Effect Of Serum From Patients With Aplasitc Anemia On Apoptosis Of CD34~+ Cells And Expression Of PAkt

Objective: to compare the effect of separating CD34+ cells from cord blood with different methods, and to observe the effect of the serum from patients with aplasitc anemia on the apoptosis of CD34+ cells and the expression of pAkt.Methods:1. Collecting 48 cord blood units, separating mononuclear cells by density centrifugal method or hydroxyethyl starch precipitation method after they were kept at room temperature (RT) for 12h, 24h or 48h respectively, purifying CD34+ cells with immunomagnetic beads, counting the number of CD34+ cells before and after separation, and then assessing cell viability by typan blue staining.2. The collected CD34+ cells were divided randomly into three groups, and were cultured with serum from healthy persons (controls, n=7), or from patients with severe AA (SAA, n=7) or non severe AA (NSAA, n=7) for 24h, respectively.3. Counting the number of CD34+ cells of each group after incubation, and assessing their viabilities by typan blue staining.4. Analysising the apoptosis of the CD34+ cells of each group with flow cytometry after incubation.5. Detecting the expression of pAkt by immunocytochemical staining.Results: 1. The recovery rates of CD34+ cells were the highest when kept for 48h at RT, lower for 12h, and the lowest for 24h (P<0.01). The recovery rates of CD34+ cells by HES precipitation method were higher than by density centrifugal method (P<0.01). There was no significant differences between the cell survival rates of each group (P>0.05).2. After incubation for 24h, the CD34+ cell concentration and viability were the lowest in the SAA group, higher in the NSAA group, and the highest in the control group (P<0.01).3. After incubation for 24h, the CD34+ cell apoptosis rates were the highest in the SAA group, lower in the NSAA group, and the lowest in the control group (P<0.01).4. After incubation for 24h, the expression of pAkt was the lowest in the SAA group, higher in the NSAA group, and was the highest in the control group (P<0.01).5. Correlation analysis indicated that there was negative correlation between the apoptosis rates of CD34+ cells and the expressions of pAkt in the cells cultured with the same serum(r=0.969, P<0.05).Conclusions:1. HES precipitation method is superior to density centrifugal method, and it is best to separate CD34+ cells when kept for 48h at RT.2. The serum of patients with AA can induce the apoptosis of CD34+ cells, and the effect of SAA is stronger than that of NSAA.3. The serum of patients with AA can downregulate the expression of pAkt, and the effect of SAA is stronger than that of NSAA.4. pAkt protein may be involved in the apoptosis of CD34+ cells induced by the serum of patients with AA.