Establishment Of The Two-Dimensional Electrophoresis Maps Of K562 Cell Treated By Patrinia Heterophylla Bunge Extract

Objectives:1.Higher resolution and reproducibility two-dimensional electrophoresis (2-DE) map of K562 cell protein is established to further understand the new mechanism of how Patrinia heterophylla Bunge worked on leukemia cell.2.To analyze the Protein expression differences after treating K562 cell with Patrinia heterophylla Bunge Extract by the relavent technics of Proteomics and provide the theoretical basis of target proteins.Methods:1.To separate the leukemia K562 cell into control group and experimental group in its logarithmic growth phase. The control group was cultured without any additions while the experimental group in the Patrinia heterophylla Bunge extract containing medium for 48 hours. Cell morphous of both group was observed under Inverted microscop and leukemia K562 cells of two group was collected separately.2.Standard lysis buffer was used for extracting total protein of two groups, and then the control group was precipitated by acetone and TCA/ acetone separately. we can choose the best mothod to prepare protein sample through comparing the 2-DE maps of three ways (standard lysis buffer method,acetone precipitation,TCA/acetone precipitation).3. the focus voltage was optimized on the basis of the traditional 2-DE focus conditions to successfully establish a 2-DE map method with a clear background and good protein separation for leukemia K562 cells 4. the significant points of difference was detected on the gel imaging by image analysis software.Results:1. Patrinia heterophylla Bunge extract could inhibit proliferation of Leukemia K562 and accelerate cell apoptosis.2.Acetone precipitation not only decreased protein decomposition, but also increased protein solubility, and could obtain the 2-DE map with higher resolution and better reproducibility.3.The optimized focusing condition improved horizontal and vertical tailing obviously, and a high-resolution 2-DE map with clearer protein spots and more complete separation was obtained.4.PDQuest7.0 software showsed that 2-DE map of experimental group changed obviously in alkine area. The average protein spots of control groups was 378±16 and the expennental group was 397±21.In 24 Protein spots selected with significant differences between the two groups,12 Protein spots were up-regulated and 9 spots were down-regulated in the control groups, only 3 spots were expressed in experimental group.Conclusions:1.Acetone precipitation combined with standard lysis buffer is more suitable for preparing the 2-DE K562 cell sample.2. properly prolonged desalinization time in isoelectric focusing (IEF) can separate whole proteins of leukemia K562 cell more effectively.