Preparation Of Anxa1 Gene Knockout And Overexpression Mice And Preliminary Study On Their Effects On Hair Follicle Growth In Mice

Anxa1 is a calcium dependent phospholipid-binding protein,which is expressed in animals,plants and microorganisms and participates in many cellular physiological processes.Because the expression of Anxa1 gene in mouse back skin changes with the growth cycle of hair follicles,we speculate that Anxa1 may be a key factor affecting the growth and development of hair follicles.In order to explore the influence of Anxa1 gene on the growth and development of mouse hair follicles,the knockout and overexpression mice of Anxa1 gene were prepared by CRISPR/Cas9 system combined with prokaryotic microinjection technique.Comparison of the growth rate of mouse back hair by depilation induction experiment.The density and diameter of hair follicles in mouse back skin were counted by microscopic observation of tissue sections.The diameter of mouse hair shaft was measured by fiber fineness meter.Detection of Ki-67 and CD34 in mouse hair follicles by immunofluorescence staining.The expression of Egf,Notch1 and Mkk7 in the skin tissues of mice back were detected by qRT-PCR or Western Blot.The phosphorylation levels of Akt1 and Erk1/2 in the back skin of mice were detected by Western Blot.In this study,Anxa1 knockout(KO)and overexpression(OE)mice were prepared by CRISPR/Cas9 technology system combined with prokaryotic microinjection.Four KO mice and five OE mice were obtained,with positive rates of 19.0% and 20.8%,respectively.After 13 days of depilation at 10 weeks of age,KO mice had developed thicker hair,while unmodified WT and OE mice had shorter hair on the back.The number of hair follicles in 10-week-old KO mice was slightly more than that in WT mice(33.6±0.5),and that in OE mice(27±0.7)was less than that in WT mice(33±1.2)(P < 0.05).The diameter of hair follicles in the back skin of KO mice aged P70(43.28±2.27?m)was larger than that of WT mice(35.76±2.11?m)(P < 0.05).The diameter of hair follicles in the back skin of OE mice(33.35±2.17?m)was smaller than that of WT mice(35.76±2.11?m).The results showed that the diameter of KO,OE and WT mice hair shaft were 21.12±1.18?m,19.24±1.87?m and 19.92±1.32?m respectively.The diameter of KO mice hair shaft was larger than that of WT mice(P < 0.05),and that of OE mice was smaller than that of WT mice.The hair follicle diameters of KO,OE and WT mice at the age of P35 were 92.29±3.22?m,73.71±2.92?m and 80.57±1.36?m respectively.The hair follicle diameters of KO mice were larger than those of WT mice(P < 0.05),while those of OE mice were smaller than those of WT mice.Ki-67 and CD34 protein aggregated in the hair follicles of KO mice at the age of P35,while Ki-67 and CD34 protein mainly concentrated in the inner and outer root sheaths of hair follicles of OE mice and WT mice.In P70 mice,the aggregation of Ki-67 and CD34 proteins in the hair follicles of KO,OE and WT mice was very low.The relative expression levels of Egf,Notch1 and Mkk7 in the dorsal skin of P35 mice were 2.15,19 and 2.78 times higher than those of WT mice,respectively.The relative expression levels of Egf,Notch1 and Mkk7 in OE mice were 0.36,0.28 and 0.48 times lower than those in WT mice,respectively.In the back skin of P70 mice,the relative expression of Egf,Notch1 and Mkk7 in KO mice was 3.09 times(P < 0.05),3.98 times and 1.47 times higher than that in WT mice.The relative expression of Egf,Notch1 and Mkk7 in OE mice was 0.16 times(P < 0.05),0.40 times and 0.57 times lower than that in WT mice.The results showed that the phosphorylation levels of Akt1 and Erk1/2 protein in KO mice were 2.67 times(P < 0.05)and1.78 times higher than those in WT mice(P < 0.05),and the phosphorylation levels of Akt1 and Erk1/2 in OE mice were 0.69 times and 0.8 times lower than those in WT mice.