Objective:To study the genetic diversity at the msp—1,msp—2,and glurp genes of Plasmodium falciparum(P.falciparum) isolates from 3 endemic areas in Thailand:Tak.Kanchanaburi and Ranong provinces.Methods:A total of 1...Objective:To study the genetic diversity at the msp—1,msp—2,and glurp genes of Plasmodium falciparum(P.falciparum) isolates from 3 endemic areas in Thailand:Tak.Kanchanaburi and Ranong provinces.Methods:A total of 144 P.falciparum isolates collected prior to Irealmenl during January.2012 to June,2013 were genotyped.DNA was extracted:allele frequency and diversity of msp-1.msp-2,and glurp genes were investigated by nested polymerase chain reaction.Results:P.falciparum isolates in this study had high rate of multiple genotypes infection(96.5%)with an overall mean multiplicity of infection of 3.21.The distribution of allelic families of msp-1was significantly different among isolales from Tak.kanchanahnri.and Ranong but not for the msp-2.K1 and MAD20 were the predominant allelic families at the msp-1 gene,whereas alleles belonging to 3D7 were more frequent at the msp-2 gene.The glurp gene had the least diverse alleles.Population structure of P.falciparum isolates from Tak and Ranong was quite similar as revealed by the presence of similar proportions of MAD20 and K1 alleles at msp-1 loci.3D7 and FC27 alleles at msp-2 loci as well as comparable mean MOI.Isolates from Kanchanaburi had different structures;the most prevalent alleles were K1 and RO33.Conclusions:The present study shows that P.falciparum isolates from Tak and Ranong provinces had similar allelic pattern of msp—1 and msp—2 and diversity but different from Kanchanaburi isolates.These allelic variant profiles are valuable baseline data for future epidemiological study of malaria transmission and for continued monitoring of polymorphisms associated with antimalarial drug resistance in these areas.展开更多
目的检测骨形态发生蛋白-2(bone morpgenetic protein 2,BMP-2)基因启动子区甲基化水平,探讨其在骨肉瘤的发生发展过程中的临床意义。方法共收集骨组织标本60例,其中骨肉瘤组织40例,良性病变骨组织10例,正常骨组织10例。应用甲基化特异...目的检测骨形态发生蛋白-2(bone morpgenetic protein 2,BMP-2)基因启动子区甲基化水平,探讨其在骨肉瘤的发生发展过程中的临床意义。方法共收集骨组织标本60例,其中骨肉瘤组织40例,良性病变骨组织10例,正常骨组织10例。应用甲基化特异性PCR法检测60例样本中BMP-2基因甲基化水平。结果骨肉瘤组织中BMP-2基因甲基化水平均低于良性病变组织和正常骨组织,差异有统计学意义(P=0.004 3;P=0.003);而良性病变组织中BMP-2基因甲基化水平低于正常骨组织,但差异无统计学意义(P=0.136)。结论骨肉瘤中BMP-2基因甲基化水平低。选择BMP-2基因甲基转移酶的抑制剂促进高甲基化的抑癌基因的激活,可抑制骨肉瘤的发生发展。展开更多
Objective: To characterize the genetic diversity of Plasmodium falciparum ( P. falciparum ) field isolates in children from Lafia, North-central Nigeria, using the highly polymorphic P. falciparum merozoite surface pr...Objective: To characterize the genetic diversity of Plasmodium falciparum ( P. falciparum ) field isolates in children from Lafia, North-central Nigeria, using the highly polymorphic P. falciparum merozoite surface protein 2 (MSP-2) gene as molecular marker. Methods: Three hundred and twenty children were enrolled into the study between 2005 and 2006. These included 140 children who presented with uncomplicated malaria at the Dalhatu Araf Specialist Hospital, Lafia and another 180 children from the study area with asymptomatic infection. DNA was extracted from blood spot on filter paper and MSP-2 genes were genotyped using allele-specific nested PCR in order to analyze the genetic diversity of parasite isolates. Results: A total of 31 and 34 distinct MSP-2 alleles were identified in the asymptomatic and uncomplicated malaria groups respectively. No difference was found between the multiplicity of infection in the asymptomatic group and that of the uncomplicated malaria group ( P >0.05). However, isolates of the FC27 allele type were dominant in the asymptomatic group whereas isolates of the 3D7 allele type were dominant in the uncomplicated malaria group. Conclusions: This study showed a high genetic diversity of P. falciparum isolates in North-central Nigeria and is comparable to reports from similar areas with high malaria transmission intensity.展开更多
Objective:To assess the quality of expressed MSP-2 and also to confirm the immune response against different domains of these proteins.Methods:Mice were immunized with a schizont extract to stimulate the immune system...Objective:To assess the quality of expressed MSP-2 and also to confirm the immune response against different domains of these proteins.Methods:Mice were immunized with a schizont extract to stimulate the immune system to make antibodies against different antigens of the late stage parasite including production of antibodies against different domains of Plasmodium falciparum(P.falciparum)MSP-2.B lymphocytes of immunized mice were extracted from the spleen and the fusion was performed using NS-1 myeloma cells and the hybridoma cells were assayed by ELISA either with a schizont extract or different domains of MSP-2 and/or by IFAT with whole schizont preparation.Fusion of NS-1 and spleen cells was performed.The positive hybrids were cloned and ELISA was applied against different dilutions.The positive clones were transferred to a small tissue culture flask and after developing they were assayed against schizont extract and the different MSP-2 domains.The positive clones were expanded to large(75 cm^2)flask and cultured under the same conditions,checking them using both ELISA and IFAT and the positive cells were frozen as soon as possible.Results:A total number of 7 fusions including 26 plates(2496 wells)were performed,of which 1336 hybrids were produced and the overall efficiency(1336/24%×100)was about 53%.ELISA was performed to detect the positive hybrids against crude schizont extract by which the highest frequency to crude schizont extract was found for the supernatant of the hybrids produced in fusion number 3(66 out of 315 hybrids).The supernatant of both B5 and Ft hybridoma cells were more positive against domain 2 of the MSP-2 recombinant protein in Western blotting test.Western blotting results also showed that different domains of the MSP-2 recombinant protein and also the MSP-2 of the P.falciparum parasite were recognized by some of the positive clones and also immune sera.Conclusions:Bringing together all the results of this study it has been confirmed that some clones have recognized both schizont extract and different domains of the MSP-2 recombinant protein and therefore confirming the quality of the MSP-2 domains.展开更多
Objective: To determine the effects of toll-like receptor 2(TLR-2) agonist, Pam3 CSK4, on cellular and humoral immune response against recombinant Mycobacterium bovis bacille Calmette-Guérin(rBCG) expressing the ...Objective: To determine the effects of toll-like receptor 2(TLR-2) agonist, Pam3 CSK4, on cellular and humoral immune response against recombinant Mycobacterium bovis bacille Calmette-Guérin(rBCG) expressing the C-terminus of merozoite surface protein-1 of Plasmodium falciparum.Methods: Six groups of mice(n=6 per group) received intraperitoneal phosphate buffered saline T80(PBS-T80), BCG or rBCG in the presence or absence of Pam3 CSK4. Enzymelinked immunosorbent assay was carried out to measure serum total IgG, IgG1, IgG2 a, and Ig G2 b production. Spleens were also harvested and splenocytes were co-cultured with rBCG antigen for in vitro determination of IL-4 and IFN-γ via enzyme-linked immunosorbent assay.Results: The production of total IgG and the isotype IgG1, IgG2 a and IgG2 b was significantly higher in rBCG-immunised mice than in the BCG and PBS-T80-immunised mice, and Pam3 CSK4 further enhanced their productions. A similar pattern was also observed in IFN-γ production. Moreover, there was no significant difference in IL-4 production in all groups either in the presence or absence of Pam3 CSK4.Conclusions: We present evidence of the adjuvant effects of TLR-2 agonist in enhancing the production of total IgG, IgG1, IgG2 a, IgG2 b, as well as IFN-γ in response to rBCG. However, the presence or absence of Pam3 CSK4 had no effect on IL-4 production.展开更多
目的探讨抑癌基因PTEN表达沉默的机制及诱导PTEN表达对白血病细胞的作用。方法应用甲基化特异性聚合酶链反应（Methylation specific PCR，MSP）检测白血病细胞系HL-60、Nalm-6、Raji、KG-1a、U937、NB4、K562中PTEN基因启动子区域甲基...目的探讨抑癌基因PTEN表达沉默的机制及诱导PTEN表达对白血病细胞的作用。方法应用甲基化特异性聚合酶链反应（Methylation specific PCR，MSP）检测白血病细胞系HL-60、Nalm-6、Raji、KG-1a、U937、NB4、K562中PTEN基因启动子区域甲基化状态；用甲基化转移酶抑制剂5-氮-2’-脱氧胞苷（5-Aza-CdR）处理白血病细胞，MSP检测甲基化状态的改变、RT-PCR检测PTEN mRNA水平的改变、瑞特染色观察细胞形态学改变、膜联蛋白V／碘化丙锭（Annexin V／PI）标记染色检测细胞凋亡。结果检测的白血病细胞系中HL-60、Nalm-6、Raji、KG-1a、U937细胞PTEN基因显示超甲基化状态，而NB4和K562细胞显示低甲基化状态；5-Aza-CdR处理HL-60和Nalm-6细胞后，PTEN基因甲基化降低、mRNA表达水平则逐渐增高，并呈剂量依赖性，细胞出现凋亡现象。结论PTEN基因启动子区异常甲基化可能导致该基因转录表达失活或沉默，甲基化抑制剂可以诱导PTEN表达，并引起白血病细胞凋亡。展开更多
基金Supported by a grant from Research Institute,Bansomdejchaopraya Rajabhat University(Grant no.2555)
文摘Objective:To study the genetic diversity at the msp—1,msp—2,and glurp genes of Plasmodium falciparum(P.falciparum) isolates from 3 endemic areas in Thailand:Tak.Kanchanaburi and Ranong provinces.Methods:A total of 144 P.falciparum isolates collected prior to Irealmenl during January.2012 to June,2013 were genotyped.DNA was extracted:allele frequency and diversity of msp-1.msp-2,and glurp genes were investigated by nested polymerase chain reaction.Results:P.falciparum isolates in this study had high rate of multiple genotypes infection(96.5%)with an overall mean multiplicity of infection of 3.21.The distribution of allelic families of msp-1was significantly different among isolales from Tak.kanchanahnri.and Ranong but not for the msp-2.K1 and MAD20 were the predominant allelic families at the msp-1 gene,whereas alleles belonging to 3D7 were more frequent at the msp-2 gene.The glurp gene had the least diverse alleles.Population structure of P.falciparum isolates from Tak and Ranong was quite similar as revealed by the presence of similar proportions of MAD20 and K1 alleles at msp-1 loci.3D7 and FC27 alleles at msp-2 loci as well as comparable mean MOI.Isolates from Kanchanaburi had different structures;the most prevalent alleles were K1 and RO33.Conclusions:The present study shows that P.falciparum isolates from Tak and Ranong provinces had similar allelic pattern of msp—1 and msp—2 and diversity but different from Kanchanaburi isolates.These allelic variant profiles are valuable baseline data for future epidemiological study of malaria transmission and for continued monitoring of polymorphisms associated with antimalarial drug resistance in these areas.
文摘目的检测骨形态发生蛋白-2(bone morpgenetic protein 2,BMP-2)基因启动子区甲基化水平,探讨其在骨肉瘤的发生发展过程中的临床意义。方法共收集骨组织标本60例,其中骨肉瘤组织40例,良性病变骨组织10例,正常骨组织10例。应用甲基化特异性PCR法检测60例样本中BMP-2基因甲基化水平。结果骨肉瘤组织中BMP-2基因甲基化水平均低于良性病变组织和正常骨组织,差异有统计学意义(P=0.004 3;P=0.003);而良性病变组织中BMP-2基因甲基化水平低于正常骨组织,但差异无统计学意义(P=0.136)。结论骨肉瘤中BMP-2基因甲基化水平低。选择BMP-2基因甲基转移酶的抑制剂促进高甲基化的抑癌基因的激活,可抑制骨肉瘤的发生发展。
基金supported by a Deutscher Akademischer Austausch Dienst (DAAD) Research Fellowship as well as a Staff Training Grant from Bells University of Technology, Ota, Nigeria
文摘Objective: To characterize the genetic diversity of Plasmodium falciparum ( P. falciparum ) field isolates in children from Lafia, North-central Nigeria, using the highly polymorphic P. falciparum merozoite surface protein 2 (MSP-2) gene as molecular marker. Methods: Three hundred and twenty children were enrolled into the study between 2005 and 2006. These included 140 children who presented with uncomplicated malaria at the Dalhatu Araf Specialist Hospital, Lafia and another 180 children from the study area with asymptomatic infection. DNA was extracted from blood spot on filter paper and MSP-2 genes were genotyped using allele-specific nested PCR in order to analyze the genetic diversity of parasite isolates. Results: A total of 31 and 34 distinct MSP-2 alleles were identified in the asymptomatic and uncomplicated malaria groups respectively. No difference was found between the multiplicity of infection in the asymptomatic group and that of the uncomplicated malaria group ( P >0.05). However, isolates of the FC27 allele type were dominant in the asymptomatic group whereas isolates of the 3D7 allele type were dominant in the uncomplicated malaria group. Conclusions: This study showed a high genetic diversity of P. falciparum isolates in North-central Nigeria and is comparable to reports from similar areas with high malaria transmission intensity.
文摘Objective:To assess the quality of expressed MSP-2 and also to confirm the immune response against different domains of these proteins.Methods:Mice were immunized with a schizont extract to stimulate the immune system to make antibodies against different antigens of the late stage parasite including production of antibodies against different domains of Plasmodium falciparum(P.falciparum)MSP-2.B lymphocytes of immunized mice were extracted from the spleen and the fusion was performed using NS-1 myeloma cells and the hybridoma cells were assayed by ELISA either with a schizont extract or different domains of MSP-2 and/or by IFAT with whole schizont preparation.Fusion of NS-1 and spleen cells was performed.The positive hybrids were cloned and ELISA was applied against different dilutions.The positive clones were transferred to a small tissue culture flask and after developing they were assayed against schizont extract and the different MSP-2 domains.The positive clones were expanded to large(75 cm^2)flask and cultured under the same conditions,checking them using both ELISA and IFAT and the positive cells were frozen as soon as possible.Results:A total number of 7 fusions including 26 plates(2496 wells)were performed,of which 1336 hybrids were produced and the overall efficiency(1336/24%×100)was about 53%.ELISA was performed to detect the positive hybrids against crude schizont extract by which the highest frequency to crude schizont extract was found for the supernatant of the hybrids produced in fusion number 3(66 out of 315 hybrids).The supernatant of both B5 and Ft hybridoma cells were more positive against domain 2 of the MSP-2 recombinant protein in Western blotting test.Western blotting results also showed that different domains of the MSP-2 recombinant protein and also the MSP-2 of the P.falciparum parasite were recognized by some of the positive clones and also immune sera.Conclusions:Bringing together all the results of this study it has been confirmed that some clones have recognized both schizont extract and different domains of the MSP-2 recombinant protein and therefore confirming the quality of the MSP-2 domains.
基金supported by the Universiti Sains Malaysia(USM)Research University(RU)Grants(No.1001/PPSK/8011100)
文摘Objective: To determine the effects of toll-like receptor 2(TLR-2) agonist, Pam3 CSK4, on cellular and humoral immune response against recombinant Mycobacterium bovis bacille Calmette-Guérin(rBCG) expressing the C-terminus of merozoite surface protein-1 of Plasmodium falciparum.Methods: Six groups of mice(n=6 per group) received intraperitoneal phosphate buffered saline T80(PBS-T80), BCG or rBCG in the presence or absence of Pam3 CSK4. Enzymelinked immunosorbent assay was carried out to measure serum total IgG, IgG1, IgG2 a, and Ig G2 b production. Spleens were also harvested and splenocytes were co-cultured with rBCG antigen for in vitro determination of IL-4 and IFN-γ via enzyme-linked immunosorbent assay.Results: The production of total IgG and the isotype IgG1, IgG2 a and IgG2 b was significantly higher in rBCG-immunised mice than in the BCG and PBS-T80-immunised mice, and Pam3 CSK4 further enhanced their productions. A similar pattern was also observed in IFN-γ production. Moreover, there was no significant difference in IL-4 production in all groups either in the presence or absence of Pam3 CSK4.Conclusions: We present evidence of the adjuvant effects of TLR-2 agonist in enhancing the production of total IgG, IgG1, IgG2 a, IgG2 b, as well as IFN-γ in response to rBCG. However, the presence or absence of Pam3 CSK4 had no effect on IL-4 production.
文摘目的探讨抑癌基因PTEN表达沉默的机制及诱导PTEN表达对白血病细胞的作用。方法应用甲基化特异性聚合酶链反应（Methylation specific PCR，MSP）检测白血病细胞系HL-60、Nalm-6、Raji、KG-1a、U937、NB4、K562中PTEN基因启动子区域甲基化状态；用甲基化转移酶抑制剂5-氮-2’-脱氧胞苷（5-Aza-CdR）处理白血病细胞，MSP检测甲基化状态的改变、RT-PCR检测PTEN mRNA水平的改变、瑞特染色观察细胞形态学改变、膜联蛋白V／碘化丙锭（Annexin V／PI）标记染色检测细胞凋亡。结果检测的白血病细胞系中HL-60、Nalm-6、Raji、KG-1a、U937细胞PTEN基因显示超甲基化状态，而NB4和K562细胞显示低甲基化状态；5-Aza-CdR处理HL-60和Nalm-6细胞后，PTEN基因甲基化降低、mRNA表达水平则逐渐增高，并呈剂量依赖性，细胞出现凋亡现象。结论PTEN基因启动子区异常甲基化可能导致该基因转录表达失活或沉默，甲基化抑制剂可以诱导PTEN表达，并引起白血病细胞凋亡。
