PHA刺激对淋巴细胞修复DNA损伤的影响

本文报道PHA刺激对淋巴细胞DNA修复的影响的实验结果。以254nm波长的UV照射细胞(30J/m~2)引起DNA损伤,以~3H]-TdR掺入实验测定非程序DNA合成,用超微量法测定细胞的NAD~+含量,并以~(35)S]-蛋氨酸掺入,聚丙烯酰胺凝胶电泳及放射自显影术测定蛋白质生物合成,其结果如下: (1)在被PHA转化的淋巴细胞内非程序DNA合成,随PHA刺激的时间加长而增高;PHA处理淋巴细胞42小时,合成的速率约增加4倍;(2)在转化的淋巴细胞内,非程序DNA合成及程序DNA合成都被N-乙基马来酰亚胺(一种DNA聚合酶α的抑制剂)抑制,表明在DNA修复过程中DNA聚合酶α可代替DNA聚合酶β发挥作用; (3)UV照射后,被PHA刺激的淋巴细胞内NAD~+含量大约减少43.2％,而对照淋巴细胞内NAD~+的含量只减少25％,似乎说明PHA刺激能促进淋巴细胞内的P-ADP-核糖化作用;(4)在受PHA刺激72小时的淋巴细胞内有多种蛋白质合成,这些细胞在UV照射后以含10μg/ml嘌呤霉素的培养基培养,则非程序DNA合成被明显抑制(P<0.01),这提示DNA修复是一需要蛋白质合成的过程。此外,在受UV照射后10-45小时的淋巴细胞内,诱导产生一种分子量大约34000道尔顿的蛋白质。 上述结果表明,当PHA使淋巴细胞从静止状态转化为增殖状态时,有多种酶被诱导。由于这些酶,如DNA聚合酶α及P-ADP-核糖聚合

EFFECT OF PHA STIMULATION ON LYMPHOCYTES REPAIRING DNA DAMAGE

This paper is to report the experimental results concerning the effect of PHA stimulation on lymphocyte DNA repair. DNA damage was induced by UV irradiating cells (30J/m2) at 254nm, the unscheduled DNA synthesis (UDS) was determined by 3H]-TdR incorporation experiment, the NAD+ of cells were measured by ultra-micro method, and the protein biosynthesis was observed by 35S]-methi onine incorporation, PAGE and autoradiography. The results are as follows:(1 ) In the lymphocytes transformed by PHA, the UDS was increased, and the increase went up with persistence of PHA stimulation. When lymphocytes were treated with PHA for 42 hours, the rate of UDS was increased by about four times; ( 2 ) In the transformed lymphocytes, the UDS and the scheduled DNA synthesis were both inhibited by N-ethylmaleimide (an inhibitor of DNA polyme rase a) . It suggested that DNA polymerase a could play a role in DNA repair instead of DNA polymeraseβ; ( 3 ) After UV irradiation, the NAD+ content in the lymphocytes stimulated by PHA was decreased by about 43.2%, and the NAD+46 content in control lymphocytes was decreased by only 25%, which seemed to show that PHA stimulation could promote poly- (ADP-ribosyl) ation in lymphocytes;( 4 ) A variety of proteins were synthesized in the lymphocytes stimulated with PHA for 72 hours. If these cells were treated with 10μg/ml puromycin in medium after UV irradiation, the UDS was inhibited obviously (p<0.01) . It suggested that DNA repair was a process requiring protein synthesis. Moreover, an approximate 34k dalton protein was induced in these cells at 10-45 hours after UV irradiation .The above-mentioned results show that when lymphocytes are transformed from quiescent state to growth state by PHA, many species of enzymes are induced .Since some of them, e.g., DNA polymerase a and poly (ADP-ribose) polymerase etc. are involved in DNA repair, the UDS is increased. Whether the UV-induced 34k protein is the x-protein associated with SOS repair, explanations can not be made until further study.