This study investigated the specific immune response of BALB/c mice that was induced by a circular RNA (circRNA) vaccine expressing the herpes simplex virus type II (HSV-2) glycoprotein D (gD). The aim was to evaluate the immunological potential of this vaccine and lay a foundation for developing an mRNA vaccine against HSV-2. PCR and homologous recombination were employed to integrate the gD gene obtained from the pT7AMP-gD ectodomain plasmid into pUC57 to generate the recombinant plasmid pUC57-circ-gD, which was then sequenced and characterized. In vitro transcription and cyclization were performed on the template DNA to generate pUC57-circ-gD mRNA. To validate the formation of circular RNA, we cleaved the pUC57-circ-gD mRNA with RNase R and employed RT-PCR to validate the cyclization. The pUC57-circ-gD mRNA was then transfected into 293T cells. After 72 h, the cell supernatant was collected, and Western blotting was employed to measure the protein level of gD. Subsequently, the mRNA was encapsulated in lipid nanoparticles (LNPs) by microfluidic encapsulation. BALB/c mice were administrated with the encapsulated mRNA, and blood was collected from the fundus venous plexus after 21 and 35 days, and from the enucleated eyeballs after 49 days. Enzyme-linked immunosorbent assay was employed to measure the titers of antibodies, including virus-neutralizing antibodies. After 49 days, spleens were harvested and assessed for secretion of interferon-gamma (IFN-γ) by solid-phase enzyme-linked immunospot. The results showed successful construction and sequencing of the recombinant plasmid. RNase R digestion confirmed the presence of circular RNAs. Western blotting of the 293T cells transfected with the mRNA showed clear specific bands. The quality of the vaccine was tested by size exclusion chromatography-high performance liquid chromatography, which showed that the purity of the vaccine was about 90%. The mRNA-LNP showcased the particle size of 82.76 nm and an encapsulation rate of approximately 98%. Following three-dose vaccination, all immunized mice exhibited steady weight gain with 100% survival rate throughout the 28-day observation period, indicating no significant acute toxicity associated with the vaccine formulation. The immunized mice showed dose-dependent increases in serum IgG antibody titer and IFN-γ secretion by splenocytes and they were resistant to virus attacks. These findings indicate good immunogenicity and persistence of the pUC57-circ-gD mRNA vaccine, providing a reference for further studies on circRNA vaccines.
Animals ; Mice, Inbred BALB C ; RNA, Circular ; Mice ; Humans ; Herpesvirus 2, Human/genetics* ; Viral Envelope Proteins/genetics* ; Antibodies, Viral/blood* ; HEK293 Cells ; Female ; Nanoparticles ; Plasmids

Objective:To explore the pathological differences of surgically resected specimens of advanced esophageal squamous cell carcinoma (ESCC) to different neoadjuvant therapies (neoadjuvant radiochemotherapy and toripalimab combined with neoadjuvant radiochemotherapy).Methods:Thirty patients diagnosed with advanced ESCC who underwent surgical operation after neoadjuvant therapy in Jiangsu Cancer Hospital from October 2020 to September 2021 were included. Among them, 15 patients received neoadjuvant radiochemotherapy (radiochemotherapy group) and 15 patients were treated with toripalimab combined with radiochemotherapy (immunotherapy combined with radiochemotherapy group). Surgically resected specimens were collected. The histopathological features of primary esophageal lesions and the responses of involved lymph nodes were analyzed and compared between two groups.Results:The major pathological response (MPR) rate in the radiochemotherapy group was 10/15, and 14/15 in the immunotherapy combined with radiochemotherapy group ( P=0.17). The pathological complete response (pCR) rate of the primary lesions in the radiochemotherapy group was 7/15, and 10/15 in the immunotherapy combined with radiochemotherapy group ( P=0.46). In the radiochemotherapy group, the incidence rate of tertiary lymphoid structure (TLS) was 7/15, and 12/15 in the immunotherapy combined with radiochemotherapy group ( P=0.02). The incidence rate of necrosis in the radiochemotherapy group was 6/15, and 1/15 in the immunotherapy combined with radiochemotherapy group ( P=0.03). In addition, the incidence rate of foam cell infiltration in the radiochemotherapy group was 6/15, and 13/15 in the immunotherapy combined with radiochemotherapy group ( P=0.01). Furthermore, the pCR rate of involved lymph nodes in the radiochemotherapy group was 7/33, and 11/12 in the immunotherapy combined with radiochemotherapy group ( P<0.001). Conclusion:Compared with the radiochemotherapy group, the incidence of TLS and foam cell infiltration is higher, the incidence of necrosis is lower and clinical efficacy of involved lymph nodes is higher in the immunotherapy combined with radiochemotherapy group, prompting that toripalimab combined with neoadjuvant radiochemotherapy exert higher synergistic immune effect.

BACKGROUND:Understanding the actions and underlying mechanisms of proteins in cel s and organisms and studies on the lumbar disc herniation (LDH)-associated proteins contribute to further clarify the LDH pathogenesis. OBJECTIVE:To analyze the serum proteome changes in LDH patients using two-dimensional gel electrophoresis-tandem mass spectrometry, and to screen the biomarkers for LDH diagnosis and treatment. METHODS:Twenty-five LDH patients were enrol ed and their serums were col ected before and after treatment. After removal of high abundant protein, the serum protein spectrum was compared after two-dimensional gel electrophoresis serum protein spectrum diagram, to look for differential protein spots. Subsequently, the differential protein spots were identified by MALDI-TOF/TOF technique combined with biology software and database retrieval. RESULTS AND CONCLUSION:Six differential protein spots were screened preliminarily, and three kinds of proteins were confirmed after mass spectrum detection, among which, acid glycoprotein was related to the LDH immune regulation and occurrence, development and targeting sites of chemical radiculitis. The expression level of acid glycoprotein was significantly decreased in LDH patients after treatment (P<0.05). These results suggest that acid glycoprotein is associated with LDH through gel electrophoresis-base proteome analysis.

BACKGROUND:Recently, there are many documents al over the world reporting hypotoxicity infection after fracture internal fixation surgery, but reports are different on whether it is necessary for chronic hypotoxicity infection internal fixation removal post surgery. There are no fixed judgment criteria of curative effects, which leads to inexact conclusion of treatment method. ＜br> OBJECTIVE:To observe the curative effects of taking rifampicin and ciprofloxacin with transfer of skin flap in the treatment of chronic hypotoxicity infection with sinus formation after fracture surgery on tibial plateau and ankle. METHODS:A total of 56 cases of chronic hypotoxicity infection after fracture surgery of tibial plateau and ankle were col ected from September 2005 to December 2012. 30 cases in the therapy group were treated with ＜br> levofloxacin and rifampicin with transfer of skin flap. 26 cases in the control group were treated with conventional intravenous antibiotics with local debridement to remove internal fixation. In both groups, the course of disease was 3 to 6 months. The erythrocyte sedimentation rate, kidney function and radiographic indices were reviewed every 1 to 3 months, and curative effects were evaluated. ＜br> RESULTS AND CONCLUSION:During the fol ow-up visit of 6-24 months, no recurrence happened to the cured and improved patients. In the therapy group, 24 cases were cured, 4 cases were improved, and 2 cases were invalid, with a total effective rate of 93%. In the control group, 11 cases were cured, 6 cases were improved and 9 cases were invalid with the total effective rate of 65%.χ2 test showed that therapeutic effects were significantly better in the therapy group than in the control group (P＜0.05). These data indicated that rifampicin and ciprofloxacin with transfer of skin flap for chronic hypotoxicity infection with sinus formation after fracture surgery of tibial plateau and ankle showed good curative effects.

Objective To investigate the protective effects of simvastatin on cobalt choride ( CoCl2 ) -induced hypoxia and reoxygenation injury on alveolar type Ⅱ cells and the underlying mechanisms.Methods CoCl2 was used to establish the hypoxia and reoxygenation injury model on AT Ⅱ cells.Blank,control and variant doses simvastatin-treated groups ( 5,10,20,30,50,100 μ mol/L) were designed in the present study.The proliferation of AT Ⅱ cells was evaluated by Cell Counting Kit-8 ( CCK-8 ) assay.The percentage of apoptotic cells was assessed by flow cytometry AV/PI double-staining.The protein levels of surfactant protein-C (SP-C) and proliferating cell nuclear antigen (PCNA) in AT Ⅱ cells was determined by Western blot.Results As compared with the control group,pretreatment with low dose (5 - 20 μmol/L),but not high dose simvastatin (50 - 100 μmol/L) markedly reduced A549 cells apoptosis,and increased their proliferation and the protein levels of SPC and PCNAin vitro.The protective effect could be reversed in vitro by L-mevalonate,a simvastatin competitive inhibitor,which indicated that the inhibition of mevalorate pathway was involved in the simvastatin induced AT Ⅱ cells function restoration.Condusion Low doses simvastatin reversed CoCl2-induced hypoxia and reoxygenation injury of AT Ⅱ cells.The inhibition of mevalonate pathway contributed to simvastatin induced AT Ⅱ cells function restoration.

Objective Experiment the effective method to improve cell injury of cerebral ischemia.MethodMake Mongolirn-Gerbil acute full cerebral ischemia model with ligation of double main carotid arteries,observe the influence of intervening abdomen injection of western medicine Nimodipin and TCM Xingnaojing on MDA and SOD in cerebral tissue of Mongolirn-Gerbil in acute period of ischemia reperfusion.ResultIt finds that Nimodipin and Xingnaojing both can markedly increase SOD activity and decrease MDA content,there’s no obvious difference between them,while there’s difference among different dosages of Xingnaojing,the effect is rising with dosage increased.ConclusionAs a Chinese drug of anti-free radical injury,Xingnaojing has similiar effect with Nimodipin.

Objective To investigate the feasibility of transient blocking of both pulmonary artery and veins for surgical treat- ment of central lung cancer with stage Ⅲ to preserve the normal pulmonary.Methods Firstly,the relation of the pulmonary artery, the lung neoplasm and the enlarged mediastinal lymph nodes was investigated.If the hilum of lung remained frozen,the pericardium was opened and the pulmonary artery,the upper and lower lobe pulmonary veins were dissected.Then those three vessels were blocked.When the pulmonary cireulation was stopped,bloodless lobectomy and pulmonary artery angioplasty and/or anastomosis were performed.Then the blockers were released,and pulmonary circulation was restored.The time of blocking was(35?15)minutes (16～66 minutes).Results All 20 patients suffer from stage Ⅲ central lung cancer,which' s the hila of lung remained frozen,re- ceived complete resection of the tumor.The normal functioning pulmonary tissue in the 20 patients was preserved instead of pneumone- ctomy.The average amount of bleeding was 256 ml(180～420 ml)during operation.All 20 patients recovered well.Conclusion Transient blocking of both pttlmonary artery and veins for surgical treatment of stage Ⅲ central lung cancer is and innovation in surgical technique,which makes the operation safe and easy.This technique may provide a chance to patients,with poor cardio-pulmonary function.In addition,this technique widens the surgical indications for patients suffering from lung cancer.

AIM To investigate the mices central nerve system function and the effect of drugs through independent activity pattern observation. METHODS By observing the influence of drugs on the independent activity patterns of mice of different testing time and testing times, sex, sound, and brightness. RESULTS The mices independent activity is related to testing times, sound and drugs applied, but not related to testing interval, sex, and brightness. CONCLUSION Individual difference of the independent activity of mice is great. The quietness of the experimental environment influences the result.