Expression Of HSulf-1 Gene In Psoriasis And HaCaT Cells And Construction Of HSulf-1 Over Expression Lentiviral Vector

Background: Psoriasis is a chronic recurrent disease associated with many factors,such as immunity,inflammation,cell proliferation and apoptosis,angiogenesis and so on.The exact pathogenesis is unknown.The main features of histopathology are keratinocyte proliferation and angiogenesis.At present,there is a certain relationship between the incidence of psoriasis and hypoxia.In psoriasis,keratinocyte proliferation may contribute to the increase in oxygen consumption,thickening of the epidermis may also lead to increased oxygen diffusion distance and reduce oxygen supply,resulting in local hypoxia.Hypoxia inducible factor 1-alpha(HIF-1α)is a key factor in hypoxic microenvironment.It is known to be involved in the development of psoriasis and is highly expressed in psoriasis and human immortalized keratinocytes(Ha Ca T cells).Human sulfatase 1(HSulf-1)is a novel tumor suppressor gene found in recent years.It is present on the cell surface and is expressed in normal tissues,and its expression is decreased or absent in most malignant tumors.Its overexpression can lead to a relative weakening of the ability of tumor cells to proliferate,migrate or invade.It has been reported that HSulf-1 is a downstream target gene of HIF-1α,and there is a hypoxia response element(HRE)site capable of binding to HIF-1α at the promoter upstream of HSulf-1 transcription initiation.Hypoxia may cause HSulf-1 abnormal activation to decrease,leading to changes in downstream signaling pathways,cell invasion and metastasis.Both psoriasis and malignant tumors are characterized by local hypoxia and cell proliferation,We guessed that HSulf-1 may play an important role in the development of psoriasis.The aim of this study was to investigate the expression of HSulf-1 and HIF-1α in psoriatic lesions,normal skin lesions and the human immortalized keratinocyte in normoxia or hypoxia.Then we will construct the HSulf-1 gene overexpression vector to study of its relationship with keratinocyte proliferation in vitro and in vivo.Objectives: To investigate the expression of HSulf-1 and HIF-1α in psoriatic lesions,normal skin lesions and in the human eukaryotic keratinocyte in normoxia or hypoxia.And construction of HSulf-1 gene overexpression vector to further study of its relationship with keratinocyte proliferation in vitro and in vivo.Methods:Part one:(1)The immunohistochemical staining of HSulf-1 and HIF-1α antibody was used to detect the skin tissue of psoriatic lesions and normal human skin.The number of positive cells was observed under high magnification.(2)Ha Ca T cells were cultured and added with different concentrations of Co Cl2(concentration of 0μmol/L,100μmol/L,300μmol/L,600μmol/L)for 24 hours,collect the total protein.HSulf-1 and HIF-1 alpha protein expression were detected by Western blotting analysis.Part two:The HSulf-1 protein was inserted into adenovirus genome to generate recombinant adenovirus p CDH-HSulf-1.The overexpression vector of HSulf-1 protein in Ha Ca T cells was detected by Western blotting analysis.Results:Part one:(1)HSulf-1 was low expression in psoriatic lesions and high expression in normal tissues.HIF-1 alpha was high expression in psoriatic lesions and low expression in normal tissues.(2)Compared with normoxia,HSulf-1 protein was low expressed and it was negatively correlated with the expression of HIF-1 alpha protein in hypoxic condition.Part two:(1)Recombinant plasmid p CDH-HSulf-1 was successfully constructed by Nhel I and Bam HI double digestion PCR and DNA sequencing.(2)Lentiviral packaging of p CDH-HSulf-1 plasmid,showed green GFP fluorescence.(3)Compared with the control group and the blank group,the expression level of HSulf-1 protein in HSulf-1 overexpression group was significantly increased(P<0.05).The stable overexpression cell lines was successfully screened.Conclusion: HSulf-1 was low expression in psoriatic lesions and high expression in normal tissues.HSulf-1 is low expression in Ha Ca T cells under hypoxia,which is consistent with the expression of tumor cells or tumor tissues.The overexpression of HSulf-1 can inhibit the proliferation of tumor cells.We hypothesize that overexpression of HSulf-1 can inhibit the proliferation of keratinocytes.We construct the overexpression vector of HSulf-1 and screen the stable overexpression cell lines that it is for further to study the effects of keratinocyte cell proliferation in vivo and in vitro.