本文選題：神經(jīng)生長因子　切入點(diǎn)：絨山羊　出處：《吉林農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：絨山羊次級(jí)毛囊的生長發(fā)育與絨毛生產(chǎn)數(shù)量和質(zhì)量息息相關(guān)。毛用哺乳動(dòng)物的毛囊的生長發(fā)育一般經(jīng)歷三個(gè)階段(休止期、生長期、退行期)周而復(fù)始的循環(huán),它是一系列信號(hào)分子相互作用相互調(diào)控的結(jié)果,在這一過程中,外跟鞘(Outer root sheath,ORS)細(xì)胞的增殖被認(rèn)為是毛囊生長發(fā)育的重要?jiǎng)恿碓粗�。近年來的研究發(fā)現(xiàn),神經(jīng)生長因子(nerve growth factor,NGF)除了在神經(jīng)系統(tǒng)發(fā)揮作用以外,還在一些非神經(jīng)系統(tǒng)發(fā)揮生物學(xué)功能,如有研究發(fā)現(xiàn)NGF可以影響小鼠毛囊的形態(tài)學(xué)變化,NGF及其受體存在于小鼠毛囊發(fā)育的不同階段,說明NGF是小鼠毛囊發(fā)育過程中至關(guān)重要的一種因子。然而,作為重要的毛絨用動(dòng)物—絨山羊的皮膚毛囊中是否存在NGF及其受體酪氨酸激酶A(tyrosine kinase A,TrkA),如果存在其功能又有哪些?絨山羊ORS細(xì)胞中是否是NGF的分泌細(xì)胞或作用靶細(xì)胞,NGF是否參與ORS細(xì)胞的增殖過程?針對(duì)上述假設(shè)本論文對(duì)此進(jìn)行了系統(tǒng)的研究,并獲得了相應(yīng)的研究結(jié)果。1.NGF及其受體TrkA在絨山羊絨毛生長周期皮膚組織中的表達(dá)利用RT-PCR方法檢測(cè)了皮膚組織中是否有NGF及其受體TrkAmRNA的表達(dá),并利用實(shí)時(shí)熒光定量PCR和Western blot的方法對(duì)毛囊生長發(fā)育三個(gè)階段皮膚組織中NGF及其受體TrkA的表達(dá)進(jìn)行定量分析。結(jié)果表明:NGF及其受體TrkA的mRNA存在于毛囊生長周期各階段的皮膚組織中;生長期皮膚組織中NGF及其受體TrkA mRNA的表達(dá)水平顯著高于休止期和退行期(p0.05);生長期皮膚組織中NGF蛋白水平顯著高于休止期和退行期(p0.05),生長期TrkA的蛋白水平與休止期沒有顯著差異,但顯著的高于退行期(p0.05)。利用HE染色觀察毛囊生長發(fā)育三個(gè)階段的絨山羊毛囊的形態(tài)。結(jié)果表明:在生長期,初級(jí)毛囊(Primary hair follicle,PF)和次級(jí)毛囊(Secondary hair follicle,SF)的數(shù)量要多于休止期和退行期,且毛囊結(jié)構(gòu)完整、清晰;在退行期,PF和SF的數(shù)量顯著降低。毛囊的結(jié)構(gòu)松散,毛乳頭(Dermal papilla,DP)、ORS和內(nèi)根鞘(Internal root sheath,IRS)分層不清晰;在休止期,PF和SF的數(shù)量少,PF的ORS和IRS層清晰可見,但SF的ORS和IRS層不清晰。利用間接免疫熒光技術(shù)對(duì)NGF和TrkA的蛋白進(jìn)行定位。結(jié)果表明:NGF和TrkA的蛋白存在于PF和SF毛囊生長周期的所有階段;在生長期,在ORS和IRS細(xì)胞中檢測(cè)到NGF的高表達(dá),并且在ORS細(xì)胞中檢測(cè)到TrkA的陽性信號(hào)。信號(hào)強(qiáng)度為生長期最高,休止期其次,退行期最低。2.NGF及其受體TrkA在體外培養(yǎng)絨山羊次級(jí)毛囊ORS細(xì)胞中的表達(dá)分離生長期絨山羊的SF,進(jìn)行ORS細(xì)胞的體外分離與培養(yǎng)。結(jié)果表明:中性蛋白酶消化法分離毛囊省時(shí)省力,毛囊周圍組織去除較干凈,細(xì)胞不易污染;分離培養(yǎng)的ORS細(xì)胞于培養(yǎng)基中2~3天貼壁,細(xì)胞多呈多角形,少數(shù)為卵圓形,特異性蛋白CK19鑒定ORS細(xì)胞的特征,ORS細(xì)胞純度在90%以上。利用間接免疫熒光技術(shù)發(fā)現(xiàn)ORS細(xì)胞中均可檢測(cè)到NGF、TrkA的免疫活性,NGF在細(xì)胞核中的信號(hào)強(qiáng)度高于細(xì)胞質(zhì)中,在細(xì)胞核中沒有TrkA的免疫活性。利用ELISA方法發(fā)現(xiàn)體外培養(yǎng)的ORS細(xì)胞可以持續(xù)分泌NGF,但隨著時(shí)間的延長,分泌量顯著下降(p0.05)。3.NGF對(duì)絨山羊次級(jí)毛囊ORS細(xì)胞增殖作用的研究通過外源性添加NGF重組蛋白及其受體阻斷劑K252a的方法研究NGF對(duì)ORS細(xì)胞增殖的影響。結(jié)果表明:20ng/mL或100ng/mL的NGF重組蛋白可促進(jìn)絨山羊次級(jí)毛囊ORS細(xì)胞的增殖,當(dāng)用20ng/mL的K252a處理后,NGF的這種促增殖作用消失。說明NGF/TrkA系統(tǒng)可以以劑量依賴的方式促進(jìn)ORS細(xì)胞的增殖。此外,我們利用細(xì)胞免疫熒光的方法研究了ORS細(xì)胞中是否存在環(huán)磷腺苷效應(yīng)元件結(jié)合蛋白(cAMP-response element binding protein,CREB)。結(jié)果表明:ORS細(xì)胞中檢測(cè)到了CREB的免疫陽性信號(hào),且細(xì)胞核中的信號(hào)強(qiáng)度強(qiáng)于細(xì)胞質(zhì)中。外源性添加20ng/mL或100ng/mL NGF重組蛋白能顯著的提高細(xì)胞中CREB的活性,且NGF提高細(xì)胞內(nèi)CREB的活性依賴于NGF/TrkA。通過研究增殖細(xì)胞核抗原(proliferating cell nuclear antigen,PCNA)mRNA的表達(dá)情況,發(fā)現(xiàn)NGF/TrkA系統(tǒng)在維持和促進(jìn)PCNA表達(dá)過程中發(fā)揮著重要作用。綜上所述,本論文證實(shí)了絨山羊毛囊組織中存在NGF及其高親和力受體TrkA的mRNA和蛋白,且在毛囊生長期顯著的高于其他時(shí)期。體外培養(yǎng)的外根鞘細(xì)胞能檢測(cè)到NGF及其受體TrkA的蛋白,且在體外條件下,外根鞘細(xì)胞能持續(xù)分泌NGF。另外我們還發(fā)現(xiàn)NGF/TrkA路徑影響轉(zhuǎn)錄因子CREB的活性,且這一路徑影響增殖相關(guān)基因PCNA的表達(dá)。本研究結(jié)果初步探討了NGF在毛囊中表達(dá)規(guī)律及其作用機(jī)制,為深入研究絨山羊絨毛生長機(jī)制提供了思路。
[Abstract]:Cashmere goat hair follicle growth and secondary villus production quantity and quality are closely related. Wool mammalian hair follicle growth generally experienced three stages (rest period, growth period, catagen) cycle, it is the interaction of a series of signal molecules control the results, in this process, with the outer sheath (Outer root sheath, ORS) cell proliferation is one of the important power source of hair growth. Recent studies have found that nerve growth factor (nerve growth, factor, NGF) in addition to play a role in the nervous system, in some non nervous system functions, such as studies have found that NGF can affect the morphological changes the mouse hair follicles, the different stages of follicle development in mouse NGF and its receptor, indicating that NGF is a kind of mouse hair follicle development process essential factor. However, as an important Stuffed with NGF and its receptor tyrosine kinase A exists in cashmere goat skin and hair follicle in animal - (tyrosine kinase A, TrkA), if there is the function of what? Cashmere goat ORS cells is the secretion of NGF cells or target cells, the proliferation of NGF cells in ORS according to the hypothesis of this? This thesis gives a systematic study, and obtained the corresponding results of the expression of.1.NGF and its receptor TrkA in the cashmere growth cycle in skin tissue using RT-PCR method to detect whether the expression of NGF and TrkAmRNA in skin tissue, expression and quantitative analysis using the method of real-time fluorescence quantitative PCR and Western blot TrkA NGF and its receptor three stages in skin tissue on the growth of hair follicles. The results showed that NGF and its receptor TrkA mRNA in various stages of hair follicle growth cycle of skin tissue ; the expression level of NGF and its receptor TrkA mRNA in anagen skin tissues was significantly higher than that in the resting phase and catagen (P0.05); the level of NGF protein in skin tissue growth period was significantly higher than that of telogen and catagen (P0.05), there was no significant difference in growth period of TrkA protein level and the resting period, but significantly higher than catagen (P0.05). HE staining was used to observe the hair follicle of cashmere goat three stages of growth and development of the form. The results showed that: in the growth period, primary follicles (Primary hair follicle PF (Secondary hair) and secondary follicles follicle, SF) number more than telogen and catagen hair follicles and structural integrity clear; in catagen, the number of PF and SF decreased significantly. The loose structure of hair follicle dermal papilla (Dermal, papilla, DP), and ORS (Internal root sheath, IRS IRS) stratification is not clear; in the rest period, the number of PF and SF, ORS and IRS PF are clearly visible, But SF ORS and IRS layer is not clear. To locate protein by indirect immunofluorescence technique on NGF and TrkA. The results show that the existence of all stages of PF and SF in the growth cycle of hair follicle NGF and TrkA protein; in the growth period, to the high expression of NGF was detected in ORS and IRS cells, and detected the positive signal of TrkA in ORS cells. The signal strength is the highest growth period and telogen second lowest.2.NGF catagen and its receptor TrkA in vitro expression of goat secondary follicles in ORS cells from growth period of cashmere goat SF in vitro, isolation and cultivation of ORS cells. The results showed that: isolating and time-saving neutral protease digestion method and tissue around the hair follicles were eliminated completely, the cells are not easy to pollution; separation of ORS cells cultured in medium 2~3 days adherent cells were polygonal, rarely ovoid, feature specific protein CK19 identification of ORS cells, The purity of ORS cells can be found in more than 90%. ORS cells NGF was detected by indirect immunofluorescence technique, TrkA immunoreactivity, NGF signal intensity in the nucleus than in the cytoplasm, no TrkA immunoreactivity in the nucleus. By using the ELISA method found that ORS cells cultured in vitro can continue to secrete NGF, but with the time prolonged secretion was significantly decreased (P0.05) study on the role of cashmere goat secondary follicle proliferation of ORS cells.3.NGF by exogenous addition of recombinant NGF protein and its receptor antagonist K252a of NGF on ORS cell proliferation effects. The results showed that the 20ng/mL or 100ng/mL of the recombinant NGF protein can promote the second hair follicle of cashmere goat ORS cells the proliferation, when using the 20ng/mL K252a treatment, NGF this proliferative effect disappeared. NGF/TrkA system can be in a dose dependent manner to promote the proliferation of ORS cells. In addition, we Methods immunofluorescence was used to study the existence of cAMP responsive element binding protein in ORS cells (cAMP-response element binding protein, CREB). The results showed that ORS cells were detected in immune CREB positive signals, and the signal intensity in the nucleus to the cytoplasm. The exogenous addition of 20ng/mL or 100ng/mL NGF recombinant protein CREB can significantly improve the cell activity and NGF increased intracellular CREB activity dependent on NGF/TrkA. through the study of proliferating cell nuclear antigen (proliferating cell nuclear antigen, PCNA) mRNA expression, we found that the NGF/TrkA system plays an important role in maintaining and promoting the expression of PCNA in the process. In conclusion, this paper confirmed the mRNA and protein the presence of NGF and its high affinity receptor TrkA of cashmere goat tissues, and in hair follicle growth period was significantly higher than that of the other cultured period. Outer root sheath cells can be detected NGF and its receptor TrkA protein, and in vitro conditions, outer root sheath cells can continue to secrete NGF. we also found the NGF/TrkA path effects of transcription factor CREB activity, and the path expression of proliferation related gene PCNA. The results of this study discussed the expression pattern and its mechanism NGF in the hair follicle, provides a way for the further study of the cashmere growth mechanism.

【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S827

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3 張微;絨山羊微量元素銅需要量的研究[D];中國農(nóng)業(yè)大學(xué);2004年

4 劉斌;絨山羊絨毛生長相關(guān)基因的篩選、鑒定和多態(tài)性分析[D];內(nèi)蒙古農(nóng)業(yè)大學(xué);2012年

5 狄冉;中國產(chǎn)絨山羊微衛(wèi)星和單核苷酸多態(tài)性研究[D];中國農(nóng)業(yè)科學(xué)院;2008年

6 白文林;中國產(chǎn)絨山羊品種父系與母系起源的分子特征及遺傳分化[D];吉林大學(xué);2009年

7 孫澤威;松嫩羊草草地放牧絨山羊營養(yǎng)限制因素的研究[D];東北師范大學(xué);2008年

8 郭旭東;絨山羊轉(zhuǎn)胰島素樣生長因子Ⅰ基因體細(xì)胞核移植胚胎的制備[D];內(nèi)蒙古大學(xué);2008年

9 袁建龍;利用體細(xì)胞核移植技術(shù)生產(chǎn)轉(zhuǎn)基因絨山羊的研究[D];內(nèi)蒙古大學(xué);2013年

10 許騰;絨山羊皮膚絨毛生長期與靜息期基因差異表達(dá)的研究[D];內(nèi)蒙古大學(xué);2013年

相關(guān)碩士學(xué)位論文 前10條

1 趙帥;細(xì)毛羊次級(jí)毛囊形態(tài)發(fā)生誘導(dǎo)期轉(zhuǎn)錄組差異表達(dá)分析[D];甘肅農(nóng)業(yè)大學(xué);2015年

2 劉善博;細(xì)毛羊次級(jí)毛囊形態(tài)發(fā)生誘導(dǎo)期差異表達(dá)LncRNA靶基因的鑒定與功能分析[D];甘肅農(nóng)業(yè)大學(xué);2016年

3 張婧婧;VEGF、L-蛋氨酸對(duì)絨山羊次級(jí)毛囊外根鞘細(xì)胞增殖和分化的影響[D];西北農(nóng)林科技大學(xué);2016年

4 王蒙;Neuropilin-1過表達(dá)轉(zhuǎn)基因絨山羊模型的建立及其相關(guān)次級(jí)毛囊信號(hào)通路的研究[D];內(nèi)蒙古大學(xué);2016年

5 季小陽;絨山羊皮膚初級(jí)毛囊與次級(jí)毛囊轉(zhuǎn)錄組比較分析[D];內(nèi)蒙古農(nóng)業(yè)大學(xué);2016年

6 張?chǎng)?雷帕霉素對(duì)轉(zhuǎn)基因絨山羊細(xì)胞中外源基因表達(dá)的影響[D];內(nèi)蒙古大學(xué);2015年

7 劉樹青;T細(xì)胞因子4(TCF-4)基因表達(dá)水平對(duì)絨山羊毛囊生長發(fā)育相關(guān)基因表達(dá)的影響[D];內(nèi)蒙古大學(xué);2015年

8 郭楊;羊絨周期性生長特異性IncRNAs的篩選[D];西北農(nóng)林科技大學(xué);2015年

9 李彥飛;用不同動(dòng)物模型對(duì)新疆南疆絨山羊主要性狀進(jìn)行遺傳分析[D];新疆農(nóng)業(yè)大學(xué);2015年

10 吳鐵成;基于絨山羊育種生產(chǎn)管理系統(tǒng)實(shí)現(xiàn)超細(xì)超長育種目標(biāo)的研究[D];甘肅農(nóng)業(yè)大學(xué);2016年

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