| BackgroundThe interaction between tumor cells and their microenviroment decides the final outcome of tumor development. Study on this kind of reciprocal interaction has recently become a focus in various areas of cancer research. The generation, evolvement, invasion and metastasis of tumor regards as a broken dynamic equilibrium between tumor and their microenvironment. The tumor microenviroment includes extracellular matrix, immediate contact cells, soluble substances such as growth factors, hormone and interstitial tissue and so on.Decorin is an important member of the small proteglycans in extracellular matrix, which distributes generally in tissues and can bind to several growth factors and cell factors to adjust cells proliferation and tissue remodeling. International tumor study fields pay close attention to it recently. More researchs indicate that decorin is related to the initiation, progression and growth of the tumor. Decorin maybe a kind of anti-tumor matter and promises to become a novel medicine for the therapy of human cancers.Decorin consists of a globular core protein and a covalently linked glycosaminoglycan chain including CS/DS,which "decorates" the surfaces of fibrillar collagen. The molecular weight is 92.5kDa, the core protein is 40kDa. Decorin is a member of PG families, so decorin is also called PGⅡand PG40.To make clear of the relation between decorin and tumor, the domestic and abroad scholars have studied liver cancer, colon cancer, ulocarcinoma, ovarian cancer, renel carcinoma and stomach cancer through the technology of straight intervertion, situ hybridization and gene transfection. Those studies indicate decorin can inhibit growth of many kinds of tumor. The mechanism maybe:1)decorin binds to transforming growth factor-β(TGF-β) to inhibit the activity of TGF-β;2)decorin increases the expression of P21, a potent inhibitor of cyclin-dependent kinases(CDKs). It is clear that TGF-βand P21 are relevant to lung cancer and decorin expression is downregulated in lung cancer, however research are lacking about the influence of decorin on lung cancer. Therefore my study chooses human adenocarcinoma cell A549 lines and nude mice transplantation tumor model as my study object to carry out three parts researches: part one. the influence of decorin on growth of human adenocarcinoma cell of lung A549 lines in vitro; part two. the influence of decorin on capability of adhesion, protraction and invasion of human adenocarcinoma of lung cells; part three. experiment researches about the target therapy of decorin treating nude mice human lung adenocarcinoma transplantation tumor A549, and some investigation about the potential mechanism, further observation about the decorin inhibition on the growth of nude mice transplantation tumor. The study aims at investigating the theoritic foundation of decorin function in the generation of adenocarcinoma of lung, and providing a new thought about the therapy of lung adenocarcinoma.Methods:1 cell cultureAdenocarcinoma cell A549 lines were cultured with decorin and cells in exponential phase of growth were selected for tests.2 The influence of decorin on the cell growth of human lung adenocarcinoma were studied by MTT.Cells in exponential phase of growth were added into decorin, the final concentration is 0, 2.5, 5, 10, 20, 30, 40μg/ml, cells were divided into 7 groups, 3 double holes were set for every group. Cells in 7 groups were cultured for 0h, 24h, 48h, 72h, 96h, 120h, then were studied by MTT. The detected wave length on enzyme mark meter was set at 490nm to calculate the survival rate of A549 cells.3 The changes of cell cycle and apoptosis were analyzed by FCM.Cells in exponential phase of growth were collected,and incubated 0-96h together with decorin in concentration of 0, 5, 10, 20, 30, 40μg/ml respectively. Collect cells in centrifuge tubes, centrifuge, wash, dye, and then place on FCM.20000 cells were tested by each specimen, and rate of testing was set at 150～200 cells/s. The software of MultiCycle for Windows was used to analyze the collected data to calculate the rate of cellular apoptosis.4 The influence of decorin on expression of mRNA were studied by RT-PCR.Cells in exponential phase of growth were collected, and incubated 24h together with decorin in concentration of 0, 10, 20, 30, 40,50μg/ml respectively.Divided into 5 groups according to the concentration of decorin and extracted and quantitated RNA which as a model of reverse transcription to synthesis cDNA chains, carried out PCR amplication by primer. Products were detected by agarose gel electrophoresis, the amount of expression of decorin mRNA were judged by intracontrast of photodensity.5 TGF-βwere studied by Elisa.Cells in exponential phase of growth were collected,were added with decorin, the final concentration was 0μg/ml in contral group, 30μg/ml in experimental group, 3 double holes were set for every group, cultured for 4d, activated specimen, established standard curves, and detected.6 P21 were studied by Western.Cells in exponential phase of growth were collected, were added with decorin, the final concentration was 0μg/ml in contral group, 30μg/ml in experimental group, extracted total protein in cells, detected protein content, then observed after polyacrylamide gel electrophoresis, dyeing and transmembrane.7 Adhesion assay, invasion assay, migration assay technique were used to detect the adhesin, invasion and migration ability of decorin on A549.Cells in exponential phase of growth were collected, were added with decorin, the final concentration was 0μg/ml in contral group,2.5μg/ml in experimental group. Adhesion assay was detectde by MTT after coating and hydration basal membrane, inculating and culturing cells. Transwell cabin was paved by Matrigel gum, cultured cells, moved by chemotaxy, fixed, dyed, counted by microscope, then calculated the rate of invasion. Coated and hydrated basal membrane, preparated and cultrued monolayer cells, by man-made scratch method, randomly calculated 5 total cellular score in 100×vision field of phase contrast microscope which migrated to the vacant space between scratchs.8 Construct transplantation tumor nude mice model to detect the targeted function of decorin in vivo.Constructed transplantation tumor nude mice model, and decorin was directly injected into transplantation tumor, then observed its effect on anti-transplantation tumor growth,including general state, local solid tumor growth inhibition rate and histopathology assay. Took tumor tissue, P21 were studied by Westem. TGF-βwere studied by Elisa.Results:1 To some extent, the inhibition effect of decorin on A549 cellular survival rate shows dose and time dependence(the inhibition was most significant at the decorin concentration of 30μg/ml for 96 h)。2 After decorin influenced on A549 cells, cells in G1 phase increased obviously, and cells in S phase decreased obviously, cells in G2 phase didn't change obviously, hint that decorin may delay the transition of tumor cells from G1 phase to S phase。After decorin influenced on A549 cells, the rate of apoptosis in A549 increased, especially at the concentration of 30μg/ml for 96 h。3 influenced by 30μg/mldecorin for 96h, amplificated by RT-PCR, the expression of decorinmRNA was obvious higher than the group without decorino。4 A549cells were influenced by 30μg/ml decorin for 96h, expression level of TGF-βprotein decreased significantly (0.584±0.107, 0.252±0.008) P＜0.001。5 Worked by 30μg/ml decorin for 96h, by Western assay, the expression level of P21 protein was obviously higher than those without decorin6 Influenced by 2.5μg/mldecorin for 4 days, the adhesive rate of A549 cells declined from 0.462±0.034 to 0.142±0.024, P＜0.001。7 Treated by 2.5μg/mldecorin for 4 days, invasive A549 cell population was 164.09±9.52, significent lower than those without decorin (229.89±10.72) P＜0.001。8 Treated by 2.5μg/mldecorin for 4 days migrating A549 cell population decreaded from 57.75±2.42 to 11.33±1.23, obviously lower than those without decorin (57.75±2.42) P＜0.001。9 The rate of successful generation of transplantation tumor in nude mice was 97.2%, average time for tumor formation was 30±3d。Treated by Decorin, the rate of tumor inhibition(%) was 49.58%。The index of tumor growth was 1.02±0.71, obviously lower than that in control group of 6.66±2.96。Weight of tumor was(1.19±0.71)g, obviously lower than that in control group of (2.36±0.46)g。Heart,liver. nephridial tissue had no obvious damage, and their structures were generally normal. Treated by decorin, the expression level of P21 protein was obviously higher than that in groups without decorin, the expression level of TGF-βprotein was obviously lower than that in groups without decorin (0.071±0.008, 0.229±0.002) P＜0.001。Conclusion1 The proliferation of A549 cell could be inhibited by decorin in vitro. The inhibition effect showed the time-dose dependence.2 Decorin inhibited the growth of adenocarcinoma cells. The potential mechanism could be relevant to up-regulation of p21, inhibition of TGF-βexpression, stoppage of transition of adenocarcinoma cell phase G1/S, enhancement of apoptosis in lung cancer cells3 Decorin inhibited tumor cell adhesion, migration and invasion in vitro.4 Decorin inhibited the xenografts growth significantly in nude mice by injection into the tumor and no toxic effects of decorin were found in heart, kidney and liver of the nude mice. |
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