The Effects Of CXCL4 Monoclonal Antibody And S100A3 On Mouse Hair Growth

Hair loss(alopecia)is a common disease.Although hair disorders are not life threatening,their profound impact on patient's social life and psychological well being is undeniable.To date,there have not been effective strategies for treating alopicea.We proposed that homeostatically regulated genes during hair regeneration in hair growth based on the cyclicity of hair growth in mammal.In this study,we used DNA microarray hybridization to analyze sequential gene expression patterns in mouse skin following hair cycle synchronization by depilation.Messenger RNA levels in mouse skin at various times after depilation were compared with those prior to depilation(resting phase).We focused on some functional genes that participate in hair growth,and prepare the genetically engineered protein and antibodies.We investigated the effect and underlying mechanism of action on mouse hair growth using these potein and antibodies.The study provides sciectific basis for developing antibody drugs of alopecia treatment,and has important significance and proctical value.The main results were listed as follows:1.We used DNA microarrays to profile m RNA expression in mouse back skin from four representative time points,and analyzed the sequential gene expression changes after depilation compared with telogen.According to their expression patterns,the changed genes were categorized into six clusters: Cluster 1,Cluster 2,Cluster 3,Cluster 4,Cluster 5,and Cluster 6.In Cluster 1,we found the expression of chemokine CXCL4(referred to as CXCL4)was significantly downregulated during hair growth,thereafter recovered.And in Cluster 4,S100A3 was significantly upregulated during hair growth,thereafer recovered.These results implied the involvement of CXCL4 and S100A3 in hair regeneration.Based on this clue,we primarily identified the m RNA expression changes of CXCL4 and S100A3 in hair regeneration using fluorescence quantitative PCR.2.We study the effect and underlying mechanism of CXCL4 monoclonal antibody(referred to as CXCL4 mab)on mouse hiar growth by blockade of CXCL4 activity with CXCL4 mab.The depilated mice in the test group were subcutaneously injected with 1 mg/kg CXCL4 mab,and the control group with the rat isotype Ig G.At 10 days after depilation,there were some hairs in the back skin in CXCL4 mab treated group,while there was no hair in control group.We observed the number of hair follicles in deep dermal areas of CXCL4 mab treated group was greater than that in the control group.And the hair length of CXCL4 mab treated mice was significantly longer than that of the contol group at 14 and 18 days after depilation.Moreover,we observed vascular branches of CXCL4 mab treated mice skin were more than that of the control group.These results indicated CXCL4 mab accelerated telogen to anagen transition,promoted hair regeneration and delayed hair follicles degeneration.3.The study discussed the hair-promoting effect mechanism of CXCL4 mab at cell proliferation,apoptosis,RNA and protein levels.The results suggested that CXCL4 mab significantly upregulated the transcription levels of several hair growth-related genes,including ?-catenin,Lef1,Wnt10 b,Ccnd2,Bmp4,Gli-2,and Vcan.The immunohistochemistry analysis indicated CXCL4 mab induced protein expression of ?-catenin and Shh,which are essential for the development and maintenance of hairs not only in embryos,but also in adults.And CXCL4 mab increased the levels of proliferation-related protein PCNA and Bcl-2 during anagen,while it reduced the expression of pro-apoptotic protein Bax and cleaved caspase-3during catagen.These results indicated that CXCL4 mab accelerated telogen to anagen transition and attenuated apoptosis of hair follicle,prolonged the anagen,and then promoted hair growth.4.We prepared genetically engineered protein S100A3 and rabbit anti-S100A3 polycolonal antibody.Calcium-binding protein S100A3 as a member of S100 protein family,shows a remarkably narrow tissue-and cell-specific expression pattern.It highly expressed in hair follicle cells and some astrocytomas.Due to high proliferation rates of both cell types,S100A3 was supposed to be involved in cell cycle progression.In the study,RNA was isolated from mouse skin.S100a3 was cloned and inserted into the prokaryotic expression vector.Then the S100A3 protein was expressed in E.coli and purified by nickel column affinity,cleavage of thrombin,and anion exchange chromtography.And high titer of rabbit polyclonal antibody against S100A3 was prepared.5.The effect of rabbit anti-mouse S100A3 polycolonal antibody on mouse hair growth was investigated.The results indicated S100A3 promoted hair growth.The expression of S100A3 depended on hair cycle.It highly expressed in hair shaft,and less in sebaceous gland.The S100A3 immuno-neutralization delayed hair follicels entry into anagen from telogen and inhibited hair shaft elongation.We also observed the number of hair follicles in deep dermal areas of S100A3 polyclonal antibody was less than that in the control group.In addition,the result of q RT-PCR indicated S100A3 neutralization significantly downregulated the transcription levels of hair growth related genes.