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BACKGROUND: The shortage of water resources and the increase of greenhouse gas emissions from soil seriously restrict the sustainable development of agriculture. Under the premise of ensuring a stable yield of winter wheat through a reasonable irrigation scenario, identifying a suitable straw returning method will have a positive effect on agricultural carbon sequestration and emission reduction in North China Plain. RESULTS: Straw burying (SR) and straw mulching (SM) were adopted based on traditional tillage under in the winter wheat growing season of 2020-2021 and 2021-2022. Three irrigation scenarios were used for each straw returning method: no irrigation (I0), irrigation 60 mm at jointing stage (I1), and irrigation of 60 mm each at the jointing and heading stages (I2). Soil moisture, soil respiration rate, cumulative soil CO2 emissions, yield, water use efficiency (WUE) and soil CO2 emission efficiency (CEE) were mainly studied. The results showed that, compared to SM, SR improved the utilization of soil water and enhanced soil carbon sequestration. SR reduced soil respiration rate and cumulative soil CO2 emissions in two winter wheat growing seasons, and increased yield by increasing spike numbers. In addition, with an increase in the amount of irrigation, soil CO2 emissions and yield increased. Under SR-I1 treatment, WUE and CEE were the highest. SR-I1 increases crop yields at the same time as reducing soil CO2 emissions. CONCLUSION: The combination of SR and irrigation 60 mm at jointing stage is a suitable straw returning irrigation scenario, which can improve water use and reduce soil CO2 emission in NCP. © 2023 Society of Chemical Industry.
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Solo , Triticum , Estações do Ano , Dióxido de Carbono/análise , Água , Carbono , Agricultura/métodos , ChinaRESUMO
BACKGROUND AND PURPOSE: The AMP-activated protein kinase (AMPK) signalling pathway is a desirable target for various cardiovascular diseases (CVD), while the involvement of AMPK-mediated specific downstream pathways and effective interventions in hyperlipidaemia-induced endothelial dysfunction remain largely unknown. Herein, we aim to identify an effective AMPK activator and to explore its efficacy and mechanism against endothelial dysfunction. EXPERIMENTAL APPROACH: Molecular docking technique was adopted to screen for the potent AMPK activator among 11 most common rare ginsenosides. In vivo, poloxamer 407 (P407) was used to induce acute hyperlipidaemia in C57BL/6J mice. In vitro, palmitic acid (PA) was used to induce lipid toxicity in HAEC cells. KEY RESULTS: We discovered the strongest binding of ginsenoside Rh4 to AMPKα1 and confirmed the action of Rh4 on AMPK activation. Rh4 effectively attenuated hyperlipidaemia-related endothelial injury and oxidative stress both in vivo and in vitro and restored cell viability, mitochondrial membrane potential and mitochondrial oxygen consumption rate in HAEC cells. Mechanistically, Rh4 bound to AMPKα1 and simultaneously up-regulated AKT/eNOS-mediated NO release, promoted PGC-1α-mediated mitochondrial biogenesis and inhibited P38 MAPK/NFκB-mediated inflammatory responses in both P407-treated mice and PA-treated HAEC cells. The AMPK inhibitor Compound C treatment completely abrogated the regulation of Rh4 on the above pathways and weakened the lowering effect of Rh4 on endothelial impairment markers, suggesting that the beneficial effects of Rh4 are AMPK dependent. CONCLUSION AND IMPLICATIONS: Rh4 may serve as a novel AMPK activator to protect against hyperlipidaemia-induced endothelial dysfunction, providing new insights into the prevention and treatment of endothelial injury-associated CVD.
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Proteínas Quinases Ativadas por AMP , Ginsenosídeos , Camundongos Endogâmicos C57BL , Animais , Ginsenosídeos/farmacologia , Ginsenosídeos/química , Proteínas Quinases Ativadas por AMP/metabolismo , Masculino , Camundongos , Hiperlipidemias/tratamento farmacológico , Hiperlipidemias/metabolismo , Humanos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Simulação de Acoplamento Molecular , Estresse Oxidativo/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Ativadores de Enzimas/farmacologia , Células CultivadasRESUMO
OBJECTIVE: To analyze the prevalence and risk factors of multiple musculoskeletal disorders (MSDs) in auto workers and the associations between MSDs at different sites. METHODS: A cross-sectional survey was conducted in 3998 workers, who were selected from a Chinese auto corporation by cluster random sampling, using the revised Nordic MSDs standard questionnaire; 3800 completed questionnaires were returned. Multinomial logistic regression analysis was performed to assess the risk factors for multiple MSDs. The logbinomial model was used to calculate the prevalence ratios (PRs) of MSDs at different sites and evaluate the associations between MSDs at different sites. RESULTS: Of the 3800 subjects, 2452 (64.5%) had MSDs at two or more sites, and 469 (12.3%) had MSDs at one site. The PRs varied from 1.5 to 6.7, with significant differences among different sites (P < 0.01). Relatively close associations were found between the MSDs at neck and shoulders, back and shoulders/waist, elbows and wrists/hands, waist and neck, wrists/hands and waist, hip and waist, knees and waist, and ankles/feet and elbows. The multinomial logistic regression analysis indicated that the highest risk factor for MSDs was poor posture, including often working in an uncomfortable posture, neck bending forward, and neck twisting (ORs = 3.39, 1.93, and 1.38), followed by labor organization, in which break between tasks could decrease the risk of MSDs at three or more sites to 31%, staff shortage, which could increase the risk of MSDs by 75%, and pushing and pulling heavy objects (> 20 kg) (OR = 1.76). CONCLUSION: Most auto workers with MSDs have multiple sites affected, and there are high associations between the MSDs at different sites. The major risk factors for multiple MSDs in auto workers include poor posture, labor organization, and heavy physical labor.
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Doenças Musculoesqueléticas/epidemiologia , Doenças Profissionais/epidemiologia , Adulto , Automóveis , Estudos Transversais , Feminino , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Doenças Musculoesqueléticas/etiologia , Doenças Profissionais/etiologia , Fatores de Risco , Inquéritos e QuestionáriosRESUMO
Objective: To investigate the clinical efficacy of arthroscopic capsular release and postoperative intra-articular infusion of cocktail combined with tranexamic acid (TXA) in the treatment of patients with frozen shoulder. Method: A total of 85 middle-aged and older patients with frozen shoulder who underwent arthroscopic capsular release and received intra-articular infusion of TXA alone (n = 28), cocktail alone (n = 26), and cocktail plus TXA (n = 31) after surgery were retrospective analyzed. The drainage volume within 24â h after surgery, postoperative length of hospital stay, postoperative complications, visual analog scale (VAS), Neer shoulder assessment scale, ASES score, and range of motion (ROM) of the shoulder joint at 1 day, 1 week, 1 month, and 3 months after surgery in all three groups were recorded and compared. Results: Postoperative length of hospital stay was significantly shorter in the cocktail + TXA and cocktail groups than that in the TXA group. Postoperative drainage volume was significantly higher in the cocktail group compared with TXA + cocktail group (P < 0.05). At 1 day and 1 week after surgery, pain was more pronounced in the TXA group, which was significantly relieved in the cocktail and the cocktail + TXA groups (P < 0.05). Pain was significantly relieved in all the three groups at 1 and 3 months after surgery. Significant functional improvement of the shoulder was achieved in all three groups at 1 week after surgery, the improvement was apparent in the cocktail + TXA groups (P < 0.05), followed by the cocktail group. At 1 month after surgery, patients in the cocktail + TXA groups obtained excellent functional recovery of the shoulder joint. At 3 months after surgery, patients in all the three groups both obtained good recovery of the shoulder joint function, and the recovery was apparent in the cocktail + TXA groups (P < 0.05). Conclusion: Arthroscopic capsular release and postoperative intra-articular infusion of cocktail combined with TXA has good safety and efficacy in the treatment of middle-age and older patients with frozen shoulder, which can reduce postoperative pain and intra-articular bleeding, promote early postoperative functional exercises and accelerate early postoperative recovery.
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OBJECTIVE: To understand the major causes of death in automobile foundry workers and investigate casting manipulations hazards to health. METHODS: A cohort study of 3529 foundry workers registered in one big automobile factory in Shiyan city of China was performed. Standardized mortality ratios (SMRs) were calculated for the main causes of death by using Chinese national mortality rates as reference. RESULTS: The cohort mortality was traced from 1980 to the end of 2005 with an accumulation of 84 999 person-years, revealed 265 deaths. The results of this study showed that the standardized mortality ratio for all subjects was 0.96 (95% CI: 0.85 approximately 1.08), which was very close to that expected on the basis of the China national mortality rates. The SMR increased with age, the SMR became greater than 1 in all groups of age 50 and higher. The results showed that malignant neoplasm (3.43%), accidents (1.16%), cerebro-vascular diseases (1.08%), cardio-vascular diseases (0.79%) were the first four illnesses that threatened workers' life span. Statistically significant mortality of malignant neoplasm (SMR = 7.87), accidents (SMR = 2.70), cardio-vascular diseases (SMR = 2.68) and digestive diseases (SMR = 2.79) were found in the foundry workers. The relative risk of malignant neoplasm for first line workers to assistant workers was 1.99 (P < 0.05). CONCLUSION: The occupational hazards in foundry factory have harmful impact on the workers' health and life span.
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Automóveis , Metalurgia , Mortalidade , Causas de Morte , China/epidemiologia , Feminino , Seguimentos , Humanos , Masculino , Doenças Profissionais/mortalidade , Exposição Ocupacional/estatística & dados numéricos , Estudos RetrospectivosRESUMO
OBJECTIVE: To study the anticancerous effect of Fuganchun 6 (FGC-6) and its immunoregulatory effect on tumor-bearing mice. METHOD: The mice inoculated by H22 cells were divided into 5 groups: model group, 5-Fu group and FGC-6 in high dose, medium dose, and low dose groups. The normal mice were also observed. These mice were treated for 10 days. The weight of tumor mass and mouse were examined. The target-cell-killing activity of NK cells. The proliferation activity of lymphocyte and the production of IL-2 of murine splenocytes were detected respectively. The serum containing FGC-6 was prepared and its inhibition effect on H22 cells was examined by MTT assay and growth curve in vitro. RESULT: Growth of tumor was inhibited markedly by FGC-6 high dose. The inhibition of serum containing FGC-6 on the proliferation of H22 cells in vitro was observerd in a dose and time-dependent manner. The target-cell-killing activity of NK cells and the production of IL-2 of murine splenocytes of model group were lower than those of normal group (P < 0.05). When compared with model group, FGC-6 in high dose elevated the two indexes above-mentioned, and also enhanced the proliferation activity of lymphocyte markedly (P < 0.05). The production of IL-2 of murine splenocytes was also improved when treated by FGC-6 in medium dose (P < 0.05). CONCLUSION: FGC-6 can inhibite the growth of H22 cells markedly and also can strengthen the immunity of H22 transplanted mouse.
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Antineoplásicos Fitogênicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Neoplasias Hepáticas Experimentais/patologia , Linfócitos/patologia , Animais , Antineoplásicos Fitogênicos/isolamento & purificação , Linhagem Celular , Combinação de Medicamentos , Medicamentos de Ervas Chinesas/isolamento & purificação , Humanos , Interleucina-2/metabolismo , Células Matadoras Naturais/imunologia , Neoplasias Hepáticas Experimentais/imunologia , Masculino , Camundongos , Plantas Medicinais/química , Baço/citologia , Baço/metabolismoRESUMO
Purpose. To evaluate aqueous humor MMP-9 levels in alkali-burned rabbit cornea following KPr implantation and their roles in RPMs formation. Methods. Left eyes of 36 rabbits received a deep corneal alkali wound. 12 corneas were implanted with KPro and the other 24 control corneas were either penetrating keratoplasty or left without keratoplasty. Aqueous humor MMP-9 and TIMP-1 levels were determined and RPMs were obtained for histopathological and ultrastructural examination. Results. Alkali exposure induced significant increase in aqueous humor MMP-9 level and the data were further enhanced by KPro implantation. By contrast, TMIP-1 levels in aqueous humor showed a decreased trend following corneal alkali burn and KPro surgery. RPMs were developed in 5 out of 10 cases of KPro successfully implanted eyes. Histopathology showed the presence of a large number of fibroblasts and collagen fibers arranged irregularly with inflammatory cells infiltration, and an ingrowth of new blood vessels in this retrokeratoprosthesis fibrous tissue. Immunohistochemical analysis showed positive stain of RPMs for both MMP-9 and TIMP-1. Aqueous humor MMP-9 levels were significantly higher in RPM group postoperatively, while TIMP-1 levels were comparatively lower than that of No-RPM group. Conclusions. Our study evidenced the potential pathophysiological role of MMP-9 expression in RPM formation following KPro implantation.
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OBJECTIVE: To study the random amplified polymorphic DNA (RAPD) characteristics of five S180, clonal cell strains with 23 primers and the biological characteristics of passage cells from S180-S2D9. METHODS: The DNA of 5 clone strains of S180 including S180-S2D9, S180-S2D7, S180-S1F11, S180-S1H10 and S180-S1B11, was amplified with RAPD-PCR using 23 single primers. The PCR products were resolved with electrophoresis on agarose gel. To analyze genomic DNA with RAPD, the cultured cells in vitro were treated with colchicine for 6 hours. Then, the chromosome numbers of 100 cells were examined under the microscope. The KM mice were injected intraperitoneally with 0.2 ml solution of S180-S2D9 cell, and the growth of ascitic fluid and the life span of the mice were observed. The cultured cells were fixed with 750 ml/L ethanol, and DNA analysis was made by Flow Cytometry. RESULTS: Three of the 23 single primers resulted in diversity between amplified products from different clonal strains. RAPD character of S180-S2D9 was analyzed by the 3 single primers; RAPD bands of the first generation, 25th generation, 50th generation and 75th generation showed no difference; ANOVA showed that the average numbers of chromosomes of four generations were of no significant difference (P>0.05). The ascites of the KM mice subjected to the intraperitoneal injection of first generation and 50th generation S180-S2D9 cells was bloody, and the survival days of mice were 13-23 d and 13-20 d respectively; ANOVA showed there was no significant difference between the first generation and 50th generation (P>0.05). DNA contents assayed by flow cytometry revealed that DNA corresponding content of the cells is individually 0.3890, 0.3542, 0.3575 and 0. 3984. These results imply that the S180-S2D9 passage cells have not been found to vary a lot. CONCLUSION: It is adaptable to give quality control to the clonal cell strains with RAPD.
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Células Clonais/patologia , Técnica de Amplificação ao Acaso de DNA Polimórfico , Sarcoma 180/genética , Sarcoma 180/patologia , Animais , Células Clonais/metabolismo , Impressões Digitais de DNA , Marcadores Genéticos , Masculino , Camundongos , Sarcoma 180/classificação , Células Tumorais CultivadasRESUMO
Cultured mouse fibroblasts were treated with rabbit liver RNA. Rat liver RNA was injected into mouse prostates. Influence of exogenous RNA upon expression of mouse albumin gene was detected by immunohistochemical staining. Nucleuses of cultured mouse fibroblasts treated with different RNAs were isolated and digested with DNase I. Mouse albumin gene was amplified by PCR to detect levels of its digestion. It was found that exogenous RNAs could increase the sensitivity of mouse albumin gene to DNase I digestion and promote its expression.
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Albuminas/biossíntese , Expressão Gênica/efeitos dos fármacos , RNA/farmacologia , Albuminas/genética , Animais , Desoxirribonuclease I/metabolismo , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Peixes , Genes/efeitos dos fármacos , Rim/química , Fígado/química , Pulmão/química , Masculino , Camundongos , Próstata/citologia , Próstata/efeitos dos fármacos , RNA/química , Coelhos , RatosRESUMO
OBJECTIVE: To examine the expression difference of mRNA of L1210 cell strains and its cloned cells and discuss the methods for quality control of cell strains. METHODS: We used SDS-PAGE to observe the difference of protein and performed in situ hybridization to examine the expression of mRNA with the use of 6 cDNA probes that were marked by biotin. RESULTS: The number of protein bands of L1210 from Beijing Cancer Institute was 32. The number of protein bands of the two cloned cells L3E11 and L3F9 was 31. The 6 cDNA probes (p16, c-fos, c-jun, c-myc, p21, and p53 mRNA) were found to be existing in Beijing Cancer Institute L1210 and two different cloned cell strains. Expression of c-myc, c-fos, p53 mRNA could distinguish L3E11 and L3F9 cloned cells.
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Leucemia L1210/genética , Proteínas de Neoplasias/análise , Proteína Supressora de Tumor p53/genética , Animais , Células Clonais , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Hibridização In Situ , Leucemia L1210/patologia , Camundongos , Camundongos Endogâmicos DBA , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteína Supressora de Tumor p53/biossínteseRESUMO
It has been shown that silencing of suppressor of cytokine signalling 1 (Socs1) or stably expressing transgenic protein Ags in antigen-presenting dentritic cells (DCs) strongly enhances antigen-specific anti-tumour immunity. However, whether the strong and long-lasting T cell responses induced by the modified DCs could modulate the immunosuppressive tumour microenvironment has not been clarified. In this study, we explored the anti-tumour immunity of DCs modified by Socs1-shRNA lentiviral transduction combined with sustained expression of TRP2 in different tumour models. We showed that transfer Socs1-silenced or tumour antigen TRP2 persistent expressed DCs, or DCs modified by combination of Socs1-silencing and sustaining TRP2 expression prior to inoculation of tumour cells delayed B16 tumour cell growth, prolonged mouse survival and increased the ratio of CD8+ T/Treg as well as the CTL activity in tumours. However, there was no significant effect on tumour growth and mouse survival rate upon tumour established. Further, we showed that tumour cell secreted IL-10 counteracted the immunity of modified DCs in established tumour model, injection of Socs1-shRNA and TRP2 antigen modified significantly inhibited growth of the established B16-IL-10(-/-) tumours. These data indicated that the high level of IL-10 within tumour microenvironment is one of factors that compromise DC vaccine functions.
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Vacinas Anticâncer/farmacologia , Células Dendríticas/transplante , Interleucina-10/metabolismo , Melanoma Experimental/terapia , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Evasão Tumoral , Microambiente Tumoral , Animais , Apresentação de Antígeno , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Interleucina-10/deficiência , Interleucina-10/genética , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Interferência de RNA , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/deficiência , Proteínas Supressoras da Sinalização de Citocina/genética , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/imunologia , Fatores de Tempo , Transdução Genética , Transfecção , Carga TumoralRESUMO
The objective of this study was to obtain better antigen specific cytotoxic T cell responses in vivo. We examined the augmented induction of antigen-specific cytotoxic T cell responses to co-administration of oligonucleotides (CpG-ODN), dimethyl dioctadecyl ammonium bromide (DDA), and Lipofectamine™ 2000 with a DNA vaccine (pVAX1-CpG-Loop) and boosting with pVAX1-CpG-Loop in BALB/c mice. The results show that Loop protein-specific T cell proliferation, cytotoxic T cell activity, and the production of CD8+ T cells and IFN-γ were enhanced after co-immunization of mice with adjuvants and pVAX1-CpG-Loop. We demonstrated that significant T cell-mediated immune responses were induced in the mice with the help of DDA, CpG-ODN and Lipofectamine™ 2000.
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Oligodesoxirribonucleotídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas Sintéticas/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Cães , Epitopos , Feminino , Hepatite Infecciosa Canina/imunologia , Imunidade nas Mucosas , Imunização Secundária , Lipídeos/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Oligodesoxirribonucleotídeos/administração & dosagem , Vacinas de DNA/imunologia , Vacinas Sintéticas/administração & dosagemRESUMO
With accumulating evidence indicating the importance of cytotoxic T lymphocytes (CTLs) in the antitumor response, strategies are being pursued to elicit augmented CD8(+) T-cell responses against tumors with tumor vaccines. Here, we report the protective efficacy of vaccine-elicited antitumor immune responses with an aggressive HBc-expressing B16-HBc melanoma, which expressed HBc as a self and model antigen, tumor model. We demonstrated that the significantly better memory responses or marked inhibition on tumor growth could be achieved after coadministration of cytokine adjuvants RANTES and Flt3L in a DNA prime-protein boost regimen. Furthermore, the augmentation of DNA prime-protein boost regimens by cytokines gene was due to the improvement the immunopotency of DNA vaccine and subsequently the augmented Ag-specific and IFN-gamma mediating CD8(+) T-cell responses after protein boosting. Hence, this study demonstrates for the first time that combinatorial use of chemotactic and potent DC-specific growth factor molecules provides a useful strategy for enhancing antitumor responses.
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Vacinas Anticâncer/imunologia , Quimiocina CCL5/imunologia , Imunização Secundária/métodos , Melanoma/prevenção & controle , Proteínas de Membrana/imunologia , Vacinação/métodos , Vacinas de DNA/imunologia , Animais , Vacinas Anticâncer/genética , Quimiocina CCL5/genética , Citocinas/metabolismo , Imunidade Celular , Incidência , Melanoma/imunologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Vacinas de DNA/genética , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
AIM: To establish a tumor model in HLA-A2.1 transgenic mice to examine the efficacy of MAGE-3 vaccine, a cell line coexpressing HLA-A 0201/K(b) and MAGE-3 is established. METHODS: B16-HLA-MAGE-3 melanoma was obtained by means of cotransfection of HLA-A 0201/K(b) and MAGE-3 to B16 melanoma. RT-PCR, FCM analysis and Western blot were used to detect the mRNA or protein of HLA-A 0201/K(b) or MAGE-3 expression in B16-HLA-MAGE-3. The ability of MAGE-3 antigen to be processed and presented in the B16-HLA-MAGE-3 cell line were observed by CTL activity detection and tumor challenge test. RESULTS: Transcription and protein expression of HLA-A 0201/H-2k(b) and MAGE-3 were demonstrated in B16-HLA-MAGE-3 cells. CTL activity of splenocytes in immunized mice against B16-HLA-MAGE-3 was detected and the growth of B16-HLA-MAGE-3 in immunized mice was also inhibited. CONCLUSION: MAGE-3 antigen is able to be processed and presented efficiently by B16-HLA-MAGE-3 melanoma cells and this cell can be employed to test HLA-A2 restricted epitope immunogenicity in the A2-transgenic mice.
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Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Antígenos HLA/imunologia , Melanoma Experimental/imunologia , Proteínas de Neoplasias/imunologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Western Blotting , Vacinas Anticâncer/genética , Vacinas Anticâncer/metabolismo , Linhagem Celular , Antígenos HLA/genética , Antígenos HLA/metabolismo , Melanoma Experimental/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The major aim of the project was to develop the virus-like particles (VLPs) displaying single or multi-epitope of hepatocellular carcinomas (HCC) in Escherichia coli and to evaluate the effect on inducing Ag-specific CD8(+) T cell response and antitumor efficacy as candidate vaccines. To this end, hepatitis B virus core (HBc) particles were used as a carrier of HCC epitopes. Four HCC epitopes MAGE-1(278-286aa), MAGE-3(271-279aa), AFP1 (158-166aa) or AFP2 (542-550aa) were fused to the 3' terminus of the truncated HBV core gene, respectively, or conjunctively. Not all recombinant plasmids led to expression of chimeric proteins in expression strain E. coli BL21 (DE3), but chimeric proteins which are expressed in inclusion bodies resulted in the formation of complete "mature" VLPs. E. coli-derived truncated HBc(1-144) chimeric protein self-assembled into VLPs that both morphologically and physically are similar to the wild-type ones and they still remained activity after purification and refolding from 6M urea solution. We also showed that they could be internalized and presented by DCs in vitro. Additionally, DCs pulsed with the chimeric HBc-VLPs could induce stronger CTL activity and greater IFN-gamma secretion by responding T cells compared with peptid-pulsed DCs. In the B16-pIR-HH tumor therapy model, the growth of established tumors was significantly inhibited by immunization using VLP-pulsed DCs, resulting in significantly higher survival rate of immunized animals. Thus, the results of the current study have demonstrated the principal possibility of using VLP on the basis of HBcAg for creation of a new type of HCC-specific immunogen.
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Carcinoma Hepatocelular/terapia , Epitopos/química , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Neoplasias Hepáticas/terapia , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Especificidade de Anticorpos/imunologia , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Carcinoma Hepatocelular/imunologia , Células Dendríticas/imunologia , Células Dendríticas/virologia , Epitopos/genética , Epitopos/imunologia , Escherichia coli/genética , Anticorpos Anti-Hepatite B/biossíntese , Antígenos do Núcleo do Vírus da Hepatite B/química , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vacinas contra Hepatite B/biossíntese , Vacinas contra Hepatite B/imunologia , Vírus da Hepatite B/química , Humanos , Imunoterapia , Interferon gama/metabolismo , Neoplasias Hepáticas/imunologia , Masculino , Antígenos Específicos de Melanoma , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Resultado do Tratamento , Células Tumorais Cultivadas , Vacinas Sintéticas/química , Vacinas Sintéticas/imunologiaRESUMO
AIM: To prepare monoclonal antibody(mAb) against human c-erbB2 and identify its specificity. METHODS: The epitope of human c-erbB2 antigen was analyzed by using computer software and a immunodominant epitope at the carboxyl-terminal was selected. A peptide consisting of 13 amino acids was synthesized and coupled with keyholelimpet hemocyanin (KLH), and then it was used to immunize BLAB/c mice. The splenocytes of the immunized mice were fused with Sp2/0 cells routinely and the hybridoma cells were selected by HAT selected culture, indirect ELISA, and immunohistochemical staining, and cloned by limiting dilution. The specificity of the mAb was identified by cross-reaction test and blocking test. RESULTS: A hybridoma cell line SC8C1, stably secreting anti-c-erbB2 mAb was obtained. The mAb SC8C1 could react to breast cancer tissue expressing c-erbB2 molecule but did not react to other c-erbB2-negative cells. The mAb will lose the activity after being blocked with synthesized 13 peptide. CONCLUSION: A anti-c-erbB2 mAb SC8C1 is prepared successfully using synthesized 13 peptide as immunogen.