Objective To investigate the expression and distribution of 5-hydroxytryptamine 7(5-HT7)receptor in brain and intestine of rats with different subtype of irritable bowel syndrome(IBS).Methods Forty-five SD rats were divided into healthy contral.diarrhea-predominant(IBS-D)and consti pation-predominant(IBS-C)IBS groups.IBS-D and IBS-C models were established by either colonic instillation of acetic acid and restraint stress or stomach irrigation with cool water(0-4℃).The expression and distribution of 5-HT7 receptor at brain and intestine were determined by immunohistochemistry and real-time PCR.The tissue concentration of cyclic AMP(cAMP)was examined by radioimmunoassay.Results Immunohistochemistry study demonstrated that the staining of 5-HT7 receptor at hippocampus and hypothalamus were strong in IBS-C and IBS-D groups compared to control group(P＜0.01),The staining of 5-HT7 receptor at ileum,proximal colon and distal colon were higher in IBS-C group than those in control group(P＜0.05).Real-time PCR revealed that the expression of 5-HT7 receptor at hippocampus and hypothalamus were higher in IBS-C and IBS-D groups than those in control group(P＜0.05).and the expression at ileum and colon were remarkably higher in IBS-C group compared to control group(P＜0.05).The concentration of cAMP at hippocampus and hypothalamus were higher in IBS-C and IBS-D groups than those in control group(P＜0.01,P＜0.05).The cAMP level at proximal and distal colon in IBS-C group was higher than those in control group(P＜0.05).Conclusion The upregulation of 5-HT7 receptor in brain and intestine may be related with the dyskinesis and visceral paresthesia of IBS-C.

Objective: To investigate the genetic stability of virus seed H₂M₂₀K₇ (K7) of live attenuated Hepatitis A virus H2 strain (HAV, H2 strain) for production of hepatitis A (Live) vaccine, lyophilized after continuous passages. Methods: The virus seed K7 of H2 strain was proliferated and passaged in KMB₁₇ cells in cell factories. Viruses of different passages were harvested after continuous passages. Virus RNA was extracted and the complete genomes of different virus passages (K7, K10, K11, K13, K15, K18) were sequenced by using next-generation deep sequencing. The mutation rates of different passages were compared. The infectivity titers of different virus passages of H2 strain were tested by ELISA. Results: The mutation rates of complete genomes of different passages were low after continuous passages of master virus seed. The structure of gene was stable and non-synonymous mutation rate was lower than 0.57%. The mutation rate of 5 ’non-coding regions was lower than 0.1%. There was no significant mutation in VP1/2 A and 2C virulence site. The infectious titers of H2 strains of different passages were within 7.76-8.50 lgCCID₅₀/ml. No statistically significant difference was found in this study. Conclusions The gene structure of the master virus seed, working seed and different passages of H₂M₂₀K₇ after subculture was stable and the mutation rate was low. No significant mutation was found in 5’non-coding regions, and the critical virulence sites such as VP1/2 A, 2B and 2C showed attenuated characteristics with low mutation rate. Virulence of the virus did not changed. The H2 strain maintained stable viral infectivity and genetic stability and comply with the requirements as virus seed for vaccine manufacturing.

Objective:To study the intestinal flora specific differences with different lesional stages of metabolic (disorder) associated fatty liver disease (MAFLD), namely simple steatosis and steatohepatitis, so as to provide a new direction for MAFLD-related intestinal flora transplantation and targeted therapy.Methods:Mice were fed with normal diet, methionine-choline deficient diet (MCD) and a high-fat high-fructose diet (HFHF) for 12 weeks to construct simple steatosis and steatohepatitis models. HE and Sirius scarlet staining was performed to observe the liver pathological changes. The qPCR method was used to evaluate inflammation and liver fibrosis factors. A fully automatic biochemical analyzer was used to detect changes in liver transaminase and blood lipids. 16S rRNA sequencing method was used to observe the intestinal flora differences in the feces of each group of mice. The comparison of means between two groups was performed by t-test, and the comparison of means between multiple groups was performed by one-way analysis of variance. Kruskal-Wallis rank sum test was used for non-normally distributed data.Results:NAFLD scores were determined with pathological sections (HE and Sirius scarlet staining) of mice liver, which showed that the inflammation and liver fibrosis scores of the MCD and HFHF groups were 2.12 ± 0.18 and 1.06 ± 0.24, and 2.22 ± 0.16 and 0.46 ± 0.10, respectively. The degree of liver inflammation and fibrosis was significantly higher in the MCD than the HFHF group ( P < 0.001 and P < 0.01). Lipid deposition was higher in the HFHF than the MCD group ( P < 0.001), and the scores were 2.36 ± 0.17 and 1.60 ± 0.24 respectively. Simultaneously, the inflammatory [tumor necrosis factor-A (TNF-a), chemokine factor-2 (CXCL-2)] and hepatic fibrosis indicators [vascular smooth muscle actin alpha (a-SMA) and connective tissue growth factor (CTGF)] had confirmed the above-mentioned results at the transcription level. Moreover, the intestinal flora diversity was reduced ( P < 0.05) in the MCD group than the HFHF group, and the Simpson and Shannon index were 0.31 ± 0.10 and 0.42 ± 0.05, and 2.03 ± 0.33 and 1.70 ± 0.28, respectively, and the differences were significant between different intestinal flora groups. The levels of Desulfovibrio, Odoribacter, and Roseburia flora were significantly increased in the HFHF than the MCD group, and the levels of Faecalibaculum, Parasutterella, Alipis, Butyricimonas_virosa, Turicibacter_sp, and Romboutsia_ilealis were significantly increased in the MCD than the HFHF group, and the difference was statistically significant ( P < 0.05). Conclusion:There are significant differences in intestinal flora diversity between simple steatosis and steatohepatitis models. Therefore, clarifying the difference between the two may provide a new direction for the stage manner treatment of MAFLD.

[Abstract] Objective:To prepare the third generation CAR-T cells targeting EGFRvⅢ (EGFRvⅢCAR-T) and to detect its specific killing effect against EGFRvⅢ+ U87 cells in vitro and in vivo. Methods: Human CD3+ T cells were transfected with lentiviral EGFRv Ⅲ/3CAR, which was generated by calcium phosphate co-precipitation of three plasmids. The expression of EGFRvⅢ/3CAR in T cells was detected by Western blotting and flow cytometry. In vitro killing effect of EGFRvⅢ/3CAR-T cells on EGFRvⅢ+ U87 cells was detected by 51Cr release assay. The secretion of cytokine IFN-γ of EGFRvⅢ/3CAR-T cells was detected by ELISA. Nude mouse xenograft model was constructed to detect the in vivo cytotoxicity of EGFRvⅢ/3CAR-T cells on xenograft tumor. Results: The EGFRvⅢ/3CAR lentivirus was successfully packaged with an average titer of 5×106 TU/ml. Western blotting showed that a protein band of approximate 58 000 molecular weight was observed in EGFRvⅢ/3CAR-T cells but absent in untransfected T cells. Flow cytometry indicated the average transduction efficiency of EGFRvⅢ/3CAR was 52.3%. 51Cr release assay showed that the specific killing effect of EGFRvⅢ/ 3CAR-T cells was positively correlated with E/T ratio (E∶T=4∶1, 8∶1, 16∶1, 32∶1). ELISA showed that cytokine IFN-γ secretion was (1 836±148.2) pg/ml, which was significantly different from that of NTT and GFP+ T cells (P<0.01). The specific killing activity of EGFRvⅢ/3CAR-T cells and IFN-γ secretion were both dependent on the expression level of EGFRvⅢ in U87 cells. The tumor growth monitoring results showed that the tumor volume of EGFRvⅢ/3CAR-T cell group was significantly different from that of GFP+ T cell group and PBS group around 3 weeks after injection (P<0.01). Conclusion: EGFRvⅢ/3CAR-T cells demonstrated specific antitumor effectagainstEGFRvⅢ+U87cellsbothinvitro and in vivo, providing basis for immunotherapyofgliomainfuture clinical use.