Isolation And Culture Of Human Foreskin Fibroblast Cell And Expression Patterns Of MICA~*001、~*008Genes In This Cell Line

A member of a novel family of the human major histocompatibility complex (MHC) class I genes termed MIC (MHC class I chain-related genes), MICA(MHC class I chain A), has been recently reported more than70highly polymorphic alleles. MICA encoded molecules deliver activating signals through the NKG2D receptor expressed on the surface of natural killer. Lots of studies have investigated associations between MICA polymorphism and susceptibility to diseases such as organ transplantation, tumors, viral infections and so on. Chlamydia trachomatis (Ct) is an obligate intracellular bacterium with a unique life cycle. It is the leading cause of preventable infectious blindness and sexually transmitted disease, which are always the sequelae of persistent Ct infection. Ct affect the biological behavior of the host cells in many aspects in a way that the mechanism remains unclear. Our previous studies have found that the MICA*001and MICA*008may be involved in Ct infection. Primary human foreskin fibroblast (HFF) is one widely used type of human fibroblast, which express little MICA on the protein level, for it’s relatively easier to be isolated and cultured. Now there are few researches on whether MICA are involved in immune control of Ct infection and the possible role of MICA in the molecular mechanisms underlying chlamydial evasion of host immune recognition.Objective:(1) To isolate and culture primary human foreskin fibroblast cells (HFF);(2) To infect HFF with Ct;(3) Transient transfection of exogenous MICA*001and*008gene into HFF respectively.Methods:(1) Isolation and culture of HFF was performed with attachment of tissue pieces. The HFF culture conditions in vivo were37℃,5%CO2and DMEM medium with15%fetal bovine serum, and the cell morphology was recorded during its growth process.(2) Immunocytochemical method was carried out for detection of vimentin in HFF, a human fibroblast-specific marker, to make HFF initially identified.(3) The detection of bacteria and mycoplasma was performed to determine whether there was microbial contamination.(4) The growth curve of HFF’s6th and12th generation was drawn, respectively, and the chromosome number and structure of the8th generation of HFF were analysed to identify the proliferation capacity and stability of the cultured HFF.(5) Giemsa staining of HFF was used to determine whether Ct infected HFF successfully when HFF had been infected by Ct for40h.(6) Lipofactamine was used to make retrovirus Plasmid pLXSN, pLXSN-MICA*001and pLXSN-MICA*008transfected into PA317packaging cells, respectively. The cell culture mediums were collected, which contained the complete virus particles of PA317-pLXSN, PA317-pLXSN-MICA*001and PA317-pLXSN-MICA*008, respectively.(7) These three culture mediums containing the complete virus particles were used to infect HFF, then after48h, total proteins of HFF were extracted. Western blot assay was carried out to examine the expression levels of the proteins encoded by pLXSN-MICA*001andpLXSN-MICA*008.Results:(1) HFF was successfully isolated from tissue pieces of human foreskin and HFF could be continuously passaged with good proliferation ability and chromosomal stability.(2) Inclusion bodies of Ct could be observed after Giemsa staining HFF, which showed that Ct had successfully infected HFF.(3) MICA*001and MICA*008gene were successfully transfected into HFF by retroviral system.(4) MICA protein expression in HFF-pLXSN-MICA*001and HFF-pLXSN-MICA*008was detected by western blot.Conclusion:(1) HFF is isolated and cultured successfully.(2) Ct can infect HFF.(3)Exogenous MICA*001and MICA*008genes were successfully expressed in HFF, respectively.