ObjectiveTo investigate the effect of Wnt5a derived from tumor-associated fibroblasts （CAFs） on the migration and invasion of gastric cancer cells. MethodsThe differentially expressed genes Wnt5a between CAFs and normal gastric fibroblasts (NGFs) in gastric cancer tissues and their corresponding normal gastric tissues using the GEO database GSE194261 dataset were screened. Immunohistochemical method was used to detect the expression of Wnt5a protein in tissue samples of clinical gastric cancer patients, and the relationship between Wnt5a protein expression and clinicopathological features of gastric cancer was analyzed. CAFs and NGFs were extracted from fresh surgical specimens of gastric cancer patients, and the expression of Wnt5a in CAFs was detected by real-time fluorescence quantitative-polymerase chain reaction and Western blot experiment. Transwell invasion and migration experiment was used to observe the effects of CAFs, inhibition of Wnt5a expression in CAFs and different concentrations of recombinant Wnt5a protein on the migration and invasion ability of gastric cancer MGC-803 and MKN-28 cell lines in vitro. ResultsThrough the screening of GEO database GSE194261 data set, it was found that Wnt5a was more expressed in CAFs than NGFs (P<0.05). Immunohistochemical results showed that the expression of Wnt5a protein in gastric cancer tissues was significantly stronger than that in normal gastric tissues (P<0.05), and the expression of Wnt5a protein was related to T stage of tumor (χ2=5.035, P<0.05), but not related to gender, age, degree of tumor differentiation, lymph node metastasis, vascular invasion and nerve invasion (P>0.05). Inhibiting Wnt5a derived from CAFs could inhibit the invasion and migration of gastric cancer cells. By stimulating gastric cancer cells with different concentrations of human recombinant Wnt5a protein, it was found that when the concentration of human recombinant Wnt5a protein was greater than 100 ng/mL, the invasion and migration abilities of MGC-803 and MKN-28 gastric cancer cells were significantly increased (P<0.05). ConclusionWnt5a is highly expressed in CAFs derived from the interstitial tissue of gastric cancer, which is related to the invasion depth of gastric cancer and can promote the invasion and migration of gastric cancer cells.
Objective To explore the possibility of constructing tissue engineered cartilage complex three-dimensional nano-scaffold with collagen type II and hyaluronic acid (HA) by electrospinning. Methods The three-dimensional porous nano-scaffolds were prepared by electrospinning techniques with collagen type II and HA (8 ∶ 1, W ∶ W), which was dissolved in mixed solvent of 3-trifluoroethanol and water (1 ∶ 1, V ∶ V). The morphology were observed by light microscope and scanning electron microscope (SEM). And the porosity, water absorption rate, contact angle, and degradation rate were detected. Chondrocytes were harvested from 1-week-old Japanese white rabbit, which was disgested by 0.25% trypsin 30 minutes and 1% collagenase overlight. The passage 2 chondrocytes were seeded on the nano-scaffold. The cell adhesion and proliferation were evaluated by cell counting kit 8 (CCK-8). The cell-scaffold composites were cultured for 2 weeks in vitro, and the biological morphology and extracelluar matrix (ECM) secretion were observed by histological analysis. Results The optimal electrospinning condition of nano-scaffold was 10% electrospinning solution concentration, 10 cm receiver distance, 5 mL/ h spinning injection speed. The scaffold had uniform diameter and good porosity through the light microscope and SEM. The diameter was 300-600 nm, and the porosity was 89.5% ± 25.0%. The contact angle was (35.6 ± 3.4)°, and the water absorption was 1 120% ± 34% at 24 hours, which indicated excellent hydrophilicity. The degradation rate was 42.24% ± 1.51% at 48 days. CCK-8 results showed that the adhesive rate of cells with scaffold was 169.14% ± 11.26% at 12 hours, and the cell survival rate was 126.03% ± 4.54% at 7 days. The histological and immunohistochemical staining results showed that the chondrocytes could grow well on the scaffold and secreted ECM. And the similar cartilage lacuma structure could be found at 2 weeks after co-culture, which suggested that hyaline cartilage formed. Conclusion The collage type II and HA complex three-dimensional nano-scaffold has good physicochemical properties and excellent biocompatibility, so it can be used as a tissue engineered cartilage scaffold.