Enteral nutrition support reduces toxicity of chemotherapy in patients with advanced or metastatic esophageal cancer / 北京大学学报(医学版)

OBJECTIVE@#To assess the impact of enteral nutrition support on response and toxicity of the first-line chemotherapy in those patients with advanced or metastatic esophageal cancer.@*METHODS@#We collected the clinical data of 118 patients with unresectable advanced or metastatic esophageal cancer who received the first-line chemotherapy in our center from July 2014 to December 2016. All these 118 esophageal cancer patients were then divided into two groups: the nutrition group (received enteral nutrition support in addition to chemotherapy) and the control group (received chemotherapy only). Differences were analyzed before and after chemotherapy in each of the nutritional indicators including Karnofsky performance status (KPS), weight, body mass index (BMI), hemoglobin (Hb), number of lymphocytes (Lymph), total protein (TP), albumin (Alb), triglycerides (TG), total cholesterol (TC) in both groups. And differences of the efficacy and toxicities of the first-line chemotherapy between the two groups were also analyzed.@*RESULTS@#(1) Weight, BMI and Hb were all significantly decreased after chemotherapy in the control group (P<0.001), while there was no significant change of weight and BMI in the nutrition group, just with Hb decrease only. However, there was no significant change of all the other nutrition indicators after chemotherapy in both groups. (2) Compared with the control group, the nutrition group had significantly lower incidence of grade 3 to 4 hematologic toxicities after chemotherapy (15.4% vs. 42.1%, P=0.004). In addition, the incidence of grade 3 to 4 nonhematologic toxicities after chemotherapy was also lower in the nutrition group but without statistical significance (0 vs. 9.2%, P=0.123). Logistic regression model was then used for multivariate analysis to identify the factors that affected the toxicity of chemotherapy in these patients, and the results showed that nutrition therapy was an independent influencing factor of grade 3 or higher hematological toxicity after chemotherapy in the patients with esophageal cancer (P=0.008, RR=6.048, 95%CI: 1.589-23.027). (3) The response rate of chemotherapy between the control group and the nutrition group had not significant difference.@*CONCLUSION@#Enteral nutrition support in addition to chemotherapy could improve nutrition status and reduce toxicity of chemotherapy in advanced or metastatic esophageal cancer patients.

Estabilshment and stemness identification of an asymmetric dividing cell line derived from mouse Lewis lung cancer cells / 基础医学与临床

Objective To establish an asymmetric dividing cell line(LLC-ASD cells) derived from mouse Lewis lung carcinoma cancer cells(LLC-parental cells),and to investigate its stemness features in order to lay a founda-tion for depth studying the function of asymmetric dividing in the cancer biology. Methods In order to obtain asymmetrically dividing LLC cells (LLC-ASD cells) derived from LLC-Parental cells,8 times of consecutive cul-ture,enrichment and collection of floating spheriod forming cells followed by 5 times of consecutive single cell clo-ning were conducted. Immunofluorescence assay was used to visualize and quantify the rate of asymmetric division in LLC-ASD cells labeled by BrdU. For comparing the stem characteristics of LLC-Parental and LLC-ASD, RT-qPCR,clonogenic assay in 6-well plate,single cell spheroid formation assay with agar in 6-well plate and 96-well-plate single cell cloning assay were conducted. In vivo,LLC-parental cells and LLC-ASD cells were subcutaneously transplanted in nude mice to determine the effect of the difference in stem cell like properties on tumorigeneicy. The same lung transplantation into tumor experiment in mice were used to compare the differences in cancer biology. Results Asymmetric dividing cells were found in LLC-ASD cell culture through the BrdU immunofluorescence assay and the rate of asymmetric division in the anaphase cells was as high as 50%。According to the clonogenic assay in 6-well plate,single cell and spheroid formation assay with agar in 6-well plate and 96-well-plate single cell cloning assay in LLC-ASD cells,the results showed that they were more prominent than those in the LLC-Parental cells(P<0.05). In vivo,the tumor metastasis potentials of LLC-ASD was enhanced than that of LLC-parental when transplanted to the C57 mice. Further,the tumorigenic potentials of LLC-ASD cells was also increased.Conclusions The asymmetric dividing cell line derived from mouse Lewis lung carcinoma cancer cell line (LLC-ASD cells) is established which exhibits stemness properties. The establishment and characterization of this model will facilitate the research on the function of asymmetric cell dividing in cancer biology.

Characteristics of Schizophrenia Patients' Homicide Behaviors and Their Correlations with Criminal Capacity / 法医学杂志

OBJECTIVES@#To explore the characteristics of schizophrenia patients' homicide behaviors and the influences of the assessments of criminal capacity.@*METHODS@#Indicators such as demographic and clinical data, characteristics of criminal behaviors and criminal capacity from the suspects whom were diagnosed by forensic psychiatry as schizophrenia （n=110） and normal mental （n=70） with homicide behavior, were collected by self-made investigation form and compared. The influences of the assessments of criminal capacity on the suspects diagnosed as schizophrenia were also analyzed using logistic regression analysis.@*RESULTS@#There were no significant statistical differences between the schizophrenic group and the normal mental group concerning age, gender, education and marital status （P>0.05）. There were significant statistical differences between the two groups concerning thought disorder, emotion state and social function before crime （P<0.05） and there were significant statistical differences in some characteristics of the case such as aggressive history （P<0.05）, cue, trigger, plan, criminal incentives, object of crime, circumstance cognition and self-protection （P<0.05）. Multivariate logistic regression analysis suggested that thought disorder, emotion state, social function, criminal incentives, plan and self-protection before crime of the schizophrenic group were positively correlated with the criminal capacity （P<0.05）.@*CONCLUSIONS@#The relevant influences of psychopathology and crime characteristics should be considered comprehensively for improving the accuracy of the criminal capacity evaluation on the suspects diagnosed as schizophrenia with homicide behavior.

Formaldehyde induces the bone marrow toxicity in mice by regulating the expression of Prx3 protein / 华中科技大学学报（医学）（英德文版）

Formaldehyde (FA) is a ubiquitous toxic organic compound, and it has been regarded as a leukemogen. However, the mechanisms by which FA induces bone marrow toxicity remain unclear. The present study was aimed to examine the bone marrow toxicity caused by FA and the mechanism involving the expression changes of peroxiredoxin3 (Prx3) in this process. The mice were divided into four groups with 6 mice per group. Animals in the control group were exposed to ambient air and those in the FA groups to different concentrations of FA (20, 40, 80 mg/m(3)) for 15 days in the separate inhalation chambers, 2 h a day. At the end of the 15-day experimental period, all mice were killed. Bone marrow cells were obtained. The level of hydrogen peroxide (H2O2), the apoptosis rate, and the activities and protein expression levels of caspase-3 and caspase-9 were determined by biochemical assay, flow cytometry and immunohistochemistry, respectively; DNA damage and Prx3 expression levels were measured by single cell gel eletrophoresis immunohistochemistry and Western blotting, respectively. The results showed that the H2O2 level and cell apoptosis rate were significantly increased in FA groups relative to the control group. Caspase-3 and caspase-9 activities and their protein expression levels were markedly increased as well. Additionally, FA also increased the rate of DNA damage and the expression level of Prx3 compared with control group. Our study suggested that a certain concentration of FA causes the bone marrow toxicity by regulating the expression of Prx3.

Formaldehyde induces the bone marrow toxicity in mice by regulating the expression of Prx3 protein / 华中科技大学学报（医学）（英德文版）

Formaldehyde (FA) is a ubiquitous toxic organic compound, and it has been regarded as a leukemogen. However, the mechanisms by which FA induces bone marrow toxicity remain unclear. The present study was aimed to examine the bone marrow toxicity caused by FA and the mechanism involving the expression changes of peroxiredoxin3 (Prx3) in this process. The mice were divided into four groups with 6 mice per group. Animals in the control group were exposed to ambient air and those in the FA groups to different concentrations of FA (20, 40, 80 mg/m(3)) for 15 days in the separate inhalation chambers, 2 h a day. At the end of the 15-day experimental period, all mice were killed. Bone marrow cells were obtained. The level of hydrogen peroxide (H2O2), the apoptosis rate, and the activities and protein expression levels of caspase-3 and caspase-9 were determined by biochemical assay, flow cytometry and immunohistochemistry, respectively; DNA damage and Prx3 expression levels were measured by single cell gel eletrophoresis immunohistochemistry and Western blotting, respectively. The results showed that the H2O2 level and cell apoptosis rate were significantly increased in FA groups relative to the control group. Caspase-3 and caspase-9 activities and their protein expression levels were markedly increased as well. Additionally, FA also increased the rate of DNA damage and the expression level of Prx3 compared with control group. Our study suggested that a certain concentration of FA causes the bone marrow toxicity by regulating the expression of Prx3.

Correlation of MDR1 and ABCG2 genetic polymorphisms with the efficacy and adverse events of irinotecan chemotherapy in patients with colorectal cancer / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the correlation of MDR1 and ABCG2 genetic polymorphisms with the efficacy and adverse events of irinotecan chemotherapy in patients with colorectal cancer (CRC).</p><p><b>METHODS</b>Clinical data of CRC patients treated with irinotecan-based chemotherapy in the Peking University Cancer Hospital between January 1996 and December 2011 were collected, and their blood samples were collected accordingly. Genomic DNA was extracted from blood samples. The following SNP detection of MDR1 and ABCG2 genes was conducted by direct sequencing method. The correlation of genetic SNPs with efficacy and toxicity of irinotecan treatment was further analyzed.</p><p><b>RESULTS</b>Allele frequencies of MDR1 2677 G>T/A, ABCG2 421 C>A, 34 G>A, 376 C>T were comparable with previous studies. Genetic SNPs results from peripheral blood samples and tumor tissues were highly consistent. Patients carrying MDR1 2677 wild type had higher clinical benefit than those carrying mutant genotype, while the differences were not significant. The progression-free survival (PFS) was longer in wild-type patients as compared to mutant-type patients in second-line chemotherapy (P=0.012). There were no significant correlations between ABCG2 421 C>A, 34 G>A, 376 C>T and chemotherapy efficacy. No significant correlations were observed between MDR1 2677 G>T/A, ABCG2 421 C>A, ABCG2 34 G>A, ABCG2 376 C>T and irinotecan-related grade 3 and 4 neutropenia or diarrhea.</p><p><b>CONCLUSION</b>MDR1 2677 G>T/A may be served as a biomarker in predicting the efficacy of irinotecan chemotherapy in patients with colorectal cancer.</p>

Mutations of KRAS and BRAF in Chinese patients with colorectal carcinoma: analyses of 966 cases / 中华病理学杂志

<p><b>OBJECTIVE</b>To identify the mutation rate of KRAS and BRAF in Chinese patients with colorectal carcinoma (CRC), and to analyze the associations between KRAS/BRAF mutations and patients' clinicopathological characteristics.</p><p><b>METHODS</b>Tumor specimens were obtained from 966 CRC patients treated in Peking University Cancer Hospital from December 2008 to January 2012. Mutation analysis of KRAS (codons 12 and 13 of exon 2) and BRAF (exon 15) was conducted by direct sequencing. The relationships between gene mutations and clinicopathological characteristics were statistically analyzed.</p><p><b>RESULTS</b>The mutation rates of KRAS and BRAF in Chinese CRC patients were 38.8% (375/966) and 4.4% (40/915), respectively. Among patients with wild-type KRAS, the mutation rate of BRAF was 7.4% (40/540). KRAS and BRAF mutations were mutually exclusive. Eight mutation types of KRAS were detected in this study with three common types G12D, G12V and G13D. Three mutation types of BRAF were detected with the most common type V600E. KRAS mutation rate was significantly higher in female, well-differentiated and right side colon tumors (all P < 0.05). Also, the mutation rate in patients ≥ 65 years was higher than that in patients < 65 years (P = 0.05). BRAF mutation rate was higher in poorly-differentiated and right side colon tumors (P < 0.05). No significant associations were observed between KRAS/BRAF mutations and tumor size, depth of invasion, lymph node metastasis and TNM staging (P > 0.05).</p><p><b>CONCLUSIONS</b>In Chinese CRC patients, KRAS mutations are associated with gender, age, tumor differentiation and primary tumor sites, while BRAF mutation is only associated with tumor differentiation and primary tumor sites. The correlations between KRAS/BRAF mutations and patients' prognosis need further investigation.</p>

Toxicity of nano silica particles on rat type I-like alveolar epithelial cells / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To access rat lung toxicity of nano silica particles.</p><p><b>METHODS</b>Transmission electron microscope was used to observe size, shape and dispersibility of two silica particles; Size of two particles in water and RPMI 1640 containing 1% FBS were measured using Zeta Potential Analyzer. LDH activities of rat type I-like alveolar epithelial cell line R3/1 cells after 6, 24 and 48 h incubation with 2.5, 5.0, 10.0, 20.0 microg/ml of two silica nano particles were detected by spectrophotometric method; protein carbonylation and MIP-2 release of R3/1 cells after 24 h incubation with 2.5, 5.0, 10.0, 20.0 microg/ml of two silica nano particles were measured using ELISA kits.</p><p><b>RESULTS</b>TEM image showed nano silica particles were round and dispersed evenly; the average sizes of the two silica particles were (43+/-4.2) and (68+/-5.7) nm. Two silica particles had similar size in water and RPMI 1640 containing 1% FBS, respectively. Both nano silica particles in 2.5 approximately 20.0 microg/ml dose range did not cause significant increase of LDH activities (P>0.05), did not elevate protein carbonylation and MIP-2 levels in R3/1 cells (P>0.05).</p><p><b>CONCLUSION</b>Two nano silica particles do not have lung toxicity in 2.5 approximately 20.0 microg/ml dose range.</p>

Construction of mammalian cell lines continuously expressing JEV structural proteins / 病毒学报

Based on the infectious clone of JEV vaccine strain SA14-14-2, prM-E genes and C-prM-E genes were cloned into pCDNA3.1 vector. The recombinant plasmid pCJE-ME was transfected into BHK-21 cells, the expressed proteins were toxic to the cell growth and accelerated the cell death. But when transfected with the plasmid pCJE-CME, the cell lines continuously expressing structural proteins could be selected with G418. And the expression products of pCJE-CME vector could be detected by ELISA, Western Blot and IFA assay. It showed that the JEV capsid protein could enhance the stability of the cell lines expressing the structural proteins. The established cell lines can make the acquirement of the virus-like particles much easier.

A study on apoptosis and apoptotic mechanisms of HL-7702 cell line induced by methylmercury / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the apoptotic effect and mechanisms of methylmercury (MeHg) on HL-7702 cell line in vitro.</p><p><b>METHODS</b>In this study, the cell apoptosis was observed by AO/EB method and FCM method; the mitochondrial membrane potential was detected by FCM; and the expression of proteins related to apoptosis was measured by immunocytochemical method.</p><p><b>RESULTS</b>After exposure to MeHg for 24 h in different doses, apoptotic rate ascended with the increasing of MeHg concentration. By AO/EB method, cell apoptotic ratio of negative control group was (2.62 +/- 0.19)%, cell apoptotic ratio of 10-50 micromol/L exposure groups were (7.97 +/- 0.64)%, (12.66 +/- 0.76)%, (19.16 +/- 0.87)%, (18.42 +/- 0.88)%, and (11.52 +/- 0.63)%, there were significant differences between the exposure and negative control groups (q values were 17.057, 32.009, 52.732, 50.373, 28.375; P<0.05). Mitochondrial membrane potential descended with the increase of MeHg, mitochondrial membrane potential of negative control group was (10.23 +/- 3.43) mV, mitochondrial membrane potential of 10-50 micromol/L exposure groups were (3.25 +/- 0.66), (3.03 +/- 0.35), (1.68 +/- 1.26), (1.69 +/- 1.13) and (1.77 +/- 0.88) mV, and there was significant differences between exposure and negative control groups (q values were 9.569, 9.871, 11.722, 11.708, 11.598; P<0.05). The expression of Bax, Bcl-2, CytC, Caspase-3 and AIF enhanced with the increase of MeHg, Bax/Bcl-2 ratio also appeared a trend of increase. Bax expression integral optical density (IOD) of negative control group was (21295.86 +/- 1969.81), Bax expression IOD of 10, 20, 30 micromol/L groups were 42807.87 +/- 4416.64, 55651.65 +/- 4662.72, and 72708.56 +/- 910.10, there were significant differences in Bax expression between 10, 20, 30 micromol/L groups and negative control group (q values were 14.191, 14.320, 33.917; P<0.05); Bcl-2 expression IOD of negative control group was (12588.33 +/- 4091.02), Bcl-2 expression IOD of 10, 20, 30 micromol/L groups were 20539.16 +/- 4906.09, 23689.97 +/- 2281.42, and 28692.80 +/- 4655.86, there were significant differences in Bcl-2 expression between 10, 20, 30 micromol/L groups and negative control group (q values were 4.322, 6.035, 8.754; P<0.05); and AIF expression IOD of negative control group was (12942.72 +/- 457.94), AIF expression IOD of 10, 20, 30, 40 micromol/L groups were 16973.57 +/- 1922.87, 29998.91 +/- 6803.58, 52467.16 +/- 1916.25 and 106342.53 +/- 1273.19, there were significant differences in AIF expression between 20, 30 and 40 micromol/L groups and negative control group (q values were 11.449, 26.530, 62.692; P<0.05).</p><p><b>CONCLUSION</b>MeHg could induce apoptosis on HL-7702 cell line in vitro. The mechanisms could be related to mitochondrial pathway in apoptosis.</p>

Effects of over-expressed Smac gene coupling with cisplatin on proliferation and apoptosis of hepatocarcinoma cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effects of over-expressed Smac gene combined with cisplatin (CDDP) on proliferation and apoptosis of hepatic carcinoma cells.</p><p><b>METHODS</b>The recombinant plasmid pcDNA3.1+-hSmac was introduced into the human hepatic carcinoma SMMC-7721 cells using a liposome-mediated method. The expression of Smac protein was detected by Western blot and flow cytometry. The cells were treated with three different doses of CDDP, 5, 15 and 25microg/ml, for 24 hours after the transfection. MTT colorimetry was used to detect the cellular growth-inhibitory effects; acridine orange-ethidium bromide fluorescent staining (AO/EB) and flow cytometry with annexin V-PI double staining</p><p><b>METHODS</b>were used to detect the changes of cell apoptosis.</p><p><b>RESULTS</b>Western blot and flow cytometry results demonstrated that the Smac protein level in SMMC-7721 cells was significantly increased after the transfection (P less than 0.01). Compared with that of the control group, the over-expressed Smac gene inhibited the cell growth and induced cell apoptosis (P less than 0.01). After being treated with CDDP, the inhibitory rates were increased significantly with increasing concentrations of CDDP compared with that of the control group, and the inhibitory rate of the CDDP-treated plus Smac group was significantly higher than that of the CDDP-treated group (P less than 0.01). The results detected by AO/EB and flow cytometry demonstrated that the apoptotic rates of CDDP-treated plus Smac group were higher than those of the CDDP-treated group (P less than 0.01). The results demonstrated that the Smac over-expression enhanced the effects of cell growth inhibition and apoptotic promotion induced by CDDP.</p><p><b>CONCLUSION</b>The pro-apoptotic Smac gene could be over-expressed in hepatocarcinoma SMMC-7721 cells and inhibit cell growth and induce apoptosis. Moreover the over-expressed Smac could enhance the chemotherapeutic sensitivity of SMMC-7721 to cisplatin. This experimental work may help in further study on the regulatory mechanism of Smac in apoptosis and improve the chemotherapeutic effect on hepatoma.</p>

Development of site-specific integration system to high-level expression recombinant proteins in CHO cells / 生物工程学报

Stable transformants for mammalian cells from gene transfer often show extreme variability in expression of the introduced transgene. This occurs from the highly variable number of copies integrated into the genome and from position effects on gene expression due to random integration. We constructed engineered CHO strains that can be used for high-level production of foreign proteins by gene-targeting. After transfecting dihydroforate reductase (DHFR)-deficient CHO cells with a newly screening vector plasmid pMCEscan, which carrying a FRT-neo*-IRES-k2tPA fusion gene and a DHFR gene, we screened colonies by k2tPA expression level. We selected 7 clones that expressed high level of k2tPA and carried one copy of the plasmid in their chromosomes. These clones showed in high level k2tPA production without amplification. So we targeted reporter gene (k2tPA) to test the basal expression ability of these cells clones. The clone, 8-1, showed the same effect to high base expression level. In this clone, the FRT-neo*-IRES-tPA gene was integrated at a transcription-active, DHFR-mediated, gene-amplifiable locus in the chromosomes. A gene-targeting vector, carrying a FRT-fused hygromycin-resistance gene, was constructed to target desired genes in chromosomal FRT by FLP recombinase-mediated site-specific recombination. Using this cell-vector system, we could reproducibly obtain high producers of recombinant proteins by gene-targeting and gene amplification. Using the site-specific integration CHO/dhfr- cell line 8-1, the expression level of k2tPA could amount to 17.1 microg/10(6) cell x 24 h.

High-level expression of the Hcc domain of Clostridium botulinum neurotoxin serotype A in Escherichia coli and its immunogenicity as an antigen / 生物工程学报

A completely synthetic gene encoding the He domain of Clostridium botulinum neurotoxin serotype A (AHc, 1287 bp, 429 aa, -50 kD) was constructed with oligonucleotides. After expressed in Escherichia coli, soluble product AHc was gained and verified by SDS-PAGE and Western blot analysis. The expressive level of recombinant AHc in E. coli was very high (36%-53% of soluble total proteins) and the purified yield was more than 30 mg/L by one-step purification. Then, the purified AHc was used to vaccinate Balb/c mice, which developed a strong and specific immune response as expected following administration of AHe protein via the subcutaneous route. Results from BoNT/A neutralization assay showed that the serum from mice vaccinated with AHc contained high titer protective antibody. These results showed that the soluble, stable and high-levelly expressive AHc not only could be produced by the prokaryotic expression system built in our lab, but also owned strong immunogenicity to prepare antitoxin for treatment and as sub-unit candidate vaccine for prophylaxis against botulinum toxin serotype A.

Enhanced Effects of BoNT/A DNA Vaccines by Electric Pulses and Bupivacaine / 中国生物工程杂志

Objective:To determine if suitable electric pulses-mediated DNA and DNA and bupivacaine complexes delivery technologies could enhance effects of botulinum neurotoxin serotype A (BoNT/A) DNA vaccines in mouse model. Methods:Vaccination of mice i.m. with plasmid DNA replicon vaccine pSCARSHc and conventional plasmid DNA vaccine pcDNASHc following electric pulses and with DNA and bupivacaine complexes. AHc-specific group antibody ELISA titers and lymphocyte proliferative responses of mice were detected and IgG1 and IgG2a isotype profiles were assayed. Results:Immune effects of DNA vaccines were enhanced following electric pulses and bupivacaine delivery. Effects of DNA vaccines following electric pulses were better than that of DNA vaccines formulated with bupivacaine,and the combined delivery technology of electric pulses and bupivacaine induced the highest level of specific antibodies and lymphocyte proliferative responses. Plasmid DNA replicon vaccine pSCARSHc induced relatively higher AHc-specific antibodies and lymphocyte proliferative responses in immunized Balb/c mice than conventional plasmid DNA vaccine pcDNASHc in these DNA delivery technologies. And vaccine pSCARSHc induced Th2/Th1-type immune responses with a general bias to Th2-type,and vaccine pcDNASHc induced Th2-type immune responses. Conclusion:Suitable electric pulses-mediated DNA and DNA and bupivacaine complexes delivery technologies could enhance effects of BoNT/A DNA vaccines in mouse model. Therefore,the methods described here potentially provide suitable strategies in developing an efficacious vaccine against botulinum neurotoxin serotype A.

Construction of DNA and RNA based on bifunctional replicon vector derived from Semliki Forest virus / 生物工程学报

DNA-based replicon expression vector pSMCTA and helper vector pSHCTA were constructed by replacing the SP6 promoter used in the original system pSFV1 and pSFV-helper2 derived from Semliki Forest virus (SFV) with the RNA polymerase II -dependent cytomegalovirus immediate early (CMV IE) enhancer/promoter and T7 promoter, and inserting BGH transcription termination and polyadenylation signal downstream 3'-untranslated region (UTR). The RNA polymerase II -dependent cytomegalovirus immediate early (CMV IE) enhancer/promoter and T7 promoter in pSMCTA and pSHCTA could drive transcription to produce replicon RNA in vivo and ex vivo. High level expression of foreign genes (GFP and LacZ) could be demonstrated by transfecting BHK21 cells with the new replicon expression vectors based on both DNA and RNA, and recombinant virus particles (RVP) be prepared by cotranfecting the expression vectors with the helper vectors. Foreign genes were also highly expressed in cells (BHK21) which were infected with RVP activated by alpha-chymotrypsin. The bifunctional replicon vectors can be used in highly efficient expression of foreign genes and preparation of RVP ex vivo, also in development of replicon vaccines and gene therapy vectors in vivo.