Effect Of TAT-HOXB4 Protein On Mobilization Of Monkeys' Peripheral Blood Hematopoietic Stem Cells

[Objective] This paper deals with the effect and the safety evaluation of TAT-HOXB4 protein on mobilization of monkeys'peripheral blood hematopoietic stem cells.[Content] This experiment contained four stages. Firstly, the TAT-HOXB4 protein's capability of permeation to the intracellular was examined, which could produce the evidence in support of this experiment. Secondly, that made preparation for the following stages of the experiment by examining the expansion capability of TAT-HOXB4 protein as a cytokines CD34+ cells in vitro. Thirdly, it examined the effect of TAT-HOXB4 protein on mobilization of monkeys'peripheral blood hematopoietic stem cells. Lastly, the monkeys'health conditions were evaluated in the past-experiment.[Method] In this experiment, the first stage was to examine the TAT-HOXB4 protein's permeation capability to the nucleus with GFP and the Western Blotting. Second, it examined TAT-HOXB4 protein's capability that expanded the CD34+ cells in human body as a cytokine co-culture with the purified CD34+ cells which was harvested by MiniMACS. It organized the monkeys into four samples, injected amount of G-CSF,HOXB4+ G-CSF,HOXB4 and physiological saline into the four samples respectively. Finally, the effect and the safety of TAT-HOXB4 protein on mobilization were evaluated and analyzed in past-experiment by examining the FACS,CBC and hepatorenal function in pre-injected and past-injected.[Result] There were two experiments to prove TAT-HOXB4 protein could permeate the nucleus'eukaryotic cells effectively and produce the biological effects. The one experiment was to mark TAT-HOXB4 protein with GFP, dye the cell-nuclear with DAPI and co-culture of them. It was found that TAT-HOXB4 protein which marked with the GFP permeated the nucleus. Another experiment was examined by the Western Blotting. There were TAT-HOXB4 protein twigs in the cell-nuclear by the TAT-HOXB4 protein culture, but it didn't contain this kind of twigs without the culture. TAT-HOXB4 protein was able to permeate the cell membrane effectively in the two experiments. It could expand the CD34+ cells with the best concentration and processing conditions of TAT-HOXB4 protein which is done from the previous experiment. There were indications that the CD34+ expanded cells were 14 times more and the mononuclear cells were 6 times more than in control sample in numbers. It was proved that TAT-HOXB4 protein could make the CD34+cells expand in vitro. To compare the effects of mobilization in the four different kinds of samples that the proportion of CD34+cells in peripheral blood mononuclear cells of sample HOXB4 is obviously bigger than those of three samples, and there was no adverse reaction to the injection with TAT-HOXB4 protein. The results of CBC and hepatorenal function indicated that all the data were in the normal range and there were no distinct differences in pre-injected and past-injected. It proved a noticeable effect of TAT-HOXB4 protein on mobilization of monkeys'peripheral blood hematopoietic stem cells and significantly better than the other three samples. It was also found that TAT-HOXB4 protein can provide a health condition in the experiment additionally.[Conclusion]This experiment proved that TAT-HOXB4 protein could permeate the eukaryotic cells and expanded CD34+ cells in vitro effectively. It can provide a very good effect and health condition on mobilization of hematopoietic stem cells which maintain surface marker in the expanded cells.