| With the development of cellular and molecular technique , scientists have discovered several stem cells, such as embryonic stem cell, neural stem cell, hemopoietic stem cell , marrow mesenchymal stem cell. And hemopoietic stem cell has been partly used in clinical . Now stem cell biology has been the focus of peopled attention. A stem cell can be defined as any cell that retains a high capacity for self - renewal throughout adult life and usually be considered to have the ability to produce daughters that undergo terminal differentiation. Stem cells can provide cell source for tissue regeneration medicine.Because of its anatomical and functional complexity, the stem cell research for the respiratory system has been developing rather slowly . If a population of multipotent stem cell exists in respiratory system, its location and function are well characterized, it is important for the use of stem cell for transplantation as a replacement of lost or diseased parts of the organ . It is also be helpful to interpret the histologenous of lung cancer and wound - repair process.We developed an in vitro injury model of human bronchial epithelium induced by fluorouracil(5 - FU) ,then observed the wound - repair process in situ and the location of bronchial stem cell by LM, TEM, AB - PAS and immunohis-tochemistry ( SP methods ). This may be the basis of dissociation and purification of stem cell. Stem cells show reduced staining with a mitochondrial fluorescent dye - Rhodaminel23, and this characteristic is involved in P - glycoprotein, a transmembrane efflux pump responsible for multidrug resistance in tumor cells. To analyze the characteristic of bronchial stem cell , we examine the condition of Rhodamine123 staining and the expression of P - glycoprotein in normal and injurious bronchial epithelium by immunofluorescence, flow cytometric method and Western - blotting. RT - PCR was applied to detection of P - glycoprotein mR-NA.Methods1. Human bronchial rings were dissociated from the uninvolved parts obtained from patients who underwent lung cancer operation and cultured on Ham F12 medium. We developed an in vitro injury model of human bronchial epithelium induced by 5 -FU. The bronchial rings were divided into two groups; the treated group were put into Ham's F12 culture medium with the Concentration of 5 - FU 12.5mg/ml . The culture dishes were incubated at 37 C. in a humidified ,5% C02 atmosphere. Then exchanged the fresh Ham's F12 culture medium after 12 hours, extracted two rings respectively at 0,3,6,9,12,24,48,72hours after exchanging medium , fixed separately for LM and TEM. The culture medium was replaced every 24 hours. We applied the same volume of saline taking place of 5 - FU as the controlled group. The other conditions were same as the treated group.2. The wound 梤epair process were dynamically observed under LM, TEM and AB - PAS staining. PCNA (proliferating cell nuclear antigen) , B1- inte-grin and Cytokeratinl9 ( CK - 19) expression levels were analyzed by immuno-histochemistry ( SP methods). The experiment procedure accorded to the KIT introduction. Using positive control from INC. PBS replaced the first antibody as negative group. Result determination: positive cell were buffy, negative cell were not coloured. PCNA is nuclear positive . B1 - integrin is positive expressed at cell membrane and CK -19 positive located in cytoplasm.3. Hoechst33342 staining: Human bronchial epithelium were stripped under microscope and spread on the smear. Hoechst33342 were dropped on the smear and incubated at 37C ,30 minutes. Hoechst33342 staining negative cells could be seen under fluorescent ultraviolet light excitation and bright visual field.4. The normal and injurious bronchial epithelial cells were obtained by enzymatic digestion ( Type XIV protease , Sigma Chemical Co. ) and analyzed by flow cytometry. 1) PI staining to identify the percentage of cells in different cell phase . 2) Rhodaminel23 staining in live cells were to contrast the percentageof negative cells in two gro...
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