Objective To observe the effect of pilose antler polypeptides(PAP)on the apoptosis of rabbit marrow mesenchymal stem cells (MSCs) differentiated into chondrogenic phenotype by interleukin 1β (IL-1β) so as to optimize the seeding cells in cartilage tissue engineering. Methods The MSCs were separated from the nucleated cells fraction of autologus bone marrow by density gradient centrifuge and cultured in vitro. The MSCs were induced into chondrogenic phenotype by transforming growth factor β1(TGF-β1) and basic fibroblast growth factor(bFGF). According to different medias, the MSCs were randomly divided into four groups: group A as black control group, group B(100 ng IL-1β)，group C(10 μg/ml PAP+100 ng IL-1β) and group D(100 ng/ml TGF-β1 +100 ng IL-1β). The samples were harvested and observed by morphology, flow cytometry analysis, RT-PCR and ELISA at 24, 48 and 72 hours. Results The intranuclear chromatin agglutinated into lump and located under nulear membranes which changed into irregular shapeat 24 hours. The intranuclear chromatin agglutinated intensifily at 48 hours. Then the nucear fragments agglutinated into apoptosic corpuscles at 72 hours in group B. The structure change of cells in groups C and D was later than that in group B, and the number of cells changed shape was fewer than that in group B. The structure change of cells in group A was not significant. The apoptosic rate of cells, the mRNA expression of Caspase-3 and the enzymatic activity of Caspase-3 gradually increased in group B, and there were significant differences compared with groups A,C and D(Plt;0.01). Conclusion Caspase-3 is involved in aoptosis of the MSCs differentiated into chondrogenic phenotype cultured in vitro. PAP could prevent from or reverse apoptosis of these MSCs by decreasing the expression of Caspase-3 and inhibiting the activity of Caspase-3.
ObjectiveTo observe and analyze the clinical phenotype and genetic characteristics of COL2A1 and COL11A1 de novo mutation (DNM) related Stickler syndrome type Ⅰ and Ⅱ patients. MethodsA family-based cohort study. From December 2023 to November 2024, 4 patients (all probands) with Stickler syndrome diagnosed by clinical and genetic testing in Department of Ophthalmology of People's Hospital of Ningxia Hui Autonomous Region and their parents (8 cases) were included in the study. The patients came from 4 unrelated families. A detailed medical history was taken, and the patients underwent best-corrected visual acuity (BCVA), refraction, and fundus color photography examinations. Systemic examinations included the oral and facial regions, skeletal, joints, and hearing. Peripheral venous blood samples were collected from the patients and their parents, and genomic DNA was extracted. Whole-exome sequencing was used to screen for pathogenic genes and their loci, which were then validated by Sanger sequencing and combined with segregation analysis in the families to identify candidate gene mutation sites. The candidate variants were assessed for pathogenicity according to the American College of Medical Genetics and Genomics (ACMG) criteria and guidelines for the classification of genetic variants. Additionally, cross-species conservation analysis was performed to determine the evolutionary conservation of wild-type amino acids, and protein three-dimensional modeling techniques were used to characterize the spatial conformational changes of the variant proteins and the alterations in their local hydrogen bond networks. ResultsAmong the 4 patients, there were 2 males and 2 females; their ages ranged from 3 to 12 years. There were 2 cases of Stickler syndrome type Ⅰ (proband of families 1 and 2) and 2 cases of type Ⅱ (proband of families 3 and 4). The diopters ranged from −8.00 to−18.00 D. BCVA ranged from no light perception to 0.6-. There were 2 cases each of vitreous membrane-like and “bead-like” opacity. Three cases showed peripapillary atrophy arcs and leopard pattern changes in the retina; one case had bilateral retinal detachment with a large macular hole in the left eye, which had previously been treated with vitrectomy surgery. One case had bilateral sensorineural hearing loss. There were 3 cases of simple micrognathia; one case had a flat nasal bridge, short nose, midface depression, and micrognathia. Two cases had excessive elbow joint extension. The phenotypes of the parents of the 4 patients were normal. Genetic testing results revealed that the probands of families 1 and 2 carried COL2A1 gene c.85+1G>C (M1) splice site variant and c.3950_3951insA (p.M1317Ifs*48) (M2) frameshift variant, respectively; the probands of families 3 and 4 carried COL11A1 gene (NM_001854.4) c.2549 G>T (p.G850V) (M3) missense variant and c.3816+6T>C (M4) splice site variant, respectively. The parents did not carry the related gene variants. Among them, M2, M3, and M4 are newly reported DNM. According to the ACMG guidelines, they were all considered likely pathogenic. The cross-species conservation analysis results showed that the wild-type amino acid of the COL11A1 gene M3 missense variant was highly conserved across multiple different species. Protein local structure modeling analysis revealed that the COL2A1 gene M2 frameshift variant and the COL11A1 gene M3 missense variant significantly altered the tertiary structure conformation of the protein, leading to abnormal spatial arrangement and hydrogen bond network in the key functional domains ConclusionThe COL2A1 gene M1 splice site variant, M2 frameshift variant, and the COL11A1 gene M3 missense variant, M4 splice site variant are respectively the potential pathogenic genes for families 1, 2, and families 3, 4; leading to the onset of Stickler syndrome type Ⅰ in families 1 and 2, and type Ⅱ in families 3 and 4.
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.
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 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.
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 observe the relationship of serum levels of homocysteine (HCY) and chemokine C-C motifligand 2 (CCL2) with cognitive impairment in COPD patients with different degrees of emphysema. Methods Sixty-twoCOPD patients identified according to emphysema phenotype classification and admitted from January 2016 to March 2017 were recruited in the study. There were 37 cases in emphysema 1-2 grade and 25 cases in emphysema 3-4 grade. Simultaneous 30 healthy subjects undergoing physical examination were recruited as control. Montreal cognitive assessment (MoCA) scale investigation and serum HCY and CCL2 test were completed. Relationship analysis was conducted on serum HCY, CCL2 levels with cognitive impairment in the COPD patients with different degrees of emphysema. Results Compared with the 1-2 grade subgroup, the PaO2 was lower, PaCO2 was higher, the plasma HCY and CCL2 levels increased in the 3-4 grade subgroup with significant differences (all P<0.05). MoCA total score and subscores were relatively low in the COPD group with emphysema than the control group (except visuospatial ability scores in the 1-2 grade subgroup). MoCA scores were statistically lower in the 3-4 grade subgroup than those in the 1-2 grade subgroup (allP<0.05). Correlation analysis showed that HCY and CLL2 levels were negatively correlated with MoCA scores and subscores (P<0.01), and HCY and CLL2 were positively correlated (bothP<0.01). The area under the receiver operating characteristic curve of HCY and CLL2 for evaluating cognitive impairment was 0.79 and 0.97, respectively. Conclusion In patients with different degrees of emphysema phenotype, serum HCY and CCL2 levels are increased in different degree, and the degree of emphysema is closely related with cognitive dysfunction.
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 study the clinicopathological features of mediastinum nodular sclerosis Hodgkin lymphoma (NSHL) in order to improve the recognition of it. MethodsThe clinical data of 3 cases of mediastinum NSHL between 2003 and 2012 were collected. Then we analyzed the carcinoma pathologic samples by pathomorphology, immunophenotypic phenotype, related gene rearrangement and situ hybridization with EBER. ResultsThe pathomorphologic results showed that broad fibrotic bands subdivided the lymphoid parenchyma into large nodules, the tumoral cells had distinct boundary with empty cytoplasm and small-to-medium-sized nucleoli, and the nodules contained inflammatory cell components. The immunophenotypic phenotype of the tumoral cells were CD15, CD30, PAX-5 and CD20 partly, but anaplastic lymphoma kinase, CD45, cytokeratin, CD79α and S-100 were not expressed. T cell receptor γ and IgH gene were no rearranged, and EBER in situ hybridization was not detected. ConclusionVarious lymphomas occur in the mediastinum and mediastinum NSHL is just one of them. Mastering its distinctive pathomorphology and immunophenotypic phenotype is highly significant for diagnosis, differential diagnosis and treatment of the disease.
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.