The Biology Characteristics Of Human Sweat Gland Cell And Human Sweat Gland Cell Derived Induced Pluripotent Stem Cell

Perspiration is one of the important functions of the skin,and the skin can maintain body temperature through perspiration.Sweat gland is a subsidiary organ of the skin and the executor of perspiration.Skin which has no sweat glands,such as scars,cannot perspire.The survival rate of large area and deep burned patient is increasing with the health care reform and the improvement of living level.The burned skin area of large area and deep burned patient even could reach up to 98%.The wound of injury skin will form the scar tissue after recovery,which does not contain sweat glands and loses the function of perspiration.It greatly affects a patient's quality of life.How to regenerate the sufficient amount of sweat gland cells to make the function of patient's skin perspiration repair is a clinical problem to be solved.For large area of the severely burned patients,it is impossible to obtain a large number of sweat gland cells.So the source of sweat gland cells is limited.What's more,sweat gland cells are somatic cells,it is hard to get a large scale passaged in vitro.This article is to get human sweat gland cell from the children's six-finger skin by surgical removal,and then make it to be iPS.This work lays the foundation for the way to get enough seeding cell for the sweat gland repair.Part I Human sweat gland cells extracted,cultured in vitro and identificationObjective:To get human sweat gland cells from the children's six-finger skin by surgical removal,culture in vitro and identify biological characteristics.Methods:Remove fat and connective tissue of childhood skin and cut into 1mm2 size.Then digested by type IV collagenase at 37 ? for 4 hours,the sweat glands were extracted under an inverted microscope.Dry affix the sweat glands to 6cm cell culture dish,culture sweat glands in 37??5%CO2 cell incubator with sweat gland cell culture medium.Cells grew out from the sweat glands after 2-3 days.The expression of sweat gland cell marker genes:CEA,CK14 and CK18 were detected by RT-PCR.The expression of marker proteins of sweat gland cells:CEA,CK14,and CK18 were detected by immunofluorescence.Results:(1)Sweat gland cells showed typical epithelial cell-like growth,tightly packed and similar flagstones-like.Cell boundary was obvious and nuclear was clear.(2)RT-PCR results showed that cells isolated and cultured expressed sweat gland cells marker genes CEA?CK14 and CK18.(3)Immunofluorescence staining technique results revealed that cells isolated and cultured expressed sweat gland cells marker proteins CEA?CK14 and CK18.Conclusion:We successfully established the system to extract sweat gland cells from children skin and culture sweat gland cells in vitro.At the same time,we complete the identification of the sweat gland cells.This work laid the foundation for further study of the sweat gland cells.Part ? Induced pluripotent stem cells derived from human sweat gland cells and their identificationObjective:Reprogram human sweat gland cell to induced pluripotent stem cell and identify its biological characteristics.Methods:After the sweat gland cells grew approximately 10 days,sweat gland cells were trypsinzed and seeded on 6cm cell culture dishes with a density of 1×105.The next day,the serum-free medium containing lent virus with four kinds of embryonic stem cell transcription factor genes was added to the sweat glands culture dishes.2-3 days later,digest and passage the cells into 6cm cell culture dish covered with trophoblastic cells.Incubated for about 10 days later,the clones which clonal and cell morphology identical to embryonic stem cell cloning were selected and seeded in 24-well plates paved with the trophoblastic cells.Cultured for about 10 days later,the clones which clonal and cell morphology identical to embryonic stem cell cloning were selected and seeded in 6cm cell culture dish paved with the trophoblastic cells.Whether the induced cells expressed genes:Oct4,Sox2,KLf4,c-Myc and Nanong was detected by RT-PCR.The expression of embryonic stem cell surface markers:Oct4,SSEA-3,SSEA-4,TRA-1-60 and TRA-1-81 were detected by immunofluorescence.Results:(1)Cell morphology changed after transfection with lentivirals,which was significant different from sweat gland cells.The clone morphology of iPS was similar to hESC,cell grew close,the boundary was not obvious,the nucleus-plasma ratio was high,the nucleolus was clear.There were a large number of cells metabolites around the clone;(2)RT-PCR results showed the iPS derived from the sweat glands cells expressed genes:Oct4,Sox2,KLf4,c-Myc and Nanong;(3)The immunofluorescence technique revealed the iPS derived from sweat gland cell expressed:Oct4,SSEA3,SSEA4,TRA-1-60 and TRA-1-81.Conclusion:After transfection of four factors,sweat gland cell could be reprogrammed and reversible differentiate into iPS which expressed marker genes and related molecules of hESC.