| Uncv BALB/c hairless mutant mice were discovered by the Laboratory Animal Center, Academy of Military Medical Sciences(AMMS) and designated Uncv hairless mice by the International Committee on Standardized Genetic Nomenclature. The offspring of Uncv hairless mice exhibit three phenotypes: normal hair, hairless and sparse hair.The hair follicle(Hair follicle, HF) is considered a mini-organ that forms via neuro ectodermal-mesodermal interactions. HF morphogenesis starts during the early embryonic stage. Uncv hairless mice can trace their phenotype to an autosomal gene mutation leading to HF developmental defects. Hair follicle morphogenesis is dependent on Wnt, Shh, Notch, m TOR, BMP and other signaling pathways that are involved in the interplay between epithelial and mesenchymal cells. The Wnt pathway plays an essential role during hair follicle induction, Shh is involved in morphogenesis and late stage differentiation, Notch signaling determines stem cell fate, m TOR signaling promotes stem cell activation during the hair regeneration process, and BMP is involved in cellular differentiation.Hair follicle stem cells(HFSCs) are pluripotent cells that are located in the bulge of the outer root sheath(ORS), HFSCs are undifferentiated primitive and label-retaining cells that can be identified by several distinct molecular markers(CD34/CD200/K15, etc.). HFSCs are adult stem cells that act as basal cells for HF development and the HF cycle and possess multipotent differentiation potential.During downstream migration, HFSCs can differentiate into HF and various groups of epithelial cells, form new hair and contribute to the formation of the HF cycle, while during upward migration, they can differentiate into sebaceous glands and epidermal cells, which are involved in skin wound healing.The Noggin gene, which was originally isolated from Xenopus embryos, plays an important role in the development of HF. When the Noggin gene was injected into Xenopus embryos, it significantly increased the embryonic head size. In vivo, the Noggin gene is important for the regulating the development and remodelling of multiple systems. In addition, the Noggin gene plays an important role in the development of HF.Keratin is a major structural protein during hair development. The keratin family exhibits differential expression in different HF cell layers notably, keratin 15(K15) is an important molecule involved in HF development, and its encoding gene employs an HFSC-specific promoter. Thus, the K15 promoter was used for Cre recombinase expression in hair follicle stem cells.In this thesis, to explore the mechanistic relationships between Noggin and the BMP and Wnt signalling pathways, as well as subsequent effects on signalling, we interfered with Noggin in vivo and in vitro.â'ˆ Construction and evaluation of Noggin gene silencing vectors.Four interference sequences were designed based on the Noggin m RNA sequence and were cloned into Lenti-KD vectors. The recombinant plasmids were transiently transfected into HEK-293 T cells to obtain recombinant viruses. Then,MC3T3-E1 cells were infected with the recombinant viruses and screened with puromycin. The gene silencing effects of the sh RNAs were evaluated using q PCR and Western blot. The q PCR results showed that all the four interference sequences exerted silencing effects on the expression of Noggin, but only sh Noggin-1(P<0.01)demonstrated a significant difference. Similarly, the Western blot results showed that among the four interference sequences, only sh Noggin-1(P<0.01) induced a significantly decrease on the expression of Noggin. Thus, we obtained a single interference sequence for the Noggin gene that can interfere with m RNA stability and affect its protein expression. This construct is useful to further study the unknown biological functions of Noggin.â'‰ Effects of Noggin gene silencing on the expression of the BMP and Wnt signalling pathways.Employing the Noggin gene silencing vectors, the BMP and Wnt signalling pathways were investigated by analysing the expression of BMP-2, BMP-4,BMPR-?A, BMP-6, BMP-7, LEF-1 and β-catenin in the MC3T3-E1 cell line using real-time quantitative PCR and Western blot techniques. Real-time PCR results showed that Noggin gene silencing had a significant impact on the expression of five genes in BMP signalling pathway: the expression of BMP-2(P<0.001), BMP-4(P<0.01), BMP-6(P<0.001) and BMP-7(P<0.001) increased, while the expression of BMPR-?A(P<0.01) decreased. The expression levels of two genes in Wnt signalling pathway, LEF-1(P<0.001) and β-catenin(P<0.001), also decreased significantly.Western blotting also showed that the expression of proteins in the two signallingpathways was affected: the expression levels of BMP-2(P<0.05), BMP-4(P<0.05),BMP-6(P<0.05) and BMP-7(P<0.05) increased significantly, while the expression ofβ-catenin(P<0.05) decreased, and in particular the expression of BMPR-?A(P<0.01)and LEF-1(P<0.001) dramatically decreased.â'Š Effects of in vivo Noggin gene interference on BMP and Wnt signalling pathway expression.In this study, F1 generation K15-Cre-Noggin Floxed/+ heterozygous mice were obtained by mating model mice in which HFSC and Cre recombinase were specifically expressed with mice in which the Noggin gene with Lox P sites was conditionally knocked out. An immunohistochemical technique was used to analyse the effects of Noggin gene interference on Noggin expression, and the results showed that Noggin was expressed in the HF bulge in the skin in both the K15-Cre positive group and K15-Cre negative group, with no notable difference between the two groups. Real-time quantitative PCR and Western blot techniques were employed to analyse the expression of BMP-4, BMP-7, LEF-1, and β-catenin in mouse skin after Noggin gene interference. At the m RNA and protein levels, the expression of Noggin(P>0.05), LEF-1(P>0.05) and β-catenin(P>0.05) showed a slight decline, while the expression of BMP-4(P>0.05) and BMP-7(P>0.05) demonstrated a small increase;however, these proteins exhibited no statistically significant differences. From this we can conclude that Noggin gene interference has no significant effect on the BMP and Wnt signalling pathways.Conclusions:â'ˆ In this study, retrovirus-mediated short hairpin RNA(sh RNA) expression vectors for Noggin were successfully constructed, and MC3T3-E1 stable cell lines that could stably and effectively inhibit Noggin gene expression were obtained. These stable cell lines formed the basis for further investigation into the effects of Noggin gene knockdown on the relevant signalling pathways.â'‰ Employing the mouse Noggin gene silencing vector described above, the effects of Noggin gene silencing on the BMP and Wnt signalling pathways were analysed in vitro using q PCR and Western blot. Five proteins in the BMP signalling pathway demonstrated significant differences at both the m RNA and protein levels;the expression of BMP-2, BMP-4, BMP-6 and BMP-7 increased, but the expression of BMPR-?A declined. Moreover, the expression of LEF-1 and β-catenin, which are members of the Wnt signalling pathway, was significantly reduced. Given all of ourresults, we can conclude that the Noggin gene may inhibit the BMP signalling pathway while activating the Wnt signaling pathway in vitro, which lays the foundation to further study the effects of the Noggin gene on the expression of the BMP and Wnt signalling pathways in vivo.â'Š In this study, F1 generation K15-Cre-Noggin Floxed/ + heterozygous mice were obtained by mating model mice in which HFSC and Cre recombinase were specifically expressed with mice in which the Noggin gene with Lox P sites was conditionally knocked out. The expression of relevant factors in the BMP and Wnt signalling pathways and the expression of the Noggin gene exhibited no differences.In the future, backcross experiments can be performed to obtain HFSC Noggin gene knockout homozygous mice. |
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