Effect Of Sdr9c7 Gene Knockout On Skin Development And Blood Coagulation In Mice

Objective:SDR9C7(also known as SDR-O)is a gene encoding member 7 of the short-chain dehydrogenase/reductase family 9C and is thought to be one of the causative genes in autosomal recessive congenital ichthyosis.We investigated the function of Sdr9c7 gene using Sdr9c7 knockout mice successfully constructed by TALAN technique,and found that newborn Sdr9c7^-/-mice showed cyanotic skin with mucus exudation and died several hours after birth;during this process,the peripheral blood vessels of newborn Sdr9c7^-/-mice became darker,wringcled and thicker and blood coagulation appeared,which inferred that Sdr9c7 gene might have an important effect on skin development and blood coagulation in mice.To address this,this study focused on the effects of Sdr9c7 gene knockout on skin barrier,skin development and blood coagulation in mice and the molecular mechanisms involved,and the results might provide some basis for further elucidation of the function of the SDR9C7 gene.Methods:(1)Sdr9c7 knockout mice were bred and genotyped:DNA was extracted from the toe tissue of newborn mice,and PCR amplification was performed.The PCR products were sequenced,and the genotypes of the mice were determined based on their base sequences and sequencing profiles.(2)The offspring mice produced by mating Sdr9c7^+/-female mice with Sdr9c7^+/-male mice were genotyped and analyzed for genotype proportions to exclude the possibility of embryonic lethality.(3)Sdr9c7^+/+and Sdr9c7^-/-embryonic and neonatal mice were phenotypically observed at E16.5 d,E18.5 d,E20.5 d and P0.(4)On site real time monitoring and bodyweight of neonates were carried out every hour after birth,and life-length of Sdr9c7^-/-neonates were determined.(5)Comparison of differences between the epidermal layers of Sdr9c7^+/+and Sdr9c7^-/-mice skin using HE staining of paraffin sections.(6)Skin permeability assay of Sdr9c7^+/+and Sdr9c7^-/-mice skin using toluidine blue solution.(7)Ultrasound experiments were performed on cornified cell envelope(CCE)prepared from the skin epidermis of Sdr9c7^+/+and Sdr9c7^-/-mice to compare the difference in fragility of CCE in the skin epidermis of Sdr9c7^+/+and Sdr9c7^-/-mice.(8)The expression of Involucrin,Loricrin and Filaggrin gene in the skin tissues of Sdr9c7^+/+and Sdr9c7^-/-mice was detected by qPCR using the Trizol method to extract RNA from Sdr9c7^+/+and Sdr9c7^-/-mice.(9)Quantitative analysis of T cells,B cells,macrophages,dendritic cells and CCR4+cells in skin tissues of neonatal Sdr9c7^+/+and Sdr9c7^-/-mice using-flow cytometry analysis method.(10)The expression of genes related to corneocyte lipid envelope(CLE)formation,such as Aloxe3,Alox12b,Tgm1,Gba and Asha1 in the skin tissues of newborn Sdr9c7^+/+and Sdr9c7^-/-mice,was examined by qPCR.(11)Expression of genes related to the control of keratinocyte cell differentiation and proliferation,such as K1,K5,K10,K14,K19,K6a,K6b and K16 in the skin epidermis of neonatal Sdr9c7^+/+and Sdr9c7^-/-mice,was examined by qPCR.(12)The apoptosis of keratinocyte cell in the skin epidermis of Sdr9c7^+/+and Sdr9c7^-/-mice at E18.5d,E20.5d and P0 was detected using flow cytometry analysis.(13)The protein expression levels of TARC in skin tissues and blood tissues of neonatal Sdr9c7^+/+and Sdr9c7^-/-mice were measured by Western blot.(14)The mRNA expression levels of TARC in skin tissues of neonatal Sdr9c7^+/+and Sdr9c7^-/-mice were detected by qPCR.Result:(1)Observations on the phenotype of newborn mice revealed that Sdr9c7^-/-neonates showed flushing and cyanotic skin 2 h after birth,and died within 6-8 h after birth.(2)Histological observation on Sdr9c7^+/+and Sdr9c7^-/-neonatal mice revealed that the stratum basale,stratum spinosum,stratum granulosum and stratum corneum were not abnormal in Sdr9c7^-/-neonatal mice,whereas the stratum corneum was thickened and did not form a'basket'structure.(3)Bodyweight monitoring and appearance photography were performed on 6 litters of neonatal mice every 1 h before lactation,and the results showed no significant weight loss in Sdr9c7^+/+and Sdr9c7^+/-neonatal mice at birth,but the body weight of Sdr9c7^-/-neonatal mice decreased by approximately 4%per hour after birth.(4)The skin permeability of Sdr9c7^+/+and Sdr9c7^-/-mice was tested using toluidine blue staining solution,and the results showed that the skin of Sdr9c7^+/+mice soaked with toluidine blue staining solution was basically not stained blue,while the skin of Sdr9c7^-/-mice was stained dark blue throughout the body.The results further confirmed that the Sdr9c7 knockout disrupted the epidermal barrier function.(5)Cornified cell envelope(CCE)in skin epidermis was prepared and subjected to ultrasound experiments,which showed that CCEs in the epidermis of Sdr9c7^-/-mice were more fragile and had increased brittleness.(6)The mRNA expression levels of genes related with CLE formation such as Aloxe3,Alox12b,Tgm1,Gba and Asha1,were examined using qPCR.The results showed that although Tgm1 mRNA levels were significantly increased in Sdr9c7^-/-mice compared with Sdr9c7^+/+mice,but there were no significant difference in mRNA expression levels of Aloxe3,Alox12b,Gba and Asha1 gene between Sdr9c7^+/+and Sdr9c7^-/-mice.It suggested that the expression of most genes involved in covalent binding did not change to compensate for the covalently bound?-OH-Cer in the skin of Sdr9c7^-/-mice,resulting in a failure of covalent binding and the inability of Sdr9c7^-/-mice to form a functional CLE.(7)The amounts of T cells,B cells,macrophages and dendritic cells in the skin of Sdr9c7^-/-mice did not differ those of Sdr9c7^+/+mice,indicating that the loss of Sdr9c7 gene did not cause inflammatory response in the skin.(8)The expression levels of Filaggrin,Involucrin and Ioricrin genes were significantly downregulated in Sdr9c7^-/-mice compared to newborn Sdr9c7^+/+mice.The downregulation of Involucrin,Loricrin and Filaggrin mRNA expression may be responsible for the increased fragility of the cornified cell envelope(CCE).(9)The expression levels of the keratinocyte cell differentiation marker protein genes as K1,K5,K10,K14,K19 and the keratinocyte cell proliferation marker protein genes as K6a,k6b,K16 mRNA were examined respectively using qPCR,and the results showed that K1,K5,K10,K14 were significantly reduced in Sdr9c7^-/-mouse skin,indicating that the deletion of Sdr9c7 gene might affect the differentiation of keratinocyte cell.(10)The apoptosis of keratinocyte cell in mouse skin epidermis at E18.5d,E20.5d and P0 was examined by flow cytometry analysis technique,and the results showed that the apoptosis of corneocyte cell in the epidermis of Sdr9c7^-/-mice was significantly increased at E18.5d,E20.5d and P0,indicating that the deletion of Sdr9c7 gene affects the apoptosis of keratinocyte cell.(11)Western blot,qPCR and flow analysis techniques were used to detect TARC expression in skin tissues of newborn mice respectively,and the results showed that TARC expression was significantly increased in the skin tissues of Sdr9c7^-/-mice.(12)The expression of thymus and activation-regulated chemokine(TARC)in blood tissues of newborn mice was examined by Western blot and flow cytometry analysis techniques respectively,and the results showed that TARC expression was significantly increased in blood tissues of Sdr9c7^-/-mice.Conclusion:The Sdr9c7 gene plays an important role in skin development.Sdr9c7 gene deletion disrupts the epidermal barrier function,causing dehydration and cyanosis in the skin of mice,and the Sdr9c7^-/-neonates died within 6-8 h after birth due to constant dehydration.Deletion of the Sdr9c7 gene caused the epidermal cornified cell envelope(CCE)to become more fragile and a non-functional corneocyte lipid envelope(CLE)formed.Sdr9c7 gene deletion affects differentiation and apoptosis of epidermal keratinocyte cell.Sdr9c7 gene deletion increased thymus and activation-regulated chemokine(TARC)expression in both skin tissue and blood tissue of Sdr9c7^-/-mice.