AIM:To construct the adenovirus vector with adiponectin (Acrp30) siRNA, and to observe its effect on the Acrp30 expression and glucose transport in 3T3-L1 adipocytes. METHODS: Mouse Acrp30 siRNA fragment was designed, synthesized and cloned into the adenovirus vector. 3T3-L1 cells were infected with the two recombinant adenoviruses, respectively. The mRNA expression and protein levels of Acrp30 in these cells were evaluated by RT-PCR and ELISA. Glucose transport was measured by 2-Deoxy-[3H]-D-glucose incorporation method. RESULTS: The recombinant adenoviruses were successfully constructed. They remarkably downregulated the expression of Acrp30 at both mRNA and protein levels in 3T3-L1 cells, and decreased the glucose transport in 3T3-L1 adipocytes (P

Objective To investigate the effect of combination of cervical manipulation with cervical conditioning exercise for the treatment of cervical spondylosis of neck type or nerve root type. Methods Seventy-two patients were randomly divided into a treatment group (37 patients) or a control group (35 patients) by using a random number table. Patients in the treatment group and in the control group were treated with combination of cervical manipulation or cervical manipulation only, respectively. At the end of treatment and 3 months, treatment effects were assessed by Neck Disability Index (NDI) and Visual Analog Scale (VAS). Results NDI scores in both of the treatment group and the control group after treatment were significantly lower han those before treatment (t=4.471 in the treatment group, t=3.599 in the control group, all P<0.05), and NDI score in the treatment group were significantly lower than that in the control group after treatment (t=-2.085,P<0.05). Follow-up at 3 months, NDI score in the treatment group was significantly lower than that in the control group (t=-4.549, P<0.05). Proportions of improved patients, as assessed by NDI items, including pain intensity, personal care, lifting, reading, headaches, concentration, work, sleeping, driving and recreation in the treatment group were significantly higher than those in the control groups (Z=-3.521,-3.331,-3.003,-3.001,-3.265,-3.198,-3.016,-3.000,-2.102,-2.971, respectively, all P<0.05). VAS scores in both of the treatment group and the control group after treatment were significantly lower than those before treatment (t=7.986 in the treatment group, t=6.507 in the control group, all P<0.05), and VAS score in the treatment group were significantly lower than that in the control group after treatment (t=-2.426,P<0.05). Follow-up at 3 months, the VAS score in the treatment group was significantly lower than that in the control group (t=-7.065, P<0.05). Proportions of improved patients in the treatment group was significantly higher than that in the control group (91.89% vs 74.29%; χ2=4.014, P=0.045). Conclusion Combination of cervical manipulation with cervical conditioning exercise may be superior to cervical manipulation only for the treatment of cervical spondylosis of neck type or nerve root type.

Objective:To construct a fusion protein of extracellular domain peptide fragment of muscle specific kinase ( MuSK) and fluorescent protein mCherry ,and used as antigen in the detection of antibodies against MuSK ( MuSKAb ) in the sera of patients with myasthenia gravis ( MG).Methods:The mCherry gene was amplified by PCR from vector pRSET-B and cloned into pGEM-T Easy Vector,and furthermore, cloned into Eukaryotic expression vector pMT /BiP/V5-His ( MuSK), which contains MuSK extracellular domain 22-452 amino acid peptide fragment gene to construct the fluorescent fusion protein gene MuSK -mCherry.The recombinant vector was subsequently transfected into drosophila S 2 cells for expression.The expressed fusion proteins were verified in confocal mi-croscope ,and used as antigen in the detection of MuSKAb in sera of MG patents in fluorescence immunoprecipitation test .Results:The fluorescent fusion protein MuSK-mCherry was successfully constructed and expressed.The MuSKAb in sera of patents with MG could be detected in fluorescence immunoprecipitation test using the constructed MuSK-mCherry fusion protein as antigen.Conclusion: It is available to use the constructed fluorescent fusion protein MuSK-mCherry as antigen in fluorescence immunoprecipitation test for the detection of MuSKAb in sera of patents with MG.

Objective To investigate the effects of mannan-binding lectin (MBL) on IL-8 and TNF-α production induced by Candida albicans ( C. albicans) in human THP1/CD14 monocytes. Methods The THP1/CD14 cells were stimulated for 24 h with heat-inactivated yeast form or hyphal form cells of C. albicans strain at the indicated ratios after pretreated with human natural MBL at concentrations ranging from 1 to 20 mg/L for 2 h. The content of IL-8 and TNF-α in culture supernatants were detected by ELISA,and the levels of IL-8 and TNF-α mRNA expressions in these cells were determined by RT-PCR. Western blot was used to detect C. albicans-induced NF-κB translocation in THP1/CDI4 cells. Results ELISA showed that secretion of IL-8 and TNF-α from THP1/CD14 cells could be induced by both yeast cells and hyphal cells. Hyphal cells proved to be much less efficient than yeast cells in stimulating production of IL-8and TNF-α by THP1/CD14 cells. The productions of IL-8 and TNF-α by THP1/CD14 cells induced with C.albicans were profoundly inhibited by MBL at higher concentrations ( 10-20 mg/L) but not MBL at lower concentrations ( 1 mg/L). RT-PCR analysis also indicated that the mRNA expressions of IL-8 and TNF-αt in THP1/CD14 cells were decreased to various extents by MBL at higher concentration, compared to the corresponding THP1/CD14 cells stimulated with C. albicans only. Similarly, MBL at higher concentration ( 20mg/L) decreased the NF-κB translocation in THP1/CD14 cells. Conclusion MBL may inhibit IL-8 and TNF-α production induced by dimorphism C. albicans in THP1/CD14 cells, suggesting that MBL can play some roles on the regulation of C. albicans immune response.

Objective:To investigate the regulatory effects and mechanism of mannose-binding lectin(MBL) on autophagy during the differentiation of 3T3-L1 adipocytes, and provide the feasibility for targeting autophagy to prevent obesity and related pathological conditions in natural immunity.Methods:3T3-L1 preadipocytes were cultured in vitro and induced to differentiation. Cell differentiation and lipid accumulation were analyzed by oil red O staining and CCK-8 was used to detect the effect of different concentrations of MBL (0, 1, 5, 10 μg/ml) on cell proliferation ability at different differentiation stages. Western blot was used to analyze the expression of MBL(10 μg/ml) on the key autophagy factors LC3B, Beclin1 and p62 protein at different stages of differentiation, and the changes of lipid droplet accumulation under the intervention of MBL were observed by oil red O staining. The protein and mRNA expression of autophagy key factors under the intervention of different concentrations of MBL were detected by Western blot and qRT-PCR. And autophagy flow analysis based on autophagic degradation was used to further illustrate the autophagic activity. The expression and phosphorylation of adenosine monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR) signaling molecules were analyzed by Western blot. Results:The results of oil red O staining showed that 3T3-L1 preadipocytes could achieve complete differentiation after 10 days of induction. CCK-8 showed that the concentration of MBL (1-10 μg/ml) in the experimental group had no effect on cell proliferation at different differentiation stages. During the differentiation of 3T3-L1 preadipocytes, Western blot and qRT-PCR showed that the expression of autophagy-related proteins and mRNA levels was enhanced in the MBL treated group, and presented a concentration-dependent relationship. Oil red O staining showed that the lipid droplets in adipocytes at different stages of differentiation are reduced to varying degrees under the intervention of MBL. Fluorescence microscopy results further confirmed that MBL enhanced the autophagy activity of adipocytes by increasing the synthesis of autophagosomes. Moreover, under the intervention of MBL, the phosphorylation level of AMPK was significantly up-regulated, while the phosphorylation level of mTOR was significantly down-regulated, also showing a concentration-dependent relationship.Conclusions:MBL accelerates the autophagy process during the differentiation of 3T3-L1 adipocytes through AMPK/mTOR signaling pathway, reduces lipid accumulation, providing a possible functional pathway for the treatment of obesity and related metabolic diseases.

Objective:To investigate the association of single nucleotide polymorphisms (SNPs) of receptor-associated protein at the synapse ( rapsyn ) with myasthenia gravis ( MG ).Methods: The genomic DNA was extracted from peripheral blood cells , sampled from 132 patients with MG and 153 control individuals.The 8 exons of rapsyn gene were amplified by PCR ,then the products of PCR sequenced directly.Each sequence was compared with wild-type rapsyn gene , and the association between mutation and clinical symptoms of MG analysed.Results:No mutation was found in the exons 1,2,4,5,6,7,and 8 of rapsyn gene both in MG patients and control group compared with the wild-type rapsyn gene.However,a new SNP,L222R[CTG>CGG(2)] or T665G,was found in exon-3.The allele and genotype frequencies of SNP L 222R met Hardy-Weinberg genetic equilibrium (P>0.05),indicating the group repre-sentativeness.The allele frequencies of G were not statistically different between patient and control groups ( P>0.05 ).There were differences in the 3 genotypes TT , TG and GG between patient ( 42.4% vs 48.5% vs 9.1%) and control ( 49.0% vs 33.3% vs 17.6%) groups ( P<0.05 ).The genotype frequencies of GG were statistically higher in control group than that in patient group , showing a recessive model of inheritance.Conclusion: The SNPs in the rapsyn gene are associated with MG in this study.L222R ( T665 G) is a new SNP found and allele G might be a protective factor for MG.

Objective To investigate the effects of mannan-binding lectin(MBL) on TNF-α production induced by peptidoglycan (PGN) and its mechanism in human THP-1/CD14 monocytes.Methods The THP-1/CD14 cells were stimulated for 24 h with PGN at the indicated ratios after pretreated with human natural MBL at concentrations ranging from 1 to 20 mg/L for 2 h.The content of TNF-α and IL-6 in culture supernatants were detected by ELISA,and the levels of TNF-α and IL-6 mRNA expressions in these cells were determined by RT-PCR.FACS was used to investigate the interaction of MBL with THP-1/CD14 cells and the impact of MBL on PGN binding to THP-1/CD14 cells.Western blot was used to detect PGN-induced NF-κB translocation in THP-1/CD14 cells.Results ELISA showed that secretion of TNF-α and IL-6 from THP-1/CD14 cells could be induced by PGN ;The productions of TNF-α and IL-6 by THP-1/CD14 cells induced with PGN were profoundly inhibited by MBL at higher concentrations (10-20 mg/L) but not MBL at lower concentrations (1 mg/L).RT-PCR analysis also indicated that the mRNA expressions of TNF-α and IL-6 in THP-1/CD14 cells were decreased by MBL at higher concentration,compared to the corresponding THP-1/CD14 cells stimulated with PGN only.FACS showed that the binding of MBL to THP-1/CD14 cells was evident in a Ca2+-dependent manner.PGN could competitively inhibit the binding of MBL to THP-1/CD14 cells.MBL could competitively inhibit the binding of PGN to THP-1/CD14 cells by binding to THP-1/CD14 cells directly.Similarly,MBL at higher concentration (20 mg/L) decreased the NF-κB translocation in THP-1/CD14 cells.Conclusion MBL may inhibit TNF-α and IL-6 production induced by PGN in THP-1/CD14 cells through NF-κB signaling pathways,suggesting that MBL can play some roles in the regulation of PGN-induced inflammatory response.

Sonic Hedgehog (SHH) signaling pathway participates in the regulation of various organs and growth of tissue cells, maintains normal function and structure in the development of embryogenesis, however, SHH signaling pathway is in a state of inhibition. Meanwhile, the abnormal regulation of SHH signaling pathway plays an important role in the occurrence, development and drug resistance of leukemia. The mechanism of SHH signaling pathway in leukemia is similar to the solid tumor. With the further understanding of SHH signaling pathway, there are many antitumor drugs targeting SHH signaling pathway. The targeting inhibitors targeted SHH signaling pathway will become a new method for the treatment of leukemia. This paper reviews the application of the inhibitors targeting SHH signaling pathway in leukemia.

Objective:To investigate the regulatory effects and mechanism of mannan-binding lectin (MBL) on adipogenic differentiation of 3T3-L1 preadipocytes.Methods:3T3-L1 preadipocytes were induced to differentiate into adipocytes in vitro, and stimulated with different concentrations of MBL (0, 1, 10, 20 μg/ml). Firstly, changes in cell proliferation ability were detected by CCK-8. Then lipid accumulation was analyzed by Oil red O staining and intracellular triglyceride content determination. Further, the expression of adipogenic differentiation-related factors PPARγ and C/EBPα at protein and mRNA levels were detected by Western blot and qRT-PCR, respectively. Finally, Western blot was used to analyze the expression and phosphorylation of Akt, a signal molecule related to adipogenic differentiation. Results:MBL at the concentrations of 0, 1, 10 and 20 μg/ml had no effect on the proliferation of 3T3-L1 preadipocytes. The level of triglyceride in MBL treatment groups decreased in a dose-dependent manner on 3 d after 3T3-L1 preadipocyte differentiation. Results of Oil red O staining showed that the number of lipid droplets in MBL treatment groups reduced significantly, and the absorbance values also decreased significantly in a concentration-dependent manner. Western blot and qRT-PCR results showed that the expression of PPARγ and C/EBPα at both protein and mRNA levels in MBL treatment groups decreased significantly in a dose-dependent manner, and the phosphorylation level of Akt was significantly down-regulated as well.Conclusions:MBL regulates the adipogenic differentiation of 3T3-L1 preadipocytes via Akt signaling pathway.

Objective:To investigate the effect of programmed death receptor (PD)-1 antibody therapy in patients with hepatitis B-associated liver cancer.Methods:Data of 29 chronically infected HBV patients with liver cancer who received PD-1 antibody combined with tyrosine kinase inhibitor in the Department of Infectious Diseases of the Fifth Medical Center of PLA General Hospital from March 2020 to January 2021 were selected. At the same time, all of the above-mentioned hepatitis B virus (HBV) patients were treated with nucleos(t)ide analogues. Patients clinical diagnostic data, laboratory test results, tumor response and the incidence of adverse reactions were collected retrospectively to understand the overall safety, therapeutic anti-tumor effect, HBV changes condition and the correlation between HBV changes and anti-tumor PD-1 antibody efficacy, high viral load treatment condition, and HBV reactivation safety issues. Statistical analysis was performed by non-parametric rank sum test.Results:Therapeutic anti-tumor effect and safety profile were good in patients. The complete remission rate was reached 27.6%. Adverse reactions were mostly mild, and the incidence of serious adverse reactions was low. After 12 weeks of follow-up, HBV DNA and hepatitis B surface antigen (HBsAg) was quantitatively decreased ( P < 0.05). HBV DNA and HBsAg were decreased more significantly in patients with progressive disease (PD), stable disease (SD) and partial response (PR) ( P < 0.05). Five patients with HBV DNA ≥ 10 4 IU/ml had responded well to the tumor treatment without serious adverse reactions. One patient had a slight increase in HBV DNA and alanine aminotransferase, while there was no HBV reactivation and correlated liver damage. Conclusion:Patients with HBV-associated liver cancer who received combined therapy have good anti-tumor efficacy and safety profile. PD-1 treatment has a certain effect on HBV. Compared with non-responders, patients with tumor response have better antiviral treatment efficacy. The safety of treatment in patients with high viral load is manageable, and there are no safety issues related to HBV reactivation.