Objective　To investigate the involvement of cell cycle negative regulator p16 in the replicative senescence of normal human diploid fibroblasts. 　Methods　 p16 cDNA and retroviral vector was introduced into normal human fibroblast 2BS cells by transfection technique. Then the effects of p16 on replicative cellular senescence of 2BS cells were examined. 　Results　 Compared with the control cells, 2BS cells transfected with p16 cDNA showed significant suppression of growth rate (decrease 50%) with cell cycle arrested at G1 phase. This phenomenon similar to that of the senescent cells, with the cellular response to stimulation by growth factor decreased 79.4%, showing the characteristics in morphology of senescent fibroblasts. 　Conclusions　 The over expression of p16 gene contributes to the process of cellular senescence of normal human diploid fibroblasts.

Antiviral nucleoside drugs represented by zidovudine, lamivudine, telbivudine, ribavirin, etc. have become the first choice drugs for clinical treatment of viral diseases such as AIDS, hepatitis B, herpes. However, the above drugs not only can inhibit virus replication, they can also interfere with the host mitochondrial mtDNA replication through similar mechanisms, leading to mitochondrial toxicity-related adverse reactions. Therefore, both the routine safety evaluation and mitochondrial toxicity evaluation are required for the development of nucleoside drugs. The research progress of mitochondrial toxicity evaluation models of nucleoside drugs are summarized for reference.

Background and purpose: Pancreatic intraepithelial neoplasia (PanIN) is thought to be a precursor lesion of infiltrating pancreatic ductal adenocarcinoma. The mutation of the phenotypic impact of K-ras G12D alone, silencing of p53 and p16 could promote this process. The role of Smad4 in this progression was poorly understood. In the present study, we investigated the role of Smad4 in the development of pancreatic tumor, based on PanIN cell line from mice with K-ras G12D mutation in order to investigate the effect of Smad4 silencing on PanIN cells in the development and malignant transformation in nude mice. Methods: Smad4 knock-down PanIN cells (PanIN-S) were established by stable transfeetion with lentiviral-mediated Smad4 RNA interference (RNAi). In xenograft model experiments, BALB/c nude mice were randomly divided into 2 groups (5 mice per group) implanted with PanIN or PanlN-S cells subcutaneously. Two weeks after tumor cells inoculation, tumor volume and weight were estimated. PCNA staining was used to evaluate cell proliferation and CD31 polyclonal antibody was used to assess micro-vessel density (MVD) in tumors. Results: Effect of siRNA of Smad4 gene in PanlN cells was confirmed by RT-PCR and Western blot. Compared with PanlN groups, there was a dramatic increase in tumor volume and weight in PanIN-S groups (P<0.05). Furthermore, immunohistochemical analysis of the harvested tumors suggested that Smad4 silencing was associated with 'increased tumor cell proliferation (PCNA reactivity) and angiogenesis (micro-vessel density, MVD). The percentage of PCNA-positive cells in the PanlN-S groups were significantly increased than PanIN groups (P<0.05). CD31 staining revealed a significant increase in the PanlN-S groups compared to the PanlN groups (P<0.05). Conclusion: Silencing of Smad4 in PanlN cells with endogenous expression of K-ras G12D, enhanced progression to invasive adenocarcinomas. Cell proliferation and vascularization may be its important mechanisms.

In this study, we successfully expanded a full length gene encoding the monooxygenase eytochrome P450 2C9 gene from human liver of Chinese Han by RT-PCR. Our findings indicated that except G-->T mutation at the 190th nucleotide site, the other nucleotide sequences are completely consistent with CYP2C9 (NM017460) in GenBank. The SDS-PAGE and Western-Blot analysis showed that the CYP 2C9 gene was successfully expressed in the host cell E. coli BL21 (DE3). Our current study lays the foundation for the evaluation of pre-clinical drug metabolism and safety in the future.
Aryl Hydrocarbon Hydroxylases ; biosynthesis ; genetics ; China ; ethnology ; Cloning, Molecular ; Cytochrome P-450 CYP2C9 ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Point Mutation ; Polymorphism, Genetic ; Recombinant Proteins ; biosynthesis ; genetics

Coronavirus is an important pathogen of humans and animals. Among them, the novel coronavirus disease (COVID-19) breaking out in 2019 has brought a fatal threat to human health. The host"s innate immune response is the host"s first line of defense against pathogen invasion, but an excessive immune response can also aggravate viral infection and pathological damage. The immune escape of coronavirus is a critical pathogenic mechanism causing death. This work mainly reviews the pathogenic mechanism of coronavirus immune escape from several aspects such as host immunosensor, interferon, cytokine and coronavirus antagonizing host immune response, which provide a theoretical reference for the development of anti-coronavirus drugs.

Autophagy is a conserved self-defense mechanism of organism, degrading the necrotic organelles and excess protein into small molecules for recycling. Autophagy plays a role in both physiological and pathological condition, influencing the expression of intracellular substance through multiple signaling pathways. Although it has been demonstrated that Ras/Raf/MEK/ERK signaling pathway was not only extensively involved in the regulation of cell growth, proliferation, differentiation and apoptosis, but was also implicated in autophagy and autophagic cell death, though its detailed mechanisms involved in regulation of autophagy has not been fully elucidated yet. This review focused on the advances of autophagy induced by Ras/Raf/MEK/ERK signaling pathway, to better understand the role of Ras/Raf/MEK/ERK signaling pathway in regulation of autophagy.

Objective:To summarize the clinical application value of color Doppler ultrasound (CDU) in the nursing observation of flap vascular circulation after free flap transplantation.Methods:From September 2018 to November 2020, 18 patients with skin defect of limbs treated by free flap transplantation in the Second Affiliated Hospital of Wenzhou Medical University were monitored by skin color, skin temperature, skin tension, capillary reaction time and CDU. When the vascular circulation disorder was found, the patients should be treated with volume expansion, rehydration, local massage of the flap, anticoagulation and microcirculation improvement. Positive drainage under the flap to prevent infection, combined with hyperbaric oxygen treatment, accurate and timely exploration of vascular crisis to avoid flap necrosis.Results:Postoperatively, CDU monitoring found that 2 cases had regional vascular circulation disorder, 1 case had complete embolization of the main vessels and perforators in the flap, the flap was thinned and changed to full thickness skin grafting, the deep tissue exposed area was healed by dressing change, and 1 case had incomplete embolization of the flap′s reflowing vein. After operation, the venous crisis was relieved and the flap survived completely.Conclusions:Comprehensive observation is needed after free flap transplantation. Combined with four routine vascular circulation indexes, CDU monitoring of flap blood flow can provide more objective, accurate and detailed basis for vascular circulation status, and can accurately find the vascular circulation disorder after free flap transplantation in early stage, and understand the vascular flow status of the flap, so as to effectively avoid the continuous progress of vascular crisis, to avoid flap necrosis and improve the survival rate of flap.

Since outbreak of the coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)at the end of 2019,hundreds of millions of people were infected and millions of people around the world were killed.SARS-CoV-2 is highly prone to present rapid mutations that can render vaccines ineffective.Drug therapy is currently an effective mean to prevent and treat COVID-19,and traditional Chinese medicine therapy based on clinical char-acteristics and syndrome differentiation has played an extremely important role in the prevention and treatment of COVID-19 in China.Therefore,the characteristics and mechanism of the various prescrip-tions via multiple approaches and targets used in the treatment of COVID-19,including drugs with func-tions of heat-clearing,exterior-resolving,aromatic flavor and damp-resolving,and deficiency-tonifying,based on the clinical experience of Chinese traditional medicine in preventing and treating COVID-19,in order to provide reference for the clinical treatment of such diseases with symptomatic prescriptions.

Since outbreak of the coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)at the end of 2019,hundreds of millions of people were infected and millions of people around the world were killed.SARS-CoV-2 is highly prone to present rapid mutations that can render vaccines ineffective.Drug therapy is currently an effective mean to prevent and treat COVID-19,and traditional Chinese medicine therapy based on clinical char-acteristics and syndrome differentiation has played an extremely important role in the prevention and treatment of COVID-19 in China.Therefore,the characteristics and mechanism of the various prescrip-tions via multiple approaches and targets used in the treatment of COVID-19,including drugs with func-tions of heat-clearing,exterior-resolving,aromatic flavor and damp-resolving,and deficiency-tonifying,based on the clinical experience of Chinese traditional medicine in preventing and treating COVID-19,in order to provide reference for the clinical treatment of such diseases with symptomatic prescriptions.

Objective To conduct lipidomics analysis of medical Marmota himalayana oil and explore its potential biological effects. Methods Marmota himalayana abdominal fat was heated and refined to produce medical marmot oil. Lipidomics analysis of two batches of Marmota himalayana oil was performed using gas chromatography-mass spectrometry (GC-MS) and ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS). The potential effects of Marmota himalayana oil were analyzed based on the results of lipid composition detection. Results GC-MS results showed that the main components of the two batches of Marmota himalayana oil were saturated fatty acids (volume fraction of about 60%) and unsaturated fatty acids (volume fraction of about 30%). The saturated fatty acids were mainly palmitic acid, stearic acid, and arachidic acid, while the unsaturated fatty acids were mainly oleic acid and linoleic acid. There were no significant differences in the volume fraction of various fatty acids between the two batches, indicating that the preparation process of Marmota himalayana oil was stable. The results of UPLC-MS negative ion mode detection showed that Marmota himalayana oil contained 15 saturated fatty acids and 18 unsaturated fatty acids, but there were significant differences in volume fraction between the two batches. The results of UPLC-MS positive ion mode detection showed that Marmota himalayana oil of both batches contained 15 diglycerides and 30 triglycerides. Conclusion This study innovatively applies GC-MS and UPLC-MS techniques to analyze the lipidomic components of medical Marmota himalayana oil, providing a reliable basis for subsequent exploration of its pharmacological activity and the establishment of strict quality control standards.