Objective To investigate the effect of microglia activation regulated by C-X3-C motif chemokine ligand 1(CX3CL1)-C-X3-C motif chemokine receptor 1(CX3CR1)pathway on memory function in hemorrhagic shock/resuscitation rats.Methods The experiment was divided into two parts.In the first part,the rats were randomly divided into sham group,model-0.5 hour group,model-1.5 hour group,model-3 hour group,10 rats in each group.There were differences in the time of hemorrhagic shock among each group.In the second part,rats were randomly divided into control group and CX3CL1 group,10 rats in each group.The rats in CX3CL1 group were treated with CX3CL1 protein factor(intraventricular injection),and the rats in control group were treated with saline.All rats were trained in Morris water maze experiments before model construction,and tests of Morris water maze experiments were carried out after 4 days of model construction.After completion,the whole brains were taken for HE staining and immunohistochemical staining.Cerebrospinal fluid was taken for detection of inflammatory cytokines,and hippocampus tissues were taken for Real-time PCR detection and Western blotting detection.Results Compared with the sham group,the escape latency of rats in model group increased,the number of platform crossings and the resident time in the third quadrant decreased.The neuronal state was impaired in HE staining in model group.In addition,compared with the sham group,the expression of ionized calcium binding adaptor molecule-1(Iba1)in the brain of the rats in model group increased,the contents of tumor necrosis factor-α(TNF-α)and interleukin(IL)-6 in the cerebrospinal fluid increased,and the M1-type microglia markers CD16,TNF-α,IL-1β and inducible nitric oxide synthase(iNOS)mRNA content increased.At the same time,compared with the sham group,the expressions of CX3CL1 and CX3CR1 in the brain of model group decreased,and the expressions of phosphorylated nuclear factor-κB(p-NF-κB)and nucleotide binding oligomerization domain(NOD)-like receptor protein 3(NLRP3)increased.However,compared with the control group,rats in CX3CL1 group had reduced escape latency,increased platform crossing times and quadrantⅢresident time,and recovered neuronal states.In addition,the expression of Iba1 in the brain of CX3CL1 group decreased,the contents of TNF-α and IL-6 in the cerebrospinal fluid decreased,the mRNA contents of M1-type microglia markers like CD16,TNF-α,IL-1β and iNOS decreased,and the mRNA contents of markers of M2-type microglia glial like CD206,transforming growth factor-β(TGF-β),arginase-1(Arg1),Chitinase 3-like protein 1(Ym 1)increased.Conclusion CX3CL1 can help inhibit the excessive activation of microglia,induce the polarization of microglia to M2 type,inhibit the polarization of M1 type,reduce the release of inflammatory cytokines,and alleviate the memory function damage induced by hemorrhagic shock/resuscitation.

OBJECTIVE: To explore the potential mechanism of resistance to axitinib in clear cell renal cell carcinoma (ccRCC), with a view to expanding the understanding of axitinib resistance, facilitating the design of more specific treatment options, and improving the treatment effectiveness and survival prognosis of patients. METHODS: By exploring the half maximum inhibitory concentration (IC₅₀) of axitinib on ccRCC cell lines 786-O and Caki-1, cell lines resistant to axitinib were constructed by repeatedly stimulated with axitinib at this concentration for 30 cycles in vitro. Cell lines that were not treated by axitinib were sensitive cell lines. The phenotypic differences of cell proliferation and apoptosis levels between drug resistant and sensitive lines were tested. Genes that might be involved in the drug resistance process were screened from the differentially expressed genes that were co-upregulated in the two drug resistant lines by transcriptome sequencing. The expression level of the target gene in the drug resistant lines was verified by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot (WB). The expression differences of the target gene in ccRCC tumor tissues and adjacent tissues were analyzed in the Gene Expression Profiling Interactive Analysis (GEPIA) public database, and the impact of the target gene on the prognosis of ccRCC patients was analyzed in the Kaplan-Meier Plotter (K-M Plotter) database. After knocking down the target gene in the drug resistant lines using RNA interference by lentivirus vector, the phenotypic differences of the cell lines were tested again. WB was used to detect the levels of apoptosis-related proteins in the different treated cell lines to find molecular pathways that might lead to drug resistance. RESULTS: Cell lines 786-O-R and Caki-1-R resistant to axitinib were successfully constructed in vitro, and their IC₅₀ were significantly higher than those of the sensitive cell lines (10.99 μmol/L, P < 0.01; 11.96 μmol/L, P < 0.01, respectively). Cell counting kit-8 (CCK-8) assay, colony formation, and 5-ethynyl-2 '-deoxyuridine (EdU) assay showed that compared with the sensitive lines, the proliferative ability of the resistant lines decreased, but apoptosis staining showed a significant decrease in the level of cell apoptosis of the resistant lines (P < 0.01). Although resistant to axitinib, the resistant lines had no obvious new replicated cells in the environment of 20 μmol/L axitinib. Nuclear protein 1 (NUPR1) gene was screened by transcriptome sequencing, and its RNA (P < 0.0001) and protein expression levels significantly increased in the resistant lines. Database analysis showed that NUPR1 was significantly overexpressed in ccRCC tumor tissue (P < 0.05); the ccRCC patients with higher expression ofNUPR1had a worse survival prognosis (P < 0.001). Apoptosis staining results showed that knockdown ofNUPR1inhibited the anti-apoptotic ability of the resistant lines to axitinib (786-O, P < 0.01; Caki-1, P < 0.05). WB results showed that knocking downNUPR1decreased the protein level of B-cell lymphoma-2 (BCL2), increased the protein level of BCL2-associated X protein (BAX), decreased the protein level of pro-caspase3, and increased the level of cleaved-caspase3 in the resistant lines after being treated with axitinib. CONCLUSION ccRCC cell lines reduce apoptosis through theNUPR1 -BAX/ BCL2 -caspase3 pathway, which is involved in the process of resistance to axitinib.
Humans ; Carcinoma, Renal Cell/metabolism* ; Axitinib/pharmacology* ; Kidney Neoplasms/metabolism* ; bcl-2-Associated X Protein ; Nuclear Proteins ; Cell Line, Tumor ; Apoptosis ; Cell Proliferation

OBJECTIVE: To investigate the toxicity management and efficacy evaluation of BCMA-chimeric antigen receptor T cells(CART) in the treatment of relapsed and refractory multiple myeloma (MM). METHODS: The efficacy and adverse reactions of 21 patients with MM who received BCMA-CART treatment at the First Affiliated Hospital of Wenzhou Medical University from December 2017 to September 2020 were evaluated, and the efficacy assessment and survival analysis for high-risk patients and non-high-risk patients were evaluated. RESULTS: After infusion of BCMA-CART cells in 21 MM patients, the number of effective cases was 17, of which the complete remission (sCR/CR) was 10, and the partial remission (VGPR/PR) was 7. The median OS time for all patients was 19.4 months, and the median PFS time was 7.9 months. The number of patients with extramedullary disease(EMD), high-risk genetics, and ISS stage Ⅲ were 5, 15 and 8, and the effective number was 3, 11 and 6, respectively. The treatment of 3 patients without high-risk factors was effective. The median OS and median PFS of patients with EMD were 14.2 and 2.5 months, respectively, which were shorter than those of patients without EMD (19.4 months and 8.9 months, respectively). The median OS and median PFS of patients with high-risk cytogenetic factors and ISS Ⅲ were not significantly different from those of non-high-risk patients. Cytokine release syndrane (CRS) occurred in 20 patients, of which 14 cases were Grade 1 CRS, while 6 were Grade 2, no CRS of Grade 3 or above occurred. IL-6 receptor inhibitors were used in 9 patients. All CRS were controlled effectively, and no patients had neurological toxicity. CONCLUSION BCMA-CART is a certain curative effect in the treatment of relapsed and refractory multiple myeloma, and the adverse reactions can be well controlled through close monitoring and timely treatment.
B-Cell Maturation Antigen ; Humans ; Immunotherapy, Adoptive/adverse effects* ; Multiple Myeloma/therapy* ; Receptors, Chimeric Antigen ; Remission Induction

TANK-binding kinase 1 (TBK1), a core kinase of antiviral pathways, activates the production of interferons (IFNs). It has been reported that deacetylation activates TBK1; however, the precise mechanism still remains to be uncovered. We show here that during the early stage of viral infection, the acetylation of TBK1 was increased, and the acetylation of TBK1 at Lys241 enhanced the recruitment of IRF3 to TBK1. HDAC3 directly deacetylated TBK1 at Lys241 and Lys692, which resulted in the activation of TBK1. Deacetylation at Lys241 and Lys692 was critical for the kinase activity and dimerization of TBK1 respectively. Using knockout cell lines and transgenic mice, we confirmed that a HDAC3 null mutant exhibited enhanced susceptibility to viral challenge via impaired production of type I IFNs. Furthermore, activated TBK1 phosphorylated HDAC3, which promoted the deacetylation activity of HDAC3 and formed a feedback loop. In this study, we illustrated the roles the acetylated and deacetylated forms of TBK1 play in antiviral innate responses and clarified the post-translational modulations involved in the interaction between TBK1 and HDAC3.

Osteoarthritis (OA) is a common degenerative disease. Its most significant pathological change is destruction of articular cartilage and the main clinical symptoms are pain and dysfunction of joints. Recent studies have shown that the expression of non-coding RNA (ncRNA) in chondrocytes can abnormally up-regulate or down-regulate and alter the activities of chondrocytes like their proliferation, migration and apoptosis, thus leading to the occurrence and development of osteoarthritis. Exosomes are extracellular vesicles with a diameter of 40-100 nm, which are secreted in intercellular fluid, act as medium of intercellular communication. They protect ncRNA, protein, lipid and other bioactive materials from enzymatic degradation by encapsulating them and transferring to sibling chondrocytes, due to their good tissue permeability. They can also improve communication between cells and regulate the activities of chondrocytes. Thus, exosomes behave like gene carriers. The ncRNA carried by exosomes can supplement or adsorb the abnormal ncRNA in chondrocytes, so as to regulate the activity of chondrocytes, and is therefore considered as a possible candidate with capabilities to repair cartilages. In this study we reviewed existing literatures related to the roles and effects of exosome miRNA, lncRNA and circRNA on osteoarthritis. We also reviewed the pathogenesis of exosome ncRNA in osteoarthritis.

Malignant bone tumors, one of the most common bone tumors includes osteosarcoma, Ewing's sarcoma, multiple myeloma, and metastatic bone tumors etc. These tumors are often accompanied by distant metastatic lesions at time of diagnosis, leading to low 5-year survival rates. At present, the use of biomarkers for early detection in order to facilitate early treatment are very limited. Therefore, most medical researchers are exploring the roles of exosomes in detecting malignant bone tumors. Exosomes are extracellular microvesicles secreted by different types of cells, which exist in a variety of body fluids. They are new intercellular information carriers that play important physiological roles. Current literature have reported that the RNA contained in exosomes (such as mRNA, miRNAs, lncRNAs and circRNAs) play important roles in the incidence as well as development of malignant bone tumors. However, the previous studies mostly focused on the roles of exosomal RNA in malignant bone tumor diseases, in tumor cell proliferation or apoptosis, transfers, evasion of immune surveillance and chemotherapy drug resistance etc. However, exosomal RNAs may function in the whole process of the disease progression via regulation networks. Our review of existing literature revealed that exosomal RNAs affacted the proliferation, metastasis, immune evasion and drug resistance of five common malignant bone tumors; Osteosarcoma, Ewing sarcoma, Chondrosarcoma, multiple myeloma, and metastatic bone tumors. Therefore, by elucidating on the mechanism of exosomal RNAs in the occurrence and development of malignant bone tumors, this study, could provide new ideas for early diagnosis, concomitant diagnosis, efficacy estimation (chemotherapy, radiotherapy and immunotherapy, etc.) as well as assessing the prognosis of malignant bone tumors.

OBJECTIVE: To investigate the effect of parathyroidectomy(PTX)on the progression of renal anemia and erythropoietin dose in maintenance hemodialysis(MHD)patients with secondary hyperparathyroidism(SHPT)of uremia. METHODS: A total of 58 MHD patients with SHPT treated with PTX were retrospectively analyzed. The changes of serum intact parathyroid hormone(iPTH), calcium(Ca), phosphorus(P), alkaline phosphatsae(AKP), hemoglobulin(Hb), hematocrit(Hct), serum ferritin(SF), transferrin saturation(TS), c-reactive protein(CRP), albumin(Alb)and Kt/V at 3 months and6 months after PTX treatment were evaluated. The Hb level at 3 and 6 months after surgery and the doses of recombinant human erythropoietin(EPO)were recorded. RESULTS: Serum iPTH decreased dramatically(before PTX 1828.88±811.2 ng/L,three months after PTX 56.75±158.4 ng/L,six months after PTX 64.52±178.5 ng/L, P<0.05); Ca, P and AKP levels also decreased significantly after PTX(P<0.05). Hb increased after PTX(before PTX 99.92±14.26 g/L, six months after PTX123.2±13.65 g/L, P<0.05); meanwhile, the dose of EPO decreased after the operation[before PTX 235.18±62.65 U/（kg·W), three months after PTX 158.78±34.24 U/（kg·W),six months after PTX 112.53±25.37 U/（kg·W), P<0.05]. CONCLUSION: PTX can effectively control secondary hyperparathyroidism, significantly improve the renal anemia, and decrease dose of erythropoietin in MHD patients with SHPT.

OBJECTIVETo investigate the effect of steadily down-regulating the expression of VE-cadherin on the chemotheraputic sensitivity of K562 cells, and explore its possible mechanism.

METHODSSpecifically targeting interference sequences carrying human VE-cadherin were designed, the recombinant lentiviral vector containing the IRES-GFP and NEO segment was constructed; recombinant lentivirus was generated by three-plasmids packing system, and transfected into K562 cells, then the cells steadily down-regulated were sorted. CCK-8 assay was performed to evaluate the VE-cadherin of chemotherapeutic (Imatinib) sensitivity of K562 cells. The apoptosis was analyzed by flow cytometry with Annexin V/7-AAD double labeling. The expressions of CD133 and ALDH1 mRNA were determined by real time PCR. The protein expressions of VE-cadherin, BCR-ABL and β-catenin were analyzed by Western blot.

RESULTSThe recombinant lentiviral vector pLB-shVEC-NEO-IRES-GFP was successfully constructed, packed into the lentivirus, then the K562 cells steadily down-regulating VE-cadherin expression was obtained. When VE-cadherin was down-rengulated in K562 cells, the proliferation rate was reduced while the the apoptosis rate was increased; the mRNA levels of CD133 and ALDH1 also were reduced; BCR-ABL fusion protein was not obviously changed; the total β-catenin protein, as well as the nuclear β-catenin protein were decreased in the K562/shVEC cells. Conclution: K562 cells are more susceptible to chemotherapy when VE-cadherin is down-regulated, that may be realized via reducing the stability and the nuclear transfer of β-catenin protein.

OBJECTIVETo investigate a modified protocol of establishing mouse Ph ALL model so as to provide a more convenient and more powerful tool for Ph ALL studies.

METHODSImmature B cells from BALB/c mice were transfected with the Mig190 retrovirus and infused into irradiated syngeneic mice. The immunophenotype was identified by flow cytometry, the BCR-ABL was identified by RT-PCR and Western blot. Leukemia cells isolated from sick mice were re-infused into syngeneic mice without irradiation.

RESULTSThe acute lymphoblastic leukemia was established in mice after Mig190 retroviral transfection, the lymphoblasts were observed by blood smears, the immunophenotype of these leukemic cells was CD19, and the BCR-ABL was detected by RT-PCR and Western blot, the immunophenotype was conformed as Ph ALL, the isolated leukemia cells infused into mice rapidly developed B-ALL.

CONCLUSIONA new protocol is established to generate mice with Ph ALL. This mouse model can provide a more simple and effective tool in comparison with the conversional protocols.

OBJECTIVETo evaluate biological effects of OCT4A gene on K562 cells and explore the molecular mechanism of K562 cell apoptosis.

METHODSTwo recombinant lentiviral vectors were constructed, which could stablely up- regulate and down- regulate OCT4A protein. Recombinant lentivirus was generated by co-transfection of three-plasmids and transfec-ted into K562 cells. The experiments were divided into 5 groups: normal, pLVX-OCT4A-ZsGreen1, pLVX vector control, PLB-OCT4A shRNA and non-specific shRNA groups. Western blot was applied to detect the expression of OCT4A protein, the cell counting kit-8 was applied to evaluate the effect of OCT4A on proliferation of K562 cells. The apoptosis and differentiation of K562 cells were detected by flow cytometry with AnnexinV/7-AAD double staining. The mRNA expressions of caspase-3,BIM,BCL-xL,BAX in K562 cells were determined by real time PCR.

RESULTSThe OCT4A fragment was amplified by reverse transcription polymerase chain reaction(RT-PCR), the 2 lentiviral vectors were successfully constructed. In comparson with those in the control group, the expression of OCT4A protein of pLVX-OCT4A-ZsGreen1 group was significantly increased, but decreased in PLB-OCT4A shRNA group. CCK-8 assay showed that the higher the content of OCT4A protein, the faster the cell proliferation. The apoptosis rate was (3.48±0.52)% of pLVX-OCT4A-ZsGreen1 group, which was lower than that of control group, while the apoptosis rate PLB-OCT4A shRNA group was (7.25±0.57)%, which was higher than that of control group (P<0.05), however, the K562 cells differentiation was not influenced(P>0.05). Compared with control group, the gene expression of Caspase-3,BIM and BAX was down-regulated(P>0.05), but a significant up-regulation of BCL-xL gene expression was observed(P<0.05).

CONCLUSIONTwo lentiviral vectors have been successfully constructed, which can stably up- and down- regulate the expression of OCT4A in K562 cells respectively. OCT4A can promote the K562 cell proliferation and inhibit the apoptosis, the mechanism may be related with up-regulation of BCL-xl expression.