Protective Effects And Mechanism Of Platelet Factor 4 On Human Bone Marrow Stromal Cells With Acute Irradiation Injury

Background and objective The recovery of tissues and organs from ionizing irradiation is critically dependent on the repopulation of resident stem cells, defined as the subset of cells with capacity for both self-renewal and differentiation. Stem cells of both hematopoietic and epithelial origin reside in defined areas of the hematopoietic inductive microenvironment. Both hematopoietic stem cells and mesenchymal stem cell populations have been shown to be involved in the repair of ionizing irradiation damage of distant epithelial as well as other hematopoietic sites through their capacity to migrate through the circulation. Bone marrow stromal cells in the hematopoietic microenvironment not only contribute to stem cell repopulation capacity but also to the maintenance of their quiescent or nonproliferative state, which allows the most primitive hematopoietic stem cells to stay in a noncycling state protected from both direct ionizing radiation-induced cell-cycle phase-specific killing and indirect cytokine and free radical mediated killing. BMSCs which regulate hematopoiesis by interacting with hematopoietic cells and extracellular matrix and secreting a large number of cytokines are the main construction of the hematopoietic inductive microenvironment. Their integral structures and biologic function are important to maintain hematopoiesis under both physiological and pathological condition. Platelet factor 4, a founding member of the CXC family of chemokines, is considered a potential radioprotector. It is reported that PF4 could inhibit the growth of hematopoietic stem/progenitor cells reversiblely, promote adhesion of hematopoietic stem/progenitor cells to endothelial cells and human bone marrow cells, protect hematopietic cells from cytotoxicity of chemotherapy, reduce the chemosensitivity of bone marrow cells to several cytotoxic agents significantly including 5-fluorouracil, etoposide, daunorubicine, adriamycin, methotrexate and vincristine, accelerate hematopoietic recovery of mice from a total body irradiation, abate the radiation damage of the bone marrow and lymphoid tissues of irradiated mice, decrease the apoptosis of bone marrow cells of irradiated mice.Methods and results This article is aimed to investigate the protective effects and mechanisms of PF4 on human bone marrow stromal cells which are irradiated by acute ionizing radiation in vitro. Human primary bone marrow stromal cells were randomly divided into four groups:â‘ irradiation protection group with PF4 (P + I),â‘¡PF4 treatment group (P),â‘¢irradiation group (I),â‘£n ormal control group (N). Cells were incubated with 1μg·ml^-1 PF4 or equivalence PBS for 12 hours before 5.0 Gy⁶⁰Co-γray irradiation and were obversed by kinds of experimental methods on suitable time points. The experiment was carried on as follows. Firstly, the 3(4, 5-dimethylthiazol-2yl) 2, 5-diphenyl-tetrazolium bromide assay was performed to test the effect of PF4 on the viability of hBMSCs after irradiation frome day 0 to day 10. Secondly, the behaviors of hBMSCs were observed by confocal microscopy dynamicly. Thirdly, cell cycle was performed with a FACScan cytometer at the time of 20 hours after irradiation. Fourthly, the protective effect of PF4 to hBMSCs from apoptosis induced by irradiation was evaluated using the Annexin V-FITC and propidium iodide (PI) assay on various time points. Lastly, the expressions of p21, CYP1A1 and PCNA mRNA were detected by reverse transcription-polymerase chain reaction at 20 hours after radiation.As a result, PF4 had neither inhibitive nor promotive action on normal hBMSCs without other teatment. However, the survival ratio was significantly higher in the P+I group (>60 % ) than in irradiation group(<40 % ). The cells proliferated slowly after irradiation and their exponential growth phase took place at d 4. Specifically, the irradiated cells treated by PF4 previously had more preferable viability, which had larger size, more integral boundary, less secretory granule in cellular and in culture medium, fewer fragmented cells, classical trabs and whirlpool appearance as classical fibroblasts contrasting with exposured cells. As the experiment shown, the living times of PF4 preincubated radiated cells was 2.5 months in contrast with 1.5 months in radiated cells along with continuing cultivation. Furthermore, the former could generate for 4～5 times approximately, while the latter for 1～2 times only. As cell cycle shown, PF4 caused a significant accumulation of exponentially growing cells in early S phase in P group and P+I group compared to I group and N group at the point of 20 h after exposure (P<0.01). There was also an accumulation of cells in G0/G1 phase under the condition of radiation compared to untreated cells with a difference from 20 to 40 percentages. Ionizing radiation affected hBMSCs as early as day 2 by reducing the percentage of living cells (91.500±0.819 %) and increasing the percentage of late apoptosis cell (3.567±0.551 %). Moreover, a marked increase of early apoptosis cells (17.133±2.854 %) was observed in further incubation. Meanwhile, there was no significant but statistical protection of PF4 in day 2 after radiation except lesser early apoptosis cells (2.233±0.208 %) and more living cells (93.033±0.379 %). However, the above effects were strickly enhanced in P+I group from the time of day 4 on (P<0.001). By the end of day 6, the distinction between P+I group and N group had not been observed statistically. As RT-PCR shown, the expression of p21 mRNA in I group ( + 0.84±0.03 fold) was up-regulated in contrast with N group (0.000±0.000) at the time of 20h after irradiation (P<0.01), as well as the one in P group ( + 0.170±0.090 fold) was down-regulated rather than I group (P<0.05). The expression of CYP1A1 mRNA in P group (ï¹£0.221±0.084 fold) and P+I group (ï¹£0.502±0.060 fold) were down-regulated in contrast with I group (+ 0.20 1±0.091 fold) (P<0.01).Conclusions First of all, it is identified that the optimal concentration of PF4 and the optimal doseage of 60Co-γray in this experiment are 1μg·ml-1 and 5.0 Gy respectively. Meanwhile, 12 hours treatment of PF4 before irradiation is defined as the minimum dosage but maximum effective administration. Second, PF4 has neither inhibitive nor promotive action on normal hBMSCs without other teatment. Third, PF4 has an obviously and reversely protective effect on normal hBMSCs accepting acute radiation injury in a dose-dependent behaviour, in which PF4 could inhibite cell apoptosis; increase the percentage and vitality of living cells; maintain the normal morphous of hBMSCs and enhance the recover of cells after acute inhibitive period. Fourth, PF4 induces S phase arrest and the mechanism may be associated with down-regulating the expression of p21 mRNA and CYP1A1 mRNA which are associated with a series of signal transduction pathway.