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Objective To construct a universal influenza mRNA vaccine and evaluate its immunogenicity.Methods The antigen sequence of hemagglutinin(HA),nucleoprotein(NP)and matrix protein 2 ectodomain(M2e)in influenza A/California/04/2009 was optimized.HA,NP and 3 tandem M2e(3M2e)were cloned into pcDNA3.1 vector,respectively.Then the mRNAs were synthesized by linearization,in vitro transcription,enzymatic capping and enzymatic tailing,and named as mRNA-HA,mRNA-NP and mRNA-3M2e,respectively.The protein expression of the 3 kinds of mRNAs in 293T cells was detected by immunofluorescence assay.Comb-mRNA vaccine was prepared by enveloped mRNA-HA,mRNA-NP and mRNA-3M2e with lipid nanoparticles,respectively,and the particle size and potential were identified.Twenty-eight 6-week-old female BALB/c mice(18～22 g)were randomly divided into LNP group(n=14)and Comb-mRNA group(n=14).Hemagglutination inhibition(HI)method and microneutralization(MN)test were used to evaluate the serum antibody titer induced by Comb-mRNA vaccines.The mice were infected by 5LD50 wild-type H1 N1 influenza virus to evaluate the protective efficacy.Results The mRNA-HA,mRNA-NP and mRNA-3M2e were successfully constructed,and the 3 mRNAs could be expressed in 293T cells.The average size of mRNA encapsulated by lipid nanoparticles was 119.53±6.5 nm,and the average potential was-8.23±1.3 mV.The geometric mean titer(GMT)of HI and MN in the Comb-mRNA group were 179.6 and 201.6,compared with the LNP group.The ratio of IFN-γ+CD4+/CD8+Tcells was increased.The Comb-mRNA group could provide protection against 5LD50 wild type influenza H1 N1 virus after 2 weeks of booster immunization.Conclusion Comb-mRNA,an influenza vaccine candidate,can induce immune responses and protect mice from influenza virus challenge.
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Objective:To investigate the expression of nuclear matrix protein 4 (NMP4) in hepatocellular carcinoma (HCC), and its relationship with clinicopathological features and survival prognosis of patients.Methods:The clinical data of 100 HCC patients who were treated with radical resection of liver cancer in the Department of Hepatobiliary Surgery of the Third Affiliated Hospital of Wenzhou Medical University from July 1, 2014 to July 1, 2019 were retrospectively analyzed. There were 63 males and 37 females, aged (58.5±10.4) years old. Immunohistochemical method was used to detect the expression of NMP4 protein in HCC cancer tissue and the corresponding adjacent normal tissue. According to the expression of NMP4 in HCC tissues, 100 patients were divided into two groups: the NMP4-positive expression group ( n=62) and the NMP4-negative expression group ( n=32). Univariate analysis was performed on the relationship between NMP4 expression and clinical pathological features as well as overall survival of HCC patients. Cox multivariate analysis was performed on the factors influencing postoperative prognosis of HCC patients. Results:Immunohistochemistry results showed that NMP4 was primarily expressed in the nucleus, the positive expression rate of NMP4 in HCC tissues was higher than that in adjacent non-cancerous tissues [62.0% (62/100) vs. 8.0%(8/100)], and the difference was statistically significant ( χ2=2.12, P=0.003). Univariate analysis revealed that the overall survival of HCC patients was correlated with the degree of tumor differentiation, tumor length, BCLC stage, number of tumor foci, vascular tumor thrombus and expression of NMP4 (all P<0.05). Cox multivariate analysis revealed that low differentiation, high BCLC stage (stage C), number of tumor foci (≥3), and positive expression of NMP4 were independent risk factors affecting postoperative survival and recurrence-free survival of HCC patients. The median overall survival and median recurrence-free survival of HCC patients in the NMP4-positive expression group were 22.3 months and 11.5 months, respectively. In contrast, that in the NMP4-negative expression group were 40.6 months and 19.4 months, respectively. The cumulative survival rate and recurrence-free survival rate of HCC patients in the NMP4-positive expression group were lower than those in the NMP4-negative expression group, and the differences were statistically significant (both P<0.05). Conclusion:Positive NMP4 expression was closely correlated with malignant biological progression and poor prognosis of HCC patients.
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@#Objective To prepare murine and rabbit polyclonal antibodies against rabies virus(RV) matrix(M) protein and compare their reactivity.Methods The prokaryotic expression vector pET-28a-M was constructed by using the cDNA of cells infected with RV CVS-11 strain as template,then transformed into E.coli BL21(DE3),and the induced by IPTG to express M protein.After nickel column affinity chromatography and dialysis renaturation,female BALB/c mice and New Zealand white rabbits were immunized with the M protein,and the whole blood was taken to separate the serum.The titers of the murine and rabbit polyclonal antibodies were detected by ELISA,and the reactivity was measured by Western blot,indirect immunofluorescence assay(IFA) and immunoprecipitation(IP).Results The plasmid pET-28a-M was constructed correctly as identified by sequencing.The titers of murine and rabbit polyclonal antibodies were 1:100 and 1:256 000respectively,and the polyclonal antibodies had reactivity with different RV strains.Conclusion The murine and rabbit polyclonal antibodies against M protein were successfully prepared,which provides important biological tools for exploring the interaction between M protein and host protein as well as studying the pathogenesis of RV.
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Objective @#To study the effect of light-emitting diode (LED) red light on the osteogenic/odontogenic differentiation of human dental pulp stem cells (hDPSCs) and its mechanism were discussed. @*Methods@#This study has been reviewed and approved by the Ethics Committee. hDPSCs were cultured by tissue block enzyme digestion. The proliferative capacity of hDPSCs was detected by the CCK-8 at days 1, 3, 5 and 7 under stimulation with 0, 1, 5 and 10 μg/mL lipopolysaccharide (LPS), and the LPS stimulatory concentration was screened. The CG group (mineralization induction), LPS+CG group, and LPS+CG+ (2, 4, 6, 8, and 10 J/cm2) LED red light groups were set. On day 7, alkaline phosphatase (ALP) staining and ALP activity were determined. Relative expression levels of the ALP, osterix (OSX), dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP) genes were measured by qRT-PCR. On day 21, alizarin red staining and calcium nodule quantitative determination were performed to screen the best light energy. The LPS+CG group and LPS+CG+LED group (optimal energy) were set up, and the secretion and expression levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were detected by ELISAs on days 1, 3, 5 and 7. The relative expression levels of the extracellular regulated protein kinases 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and extracellular regulated protein kinases 5 (ERK5) proteins and their phosphorylated proteins in the MAPK signaling pathway were detected by Western blots. After the pathway was blocked, the relative expression levels of the ALP, OSX, DMP-1, and DSPP proteins after LED red light irradiation on day 7 were detected by Western blots.@*Results@# CCK-8 assays showed that the proliferation of hDPSCs induced by 10 μg/mL LPS was lower than that of the 0, 1, and 5 μg/mL groups on the 5th and 7th days (P<0.05), and 10 μg/mL was selected as the LPS stimulatory concentration in the follow-up experiment. ALP staining, ALP activity, gene expression levels of ALP, OSX, DMP-1 and DSPP and calcium nodule quantification in the LPS+CG+4 J/cm2 group were higher than those in the other treatment groups (P<0.05). 4 J/cm2 LED red light had the strongest ability to promote osteogenic/odontogenic differentiation and was used as the LED light energy density in subsequent experiments. ELISA showed that the secretion and expression levels of TNF-α and IL-1β in the LPS+CG+LED group were lower than those in the LPS+CG group on the 5th and 7th days (P<0.05). Western blot analysis showed that 4 J/cm2 LED red light promoted the expression levels of the p-ERK1/2, p-p38, p-JNK and p-ERK5 proteins. After the MAPK pathway was blocked, the expression levels of the ALP, OSX, DMP-1, and DSPP proteins in the LPS+CG+LED+U0126 (ERK1/2 inhibitor), SP600125 (JNK inhibitor), and BIX02189 (ERK5 inhibitor) groups were lower than those in the LPS+CG+LED group (P<0.001). The protein expression levels of ALP, OSX and DMP-1 in the LPS+CG+LED+SB203580 (p38 inhibitor) group were not significantly different from those in the LPS+CG+LED group (P>0.05).@*Conclusion@#In inflammatory conditions, LED red light promotes osteogenic/odontogenic differentiation of hDPSCs. This effect may be attributed to enhancement of the ERK1/2, JNK, and ERK5 signaling pathways, which reduces the production of the inflammatory cytokines TNF-α and IL-1β.
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Dermatomyositis(DM)is an autoimmune disease often complicated with malignant tumors.More than 50%of DM patients have myositis specific autoantibodies in their bodies.DM specific autoantibodies[including anti-migration inhibitory factor(Mi)-2 antibody,anti-nuclear matrix protein(NXP)-2 antibody,anti-transcription intermediary factor(TIF)1-γ antibody,and anti-small ubiquitin like modifier activating enzyme(SAE)antibody]play important roles in the pathogenesis of malignancy associated DM.Revealing the role of DM specific autoantibodies in the development of malignant tumors in DM patients can provide important evidence for accurately assessing the risk of developing malignant tumors in DM patients,and also provide new ideas for clinical diagnosis of DM and precise treatment.
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Objective To explore the effects of transurethral en-blocresection of bladder tumor(TeURBT)on urination function,serum chitinase protein 40(YKL-40),bladder specific nuclear matrix protein-1(BLCA-1),and p-oxyfossase-1(PON-1)levels in patients with non-muscle invasive bladder cancer(NMIBC).Methods 74 patients with NMIBC admitted to our Hospital from January 2019 to May 2022 were divided into TeURBT group and transurethral resection of bladder tumor(TURBT)group by random lottery,with 37 cases in each group.The effective tumor clearance rate,surgery and urination function indexes were compared between the two groups.Blood samples were collected before surgery,3 months,6 months and 12 months after surgery to determine the levels of serum YKL-40,BLCA-1 and PON-1,and the recurrence rate was analyzed at 12 months of follow-up.Results The effective resection rate of bladder tumor was 100.00％in both groups.The operative time in TeURBT group was significantly longer than that in TURBT group(P＜0.05),and the intraoperative blood loss,catheter indwelling/bladder irrigation/hospitalization time in TeURBT group were significantly less than those in TURBT group(P＜0.05).Urinary output and maximum urinary flow per second in both groups significantly increased on postoperative day 14(P＜0.05),but there was no statistically significant difference in the above indexes between the two groups on postoperative day 14(P＞0.05).The levels of serum YKL-40 and BLCA-1 in the two groups at 3,6 and 12 months after surgery significantly decreased(P＜0.05),while the level of PON-1 significantly increased(P＜0.05).There were no statistically significant differences in serum YKL-40 and PON-1 levels between the two groups at 3 and 6 months after surgery(P＞0.05),while serum YKL-40 and BLCA-1 levels in the TeURBT group were significantly lower than those in the TURBT group at 12 months after surgery(P＜0.05),and PON-1 levels were significantly higher than those in the TURBT group(P＜0.05).The stages in the TeURBT group could be accurately diagnosed after operation,and the postoperative pathological stages were Ta stage in 24 cases and T1 stage in 13 cases.The total incidence of postoperative complications in TeURBT group and cumulative incidence of recurrence in 1-year follow-up were 5.40％,which was significantly lower than 24.32％and 21.62％in TURBT group(P＜0.05).Conclusion The effective resection rate of TeURBT and TURBT in the treatment of NMIBC tumor is similar,which can effectively improve the urination function of patients,downregulation of serum YKL-40 and BLCA-1 levels,and up-regulation of PON-1 levels.Compared with TURBT,TeURBT has advantages of less intraoperative blood loss,fewer postoperative complications,faster recovery,and lower recurrence rate.
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Matrix protein p17 is a structural protein of human immunodeficiency virus（HIV）. It not only plays a key role in multiple stages of HIV life cycle，but also is closely associated with HIV-related lymphoma，neurocognitive impairment and breast cancer. This article reviews the role of matrix protein p17 in HIV infection and HIV-related diseases.
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Objective:To explore the changes of immune microenvironment and prognosis of bladder cancer patients with positive urinary nuclear matrix protein 22 (NMP22).Methods:Retrospective analysis was made on 86 patients who were diagnosed with bladder cancer in Xuzhou Central Hospital from January 2019 to September 2020. All patients were tested for urinary NMP22 by colloidal gold method. The patients with positive test results were NMP22 positive group, and the patients with negative test results were NMP22 negative group. The expression of CD8, programmed cell death-ligand 1 (PD-L1), programmed cell death protein-1 (PD-1) and PanCK were detected by multiple fluorescent immunohistochemical method on the pathological tissue sections of all enrolled patients with bladder cancer after surgery. Follow-up data of enrolled patients were collected after discharge, and univariate and multivariate Cox analysis was performed on the follow-up data.Results:There were 69 patients in the NMP22 positive group and 17 patients in the NMP22 negative group. The percentage of CD8 and PD-L1 positive cells in NMP22 positive group was significantly higher than that in NMP22 negative group, and the difference was statistically significant (all P<0.05). Univariate analysis showed that tumor stage was correlated with bladder cancer progression ( HR=2.67, P=0.017). Multivariate analysis showed that positive NMP22 was significantly correlated with bladder cancer recurrence and disease progression (all P<0.05). Conclusions:The density of CD8 + T cells and PD-L1 in tumor parenchyma of urinary NMP22 positive bladder cancer patients was higher than that of NMP22 negative patients. Urinary NMP22 positive can be one of the bad prognostic factors of bladder cancer, and the patients with NMP22 positive should strengthen reexamination.
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{L-End}Objective To analyze the local muscle response under continuous ergonomic workload by simulating manual lifting, and to screen the sensitive metabolic biomarkers during fatigue process. {L-End}Methods A total of 13 healthy male volunteers were selected as the study subjects using simple random sampling method. Study subjects underwent repetitive simulated manual lifting for four periods (T1 to T4), each lasting 12 minutes. The degree of work-related fatigue in the forearm, upper arm, shoulder, back, and leg muscles, and the whole body was accessed using Borg 6-20 Rating of Perceived Exertion (RPE) Scale. The venous blood samples were collected from elbow between each two periods to detect the levels of eight metabolic biomarkers: ammonia, lactate, creatine kinase, lactate dehydrogenase (LDH), C-terminal telopeptide of type Ⅰ collagen (CTX-Ⅰ), C-terminal telopeptide of type Ⅱ collagen (CTX-Ⅱ), cartilage oligomeric matrix protein (COMP), and calcium ions. {L-End}Results The RPE scores of the study subjects for the muscles of five body parts and the whole body increased with the increasing lifting periods (all P<0.01). Fatigue was observed in all target muscles, with overall body fatigue occurring in the T2 period. The levels of ammonia, lactate, creatine kinase, LDH, COMP, and calcium ions in the serum of study subjects were higher in the T1 to T4 periods than in the T0 period (all P<0.05). The serum CTX-Ⅰ level was higher in the T1 and T3 periods than that in the T0 period (all P<0.05) , and the serum CTX-Ⅱ level was higher in the T1, T2 and T4 periods than that in the T0 period (all P<0.05). The level of these eight serum metabolic biomarkers fluctuated during the T1 to T4 periods. The serum creatine kinase level increased with the period of lifting (all P<0.05). The serum lactate level was higher in the T3 period than those in the T1 and T2 periods (all P<0.05). The serum LDH and calcium ion levels were higher in the T2 to T4 periods than that in the T1 period (all P<0.05). The serum COMP level was higher in the T2 and T3 periods than that in the T1 period (all P<0.05). Except for CTX-Ⅰ, the levels of other seven metabolic markers in serum were higher in individuals after fatigue than before fatigue (all P<0.05). {L-End}Conclusion Serum metabolic biomarkers such as ammonia, lactate, creatine kinase, calcium ions, LDH, CTX-Ⅱ, and COMP exhibit significant changes before and after fatigue. These metabolic biomarkers could be used as sensitive biomarkers for evaluating muscle fatigue during repetitive works.
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COVID-19 is caused by coronavirus SARS-CoV-2. Current systemic vaccines generally provide limited protection against viral replication and shedding within the airway. Recombinant VSV (rVSV) is an effective vector which inducing potent and comprehensive immunities. Currently, there are two clinical trials investigating COVID-19 vaccines based on VSV vectors. These vaccines were developed with spike protein of WA1 which administrated intramuscularly. Although intranasal route is ideal for activating mucosal immunity with VSV vector, safety is of concern. Thus, a highly attenuated rVSV with three amino acids mutations in matrix protein (VSVMT) was developed to construct safe mucosal vaccines against multiple SARS-CoV-2 variants of concern. It demonstrated that spike protein mutant lacking 21 amino acids in its cytoplasmic domain could rescue rVSV efficiently. VSVMT indicated improved safeness compared with wild-type VSV as the vector encoding SARS-CoV-2 spike protein. With a single-dosed intranasal inoculation of rVSVΔGMT-SΔ21, potent SARS-CoV-2 specific neutralization antibodies could be stimulated in animals, particularly in term of mucosal and cellular immunity. Strikingly, the chimeric VSV encoding SΔ21 of Delta-variant can induce more potent immune responses compared with those encoding SΔ21 of Omicron- or WA1-strain. VSVMT is a promising platform to develop a mucosal vaccine for countering COVID-19.
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Ebola virus (EBOV) is an enveloped negative-sense RNA virus and a member of the filovirus family. Nucleoprotein (NP) expression alone leads to the formation of inclusion bodies (IBs), which are critical for viral RNA synthesis. The matrix protein, VP40, not only plays a critical role in virus assembly/budding, but also can regulate transcription and replication of the viral genome. However, the molecular mechanism by which VP40 regulates viral RNA synthesis and virion assembly/budding is unknown. Here, we show that within IBs the N-terminus of NP recruits VP40 and is required for VLP-containing NP release. Furthermore, we find four point mutations (L692A, P697A, P698A and W699A) within the C-terminal hydrophobic core of NP result in a stronger VP40-NP interaction within IBs, sequestering VP40 within IBs, reducing VP40-VLP egress, abolishing the incorporation of NC-like structures into VP40-VLP, and inhibiting viral RNA synthesis, suggesting that the interaction of N-terminus of NP with VP40 induces a conformational change in the C-terminus of NP. Consequently, the C-terminal hydrophobic core of NP is exposed and binds VP40, thereby inhibiting RNA synthesis and initiating virion assembly/budding.
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Humanos , Ebolavirus/fisiología , Células HEK293 , Células HeLa , Proteínas de la Nucleocápside/metabolismo , ARN Viral/metabolismo , Proteínas de la Matriz Viral/metabolismo , Virión/metabolismo , Ensamble de VirusRESUMEN
Objective@#To analyze changes in proteoglycan and its correlation with alveolar bone resorption in periodontitis. @*Methods @#Twelve eight-week-old C57BL/6J male mice were selected, and the periodontitis model was established by ligating the right maxillary second molar with 6-0 silk thread. The nonligated part of the left maxilla was used as the control. The mice were killed 14 days after the operation. Micro-CT was used to assess alveolar bone resorption. HE staining was used to observe the alveolar bone profile, and TRAP staining was conducted to examine the positive rate of osteoclasts. The expression of proteoglycan-related genes, such as aggrecan (ACAN), biglycan (BGN), versican (VCAN), decorin (DCN), osteoclast-related genes, such as cathepsin K (CTSK), matrix metalloprotein-9 (MMP-9), and receptor activator of nuclear factor kappa-B ligand (RANKL), and inflammation-related genes, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), was detected by real-time quantitative PCR. Additionally, the correlation of the expression of proteoglycans with osteoclast-related genes and inflammation-related genes was evaluated by Pearson correlation analysis.@* Results@#The resorption of alveolar bone on the periodontitis side increased. TRAP staining showed that the number of osteoclasts was substantially increased in the maxilla with periodontitis. Real-time quantitative PCR demonstrated that compared with the control side, the expression of proteoglycan-related genes, such as ACAN, BGN, and DCN, was decreased, whereas the expression of the VCAN gene was significantly increased in the periodontitis side. Meanwhile, the expression of osteoclast-related genes, such as CTSK, MMP-9, and RANKL, and inflammation-related genes, such as IL-1β, IL-6, and TNF-α, was markedly increased in the periodontitis side (P<0.05). Pearson correlation analysis indicated a negative correlation between the expression of proteoglycans and the mRNA levels of osteoclast-related genes and inflammation-related genes (P<0.05). @*Conclusion @#The expression of proteoglycan was closely related to alveolar bone resorption in a periodontitis model.
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Objective:To evaluate the immunogenicity of a novel influenza virus mRNA vaccine based on conserved antigens delivered by lipopolyplex (LPP) platform in a mouse model.Methods:Four copies of genes coding for extracellular domain of matrix 2 protein (M2e) and nucleoprotein (NP) of influenza A virus were synthetized after codon optimization. The fusion antigens were transcribed in vitro and delivered by LPP platform, named as LPP-4M2eNP. Expression of M2e and NP in eukaryotic cells was detected by immunofluorescence assay (IFA). BALB/c mice were inoculated intramuscularly twice with 10 μg or 30 μg LPP-4M2eNP vaccine at an interval of four weeks. Antibody response was detected by ELISA and cellular-mediated immunity (CMI) was detected by enzyme-linked immunospot assay (ELISPOT). Results:IFA showed that NP and M2e were expressed correctly in eukaryotic cells. Single dose immunization could induce significant antigen (NP, M2e)-specific CMI and antigen (NP, M2e)-specific antibody response was induced in mice with Th1 type bias after boost immunization. Moreover, NP-specific CMI was increased significantly after the second immunization, while no significant change in M2e-specific CMI was observed.Conclusions:Stronger CMI was triggered in mice by single dose of LPP-4M2eNP vaccine. Furthermore, robust humoral and cellular immune responses were induced after boost immunization. This study suggested that LPP-4M2eNP vaccine, which based on conserved antigen of influenza A and delivered by LPP platform, had great potential for development and application.
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In the present study, the clinical features of a patient with autosomal recessive hypophosphatemic rickets 1 caused by dentin matrix protein 1(DMP1)gene mutation and her family members were investigated. DMP1 gene from peripheral blood was sequenced by Sanger sequencing, and the known mutation was verified among her family members and 250 healthy populations. The proband was a 42-year-old female with bone deformity of both lower limbs, bone pain, and short stature. The results of X-rays and laboratory examination were consistent with the hypophosphatemic rickets reported before. A homozygous mutation(c.2T> C)in DMP1 was identified by Sanger sequencing in the proband, her son and daughter were heterozygous for c. 2T> C.
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Background:Cartilage oligomeric matrix protein (COMP), is an extracellular matrix (ECM) non-collagenous glycoprotein that is mainly localized within the cartilage, and also be found in tendon and synovium.RecentstudiesinwestandAsiaPacificregionhasshownthatCOMP, is a prognostic marker in Rheumatoid arthritis(RA).Objective:To correlate serum COMP levels with disease severity and cartilage destruction in rheumatoid arthritis.Methods:The study was conducted in Department of Pathology and Rheumatology, Ziauddin University Hospital, Karachi from June 2018 to May 2019. Patients were recruited as per American College of Rheumatology (ACR) 2010 classification criteria. The study populationconsists of 88 healthy subjects and 88 RA patients. Sandwich ELISA technique was used to assess serum COMP level. Other inflammatory markers such as erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) antibodies like rheumatoid factor, and anti-cyclic citrullinated protein (anti-CCP) were also assessed. Results were analyzed using SPSS-20 and P-value ≤0.05 was considered as significant.Results: Serum COMP levels were significantly higher in RA patients 51.35ng/ml than controls 21.454ng/ml with significant p value=<0.0001. There was strong positive correlation between COMP level and disease severity in RA patients with moderate as well as high disease activity score (DAS) with significant p value. Serum COMP showed 96% sensitivity and 83% specificity at level of 27.01ng/ml for diagnosis of RA.Conclusions:COMP has significant positive correlation with severity of RA. Serum COMP can be utilized as a biomarker to quantify cartilage destruction in RA patients
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BACKGROUND: Bisphosphonates are a novel inhibitor of bone resorption that can inhibit the activity and function of osteoclasts. OBJECTIVE: To observe the effects of sodium ibandronate on the expression of dentin matrix protein 1, Caspace3, Bcl-2 and Bax in condylar cartilage in osteoporosis rats. METHODS: Thirty-six female rats were randomly divided into sham group, osteoporosis group and sodium ibandronate group, twelve in each group. The sham group did not excise ovaries during surgery. Bilateral ovaries of rats were removed in the osteoporosis and sodium ibandronate groups. On the 7th day after operation, rats in the sodium ibandronate group were intraperitoneally given sodium ibandronate 10 µg/kg, once every 7 days. After 90 days, the rat ovaries were taken. Bone mineral density was measured in each group. The changes of condylar cartilage were observed by toluidine blue staining and TUNEL staining. The expression of dentin matrix protein 1 protein was detected by immunohistochemistry. The levels of Caspase3, Bcl-2 and Bax were detected by western blot assay. The study protocol was approved by the Animal Ethics Committee of Nanhua Hospital in China with the approval No. SLXD_201804010. RESULTS AND CONCLUSION: Compared with the sham group or sodium ibandronate group, the bone mineral density in the osteoporosis group was significantly decreased (P < 0.05). The results of toluidine blue staining showed that the hypertrophic layer of condylar cartilage in the sodium ibandronate group was significantly thicker than that in the osteoporosis group. Compared with the sham group or sodium ibandronate group, the number of apoptotic cells in condylar cartilage and subchondral bone increased significantly in the osteoporosis group (P < 0.05). The expression of dentin matrix protein 1 protein was significantly lower in the osteoporosis group than the sham group, but it increased after treatment with sodium ibandronate (P < 0.05). Compared with the sham group, the expression of Caspase 3 and Bax in the osteoporosis group increased significantly, and the expression of Bcl-2 decreased. However, treatment with sodium ibandronate decreased the expression of Caspase 3 and Bax and increased the expression of Bcl-2 significantly. Overall, our findings reveal that sodium ibandronate can inhibit the apoptosis of condylar chondrocytes and the number of osteoclasts in osteoporotic state, which may be related to the regulation of Caspase 3, Bcl-2, Bax and dentin matrix protein 1 expression.
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BACKGROUND: Dental pulp stem cells can differentiate into dentin under appropriate induction conditions, which are important seed cells i n dental tissue engineering. However, the commonly used inducers are chemical agents, which are not available for in vivo application. Mesenchymal stem cells can differentiate with the material hardness, and the physical property-induced cell differentiation is little reported. OBJECTIVE: To observe the extension characteristics and dentin differentiation potential of dental pulp stem cells from human deciduous teeth on the stiff matrix surface. METHODS: Dental pulp stem cells from naturally shed deciduous teeth were isolated, cultured and identified. Four solid gel matrixes with elasticity modulus of (9.12±0.94), (27.18±3.55), (59.37±4.05) and (86.45±5.33) kPa were made using low melting point agarose. The extension ability of passage 4 dental pulp stem cells on the surface of the above solid matrixes was detected by two-dimensional clone formation and cell scratch tests. The protein expression levels of dentin matrix protein-1, dentin phosphoprotein and dentin sialoprotein were detected by western blot assay. RESULTS AND CONCLUSION: Dental pulp stem cells from human deciduous teeth seeded on the gel matrix with extremely low and low hardness almost existed as cell clones with neat edges, and cell spreading and extension were rare. When seeded on the gel matrix with moderate and high hardness, the cloned edge of deciduous dental pulp stem cells spread and extended obviously. The cell body became large and the cell edge extended significantly. The cell scratch test revealed the similar phenomenon. When seeded on the gel matrix with moderate and high hardness, dental pulp stem cells from human deciduous teeth exhibited high expression levels of of dentin matrix protein-1, dentin phosphoprotein and dentin sialoprotein. In summary, with the increase of matrix hardness, the abilities of extension and differentiation into dentin of dental pulp stem cells from human deciduous teeth are increased gradually, which provides a method for dental tissue engineering.
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OBJECTIVE：To investigate the effects of Aconitum injection on cartilage oligomeric matrix protein （COMP）， encoded protein by tumor suppressor gene p53（p53 protein）and bone morphogenetic protein 2（BMP-2）in knee osteoarthritis （KOA）model rabbit ，so as to explore the mechanism of the injection in the treatment of KOA. METHODS ：Totally 24 rabbits were randomly divided into blank group ，model group ，Sodium hyaluronate group and Aconitum injection group ，with 6 rabbits in each group. KOA model was established by injecting 2％ papain-0.03 mol/L L-cysteine solution into the articular cavity of rabbits in model group ，sodium hyaluronate group and Aconitum injection group at the 1st，4th and 7th day ，respectively，except the blank group. At the 1st，4th and 7th day after modeling succeeded ，0.1 mL/kg of normal saline ，Sodium hyaluronate injection and Aconitum injection were injected into the articular cavity of rabbits ，respectively. The cartilage tissue of knee joint was taken from above 4 groups，and the contents of COMP and p 53 protein were detected by ELISA. The cartilage morphology of rabbit knee joint was observed by naked eye. The cartilage of the knee joint was collected and stained by HE staining ，and then the histomorphology changes were observed by light microscope ；Mankin scoring was conducted. The two-step method of PV was used to make the immunohistochemical specimens of knee joint cartilage ，and the relative expression of BMP- 2 was detected. RESULTS ：Compared with blank group ，the edge of cartilage was damaged and the cartilage surface was damaged in the model group. The results of histomorphology observation showed that the joint tissue structure was obviously irregular ，the distribution of chondrocytes was disordered with morphological changes ，and the Mankin score was significantly increased （P＜0.05）；the contents of COMP and cancer cells by indirect inhibition of RAD 51-mediated re - . Suppression of ERCC 1 and RAD51 expression through ERK 1/2 inactivation is essen - tial in emodin-mediated cytotoxicity in human non-small 。E-mail： cell lung cancer cells. p53 protein in knee joint fluid were increased significantly ，while the relative expression of BMP- 2 in knee joint tissue decreased significantly （P＜0.05）. Compared with model group ，the appearance ，histomorphology changes of knee joint cartilage in administration groups were improved，Mankin scores were significantly decreased （P＜0.05）；the contents of COMP and p 53 protein in the knee joint fluid were decreased significantly ，and the relative expression of BMP- 2 in the knee joint tissue were increased significantly （P＜0.05），but there was no significant difference between Aconitum group and Sodium hyaluronate group （P＞0.05）. CONCLUSIONS ：Aconitum injection can improve synovitis inflammation ，delay articular cartilage degeneration ， promote cartilage repair and protect joints of KOA model rabbits. The mechanism may be related to inhibiting COMP secretion ， decreasing p 53 protein expression and promoting BMP- 2 release.
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OBJECTIVE@#To detect the expression of cartilage oligomeric matrix protein (COMP) in the synovial chondromatosis of the temporomandibular joint (TMJSC), and to discuss the possible interactions between COMP, transforming growth factor (TGF)-β3, TGF-β1 and bone morphogenetic protein-2 (BMP-2) in the development of this neoplastic disease.@*METHODS@#Patients in Peking University School and Hospital of Stomatology from January 2011 to February 2020 were selected, who had complete medical records, TMJSC was verified histologically after operation. The expressions of COMP, TGF-β3, TGF-β1 and BMP-2 in the TMJSC of the temporomandibular joint were detected by immunohistochemistry and quantitative real-time PCR (RT-PCR) at the protein level and mRNA level respectively, compared with the normal synovial tissue of temporomandibular joint. The histological morphology, protein expression and distribution of TMJSC tissues were observed microscopically, and the positive staining proteins were counted and scored. SPSS 22.0 statistical software was used to analyze the expression differences between the related proteins in TMJSC tissue and the normal synovial tissue of temporomandibular joint and to compare their differences. P < 0.05 indicated that the difference was statistically significant.@*RESULTS@#Immunohistochemical results showed that the positive expression of COMP in TMJSC tissues was mostly found in synovial tissues and chondrocytes adjacent to synovial tissues, and the difference was statistically significant, compared with the normal temporomandibular joint synovial tissues. The positive expression of COMP was significantly different between recurrent TMJSC and non-recurrent ones. The positive expressions of TGF-β3, TGF-β1 and BMP-2 were higher than the normal synovial tissue, and were also mostly found in the synovial cells and adjacent chondrocytes, which was further confirmed by Western blot. According to the RT-PCR results, the expressions of COMP, TGF-β3, TGF-β1 and BMP-2 in TMJSC were higher than those in the normal synovial tissue.@*CONCLUSION@#The expression of COMP in TMJSC of temporomandibular joint increased significantly, compared with the normal synovial tissue. There may be interactions between COMP and cytokines related to the proliferation and differentiation, like TGF-β3, TGF-β1 and BMP-2, which may play a potential role in the pathogenesis of TMJSC.
Asunto(s)
Humanos , Proteína de la Matriz Oligomérica del Cartílago/genética , Condromatosis Sinovial , Membrana Sinovial , Articulación Temporomandibular , Factor de Crecimiento Transformador beta3RESUMEN
Fractures are frequently occurring diseases that endanger human health. Crucial to fracture healing is cartilage formation, which provides a bone-regeneration environment. Cartilage consists of both chondrocytes and extracellular matrix (ECM). The ECM of cartilage includes collagens and various types of proteoglycans (PGs), which play important roles in maintaining primary stability in fracture healing. The PG form of dentin matrix protein 1 (DMP1-PG) is involved in maintaining the health of articular cartilage and bone. Our previous data have shown that DMP1-PG is richly expressed in the cartilaginous calluses of fracture sites. However, the possible significant role of DMP1-PG in chondrogenesis and fracture healing is unknown. To further detect the potential role of DMP1-PG in fracture repair, we established a mouse fracture model by using a glycosylation site mutant DMP1 mouse (S89G-DMP1 mouse). Upon inspection, fewer cartilaginous calluses and down-regulated expression levels of chondrogenesis genes were observed in the fracture sites of S89G-DMP1 mice. Given the deficiency of DMP1-PG, the impaired IL-6/JAK/STAT signaling pathway was observed to affect the chondrogenesis of fracture healing. Overall, these results suggest that DMP1-PG is an indispensable proteoglycan in chondrogenesis during fracture healing.