目的探讨基因枪转导突变特异性K-ras si RNA对胰腺癌细胞生长的抑制作用。方法根据胰腺癌细胞系突变特征,设计突变特异性K-ras si RNA;应用基因枪将si RNA转导入胰腺癌细胞系,提取总RNA和胞浆蛋白;应用逆转录-聚合酶链反应(RT-PCR)和免...目的探讨基因枪转导突变特异性K-ras si RNA对胰腺癌细胞生长的抑制作用。方法根据胰腺癌细胞系突变特征,设计突变特异性K-ras si RNA;应用基因枪将si RNA转导入胰腺癌细胞系,提取总RNA和胞浆蛋白;应用逆转录-聚合酶链反应(RT-PCR)和免疫印迹法(Westernblot)方法,检测基因枪转导突变特异性K-ras si RNA对突变型K-ras基因表达的干扰作用;行细胞爬片K-ras p21蛋白免疫组织化学染色,探讨si RNA对K-ras p21蛋白表达的抑制作用;采用CCK-8(cell counting kit-8)活细胞计数法,绘制转基因前后细胞生长曲线,观察si RNA的抑瘤效果。结果RT-PCR结果显示,K-ras突变特异性si RNA,能有效抑制K-ras基因表达(P<0.05);Westernblot和免疫组织化学结果表明,K-ras p21蛋白的表达明显降低(P<0.05);细胞生长曲线显示,转导si RNA组较对照组细胞生长明显受抑制(P<0.05)。结论基因枪可以有效地对小片段双链si RNA进行细胞内转导;突变特异性K-ras si RNA,可显著下调胰腺癌细胞系突变型K-ras mRNA及K-ras p21蛋白表达,并能有效抑制肿瘤细胞的增殖。展开更多
AIM:To study the role of Twist gene in gastric cancer by gene silencing,including the potential of induction of apoptosis,cell cycle arrest,and proliferation inhibition in human malignant gastric SGC7901 cells.METHODS...AIM:To study the role of Twist gene in gastric cancer by gene silencing,including the potential of induction of apoptosis,cell cycle arrest,and proliferation inhibition in human malignant gastric SGC7901 cells.METHODS:The expression level of Twist in gastric cancer samples was measured by immunohistochemistry.The effects of Twist gene silencing were detected at both m RNA and protein levels by RT-PCR and Western blot.We also evaluated the cell proliferation and apoptosis by CCK-8 assay and flow cytometry.We determined the activity of caspase-3 and caspase-9 with a caspase activity assay kit.Cell cycle distribution was analyzed by flow cytometry.Cell migration and invasion ability was evaluated by wound scratch assay and Boyden chamber assay.RESULTS:Twist protein was highly expressed in gastric cancer samples.Twist gene silencing significantly induced apoptosis,cell cycle arrest at G0/G1 phase,proliferation inhibition,and reduced the ability of migration and invasion in human gastric cancer SGC7901 cells.Meanwhile,both caspase-3 and caspase-9 were activated.CONCLUSION:The Twist gene could serve as a potential molecular target for gene therapy of gastric cancer with targeted small interfering RNA.展开更多
The full-length c DNA of functionally-unknown salivary protein C002 in Schizaphis graminum was cloned using rapid amplification of c DNA ends（RACE） and designated as Sg C002（Gen Bank accession no. KC977563）. It is...The full-length c DNA of functionally-unknown salivary protein C002 in Schizaphis graminum was cloned using rapid amplification of c DNA ends（RACE） and designated as Sg C002（Gen Bank accession no. KC977563）. It is 767 bp long and encodes a protein of 190 amino acid residues with a predicted mass of 21.5 k Da and a predicted cleavage site of N-terminal signal peptide between the 24 th and the 25 th residues. Sg C002 is specifically expressed in salivary gland with the highest level at the 2nd instar. Introducing Sg C002-specific 476-si RNA, but not 546-si RNA to aphids through artificial diet significantly suppressed Sg C002 expression. Silencing Sg C002 gene led to lethality of the aphid on wheat plants, but not on pure artificial diet. Our study demonstrated that artificial diet-mediated RNAi can be a useful tool for research on the roles of genes in aphid salivary gland, and also provided new insights into the characteristics of C002 in wheat aphids.展开更多
The Maf family of transcription factors is characterized by a typical b Zip structure; these transcription factors act as important regulators of the development and differentiation of many organs and tissues, includi...The Maf family of transcription factors is characterized by a typical b Zip structure; these transcription factors act as important regulators of the development and differentiation of many organs and tissues, including the kidney. The Maf family consists of two subgroups that are characterized according to their structure: large Maf transcription factors and small Maf transcriptionfactors. The large Maf subgroup consists of four proteins, designated as MAFA, MAFB, c-MAF and neural retina-specific leucine zipper. In particular, MAFA is a distinct molecule that has been attracting the attention of researchers because it acts as a strong transactivator of insulin, suggesting that Maf transcription factors are likely to be involved in systemic energy homeostasis. In this review, we focused on the regulation of glucose/energy balance by Maf transcription factors in various organs.展开更多
目的探讨以si RNA为载体沉默骨髓间充质干细胞(MSC)中核因子E2相关因子2(Nrf2)基因后细胞移植对大鼠心肌梗死后心肌纤维化和心室重构的影响及可能机制。方法建立心肌梗死大鼠模型,随机分为沉默Nrf2基因的MSC移植组(MSCNrf2-/-组)、过表...目的探讨以si RNA为载体沉默骨髓间充质干细胞(MSC)中核因子E2相关因子2(Nrf2)基因后细胞移植对大鼠心肌梗死后心肌纤维化和心室重构的影响及可能机制。方法建立心肌梗死大鼠模型,随机分为沉默Nrf2基因的MSC移植组(MSCNrf2-/-组)、过表达Nrf2基因的MSC移植组(MSCNrf2+/+组)和生理盐水转染MSC移植组(对照组),每组12只。细胞移植后28天,采用Masson染色检测心肌梗死边缘区胶原沉积含量和纤维化程度,Western blot检测梗死心肌Nrf2和血红素氧合酶1(HO-1)蛋白表达水平,超声心动图评价梗死后心功能。结果 si RNA-Nrf2转染MSC后,Nrf2蛋白表达明显减少。移植后第28天,MSCNrf2-/-组心肌组织纤维化程度较对照组加重,MSCNrf2+/+组心肌组织纤维化程度较对照组减轻(P<0.05);MSCNrf2-/-组梗死心肌中Nrf2、HO-1蛋白表达较对照组下降(P<0.05),而MSCNrf2+/+组梗死心肌中Nrf2、HO-1蛋白表达较对照组增加(P<0.05);超声心动图结果显示,与对照组比较,MSCNrf2-/-组左心室舒张期末内经(LVEDD)和左心室收缩期末内经(LVESD)增大,左心室射血分数(LVEF)下降(P<0.05),MSCNrf2+/+组LVEDD和LVESD均减小,LVEF无下降(P<0.05)。结论 si RNA-Nrf2可有效干扰MSC中Nrf2蛋白的表达,降低外源性MSC对梗死心脏的修复能力,增加心肌梗死区胶原沉积,进而促进心室重构,降低心功能。由此推测Nrf2信号通路在干细胞移植治疗心肌梗死后心肌重构和纤维化中起了关键作用。展开更多
The lentivirus-mediated u PA interference in the proliferation, apoptosis, and secretion of osteoarthritic chondrocytes was examined in this study. Cells were obtained from the cartilage tissues of New Zealand white r...The lentivirus-mediated u PA interference in the proliferation, apoptosis, and secretion of osteoarthritic chondrocytes was examined in this study. Cells were obtained from the cartilage tissues of New Zealand white rabbits. They were cultured with interleukin（IL）-1β（10 ng/m L） for 24 h and then divided into three groups： u PA-si RNA group（cells transfected with u PA-si RNA lentiviruses）, blank control group（untreated cells）, and negative control group（cells transfected with empty vectors）. Western blotting and real-time quantitative reverse transcription-PCR（RT-QPCR） were performed to detect the protein and m RNA expression levels of u PA, MMP-1, MMP-3, MMP-9, MMP-10, MMP-13 and MMP-14 in osteoarthritic chondrocytes. Cell Counting Kit-8, flow cytometry, and colony formation assay were used to examine the proliferation and apoptosis of chondrocytes. The results showed that after u PA-si RNA transfection, the protein and mR NA expression levels of uP A, MMP-1, MMP-3, MMP-9, MMP-10, MMP-13, and MMP-14 were significantly decreased（P〈0.05 for MMP-1, MMP-9, MMP-10 and MMP-14, P〈0.01 for u PA, MMP-3 and MMP-13）. Cell proliferation and colony formation rate were significantly higher and the cell apoptosis rate was significantly lower in u PA-si RNA group than in control groups（P〈0.01）. The proportion of cells in G0/G1 phase was markedly increased and that in the S phase decreased, and the cell cycle was arrested at the G1/S phase in the control group. In the u PAsi RNA group, the proportion of cells in the S phase was significantly increased, resulting in a different proportion of cells in cell cycle phase（P〈0.01）. It was suggested that the down-regulation of uP A gene could inhibit the expression of MMPs protein and cell apoptosis, increase the proliferation and colony formation of osteoarthritic chondrocytes.展开更多
目的:评估叉头框蛋白M1(forkhead box protein M1,FOXM1)在结直肠癌(colorectal cancer,CRC)的发生发展中的作用。方法:研究103例原发CRC和配对的正常组织标本,探讨FOXM1表达变化的潜在机制及其对体外CRC细胞模型的增殖和转移的影响。结...目的:评估叉头框蛋白M1(forkhead box protein M1,FOXM1)在结直肠癌(colorectal cancer,CRC)的发生发展中的作用。方法:研究103例原发CRC和配对的正常组织标本,探讨FOXM1表达变化的潜在机制及其对体外CRC细胞模型的增殖和转移的影响。结果:103例CRC组织染色后FOXM1阳性率为85.44%(88/103),癌旁正常组织中阳性率为20.39%(21/103)。两组间的FOXM1的表达差异具有统计学意义(P<0.001);FOXM1沉默能抑制结肠癌细胞的增殖,且侵袭和迁移也明显受抑。结论:CRC的发病机制可能是由FOXM1介导,FOXM1可能代表CRC分子靶向治疗的选择性靶点。展开更多
目的:研究基质金属蛋白酶-2(MMP-2)si RNA对口腔鳞状细胞癌Tca8113细胞凋亡的作用。方法:采用MMP-2 si RNA慢病毒感染Tca8113,q PCR检测MMP-2表达,MTT检测细胞活性,Annexin-V单染检测细胞凋亡。结果:MMP-2 si RNA慢病毒成功抑制Tca8113...目的:研究基质金属蛋白酶-2(MMP-2)si RNA对口腔鳞状细胞癌Tca8113细胞凋亡的作用。方法:采用MMP-2 si RNA慢病毒感染Tca8113,q PCR检测MMP-2表达,MTT检测细胞活性,Annexin-V单染检测细胞凋亡。结果:MMP-2 si RNA慢病毒成功抑制Tca8113中MMP-2 m RNA表达。MMP-2干扰组细胞活性显著低于阴性病毒组(P<0.01),且细胞凋亡率高于阴性对照组(P<0.01)。结论:MMP-2干扰能促进Tca8113凋亡,起到抗肿瘤效应。展开更多
文摘AIM:To study the role of Twist gene in gastric cancer by gene silencing,including the potential of induction of apoptosis,cell cycle arrest,and proliferation inhibition in human malignant gastric SGC7901 cells.METHODS:The expression level of Twist in gastric cancer samples was measured by immunohistochemistry.The effects of Twist gene silencing were detected at both m RNA and protein levels by RT-PCR and Western blot.We also evaluated the cell proliferation and apoptosis by CCK-8 assay and flow cytometry.We determined the activity of caspase-3 and caspase-9 with a caspase activity assay kit.Cell cycle distribution was analyzed by flow cytometry.Cell migration and invasion ability was evaluated by wound scratch assay and Boyden chamber assay.RESULTS:Twist protein was highly expressed in gastric cancer samples.Twist gene silencing significantly induced apoptosis,cell cycle arrest at G0/G1 phase,proliferation inhibition,and reduced the ability of migration and invasion in human gastric cancer SGC7901 cells.Meanwhile,both caspase-3 and caspase-9 were activated.CONCLUSION:The Twist gene could serve as a potential molecular target for gene therapy of gastric cancer with targeted small interfering RNA.
基金supported by the National Natural Science Foundation of China (30971920,31371946)the International Cooperation Project between China and Belgium (2010DFA32810)the Earmarked Fund for Modern Agro-Industry Technology Research System,China (CARS3)
文摘The full-length c DNA of functionally-unknown salivary protein C002 in Schizaphis graminum was cloned using rapid amplification of c DNA ends（RACE） and designated as Sg C002（Gen Bank accession no. KC977563）. It is 767 bp long and encodes a protein of 190 amino acid residues with a predicted mass of 21.5 k Da and a predicted cleavage site of N-terminal signal peptide between the 24 th and the 25 th residues. Sg C002 is specifically expressed in salivary gland with the highest level at the 2nd instar. Introducing Sg C002-specific 476-si RNA, but not 546-si RNA to aphids through artificial diet significantly suppressed Sg C002 expression. Silencing Sg C002 gene led to lethality of the aphid on wheat plants, but not on pure artificial diet. Our study demonstrated that artificial diet-mediated RNAi can be a useful tool for research on the roles of genes in aphid salivary gland, and also provided new insights into the characteristics of C002 in wheat aphids.
基金Supported by A Grant-in-Aid for Scientific Research of Japan(C17591443:Tsuchiya M,C26461243:Tsuchiya K)
文摘The Maf family of transcription factors is characterized by a typical b Zip structure; these transcription factors act as important regulators of the development and differentiation of many organs and tissues, including the kidney. The Maf family consists of two subgroups that are characterized according to their structure: large Maf transcription factors and small Maf transcriptionfactors. The large Maf subgroup consists of four proteins, designated as MAFA, MAFB, c-MAF and neural retina-specific leucine zipper. In particular, MAFA is a distinct molecule that has been attracting the attention of researchers because it acts as a strong transactivator of insulin, suggesting that Maf transcription factors are likely to be involved in systemic energy homeostasis. In this review, we focused on the regulation of glucose/energy balance by Maf transcription factors in various organs.
文摘目的探讨以si RNA为载体沉默骨髓间充质干细胞(MSC)中核因子E2相关因子2(Nrf2)基因后细胞移植对大鼠心肌梗死后心肌纤维化和心室重构的影响及可能机制。方法建立心肌梗死大鼠模型,随机分为沉默Nrf2基因的MSC移植组(MSCNrf2-/-组)、过表达Nrf2基因的MSC移植组(MSCNrf2+/+组)和生理盐水转染MSC移植组(对照组),每组12只。细胞移植后28天,采用Masson染色检测心肌梗死边缘区胶原沉积含量和纤维化程度,Western blot检测梗死心肌Nrf2和血红素氧合酶1(HO-1)蛋白表达水平,超声心动图评价梗死后心功能。结果 si RNA-Nrf2转染MSC后,Nrf2蛋白表达明显减少。移植后第28天,MSCNrf2-/-组心肌组织纤维化程度较对照组加重,MSCNrf2+/+组心肌组织纤维化程度较对照组减轻(P<0.05);MSCNrf2-/-组梗死心肌中Nrf2、HO-1蛋白表达较对照组下降(P<0.05),而MSCNrf2+/+组梗死心肌中Nrf2、HO-1蛋白表达较对照组增加(P<0.05);超声心动图结果显示,与对照组比较,MSCNrf2-/-组左心室舒张期末内经(LVEDD)和左心室收缩期末内经(LVESD)增大,左心室射血分数(LVEF)下降(P<0.05),MSCNrf2+/+组LVEDD和LVESD均减小,LVEF无下降(P<0.05)。结论 si RNA-Nrf2可有效干扰MSC中Nrf2蛋白的表达,降低外源性MSC对梗死心脏的修复能力,增加心肌梗死区胶原沉积,进而促进心室重构,降低心功能。由此推测Nrf2信号通路在干细胞移植治疗心肌梗死后心肌重构和纤维化中起了关键作用。
基金supported by grants from the National Natural Science Foundation of China(Nos.81160225,812604531,and 81360451)the Xinjiang Bingtuan Special Program of Medical Science(Nos.2011BC004,2013BA020,and 2014BC003)
文摘The lentivirus-mediated u PA interference in the proliferation, apoptosis, and secretion of osteoarthritic chondrocytes was examined in this study. Cells were obtained from the cartilage tissues of New Zealand white rabbits. They were cultured with interleukin（IL）-1β（10 ng/m L） for 24 h and then divided into three groups： u PA-si RNA group（cells transfected with u PA-si RNA lentiviruses）, blank control group（untreated cells）, and negative control group（cells transfected with empty vectors）. Western blotting and real-time quantitative reverse transcription-PCR（RT-QPCR） were performed to detect the protein and m RNA expression levels of u PA, MMP-1, MMP-3, MMP-9, MMP-10, MMP-13 and MMP-14 in osteoarthritic chondrocytes. Cell Counting Kit-8, flow cytometry, and colony formation assay were used to examine the proliferation and apoptosis of chondrocytes. The results showed that after u PA-si RNA transfection, the protein and mR NA expression levels of uP A, MMP-1, MMP-3, MMP-9, MMP-10, MMP-13, and MMP-14 were significantly decreased（P〈0.05 for MMP-1, MMP-9, MMP-10 and MMP-14, P〈0.01 for u PA, MMP-3 and MMP-13）. Cell proliferation and colony formation rate were significantly higher and the cell apoptosis rate was significantly lower in u PA-si RNA group than in control groups（P〈0.01）. The proportion of cells in G0/G1 phase was markedly increased and that in the S phase decreased, and the cell cycle was arrested at the G1/S phase in the control group. In the u PAsi RNA group, the proportion of cells in the S phase was significantly increased, resulting in a different proportion of cells in cell cycle phase（P〈0.01）. It was suggested that the down-regulation of uP A gene could inhibit the expression of MMPs protein and cell apoptosis, increase the proliferation and colony formation of osteoarthritic chondrocytes.
文摘目的:评估叉头框蛋白M1(forkhead box protein M1,FOXM1)在结直肠癌(colorectal cancer,CRC)的发生发展中的作用。方法:研究103例原发CRC和配对的正常组织标本,探讨FOXM1表达变化的潜在机制及其对体外CRC细胞模型的增殖和转移的影响。结果:103例CRC组织染色后FOXM1阳性率为85.44%(88/103),癌旁正常组织中阳性率为20.39%(21/103)。两组间的FOXM1的表达差异具有统计学意义(P<0.001);FOXM1沉默能抑制结肠癌细胞的增殖,且侵袭和迁移也明显受抑。结论:CRC的发病机制可能是由FOXM1介导,FOXM1可能代表CRC分子靶向治疗的选择性靶点。
文摘目的探讨通过si RNA干扰β-catenin对亚砷酸钠致大鼠体内砷水平和肺组织病变的影响。方法将32只健康成年SPF级SD大鼠按体重随机分为对照(超纯水)组、染砷空白对照组、染砷+β-catenin干扰组和染砷+阴性转染对照组,每组8只,雌雄各半。采用自由饮水方式染毒亚砷酸钠溶液(11.25 mg/kg),连续染毒16周;在最后1周,腹腔注射β-catenin si RNA转染试剂和阴性对照转染试剂(0.49μg/g),连续1周。观察大鼠肺组织病理学改变,检测大鼠血砷、尿砷和肺组织砷水平。结果染砷第16周,染砷空白对照组、染砷+β-catenin干扰组和染砷+阴性转染对照组大鼠体重较对照组减轻(P<0.05)。染砷+β-catenin干扰组大鼠肺组织病理损伤较染砷空白对照组和染砷+阴性转染对照组严重,出现大部分纤维化,肺泡结构几乎消失。染砷空白对照组、染砷+β-catenin干扰组和染砷+阴性转染对照组大鼠血砷、尿砷和肺组织砷水平较对照组增加(P<0.05)。染砷+β-catenin干扰组大鼠血砷、尿砷和肺组织砷水平较染砷+阴性转染对照组增加(P<0.05);其中雄性大鼠肺组织砷含量较染砷空白对照组增加(P<0.05)。结论长期砷暴露致大鼠肺组织病变;而干扰β-catenin基因后可能增加砷的蓄积,加重大鼠肺组织病变。
文摘目的:研究基质金属蛋白酶-2(MMP-2)si RNA对口腔鳞状细胞癌Tca8113细胞凋亡的作用。方法:采用MMP-2 si RNA慢病毒感染Tca8113,q PCR检测MMP-2表达,MTT检测细胞活性,Annexin-V单染检测细胞凋亡。结果:MMP-2 si RNA慢病毒成功抑制Tca8113中MMP-2 m RNA表达。MMP-2干扰组细胞活性显著低于阴性病毒组(P<0.01),且细胞凋亡率高于阴性对照组(P<0.01)。结论:MMP-2干扰能促进Tca8113凋亡,起到抗肿瘤效应。
