ObjectiveTo investigate the regulatory effect of resveratrol (RES) on the extracellular matrix (ECM) expression of nucleus pulposus cells (NPC), and its relative molecular mechanism.MethodsTen patients receiving discectomy were collected, of which 5 patients were young with spinal burst fracture, classified as control group; the rest 5 patients were senile with lumbar disc herniation, classified as degenerative group. The nucleus pulposus tissue of 2 groups were collected, the in situexpression of β-catenin was detected by immunohistochemistry, and the protein expressions of collagen type Ⅱ and Aggrecan were detected by Western blot. The NPC were isolated and cultured from degenerative nucleus pulposus tissues. RES treated the third-passage NPC with (group B) or without IL-1β (group C), to further determine the protein expressions of collagen type Ⅱ and Aggrecan by Western blot, the unstimulated cells were set up as blank control group (group A). Moreover, NPC treated with small interfering RNA (siRNA) targeted silent SIRT1 or β-catenin were used to determine the protein and gene expressions of β-catenin and SIRT1 by Western blot and real-time fluorescence quantitative PCR. In addition, the third-passage NPC treated with complete medium (group 1), IL-1β (group 2), RES+IL-1β (group 3), and SIRT1-siRNA+RES+IL-1β (group 4) for 24 hours were used to detect the nuclear translocation of β-catenin by cell immunofluorescence staining. Finally, the third-passage NPC treated with complete medium (group Ⅰ), IL-1β (group Ⅱ), IL-1β+β-catenin-siRNA (group Ⅲ), IL-1β+RES (group Ⅳ), and IL-1β+RES+SIRT1-siRNA (group Ⅴ) for 24 hours were used to detect the protein expressions of collagen type Ⅱ and Aggrecan by Western blot.ResultsImmunohistochemical staining and Western blot detection showed that when compared with control group, the cell proportion of expression of β-catenin were significantly increased in degenerative group (t=4.616, P=0.010); the protein expression of β-catenin was also significantly increased and the protein expressions of collagen type Ⅱ and Aggrecan were significantly decreased (P<0.05). In cytology experiments, the protein expression of β-catenin in group B was significantly higher than that in groups A and C, and the protein expressions of collagen type Ⅱ and Aggrecan in group B were significantly lower than those in groups A and C (P<0.05). After transfection of siRNA, the protein expressions of SIRT1 and β-catenin significantly decreased (P<0.05). The results of cell immunofluorescence staining further confirmed that when compared with group 3, after the SIRT1 was silenced by siRNA in group 4, the attenuated nuclear translocation of β-catenin by RES treatment was aggravated. Western blot results showed that the protein expressions of collagen type Ⅱ and Aggrecan in group Ⅱ were significantly lower than those in group Ⅰ(P<0.05); after transfection of β-catenin-siRNA in group Ⅲ, the degradation of ECM by IL-1β was obviously inhibited, the protein expressions of collagen type Ⅱ and Aggrecan were significantly increased when compared with group Ⅱ (P<0.05); after transfection of SIRT1-siRNA in group Ⅴ, the protective effect of RES on the degradation of ECM was inhibited, the protein expressions of collagen type Ⅱ and Aggrecan were significantly decreased when compared with group Ⅳ (P<0.05).ConclusionRES regulates the ECM expression of NPC via Wnt/β-catenin signaling pathway, which provide a new idea for intervertebral disc degeneration disease treatment.
Objective To investigate the effect of porcine small intestinal submucosa extracellular matrix (PSISM) on the vitality and gene regulation of hepatocyte so as to lay the experimental foundation for the application of PSISM in liver tissue engineering. Methods The experiment was divided into two parts: ① BRL cells were cultured with 50, 100, and 200 μg/mL PSISM-medium which were prepared by adding PSISM into the H-DMEM-medium containing 10%FBS in groups A1, B1, and C1, and simple H-DMEM-medium served as a control (group D1); ② BRL cells were seeded on 1%, 2%, and 3% PSISM hydrogel which were prepared by dissolving PSISM in sterile PBS solution containing 0.1 mol/L NaOH in groups A2, B2, and C2, and collagen type I gel served as a control (group D2). At 1, 3, and 5 days after culture, the morphology and survival of liver cells were detected by the Live/Dead fluorescent staining. The cell vitality was tested by cell counting kit-8 (CCK-8) assay. And the relative expressions of albumin (ALB), cytokeratin 18 (CK18), and alpha-fetoprotein (AFP) in hepatocytes were determined by real-time fluorescent quantitative PCR (RT-qPCR). Results The Live/Dead fluorescent staining showed the cells survived well in all groups. CCK-8 results displayed that the absorbance (A) value of group C1 was significantly higher than that of group D1 at 5 days after culture with PSISM-medium, and there was no significant difference between groups at other time points (P>0.05). After cultured with PSISM hydrogels, theA values of groups A2, B2, and C2 were significantly higher than those of group D2 at 3 and 5 days (P<0.05), theA value of group A2 was significantly higher than that of groups B2 and C2 at 5 days (P<0.05), but there was no significant difference between groups at other time points (P>0.05). RT-qPCR showed that the relative expressions of ALB and CK18 mRNA significantly increased and the relative expression of AFP mRNA significantly decreased in groups A1, B1, and C1 when compared with group D1 (P<0.05). The relative expression of CK18 mRNA in group C1 was significantly lower than that in groups A1 and B1 (P<0.05). The relative expressions of ALB and CK18 mRNA were significantly higher and the relative expression of AFP mRNA was significantly lower in groups A2, B2, and C2 than group D2 (P<0.05); the relative expression of CK18 mRNA in group A2 was significantly higher than that in group B2 (P<0.05), and the relative expression of AFP mRNA in group A2 was significantly lower than that in group C2 (P<0.05), but no significant difference was found between other groups (P>0.05). Conclusion PSISM has good compatibility with hepatocyte and can promote the vitality and functional gene expression of hepatocyte. PSISM is expected to be used as culture medium supplement or cell carrier for liver tissue engineering.
ObjectiveTo investigate effect of heart tissue-derived extracellular matrix(ECM) on the differentiation, proliferation and apoptosis of cardiosphere-derived cells(CDC) in vitro. MethodsCDCs were cultured by cardiac explant methods. ECM was prepared by decelluariztion procedure. CDCs were cultured on ECM coated dishes or conventional fibrin (FN) coated dishes. Then we compared the differentiation rate, proliferation, and apoptosis rate of CDC between the two groups in vitro. ResultsECM could significantly promote CDC differentiating into vascular endothelial cell, cardiac muscle cell or smooth muscle cell (0.060±0.002 vs. 0.043±0.002, P < 0.001; 0.082±0.003 vs. 0.051±0.002, P < 0.001; 0.055±0.002 vs. 0.034±0.001, P < 0.001). ECM also significantly promoted the proliferation of CDC and reduced the apoptosis and necrosis rate of CDC in vitro (0.052±0.002 vs. 0.025±0.001, P < 0.001). ConclusionWe obtained c-kit+ CDCs, effectively remove the cellular components of heart tissue-derived ECM and preserved the composition and structure of ECM. ECM can promote the differentiation of CDC to vascular endothelial cell, cardiac muscle cell or smooth muscle cell, promote the proliferation of CDCs and decrease CDC apoptosis and necrosis rate in vitro.
Objective To investigate the proteomic changes among normal skin tissues in young and elderly people and cutaneous squamous cell carcinoma (cSCC) tissues in elderly patients with cSCC, find proteins associated with skin aging and cSCC, and provide a new basis for target screening for cSCC therapy. Methods Five cSCC tissue samples from 5 elderly patients with cSCC and 10 normal skin tissue samples from 5 young and 5 elderly people removed during surgery between January 2019 and December 2020 in West China Hospital of Sichuan University were selected. The differences in tissue morphology and structure were observed by hematoxylin-eosin staining, and the whole protein group was qualitatively and quantitatively analyzed by pressure cycle technique. Results With aging, the structure of skin tissues underwent corresponding changes, including thinning of the skin, increased collagen fiber density, and more organized arrangement. Compared to normal skin tissue, cSCC tissue exhibited epithelial cell dysplasia, atypical mitoses, nest-like distribution of cancer cells, infiltration of inflammatory cells, and formation of keratin pearls. Proteomic analysis identified 3008 specific proteins, and there were 37 proteins with common differential expression. Further screening of databases identified 8 proteins derived from the extracellular matrix, primarily involved in morphological structure formation, tensile strength, interaction with platelet-derived growth factors and receptors, collagen degradation metabolism, and cell adhesion. Conclusions Aging leads to changes in skin structure. The changes of tissue structure caused by aging lead to the weakening of skin barrier function. At the same time, aging leads to the down-regulated expression of protein with the function of inhibiting tumor progression and the up-regulated expression of protein with the function of promoting tumor progression in extracellular matrix. These changes may affect the occurrence and development of cSCC by affecting the regulation mechanism of tumor extracellular microenvironment.
Small intestinal submucosa (SIS) is a natural decellularized extracellular matrix material. Due to its excellent biocompatibility, unique biomechanical properties and biological activity, it has been widely used as a scaffold in regenerative medicine. This article reviews the recent progress in the characterization and medical application of SIS respectively. The specific biological properties of the SIS, as well as its interaction with cells, are highlighted. Some of the SIS products and clinical cases are also reviewed and discussed.
ObjectiveTo review the application of cell derived decellularized extracellular matrix (CDM) in tissue engineering. Methods The literature related to the application of CDM in tissue engineering was extensively reviewed and analyzed. Results CDM is a mixture of cells and their secretory products obtained by culturing cells in vitro for a period of time, and then the mixture is treated by decellularization. Compared with tissue derived decellularized extracellular matrix (TDM), CDM can screen and utilize pathogen-free autologous cells, effectively avoiding the possible shortcomings of TDM, such as immune response and limited sources. In addition, by selecting the cell source, controlling the culture conditions, and selecting the template scaffold, the composition, structure, and mechanical properties of the scaffold can be controlled to obtain the desired scaffold. CDM retains the components and microstructure of extracellular matrix and has excellent biological functions, so it has become the focus of tissue engineering scaffolds. ConclusionCDM is superior in the field of tissue engineering because of its outstanding adjustability, safety, and high bioactivity. With the continuous progress of technology, CDM stents suitable for clinical use are expected to continue to emerge.
ObjectiveTo understand the research progress of the matrix metalloproteinases (MMPs) family in regulating the development of hepatocellular carcinoma (HCC) and its mechanism, in order to provide a reference for the basic research and clinical diagnosis and treatment of HCC. MethodThe relevant literature on the regulation of HCC occurrence, development, and mechanisms by MMPs both domestically and internationally in recent years was reviewed. ResultsThe extracellular matrix (ECM) microenvironment of HCC cells determined the invasiveness and degree of metastasis of tumor cells. The degradation and remodeling of ECM during epithelial mesenchymal transition (EMT) were the main factors contributing to the invasion and metastasis of HCC. The abnormal expression of most members of the MMPs family could lead to ECM breakdown, cell invasion and attachment, and markedly accelerate the process of EMT, thereby promoting the invasion and metastasis of HCC cells. At present, there were many MMPs related to the development of HCC, including MMP-1, 2, 3, 7, 9, 12, 13, 14. The relevant research on the relation between MMP-8, 10, 11, 15, 16, 20, 21, 26 or 28 and the development of HCC was relatively limited, while the exact research on the relationship between the MMP-17, 19, 23, 24, 25 or 27 and HCC development had not been retrievaled. ConclusionsThe MMPs family members (especially MMP-2, 3, 7, 9, 10, 12) play a crucial role in the progression of HCC, including proliferation, invasion, and metastasis. Further exploration of the potential intrinsic relation between all members of the MMPs family members and the development of HCC is crucial for predicting HCC metastasis potentiality and prognosis, as well as developing new or improved targeted anti-cancer therapies for HCC.
ObjectiveTo investigate the expression of Runt-related transcription factor 1 (RUNX1) in gastric cancer and its correlation with clinicopathological features, prognosis and tumor cell invasion ability. Methods① Database analysis: the expression of RUNX1 in gastric cancer and adjacent tissues were analyzed by TCGA and GEO database. Kaplan-Meier Plotter database was used to analyze the correlation between RUNX1 expression level and overall survival (OS) of gastric cancer patients. GO analysis and KEGG pathway enrichment were used to analyze the possible functions and signaling pathways of RUNX1 in gastric cancer, and gene correlation was verified by GEPIA database. ② Clinical case validation: the cancer tissues and adjacent tissues of 62 patients with gastric cancer admitted to the Second Hospital of Lanzhou University from June 2018 to December 2019 were retrospectively collected for immunohistochemical staining, HE staining and Sirius red staining, and the relation between RUNX1 expression and clinicopathological features and prognosis of patients was explored. ③ Cell experiment: we knocked down RUNX1 by using small interfering RNA, and then analyzed the relation between RUNX1 and the invasion ability of gastric cancer cells by Transwell assay. Results① Database analysis: RUNX1 was highly expressed in gastric cancer tissues and negatively correlated with OS (P<0.001). GO analysis and KEGG pathway enrichment analysis showed that RUNX1 was not only involved in the construction of collagen in extracellular matrix (ECM), but also significantly enriched in ECM-receptor interaction pathway. The results of GEPIA gene correlation analysis showed that RUNX1 was positively correlated with gene expression involved in ECM-receptor interaction pathway (P<0.05). ② Clinical case validation: the results of immunohistochemical staining showed that RUNX1 was relatively highly expressed in gastric cancer tissues, and the high expression of RUNX1 was a risk factor affecting the postoperative OS of gastric cancer patients (RR=5.074, P=0.034); the expression of RUNX1 in gastric cancer tissues was positively correlated with red staining area of Sirius red staining (r=0.46, P<0.001). ③ Cell experiment: invasion experiments confirmed that the number of invasive AGS or HGC27 cells in si-001 group and si-002 group decreased after RUNX1 knockdown. ConclusionRUNX1 is highly expressed in gastric cancer and suggests a worse survival prognosis, and it is possible that RUNX1 promotes the development of gastric cancer by activating the ECM-receptor interaction pathway.
Aortic aneurysm and dissection are critical cardiovascular diseases that threaten human life and health seriously. No pharmacological treatment can effectively prevent disease progression. The imbalance of aortic wall cells and non-cellular components leads to structural or functional degeneration of the aorta, which is a prerequisite for disease occurrence. As the important non-cellular component, extracellular matrix (ECM) is crucial to maintain the aortic structure, function, and homeostasis. Abnormal production of elastin and collagen, destruction of cross-linking between elastic fibers and collagen fibers, and the imbalance of metalloproteinase and inhibitors leads to excessive degradation of ECM proteins, all of which have destroyed the structure and function of aorta. It will provide more ideas for disease prevention and treatment by learning ECM proteins and their metabolic mechanism. Here, we focus on the ECM proteins that have been reported to be involved in aortic aneurysm and dissection, and discuss the regulatory mechanism of metalloproteinase and inhibitors.
ObjectiveTo investigate the differential expression of transient receptor potential vanilloid receptor 4 (TRPV4) protein in the osteoarthritis (OA) and normal cartilages, and explore the role of TRPV4 in the prevention and treatment of OA.MethodsThe cartilage tissues from the patients of knee OA (OA group) and femoral neck fracture (control group) were taken. In OA group, there were 6 males and 9 females; the age ranged from 55 to 78 years (mean, 69 years); the Kellgren-Lawrence (K-L) score was 3.0±0.8. In control group, there were 5 males and 10 females; the age ranged from 57 to 91 years (mean, 71 years). There was no significant difference in gender and age between the two groups (P>0.05). Western blot, real-time fluorescence quantitative PCR, Masson staining, and immunohistochemical staining were used to detect the difference in protein and mRNA expressions of TRPV4 between the OA and normal cartilages. Then the relationship between the K-L score of OA and the rate of TRPV4-positive cells was analyzed.ResultsThe relative expression of TRPV4 protein and mRNA in OA group were 0.454±0.199 and 2.951±1.200, which were higher than those in control group (0.165±0.074, 1.437±0.682). The difference in relative expression of TRPV4 protein was significant (t=2.718, P=0.026). Histology observation showed that the chondrocytes arranged disorderly in OA group, the structure of extracellular matrix was abnormal, and the cartilage defect reached the deep layer. There were more TRPV4-positive cells in the degenerated tissue, and the rate of TRPV4-positive cells was 37.353%±13.496%. The chondrocytes were arranged well in control group, and the rate of TRPV4-positive cells was only 9.642%±3.284%. There was a significant difference between the two groups (t=7.491, P=0.000). The rate of TRPV4-positive cells in OA group was positively correlated with the OA K-L score (r=0.775, P=0.001).ConclusionThe TRPV4 expression increased in OA cartilages that may contribute to the development of OA.