The Mechanism Of TGF-? Pathway In Regulating The Proliferation Of Lung Airway Epithelial Stem Cells By Fibroblast

Objective: Varying degrees of the respiratory system pathologic changes and dysfunction in lung diseases are closely related with the altered proliferation and differentiation of lung stem cells and the homeostasis disbalance. Consequently, the repair mechanism of lung stem cells acts an important role in the therapy of respiratory system diseases. According to the anatomical structure, the appearance of airway is covered with lung epithelial cells, which not only maintains the epithelial structure integrity of the airway and pulmonary but also keeps the stability of the lung function. After the lung injury, the quantity and function of the lung epithelial cells are maintained and recovered by the self-renewing and wound-healing of the lung stem cells. There are various mechanisms of repairing the injured lung epithelial cells. Extrapulmonary stem cells, which derived from bone marrow, peripheral blood, embryo and so on, can come into play through migrating to the injured lung tissue. However, intrapulmonary stem cells can play a better role than extrapulmonary stem cells during repairing process. Intrapulmonary stem cells include basal cells, Club cells, bronchioalveolar stem cells and type II alveolar cells currently known. The function of lung stem cells is closely regulated by microenvironment which including fibroblasts, blood capillary, airway smooth muscle cells, etc. TGF-?(transforming growth factor beta, TGF-?) is secreted by fibroblasts and TGF-? has the ability of promoting the stem cell differentiation and inhibiting proliferation. Nevertheless, whether fibroblasts can regulate lung stem cells by TGF-? pathway has not been reported. In this study, we use molecular and cellular biology to investigate the mechanism of TGF-? pathway in the proliferation of lung airway epithelial stem cells regulated by lung fibroblasts(MLg cells), which provide an innovative therapeutic target for the lung disease.Method:1. We used enzymatic digestion to separate lung airway epithelial cells from mice and sorted the airway epithelial stem cells by Flow Cytometry with special antigen. 2. Lung airway epithelial stem cells were divided into the experimental group and the control group. We added growth factors(FGF1?FGF2?FGF10?HGF), TGF-? inhibitor SB431542, lung fibroblasts, lung fibroblasts+TGF-? inhibitor SB431542 in different experimental groups during cell culture to study the proliferation airway epithelial stem cells through fluorescence microscopy. The control group was only added basal medium. 3. The knock-out mice of TGF-?(TGF-?R-lung stem cells, SPC-Cre/TGF-?Rf/f) were established as experimental groups and the gene none knock-out mice were set as control groups. We selected and cultured the lung epithelial cells from these two groups in vitro. We added the basal medium and TGF-? inhibitor SB431542 in experimental group and control group during cell culture to study the proliferation airway epithelial stem cells through fluorescence microscopy and statistical analysis was carried out on the number of stem cell cloning. We cultured the lung fibroblasts MLg. The control group was added basal medium while the exprimental group was added TGF-? inhibitor SB431542. After 48 hrs, we observe the quantity and morphology of MLg cells in these two groups. We isolated the whole RNA and studied the growth factor(FGF1, FGF2, FGF4, FGF10, HGF) gene expression levels in the MLg cells after adding the TGF-? inhibitor SB431542 by Real-time PCR assay.Result:1. The result of Flow Cytometry selection: Firstly,we removed cell debris and 7-AAD positive marker of dead cells and separated lung living cells. Secondly, we removed CD31/34/45-APC-CY7 positive marker of endothelial cells, stromal cells and hematopoietic cells and kept Ep CAM-PE- CY7 positive marker cells as epithelial cells. Lastly, we chose Sca-1-APC and GFP-FITC double positive marker of epithelial cells as airway epithelial stem cells. 2. The growth factors secreted from MLg cells has the ability of promoting the proliferation of airway epithelial stem cells. 3. There is no direct correlation between TGF-? pathway and the proliferation of airway epithelial stem cells. 4. MLg cells accelerate the proliferation of airway epithelial stem cells via inhibiting TGF-? pathway. 5. TGF-? inhibitor SB431542 has no clearly influence on the morphology and quantity of MLg cells. 6. TGF-? inhibitor SB431542 can up-regulate the expression level of growth factors which secreted by MLg cells.Conclusion: Lung fibroblasts(MLg cells) can facilitate the proliferation of airway epithelial stem cells through supressing TGF-? signaling pathway and up-regulating growth factors(FGFs?HGF) screted by lung fibroblasts. We can come to a conclusion that TGF-? pathway plays a significant part in the proliferation of lung airway epithelial stem cells. In addition, the function of lung stem cells is closely regulated by fibroblasts in microenvironments.