The Study Of The Effect and Mechanism Of Chemotherapy Plus Granulocyte Colony Stimulating Factor (DAG) for Refractory Paroxysmal Nocturnal Hemoglobinuria In Vitro
Authors: Shao Zonghong , Dong Xifeng , Fu Rong 
Published: October 2019

Objective
To compare the response of GPI-AP negative or positive bone marrow mononuelear cells(BMMNCs) from PNH patients to DAG in vitro and explore the related mechanism.

Methods
Seventeen PNH patients as well as fourteen normal controls were enrolled. CD59-/CD59+ cells were sorted by magnetic activated cell-sorting system. Then the cells were incubated in IMDM medium containing several hemapoitic growth factors with DAG or G-CSF for 48h in vitro. The cell cycle kinetics and apoptosis of these cells were detected by flow cytometry(FCM). The expressions of CD114 on CD34+CD59- and CD34+CD59+ bone marrow cells(BMC) after incubated with G-CSF were measured by FCM. And another 14 PNH peripheral blood samples were obtained, the expression of CD44/CD49d on CD59- and CD59+ cells were analyzed by FCM respectively. The mRNA of CD114 and CD44/CD49d was also tested in 22 PNH patients vs 14 controls and CD59- vs CD59+ cells from 14 PNH patients by Q-PCR.

Results
After incubated with DAG for 48h in vitro, the death rate and apoptosis rate for GPI-AP negative and positive cells(CD59-/CD59+ BMMNCs cells): for CD59- BMMNCs, compared with control group, the death rate of DAG group increased (27.29±22.04% vs 19.10±20.93%), apoptosis rate also increased(10.55±12.34% vs 7.2±6.76%), there was no significant difference for them; for CD59+ BMMNCs, compared with control, the death rate of cells from DAG group increased significantly (31.89±26.75% vs 12.83±18.92%)(P<0.05), whereas there was no significant difference for the apoptosis rate (9.66±7.96% vs 6.31±1.32%); for the CD59- and CD59+ BMMNCs, the death rate was significant higher than apoptosis rate respectively(P<0.05). For cell cycle kinetics, there was no significant difference between the two kinds of BMMNCs. As to the percentage of CD114, compared with control group, it increased significantly in CD34+CD59+ BMMNCs from G-CSF group (48.12±41.20% vs 12.84±15.32%) (P<0.05), whereas there was no significant difference for CD34+CD59- BMMNCs (41.76±44.62% vs 26.79±41.62%). And the variation of CD114 for CD34+CD59+ BMMNCs was higher than that for CD34+CD59- BMMNCs(33.97±36.03% vs 14.88±27.02%)(P<0.05). The expression of CD44/CD49d protein: the expression of CD44 for CD59+was higher than that for CD59- cells(97.66±4.21% vs 93.46±9.52%, P<0.05); and there was no significant difference for CD49d expression in CD59- and CD59+ cells(38.46±27.37% vs 43.79±24.77%). The mRNA expressions of CD114, CD44 and CD49d in 22 PNH patients compared with 15 control and CD59- cells compared with CD59+ cells from 14 PNH patients : for CD114, its mRNA expression was higer for CD59+ cells compared with that for CD59- cells(2.78±2.52 vs 1.69±2.34, P<0.05), but there was no significant difference for CD114 in PNH patients and controls; for CD44, the significant difference exited for PNH patients compared with controls and CD59- cells compared with CD59+ cells(1.73±2.20 vs 3.80±3.87, P<0.05; 0.82±0.75 vs 2.38±2.42, P<0.05); for CD49d, no significant difference exited for PNH patients compared with controls and CD59- cells compared with CD59+ cells(2.83±2.62 vs 2.56±3.04; 1.74±2.60 vs 1.94±3.02).

Conclusions
In vitro, effect of DAG was similar on CD59- and CD59+ BMMNCs, the style of death was necrosis not apotosis and the cell cycle was not influenced by DAG. The variation of CD114 for CD34+CD59- after G-CSF stimulation was less than that in CD34+CD59+ cells, and mRNA of CD114 was lower in CD59- cells compared with CD59+ cells, which may indicating the mechanism for the remission of PNH patients after DAG chemotherapy. The protein and mRNA of CD44 was lower in PNH patients and CD59- cells compared with control and CD59+ cells respectively, which may explain the inferior growth of PNH cells, because they can not fully use the BM microenvironment.

Disclosures:
No relevant conflicts of interest to declare.