We wished to screen the cell line that stably expresses the HPV18E5 protein, and to ascertain the influence of HPV18E5 protein on cell proliferation and the cell cycle. The HPV18E5 gene was amplified by the polymerase chain reaction. Then, the His-tag pSecTag-HPV18E5 eukaryotic expression vector was constructed by digestion ligation and connection. The recombinant plasmid was transfected into Balb/c3T3 cells with lipofectamine, and positive cell lines were screened by a culture medium containing bleomycin. HPV18E5 expression in cells was confirmed by western blotting and immuno-enzymatic methods. The influence of HPV18E5 on cell proliferation and the cell cycle were detected by Cell Counting Kit-8 and flow cytometry, respectively. The pSecTag-HPV18E5 eukaryotic expression vector was constructed. After 21-day selection in a culture medium containing 400 μg/mL bleomycin, stably expressing HPV18E5 protein cells were harvested. Compared with control groups, cell proliferation in HPV18E5 stably expressed cells was obviously increased, as was the S phase in the cell cycle. Our results suggested that HPV18E5 influences cell proliferation and the cell cycle. Our study has laid the foundation of the biologic properties of HPV18E5 protein, which will aid further studies on the mechanism of action of carcinogenesis.
Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Human papillomavirus 18 ; genetics ; metabolism ; Humans ; Oncogene Proteins, Viral ; genetics ; metabolism ; Papillomavirus Infections ; physiopathology ; virology ; Transfection

Objective To assess the growth of monochorionic diamniotic (MCDA) twins discordant for birth weight during their first 24 months of life.Methods Clinical data and growth parameters from birth to 24 months of age of 31 pairs(62 cases) of birth weight-discordant MCDA twins (≥ 25％ difference in birth weight) who were born alive in the First Affiliated Hospital,Sun Yat-sen University between January 1,2010 and June 30,2016 were retrospectively analyzed.Each pair of twins was divided into the large birth weight group (31 cases) and the small birth weight group (31 cases).All datas were statistically analyzed with Chisquare test,paired t test,analysis of variance or LSD-t test.Results (1) The incidence of birth defects and hypoproteinemia in the small birth weight group were higher than that in the large birth weight group [29.0％ (9/31) vs 0.0％ (0/31),54.8％ (17/31) vs 25.8％ (8/31);x2=8.319 and 5.429;both P ＜ 0.05].(2) The standard deviation scores (SDS) of weight,length and head circumference at birth and 1,6,12,18 and 24 months of age of the small birth weight group were lower than those of the large birth weight group [birth:(-2.00 ± 0.66) vs (-0.04±0.60),(-1.83±1.13) vs (-0.37±0.83),(-1.42±1.03) vs (0.17±0.84),t=17.214,8.390 and 7.759;1 month:(-1.77±0.81) vs (-0.60±0.65),(-2.36±1.20) vs (-0.94±0.74),(-1.71±1.26) vs (-0.44± 1.09),t=9.424,9.059 and 7.197;6 months (-1.00±0.84) vs (-0.09±0.56),(-1.31 ± 1.22) vs (-0.04±0.80),(0.30±1.51) vs (1.11 ± 1.20),t=-7.578,7.988 and 6.091;12 months:(-0.34±1.06) vs (0.47±0.79),(-1.00±0.92) vs (-0.14±0.73),(-0.16±0.76) vs (0.49±0.58),t=5.747,7.155 and 5.664;18 months:(-0.06±0.95) vs (0.74±0.66),(-0.92± 1.07) vs (-0.24±0.92),(-0.32±0.72) vs (0.29±0.66),t=6.153,4.496 and 3.877;24 months:(0.20±0.79) vs (0.88±0.62),(-0.66±0.59) vs (0.01 ±0.67),(-0.37±0.60) vs (0.34±0.68),t=5.317,4.800 and 4.905;all P ＜ 0.001].However,the changes in SDS (△ SDS) of weight,length and head circumference from birth to 24 months of age and the incidence of △ SDS ＞ 0.67 were significantly higher in the small birth weight group than those in the large birth weight group [△SDS:(2.20± 1.10) vs (0.92±0.91),(1.17± 1.21) vs (0.37± 1.14),(1.05± 1.07) vs (0.16±0.89),t=8.422,3.918 and 3.547,all P ＜ 0.001;△SDS ＞ 0.67:93.5％ (29/31) vs 61.3％ (19/31),61.3％ (19/31) vs 35.5％ (11/31),61.3％ (19/31) vs 29.0％ (9/31),x2=9.226,4.133 and 6.53 1,all P ＜ 0.05].(3) The difference in SDS for weight,length and head circumference between the large and small birth weight twins at 24 months of age were significantly lower than those at birth [(0.68 ± 0.71) vs (1.95 ± 0.63),(0.67 ± 0.77) vs (1.46± 0.97),(0.71 ± 0.80) vs (1.60±1.15);all P ＜ 0.05].Conclusions Growth differences are found between birth weight-discordant MCDA twins all the way to 24 months of age from birth at a reduced trend.

Objective To investigate the effectiveness of intravenous thrombolytic therapy mode led by fast-track specialist nurses on patients with acute ischemic stroke(AIS).Methods This study involved 124 AIS patients who underwent intravenous thrombolytic therapy in the Department of Emergency of our hospital from March 2021 to February 2023.Among the patients,61 admitted between March 2021 and February 2022 received conventional AIS thrombolytic therapy were assigned to a control group.While the 63 patients who received AIS thrombolytic therapy under the specialist nurse-led intravenous thrombolytic therapy mode between March 2022 and February 2023 were assigned to an observation group.The two groups were compared in terms of the time from admission to completion of CT examination,time for signing the informed consent for thrombolytic therapy,door to needle time and percentage of DTN<60 minutes,as well as the post-thrombolysis scores according to the National Institute of Health Stroke Scale(NIHSS)and satisfaction to medical consultation.Results The observation group exhibited a significantly shorter time from admission to completion of CT examination,a shorter time for signing an informed consent for thrombolytic therapy,a shorter door to needle time and a higher percentage of DTN<60 minutes,all with significant difference in comparison with those in the control group.After thrombolysis,the NIHSS score of the observation group decreased more than that of the control group(P<0.05).The patients and their families in the observation group reported significantly higher satisfaction compared to those in the control group(both P<0.05).Conclusion The fast-track specialist nurse-led intravenous thrombolytic therapy mode demonstrates the superiority in reduction of the time from admission to completion of CT examination,time for signing an informed consent for thrombolytic therapy,door to needle time and the NIHSS scores,higher percentage of DTN<60 minutes as well as improvement of patient satisfaction.

Precise orchestration of cell fate determination underlies the success of scaffold-based skeletal regeneration.Despite extensive studies on mineralized parenchymal tissue rebuilding,regenerating and maintaining undifferentiated mesenchyme within calvarial bone remain very challenging with limited advances yet.Current knowledge has evidenced the indispensability of rebuilding suture mesenchymal stem cell niches to avoid severe brain or even systematic damage.But to date,the absence of promising therapeutic biomaterials/scaffolds remains.The reason lies in the shortage of fundamental knowledge and methodological evidence to understand the cellular fate regulations of scaffolds.To address these issues,in this study,we systematically investigated the cellular fate determinations and transcriptomic mechanisms by distinct types of commonly used calvarial scaffolds.Our data elucidated the natural processes without scaffold transplantation and demonstrated how different scaffolds altered in vivo cellular responses.A feasible scaffold,polylactic acid electrospinning membrane(PLA),was next identified to precisely control mesenchymal ingrowth and self-renewal to rebuild non-osteogenic suture-like tissue at the defect center,meanwhile supporting proper osteointegration with defect bony edges.Especially,transcriptome analysis and cellular mechanisms underlying the well-orchestrated cell fate determination of PLA were deciphered.This study for the first time cellularly decoded the fate regulations of scaffolds in suture-bony composite defect healing,offering clinicians potential choices for regenerating such complicated injuries.

Objective To construct the eukaryotic expression vector of human papillomavirus type18E5 and EGFP fusion gene,to analyze E5 expression in BALB/c3T3 cell.Methods The HPV18E5 and EGFP gene were amplified by polymerase chain reaction,and pSecTag-HPV18 E5-EGFP fusion gene eukaryotic expression vector was constructed by the method of digestion ligation and transformation.The recombinant plasmid was transfected into BALB/c3T3 cells with lipofectamine and the expressed product was detected by RT-PCR,the aim gene expression was observed under fluorescence microscope.Results The success in construction of pSecTag-HPV18 E5-EGFP fusion gene eukaryotic expression vector,RT-PCR result showed that HPV18E5 gene could be expressed in BALB/c 3T3 cell,fluorescent light could be observed under fluorescence microscope.Conclusion pSecTag-HPV18 E5-EGFP fusion gene eukaryotic expression vector has been successfully constructed,the recombinant plasmid could be expressed in BALB/ c3T3 cell.

Autoimmune hepatitis(AIH)is a type of chronic hepatitis caused by the attack of hepatocytes by the autoimmune system,and with the prolongation of disease course,it may gradually progress to liver cirrhosis and even hepatocellular carcinoma.Although great achievements have been made in the understanding and treatment of AIH,its etiology and pathogenesis still remain unclear.T cells play a crucial role in the development and progression of AIH,and by focusing on follicular helper T cells,this article elaborates on the research advances in follicular helper T cells in AIH,in order to provide new ideas and strategies for the clinical treatment of AIH.

BACKGROUND: Lung cancer is the second most common cancer worldwide, with non-small-cell lung cancer (NSCLC) accounting for the majority of cases. Patients with NSCLC have achieved great survival benefits from immunotherapies targeting immune checkpoints. Glucocorticoids (GCs) are frequently used for palliation of cancer-associated symptoms, as supportive care for non-cancer-associated symptoms, and for management of immune-related adverse events (irAEs). The aim of this study was to clarify the safety and prognostic significance of glucocorticoid use in advanced patients with NSCLC treated with immune checkpoint inhibitors (ICIs). METHODS: The study searched publications from PubMed, Embase, Cochrane Library, Web of Science, China Biology Medicine disc, Chinese National Knowledge Infrastructure, Wanfang Data, and Chinese Science and Technology Journal Database up to March 1st, 2022, and conducted a meta-analysis to assess the effects of glucocorticoid use on overall survival (OS) and progression-free survival (PFS) in NSCLC patients treated with ICIs through the available data. The study calculated the pooled hazard ratios (HRs) and 95% confidence intervals (CIs). RESULTS: This study included data from 25 literatures that were mainly retrospective, with 8713 patients included. Patients taking GCs had a higher risk for tumor progression and death compared with those not taking GCs (PFS: HR = 1.57, 95% CI: 1.33-1.86, P <0.001; OS: HR = 1.63, 95% CI: 1.41-1.88, P <0.001). GCs used for cancer-associated symptoms caused an obviously negative effect on both PFS and OS (PFS: HR = 1.74, 95% CI: 1.32-2.29, P <0.001; OS: HR = 1.76, 95% CI: 1.52-2.04, P <0.001). However, GCs used for irAEs management did not negatively affect prognosis (PFS: HR = 0.68, 95% CI: 0.46-1.00, P = 0.050; OS: HR = 0.53, 95% CI: 0.34-0.83, P = 0.005), and GCs used for non-cancer-associated indications had no effect on prognosis (PFS: HR = 0.92, 95%CI: 0.63-1.32, P = 0.640; OS: HR = 0.91, 95% CI: 0.59-1.41, P = 0.680). CONCLUSIONS In advanced NSCLC patients treated with ICIs, the use of GCs for palliation of cancer-associated symptoms may result in a worse PFS and OS, indicating that they increase the risk of tumor progression and death. But, in NSCLC patients treated with ICIs, the use of GCs for the management of irAEs may be safe, and the use of GCs for the treatment of non-cancer-associated symptoms may not affect the ICIs' survival benefits. Therefore, it is necessary to be careful and evaluate indications rationally before administering GCs in individualized clinical management.
Humans ; Carcinoma, Non-Small-Cell Lung/drug therapy* ; Glucocorticoids/therapeutic use* ; Immune Checkpoint Inhibitors/therapeutic use* ; Lung Neoplasms/drug therapy* ; Retrospective Studies

Objective:To investigate the expression of Macrophage migration-inhibitory factors (MIF) in hepatocellular carcinoma (HCC) tissues and its interaction with ERK1/2 signaling pathway, so as to establish a theoretical basis for further studying the molecular mechanism of MIF promoting HCC.Methods:From February 2020 to August 2021, 52 cases of hepatocellular carcinoma (HCC) tissues based on hepatitis B cirrhosis (HBV-LC) and 52 cases of adjacent tissues in Tianjin Medical University Cancer Hospital and 940th Hospital of Joint Logistic Support Force of PLA were collected as the experimental group, including 39 males and 13 females, aged 35-65 years. And 20 cases of normal liver tissue were selected as the control group. Immunohistochemistry was used to detect the expression of MIF, ERK1/2 and p-ERK1/2 proteins in liver tissues of the two groups, and in situ hybridization was used to detect the expression of ERK1/2 nucleic acid in liver tissues of the two groups.HepG2 HCC cells and L-02 normal hepatocytes were co-cultured with different concentrations of rMIF, the expression and phosphorylation levels of ERK1/2 and JNK1 proteins in the two kinds of liver cells were detected by Western-blot, and the expression levels of ERK1/2 nucleic acids in the two kinds of liver cells were detected by RT-PCR. One-way ANOVA was used for measurement data and χ 2 test was used for counting data. Results:The expressions of MIF, ERK1/2, p-ERK1/2 and ERK1/2 mRNA were significantly increased in HCC and para-cancer tissues (the expression of MIF in HCC group was 78.8%, and that in adjacent group was 75.0%; ERK1/2 80.8% in HCC group and ERK1/2 71.8% in paracancerous group. The expression of p-ERK1/2 75.0 % in HCC group and 46.2% in paracancerous group were respectively detected. ERK1/2 mRNA was expressed in HCC group 76.9%, ERK1/2 mRNA expression in paracancerous group 78.8%), and the differences were statistically significant compared with normal liver tissues ( P<0.05), but there was no significant difference between HCC and para-cancer tissues ( P>0.05). The expressions of ERK1/2, p-ERK1/2 and ERK1/2 mRNA in HepG2 HCC cells were significantly increased with the increase of rMIF concentration, and the increase was most obvious when rMIF concentration was 200 ng/ml, and the difference was statistically significant compared with L-02 normal hepatocytes ( P<0.05). Conclusion:MIF, ERK1/2 and p-ERK1/2 are highly expressed in HCC tissues and HepG2 HCC cells, suggesting that MIF promotes the occurrence and development of hepatocellular carcinoma through ERK1/2 signaling pathway.

Autoimmune hepatitis （AIH） is a type of chronic hepatitis caused by the autoimmune system attacking hepatocytes， and its chronic progression may lead to liver cirrhosis and even hepatocellular carcinoma. Currently， pharmacotherapy and liver transplantation are the main treatment methods for AIH， but both methods have their own limitations， which limits the clinical benefits of patients. Therefore， it is a critical issue to search for new therapeutic agents and methods. Recent studies have shown that mesenchymal stem cells （MSC） and their exosomes can improve the symptoms of patients with AIH by suppressing inflammatory response， enhancing the regeneration of hepatocytes， and regulating the immune system and thus have wide application prospects in the treatment of AIH. By summarizing related articles， this article reviews the possible mechanisms and application of MSC and their exosomes in the treatment of AIH， in order to provide new ideas for the clinical treatment of AIH.

Advanced gastric cancer(AGC)includes locally unresectable gastric cancer(GC),metastatic GC,and postoperative recurrent GC.Due to delayed diagnosis and lack of effective treatment for AGC,the median survival time of AGC patients is only 6-12 months.At present,the main treatment goal of AGC is to improve symptoms and prolong the survival time of patients receiving sequential chemotherapy.Although the therapeutic effect of systemic therapy on AGC is gradually becoming apparent,the patient's prognosis is far from expected.In addition,targeted therapy and novel immunotherapy have drawbacks such as high incidence of drug resistance,high toxic side effects,and heavy economic burden on patients.Therefore,finding new therapeutic targets and developing anti-tumor drugs is a key issue that urgently needs to be addressed.According to reports,abnormal activation of the hepatocyte growth factor(HGF)/cellular-mesenchymal epithelial transition factor(c-MET)pathway plays a crucial role in the progression of GC and the occurrence of multi-line resistance and may be a potential therapeutic target for GC.In recent years,some HGF/c-MET-targeting small molecule tyrosine kinase inhibitors(TKIs)have been found to show good clinical effects in the treatment of GC.Meanwhile,new HGF/c-MET inhibitors(such as monoclonal antibodies,bispecific antibodies,antibody drug conjugates,etc.)have shown good anti-tumor activity in preclinical studies,but they are all at different stages of clinical research,and their efficacy and safety still need further confirmation.This review elaborates on the latest research progress of HGF/c-MET inhibitors in the treatment of AGC and discusses the main reasons and strategies for drug resistance,aiming to provide better guidance for the treatment of AGC and provide reference for future research.