Expression And Significance Of Oct3/4 And Bcl-2 In Process Of The Proliferation And Differentiation Of Tracheal Stem Cells

ObjectiveStem cells have been regarded as undifferentiated cells capable of proliferation , self - renewal, production of a large number of differentiated progeny, according to histological origin of stem cells,it contains embryonic stem cells and adult stem ceUs. That tissue - specific stem cells reside in certain adult tissue has been clearly documented, such adult stem cells are responsible for regenerating damaged tissue and maintaining tissue homeostasis . Why stem cells possess multidifferentiation, maintain undifferentiated state? The mechanisms are not clear. In the researches of embryonic stem cells, researchers find Oct3/4 plays an important role in maintaining the undifferentiated state of embryonic stem cells . Oct3/4 is the transcription factor of POU family, it expresses in embryonic stem cells ,but not in differentiated cells. Bcl -2 is a classic anti -apoptosis factor, it can suppress the apoptosis of proliferative cells and prolong the survival time of non - proliferative cells. Stem cells could proliferation and differentiation in vitro. If we control the mechanisms of undifferentiated state of stem cells, we could make stem cells maintaining undifferentiated state in order to solve the insufficiency of donators in cellular transplant. Our laboratory has completed the localization, enriching and characteristic analysis of tracheal stem cells. In order to investigate the multi - differentiated potency and undifferentiated state of tracheal stem cells,we constructed a tracheal injury model in Wistar rats and human beings treated with 5 - FU. Using light microscope, electronic microscope,immunohistochemistry and immunofluorescence methods observing the expression of Oct3/4 and Bel -2 in tracheal stem cells and investigating the significances.Materials and methods1. Tissue samples1. 1 5 - FU treatment and recovery:250g Wistar rats provided by the animal center of the China Medical University . The animals were sacrificed by intraperitoneal injection of 10% Chloral Hydrate 0.4ml/100g ,tracheas were excised aseptically, and then cut into 3 -4mm wide rings, cilia swinged well under inverted microscope. The tracheal rings cultured in DMEM/F12 medium with 5 -FU,with 5% CO2 for 12 hours, then exchange the fresh DMEM/F12 culture medium, extracted the rings at 0% 3%6%9X12%24%48 J2 hours,fixed for LM. Take 0x3x9x24x48 hours and normal tracheal tissues to obtain epithelial cells . Stored in - 10X1.1. 2 An in vitro injury model of human bronchial epithelium induced by 5 -FU:Human bronchial epithelial were dissociated from the uninvolved parts obtained from patients who underwent lung cancer operation and cultured in DMEM/F12 medium for 12 hours then exchanged the fresh DMEM/F12 medium, extracted the tissues at 0%3x9%12%24s48 hours,fixed for LM.2. Methods2. 1 HE staining.2.2 Immunohistochemistry for Oct3/4Immunohistochemistry staining was performed using a Ultro - sensitive S -P kit according to the instruction provided by the manufacturer. Color was developed using DAB/H2O solution.2. 3 Indirect immunofluorescence double staining for ABCG2 and Bel -2Introduction two - step methods, the first antibody was ABCG2 (1:100) , IgG marked by TRITC;the second antibody was Bel -2( 1;100) ,IgG marked by FITC. DAPI after staind cell nuclei.2.4 Western blotting detects the expression of Oct3/4 and Bel -2Extracted total protein of epithelial cells. Antibodies were Oct3/4 and Bel -2(1;400) ,DAB coloration . (3 - actin acts as intra - control.ResultNormal trachea epithelial is pseudostratified columnar ciliated epithelium, differentiated cells didn' t express Oct3/4, Bel - 2 expressed weakly;after 5 -FU treated for 12 hours, the whole tracheal epithelium desquamated with trifle naked — nucleus — like cells nailing just above the basement membrane alternation , both Oct3/4 and Bel - 2 started to express obviously;3 - 6 hours after removing 5 - FU, extreme squamous epithelium became to flat epithelium with a densely staining nucleus;9 - 12 hours afer removing 5 - FU, flat epithelium gradually differentiated into cuboidal epithelium and the number of cells increased, during this time the expression of Oct3/4 and Bel - 2 offered an ascendant tendency, both of them reached the climax of their expressions at 12 hours;after 24 hours there could be seen the cilia under light microscope, the expression of Oct3/4 decedented obviously;48 hours later, we observed that the tracheal rings were completely covered with pseudostratified columnar ciliated epithelium, only a few sporadic cells expressed Oct3/4. The expression of Bel -2 was decreased after 12 hours, compared with Oct3/4, the decency was smoothly ,until 48 hours there remained have little expression of Bel -2.The results of Western blotting were coincident with immunohistochemistry and immunofluorescence assays.ConclusionTracheal stem cells contain Oct3/4. At the early stage of tracheal stem cells Oct3/4 started to express, along with the differentiation of stem cells the expression of Oct3/4 gradually decreased. It suggested that Oct3/4 play an important role in maintaining undifferentiated stage of tracheal stem cells.Bel - 2 began to express when tracheal stem cells started to proliferate, a-long with differentiation the expression of Bel - 2 decreased. It suggested thatBel - 2 participate in maintaining the homeostasis of tracheal stem cells and keeping the undifferentiated stage of tracheal stem cells.