The Role Of Autophagy In Human Mesenchymal Stem Cells In Response To Irradiation

Objective: To investigate the feature and role of autophagy in human mesenchymal stem cells(hBMMSCs) in response to irradiation.Methods: In this study hBMMSCs were irradiated by sing fraction X-ray in vitro.1. (1) The cultured hBMMSCs were randomized into different irradiation doses and culture durations after irradiation groups. The mRNA expression of autophagy-related genes Beclinl and microtubule-associated protein 1 light chain 3(MAPLC3 or LC3)in hBMMSCs of each group was analyzed by reverse transcription-polymerase chain reaction(RT-PCR).(2) The apoptosis and necrosis rate were assessed by Annexin V and propidium (PI) staining in hBMMSCs at 4h after X-ray irradiation with 0,2,4,8Gy.2. (1)The autophagy reaction of hBMMSCs was regulated by autophagy inhibitor hydroxychloroquine (HCQ) and inducer rapamycin(RAP) in irradiational stress. The cultured hBMMSCs were randomized into 6 groups: normal control group, irradiation group, HCQ treatment group, rapamycin treatment group, HCQ and irradiation treatment group, rapamycin and irradiation treatment group. (2) The morphological changes of autophagy were observed by transmission election microscopy in hBMMSCs of each group. (3) The mRNA expression of Beclinl and LC3 was analyzed by RT-PCR in hBMMSCs of each group. (4) The apoptosis rate was assessed by Annexin V and propidium (PI) staining in hBMMSCs of each group.Results:(1) The mRNA expression of Beclinl and LC3 in hBMMSCs irradiated with a single dose of 2,4,8Gy was significantly higher in a dose-dependent manner than in normal control group hBMMSCs at 2h after irradiation. In addition, the expression of the above two genes was distinctly increased at 2h after 8Gy X-ray irradiation, reached a peak at 4h, and gradually decreased at 8h, then returned to the normal level at 12h. (2) The apoptosis rate of hBMMSCs was markedly decreased while the necrosis and whole dead rate were slowly increased in a dose-dependent manner. In addition, the change of apoptosis rate was more significant than necrosis rate. (3) The proportion of hBMMSCs with autophagic vacuoles in HCQ treatment group was lowest, which was similar to that of normal control group(P>0.05), while the proportion in rapamycin and irradiation treatment group was the highest and that of rapamycin treatment group was the second. The proportion in HCQ and irradiation treatment group which was not significantly different from that of irradiation group(P>0.05) was obviously higher than that in normal control group(P<0.05), but distinctly lower than in rapamycin treatment group and rapamycin and irradiation treatment group(P<0.05). (4)) The mRNA expression of Beclinl and LC3 in hBMMSCs in HCQ treatment group was lowest, which was similar to that of normal control group(P>0.05), while the expression in rapamycin and irradiation treatment group was the highest and that of rapamycin treatment group was the second. The mRNA expression of Beclinl in HCQ and irradiation treatment group was not significantly different from that of irradiation group(P>0.05), while the expression of LC3 was slightly lower(P<0.05). And the expression of two genes in HCQ and irradiation treatment group was markedly higher than that in normal control group(P<0.05), but obviously lower than in rapamycin treatment group and rapamycin and irradiation treatment group(P<0.05). (5) HCQ treatment group and rapamycin treatment group were similar to normal control group in the apoptosis rate(P>0.05), which was the lowest. The apoptosis rate in irradiation group was obviously higher than that in normal control group(P<0.05). The apoptosis rate in HCQ and irradiation treatment group which was highest was dramaticly increased compared with irradiation group(P<0.05), while that in rapamycin and irradiation treatment group was obviously decresed(P<0.05) which was still higher than in normal control group(P<0.05). In addition, the change of apoptosis rate in HCQ and irradiation treatment group was more significant than that in rapamycin and irradiation treatment group compared with irradiation group.Conclusions:(1)The level of autophagy in cultured hBMMSCs in vitro is very low in the basal state. (2) Irradiation may induce the autophagy in cultured hBMMSCs in vitro. (3)The principal mechanism of irradiation injury may be apoptosis in short time(4h) after irradiation. (4) HCQ could not influence the level of autophagy and apoptosis rate in physiological status,while rapamycin could also not impact on the apoptosis rate though it may obviously improve the level of autophagy. (5) HCQ could promote the irradiation induced hBMMSCs apoptosis by inhibition of the autophagy reaction in irradiational stress, while rapamycin may reduce the apoptosis by increase of the level of autophagy. (6)Autophagy could contribute to protecting hBMMSCs from irradiation injury, and it may be related to the radioresistant of hBMMSCs.