PURPOSE: To investigate the regulatory role of nerve injury-induced protein 1 (NINJ1) in the anti-inflammatory function of human umbilical cord mesenchymal stem cells (hUC-MSCs) co-stimulated by interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). METHODS: hUC-MSCs were expanded in vitro using standard protocols, with stem cell characteristics confirmed by flow cytometry and multilineage differentiation assays. The immunomodulatory properties and cellular activity of cytokine-co-pretreated hUC-MSCs were systematically evaluated via quantitative reverse transcription RT-qPCR, lymphocyte proliferation suppression assays, and Cell Counting Kit-8 viability tests. Transcriptome sequencing, Western blotting and small interfering RNA interference were integrated to analyze the regulatory mechanisms of NINJ1 expression. Functional roles of NINJ1 in pretreated hUC-MSCs were elucidated through gene silencing combined with lactate dehydrogenase release assays, Annexin V/Propidium Iodide apoptosis analysis, macrophage co-culture models, and cytokine Enzyme-Linked Immunosorbent Assay. Therapeutic efficacy was validated in a cecal ligation and puncture-induced septic mouse model: 80 mice were randomly allocated into 4 experimental groups (n=20/group): sham group (laparotomy without cecal ligation); phosphate-buffered saline-treated group (cecal ligation and puncture (CLP) + 0.1 mL phosphate-buffered saline); hUC-MSCs (small interfering RNA (siRNA)-interferon-gamma and tumor necrosis factor-alpha co-stimulation (IT))-treated group (CLP + hUC-MSCs transfected with scrambled siRNA); and hUC-MSCs (siNINJ1-IT)-treated group (CLP + hUC-MSCs with NINJ1-targeting siRNA). RESULTS: hUC-MSCs demonstrated compliance with International Society for Cellular Therapy criteria, confirming their stem cell identity. IFN-γ/TNF-α co-pretreatment enhanced the immunosuppressive capacity of hUC-MSCs, accompanied by the reduction of cellular viability, while concurrently upregulating pro-inflammatory cytokines such as interleukin-6 and interleukin-1β. This co-stimulation significantly elevated NINJ1 expression in hUC-MSCs, whereas genetic silencing of NINJ1 effectively suppressed pro-inflammatory cytokine production and attenuated damage-associated molecular patterns release through inhibition of programmed plasma membrane rupture. Furthermore, the NINJ1 interference potentiated the ability of cytokine-pretreated hUC-MSCs to suppress LPS-induced pro-inflammatory responses in RAW264.7 macrophages. In cecal ligation and puncture-induced sepsis model, NINJ1-silenced hUC-MSCs exhibited enhanced therapeutic efficacy, manifested by reduced systemic inflammation and multi-organ damage. CONCLUSION Our findings shed new light on the immunomodulatory functions of cytokine-primed MSCs, offering groundbreaking insights for developing MSC-based therapies against inflammatory diseases via interfering the expression of NINJ1.
Mesenchymal Stem Cells/drug effects* ; Animals ; Interferon-gamma/pharmacology* ; Tumor Necrosis Factor-alpha/pharmacology* ; Humans ; Mice ; Umbilical Cord/cytology* ; Cells, Cultured ; Apoptosis ; Male

Objective:To analyze the causal relationship between intestinal flora and vitiligo by two-sample Mendelian randomization (MR) analysis based on "co-diseases of skin and gut" in TCM.Methods:Genome-wide association study (GWAS) data of intestinal flora samples and vitiligo samples were obtained from the databases of MiBioGen and IEU OpenGWAS Project, respectively. Intestinal flora was used as exposure factor, vitiligo as outcome, and single nucleotide polymorphism (SNP) associated with various intestinal floras was used as instrumental variable. After screening qualified instrumental variable in this study, inverse variance weighting (IVW) was used for MR analysis to investigate the potential causal relationship between intestinal flora and vitiligo, MR-Egger regression, weighted median estimator (WME), weighted mode (WM), and simple mode (SM) were used as supplementary methods for IVW. The Cochran's Q test, MR-Egger intercept test, MR-PRESSO and leave one-out analysis were used for sensitivity analysis.Results:Euryarchaeota (IVW method: OR<1, P<0.05) were the protective factors for the occurrence of vitiligo, and Clostridialesvadin-BB60group (IVW method: OR>1, P<0.05) and Subdoligranulum (IVW method: OR>1, P<0.05) were the risk factors for the occurrence of vitiligo. No heterogeneity effect was found by the Cochran's Q test ( P>0.05), no horizontal pleiotropy was found by the MR-Egger intercept test ( P>0.05), no outliers were found in the MR-PRESSO analysis ( P>0.05), and the results of leave-one-out analysis indicated that the causal effects of the 3 identified intestinal floras on vitiligo were not driven by any single SNP. Conclusions:There are causal effects between some intestinal floras and vitiligo, but the specific mechanisms still need to be further studied. The gut microbiota affects the onset and treatment of vitiligo. Using TCM to regulate the gut microbiota may have a good therapeutic effect on treating vitiligo, providing a direction for clinical diagnosis and treatment of vitiligo.

Objective:To investigate the effects of Kanglaite injection combined with chemotherapy on quality of life and myelosuppression in patients with non-small cell lung cancer (NSCLC).Methods:The clinical data of 60 patients with NSCLC who received treatment at Zhejiang Jinhua Guangfu Tumor Hospital from August 2018 to February 2022 were retrospectively analyzed. These patients were divided into an experimental group and a control group, with 30 patients in each group according to the treatment methods used. The patients in the experimental group received the Kanglaite injection in combination with chemotherapy using gemcitabine and cisplatin, whereas the patients in the control group were treated solely with chemotherapy with gemcitabine and cisplatin. The quality of life was evaluated using the Karnofsky Performance Status score. Before and after treatment, a comparison was made between the two groups in terms of Karnofsky Performance Status score, the incidence of adverse reactions (such as myelosuppression), and clinical efficacy.Results:After treatment, the disease control rate and objective response rate in the experimental group were 86.67% (26/30) and 60.00% (18/30), respectively, which were significantly higher than 60.00% (18/30) and 30.00% (9/30) in the control group ( χ2 = 4.18, 4.31, both P < 0.05). Prior to treatment, the Karnofsky Performance Status scores in the experimental and control groups were (70.68 ± 3.75) points and (70.29 ± 5.11) points ( t = 0.34, P = 0.790), respectively. After treatment, the Karnofsky Performance Status scores in the experimental group were significantly higher than those in the control group [(67.02 ± 5.87) points vs. (62.37 ± 3.59) points, t = -5.29, P < 0.05]. The incidence of adverse reactions in the experimental group was significantly higher than that in the control group [40.00% (12/30) vs. 36.67% (11/30), χ2 = 0.07, P = 0.790). Additionally, the incidence of myelosuppression in the experimental group was significantly lower than that in the control group [56.67% (17/30) vs. 86.67% (26/30), χ2 = 6.90, P = 0.030]. Conclusion:Compared with chemotherapy alone, Kanglaite injection combined with chemotherapy can effectively relieve the clinical symptoms of patients with advanced non-small cell lung cancer, leading to improved prognosis.

Objective:To investigate the effects of poly(A) tails with different lengths on mRNA expression in vitro and the passage stability of transcription template with poly (A) tail in Escherichia coli ( E. coli). Methods:Plasmids with poly(A) tails of 38, 60, 103, 125 and 126 (60 nt+ 6 nt spacer+ 60 nt) nt were designed and constructed. Then the plasmids were linearized by single enzyme digestion and used as transcription template for preparing enhanced green fluorescent protein (EGFP)-mRNA. EGFP-mRNA containing poly(A) tails of different lengths were transfected into 293T cells and the expression of EGFP was detected by flow cytometry. As to stability test, the template plasmids with poly (A) tail of 125 and 126 nt were transformed into E. coli TransStbl3 and Top10 competent cells. Seven clones were selected for culture and plasmid extraction, and then the plasmids were digested by restriction enzyme and detected by capillary electrophoresis. For passage stability, three correctly sequenced clones of each group were selected for continuous passage at 37℃, and the plasmids were extracted and digested every two generations for capillary electrophoresis. At the same time, the correctly sequenced clones of 125 nt group were also passaged at 30℃, and the plasmids were also extracted and digested every two generations for capillary electrophoresis. Results:The transcription templates with poly(A) tail of different lengths were successfully constructed. Flow cytometry showed that the fluorescence expression of the template plasmids with poly (A) tail of 103 and 125 nt were significantly higher than that of 38 and 60 nt. The fluorescence expression of the plasmid with poly (A) tail of 126 nt was significantly higher than that of all other groups. The percentages of stable sequences of the template plasmid with poly(A) tail of 125 nt in TransStbl3 and Top10 competent cells were 76% and 91%, respectively. The results of continuous passage showed that poly(A) tail of 125 nt could be stable to the 4th generation at 37℃ in both TransStbl3 and Top10 competent cells, and stable to the 16th and 10th generations at 30℃. The percentages of stable sequences of the template plasmid with poly(A) tail of 126 nt in TransStbl3 and Top10 competent cells were 95% and 48%, respectively. The results of continuous passage showed that poly(A) tail of 126 nt could be stable to the 12th generation at 37℃ in both TransStbl3 and Top10 competent cells.Conclusions:The length and composition of poly(A) tail in mRNA affected the expression of target protein. Adding a spacer with a length of 6 nt to poly(A) tail and low temperature culture were both helpful to improve the stability of the template plasmid, which provided a reference for the design and preparation of in vitro transcription template of mRNA vaccine.

Objective:To prepare a recombinant hemagglutinin trimer (HA-Tri) vaccine against influenza viruses and to study its immunogenicity in a mouse model.Methods:A stable CHO cell line that could express HA-Tri was constructed. Western blot, single radial immunodiffusion, protein particle size detection and N-glycosylation site analysis were performed for qualitative and quantitative analysis of the recombinant protein. According to the different treatment conditions such as dosage and adjuvant, BALB/c mice were divided into 11 groups and subjected to consistent immunization procedures. Serum neutralizing antibody titers were measured on 56 d after the first immunization to evaluate the immunogenicity of HA-Tri.Results:The constructed CHO cells could secret and express HA-Tri proteins. The HA-Tri proteins were biologically active and capable of forming precipitation rings in the single radial immunodiffusion. The particle size of HA-Tri was approximately 18.79 nm and 10 N-glycosylation sites were detected, including high mannose, complex glycoforms and heterozygous glycoforms. After prime-boost immunization, there was no statistically significant difference in the titers of neutralizing antibodies induced in mice by 3.75 μg of HA-Tri in combination with RFH01 adjuvant and 15 μg of monovalent vaccine stock solution ( P=0.431 2, U=36). Serum antibody titers in the HA-Tri+ RFH01 groups were higher than those in the corresponding HA-Tri groups without RFH01 adjuvant, and the highest titer was induced in the 15 μg HA-Tri+ RFH01 group, which was 1 280. Conclusions:The recombinant HA-Tri protein was successfully prepared. HA-Tri in combination with RFH01 adjuvant could induce humoral immune responses against influenza viruses in BALB/c mice, which would provide reference for the development of influenza virus recombinant subunit vaccines.

Objective:To evaluate the immunogenicity of a quadrivalent subunit vaccine combined with RFH01 adjuvant in a mouse model.Methods:Identification tests were performed on four monovalent influenza virus subunit vaccine stock solutions according to the methods described in Part 3 of the Chinese Pharmacopoeia 2020 Edition. In the study of the quadrivalent subunit vaccine combined with RFH01 adjuvant, 460 female BALB/c mice (6-8 weeks old) were randomly divided into 46 groups including experimental groups, vaccine control group, negative control group and blank group with 10 mice in each group. In the study of the quadrivalent subunit vaccine in old and young mice, 80 female 10-month-old and 80 female 10-week-old BALB/c mice were randomly divided into 16 groups ( n=10) including monovalent influenza virus vaccine group, quadrivalent subunit vaccine group, quadrivalent subunit vaccine+ RFH01 adjuvant group, chicken embryo quadrivalent split vaccine control group and PBS group. All mice were immunized by intramuscular injection. At 21 d after the primary immunization, a booster immunization was conducted using the same strategy. Blood samples were collected at 21 d and 42 d after the primary immunization for serum separation. Haemagglutination inhibition (HI) test was performed to detect the antibody levels in mouse serum samples. Results:After the booster immunization, the positive conversion rates in all vaccine+ RFH01 adjuvant groups reached 100%, and the geometric mean titers (GMTs) of serum antibodies were significantly higher than those of the vaccine groups without RFH01 adjuvant. There were significant differences in serum antibody titers between the monovalent/quadrivalent subunit vaccine groups with and without RFH01 adjuvant. After the booster immunization, the titers of serum antibodies against H1N1, H3N2, B/Victoria and B/Yamagata in the 10-week-old mice were significantly higher than those in the 10-month-old mice.Conclusions:The monovalent and quadrivalent influenza virus vaccines in combination with RFH01 adjuvant could elicit higher antibody titers in young (6-10 weeks old) and old (10 months old) mice, showing good immunogenicity.

Objective:To explore the types, gene mutation types and constituent ratio of thalassemia in Xiaoshan area of Hangzhou, and to analyze the value of test index of erythrocyte parameter calculation formula (Matos & Carvalho Index, MCI) in differential diagnosis.Methods:Using the method of retrospective analysis, 150 cases of thalassemia and 124 cases of iron deficiency anemia treated in Xiaoshan Hospital Affiliated to Hangzhou Normal University from January 2019 to April 2021 were included in the study to collect the diagnostic results of α and β-thalassemia genes, ferritin and erythrocyte parameters, the test indexes were compared and analyzed, and the value of MCI in differentiating thalassemia from iron deficiency anemia was analyzed.Results:In 150 cases of thalassemia, there were 58 cases of α-thalassemia (38.67%), of which 8 cases (5.33%) were complicated with iron deficiency; there were 88 cases of β-thalassemia (58.67%), of which 3 cases (2.00%) were complicated with iron deficiency; αβ-compound thalassemia occurred in 4 cases (2.67%). The proportion of α-thalassemia was lower than β-thalassemia, the difference was statistically significant (χ 2 = 12.01, P = 0.001) . Four kinds of α-thalassemia allele were detected in 58 cases of α-thalassemia, mainly -- SEA, a total of 48 cases (82.76%); eight gene mutation types were detected in 88 cases of β-thalassemia, of which CD41-42 was the most, a total of 33 cases (37.50%). The basic parameters of β-thalassemia, red blood cell (RBC), hemoglobin (HGB) and red blood cell specific volume (HCT) were lower than those of α-thalassemia ( t = - 2.88, - 3.49, - 4.33, P < 0.05); mean hemoglobin concentration (MCHC) and red cell volume distribution width (RDW) were higher than those of α-thalassemia ( t = 3.22, 2.43, P < 0.05). The MCI value of thalassemia group was significantly higher than that of iron deficiency anemia group (23.14 ± 1.73 vs 20.47 ± 1.45, t = 13.61, P < 0.001). The area under the curve of MCI (AUC) = 0.885. The diagnostic cut-off point was set according to the maximum Jorden index method (0.594). The diagnostic cut-off point in this study was 21.375, with a sensitivity of 82.0% and specificity of 77.4%. Conclusions:The genotyping of thalassemia in Xiaoshan area of Hangzhou indicates that β-thalassemia is dominant, with the most mutations in CD41-42 genotype, followed by α-thalassemia, with -- SEA genotype mutation most. MCI has great screening value in identifying small cell anemia.

Objective:To express the head domain of influenza A virus hemagglutinin (HA) in a prokaryotic expression system and to evaluate its immunogenicity.Methods:The genes encoding the HA head domains of H1N1 and H3N2 influenza viruses were cloned into pET-22b(+ ) prokaryotic expression plasmid. After the induction with IPTG, the fusion proteins rH1N1-HA and rH3N2-HA containing HA head domain and His-tag were expressed and obtained from E. coli BL21. SDS-PAGE and Western blot was used to verify the expression of the recombinant proteins. Rabbits were immunized with multiple doses of the purified recombinant proteins to obtain polyclonal antibodies against the HA head domains of H1N1 and H3N2. The immunogenicity of the recombinant proteins was evaluated in BALB/c mice. Results:rH1N1-HA and rH3N2-HA induced protective antibodies (geometric mean titer ≥40) in mice and could be used as protective antigens. Polyclonal antibodies against rH1N1-HA and rH3N2-HA could be used as important materials for Western blot, ELISA and other immunological assays.Conclusions:The HA head domains prepared in this study could be used as protective antigens to induce protective antibodies in mice. Polyclonal antibodies against the HA head domains could be used for immunological and serological studies of influenza A viruses.

BACKGROUND: Zirconia inlays have good mechanical, biocompatible and aesthetic properties in the field of dental prosthodontics, but there is no consensus on the standards of zirconia inlays for clinical cavity design, dental preparation and selection of bonding materials. OBJECTIVE: To investigate the stress distribution and characteristics of the bonding interface, tooth and periodontal tissues of the zirconia inlay model after 3 M RelyX Unicem or vario-link resin adhesive bonding. METHODS: Micro-CT was used to scan the right mandibular third molars. A three-dimensional finite element model of MOD zirconia inlay with different adhesives (3 M RelyX Unicem resin binder, vario-link resin binder) and cavity types (2, 3, 4 mm in the depth of the joint cavity) was constructed by software Mimics, Goemagic Studio and NX 10.0. Using ANSYS Workbench mesh generation, the stress distribution of each model was analyzed after loading 10 N·mm torque, 45° 175 N, 90° 600 N. RESULTS AND CONCLUSION: (1) After loading 10 N·mm torque, the bonding agent equivalent stress and root surface equivalent stress of the model with 3 mm cavity depth were largest, so the stress on the bonding interface, crown and root was largest. (2) After loading 175 N at 45° lingual direction, the bonding interface of the model with 4 mm cavity depth undertook a higher stress. The stress values of the root and periodontal tissue of 3 M RelyX Unicem resin bonding agent model with the same cavity depth were higher. (3) After loading 600 N at 90°, the root force of the model with 3 mm cavity depth was largest. The crown, root and periodontal tissue of the 3 M RelyX Unicem resin bonding agent model with the same cavity depth undertook a higher stress. The bonding interface of the vario-link resin bonding agent model was under greater stress. (4) The regions of stress concentration areas included the bifurcation zone of the root, the inlay edge line, the roof of pulp chamber, the gingival wall, 1/3 mesial part of the buccal surface near the neck, and 1/3 mesial part of the lingual surface near the neck. (5) All these findings indicate that vario-link resin adhesive and 3 M RelyX Unicem resin adhesive are suitable for the bonding of zirconia inlay, but the vario-link adhesive strength is larger and the bonding interface stress is larger. The factors such as cavity design, residual tooth tissue resistance, retention shape, and periodontal support should be considered comprehensively. The optimization of cavity design, tooth preparation, cushion bottom and high inlay should be adopted, in order to improve the long-term repair effect of zirconia inlay. However, further clinical trials are needed to verify the results of three-dimensional finite element model.

Objective To explore the change of miRNA-16(miR-16) level in the patients with multiple my-eloma(MM ) and its relationship with the Mayo risk stratification and prognosis judgment .Methods Each 10 mL of bone marrow samples was collected from 31 cases of MM in the hematology department of the First Af-filiated Hospital of Guangxi Medical University from August 2016 to January 2017 ,and each 5 mL of bone marrow sample was collected from 5 healthy bone marrow donors as the healthy control .The real time fluores-cence quantitative PCR(qRT-PCR) was used to analyze relative the change of miR-16 relative expression level and fluorescence in situ hybridization(iFISH) was used to detect the abnormal gene .Then the relationship be-tween miR-16 relative expression level with Mayo based risk stratification ,and the difference of miR-16 rela-tive expression level between before and after self treatment were statistically analyzed .Results The miR-16 relative expression level in MM patients was increased and positively correlated with the Mayo risk stratifica-tion .Meanwhile in the MM patients with disease progression ,the miR-16 expression was increased . Conclusion miR-16may serve as the indicator for judging the recurrence or progression of MM ,and guides the clinical personalized therapy of MM according to its expression level change .