| ObjectiveStem cells have been regarded as undifferentiated cells capable of proliferation , self - renewal, production of a large number of differentiated progeny, and regeneration of tissue. Regardless of stem cell origin, most cell turnover within a tissue is likely accomplished by tissue specific, transiently - amplifying cells periodically recruited from the stem compartment. That tissue - specific stem cells reside in certain adult tissue has been clearly documented, yet their paucity in parent tissue , heterogeneity, and technical difficulties in identifying them . Such adult tissue stem cells are responsible for regenerating damaged tissue and maintaining tissue homeostasis, for example physiological replenishment of skin and blood cells. In some cases such as hematopoiesis, the stem cells can be highly enriched and markers that distinguish these cells have been well characterized . In other cases in which the extensive regenerative capacity of stem cells hasn t been documented, such as in the airway, stem cell markers still remain to be identified. Most stem cells enrichment protocols rely on the fluorescence -activated cell sorter (FACS) and use sets of antibodies to cell surface proteins. An alternative method for identifying both murine and human hematopoietic stem cells was recently developed and is based on the efflux of the DNA binding dye, Hoechst33342. This small cell population , termed the side population(SP) ,is i-dentified by a characteristic pattern of fluorescence after staining with Ho-echst33342 detected in both far red and blue emission channels. The SP has been isolated from both mouse and human bone marrow. In addition to enriching for HSC, the SP protocol can be used to identify stem cells in other tissues including skeletal muscle and epidermis. The ATP - binding cassette transporter AB-CG2/Bcrpl mediates the SP phenotype.We constructed a tracheal injury model in Wister rats treated with 5 - FU. Using light microscope, electronic microscope, immunohistochemistry, Ho-echst3332 staining and RT - PCR methods investigated the process of regeneration to localize the tracheal stem cells.Materials and methods(-)5 - FU treatment and recovery;2weeks Wistar rats provided by the animal center of the China Medical University . The animals were sacrificed by cervical vertebra dearticulation; tracheas were excised aseptically, and then cut into 3 - 4mm wide rings, cilia swinged well under inverted microscope, divided into two groups of control and treatment.Control: the tracheal rings cultured in F -12 medium with the same content of Saline with 5% C02, extracted the rings at 3, 6, 9, 12, 24,48hours, fixed for LM and EM.Treatment: the trachea rings cultured in F -12 medium with the same content of 5 - FU, with 5% C02 extracted the rings at 3,6,9,12hours, 12 hours later then exchanged the fresh F - 12 culture medium , extracted the rings at 3,6, 9,12,24,48hours,fixed for LM and EM.1.The tissue fixed by 4FG, LM samples were dehydrated, and embedded in paraffin; Tissues were sectioned into 4um thick slides stained with haematox-ylin and eosin.2. TEM samples were fixed in 2.5% glutaral in PBS, postfixes in 1% osmium tetroxide, dehydrated embedded in Epon 812, ultrathin sections for TEM.3. Analysis by immunohistochemistry measure monoclonal antibodies a-gainst the proliferating cell nuclear antigen ( PCNA) ; paraffin embedded sections , stained by DAB . PBS replaced the antigens as negative control group.() Preparation of the smear of the trachea epithelium1. Tracheal rings after 5 - FU treated for 12hours were subjected to mechanical to obtain epithelial cells then incubated in the F - 12. Stained with trypan -blue to examine the vitality of the cells.2. Hoechst 33342 staining:Epithelial cells smear of 5 -FU treated trachealrings were stained with 5ug/ml Hoechst 33342 (Sigma) for 30minutes then observed under the 01ympasBX51 fluorescence contrast microscope.3. Indirect immunofluorescence with PCNA.4. RT - PCR analysis. RNA was prepared according t... |
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