1.Establishment of a hepatic carcinoma cell line with stable ClC-3 silencing by lentivirus-mediated RNA interference and its influence on invasion and migration
Bin XU ; Jialin LIN ; Jingwen SHI ; Shisi WANG ; Jie YU
Chinese Pharmacological Bulletin 2015;(8):1101-1107
Aim To establish a hepatic carcinoma cell line with stable voltage-gated chloride channel 3 ( ClC-3 ) gene silencing through the lentivirus-mediated short-hairpin RNA ( shRNA ) method and investigate the effects of gene silencing on invasion and migration. Methods Three lentiviral vectors coding shRNA tar-geting ClC-3 gene were constructed, the recombinant plasmids were packaged into mature lentivirus by 293FT cells, and then the lentiviruses were harvested, concentrated and titrated. MHCC97H cells were infec-ted with the recombinant lentiviruses and then were se-lected to obtain cell lines stably expressing ClC-3 shR-NA. The efficiency of ClC-3 mRNA and protein ex-pression interference were determined by real-time flu-orescence quantitative PCR and Western blot, respec-tively. The effects of ClC-3 gene interference on inva-sion and migration of MHCC97 H cells were performed by Transwell chamber assays with or without Matrigel and cell scratch assay. Results The recombinant lentiviral vectors were successfully constructed and four lentiviruses were acquired after packaged by 293 FT cells. One negative control cell line and three cell lines with ClC-3 gene interference ( MHCC97 H/shClC-3-1 , shClC-3-2 and shClC-3-3 ) were successfully construc-ted after MHCC97 H cells were infected with lentivirus-es. The expression level of ClC-3 mRNA and protein in three ClC-3-silenced cells were obviously lower than the negative control cells ( P <0. 01 ) , MHCC97 H/shClC-3-2 cells showed the greatest inhibition of ClC-3 mRNA and protein expressions. As compared with the negative control cells, the ClC-3 gene interference sig-nificantly decreased invasion and migration of MH-CC97 H cells in vitro ( P <0. 01 ) . Conclusion The hepatic carcinoma cell lines with stable ClC-3 gene si-lencing were successfully established and the ClC-3 gene interference could significantly inhibit invasion and migration of MHCC97H cells.
2.Efficacy of Yiqi-Tuomin decoction combined with triamcinolone acetonide nasal spray on allergic rhinitis and its effect on Th1/Th2 balance
International Journal of Traditional Chinese Medicine 2019;41(6):565-568
Objective To investigate the effect of Yiqi-Tuomin decoction combined with triamcinolone acetonide nasal spray on Th1/Th2 balance in allergic rhinitis (AR) and to observe the clinical efficacy.Methods A total of 100 patients who met the inclusion criteria were divided into two groups by random number table method,50 in each group.The control group was treated with triamcinolone acetonide nasal spray.The observation group was given Yiqi-Tuomin decoction on the basis of the control group.Both groups were treated for 4 weeks and followed up for 3 months.Clinical symptom score was performed before and after treatment.The serum level of interferon-γ (IFN-γ) and IL-4 was detected by ELISA,and the ratio of IFN-γ/IL-4 was calculated.The adverse reaction and recurrence rate during treatment was observed and recorded,and the clinical efficacy was evaluated.Results The total effective rate was 94.0% (47/50) in the observation group and 80.0% (40/50) in the control group.There was significant difference between the two groups (x2=4.332,P=0.037).After treatment,the serum IFN-γ (95.15 ± 18.49 ng/L vs.83.85 ± 16.20 ng/L,t=3.250) and the ratio of IFN-γ/IL-4 (1.35 ± 0.21 vs.0.93 ± 0.19,t=5.878) significantly increased (P<0.01),while serum level of IL-4 (71.14 ± 14.15 ng/L vs.89.8 6 ± 17.52 ng/L,t=10.487) significantly decreased in the observation group (P<0.01).After treatment,the score of nasal obstruction,rhinocnesmus,sneezing and nasal discharge in the observation group were significantly lower than those in the control group (t=11.229,8.921,8.253,10.464,respectively,all Ps<0.01).Conclusions The Yiqi-Tuomin decoction combined with triamcinolone acetonide nasal spray can significantly improve the clinical symptoms of AR patients,correct the imbalance of Th1/Th2 ratio and improve the immunity of the body.
3.Identification of Medium-Length Antineurofilament Autoantibodies in Patients with Anti-N-Methyl-D-Aspartate Receptor Encephalitis
Shisi WANG ; Cancan XU ; Xiaobo SUN ; Yifan ZHOU ; Yaqing SHU ; Shangzhou XIA ; Zhengqi LU ; Wei QIU ; Xiaofen ZHONG ; Lisheng PENG
Journal of Clinical Neurology 2020;16(3):470-479
Background:
and Purpose: Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a severe central nervous system disorder mediated by NMDAR antibodies that damages neurons. We investigated the correlation between cytoskeletal autoantibodies and the clinical severity in patients with anti-NMDAR encephalitis.
Methods:
Non-NMDAR autoantibodies were identified by screening matched cerebrospinal fluid (CSF) and the serum samples of 45 consecutive patients with anti-NMDAR encephalitis and 60 healthy individuals against N-methyl-D-aspartate receptor 1-transfected and nontransfected human embryonic kidney 293T cells. Immunocytochemistry was performed to assess antibody binding in rat brain sections and primary cortical neurons. Cell-based assays and Western blotting were applied to identify autoantibodies targeting medium neurofilaments (NFMs). We compared clinical characteristics between patients with NMDAR encephalitis who were positive and negative for anti-NFM-autoantibodies.
Results:
Anti-NFM autoantibodies were detected in both the serum and CSF in one patient (2%) and in the serum only in six patients (13%). No antibodies were detected in the serum of healthy controls (7/45 vs. 0/60, p=0.0016). Four of the seven patients with anti-NFM autoantibodies in serum were children (57%), and three (43%) had abnormalities in brain magnetic resonance imaging. These patients responded well to immunotherapy, and either no significant or only mild disability was observed at the last follow-up. Anti-NMDAR encephalitis did not differ with the presence of anti-NFM autoantibodies.
Conclusions
Anti-NFM autoantibodies may be present in patients with anti-NMDAR encephalitis, indicating underlying neuronal damage. A large cohort study is warranted to investigate the clinical differences between patients with NMDAR encephalitis according to their antiNFM antibody status.
4.Myelin Oligodendrocyte Glycoprotein-IgG Contributes to Oligodendrocytopathy in the Presence of Complement, Distinct from Astrocytopathy Induced by AQP4-IgG.
Ling FANG ; Xinmei KANG ; Zhen WANG ; Shisi WANG ; Jingqi WANG ; Yifan ZHOU ; Chen CHEN ; Xiaobo SUN ; Yaping YAN ; Allan G KERMODE ; Lisheng PENG ; Wei QIU
Neuroscience Bulletin 2019;35(5):853-866
Immunoglobulin G against myelin oligodendrocyte glycoprotein (MOG-IgG) is detectable in neuromyelitis optica spectrum disorder (NMOSD) without aquaporin-4 IgG (AQP4-IgG), but its pathogenicity remains unclear. In this study, we explored the pathogenic mechanisms of MOG-IgG in vitro and in vivo and compared them with those of AQP4-IgG. MOG-IgG-positive serum induced complement activation and cell death in human embryonic kidney (HEK)-293T cells transfected with human MOG. In C57BL/6 mice and Sprague-Dawley rats, MOG-IgG only caused lesions in the presence of complement. Interestingly, AQP4-IgG induced astroglial damage, while MOG-IgG mainly caused myelin loss. MOG-IgG also induced astrocyte damage in mouse brains in the presence of complement. Importantly, we also observed ultrastructural changes induced by MOG-IgG and AQP4-IgG. These findings suggest that MOG-IgG directly mediates cell death by activating complement in vitro and producing NMOSD-like lesions in vivo. AQP4-IgG directly targets astrocytes, while MOG-IgG mainly damages oligodendrocytes.