中表達(dá)，并進(jìn)一步應(yīng)用免疫組織化學(xué)染色法確定AIF在小鼠毛囊組織中的表達(dá)定位，其次通過(guò)免疫熒光染色檢測(cè)AIF和親環(huán)素A(Cyclophilin A，CypA)在毛囊細(xì)胞凋亡(TUNEL染色)過(guò)程中的時(shí)空表達(dá)及其與Caspase信號(hào)之間的關(guān)系。此外，通過(guò)PCR法檢測(cè)膜受體通路相關(guān)凋亡因子在毛囊周期中mRNA水平的表達(dá)變化。最后我們通過(guò)小鼠腹腔注射Cyclosporine A、Caspase3Inhibitor (Ac-DEVD-CHO)和Calpain Inhibitor I等三種線(xiàn)粒體凋亡信號(hào)通路抑制劑，通過(guò)Western blot法從蛋白質(zhì)水平上檢測(cè)并分析了Caspase級(jí)聯(lián)信號(hào)和AIF信號(hào)之間的調(diào)控關(guān)系。 結(jié)果 PCR和Western blot結(jié)果顯示AIF在小鼠毛囊組織中呈陽(yáng)性表達(dá)。免疫組織化學(xué)染色結(jié)果顯示AIF表達(dá)于小鼠毛囊的外毛根鞘、內(nèi)毛根鞘、毛乳頭及隆突部位，另外在小鼠皮膚的表皮、皮脂腺、皮下肌肉層中也呈陽(yáng)性表達(dá)。此外，PCR結(jié)果顯示膜受體通路凋亡因子TRAIL、TNF-α和Fas L在轉(zhuǎn)錄水平上也參與調(diào)控毛囊周期。TUNEL和AIF/CypA共染結(jié)果發(fā)現(xiàn)AIF和CypA在生長(zhǎng)期和退行期大量進(jìn)入毛囊細(xì)胞核中，同時(shí)發(fā)現(xiàn)TUNEL陰性的細(xì)胞中AIF和CypA也大量的進(jìn)入細(xì)胞核。最后給小鼠注射CyclosporineA、Caspase3Inhibitor (Ac-DEVD-CHO)和Calpain Inhibitor I三種抑制劑，結(jié)果發(fā)現(xiàn)前兩者能夠有效延遲Catagen，但不能阻斷Catagen，后者既不能延遲也不能阻斷Catagen，通過(guò)Western blot結(jié)果發(fā)現(xiàn)CyclosporineA可以抑制Caspase信號(hào)級(jí)聯(lián)同時(shí)阻斷AIF信號(hào)入核，，Caspase3Inhibitor (Ac-DEVD-CHO)可以阻斷Caspase3同時(shí)激活A(yù)IF信號(hào)通路；而CalpainInhibitor I可以阻止AIF從線(xiàn)粒體釋放同時(shí)激活Caspase級(jí)聯(lián)信號(hào)。 結(jié)論 （1）AIF在小鼠毛囊組織中表達(dá)，其表達(dá)部位包括小鼠毛囊的外毛根鞘、內(nèi)毛根鞘、毛乳頭及隆突部位。（2）AIF進(jìn)入毛囊細(xì)胞核切割DNA事件的發(fā)生早于Caspase3的激活，AIF是毛囊細(xì)胞凋亡生物學(xué)行為的早期調(diào)控因子。（3）Cyclosporine A和Caspase3Inhibitor (Ac-DEVD-CHO)可以有效延遲毛囊退行期，但不能阻止退行期的到來(lái)；Calpain Inhibitor I可以抑制AIF信號(hào)通路，但同時(shí)激活Caspase信號(hào)級(jí)聯(lián)。（4）非Caspase信號(hào)通路即AIF通路和Caspase級(jí)聯(lián)信號(hào)通路相輔相成、互相彌補(bǔ)。（5）毛囊的退行是細(xì)胞膜表面凋亡受體信號(hào)通路和內(nèi)源性線(xiàn)粒體通路，以及Caspase級(jí)聯(lián)信號(hào)通路和非Caspase信號(hào)即AIF通路協(xié)同調(diào)控的結(jié)果。
[Abstract]:Purpose The aim of this study was to investigate the expression of apoptosis inducing factor Apotosisi inducing factor (AIFF) in mouse hair follicles and its effect on hair follicle cycle. Especially the effect of hair follicle cell apoptosis in receding stage provides theoretical basis for the study of hair follicle biology and provides potential ideas for the treatment of hair related diseases. Method First we isolated pure hair follicle tissue from C57BL / 6 mouse back skin by enzyme digestion and used it as raw material. The total RNA and protein were extracted. Firstly, PCR and Westernblot were used to detect the expression of AIF in hair follicles. Immunohistochemical staining was used to determine the expression of AIF in mouse hair follicles, and then AIF and cyclophilin A were detected by immunofluorescence staining. The temporal and spatial expression of CypA and its relationship with Caspase signal in the process of hair follicle apoptosis and Tunel staining. The expression of mRNA in hair follicle cycle was detected by PCR. Finally, Cyclosporine A was injected intraperitoneally into mice. Caspase3Inhibitor (Ac-DEVD-CHO) and Calpain Inhibitor I are three kinds of mitochondrial apoptosis signal pathway inhibitors. The regulatory relationship between Caspase cascade signal and AIF signal was detected and analyzed at protein level by Western blot method. Results The results of PCR and Western blot showed that the expression of AIF was positive in the mouse hair follicles, and the expression of AIF was found in the outer hair root sheath of the mouse hair follicles by immunohistochemical staining. The inner hair root sheath, dermal papilla and protuberance were also positive in epidermis, sebaceous gland and subcutaneous muscle layer of mouse skin. In addition, the results of TRAIL showed that the membrane receptor pathway apoptosis factor TRAIL. TNF- 偽 and Fas. L was also involved in the regulation of hair follicle cycle. Tunel and AIF/CypA co-staining results showed that AIF and CypA entered the hair follicle nucleus in the growth and receding stages. At the same time, it was found that AIF and CypA in TUNEL negative cells also entered the nucleus in large quantities. Finally, CyclosporineA was injected into mice. Caspase3Inhibitor (Ac-DEVD-CHO) and Calpain Inhibitor I inhibitors. The results showed that the first two could effectively delay Catagenbut could not block Catagen.The latter could neither delay nor block Catagen. The results of Western blot showed that CyclosporineA could inhibit the cascade of Caspase signals and block the entry of AIF signals into the nucleus. Caspase3Inhibitor (Ac-DEVD-CHO) can block Caspase3 and activate AIF signaling pathway. CalpainInhibitor I could prevent AIF from releasing from mitochondria and activate the Caspase cascade signal. Conclusion AIF was expressed in mouse hair follicles including outer hair root sheath and inner hair root sheath of mouse hair follicle. The occurrences of DNA in the hair follicle nuclear cleavage occurred earlier than the activation of Caspase3. AIF is the early regulatory factor for the biological behavior of hair follicle cell apoptosis. Cyclosporine A and Caspase3Inhibitor (. Ac-DEVD-CHO can effectively delay the hair follicle receding period. However, it cannot prevent the coming of receding period; Calpain Inhibitor I inhibited AIF signaling pathway. But at the same time, the activation of Caspase signal cascade. 4) Non-#en1# signal pathway, that is, AIF pathway and Caspase cascade signal pathway complement each other. The receding of hair follicles is the signal pathway of apoptosis receptor and the endogenous mitochondrial pathway on the surface of cell membrane. And the result of coordinated regulation of Caspase cascade signal pathway and non-Caspase signal, that is, AIF pathway.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R758.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 葉麗華;施U

本文編號(hào)：1395567