| IntroductionPulmonary sclerosing hemangioma (PSH) is a rare lung tumor so far failed to determine its source and nature. PSH morphology varied, but the tumor cells are mainly two:one is polygonal mesenchymal-like tumor cell in solid area, the other is covered in the nipple surface, vascular tumor-like area clearance and consolidation area cracks within the cells, they showed mostly low cuboidal, single arrangement. The TTF-1 (thyroid transcription factor-1) co-expression of two types of cells and by cloning analysis, we found two kinds of cells have the same clone, and are derived from the epithelial cell, and the polygonal cells are immature.Although most scholars believe that the tumor had mild clinical course, is "benign", but with invasion and metastasis and recurrence report.But why the formation of polygonal cells as interstitial cells in the way of arrangement? Why does not exist their corresponding cells in normal tissues then?Epithelial mesenchymal transition (epithelial-mesenchymal transition, EMT) is the phenomenon that epithelial cells in the interaction with the surrounding stroma gradually get the characteristics of interstitial cells. It not only exits in embryogenesis process, but also exists in a variety of inflammatory or neoplastic lesions. Its main feature is the loss of epithelial cell polarity and the acquisition of mesenchymal characteristics. Then do these transitions also occur in the polygonal stromal cells?In this study we first detected the expression of stem cell markers and proteins associated with lung development, to further prove its origin and nature, and then examined the expression of EMT-related factors to explore the formation mechanism of PSH.Materials and Methods1. Patients and specimensWe collected 35 cases of sclerosing hemangioma from the First Clinical College of China Medical University and Tianjin Cancer Hospital. Female 26 and male 9 are inclued, these cases all showed masses in X-ray, and were in the peripheral lung, diameter 0.6-5cm, and not found multiple foci and metastasis.2. ImmunohistochemistryThe mouse anti-human monoclonal antibodies Notch3, Notch4, Stro-1, p75 NGFR and Nestin were purchased from Abcam company,USA; The mouse anti-human monoclonal antibodies Axin was from Santa company,USA; To-use mouse anti-human monoclonal antibodies TTF-1, TGF-β1, E-cadherin,β-catenin, Vimentin, Actin (sm), MMP2 and MMP9, and SP staining kit and DAB color reagent were purchased from Fuzhou, Maixin Company. The specimens were made into 4μm serial sections, after dewaxing, removing benzene, and after hydration, streptavidin avidin-peroxidase (SP) immunohistochemistry could be performed.3. Laser capture target cells, RNA extractionThe 18 specimens were cut into 6μm serial sections, laid on a thin polyethylene foil slide, dewaxing, staining, and graded alcohol, and then put into xylene for 10 minutes. When the slide dried, place it in the LCM (LEICA LMD6000) microscope system loading platform. Locating beam calibration in chosen cells, emit laser beam to the selected target cells, and the cells will fall into an EP tube cover with the lysis buffer.We respectively capture the cubic cells and polygonal cells. More than 10,000 cells were captured for each specimen.Then we extract RNA from the captured cells.4. RT-PCR We extract RNA of polygonal cells and cuboidal cells, and then RT-PCR was performed. After electrophoresis with 1.5%agars gel, the bands of PCR products were visualized using Biolmaging Systems. The greyscales of PCR products were measured. The relative expression quantity was scored as ratios of mRNA and P-actin staining intensities.5. Statistical analysisAll statistical calculations were performed by SPSS version 13.0 for Windows software. P values less than 0.05 were considered statistically significant.Results1,Immunohistochemical staining1 Stem cell markers Stro-1, p75 NGFR, and Nestin were no significant expression, Notch3 and Notch4 in the polygonal cells in a relatively high expression.The expression of Notch3 and Notch4 in the polygonal cells is much higher than in the surface cubic cells (P<0.05).2,The expression of Axin and C-myc35 patients with PSH in the Axin cubic cells, mainly in positive expression of polygonal cells only see a few cells were positive. The C-myc expression in polygonal cells was significantly stronger in cubic cells (P<0.05).3,The expression of E-cadherin,β-cateninTwo kinds of cells in 35 cases of PSH showed strong positive expression of TTF-1, the surface cubic cells, showed a strong membrane expression of E-cadherin, P-catenin, in some cases expression of P-catenin was in membrane accompanied by cytoplasma. In the polygonal cells, E-cadherin membrane expression is not expressed or weakly positive;β-catenin membrane expression defect with some of the majority of cytoplasm. 4,The expression of other markersMesenchymal cell marker Vimentin expression mainly in the polygonal cells, a small amount of expression in the surface cells. In both cell growth factor, we can see that TGF-β1, IGF-1, and the expression of MMP2 and MMP9, but relatively weak cubic cells2,RT-PCR1. By PCR we have notyet check out the expression of Stro-1 and Nestin, Sca-1, expression of ABCG2 and OCT4 was weak,there was no significant difference in two kinds of cells.2. The expression of E-cadherin higher in the cubic cells than polygonal cells (P <0.05).3. The expression of Notch3, Jaggedl, C-myc, Twist and Snail in polygonal cells was significantly higher than in cubic cells (P<0.05)Conclusions1. Two kinds of cells are derived from alveolar epithelial cells,2. The formation of the special structure of PSH has relationship with the epithelial mesenchymal transition;3. The formation of e polygonal cells may be the result of epithelial mesenchymal transition.
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