目的利用昆虫细胞/杆状病毒系统表达细粒棘球蚴Eg95-Eg.ferritin融合蛋白,用于开发包虫病新型疫苗以及建立相关血清学诊断方法等研究。方法从细粒棘球蚴包囊中分离原头节,超声粉碎后提取总RNA为模板,通过RT-PCR扩增细粒棘球蚴Eg95和Eg.f...目的利用昆虫细胞/杆状病毒系统表达细粒棘球蚴Eg95-Eg.ferritin融合蛋白,用于开发包虫病新型疫苗以及建立相关血清学诊断方法等研究。方法从细粒棘球蚴包囊中分离原头节,超声粉碎后提取总RNA为模板,通过RT-PCR扩增细粒棘球蚴Eg95和Eg.ferritin基因,采用基因拼接法将Eg95和Eg.ferritin融合,将该融合基因Eg95-Eg.ferritin插入到p Fast Bac DUAL载体中,构建重组转座载体后转化DH10Bac感受态细胞,获得重组Bacmid质粒后转染Sf-9昆虫细胞,传毒3代,对表达蛋白进行Western blot鉴定。结果成功克隆了Eg95和Eg.ferritin基因,通过柔性氨基酸linker成功获得了融合基因Eg95-Eg.ferritin,经PCR和酶切鉴定成功构建了重组质粒p Fast Bac DUAL-Eg95-Eg.ferritin,Western blot结果证实表达蛋白能够被包虫病人标准阳性血清识别。结论在Bac-to-Bac杆状病毒表达系统中成功表达了细粒棘球蚴Eg95-Eg.ferritin融合蛋白,与包虫病人标准阳性血清具有良好的反应性。展开更多
目的探讨小分子干扰RNA(si RNA)靶向沉默CDX2基因后对白血病细胞K562增殖、凋亡的影响,并检测BCR-ABL及相关凋亡蛋白表达量的变化,探讨其可能的作用机制。方法根据前期实验结果,成功筛选出能够特异性有效靶向沉默CDX2基因的si RNA序列(C...目的探讨小分子干扰RNA(si RNA)靶向沉默CDX2基因后对白血病细胞K562增殖、凋亡的影响,并检测BCR-ABL及相关凋亡蛋白表达量的变化,探讨其可能的作用机制。方法根据前期实验结果,成功筛选出能够特异性有效靶向沉默CDX2基因的si RNA序列(CDX2-si RNA)和相应的阴性对照序列(CDX2-si RNA-NC),应用罗氏X-treme GENE HP DNA Transfection Reagent转染K562细胞,流式细胞术检测CDX2沉默表达后对K562细胞凋亡的影响;RT-PCR法和Western Blot法分别检测BCR-ABL、Caspase-9、Bax m RNA和蛋白表达量变化。结果 MTT及流式细胞术检测显示,CDX2沉默表达后,K562细胞增殖能力减弱,凋亡率明显增加;RT-PCR和Western Blot显示,与正常细胞组和CDX2-si RNA-NC组相比,CDX2沉默表达后,CDX2-si RNA组的BCR-ABL m RNA和蛋白表达量明显降低,Caspase-9、Bax m RNA和蛋白表达量明显升高(均P<0.05)。结论 CDX2-si RNA能够明显促进白血病细胞K562凋亡,其机制可能与其抑制BCR-ABL表达,增强Caspase-9、Bax的活性有关。展开更多
Background Menin is a ubiquitously expressed protein encoded by the multiple endocrine neoplasia type 1 （MEN1）gene. Besides its importance in endocrine organs, menin has been shown to interact with the mixed lineage...Background Menin is a ubiquitously expressed protein encoded by the multiple endocrine neoplasia type 1 （MEN1）gene. Besides its importance in endocrine organs, menin has been shown to interact with the mixed lineage leukemia （MLL） protein, a histone H3 lysine 4 methyltransferase, and plays a critical role in hematopoiesis and leukemogenesis.Previous studies have shown that menin promotes transforming growth factor beta （TGF-β） signaling in endocrine cells.However, little is known regarding the impact of TGF-β pathway on menin in hematopoietic system. Here, with leukemia cell lines generated from conditional MEN1 or TGF-p receptor （TβRII） knockout mouse models, we investigated the possible cross-talk of these two pathways in leukemia cells.Methods MEN1 or TβRII conditional knockout mice were bred and the bone marrow cells were transduced with retroviruses expressing oncogeneic MLL-AF9 （a mixed lineage leukemia fusion protein） to generate two leukemia cell lines. Cell proliferation assays were performed to investigate the effect of TGF-β treatment on MLL-AF9 transformed leukemia cells with/without MEN1 or TβRII excision. Menin protein was detected with Western blotting and mRNA levels of cell proliferation-related genes Cyclin A2 and Cyclin E2 were examined with real-time RT-PCR for each treated sample.In vivo effect of TGF-p signal on menin expression was also investigated in mouse liver tissue after TβRII excision.Results TGF-β not only inhibited the proliferation of wild type MLL-AF9 transformed mouse bone marrow cells, but also up-regulated menin expression in these cells. Moreover, TGF-P failed to further inhibit the proliferation of Men1-null cells as compared to Men1-expressing control cells. Furthermore, excision of TβRII, a vital component in TGF-β signaling pathway, down-regulated menin expression in MLL-AF9 transformed mouse bone marrow cells. In vivo data also confirmed that menin expression was decreased in liver samples of conditional TβRII knockout mice after TβRII excision.Conclusion These results provided the first piece of evidence of cross-talk between menin and TGF-β signaling pathways in regulating proliferation of leukemia cells, suggesting that manipulating the cross-talk of the two pathways may lead to a novel therapy for leukemia.展开更多
文摘目的利用昆虫细胞/杆状病毒系统表达细粒棘球蚴Eg95-Eg.ferritin融合蛋白,用于开发包虫病新型疫苗以及建立相关血清学诊断方法等研究。方法从细粒棘球蚴包囊中分离原头节,超声粉碎后提取总RNA为模板,通过RT-PCR扩增细粒棘球蚴Eg95和Eg.ferritin基因,采用基因拼接法将Eg95和Eg.ferritin融合,将该融合基因Eg95-Eg.ferritin插入到p Fast Bac DUAL载体中,构建重组转座载体后转化DH10Bac感受态细胞,获得重组Bacmid质粒后转染Sf-9昆虫细胞,传毒3代,对表达蛋白进行Western blot鉴定。结果成功克隆了Eg95和Eg.ferritin基因,通过柔性氨基酸linker成功获得了融合基因Eg95-Eg.ferritin,经PCR和酶切鉴定成功构建了重组质粒p Fast Bac DUAL-Eg95-Eg.ferritin,Western blot结果证实表达蛋白能够被包虫病人标准阳性血清识别。结论在Bac-to-Bac杆状病毒表达系统中成功表达了细粒棘球蚴Eg95-Eg.ferritin融合蛋白,与包虫病人标准阳性血清具有良好的反应性。
文摘目的探讨小分子干扰RNA(si RNA)靶向沉默CDX2基因后对白血病细胞K562增殖、凋亡的影响,并检测BCR-ABL及相关凋亡蛋白表达量的变化,探讨其可能的作用机制。方法根据前期实验结果,成功筛选出能够特异性有效靶向沉默CDX2基因的si RNA序列(CDX2-si RNA)和相应的阴性对照序列(CDX2-si RNA-NC),应用罗氏X-treme GENE HP DNA Transfection Reagent转染K562细胞,流式细胞术检测CDX2沉默表达后对K562细胞凋亡的影响;RT-PCR法和Western Blot法分别检测BCR-ABL、Caspase-9、Bax m RNA和蛋白表达量变化。结果 MTT及流式细胞术检测显示,CDX2沉默表达后,K562细胞增殖能力减弱,凋亡率明显增加;RT-PCR和Western Blot显示,与正常细胞组和CDX2-si RNA-NC组相比,CDX2沉默表达后,CDX2-si RNA组的BCR-ABL m RNA和蛋白表达量明显降低,Caspase-9、Bax m RNA和蛋白表达量明显升高(均P<0.05)。结论 CDX2-si RNA能够明显促进白血病细胞K562凋亡,其机制可能与其抑制BCR-ABL表达,增强Caspase-9、Bax的活性有关。
基金This work was supported by grants from the National Institutes of Health in USA （R01-CA-100912 and R01-CA-113962）, a Fellowship from China Scholarship Council and National Natural Science Foundation of China （No. 30600697）.Acknowledgement： We thank Dr. Harold Moses at Vanderbilt University for providing the mice with the floxed TβRII alleles.
文摘Background Menin is a ubiquitously expressed protein encoded by the multiple endocrine neoplasia type 1 （MEN1）gene. Besides its importance in endocrine organs, menin has been shown to interact with the mixed lineage leukemia （MLL） protein, a histone H3 lysine 4 methyltransferase, and plays a critical role in hematopoiesis and leukemogenesis.Previous studies have shown that menin promotes transforming growth factor beta （TGF-β） signaling in endocrine cells.However, little is known regarding the impact of TGF-β pathway on menin in hematopoietic system. Here, with leukemia cell lines generated from conditional MEN1 or TGF-p receptor （TβRII） knockout mouse models, we investigated the possible cross-talk of these two pathways in leukemia cells.Methods MEN1 or TβRII conditional knockout mice were bred and the bone marrow cells were transduced with retroviruses expressing oncogeneic MLL-AF9 （a mixed lineage leukemia fusion protein） to generate two leukemia cell lines. Cell proliferation assays were performed to investigate the effect of TGF-β treatment on MLL-AF9 transformed leukemia cells with/without MEN1 or TβRII excision. Menin protein was detected with Western blotting and mRNA levels of cell proliferation-related genes Cyclin A2 and Cyclin E2 were examined with real-time RT-PCR for each treated sample.In vivo effect of TGF-p signal on menin expression was also investigated in mouse liver tissue after TβRII excision.Results TGF-β not only inhibited the proliferation of wild type MLL-AF9 transformed mouse bone marrow cells, but also up-regulated menin expression in these cells. Moreover, TGF-P failed to further inhibit the proliferation of Men1-null cells as compared to Men1-expressing control cells. Furthermore, excision of TβRII, a vital component in TGF-β signaling pathway, down-regulated menin expression in MLL-AF9 transformed mouse bone marrow cells. In vivo data also confirmed that menin expression was decreased in liver samples of conditional TβRII knockout mice after TβRII excision.Conclusion These results provided the first piece of evidence of cross-talk between menin and TGF-β signaling pathways in regulating proliferation of leukemia cells, suggesting that manipulating the cross-talk of the two pathways may lead to a novel therapy for leukemia.
