应用RNA干扰技术抑制K562细胞BCR-ABL基因表达及诱导细胞凋亡

目的运用RNAi技术,设计针对BCR-ABL基因融合位点的。iRNAs,研究其干扰后细胞变化。方法针对BCR-ABL基因的融合位点设计siRNAs,用脂质体法转染K562细胞,进行MTY法、RT-PCR及流式细胞仪检测,观察其干扰效果。结果转染组与对照组相比细胞增殖减少,BCR-ABL mRNA表达量降低,并有明显的细胞凋亡。结论运用RNAi技术,可以有效地抑制BCR-ABL的表达,诱导细胞凋亡,为白血病的分子机制研究和基因治疗奠定基础。     

发夹式siRNA对hDNMT1基因表达的抑制作用

目的研究发夹式siRNA对hDNMT1基因表达的抑制作用.方法根据hDNMT1全长cDNA序列设计和合成与其互补的发夹式siRNA序列,并以发夹式siRNA序列为模板经体外合成siRNA表达盒(SECs).SECs通过脂质体载体转染结直肠癌细胞株HCT116后,经半定量RT-PCR 法检测hDNMT1 mRNA的表达水平. 结果发夹式siRNA 可以特异性地抑制hDNMT1 基因.结论通过SECs产生的发夹式siRNAs 可以特异性抑制hDNMT1 基因的表达;本研究为通过构建表达发夹式siRNA载体抑制hDNMT1基因表达的研究奠定了基础.     

PSD95 Gene Specific siRNAs Attenuate Neuropathic Pain through Modulating Neuron Sensibility and Postsynaptic CaMKIIα Phosphorylation

Objective To observe the effects of PSD95 gene specific siRNAs on neuropathic pain relief, neuron viability, and postsynaptic calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) phosphorylation in vitro and in vivo. Methods Gene-specific siRNAs of rat PSD95 were synthesized chemically for transfection. Adult male Sprague-Dawley (SD) rats were randomly divided into 3 groups: nave group (n=6), sham group (n=6), and sciatic nerve chronic constriction injury (CCI) group (n=24). The CCI group was further divided into 4 groups (n=6 in each group), which were pretreated with normal saline, transfection vehicle, negative control siRNAs, and PSD95 gene specific siRNAs respectively. All the subgroups received corresponding agents intrathecally for 3 days, started one day before the CCI of sciatic nerve. Both mechanical allodynia and thermal hyperalgesia were measured on post-operative day 3 and 7. PSD95 gene silenced NG108-15 cells were further stimulated by glutamate, with the cell viability and the expression/phosphorylation of CaMKIIα measured by MTT cell proliferation assay and Western blot, respectively. Results The siRNAs decreased PSD95 mRNA level significantly both in vivo and in vitro. Neuropathic pain rats pretreated with PSD95 gene specific siRNAs exhibited significant elevation in the mechanical withdrawal threshold and paw withdrawal thermal latency, without affecting the baseline nociception. PSD95 gene silencing enhanced neuronal tolerance against the glutamate excitotoxicity, meanwhile the phosphorylation of CaMKIIα Thr286 was attenuated. Conclusion Pre-emptive administration of PSD95 gene specific siRNAs may attenuate the central sensitization CaMKIIα-related signaling cascades, leading to the relief of neuropathic pain.