Background Notch moIecuIes, Notch Iigands and effectors. DeIta4 is a newIy-discovered Notch Iigand which has received the attention of few detaiIed studies. This study sought to expIore the bioIogicaI function of DeIta4 and observe its effects on 32D ceII differentiation. Methods DeIta4-expressing vector pTracer. CMV. DeIta4. FLAG was constructed using moIecuIar bioIogicaI techniques. CHO ceIIs stabIy transfected with pTracer. CMV. DeIta4. FLAG were confirmed to have a DeIta4 protein band via Western bIotting. High-expression DeIta4-CHO cIones were seIected for the foIIowing functionaI studies. Notch1-CHO and Notch2-CHO were used as host ceIIs. After transientIy transfecting with transition protein 1 (TP1), DeIta4 activity was compared in both cell Iines by means of Iuciferase anaIysis. CHO ceIIs were incubated with Notch1-32D cells that had been transfected with Notch1 and were observed for granuIocyte coIony-stimuIating factor (G-CSF)-induced differentiation. Jagged2-CHO and DeIta4-CHO cells transfected with the Notch Iigands Jagged2 and DeIta4, respectiveIy, were incubated with Notch1-32D cells to observed inhibition of Notch on G-CSFinduced differentiation. Results The vector ptracer. CMV. DeIta4. FLAG was constructed successfuIIy. CHO cells were stabIy transfected with the vector ptracer. CMV. DeIta4. FLAG. Two CHO ceII Iines expressing DeIta4 at high IeveIs were seIected for use in the study. DeIta4 was found to induce signaI activity via both Notch1 and Notch2 and the induction of signaIing activity was stronger in Notch2 ceIIs than in Notch1 ceIIs. Compared with other Notch Iigands, DeIta4 was sIightIy weaker than Jagged2, but stronger than DeIta1 and Jagged1 in terms of Notch1 Iigands. In terms of Notch2, DeIta4 had a strong signaIing activity, but was weaker than DeIta1, Jagged1, and Jagged2. Jagged2 couId inhibit Notch1-32D cell differentiation induced by G-CSF, but DeIta4 couId not. Conclusions DeIta4 induces both Notch1 and Notch2 activity and is a Iigand for both of them. The effect of DeIta4 is stronger on Notch2 than that on Notch1. Jagged2 can inhibit Notch1-32D cell differentiation induced by G-CSF, but DeIta4 cannot.

目的 探讨Notch信号传导系统的作用机制及其对造血系统的影响。方法 将报告基因TP1瞬时转染Notch1-CHO和Notch2-CHO细胞后，分别加入转染有不同Notch配体Delta1、Delta4、Jagged1、Jagged2的CHO细胞及正常未转染的CHO细胞，使Notch分子与其配体充分结合并发挥作用，加入发光底物PGL-100，用Lumat测定发光情况。将Notch1-32D细胞加入含G-CSF培养液的CHO、Jagged2-CHO及Delta4-CHO细胞中，观察Notch1-32D细胞分化及分化抑制情况。结果 5种Notch配体与Notch1结合后均能引起荧光素酶活性增高，Jagged2-CHO、Delta4-CHO1-4、Delta4-CHO1-5尤为明显。对Notch2来讲，5种配体均可引起TP1活性的增高。在G-CSF存在下，Notch1-32D细胞可分化为成熟的粒细胞；通过分化抑制实验发现Jagged2能抑制G-CSF引起的Notch1-32D细胞分化，但Delta4不能抑制GCSF引起的Notch1-32D细胞分化。结论 Jagged2、Delta4为Notch1分子的配体，Delta4不能抑制G-CSF引起的Notch1-32D 细胞分化，但Jagged2能抑制G-CSF引起的Notch1-32D细胞分化。

目的：探讨Delta4基因在Notch传导机制中的作用。方法：构建pTraeer. CMV. Delta4．FLAG载体，瞬时转染COS7细胞、稳定转染CHO细胞，筛选高表达Delta4的稳定CHO细胞系。用Lueiferase分析，观察并比较Delta4对Notchl-CHO及Notch2-CHO两个宿主细胞的信号功能活性，与Dehal、Jaggedl及Jagged2比较，从而对此基因定性并获知其信号功能活性水平。结果：Western-blot证实pTraeer．CMV．Delta4．FLA G可瞬时转染COS7细胞及稳定转染CHO细胞，并根据Delta4蛋白表达条带的强弱筛选高表达Delta4-CHO细胞系。Delta4对Notchl及Notch2均具有信号功能活性，且对后者的活性功能水平大于前者。对Notchl，Delta4的功能活性略低于Jagged2，但高于Dehal及Jaggedl；对Notch2，Delta4的功能活性低于Deltal、Jaggedl及Jagged2，但亦具有较强的活性水平。结论：建立的Delta4一CHO细胞系功能稳定，Delta4为Notchl及Notch2的配体，且对Notch2的活性水平高于Notchl。

Notch信号传导系统在介导造血细胞增殖与分化过程中具有重要作用。通过分子克隆技术成功构建含标记基因FLAG的Notch配基Delta4的高表达载体pTracer. CMV. Delta4. FLAG，瞬时转染COS7细胞，48h后收获细胞并制备融合蛋白，SDS-PAGE凝胶电泳和Western．blot证实后，将其稳定转染CHO细胞，Western-blot鉴定并筛选高表达Delta4的Delta4-CHO1-4及Delta4．CHO1.5细胞株，利用Luciferase分析方法进行Delta4功能性研究。研究结果发现Delta4对Notchl及Notch2均具有信号功能活性，且对后者的活性功能水平大于前者，说明Delta4为Notchl及Notch2的配基。与其他配基功能比较：对Notchl，Delta4的功能活性高于Deltal及Jaggedl，但略低于Jagged2；对Notch2，Delta4的功能活性低于Deltal、Jaggedl及Jagged2，但亦具有较强的活性水平。