| BackgroundProstaglandin(PG)is a kind of biologically active substance composed of twenty-carbon unsaturated fatty acids.It is widely present in the body fluids and tissues of mammals,and has an important regulatory effect on multiple systems of the human body.At present,it is known that there are more than 20 different molecular structures of PG in the human body.According to the structure of carbon ring and side chain,they can be divided into 9 types,A to I.They are used in inducing a series of cell physiological activities and regulating cell proliferation,differentiation,apoptosis,etc.It plays a key role in female reproductive function,platelet aggregation,cardiovascular and digestive system balance.In addition,PG is also involved in the pathological process of inflammation,pain,pigmentation,itching,and tumors.Prostaglandin receptors belong to the family of transmembrane G protein-coupled receptors and are widely distributed.Each PG has its corresponding specific receptor.The prostaglandin receptors currently known to be expressed in mammals include the following types,which are PGD2 receptors(DP1,DP2)and the four subtypes of PGE2 receptors(EP1,EP2,EPS and EP4),PGF2α receptor(FP),PGI2 receptor(IP)and TXA2 receptor(TP).Keratinocytes(KC)differentiate from the ectoderm and are the main constituent cells of the epidermis,accounting for more than 80%of the epidermal cells,and can produce keratin during the differentiation process.According to the different morphological structures caused by cell differentiation,Keratinocytes can be divided into 5 layer from the buttom to the top.They are stratum basale,stratum spinosum,stratum granulosum,stratum lucidum and stratum corneum.KC can maintain the integrity and normal penetration function of the epidermis,and exert a physical barrier function through the keratin,mucopolysaccharide and other substances produced.KC can also regulate various functions of various cells in the epidermis by synthesizing a variety of autocrine and paracrine factors.At the same time,KC also participates in the body’s innate immunity and acquired immune response,and play an important role in the skin’s immune barrier.The PG in the skin is mainly produced by epidermal KC.The half-life of PG is very short.In the case of inflammation,trauma,ultraviolet radiation,etc.,PG synthesized by KC can be quickly released outside the cell,and bind to its neighboring membrane receptors in an autocrine or paracrine manner,and promote proliferates,differentiates,and apoptosis of KC through different signal pathways.Recent studies have shown that there are multiple PG receptors in skin melanocytes.In addition,latanoprost can promote melanocyte(MC)proliferation,tyrosinase activity and melanin synthesis,and has been used in the clinical treatment of vitiligo.Our previous studies have confirmed that PGE2 and PGF2α receptors are all positively expressed in human MC and cell lines.However,PGE2 and PGF2α receptors related research of KC are rare.In addition,the co-culture model of human KC and MC also suggests that KC play an important role in regulating function of MC and mediating skin pigmentation.To this end,this study used immunofluorescence double-labeled staining technology to conduct a preliminary study on the expression and localization of PGE2 and PGF2α receptors in normal human skin tissue KC.The results are now reported as follows.ObjectiveThe immunofluorescence double-labeling staining technique was used to label KC in normal human foreskin skin tissue,and the expression and localization of PGE2 and PGF2α receptors in KC were observed by laser scanning confocal microscope,and the effect of PG on the pathophysiology of KC was investigated.The regulatory significance of the process.Methods1.Normal human foreskin skin tissue is derived from specimens after circumcision in the Urology Department of the First Affiliated Hospital of Zhengzhou University.There was no organic disease and lesions in the skin tissues taken,and no medication history,from patients who do not have family history of skin diseases.Make the skin tissue into frozen sections for later use.2.The collected skin tissue was fixed,dehydrated,frozen,grouped,and then frozen for later use.Place the frozen sections in a 37℃ oven for 1 hour to remove residual OCT embedding agent After 2%Triton X-100 was ruptured at room temperature,10%goat serum blocking solution was added and incubated in a 37℃incubator.After 1 hour,aspirate the blocking solution,and add the primary antibody mouse anti-human keratin 10 antibody with a working concentration of 1:500,and the target primary antibody mixed working solution rabbit anti-human EP1,EP2,EP3,EP4,FP antibody.Incubate the processed sections in a humidified box at 37℃ in a constant temperature box,and in a refrigerator at 4℃ overnight.Wash away the primary antibody with PBS.Sort the slides,add fluorescent Alexa Fluor(?)488 and Alexa Fluor(?)594 secondary antibody working solutions(both at a concentration of 1:200)under dark conditions,and incubate them in a humid box at 37℃ for 1.5 hours.Wash off the secondary antibody with PBS,add DAPI dropwise in the dark,and incubate at room temperature for 10 minutes.DAPI was washed away with PBS,and the anti-fluorescence quencher was added to the coverslip for mounting.3.Observe the localization of PGE2 and PGF2α receptors in human skin tissue KC under a laser scanning confocal microscope.Results1.Expression and localization of of PGE2 receptors in KC of normal human foreskin skin tissueThe PGE2 receptors in KC of normal human foreskin skin tissue was stained with Alexa Fluor(?)594,which was red fluorescent.Observed by laser scanning confocal microscope,EP1,EP2,EP3,and EP4 receptors were all positively expressed,The positive staining is located in the cell membrane and cytoplasm of KC;KC is labeled with mouse anti-human keratin 10 primary antibody and stained with Alexa Fluor(?)488,showing green fluorescence,and positive staining is located in the cell membrane and cytoplasm;DAPI labeled cell nucleus,showing blue Fluorescence:The receptor proteins of EP1～EP4 are co-labeled with the keratin 10 labeled protein,and they show orange or yellow fluorescence.The above receptor proteins are expressed in EP2,EP3 and EP4,while the expression of EP1 is relatively weak.The expression sites are mainly in the stratum spinosum and stratum granulosum.2.Expression and localization of of PGF2α receptors in KC of normal human foreskin skin tissueThe PGF2α receptors in KC of normal human foreskin skin tissue was stained with Alexa Fluor(?)594,which showed red fluorescence.Observed by laser scanning confocal microscope,the FP receptors was positively expressed,and the positive staining was located in the KC cell membrane and Cytoplasm;KC is labeled with mouse anti-human keratin 10 primary antibody and stained with Alexa Fluor(?)488,showing green fluorescence,and positive staining is located in the cell membrane and cytoplasm;DAPI labeled cell nucleus,showing blue fluorescence;FP receptor protein and keratin All the 10 labeled proteins showed yellow fluorescence after co-labeling,and the expression site was obvious in the stratum spinosum.Conclusions1.There are PGE2 and PGF2α receptors in the KC cell membrane and cytoplasm of normal skin tissues.Among them,the expression of EP2,EP3,EP4 and FP is obvious,and EP1 is weak,and the expression sites are mainly in the stratum spinosum and stratum granulosum.2.KC can participate the pathophysiological regulation mechanism of inflammation,pigmentation,wound healing and other pathophysiological control mechanisms through PGE2 and PGF2α receptors related signal pathways. |
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