Let-7b通过其靶基因-TGFβR Ⅰ调控羊驼毛生长的研究

发布时间：2018-04-25 09:12

本文选题：毛发生长 + 羊驼　；参考：《山西农业大学》2015年硕士论文

【摘要】：羊驼背部毛发质佳,细、长、密度高且生长周期短,但耳部毛发粗、短、稀疏且生长周期长。研究显示TGF-β/Smads信号通路在皮肤发育及毛发生长过程中有一定的调控作用。为了研究TGFβR I对羊驼毛囊循环和毛发生长的影响,同时从microRNA水平探索TGFβR I与let-7b的靶向关系以及调控方式,实验以青年雄性羊驼为研究对象,首先利用实时荧光定量PCR技术(Real-time PCR, RT-PCR)对TGFβR I在羊驼背部、耳部皮肤的表达量差异进行检测,利用石蜡免疫组织化学技术(Paraffin Immunohistochemical Technique, IHC-P)对TGFβR I蛋白在羊驼皮肤中进行定位；其次,对let-7b在羊驼耳部和背部皮肤的表达差异进行验证,然后借助双荧光报告载体对let-7b和TGFβR I的靶向关系进行检测;最后,利用let-7b真核表达载体转染羊驼皮肤成纤维细胞,用RT-PCR和Western blot技术检测TGFβR I基因和蛋白在转染细胞中的表达水平,旨在探索let-7b对TGFβR I的调控作用方式。实验结果如下：(1)对内源性TGFβR I基因进行检测：RT-PCR结果显示TGFβR I基因在羊驼耳部皮肤相对表达量显著高于背部皮肤(P0.01);IHC-P结果显示,TGFβR I在毛囊的毛根鞘部和毛母质阳性反应明显,毛乳头阳性反应较弱,显示TGFβR I蛋白在毛囊的特定部位发挥作用,数据分析显示耳部皮肤阳性反应物的平均光密度值显著高于背部(P0.05)。(2) RT-PCR实验对羊驼耳部和背部皮肤let-7b的表达水平进行检测,结果显示let-7b在背部皮肤的表达量显著高于耳部(P0.01),与之前研究报道相符。(3)利用双荧光素报告载体检测1et-7b与TGFβR I是否具有具有显著的靶向关系,结果显示实验组荧光素表达量下降,表明let-7b与TGFβR I具有明显的靶向关系,即TGFβR I是let-7b的靶基因。(4)培养羊驼皮肤成纤维细胞,利用细胞免疫组织化学技术(Cell Immunohisto-chemical Technique, ICC)法对TGFβR I进行定位检测。结果显示,羊驼皮肤成纤维细胞中存在TGFβR I蛋白的表达。(5)let-7b真核表达载体转染羊驼皮肤成纤维细胞,实现let-7b在细胞中的过表达。RT-PCR检测实验组和对照组中靶基因TGFβR I的mRNA水平,结果显示差异不显著；Western blot检测靶基因TGFβR I的蛋白表达水平,结果显示实验组中TGFβR I蛋白表达量显著下降(P0.05)。以上实验结果提示：1、TGFβR I对羊驼毛发的生长具有调控作用,而在耳部高表达、背部低表达提示可能对毛发的伸长有一定的抑制作用。2、let-7b与TGFβR I具有明显的靶向关系,即TGFβR I是let-7b的靶基因。3、let-7b可能通过转录阻遏下调TGFβR I蛋白的表达而调控羊驼毛的生长。
[Abstract]:The hair on the back of alpaca is of good quality, fine, long, high density and short growth cycle, but the ear hair is thick, short, sparse and long growth cycle. The results show that TGF- 尾 -Smads signaling pathway plays a role in the regulation of skin development and hair growth. In order to study the effect of TGF 尾 RI on hair cycle and hair growth of alpaca, and to explore the target relationship and regulation between TGF 尾 RI and let-7b from the microRNA level, the young male alpaca was studied. The expression of TGF 尾 RI in the dorsal and ear skin of alpaca was detected by real-time fluorescence quantitative PCR technique (RT-PCR), and the expression of TGF 尾 RI protein in alpaca skin was detected by paraffin immunohistochemical technique (IHC-PX). The difference of let-7b expression in the ear and back skin of alpaca was verified, and the targeting relationship between let-7b and TGF 尾 RI was detected by double fluorescent report vector. Finally, the eukaryotic expression vector of let-7b was used to transfect the alpaca skin fibroblasts. The expression of TGF 尾 RI gene and protein in transfected cells was detected by RT-PCR and Western blot. The aim of this study was to explore the regulation of let-7b on TGF 尾 RI. The results showed that the relative expression of TGF 尾 R I gene in the ear skin of alpaca was significantly higher than that in the dorsal skin (P 0.01). The positive reaction of TGF- 尾 R I in the hair root sheath and hair matrix was significant. The positive reaction of dermal papilla was weak, indicating that TGF 尾 R I protein played a role in the specific part of hair follicle. Data analysis showed that the average optical density of the positive reactants in the ear was significantly higher than that in the back (P 0.05)) RT-PCR test was used to detect the expression level of let-7b in the ear and back skin of alpaca. The results showed that the expression of let-7b in the skin of the back was significantly higher than that in the ear (P 0.01), which was consistent with previous reports.) the double fluorescein report vector was used to detect whether there was a significant targeting relationship between 1et-7b and TGF 尾 RI. The results showed that the expression of fluorescein in the experimental group was decreased. The results showed that let-7b and TGF 尾 RI had a significant targeting relationship, that is, TGF 尾 RI was the target gene of let-7b) and cultured alpaca skin fibroblasts. The localization of TGF 尾 RI was detected by cell Immunohisto-chemical technique. The results showed that the expression of TGF 尾 R I protein was detected in alpaca skin fibroblasts. The eukaryotic expression vector was transfected into alpaca skin fibroblasts. The overexpression of let-7b in the cells was detected by RT-PCR. The mRNA level of target gene TGF 尾 RI was detected in the experimental and control groups. The results showed that there was no significant difference between the two groups. Western blot was used to detect the protein expression of TGF 尾 RI. The results showed that the expression of TGF 尾 RI protein decreased significantly in the experimental group (P 0.05). The above results suggest that TGF- 尾 R I can regulate the growth of alpaca hair, while high expression in ear and low expression in back suggest that TGF- 尾 R I may have some inhibitory effect on hair elongation. 2TGF- 尾 R I may have an obvious target relationship with TGF 尾 R I. That is to say, TGF 尾 RI is the target gene of let-7b. 3let-7b may regulate the growth of alpaca hair by down-regulating the expression of TGF 尾 RI protein.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S829.9

【共引文献】