Objective To summarize the role of cellular senescence and senescent secretary phenotype in the intervertebral disc (IVD) degeneration. Methods Relevant articles that discussed the roles of cellular senescence in the IVD degeneration were extensively reviewed, and retrospective and comprehensive analysis was performed. The senescent phenomenon during IVD degeneration, senescent secretary phenotype of the disc cells, senescent pathways within the IVD microenvironment, as well as the anti-senescent approaches for IVD regeneration were systematically reviewed. Results During aging and degeneration, IVD cells gradually and/or prematurely undergo senescence by activating p53-p21-retinoblastoma (RB) or p16INK4A-RB senescent pathways. The accumulation of senescent cells not only decreases the self-renewal ability of IVD, but also deteriorates the disc microenvironment by producing more inflammatory cytokines and matrix degrading enzymes. More specific senescent biomarkers are required to fully understand the phenotype change of senescent disc cells during IVD degeneration. Molecular analysis of the senescent disc cells and their intracellular signaling pathways are needed to get a safer and more efficient anti-senescence strategy for IVD regeneration. Conclusion Cellular senescence is an important mechanism by which IVD cells decrease viability and degenerate biological behaviors, which provide a new thinking to understand the pathogenesis of IVD degeneration.
ObjectiveTo investigate the influences of lactic acid (LA), the final degradation product of polylactic acid (PLA) on the prol iferation and osteoblastic phenotype of osteoblast-l ike cells so as to provide theoretical basis for bone tissue engineering. MethodsRos17/2.8 osteoblast-l ike cells were harvested and divided into 3 groups. In groups A and B, the cells were cultured with the medium containing 4, 8, 16, 22, and 27 mmol/L L-LA and D, L-LA, respectively. In group C, the cells were cultured with normal medium (pH7.4). The cell prol iferation was determined with MTT method after 1, 3, and 5 days. The relative growth ratio (RGR) was calculated, and the cytotoxicity was evaluated according to national standard of China. In addition, the alkal ine phosphatase (ALP) activity of cells cultured with medium containing 4 mmol/L L-LA (group A), 4 mmol/ L D, L-LA (group B), and normal medium (group C) after 1 and 5 days were detected with ALP kits, and the relative ALP ratio (RAR) was calculated; after 21 days, the calcium nodules were tested with von Kossa staining method, and were quantitatively analyzed. ResultsWhen LA concentration was 4 mmol/L, the mean RGR of both groups A and B were all above 80%, and the cytotoxic grades were grade 0 or 1, which meant non-cytotoxicity. When LA concentration was 8 mmol/L and 16 mmol/ L, groups A and B showed cytotoxicity after 5 days and 3 days, respectively. When LA concentration was above 22 mmol/L, cell prol iferations of groups A and B were inhibited evidently after 1-day culture. At each LA concentration, RGR of group A was significantly higher than that of group B at the same culture time (P<0.05) except those at 4 mmol/L after 1-day and 3-day culture. After 1 day, the RAR of group A was significantly higher than that of group B on 1 day (144.1%±3.2% vs. 115.2%±9.8%, P<0.05) and on 5 days (129.6%±9.8% vs. 78.2%±6.9%, P<0.05). The results of von Kossa staining showed that the black gobbets in group A were obviously more than those of groups B and C. The staining area of group A (91.2%±8.2%) was significantly higher than that of groups B (50.3%±7.9%) and C (54.2%±8.6%) (P<0.05). ConclusionThe concentration and composition of LA have significant effects on the cell proliferation and osteoblastic phenotype of osteoblast-l ike cells.
ObjectiveTo explore the composition of intestinal microbiota between patients with fixed airflow obstruction asthma, reversible airflow obstruction asthma, and healthy control, and analyze the correlation between key differential bacterial distribution and clinical characteristics. MethodsFifteen patients with fixed airflow obstruction asthma (FAO) and 13 patients with reversible airflow obstruction asthma (RAO) were included, along with 11 matched healthy control subjects. Clinical data were collected, and lung function tests and induced sputum examination were performed. Blood and stool samples were tested to compare the gut microbiota status among the groups, and analyze the relationship between gut microbiota abundance and patients' blood routine, IgE levels, lung function, and induced sputum. Results The dominant bacterial compositions were similar in the three groups, but there were differences in the abundance of some species. Compared to the RAO group, the FAO group showed a significant increase in the genera of Bacteroides and Escherichia coli, while Pseudomonas was significantly decreased. The phylum Firmicutes was negatively correlated with the course of asthma, while the phylum Bacteroidetes and genus Bacteroides were positively correlated with the asthma course. Bacteroidetes was negatively correlated with Pre-BD FEV1/FVC, Pseudomonas was positively correlated with Pre-BD FEV1, Escherichia coli was negatively correlated with Post-BD FEV1/FVC, and Bacteroides was negatively correlated with Post-BD MMEF. The class Actinobacteria and the order Actinomycetales were negatively correlated with peripheral blood EOS%, while the order Enterobacteriales and the family Enterobacteriaceae were positively correlated with peripheral blood IgE levels. Furthermore, Actinobacteria and Actinomycetales were negatively correlated with induced sputum EOS%. Conclusions There are differences in the gut microbiota among patients with fixed airflow obstruction asthma, reversible airflow obstruction asthma, and healthy individuals. Bacteroides and Escherichia coli are enriched in the fixed airflow obstruction asthma group, while the Firmicutes are increased in the reversible airflow obstruction asthma group. These three microbiota may act together on Th2 cell-mediated inflammatory responses, influencing the process of airway remodeling, and thereby interfering with the occurrence of fixed airflow obstruction in asthma.
ObjectiveTo identify 3 the disease-causing genes and mutations of Leber congenital amaurosis (LCA), and to study the correlation of phenotype and genotype. MethodsA retrospective study. Four LCA patients and seven family members who were diagnosed by eye examination in Ning Xia Eye Hospital of People's Hospital of Ningxia Hui Autonomous Region from January to December 2021 were included in the study. Four patients were from 3 unrelated families. Detailed collection of medical history and family history were received. Related ophthalmologic examination were collected and genomic DNA was extracted from peripheral blood. Whole-exome sequencing method was used for genetic diagnosis. The identified variant was confirmed with Sanger sequencing. Potential pathogenic mutation was analyzed using software and conserved domain analysis and performed co-separated analysis between the family member and the proband. ResultsOf the 4 patients, 1 patient was males and 3 patients were females; the age was from 4 to 18 years. Nystagmus were seen in 3 cases, finger pressing eyes and night blindness was seen in 1 cases; electroretinogram showed 4 cases of extinction or near extinction. The foveal reflection was visible in all eyes, and there was no obvious abnormality in the peripheral retina. One eye had strong reflection signal with raised ellipsoid in macular area; two eyes had weak reflection signal faintly visible between retinal layers; 1 eye had increased blood vessel branches, peripheral retinal non-perfusion area with capillary leakage; annular strong autofluorescence in macular area 4 eyes. No obvious abnormality was found in the phenotypes of family members. Genetic testing showed that the proband of pedigree 1 (Ⅱ-1) was found a homozygous missense mutation in c.640A>T (p.C214S) (M1) of PRPH2 gene. The proband of pedigree 2 (Ⅱ-2) was found compound heterozygous mutation in c.1256G>A(p.R419Q) (M2) and c.1A>C (p.M1L) (M3) of TULP1 gene. The proband 3 (Ⅱ-1) and her sister (Ⅱ-2) were both found compound heterozygous mutation in c.1943T>C (p.L648P) (M4) and c.380C>T (p.P127L) (M5) of GUCY2D gene. The parents and sister (Ⅱ-1) of the proband in family 2 and the parents of the proband in family 3 were all carriers of the corresponding heterozygous variant. M1, M3, M4, M5 were novel mutations and unreported. The genotype and disease phenotype were co-segregated within the family. According to the analysis of pedigree and genetic testing results, all 3 families were autosomal recessive inheritance. The amino acid conservation analysis found that M1, M2, M3, M4, and M5 were highly conserved among species. The results of bioinformatics analysis were all pathogenic variants. ConclusionsPRPH2 gene M1, TULP1 gene M3, and GUCY2D gene M4, M5 were novel mutations and not been reported in the literature and database. This research expanded the gene mutation spectrum of LCA. The patients with LCA have available characterristics, including onset age, varying ocular fundus and severe visual impairment.
Objective To investigate the effects of in-vitro monolayer culture and three-dimensional (3-D) alginate microsphere culture on the differentiation of normal human nucleus pulposus cells (NPCs), and to discuss the regulatory mechanism of restoring the phenotype of dedifferentiated NPCs by culturing resveratrol (RES) in 3-D alginate microsphere. Methods Normal human nucleus pulposus tissues were harvested for culture and identification of NPCs from 6 patients with burst lumbar vertebra fracture. NPCs at passages 1, 3, 5, and 7 in the in-vitro monolayer culture were harvested to observe the morphology, cell aging, and proteoglycan expression. The cell proliferation rates of NPCs at passage 1 in-vitro in monolayer culture and in 3-D alginate microsphere culture were detected. NPCs at passage 7 were randomly divided into 3-D alginate microsphere control group (group A), RES group (group B), silent mating type information regulation 2 homolog 1 (SIRT1)- small interfering RNA (siRNA) + RES group (group C), and negative control-siRNA + RES group (group D); and NPCs in the in-vitro monolayer culture was monolayer control group (group E). After corresponding treatment, Western blot was used for determining the protein expressions of SIRT1, Aggrecan, and collagen type II; real-time fluorescence quantitative PCR was used for detecting SIRT1 mRNA expression. Results The cultured cells were identified to be NPCs. Morphological observation, senescence-associated β-galactosidase (SA-β-gal) staining, and toluidine blue staining showed that dedifferentiation of normal NPCs tended to occur under continuous in-vitro monolayer culture, which was more obvious with increase of passage number. NPCs in 3-D alginate microsphere culture showed significantly lower proliferation rate than NPCs in the in-vitro monolayer culture (P lt; 0.05), but it could significantly improve the protein expressions of collagen type II and Aggrecan in dedifferentiated NPCs, showing significantly difference between groups E and A (P lt; 0.05). The protein expressions of SIRT1, collagen type II, and Aggrecan in group B were significantly improved when compared with that in group A (P lt; 0.05). Real-time fluorescence quantitative PCR and Western blot showed that the expressions of SIRT1 mRNA and proteins in group C were significantly inhibited after transfected with SIRT1-siRNA when compared with those in groups B and D (P lt; 0.05), and the protein expressions of collagen type II and Aggrecan in group C were significantly lower than those in groups B and D (P lt; 0.05). Conclusion Continuous in-vitro monolayer culture could efficiently cultivate numerous seeding NPCs, but it is liable to dedifferentiate. In 3-D alginate microsphere culture, RES could restore the phenotype of dedifferentiated NPCs and synthesize more extracellular matrix, which is related to the regulation of SIRT1.
Objective To investigate the role of bone morphogenetic protein 2 (BMP-2) combined with hypoxic microenvironment in chondrogenic phenotype differentiation of bone marrow mesenchymal stem cells (BMSCs) of rat in vitro. Methods BMSCs were harvested from 4-week-old female Sprague Dawley rats. BMSCs at passage 2 were divided into 4 groups according different culture conditions: normoxia control group (group A), normoxia and BMP-2 group (group B), hypoxia control group (3% oxygen, group C), and hypoxia and BMP-2 group (group D). Then the cellular morphology was observed under inverted phase contrast microscope. Alcian blue immunohistochemical staining was used to detect the glycosaminoglycans (GAG), Western blot to detect collagen type II and hypoxia-inducible factor 1α (HIF-1α), and RT-PCRto detect the expressions of chondrogenic related genes, osteogenic related genes, and hypoxia related genes. Results At 21 days after induction of BMP-2 and hypoxia (group D), BMSCs became round, cell density was significantly reduced, and lacuna-l ike cells were wrapped in cell matrix, while the changes were not observed in groups A, B, and C. Alcian blue staining in group D was significantly bluer than that in other groups, and staining became darker with induction time, and the cells were stained into pieces of deeply-stained blue at 21 days. Light staining was observed in the other groups at each time point. The expression level of collagen type II protein in group D was significantly higher than those in other groups (P lt; 0.05). HIF-1α protein expression levels of groups C and D were significantly higher than those of groups A and B (P lt; 0.05). The expressions of collagen II α1 (COL2 α1) and aggrecan mRNA (chondrogenic related genes) were highest in group D, while the expressions of COL1 α1, alkaline phosphatase, and runt-related transcri ption factor 2 mRNA (osteogenic related genes) were the highest in group B (P lt; 0.05). Compared with groups A and B, HIF-1α (hypoxic related genes) in groups C and D significantly increased (P lt; 0.05). Conclusion BMP-2 combined with hypoxia can induce differentiation of BMSCs into the chondrogenic phenotype, and inhibit osteoblast phenotype differentiation. HIF-1α is an important signaling molecule which is involved in the possible mechanism to promote chondrogenic differentiation process.
In this study, we aimed to investigate the influences of conditioned medium from human umbilical vein endothelial cells (HUVEC) on cancer stem cell phenotype of human hepatoma cells. HUVEC and human hepatoma cells (MHCC97H) were cultured, respectively, and then the MHCC97H cells were co-cultured with conditioned medium from HUVEC (EC-CM) with Transwell system. Anti-cancer drug sensitivity, colony-formation, migration/invasion ability, expression of cancer stem cell marker and sphere formation were performed to determine the cancer stem cell phenotype in MHCC97H cells. We found that MHCC97H cells co-cultured with EC-CM exhibited significantly higher colony-formation ability and lower sensitivity of anti-cancer drugs 5-FU and Cis. Transwell assay showed that treatment with EC-CM obviously increased migration and invasion of MHCC97H cells. Moreover, increased sphere forming capability and expression of CD133 in MHCC97H cells were observed after co-cultured with EC-CM. These results suggested that EC-CM could promote cancer stem cell phenotype of hepatoma cells.
ObjectiveTo summarize the research progress of hepatocellular carcinoma (HCC) based on tumor microenvironment immunophenotyping.MethodThe related literatures of basic and clinical studies on HCC immunophenotyping in the recent years were reviewed.ResultsHCC could be divided into different immunophenotypes based on tumor microenvironment, and it showed different immune molecular characteristics, immune cell infiltration characteristics, and anti-tumor ability. At the same time, the HCC immunophenotype was significantly associated with patients’ survival and had been proved to be able to better evaluate the prognosis of HCC patients. According to the relevant molecular characteristics in the HCC immune microenvironment, it could provide guidance for the drug regimen of immunotherapy.ConclusionHCC immunophenotyping is still in the early stage of research, and its clinical application value has been preliminarily shown for the evaluation of patients’ prognosis and immunotherapy decision-making, which is a new idea of individualized treatment of HCC in the future.
ObjectiveTo explore the relationship of obesity with asthma control and airway inflammatory phenotype. MethodsA cross-sectional prospective study was conducted on 101 patients with asthma. Asthma control level was assessed by Asthma Control Test (ACT) and GINA. Furthermore, height and weight were measured and body mass index (BMI) was calculated. Lung function and sputum induction were performed, and differential cell count was obtained from induced sputum and peripheral blood. ResultsNinety eligible patients were divided into 3 groups as a normal-weight group (n=54), an over-weight group (n=21) and an obesity group (n=15). The asthma control levels were different among three groups (P=0.019 for ACT and P=0.014 for GINA, respectively). BMI was positively related to the number of neutrophils in induced sputum (r=0.29, P=0.039). Increased BMI deteriorated asthma control levels assessed by ACT[OR=1.84, 95% CI (1.04, 3.23), P=0.035] and GINA[OR=2.27, 95% CI (1.27, 4.07), P=0.006] in a dose-response manner. Obesity indicated poor asthma control assessed by ACT (P=0.015) and GINA (P=0.008) after adjusting for age, sex, duration of asthma, FEV1%pred, smoking, and the number of neutrophils in peripheral blood. ConclusionsIn Chinese individuals with asthma, neutrophilic inflammatory phenotype dominates the airway inflammation of obesity-associated asthma. Obesity is a risk factor that deteriorates asthma control level in a significant dose-response manner.
ObjectiveTo study the relation between the clinical phenotype and neurological developmental quotient in children with epilepsy and GPR98 gene mutation. MethodsGenomic DNA was extracted from peripheral blood lymphocytes of the probands and other available members in the epilepsy families.Clinical datas and screened for mutations by next-generation sequencing conbined target sequencing technology and PCR and direct DNA sequencing were collected.Then, the relations between the clinical phenotype and developmental quotient in children with epilepsy and GPR98 gene mutation was analyzed. ResultsSeven novel GPR98 gene mutations were found in seven probands in 65 families, including six heterozygote missense mutations (c.6083C <、c.1969A < C、c.17531C < T、c.9069G < C、c.6661G < A and c.18496A < C) and one nonsense mutation (c.14224G < T). One of their parents carried the same GPR98 gene mutation as the proband's. The initial symptom of six cases was afebrile seizures and one showed febrile seizure, in which the main type seizure was generalized seizure.Moreover, was were significant difference between children with epilepsy and GPR98 gene mutations and healthy children in developmental quotient test(P < 0.01). ConclusionsThe main type of seizures in children with epilepsy and GPR98 gene mutations is generalized seizure. Furthermore, GPR98 gene mutations may be associated with psychomotor retardation.