A real-time RT-PCR （RT-qPCR） assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequ...A real-time RT-PCR （RT-qPCR） assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequences encoding the nucleocapsid protein from the Tahyna virus. Primers and probes were selected in conserved regions by aligning genetic sequences from various Tahyna virus strains available from GenBank. The sensitivity of the RT-qPCR approach was compared to that of a standard plaque assay in BHK cells. RT-qPCR assay can detect 4.8 PFU of titrated Tahyna virus. Assay specificities were determined by testing a battery of arboviruses, including representative strains of Tahyna virus and other arthropod-borne viruses from China. Seven strains of Tahyna virus were confirmed as positive; the other seven species of arboviruses could not be detected by RT-qPCR. Additionally, the assay was used to detect Tahyna viral RNA in pooled mosquito samples. The RT-qPCR assay detected Tahyna virus in a sensitive, specific, and rapid manner; these findings support the use of the assay in viral surveillance.展开更多
Based on the Culex flavivirus (CxFV) E gene sequences in GenBank, CxFV-specific primers and probes were designed for real-time reverse transcription-polymerase chain reaction (RT-qPCR). The specificity test revealed t...Based on the Culex flavivirus (CxFV) E gene sequences in GenBank, CxFV-specific primers and probes were designed for real-time reverse transcription-polymerase chain reaction (RT-qPCR). The specificity test revealed that CxFV could be detected using RT-qPCR with the specific CxFV primers and probes; other species of arboviruses were not detected. The stability test demonstrated a coefficient of variation of <1.5%. A quantitative standard curve for CxFV RT-qPCR was established. Quantitative standard curve analysis revealed that the lower detection limit of the RT-qPCR system is 100 copies/mu L. Moreover, RT-qPCR was used to detect CxFV viral RNA in mosquito pool samples. In conclusion, we established a real-time RT-PCR assay for CxFV detection, and this assay is more sensitive and efficient than general RT-PCR. This technology may be used to monitor changes in the environmental virus levels.展开更多
目的建立real time逆转录聚合酶链反应(RT-PCR)检测BDNF mRNA基因表达的方法.方法提取脑缺血组织的总RNA,进行RT-PCR扩增BDNF mRNA特异性片段,扩增产物重组到质粒上并测序,建立real time RT-PCR检测BDNF mRNA表达水平方法.结果重组的质...目的建立real time逆转录聚合酶链反应(RT-PCR)检测BDNF mRNA基因表达的方法.方法提取脑缺血组织的总RNA,进行RT-PCR扩增BDNF mRNA特异性片段,扩增产物重组到质粒上并测序,建立real time RT-PCR检测BDNF mRNA表达水平方法.结果重组的质粒经酶切和测序,目的片段已插入到载体内,得到real time RT-PCR动力学曲线.结论成功建立real time RT-PCR检测BDNF mRNA基因表达的方法.展开更多
A preliminary study from our research group showed that picroside II inhibited neuronal apop- tosis in ischemic penumbra, reduced ischemic volume, and improved neurobehavioral function in rats with cerebral ischemia. ...A preliminary study from our research group showed that picroside II inhibited neuronal apop- tosis in ischemic penumbra, reduced ischemic volume, and improved neurobehavioral function in rats with cerebral ischemia. The aim of the present study was to validate the neuroprotective effects of picroside II and optimize its therapeutic time window and dose in a rat model of cerebral ischemia. We found that picroside Ⅱ inhibited cell apoptosis and reduced the expression of neuron-specific enolase, a marker of neuronal damage, in rats after cerebral ischemic injury. The optimal treatment time after ischemic injury and dose were determined, respectively, as follows： （1） 2.0 hours and 10 mg/kg according to the results of toluidine blue staining; （2） 1.5 hours and 10 mg/kg according to early apoptotic ratio by flow cytometry; （3） 2.0 hours and 10 mg/kg according to immunohistochemical and western blot analysis; and （4） 1.5 hours and 10 mg/kg according to reverse transcription polymerase chain reaction. The present findings suggest that an intraperitoneal injection of 10 mg/kg picroside II 1.5-2.0 hours after cerebral ischemic injury in rats is the optimal dose and time for therapeutic benefit.展开更多
Objective: To establish a fluoregenic probe quantitative RT-PCR (FQ-RT-PCR) method for detection of the expression of MDR1 gene in tumor cells and to investigate the expression of MDR1 gene in patients with lung cance...Objective: To establish a fluoregenic probe quantitative RT-PCR (FQ-RT-PCR) method for detection of the expression of MDR1 gene in tumor cells and to investigate the expression of MDR1 gene in patients with lung cancer. Methods: The fluorogenic quantitative RT-PCR method for detection of the expression of MDR1 gene was established. K562/ADM and K562 cell lines or 45 tumor tissues from patients with lung cancer were examined on PE Applied Biosystems 7700 Sequence Detection machine. Results: the average levels of MDR1 gene expression in K562/ADM cells and K562 cells were (6.86±0.65)× 107 copies/μg RNA and (8.49±0.67)×105 copies/μg RNA, respectively. The former was 80.8 times greater than the latter. Each sample was measured 10 times and the coefficient variation (CV) was 9.5% and 7.9%, respectively. Various levels of MDR1 gene expression were detected in 12 of 45 patients with lung cancer. Conclusion: Quantitative detection of MDR1 gene expression in tumor cells was achieved by using FQ-RT-PCR. FQ-RT-PCR is an accurate, and sensitive method and easy to perform. Using this method, low levels of MDR1 gene expression could be detected in 24% of the patients with lung cancer.展开更多
目的:相对定量检测Runx2mRNA在甲状腺乳头状癌和腺瘤中的表达,并探讨其在甲状腺乳头状癌的发生发展及与钙化的意义。方法:相对定量real time RT-PCR检测14例甲状腺乳头状癌和14例甲状腺腺瘤中Runx2mRNA的表达。结果:癌组和腺瘤组的ΔCT...目的:相对定量检测Runx2mRNA在甲状腺乳头状癌和腺瘤中的表达,并探讨其在甲状腺乳头状癌的发生发展及与钙化的意义。方法:相对定量real time RT-PCR检测14例甲状腺乳头状癌和14例甲状腺腺瘤中Runx2mRNA的表达。结果:癌组和腺瘤组的ΔCT值分别为2.395±0.302和5.028±1.179,两样本均数差别的t检验示两组之间差异有统计学意义(P<0.01);癌组和腺瘤组的2-ΔΔCT分别为7.826±5.004和1,两样本均数差别的t检验示两组之间差异有统计学意义(P<0.01)。癌组和腺瘤组内以钙化分组ΔCT结果示P>0.05,组间差异均无统计学意义。癌组按肿瘤大小<1cm和≥1cm分组,ΔCT值分别为2.629±0.300和2.212±0.124,P<0.05;2-ΔΔCT值分别为167.33±33.823和221.69±18.843,P<0.01。癌组和腺瘤组以及癌组内钙化分组TSH水平比较均P>0.05。结论:Runx2在甲状腺乳头状癌中的表达高,并与癌大小有关,在较大的癌中表达较高。Runx2与微钙化的关系,可能与甲状腺乳头状癌内钙化灶的产生及癌的发生发展有关,在其他的恶性肿瘤(如乳腺癌、前列腺癌、骨肉瘤)也有相关研究。展开更多
目的:检测miR-224与miR-135a两种microRNA(miRNA)在非小细胞肺癌中的表达,探讨其与肺癌临床病理的关系。方法:采用Real time RT-PCR法对31例非小细胞肺癌组织及癌旁正常肺组织的miR-224与miR-135a进行定量分析,结果由2(-△△CT)处理,并...目的:检测miR-224与miR-135a两种microRNA(miRNA)在非小细胞肺癌中的表达,探讨其与肺癌临床病理的关系。方法:采用Real time RT-PCR法对31例非小细胞肺癌组织及癌旁正常肺组织的miR-224与miR-135a进行定量分析,结果由2(-△△CT)处理,并分析与临床病理资料的关系。结果:相对内参U6,miR-224在非小细胞肺癌组织中表达量为4.2761±0.8731,在正常肺组织中的表达量为0.8967±0.2154,两者相比P<0.05,miR-135a在非小细胞肺癌组织中表达量为0.3551±0.0985,在正常肺组织中的表达量为1.7443±0.3125,两者相比P<0.05。miR-224与miR-135a的表达与非小细胞肺癌的临床分期、病理分级密切相关。结论:miR-224高表达及miR-135a低表达与非小细胞肺癌的临床分期、病理分级密切相关,miR-224有可能作为非小细胞肺癌的重要肿瘤标志物。展开更多
运用含有SYBR Green I的Real Time RT-PCR法分析SS基因在一年生三七根、茎、芦头3个部位中转录水平的相对表达差异。统计分析表明SS基因在根中的表达量最高。本研究取得了特异性高、重复性好的结果,标准曲线斜率均在-3.33～-4范围内,扩...运用含有SYBR Green I的Real Time RT-PCR法分析SS基因在一年生三七根、茎、芦头3个部位中转录水平的相对表达差异。统计分析表明SS基因在根中的表达量最高。本研究取得了特异性高、重复性好的结果,标准曲线斜率均在-3.33～-4范围内,扩增效率均在95%～100%之间,熔解曲线分析显示产物特异性的单一峰,为Real Time RT-PCR技术用于三七植物基因的差异表达分析建立了相应的技术平台。展开更多
本实验旨在探讨奶牛输卵管上皮细胞中是否存在前列腺素E2受体EP2和EP4,且该受体mRNA表达是否受雌激素(E2)的调控。将前列腺素类化合物PGE2和受体选择性激动剂(butaprost)按10-9mol/L-10-5mol/L的浓度分别作用于体外培养的奶牛输卵管上...本实验旨在探讨奶牛输卵管上皮细胞中是否存在前列腺素E2受体EP2和EP4,且该受体mRNA表达是否受雌激素(E2)的调控。将前列腺素类化合物PGE2和受体选择性激动剂(butaprost)按10-9mol/L-10-5mol/L的浓度分别作用于体外培养的奶牛输卵管上皮细胞,应用Elisa方法检测细胞中第二信使cAMP量的变化。然后,将E2作用于体外培养的奶牛输卵管上皮细胞,应用real time RT-PCR技术检测EP2和EP4受体mRNA表达量的变化。Elisa实验结果显示,前列腺素类化合物PGE2和受体选择性激动剂(butaprost)可引起奶牛输卵管上皮中cAMP量的变化,且cAMP量具有对PGE2和butaprost浓度依存性的变化规律,表明奶牛输卵管上皮细胞中存在前列腺素受体EP2和EP4。Real time RT-PCR实验结果表明,奶牛输卵管上皮细胞中存在前列腺素受体EP2和EP4,且E2对EP2和EP4受体mRNA的表达具有调控作用,低浓度(10-12mol/L)可提高该受体mRNA的表达量。展开更多
目的检测高迁移率族蛋白B1(HMGB1)在非小细胞肺癌(NSCLC)组织中的表达,探讨其与临床病理学特征的关系。方法采用Real time RT-PCR法及免疫组织化学染色法检测64例NSCLC组织及20例癌旁正常肺组织中HMGB1mRNA和蛋白表达,并分析与临床病理...目的检测高迁移率族蛋白B1(HMGB1)在非小细胞肺癌(NSCLC)组织中的表达,探讨其与临床病理学特征的关系。方法采用Real time RT-PCR法及免疫组织化学染色法检测64例NSCLC组织及20例癌旁正常肺组织中HMGB1mRNA和蛋白表达,并分析与临床病理资料的关系。结果 NSCLC组织中HMGB1阳性率显著高于正常肺组织,HMGB1的平均光密度值和HMGB1mRNA的相对转录水平均显著高于正常肺组织,差异均有统计学意义(P<0.01)。HMGB1在鳞癌组织中的平均光密度值显著高于腺癌组织,在有淋巴结转移NSCLC组织中的平均光密度值显著高于无淋巴结转移NSCLC组织,差异均有统计学意义(P<0.01和P<0.05)。HMGB1在组织学低分化组表达高于高分化组,随着临床分期增高HMGB1表达升高。HMGB1在NSCLC组织中的表达与患者年龄、性别无关。结论 HMGB1高表达可能与NSCLC的发生、发展及预后不良有关,检测HMGB1表达水平对NSCLC早期诊断及预后的综合评价有一定指导意义。展开更多
Objective: To explore the antiproliferative activity and apoptosis in cells caused by active compounds present in plants using different techniques. Methods: We investigated the antiproliferative effects of methanolic...Objective: To explore the antiproliferative activity and apoptosis in cells caused by active compounds present in plants using different techniques. Methods: We investigated the antiproliferative effects of methanolic extracts from different parts of seven plants on A-549(lung cancer) cells and primary cell culture(chick embryo fibroblast cells, as normal cells) using MTT assay and the potent plant was fractioned further. All these fractions were screened again for anti-proliferative activity. DNA fragmentation and DAPI staining were used to study apoptosis. Quantitative real-time was used to investigate the expression of apoptoticrelated genes. LC-MS and 1 H-NMR techniques were used to identify the active compounds present. EnzCheck caspase-3 assay kit was used to measure caspase-3 activity. Results: Methanolic extract of Vitex negundo(V. negundo) was selected as a potent fraction. Among all fractions screened, ethylacetate fraction of V. negundo was selected as the most potent antiproliferative fraction and phytochemical analysis of the extract revealed the presence of secondary metabolites. Ethaylacetate fraction of V. negundo was found to cause characteristic apoptotic morphological changes and generation of ROS in A-549 cells. Ethaylacetate fraction of V. negundo also induced apoptosis in A-549 which was supported by DNA fragmentation and DAPI staining. To investigate the molecular mechanism behind the cytotoxic effect of ethaylacetate fraction of V. negundo, quantitative real-time PCR was used to measure expression levels of p53, bax, bcl2, casp-3 and casp-9. Using LC-MS and 1 H-NMR techniques, cytotoxic compounds(luteolin and p-hydroxy benzoic acid) were identified which increased casp-3 activity in a dose and time-dependent manner in A-549 treated cells. Conclusions: It is concluded from the present study that V. negundo is capable of triggering growth-inhibitive and apoptosis effects in A-549 cells, signifying that V. negundo may possesses anti-lung cancer activity.展开更多
基金supported by the National Science and Technology Major Project of the Ministry of Science and Technology of China(No.2013ZX10004-101)
文摘A real-time RT-PCR （RT-qPCR） assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequences encoding the nucleocapsid protein from the Tahyna virus. Primers and probes were selected in conserved regions by aligning genetic sequences from various Tahyna virus strains available from GenBank. The sensitivity of the RT-qPCR approach was compared to that of a standard plaque assay in BHK cells. RT-qPCR assay can detect 4.8 PFU of titrated Tahyna virus. Assay specificities were determined by testing a battery of arboviruses, including representative strains of Tahyna virus and other arthropod-borne viruses from China. Seven strains of Tahyna virus were confirmed as positive; the other seven species of arboviruses could not be detected by RT-qPCR. Additionally, the assay was used to detect Tahyna viral RNA in pooled mosquito samples. The RT-qPCR assay detected Tahyna virus in a sensitive, specific, and rapid manner; these findings support the use of the assay in viral surveillance.
基金supported by grants from the Development Grant of State Key Laboratory of Infectious Disease Prevention and Control(2012SKLID204,2015SKLID505)the Ministry of Science and Technology of People’s Republic of China(No.2013ZX10004101)
文摘Based on the Culex flavivirus (CxFV) E gene sequences in GenBank, CxFV-specific primers and probes were designed for real-time reverse transcription-polymerase chain reaction (RT-qPCR). The specificity test revealed that CxFV could be detected using RT-qPCR with the specific CxFV primers and probes; other species of arboviruses were not detected. The stability test demonstrated a coefficient of variation of <1.5%. A quantitative standard curve for CxFV RT-qPCR was established. Quantitative standard curve analysis revealed that the lower detection limit of the RT-qPCR system is 100 copies/mu L. Moreover, RT-qPCR was used to detect CxFV viral RNA in mosquito pool samples. In conclusion, we established a real-time RT-PCR assay for CxFV detection, and this assay is more sensitive and efficient than general RT-PCR. This technology may be used to monitor changes in the environmental virus levels.
文摘目的建立real time逆转录聚合酶链反应(RT-PCR)检测BDNF mRNA基因表达的方法.方法提取脑缺血组织的总RNA,进行RT-PCR扩增BDNF mRNA特异性片段,扩增产物重组到质粒上并测序,建立real time RT-PCR检测BDNF mRNA表达水平方法.结果重组的质粒经酶切和测序,目的片段已插入到载体内,得到real time RT-PCR动力学曲线.结论成功建立real time RT-PCR检测BDNF mRNA基因表达的方法.
基金supported by the National Natural Science Foundation of China,No.81041092,81274116
文摘A preliminary study from our research group showed that picroside II inhibited neuronal apop- tosis in ischemic penumbra, reduced ischemic volume, and improved neurobehavioral function in rats with cerebral ischemia. The aim of the present study was to validate the neuroprotective effects of picroside II and optimize its therapeutic time window and dose in a rat model of cerebral ischemia. We found that picroside Ⅱ inhibited cell apoptosis and reduced the expression of neuron-specific enolase, a marker of neuronal damage, in rats after cerebral ischemic injury. The optimal treatment time after ischemic injury and dose were determined, respectively, as follows： （1） 2.0 hours and 10 mg/kg according to the results of toluidine blue staining; （2） 1.5 hours and 10 mg/kg according to early apoptotic ratio by flow cytometry; （3） 2.0 hours and 10 mg/kg according to immunohistochemical and western blot analysis; and （4） 1.5 hours and 10 mg/kg according to reverse transcription polymerase chain reaction. The present findings suggest that an intraperitoneal injection of 10 mg/kg picroside II 1.5-2.0 hours after cerebral ischemic injury in rats is the optimal dose and time for therapeutic benefit.
基金a grant from the National New Technology Program (No. 1998-345).
文摘Objective: To establish a fluoregenic probe quantitative RT-PCR (FQ-RT-PCR) method for detection of the expression of MDR1 gene in tumor cells and to investigate the expression of MDR1 gene in patients with lung cancer. Methods: The fluorogenic quantitative RT-PCR method for detection of the expression of MDR1 gene was established. K562/ADM and K562 cell lines or 45 tumor tissues from patients with lung cancer were examined on PE Applied Biosystems 7700 Sequence Detection machine. Results: the average levels of MDR1 gene expression in K562/ADM cells and K562 cells were (6.86±0.65)× 107 copies/μg RNA and (8.49±0.67)×105 copies/μg RNA, respectively. The former was 80.8 times greater than the latter. Each sample was measured 10 times and the coefficient variation (CV) was 9.5% and 7.9%, respectively. Various levels of MDR1 gene expression were detected in 12 of 45 patients with lung cancer. Conclusion: Quantitative detection of MDR1 gene expression in tumor cells was achieved by using FQ-RT-PCR. FQ-RT-PCR is an accurate, and sensitive method and easy to perform. Using this method, low levels of MDR1 gene expression could be detected in 24% of the patients with lung cancer.
文摘目的:相对定量检测Runx2mRNA在甲状腺乳头状癌和腺瘤中的表达,并探讨其在甲状腺乳头状癌的发生发展及与钙化的意义。方法:相对定量real time RT-PCR检测14例甲状腺乳头状癌和14例甲状腺腺瘤中Runx2mRNA的表达。结果:癌组和腺瘤组的ΔCT值分别为2.395±0.302和5.028±1.179,两样本均数差别的t检验示两组之间差异有统计学意义(P<0.01);癌组和腺瘤组的2-ΔΔCT分别为7.826±5.004和1,两样本均数差别的t检验示两组之间差异有统计学意义(P<0.01)。癌组和腺瘤组内以钙化分组ΔCT结果示P>0.05,组间差异均无统计学意义。癌组按肿瘤大小<1cm和≥1cm分组,ΔCT值分别为2.629±0.300和2.212±0.124,P<0.05;2-ΔΔCT值分别为167.33±33.823和221.69±18.843,P<0.01。癌组和腺瘤组以及癌组内钙化分组TSH水平比较均P>0.05。结论:Runx2在甲状腺乳头状癌中的表达高,并与癌大小有关,在较大的癌中表达较高。Runx2与微钙化的关系,可能与甲状腺乳头状癌内钙化灶的产生及癌的发生发展有关,在其他的恶性肿瘤(如乳腺癌、前列腺癌、骨肉瘤)也有相关研究。
文摘目的:检测miR-224与miR-135a两种microRNA(miRNA)在非小细胞肺癌中的表达,探讨其与肺癌临床病理的关系。方法:采用Real time RT-PCR法对31例非小细胞肺癌组织及癌旁正常肺组织的miR-224与miR-135a进行定量分析,结果由2(-△△CT)处理,并分析与临床病理资料的关系。结果:相对内参U6,miR-224在非小细胞肺癌组织中表达量为4.2761±0.8731,在正常肺组织中的表达量为0.8967±0.2154,两者相比P<0.05,miR-135a在非小细胞肺癌组织中表达量为0.3551±0.0985,在正常肺组织中的表达量为1.7443±0.3125,两者相比P<0.05。miR-224与miR-135a的表达与非小细胞肺癌的临床分期、病理分级密切相关。结论:miR-224高表达及miR-135a低表达与非小细胞肺癌的临床分期、病理分级密切相关,miR-224有可能作为非小细胞肺癌的重要肿瘤标志物。
文摘运用含有SYBR Green I的Real Time RT-PCR法分析SS基因在一年生三七根、茎、芦头3个部位中转录水平的相对表达差异。统计分析表明SS基因在根中的表达量最高。本研究取得了特异性高、重复性好的结果,标准曲线斜率均在-3.33～-4范围内,扩增效率均在95%～100%之间,熔解曲线分析显示产物特异性的单一峰,为Real Time RT-PCR技术用于三七植物基因的差异表达分析建立了相应的技术平台。
文摘目的:研究转化生长因子-β1(TGF-β1)在外阴癌变过程中的表达及其临床意义。方法:采用realtime RT-PCR及免疫组化方法检测60例外阴鳞癌组织、60例外阴上皮内瘤样病变及10例正常外阴组织标本中TGF-β1 mRNA及其蛋白的表达情况。结果:Real time PCR检测发现,TGF-β1在外阴鳞状细胞癌中的表达量明显低于在外阴上皮内瘤样病变及正常组织中的表达量,且差异有统计学意义(P<0.05)。免疫组化结果显示,正常外阴皮肤组的TGF-β1表达阳性率高于外阴上皮内瘤样病变组及外阴鳞状细胞癌组,与两者相比有显著差异(P<0.05)。外阴上皮内瘤样病变及外阴鳞状细胞癌中TGF-β1表达的阳性率无显著差异(P>0.05)。TGF-β1的表达与外阴鳞状细胞癌患者的组织分化程度及肿瘤分期均无关(P>0.05)。结论:TGF-β1的表达可能与外阴癌变的过程有关,与外阴鳞状细胞癌的临床分期和病理类型无关。
文摘本实验旨在探讨奶牛输卵管上皮细胞中是否存在前列腺素E2受体EP2和EP4,且该受体mRNA表达是否受雌激素(E2)的调控。将前列腺素类化合物PGE2和受体选择性激动剂(butaprost)按10-9mol/L-10-5mol/L的浓度分别作用于体外培养的奶牛输卵管上皮细胞,应用Elisa方法检测细胞中第二信使cAMP量的变化。然后,将E2作用于体外培养的奶牛输卵管上皮细胞,应用real time RT-PCR技术检测EP2和EP4受体mRNA表达量的变化。Elisa实验结果显示,前列腺素类化合物PGE2和受体选择性激动剂(butaprost)可引起奶牛输卵管上皮中cAMP量的变化,且cAMP量具有对PGE2和butaprost浓度依存性的变化规律,表明奶牛输卵管上皮细胞中存在前列腺素受体EP2和EP4。Real time RT-PCR实验结果表明,奶牛输卵管上皮细胞中存在前列腺素受体EP2和EP4,且E2对EP2和EP4受体mRNA的表达具有调控作用,低浓度(10-12mol/L)可提高该受体mRNA的表达量。
文摘目的检测高迁移率族蛋白B1(HMGB1)在非小细胞肺癌(NSCLC)组织中的表达,探讨其与临床病理学特征的关系。方法采用Real time RT-PCR法及免疫组织化学染色法检测64例NSCLC组织及20例癌旁正常肺组织中HMGB1mRNA和蛋白表达,并分析与临床病理资料的关系。结果 NSCLC组织中HMGB1阳性率显著高于正常肺组织,HMGB1的平均光密度值和HMGB1mRNA的相对转录水平均显著高于正常肺组织,差异均有统计学意义(P<0.01)。HMGB1在鳞癌组织中的平均光密度值显著高于腺癌组织,在有淋巴结转移NSCLC组织中的平均光密度值显著高于无淋巴结转移NSCLC组织,差异均有统计学意义(P<0.01和P<0.05)。HMGB1在组织学低分化组表达高于高分化组,随着临床分期增高HMGB1表达升高。HMGB1在NSCLC组织中的表达与患者年龄、性别无关。结论 HMGB1高表达可能与NSCLC的发生、发展及预后不良有关,检测HMGB1表达水平对NSCLC早期诊断及预后的综合评价有一定指导意义。
文摘Objective: To explore the antiproliferative activity and apoptosis in cells caused by active compounds present in plants using different techniques. Methods: We investigated the antiproliferative effects of methanolic extracts from different parts of seven plants on A-549(lung cancer) cells and primary cell culture(chick embryo fibroblast cells, as normal cells) using MTT assay and the potent plant was fractioned further. All these fractions were screened again for anti-proliferative activity. DNA fragmentation and DAPI staining were used to study apoptosis. Quantitative real-time was used to investigate the expression of apoptoticrelated genes. LC-MS and 1 H-NMR techniques were used to identify the active compounds present. EnzCheck caspase-3 assay kit was used to measure caspase-3 activity. Results: Methanolic extract of Vitex negundo(V. negundo) was selected as a potent fraction. Among all fractions screened, ethylacetate fraction of V. negundo was selected as the most potent antiproliferative fraction and phytochemical analysis of the extract revealed the presence of secondary metabolites. Ethaylacetate fraction of V. negundo was found to cause characteristic apoptotic morphological changes and generation of ROS in A-549 cells. Ethaylacetate fraction of V. negundo also induced apoptosis in A-549 which was supported by DNA fragmentation and DAPI staining. To investigate the molecular mechanism behind the cytotoxic effect of ethaylacetate fraction of V. negundo, quantitative real-time PCR was used to measure expression levels of p53, bax, bcl2, casp-3 and casp-9. Using LC-MS and 1 H-NMR techniques, cytotoxic compounds(luteolin and p-hydroxy benzoic acid) were identified which increased casp-3 activity in a dose and time-dependent manner in A-549 treated cells. Conclusions: It is concluded from the present study that V. negundo is capable of triggering growth-inhibitive and apoptosis effects in A-549 cells, signifying that V. negundo may possesses anti-lung cancer activity.
