NGF在绒山羊皮肤组织中的表达及其对次级毛囊外根鞘细胞增殖作用的研究

发布时间：2018-04-04 12:40

本文选题：神经生长因子　切入点：绒山羊　出处：《吉林农业大学》2017年博士论文

【摘要】：绒山羊次级毛囊的生长发育与绒毛生产数量和质量息息相关。毛用哺乳动物的毛囊的生长发育一般经历三个阶段(休止期、生长期、退行期)周而复始的循环,它是一系列信号分子相互作用相互调控的结果,在这一过程中,外跟鞘(Outer root sheath,ORS)细胞的增殖被认为是毛囊生长发育的重要动力来源之一。近年来的研究发现,神经生长因子(nerve growth factor,NGF)除了在神经系统发挥作用以外,还在一些非神经系统发挥生物学功能,如有研究发现NGF可以影响小鼠毛囊的形态学变化,NGF及其受体存在于小鼠毛囊发育的不同阶段,说明NGF是小鼠毛囊发育过程中至关重要的一种因子。然而,作为重要的毛绒用动物—绒山羊的皮肤毛囊中是否存在NGF及其受体酪氨酸激酶A(tyrosine kinase A,TrkA),如果存在其功能又有哪些?绒山羊ORS细胞中是否是NGF的分泌细胞或作用靶细胞,NGF是否参与ORS细胞的增殖过程?针对上述假设本论文对此进行了系统的研究,并获得了相应的研究结果。1.NGF及其受体TrkA在绒山羊绒毛生长周期皮肤组织中的表达利用RT-PCR方法检测了皮肤组织中是否有NGF及其受体TrkAmRNA的表达,并利用实时荧光定量PCR和Western blot的方法对毛囊生长发育三个阶段皮肤组织中NGF及其受体TrkA的表达进行定量分析。结果表明:NGF及其受体TrkA的mRNA存在于毛囊生长周期各阶段的皮肤组织中;生长期皮肤组织中NGF及其受体TrkA mRNA的表达水平显著高于休止期和退行期(p0.05);生长期皮肤组织中NGF蛋白水平显著高于休止期和退行期(p0.05),生长期TrkA的蛋白水平与休止期没有显著差异,但显著的高于退行期(p0.05)。利用HE染色观察毛囊生长发育三个阶段的绒山羊毛囊的形态。结果表明:在生长期,初级毛囊(Primary hair follicle,PF)和次级毛囊(Secondary hair follicle,SF)的数量要多于休止期和退行期,且毛囊结构完整、清晰;在退行期,PF和SF的数量显著降低。毛囊的结构松散,毛乳头(Dermal papilla,DP)、ORS和内根鞘(Internal root sheath,IRS)分层不清晰;在休止期,PF和SF的数量少,PF的ORS和IRS层清晰可见,但SF的ORS和IRS层不清晰。利用间接免疫荧光技术对NGF和TrkA的蛋白进行定位。结果表明:NGF和TrkA的蛋白存在于PF和SF毛囊生长周期的所有阶段;在生长期,在ORS和IRS细胞中检测到NGF的高表达,并且在ORS细胞中检测到TrkA的阳性信号。信号强度为生长期最高,休止期其次,退行期最低。2.NGF及其受体TrkA在体外培养绒山羊次级毛囊ORS细胞中的表达分离生长期绒山羊的SF,进行ORS细胞的体外分离与培养。结果表明:中性蛋白酶消化法分离毛囊省时省力,毛囊周围组织去除较干净,细胞不易污染;分离培养的ORS细胞于培养基中2~3天贴壁,细胞多呈多角形,少数为卵圆形,特异性蛋白CK19鉴定ORS细胞的特征,ORS细胞纯度在90%以上。利用间接免疫荧光技术发现ORS细胞中均可检测到NGF、TrkA的免疫活性,NGF在细胞核中的信号强度高于细胞质中,在细胞核中没有TrkA的免疫活性。利用ELISA方法发现体外培养的ORS细胞可以持续分泌NGF,但随着时间的延长,分泌量显著下降(p0.05)。3.NGF对绒山羊次级毛囊ORS细胞增殖作用的研究通过外源性添加NGF重组蛋白及其受体阻断剂K252a的方法研究NGF对ORS细胞增殖的影响。结果表明:20ng/mL或100ng/mL的NGF重组蛋白可促进绒山羊次级毛囊ORS细胞的增殖,当用20ng/mL的K252a处理后,NGF的这种促增殖作用消失。说明NGF/TrkA系统可以以剂量依赖的方式促进ORS细胞的增殖。此外,我们利用细胞免疫荧光的方法研究了ORS细胞中是否存在环磷腺苷效应元件结合蛋白(cAMP-response element binding protein,CREB)。结果表明:ORS细胞中检测到了CREB的免疫阳性信号,且细胞核中的信号强度强于细胞质中。外源性添加20ng/mL或100ng/mL NGF重组蛋白能显著的提高细胞中CREB的活性,且NGF提高细胞内CREB的活性依赖于NGF/TrkA。通过研究增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)mRNA的表达情况,发现NGF/TrkA系统在维持和促进PCNA表达过程中发挥着重要作用。综上所述,本论文证实了绒山羊毛囊组织中存在NGF及其高亲和力受体TrkA的mRNA和蛋白,且在毛囊生长期显著的高于其他时期。体外培养的外根鞘细胞能检测到NGF及其受体TrkA的蛋白,且在体外条件下,外根鞘细胞能持续分泌NGF。另外我们还发现NGF/TrkA路径影响转录因子CREB的活性,且这一路径影响增殖相关基因PCNA的表达。本研究结果初步探讨了NGF在毛囊中表达规律及其作用机制,为深入研究绒山羊绒毛生长机制提供了思路。
[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.

【学位授予单位】：吉林农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S827

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