Screening Key Genes Of Collagen Hydrogel Induce Chondrogenesis Based On The Literature Mining And Bioinformatics Technology

Objective:Key genes of materials induce chondrogenesis were screened and the related signaling pathways of materials on chondrogenic differentiation of stem cells was investigated by investigating the expression of related signaling pathways. Further study demonstrated the impact of collagen hydrogel on chondrogenic differentiation in vitro by investigating the expression of related signaling pathways and molecular mechanisms.Methods:In this study, we reviewed the papers of induce chondrogenesis in recent 10 years, and at the same time, using GENEVESTIGATOR software to analyze the related genes of material induced chondrogenesis. Then, we further analyzed the signaling pathway of related gene material induced chondrogenesis by the method of biomedical informatics. The genes of signaling pathways ECM-receptor interaction and TGF-β signaling pathways, such as ITGB1, TNC, COMP, COL5A1, COL6A2, SMAD6, FST and ID3, may be the key genes of materials induce chondrogenesis. Finally, combined with the gene/protein-gene/protein interaction network and the data of related gene microarrays on chondrogenic induction through the GEO public database platform, has showed some gene expression have certain genetic diversity. However, the published data in the material induced chondrogenesis was less and short experimental period. Thus, we hypothesized that materials may induce chondrogenesis by activating receptors of cells, enhance the interaction of cells and extracellular matrix. Further more, to stimulate the secretion of growth factor and build the microenvironment of induced chondrogenesis.In order to further study the molecular mechanism of collagen hydrogel induce chondrogenesis, in vitro model of chondrogenic induction was constructed by BMSCs isolated and cultured in vitro to study the effect of collagen hydrogel on chondrogenic differentiation of BMSCs by investigating related signaling pathways. Groups were designed:control group (B); the growth factor group (BT); the collagen materials group (BC); the positive control group (BCT). Then, after 7 days,14 days,21 days and 28 days of culture, the samples in all groups were taken out for analysis. Finally, the samples following various indicators:(1)cell proliferation; (2) cellmorpholopy; (3) cell viability assay; (4) Phalloidin/hoechst33258 fluorescence staining were for observation of cell distribution and dynamics of fibrous actin (F-actin);(5)Total glycosaminoglycan (GAG) secretion was examed by safranin O staining and DMMB method; (6) immunohistochemical detection; (7) qRT-PCR was used to detect the gene expression of cartilage specific gene; (8) the protein levels detection of related signal pathway of protein expression by western blot method.Results:We investigated the key genes and its related signaling pathways of materials induce BMSCs into cartilage by in vitro model of chondrogenic induction. (1) BMSCs cultured monolayer on collagen hydrogels surface in vitro, the results of cell proliferation test and cell activity test showed that the collagen hydrogel have good biocompatibility, can amount to promote BMSCs in collagen hydrogels surface anchoring and reunion growth. qRT-PCR detection results show that the early stage of the collagen hydrogels surface coating to induce BMSCs differentiation into cartilage cells direction, but cell proliferation ability gradually decline in 21 days with the extension of incubation time. The initial formation of the characteristics of cartilage cells appear to show the characteristics of cartilage COLⅡ gene expression, ACAN and COMP dropped significantly, by contrast, COLⅠ expression.(2)By means of histological tests (including HE staining, safranin O staining, calcein-AM/PI staining and phalloidin/hoechst33258 staining) and biochemical tests (including DNA content and GAG content detection), we investigated that the inductive results of BC group were superior or similar to the BT group, and the best inductive results were showed in BTC group, indicating that the collagen hydrogel material has the similar effect of chondrogenic induction compared with the growth factors. The secretion of proteoglycans and collagen type Ⅱ in BC group were superior or similar to the BT group by immunohistochemistry and qPCR detection, indicating that the collagen hydrogel material has the similar effect of chondrogenic induction compared with the growth factors. However, lower secretion of collagen type Ⅰ in BC group was showed with respect to the B and BT groups, suggesting that collagen hydrogel material in maintaining cartilage phenotype and preventing dedifferentiation was more effective. The results of PCR showed that the gene expression of signaling pathways in the BT, BC and BTC groups were higher compared with the B group, the highest gene expression of signaling pathways was investigated in the BTC group in general. The protein levels of ITGB1, TNC, SMAD6, SMAD2 and SMAD3 were detected by western blot, the research indicated that the protein expression of BC group were superior and similar to the BCT group, higher compared with the B group and BT group.Conclusion:The results showed good profit to the directed differentiation of BMSCs to cartilage cells and the synthesis of extracellular matrix with the cells were round or oval by HE dyeing, after embedded in collagen hydrogel and induced in vitro for 21 days without growth factor. The molecular mechanism of collagen hydrogel induce chondrogenesis maybe:the collagen hydrogel may induce chondrogenesis by activating the integrin β1 (ITGB1) receptor of BMSCs surface, ITGB1 is the key gene of signaling pathways ECM-receptor interaction, activating the expression of TNC and SMAD6, and then to regulate the downstream of TGF-βsignaling pathway to finally produce cartilage specific genes and proteins.