Proteomics Study On Vulva Lichen Sclerosus-associated Proteins And The Study On Correlated Factors With The Corium Lesion

Objective:Vulva Lichen sclerosus is a nonneoplasia epithelial disorders of vulva skin and mucosa that occurs in females at any age,especially the women in middle-aged and older-aged.It is characterized by depigmentation and leucismus in the skin tissues of vulva and perianal area and pathologically marked by epidermal atrophy,dermal collagen fiber hyaline degeneration and iso-substance.The presenting symptom of vulva lichen sclerosus is refractory pruritus vulvae which is obvious duing sleep.The treatment effectiveness is poor and the attack is recurrent.The lesion develops very chronically and leads to many bad outcomes,including scar in the vulva clitoris,conglutination and narrow of ostium vaginae as well as psychosexual disorder.The cause of vulva lichen sclerosus is not very clear.The pathogenesis might correlate with immunity,hormone,heredity,cell-cycled regulation and infection too,oxidative stress and local lesion.Autoimmunity acts a great role in all of the factors and holds the scholars’ s attentions of all over of the world.Some related genes and proteins were found with the progress of molecular biology research,but many truely mechanisms witch in the lichen were still unknown.Proteomics is a newly emerging experimental technology which research on the proteome from the overall perspective to analyze the intracellular protein construction and avtivty regularity dynamically.Proteomics can screen protein molecule that is specific for diseases by comparing the proteins between normal and impaired individuals.Proteomics can screen several proteins simultaneously by high-flux method and has reached to an unprecedented scale and speed in the research of the mechanisms of life activities.Though proteomics is a emerging technology,it has been a competent method to study the cellular proliferation,differentiation,abnormal transformation and tumor formation.In this study,Two-dimensional fluorescence difference gel electrophoresis(2D-DIGE)and matrix assisted laser desorption ionization time-of-flight(MALDI-TOF)mass spectrometry were used to identify the expressed proteins that significant differentially between the tissues of the vulva lichen sclerosus and the normal vulva skin.Then we validated and localized some differentially expressed proteins,and testified the consistency of the differential proteins by western blot and by immuno-histochemisty.Cell culture,primary cells proliferate 、 MTS 、immuno-cytochemistry and QRT-PCR were used to detect the effect of Galectin-7 on the collagen synthesized by the fibroblast in the normal vulva skin,and explore the correlated mechanisms underlying the dermal lesions.Methods:1、Total proteins extraction,quantification and purification:The tissue of vulva lichen sclerosus(n=6)and normal vulva(n=6)were collected.According to the protocols we uesd 2D Clean-up KitThe extracte the total proteins.The concentration of total proteins were assessed according to the directions of 2D Quant Kit.2 、Two-dimensionals difference gel electrophoresis and images analysis(2D-DIGE):Extracted total protein were processed on first dimensional immobilized pH gradients isoelectric focusing(IEF)after fluorescently labeling,and then processed on second dimensional SDS polyacrylamide gel electrophoresis after the balance of gels.We scanned the gels and then anylazed after electrophoresis.3 、 MALDI-TOF mass spectrometry and bioinformatic analysis:The samples of the gels were prepared and placed in the mass spectrometer for identification after spotting.Bioinformatics of the analyzed by selected proteins was analyzed by quering EMBL-EBI database.4 、Galectin-7,Prx4,Annexin A2 and Dermatopontin(DPT)expressions in the vulva lichen sclerosus(n=15)and normal vulva(n=10)were analyzed by immunohistochemistry streptavidin-perosidas(SP)method.5 、 Galectin-7,Prx4,Annexin A2 and DPT expressions in the vulva lichen sclerosus(n=15)and normal vulva(n=10)were analyzed by western blot.6、Fibroblasts in the normal vulva were separated,cultured and immunocytochemistry identifies the cell.7 、 The effect ofGalectin-7 on the proliferation of fibroblasts in the normal vulva was detected by MTS.8 、 The effect of Galectin-7 on the mRNA expressions of typeⅠ and type Ⅲprocollagen synthesized by fibroblasts in normal vulva were detected by Rt-PCR.Results:1、One hundred and fourteen significantly different protein points were cutted by 2D-DIGE,107 of which were identified.Ninty-nine differential proteins were aquired after removing the reduplicative proteins.40 proteins up-regulate in the VLS and 52 proteins down-regulate in the VLS.2 、 The function of biological and the localization of subcellular,witch 92 differential proteins were aquired by searching the EMBL-EBI database.The biological functions of differential proteins were various,which included apoptosis(25.01%),signal transduction(3.26%),metabolic enzyme(24.59%),transport(9.78%)and cytoskeleton(32.61%)as well as cell adhesion(4.35%).The subcellular localization analysis showed that 77 proteins were located in cytoplasm(63.7%),10 proteins were located in cell nucleus(10.87%)and the other 5 proteins were located in cell membrane(5.43%).3、The galectin-7,Prx4,Annexin A2 and DPT were expressed in the full thickness of the normal vulva and were located in the cytoplasm and nucleus.The major positive cells in the vulva lichen sclerosus were located in the cuticular layer and few of them were in the corium.The expression of Galectin-7 in the vulva lichen sclerosus were significantly higher than that in the normal vulva(P<0.05),whereas the expression of Anne A2,Prx4 and DPT in the vulva lichen sclerosus were significantly lower than that in the normal vulva(P<0.05),that were the same with the proteomics results.4、The picked protein level of the Annexin A2,Prx4 and DPT in the vulva lichen sclerosus was significantly lower than that in the normal vulva.The relative protein level of the Galectin-7 compare the vulva lichen sclerosus to the normal vulva was significantly higher.5、Galectin-7 can either improve or inhibit the proliferation of the fibroblasts in vitro.The effects of Galectin-7 on the proliferation of fibroblasts were changed with the concentration of Galectin-7.Galectin-7 could inhibit the proliferation of fibroblasts when the concentration is higher than 0.5μg/ml.6、The results of PCR showed that m RNA of TypeⅠ and Ⅲ procollagensynthesized by fibroblasts were both increased under the effect of Galectin-7 and this effect was positively correlated with the concentration of Galectin-7.The ratio of mRNA level in tpyeⅠprocollagen to that in type Ⅲ procollagen is negtively correlated with the concentration of Galectin-7.Conclusions : 1 、 Ninty-two differential proteins were screend using proteomics technology.Forty differential proteins were up-regulated and fifty-two differential proteins were down-regulated in the vulva lichen sclerosus.2 、 The expression of Galectin-7 compared the vulva lichen sclerosus to the normal vulva skin was significantly higher,whereas the expression of Annex A2,Prx4 and DPT were significantly lower in the vulva lichen sclerosus than that in the normal vulva,which were the same to the proreomic.3 、 Galectin-7 could either enhance or inhibit the proliferation of vulva fibroblasts.It enhanced the proliferation of vulva fibroblasts when the concentration of Galectin-7 was lower than 0.5μg/ml and inhibited the proliferation of vulva fibroblasts when the concentration was higher than 0.5μg/ml.4、Galectin-7 could increase the collagen protein synthesized by vulva skin fibroblasts and reduce the ratio of type Ⅰcollagen protein to typeⅢcollagen protein.As the Galectin-7concentration increased,the synthesis of collagen protein was increased whereas the the ratio of type Ⅰcollagen protein to typeⅢcollagen protein was reduced.