Study On The Role And Mechanism Of GasderminA3 Gene In Mouse Hair Follicle In The Conversion Process From Anagen To Catagen

Hair follicle is one of the basic structure and the the unit of skin, as well as the epidermal appendage. In mammals, the hair is keeping loss and regeneration cycle by cycle, and lasts from birth to death of the individual. Hair follicle cycle consists of three phases: growth phase (Anagen), regression(Catagen) and resting (Telogen) [1,2].GSDMDC family[3] is a recently newly discovered protein domain containing Gasdermin superfamily, including DFNA5, DFNA5L, GSDM, GSDML and MLZE five members. There are three members in GSDM family, which are GSDMa1, GSDMa2, GSDMa3, originally identified as the candidate causative gene for several mouse skin mutants, suggesting that GSDM family and its related genes have a trend that expressing in the epithelium[3]. Previous studies have shown that GSDMa3 plays an important role in the growth of hair, its mutations will lead to be abnormal of the hair growth[4]; A series of hair depilation are relevant with its mutations, such as Runkel et al[5] used positioning cloning strategy for identifying 2 alopecia (reduced Coat2, Rco2), GSDMa3 is a region of mutation hotspot, its mutations can cause many types of alopecia, such as Rim3[6], Rco2, Reden, Bsk, Dfl[7], Fgn.So, what hair follicle structure changes will be caused and the way to hair abnormality after GSDMa3 gene mutation? Is there any change of this gene when there is some external interference?Using paraffin section and immunohistochemical S-P, RT-PCR, western to detect the changes of factors in Wnt signal pathway and apoptosis signal pathway in the hair follicle before and after GSDMa3 mutation,to explore GSDMa3 gene is whether involved in Wnt signaling pathway and the apoptosis pathway, in order to regulate the growth of hair follicle; TUNEL is used to detect DNA fragmentation before and after GSDMa3 gene mutation in the hair follicle cells, supposing that after GSDMa3 gene mutation, apoptosis of the hair follicle will be change; Using TNF-a which is able to induce apoptosis to induce hair follicle apoptosis with subcutaneous injection, and then detecting apoptotic factors and GSDMa3 gene expression through a series of means and methods; Injecting with GSDMa3 gene expression plasmid in normal C57BL / 6 (B6) mice and GSDMa3 gene mutant mice, to detect morphological changes of hair follicle and apoptosis of molecular changes by a series of methods, to certificate that GSDMa3 gene is involved in hair follicle apoptosis pathway, and changes apoptosis of hair follicle. The main results are as follows:Detecting the morphologic changes of hair follicle before and after the GSDMa3 gene mutation. On postnatal 8d(P8), compared with B6 mice, the hair follicle of GSDMa3 gene mutated mice lengthened obviously, the longitude was more vertical than B6 mice,and the epidermis and the dermis of these mutated mice were thickened. Otherwise, On P11,their hair follicle in the midanaphase of anagen grew progressively, the longitude was more vertical and the epidermis and dermis were more thicher. On P16 and P18, the hair shafts were on telogen and shortened, the hair bulbs were atrophied, and both the dermis and epidermis were thinned. However, compared with the same phage of B6 mice, although the hair bulbs of GSDMa3 gene mutated mice also atrophied, the length of hair shaft did not reduced, the dermis and epidermis remained thick, and hair follicle's longitude became more vertical. Moreover on P24, the hair follicle of B6 was on telogen, and their length became the shortest in the hair cycle, leaving the dermal papilla and hair shaft while the hair follicle's morphology of GSDMa3 gene mutated mice at that time kept the same as pristine categen, failing to transfer into the phage of telogen.②Using IHC,RT-PCR,western to detect that after GSDMa3 gene mutation,β-catenin expression was significantly higher than C57BL/6 mouth in any point of the hair follicle,whether lower expression of Wnt 5a;③Using IHC,RT-PCR,western to detect that after GSDMa3 gene mutation, Caspase-3 expression was significantly lower than C57BL/6 mouth in any point of the hair follicle. TUNEL was used to detect after GSDMa3 gene mutation, DNA fragment in the inner and outer root sheath of hair follicle was significantly lower than before mutation;④Using exogenous injection of TNF-a into B6 mice,with GSDMa3 gene increased, apoptosis was significantly in some factors, and signs of apoptosis were more obvious. While only part of GSDMa3 gene and Caspase-3 were increased in GSDMa3 gene mutant mice.⑤Using exogenous injection of GSDMa3 gene expression plasmid, either B6 mice or GSDMa3 gene mutant mice, with GSDMa3 gene increased, apoptosis was significantly increased in some factors, and signs of apoptosis are more obvious. In summary, the issue infered that, GSDMa3 gene mutation changed the growth and the cycle of hair follicle by changing the Wnt signaling pathway and apoptosis pathway.