Transdifferentiation Of Hair Follicle Stem Cells Induced By Stroma Of Limbus Corneae

Hair follicle stem cells (HSCs) is a bond of immature cells which have features as follows: slow-division, high-generation and self-renewalless. The clonality of these cells is big. These cells can generate into transit amplifying cells, repair the tissue, and maintain the self-renewal under the condition of stimulation or injure. Oshima confirmed that the hair follicle stem cells located in the bulge by euphenics, and informed that the stem cells in the regional of bulge can also padding the renewal of the epiderm and glandulae sebaceae. Recently, the research of the hair follicle and it's cycle developed fast, the location of HSCs is getting clear day after day, and the sorting of HSC in vitro also began.The repair of damaged cornea is limitted by source and repellion, so some researchers change the direction to transdifferenriation to obtain the homobody of cornea. At present, this research has got some heartening progress.We induced the HSC through stroma of limbus corneae, and studied the possibility of transdifferentiation from HSCs to corneal epithelioid cells. The main methods and results are as follows:①The tissue location of HSCsWe used CD34,α6-integrin to mark the HSCs by immunohistochemistry(IHC), and detected their positive expression in nearly all the outer root-sheath(ORS), especially hair follicle bulge region. Our results reminded us that HSCs are mainly located in the region of bulge.②Cultivation and indentification of HSCsWe got the chap skin of SD rats on the condition of sterilities, and cleaned them with D-Hanks, then isolated and cultured the tissue of hair follicle bulge. And changed the medium when found the cells'creeption, about every 1～2 days.We passaged the cultured cells as their 80% confluence, and detected CD34,α6-integrin and K12 by immunocytochemistry(ICC). CD34,α6-integrin both expressed positively, but K12 was negative.We concluded that we cultured the HSCs sucessfully, and it's pure without corneal epithelioid-like cells.③The tissue location,cultivation and identification of corneal epithelioid cells. We used K12 to mark the corneal epithelioid cells by immunohistochemistry (IHC), and detected its positive expression in the outer layer of epithelium corneae.We got the eye ball of SD rats on the condition of sterilities, and cleaned them with D-Hanks, then isolated and cultured the cornea. And changed the medium when found the cells'creeption, about every 1～2 days.We detected K12 by immunocytochemistry (ICC).④Induction of HSCs and identification of the cells after transdifferentiationWe induced the HSCs with the condition medium(CM) which was prepared by ourselves. Then detected K12 factor in those cells by ICC,RT-PCR and flow cytometry(FCM).The results were as follows:K12 expressed positively in the cells after induced, its mRNA was also present in these cells, and a considerable rate of the K12-positive cells was also observed by FCM.Induced by homogenate of limbus corneae, hair follicle cells can differentiate into corneal epithelium-like cells in vitro. And our research offers new way for clinical treatment.