ObjectiveTo construct a hepatocellular carcinoma (HCC) cell line stably transfected with linamarase (lis) gene from cassava, and to study its biological characteristics. MethodsLis DNA was amplified from cassava by PCR and cloned into the pcDNA3.1(+) plasmid. Then the recombinant plasmid pcDNA3.1/lis was transfected into human HCC HepG2 cell line using lipofectamineTM 2000 and G418 selection. The stably transfected cell lines HepG2/lis was identified by immunofluorescence, RT-PCR, and Western blot. The enzyme activity of lis in cells was assayed by Lambert method. The characteristics of the new cell lines were checked by several methods: the cell growth curve was observed by MTT, cell cycle was analyzed by flow cytometry, and characteristics of tumor formation in vivo were detected in nude mice. ResultsLis from the cassava could stably integrate into eukaryotic cells, and package the same protein as lis with β-glucosidase activity. The stable integration of lis in cells did not interfere with cell morphology, growth characteristics, cell cycle, and tumorigenesis in vivo significantly. ConclusionsA new HCC cell lines transfected with lis is successfully established, which may lay an experimental foundation for the study of HCC treatment by using lis suicide gene system in future.
Objective To construct replication-defective adenovirus containing tk gene (ADV-tk). Methods Recombinant adenovirus of ADV-tk was constructed using homologous recombination in cells. After the interested tk gene fragment in the recombinant plasmid obtained was confirmed by PCR, the titre of purified recombinant adenovirus was detected. In vitro study, tk gene in SMMC7721 cells transfected by ADV-tk was investigated by RT-PCR. In vivo study, ADV-tk was injected intraperitoneally into BALB/c nude mice with liver cancer and apoptosis cells in tumor were observed. Results Recombinant adenovirus containing ADV-tk was proved successfully. The titre of purified recombinant adenovirus was 1.4×1010 pfu/ml. In vitro study, tk was integrated and expressed by SMMC-7721 cells. In vivo study, with the injection of ADV-tk, apoptosis cells in tumor increased. Conclusion A replication-defective adenovirus containing tk gene is successfully constructed, which may useful for further research on tumor suicide gene therapy with ADV-tk.
Objective To observe the influence of connexin 43 (Cx43) on bystander effects induced by cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-tk) coexpression suicide genes system in human cholangiocarcinoma QBC939 cells and transplantation tumors in nude mice. Methods In vitro, the CD+tk+ and CD+tk+Cx+ cells were respectively treated with 5-fluorocytosine (5-FC) and ganciclovir (GCV). The cytotoxic efficacy was evaluated by microculture tetrajolium test (MTT) method. In order to investigate the influence of Cx43 on bystander effects, the volumes of transplantation tumors of the CD+tk+ and CD+tk+Cx+ cells were measured before and after application of 5-FC and GCV. Results CD and tk gene were stably expressed in transfected QBC939 cells. Increasing expression of Cx43 was determined by testing for the presence of Cx43 mRNA by RT-PCR and the presence of Cx43 protein by Western blot in CD+tk+Cx+ cells. The killing effect of 5-FC and GCV on CD+tk+Cx+ cells was more effective than that on CD+tk+ cells both in vitro and in vivo. Conclusion Double suicide genes system CD/5-FC+tk/GCV could induce remarkable killing effect on cholangiocarcinoma cells in vitro and transplantation tumors in vivo. The cotransfection of Cx43 gene is able to enhance the bystander effects and the inhibition of carcinoma cells.
Objective To design and construct the eukaryotic expressed vector of suicide genes, which contained 5 copies of hypoxia-responsive element (5HRE), promoter of alpha-fetoprotein gene (AFPp） and nitroreductase from Escherichia coli. Methods The constructing processes were as follows: ①The design of primer: Suicide genes of NTR in the Escherichia coli, which contained 6his-tag gene (6his-tag), were cloned by overlapping PCR. ②The construction of 5HRE: The single strand of synthetized HRE oligonucleotide was annealed, and 5HRE was constructed by multiple recombinant clone. ③The recombination of NTR gene, 5HRE, AFPp and pIRES2-EGFP: pIRES2-EGFP, which had removed the instant early promoter of cytomegalovirus, was recombined with NTR gene, 5HRE, AFPp. In this way, the eukaryotic expressed vector of pIRES2-EGFP-5HRE-AFPp-NTR, which carried NTR gene, 5HRE, AFPp was finally constructed. Results NTR gene, which contained the fusion of 684-base pair and 6his-tag gene, was cloned successfully, and its sequence was coincident with the result published by Genbank. A 221-base pair of 5HRE was also constructed, which was in accordance with the expected sequences. The integrity of the eukaryotic expressed vector was verified by restriction enzyme digestion and DNA sequence analysis, respectively. Conclusion The eukaryotic expressed vector of pIRES2-EGFP-5HRE-AFPp-NTR is successfully constructed, which may be used for its further investigation in vitro.
Objective To study the effects of pcDNA3/AFP/TK/Angio fusion gene targeting therapy for human primary liver cancer in nude mice implanted with SMMC-7721. Methods Human liver cancer cell line SMMC-7721 was implanted subcutaneously in nude mice to establish experiment model. Animals bearing liver cancer were randomly divided into five groups: control group， vector group， GCV (ganciclovir) group， pcDNA3/TK/Angio group; pcDNA3/AFP/TK/Angio group. Different plasmids were directly injected into tumors and GCV was intraperitoneally administrated simultaneously according to different groups. The growth of tumors was observed and the pathology was examined as well. Serum AFP level was measured by radioimmunology, the ultrastructural change of tumor cells was studied by using electron microscopy, the expressions of MVD and VEGF were respectively detected with immunohistochemistry and the cell apoptosis in situ was detected by TUNEL. Results The success rate to establish subcutaneous implanted liver cancer model in nude mice was 100%. The tumor volume, serum AFP level, VEGF and MVD expressions of pcDNA3/TK/Angio group and pcDNA3/AFP/TK/Angio group were lower than those in control group, vector group and GCV group (P＜0.05) and more apoptosis cells could be observed. While the tumor volume, serum AFP level, VEGF and MVD expressions of pcDNA3/AFP/TK/Angio group was lower than those in pcDNA3/TK/Angio group (P＜0.05); and apoptosis index was higher than that of the latter (P＜0.05).Conclusion pcDNA3/AFP/TK/Angio fusion gene inhibits the growth of tumor remarkably and becomes a promising new biological agent to treat human primary liver cancer.
Objective To investigate the suicide related factors of the teenagers after trauma and further discuss the prevalence and related factors of suicide idea plan and behavior. Methods Using the general information questionnaire and the Youth Risk Behavior Survey (YRBS) to investigate the primary and secondary students in the nine worst-hit areas in Wenchuan earthquake of 2008, with cluster random sampling and the class as a sampling unit, to extract random 110 primary and middle schools and gather their general information, disaster-related cases and scores of suicide in YRBS. According to the different types of data, we used descriptive statistics,t test,Chi square test and Logistic regression analysis and respectively use single factor analysis and multivariate logistic regression analysis for suicide idea, plan and behavior. Results A total of 7 833 questionnaires were sent out and a total of 7 521 questionnaires were collected and the recovery rate was 96. 02%. Among them, there were 6 875 valid questionnaires (91.41%) and 636 invalid questionnaires. According to the investigation, the prevalence of the primary and secondary students which had seriously considered suicide was 6. 90%, which had a plan to commit suicide was 4.00% and which tried to take over the action of suicide was 2.70%. With general information for the independent variable, logistic regression analysis showed that whether there had seriously considered suicide as the dependent variable: age (OR=1.178, 95%CI 1.098 to 1.178) and changed in family structure after the earthquake (OR=1.360, 95%CI 1.360 to 1.085) were risk factors for post disaster the teenagers with suicide idea; whether there had been plan to commit suicide as the dependent variable: age (OR=1.099, 95%CI 1.050 to 1.150), the history of being buried in ruin (OR=2.155, 95%CI to 1.104 to 4.205) and changed in family structure after the earthquake (OR=1.495, 95%CI 1.128 to 1.981) were risk factors for post disaster the teenagers with suicide plan; whether took over the action of suicide as the dependent variable: male (OR=1.513, 95%CI 1.122 to 2.039) and changed in family structure after the earthquake (OR=1.555, 95%CI 1.112 to 2.175) were risk factors for post disaster the teenagers with suicide behavior. Conclusion Age and changed in family structure after the earthquake were the risk factors for suicide idea and age was positively correlated with suicidal idea. Age, had the history of being buried in ruin and changed in family structure after the earthquake were the risk factors for suicide plan and age was positively associated with suicide plan. Male and changed in family structure after the earthquake were the risk factors for suicide behavior. Among them, changed in family structure after the earthquake was the common risk factor. So there should be targeted psychological assessment and psychological rehabilitation intervention to avoid the risk of suicide among the teenagers after the disaster.
Objective To systematically review injury, death, and their causes in elderly people in China from 2000 to 2020 and to prevent and reduce the occurrence of injuries and death. Methods The CNKI, VIP, WanFang Data, PubMed, SinoMed, and Web of Science databases were searched to collect studies on injury and death among elderly people over 60 years of age who resided in China from January 2000 to December 2020. Two reviewers independently screened the literature, extracted data, and assessed the risk of bias of the included studies. A meta-analysis was then performed using R 4.1.2 software. Results A total of 41 studies with 187 488 subjects were included, including 125 million elderly individuals. The pooled injury mortality rate was 135.58/105 (95%CI 113.36/105 to 162.14/105, P<0.001). Subgroup analysis showed that male injury death (146.00/105, 95%CI 116.00 to 183.74, P=0.001) was significantly higher than that of females (127.90/105, 95%CI 102.31 to 159.88, P=0.001) and that overall injury mortality increased exponentially with age (R2=0.957), especially in those over 80 years old. The spatial distribution showed that the injury death rate in the central region was higher than that in the east and west and higher in the countryside than in the city. The time of death distribution showed that after China became an aging society (2000-2020), the time of death was significantly later than before (1990-2000). There were more than 12 types of injuries that caused death, the top three of which were falling, traffic accidents, and suicide. Conclusion From 2000 to 2020, the injury mortality rate of the elderly people in China initially increase and then slightly decrease. The phenomenon affects more men than women, especially those beyond the age of 80. Regional differences are identified, and the types of injuries that cause death are mainly falls, traffic accidents, and suicide. Due to the limited quantity and quality of the included studies, more high-quality studies are needed to verify the above conclusion.