Screening Of Molecular Markers For Epidermal Stem Cells In Mouse By Double Immunofluorescence Labeling Technique

Background:The skin is the largest organ in human body, which is constantly renewed throughout life. It Plays an important role in steady of intracellular environment and wound healing after injury. The epidermal stem cell is the most important of all. Epidermal stem sells are tumor initiating cells and gene therapy targets, and they play an important role in injury recovering and homeostasis. So epidermal stem sells have becoming the hot spot in stem cells research domain. The fundamental problem is how to isolate, purify, culture and identify epidermal stem sells from skin tissues. But until now, there is no specific molecular markers which were accepted by authority. Therefore, we must confirm some reliable molecular markers before separation of epidermal stem cells. A reliable way to identify the stem cells and their location in the stem cell niche is to take advantage of the label-retaining cell(LRC) approach. We can obtain the LRCs using 5-bromodeoxyuridine (BrdU) as tracer, which compete with the endogenous thymidylic aicd. Through subcutaneous or intraperitoneal injection method, BrdU mix with a DNA in the S period (DNA synthesis period) and retain the DNA synthesis label after a prolonged chase period. The stem cells retain the label after a long chase due to their slow-cycling, quiescent nature, so these LRCs which were detected by the specific BrdU antibody are thought represent the epidermal stem cell subpopulation.Objective:To screen the reliable molecular markers for epidermal stem cells in mouse by using double immunofluorescence labeling technique between the label-retaining cells(LRCs) and some molecular markers. Methods:1,5-day-old (P5) KUNMING mice were intraperitoneally injected on alternate days with BrdU twice daily, at 9 a.m. and 4 p.m. for 7 consecutive days.2,The tissue of the mice were harvested and sliced after 7days,30days,60days,90days. The paraffin section were stained by the monoclonal antibodies of anti-Brdu.3,The frozen section after 90days were separately stained with polyclonal antibodies of anti-β1 integrin,P63,CD34 and monoclonal antibodies of anti-Brdu.4,Observe under the fluoroscope (200×).The ten microscopic fields were selected in random in every group, the images were captured and merged to count the percentage of the positive cells.Results:1,After 90 days, a fewer of label-retaining cells are found in a region of the outer root sheath that lies adjacent to the insertion of the arrector pili muscle, known as the bulge region.2,The percentage of double immunofluorescence cytochemical stained positive cells ofβ1 integrin to BrdU in allβ1-positive cells was 81%,which in all BdrU-positive cells was 94%; The percentage of double immunofluorescence cytochemical stained positive cells of P63 to BrdU in all P63-positive cells was 65%,which in all BdrU-positive cells was 86%; The percentage of double immunofluorescence cytochemical stained positive cells of CD34 to BrdU in all CD34-positive cells was 30%,which in all BdrU-positive cells was 62%.Conclusion:β1 integrin is the best molecular marker for epidermal stem cells in these molecular markers. The fuction of P63 is the second.