AIM: To construct a recombinant retrovirus vector carrying hTERT for establishing UCBMSCs with hTERT(hTERT-MSCs) to overcome their limited life span and detecting whether telomerized UCBMSCs line maintained long-term self-renewal and differentiation capacity.METHODS: The whole cDNA was generated by PCR amplifications from the plasmid pEGFP-hTERT-C1.The hTERT segments were subcloned into pLNCX2.The target cells were infected with these retroviral particles.The stably transfected cells were selected by neomycin and expanded life span which were designated hTERT-MSCs was observed.The expression of hTERT in mRNA level was detected by RT-PCR and the telomerase activity was measured by TRAP(PCR)-ELISA assay.The hTERT-MSCs were induced with 5-azacytidine to cardiac muscle cells and the specific marker of myocardiocyte was detected.RESULTS: The constructed plasmids were digested with restriction endonucleases(BglⅡand NotⅠ).Two characteristic segments including 6.1 kb and 3.6 kb were obtained.The hTERT-MSCs expressed hTERT in mRNA level.The telomerase activity of hTERT-MSCs was positive.The growth kinetics of hTERT-MSCs was higher than those in UCBMSCs.The hTERT-MSCs were induced to myocardiocyte.CONCLUSION: The hTERT recombinant retrovirus vector has been successfully constructed.The hTERT gene activates the telomerase and prolongs the life-span of cells.No effect of hTERT gene on some type of differentiation potential of MSCs is present.

BACKGROUND:Human mesenchymal stem cells (hMSCs) become aging and even die after several passages. Some investigations have shown that telomere has a close correlation with life span of the cells. Whether the ectopic expression of human telomerase reverse transcriptase (hTERT) could induce the activity of the telomerase, maintain the length of telomere, and finally prolong the life cycle of MSCs without losing their multipotent differentiation capacity is still uncertain.OBJECTIVE: To observe the influence of the ectopic expression of hTERT on the telomerase activity and cell life cycles of hMSCs.DESIGN: Repetitive measurement trails.SETTING: Research Center of Stem Cell, Zhengzhou University Medical College.MATERIALS: The experiment was conducted in the Research Center of Stem Cell, Zhengzhou University Medical College from October 2003 to December 2005. hMSCs were obtained from 20 healthy donators from the Department of Pediatric Surgery and Outpatient, the Third and First Affiliated Hospitals of Zhengzhou University. Enhanced green fluorescent protein plasmid (pEGFP-C1) and pEGFP-hTERT were provided by Dr. Chantal Autexier of Canada. DH5α strain provided by Dr. Hou Wei-hong, the Key Molecular Medical Laboratory of Zhengzhou University Medical College.METHODS.: Under sterile condition, 2 mL bone marrow of sternum of healthy donors were harvested, and prepared after centrifugalization,dilution and passage.① Transfection of pEGFP-hTERT into hMSCs and the screening and amplification of resistance cloning:The 5th passage cells were seeded in a 24-well plate,and transfected by pEGFP-hTERT with lipofectamine method.The cells were divided into four groups including untransfected group,lipofectamine group,pEGFP-C1 group and pEGFP-hTERT group. Resistance cloning screen and amplification was performed by G418. ②hTERT mRNA expression and detection of telomerase activity:RT-PCR and PCR-ELISA were used to detect the hTERT mRNA expressions of the fifth passage hMSCs transfected with pEGFP-hTERT, and pEGFP-C1, the untransfected tenth passage hMSCs and K562 cells (positive control), and the telomerase activity of the fifth and thirtieth passage hMSCs transfected with pEGFP-hTERT,the fifth pEGFP-C1-transfected cells and the tenth passage untransfected cells. ③Karyotype analysis of hTERT-transfected MSCs: Chromosome analysis was performed by conventional Giemsa staining.④Inducement of differentiation from telomerase-positive MSCs into neuron-like cells and RE-PCR identification:The transfected MSCs were cultured in a medium containing epidermal growth factor and basic fibroblast growth factor, which could induce the cells differentiate into neuron-like cells. The culture solution was changed every 3 days, and the changes in cell growth condition and morphologic characteristics were observed under an inverted microscope. The microtubule associate protein (MAP2) and neurofliament subunit M (NF-M) were identified by RT-PCR.MAIN OUTCOME MEASURES:①hMSCs transfection with different kathion liposomes and the screening and amplification of resistance cloning; ②hTERT mRNA expressions of each group and detection of telomerase activity; ③Karyotype analysis of pEGFP-hTERT-transfected MSCs; ④ Induction of differentiation from telomerase-positive MSCs into neuron-like cells and RE-PCR identification.RESULTS: ①With the decrease of G418 concentration, the cells in the untransfected and lipofectamine groups died, and stably EGFP expressed MSCs were obtained; after G418 screening, there was a cell clone undergone 35 passages and continued to proliferate, whose appearance and growth characteristics were similar to the untransfected MSCs observed under inverted microscope. ②The fifth passage pEGFP-C1-transfected hMSCs and tenth passage untransfected hMSCs remained telomerase-negative, but the K562 and fifth passage hTERT-transfected cells showed positive telomerase activity. ③The telomerase activity of the fifth and thirtieth passage hTERT-transfected cells was positive. ④The hTERT-MSCs at passage 10, 20 and 30 had 23 pairs of chromosomes, and two X chromosomes. So they were still normal diploid with normal chromosome appearance and number. ⑤Many hTERT-transfected MSCs had the typical appearance of neuron-like cells. RT-PCR analysis showed that th expressions of MAP2 and NF-M were increased.CONCLUSION:Ectopic expression of the hTERT gene is found in hMSCs,and can induce the telomerase activity of hMSCs.The ectopic expression of the hTERT gene in hMSCs could extend the life spans of cells and maintain their multipotent differentiation capacity.

Objective To identify differentially expressed N-linked glycoproteins between hepatocellular carcinoma ( HCC) and adjacent non-tumorous liver tissues .Methods N-linked glycoproteome was extracted by multi-lectin affinity chromatography comprising concanavalin A (ConA), lentil lectin (LCH), and snowdrop lectin (GNA) and subsequently subjected to two-dimensional electrophoresis ( 2DE ) and mass spectrometry ( MS ) for identification of differential glycoproteins between 10 pairs of HCC and adjacent non-cancer tissue .Western blotting was used to verify different expression of human liver carboxylesterase 1 (hCE1), haptoglobin (HP)and cathepsin D (CD).Invasion potential in vitro was examined after si-RNA mediated CD gene scilencing .Results LC-ESI-MS/MS identified a total of 28 differentially expressed glycoproteins (14 up-regulation and 14 down-regulated).Western blotting detected consistent down-regulation of hCE1 and HP, and up-regulation of pro-cathepsin D (pCD) in HCC.Up-regulation of ConA-binding CD (ConA-CD), however , was verified in HCC only after ConA-CD enrichment by ConA chromatography .Down-regulation of CD expression mediated by CD-siRNA markedly inhibited the in vitro invasive potential of SNU449 and SNU473.Conclusion Dysregulation of HP , hCE1 expression and alteration of glycans linked to CD may play crucial roles in pathogenesis of HCC.

Objective To discuss the feasibility of biodegradable covered magnesium alloys stent for a rabbit model of lateral aneurysm in the common carotid artery (CCA). Methods The left CCA was ligated in 20 conventional New Zealand rabbits. MRA of the neck was performed 1 month after ligation. The rabbits with thickening of the right CCA and non-or slight thickening of the bilateral vertebral arteries were selected for lateral aneurysm model making. The venous pouch and the right CCA wall by discontinuous extroversion sutures, to form a lateral aneurysm model. The biodegradable covered magnesium alloys stents or Willis covered stents were inserted two weeks after model making. Angiography was performed prior to the procedure, 3, 6 and 12 months after stent implantation to evaluate the disappearance of the aneurysms and patency of the right CCA. The degradation behaviour is invastagated with molybdenum target 2 weeks, 1, 2, 4, 6,9 and 12 months after stent placement. Results The left CCAs were successful ligated in all rabbits. MRA 1 month after ligation showed thickening of the right CCA and non-thickening of the bilateral vertebral arteries in 17 of 20 rabbits. In these animals, the CCA lateral aneurysm model was deemed successful, and biodegradable covered magnesium alloys stents and Willis covered stents were implanted in 9 and 8 aneurysms, respectively. DSA after biodegradable covered magnesium alloys stent placement displayed disappearance of the aneurysms and patency of the CCA in all 9 rabbits during follow-ups. DSA 3 months after Willis stent placement displayed patency of the CCA in 7 rabbits and occlusion of the artery in one. No occlusion of the right CCA was observed on angiography at 6 and 12 months. The degradation of the biodegradable covered magnesium alloys stent was investigated with molybdenum target during follow-ups, and no changes was observed in Willis covered stent. Conclusion Biodegradable covered magnesium alloys stent is a feasible approach for the treatment of a lateral aneurysm in the right CCA.

OBJECTIVE: To assess the association of CYP2C19 and CYP3A5 gene polymorphisms with the risk of myocardial infarction. METHODS: Five hundred patients with myocardial infarction and 500 healthy controls were randomly selected. Fluorescent PCR and Sanger sequencing were used to detect the CYP2C19 and CYP3A5 gene polymorphisms. Logistic regression was used to analyze the correlation between the polymorphisms and myocardial infarction. Quanto software was used to evaluate the statistical power. RESULTS: The two groups had significant difference in the frequency of AG, GG genotypes and A allele of the CYP2C19 gene rs4986893 locus and the AA, AG, GG genotypes and G allele of the CYP3A5 gene rs776746 locus ( P<0.05), but not in the frequency of genotypes and alleles of CYP2C19 gene rs4244285 and rs12248560 loci, and the AA genotype of the rs4986893 locus. After correction for age, gender, and body mass index, Logistic regression indicated that the AG genotype and A allele of the CYP2C19 gene rs4986893 locus, and the GG genotype and G allele of CYP3A5 gene rs776746 locus are associated with susceptibility of myocardial infarction, while rs4986893 GG genotype and AA and AG genotypes of rs776746 may confer a protective effect. Based on the sample size and allele frequency, analysis with Quanto software suggested that the result of this study has a statistical power of 99%. CONCLUSION CYP2C19 and CYP3A5 gene polymorphisms may increase the risk for myocardial infarction.
Cytochrome P-450 CYP2C19/genetics* ; Cytochrome P-450 CYP3A/genetics* ; Gene Frequency ; Genotype ; Humans ; Myocardial Infarction/genetics* ; Polymorphism, Genetic ; Polymorphism, Single Nucleotide

Objective:To evaluate the quality, content and feasibility of health information on breast cancer treatment in Chinese websites, for reference in standardizing online health information.Methods:In January 2022, three Chinese search engines, namely Baidu, Sogou and Haosou, were used to search with " breast cancer" and " breast cancer treatment" as keywords. The first 50 links identified by each engine were accessed to screen breast cancer treatment health information websites. Subsequently, the DISCERN scoring tool was adopted to evaluate the quality of health information on the websites(16 items). The information content(10 items)assessment was conducted by referring to the " Guidelines and Specifications for Breast Cancer Diagnosis and Treatment of the Chinese Anti-Cancer Association(2019 Edition)". The feasibility of website health information was evaluated with Patient Education Materials Assessment Tool(PEMAT).Results:A total of 19 websites were included, and their service mode was mainly active information service(14 websites). The overall scoring for the quality of health information about breast cancer treatment was 2.0, with the highest score of " clarity of purpose" (3.4). According to the assessment results of information content, these websites were divided into 4 grades: excellent(3), good(11), fair(3), and poor(2). All the websites contained the information on clinical manifestations, etiology, examination and treatment of breast cancer. The overall feasibility of website health information was 44.5%, indicating incomplete feasibility.Conclusions:The Chinese website′s health information on breast cancer treatment is poor in quality and implementation. Hence, it′s urgent to further improve and standardize online health information, establishing a unified evaluation standard and actively exploring the new mode of " Internet+ Medical" service.

Objective:To analyze the research status and comment of radiation-induced heart injury based on relevant literature analysis in China.Methods:A literature database was established with studies published from 1983 to 2021 as the data source from "China National Knowledge Infrastructure (CNKI) database", "Wanfang Database", "VIP Chinese Science and Technology Journal Database" and "China Biology Medicine Disc". The publication date, type and journal distribution of studied related to radiation-induced heart injury were quantitatively analyzed.Results:A total of 300 literatures were included. The chronological distribution of literatures: from 1983 to 2021, the number of literatures related to radiation-induced heart injury was gradually increased, and the overall trend was on the rise. The top three publication journals were Chinese Journal of Radiation Oncology, Chinese Journal of Radiation Medicineand Protection, and Chinese Journal of Cancer Prevention and Treatment. Literature type distribution: clinical research accounted for 48.50%, 25.58% for review, and 17.61% for animal experiment, respectively. The distribution of clinical research types: lung cancer, esophageal cancer and breast cancer were the most, followed by thymoma and lymphoma. In animal experiments, rat was the most frequently studied animal model, and the maximum radiation dose was 20 Gy. Conclusions:The number of studies related to radiation-induced heart injury is gradually increasing in China. The quantity of clinical studies is higher than that of animal experiments, most clinical studies focus on chest tumors, but there is great potential in animal experimental research. We still need to explore the pathogenesis of radiation-induced heart injury and develop drug prevention and intervention strategies.