Study On The Location Of Tracheal Stem Cells And The Dynamic Change Of Shh In The Injury-repair Process Of Rat's Trachea In Vivo

IntroductionA stem cell can be defined as any proliferated cell which retains a high capacity for self - renewal and can give birth to high differentiated daughter cells in determinate condition. Stem cells can be divided into two groups: embryonic stem cells and adult stem cells. Scientists have discovered several adult stem cells, such as hemopoietic stem cell, bone marrow mesenchymal stem cell, neural stem cell, muscle stem cell, cornea stem cell, et al. Now stem cells have been used for the treatment of hematology, heart, hepatic, neural diseases and et al. Because the tracheal stem cell has no special superficial markers and there are no suitable models for the study of tracheal stem cell in vivo , so the process of proliferation and differentiation about tracheal stem cell is still unknown.We developed an injury - repair model of rat tracheal epithelium induced by fluorouracil (5 - FU) in vivo. 5 - FU belongs to anti - pyrimidine drugs , can inhibit the DNA synthesis through blocking thymine nucleotide synthetase. It leads to cellular degenertion and necrosis in proliferative cell of every phase, especially in S phase. But it was not sensitive to cells in G0 phase. So it is suitable to study stem cell which is in G0 phase. Hoechst33342 belongs to living -cell fluorescent dye of Bisbenzimid which has high permeability to the cell membrane , it can embed in DNA and can sent out two kinds of fluorescence, 405nm and 675nm, after being stimulated by ultraviolet light. We can observe Ho-echst33342 positive cells which sent off bright blue fluorescence under fluores-cence microscope. When analyzed by flow cytometer, the cells which show weak or no staining of fluorescent dye have high activity of stem cells and are called side population (SP) cells. We observed the injury -repair process in situ and the location of tracheal stem cell by LM, fluorescence staining and immunohisto-chemistry(SP methods). ABCG2/Bcrpl belongs to the family of ABC transport protein, it is high expressed in different source of SP cells. The cells from mice skeletal muscle and rhesus bone marrow all expressed ABCG2/ Bcrpl. So AB-CG2/ Bcrpl can be regarded as the marker of SP cells. And it is related with the characteristics of stem cells which show reduced or no staining with a fluorescent dye - Hoechst33342. We study on the expression of ABCG2 in the levels of protein and mRNA by immunofluorescence and RT - PCR. The proliferation and differentiation of stem cells in vivo are regulated by many factors including endogenous and ectogenous regulating. Shh is a member of Hh signal conducting pathway family and belongs to endogenous regulation. It can control the developing pattern of cells and procees signal conducting by continue inhibition of each other. It can lead to serious pulmonary and bronchial defection if loss of the function of Shh during embryonic period. But Shh isn t expressed in the adult lung. Shh has important function on injury - repair process of some constitutions and organs, such as in that of cornea, lens and bone. And there are no expression in there normal tissues. We observed the change of Shh signal conducting pathway during the process of proliferation and differentiation of tracheal stem cells.Methods1. The establishment of injury - repair models of rat trachea in vivo: The rat was anesthetized by 10% chloral hydrate then the trachea was exposed. The tra-chea was cut in the distal part. The rats were divided into two groups. The proximal part of the trachea in the treated group was injured by 5 - FU(25mg/ml). The other part of the trachea was intubated to keep breathing. 5 - FU was removed 30 minutes later and the trachea was flushed with Saline. The rats were killed at 0%3 ^6^9^ 12 ^24 ^48 h respectively after 5 - FU was removed. The tra-chea was cut and fixed in 10% neutral formalin for LM. We apply the same volume of PBS taking place of 5 - FU as the control group. The other conditions were the same as the treated group.2. The injury - repair process were dynamically observed under LM, PCNA ( proliferating cell nuclear antigen) expression level was analyzed by immunohis-tochemistry (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 was brown, negative cell was not coloured. PCNA is positive in nuclear.3. Hoechst33342 fluorescence staining:The rat' s tracheal epithelium was stripped under anatomic microscope and spread on the smear which had been treated by poly - lysine. Hoechst33342 were dropped on the smear and incubated at room tempreture for 30 minutes. Then the smear was washed by PBS for three times. The results were observed under fluorescence microscope.4. The expression of ABCG2; We study on the expression of ABCG2 in the levels of protein and mRNA by immunofluorescence and RT - PCR.5. The expression of Shh signal conducting pathway: The change of Shh signal conducting pathway was observed during the process of proliferation and differentiation of the tracheal stem cells by RT - PCR.Results1. Most epithelial cells exfoliated after 30 minutes'treatment by 5 -FU and the basal membrane was exposed. A few nail - like cells which looked like naked nuclei were randomly distributed on the basement membrane. PCNA negative staining suggested that these were Go phase cells. Among them, there were some Hoechst33342 negative cells or weak - staining cells.2. After 5 - FU was removed , the tracheal epithelium recovered. At 3 ~ 6h the cells became flat in morphology; At 9 ~ 12h , cuboidal and columnar epithelium appeared , and the number of the cells increased. At 24h, the epithelial cells connected with each other and the ciliated cells can be seen. Finally, at 48 h the pseudostratified mucociliary epithelium was restored the similar as origi-...