重组人生长激素对人结直肠癌细胞株放疗敏感性的影响

The effect of recombinant human growth hormone on colorectal cancer cell lines radiosensitivity

目的:观察重组人生长激素(rhGH)是否会降低结直肠癌细胞株对放疗的敏感性,并探讨其与细胞凋亡以及DNA损伤修复的关系。方法:选择生长激素受体(GHR)阳性表达的HCT-8和阴性表达的LOVO细胞,各分为单纯放疗组、rhGH+放疗组、生长激素受体中和性抗体(GHRA)+rhGH+放疗组。放疗剂量分别为2 Gy、4Gy和8 Gy;rhGH浓度为100 ng/mL;GHRA浓度为0.2μg/mL。应用克隆形成试验评估放疗敏感性,用流式细胞仪检测细胞凋亡,应用彗星电泳法检测细胞DNA损伤。结果:经过rhGH干预的HCT-8细胞放疗后,其克隆形成率较单纯放疗组显著提高,在8 Gy放疗下尤为明显(52.1±2.9)%vs(21.0±2.7)%,P<0.001。经GHRA预处理封闭GHR之后,这种作用消失。rhGH干预并不改变LOVO细胞接受放疗后的克隆形成率。rhGH干预显著减少了由放疗引起的HCT-8结直肠癌细胞的凋亡(2 Gy,P=0.042;4 Gy,P=0.013;8 Gy,P<0.001);经过中和性抗体预处理封闭GHR之后,这种作用消失。与空白对照相比,rhGH的干预使HCT-8细胞DNA初始受损的程度显著下降(O live尾时刻21.53±2.88vs36.56±3.93,P=0.003),并在到达平台期后,其所处的水平与单纯放疗相比也明显下降(5.5±0.42vs9.07±0.84,P=0.012);在使用GHRA预处理封闭细胞表面GHR后,再加入rhGH,这种作用则消失。结论:rhGH降低GHR(+)的HCT-8细胞对放疗的敏感性,GH与GHR结合,显著增强了GHR(+)结直肠癌细胞对放疗诱导的DNA损伤的修复能力。

Objective： To investigate the effect of recombinant human growth hormone （rhGH） on colorectal cancer cell lines radiosensitivity. Methods：The GHR positive HCT-8 and GHR negative LOVO cell lines were used and each cell set was divided into 3 groups, which were radiation along, radiation ＋ rhGH group and radiation ＋ rhGH ＋ GHRA （GHR neutralizing antibodies） group. The classical colony forming assay was performed to measure the post-radiation colorectal cancer cell proliferation as an indicator of radiosensitivity. Flow cytometry was used to detect the radiation induced apoptosis. Comet assay was performed to detect the radiation induced DNA damage. The radiation dose was 2 Gy, 4 Gy, and 8 Gy respectively. The rhGH concentration used was 100 ng/mL while GHRA was 0.2 ug/ mL. Results：The colony formation rate was significantly enhanced in HCT-8 cells pre-incubated with rhGH compared to the radiation group cells. Under the radiation of high dose （8 Gy） , this effect was more obvious （52.1 2.9 vs 21.0 2.7, P 〈 0. 001 ） with a dose dependent manner. When GHR was blocked with neutralizing antibodies, this protective effect was eliminated. By contrast, rhGH pre-incubation did not change the colony formation rate in LOVO cells. GH pre-incubation significantly reduces radiation induced HCT-8 cell apoptosis and this protective effect was eliminated by GHRA. rhGH intervention reduced the early HCT-8 cell DNA damage（21.53 ± 2.88 vs 36.56 ± 3.93 ; P = 0. 003 ）. This effect was eliminated by GHRA pre-treatment. Conclusion ：The colony formation assay shows the protective effect of GH on colorectal cancer cells after radial exposure. GHR is required in this protective signaling.