本文選題：毛發(fā)生長 + 羊駝��； 參考：《山西農(nóng)業(yè)大學》2015年碩士論文

【摘要】：羊駝背部毛發(fā)質(zhì)佳,細、長、密度高且生長周期短,但耳部毛發(fā)粗、短、稀疏且生長周期長。研究顯示TGF-β/Smads信號通路在皮膚發(fā)育及毛發(fā)生長過程中有一定的調(diào)控作用。為了研究TGFβR I對羊駝毛囊循環(huán)和毛發(fā)生長的影響,同時從microRNA水平探索TGFβR I與let-7b的靶向關系以及調(diào)控方式,實驗以青年雄性羊駝為研究對象,首先利用實時熒光定量PCR技術(Real-time PCR, RT-PCR)對TGFβR I在羊駝背部、耳部皮膚的表達量差異進行檢測,利用石蠟免疫組織化學技術(Paraffin Immunohistochemical Technique, IHC-P)對TGFβR I蛋白在羊駝皮膚中進行定位；其次,對let-7b在羊駝耳部和背部皮膚的表達差異進行驗證,然后借助雙熒光報告載體對let-7b和TGFβR I的靶向關系進行檢測;最后,利用let-7b真核表達載體轉染羊駝皮膚成纖維細胞,用RT-PCR和Western blot技術檢測TGFβR I基因和蛋白在轉染細胞中的表達水平,旨在探索let-7b對TGFβR I的調(diào)控作用方式。實驗結果如下：(1)對內(nèi)源性TGFβR I基因進行檢測：RT-PCR結果顯示TGFβR I基因在羊駝耳部皮膚相對表達量顯著高于背部皮膚(P0.01);IHC-P結果顯示,TGFβR I在毛囊的毛根鞘部和毛母質(zhì)陽性反應明顯,毛乳頭陽性反應較弱,顯示TGFβR I蛋白在毛囊的特定部位發(fā)揮作用,數(shù)據(jù)分析顯示耳部皮膚陽性反應物的平均光密度值顯著高于背部(P0.05)。(2) RT-PCR實驗對羊駝耳部和背部皮膚let-7b的表達水平進行檢測,結果顯示let-7b在背部皮膚的表達量顯著高于耳部(P0.01),與之前研究報道相符。(3)利用雙熒光素報告載體檢測1et-7b與TGFβR I是否具有具有顯著的靶向關系,結果顯示實驗組熒光素表達量下降,表明let-7b與TGFβR I具有明顯的靶向關系,即TGFβR I是let-7b的靶基因。(4)培養(yǎng)羊駝皮膚成纖維細胞,利用細胞免疫組織化學技術(Cell Immunohisto-chemical Technique, ICC)法對TGFβR I進行定位檢測。結果顯示,羊駝皮膚成纖維細胞中存在TGFβR I蛋白的表達。(5)let-7b真核表達載體轉染羊駝皮膚成纖維細胞,實現(xiàn)let-7b在細胞中的過表達。RT-PCR檢測實驗組和對照組中靶基因TGFβR I的mRNA水平,結果顯示差異不顯著；Western blot檢測靶基因TGFβR I的蛋白表達水平,結果顯示實驗組中TGFβR I蛋白表達量顯著下降(P0.05)。以上實驗結果提示：1、TGFβR I對羊駝毛發(fā)的生長具有調(diào)控作用,而在耳部高表達、背部低表達提示可能對毛發(fā)的伸長有一定的抑制作用。2、let-7b與TGFβR I具有明顯的靶向關系,即TGFβR I是let-7b的靶基因。3、let-7b可能通過轉錄阻遏下調(diào)TGFβR I蛋白的表達而調(diào)控羊駝毛的生長。
[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.
【學位授予單位】：山西農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S829.9

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