凋亡诱导因子（AIF）在小鼠毛囊周期中的表达调控研究

发布时间：2018-01-08 04:13

本文关键词：凋亡诱导因子（AIF）在小鼠毛囊周期中的表达调控研究　出处：《吉林大学》2015年博士论文　论文类型：学位论文

【摘要】：目的本研究旨在探究凋亡诱导因子(Apotosisi inducing factor, AIF)是否在小鼠毛囊组织中表达以及该表达对毛囊周期、特别是退行期毛囊细胞凋亡的影响，为毛囊生物学研究提供理论依据，也为毛发相关疾病的治疗提供潜在的思路。方法首先我们通过酶消化法从C57BL/6小鼠背部皮肤组织中分离得到了纯净的毛囊组织，并以此为原料，从中提取总RNA和蛋白质。首先应用PCR和Westernblot等技术，检测AIF是否在毛囊组织中表达，并进一步应用免疫组织化学染色法确定AIF在小鼠毛囊组织中的表达定位，其次通过免疫荧光染色检测AIF和亲环素A(Cyclophilin A，CypA)在毛囊细胞凋亡(TUNEL染色)过程中的时空表达及其与Caspase信号之间的关系。此外，通过PCR法检测膜受体通路相关凋亡因子在毛囊周期中mRNA水平的表达变化。最后我们通过小鼠腹腔注射Cyclosporine A、Caspase3Inhibitor (Ac-DEVD-CHO)和Calpain Inhibitor I等三种线粒体凋亡信号通路抑制剂，通过Western blot法从蛋白质水平上检测并分析了Caspase级联信号和AIF信号之间的调控关系。结果 PCR和Western blot结果显示AIF在小鼠毛囊组织中呈阳性表达。免疫组织化学染色结果显示AIF表达于小鼠毛囊的外毛根鞘、内毛根鞘、毛乳头及隆突部位，另外在小鼠皮肤的表皮、皮脂腺、皮下肌肉层中也呈阳性表达。此外，PCR结果显示膜受体通路凋亡因子TRAIL、TNF-α和Fas L在转录水平上也参与调控毛囊周期。TUNEL和AIF/CypA共染结果发现AIF和CypA在生长期和退行期大量进入毛囊细胞核中，同时发现TUNEL阴性的细胞中AIF和CypA也大量的进入细胞核。最后给小鼠注射CyclosporineA、Caspase3Inhibitor (Ac-DEVD-CHO)和Calpain Inhibitor I三种抑制剂，结果发现前两者能够有效延迟Catagen，但不能阻断Catagen，后者既不能延迟也不能阻断Catagen，通过Western blot结果发现CyclosporineA可以抑制Caspase信号级联同时阻断AIF信号入核，，Caspase3Inhibitor (Ac-DEVD-CHO)可以阻断Caspase3同时激活AIF信号通路；而CalpainInhibitor I可以阻止AIF从线粒体释放同时激活Caspase级联信号。结论（1）AIF在小鼠毛囊组织中表达，其表达部位包括小鼠毛囊的外毛根鞘、内毛根鞘、毛乳头及隆突部位。（2）AIF进入毛囊细胞核切割DNA事件的发生早于Caspase3的激活，AIF是毛囊细胞凋亡生物学行为的早期调控因子。（3）Cyclosporine A和Caspase3Inhibitor (Ac-DEVD-CHO)可以有效延迟毛囊退行期，但不能阻止退行期的到来；Calpain Inhibitor I可以抑制AIF信号通路，但同时激活Caspase信号级联。（4）非Caspase信号通路即AIF通路和Caspase级联信号通路相辅相成、互相弥补。（5）毛囊的退行是细胞膜表面凋亡受体信号通路和内源性线粒体通路，以及Caspase级联信号通路和非Caspase信号即AIF通路协同调控的结果。
[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- 伪 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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R758.7

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