叶酸防治大剂量甲氨喋呤化疗并发大鼠小肠粘膜炎的实验研究

Experimental study on the prevention and curative effect of folic acid on high-dose Methotre-xate(HDMTX)induced mucositis in rats

目的 观察叶酸对大剂量甲氨喋岭（HDMTX）化疗并发Wistar大鼠小肠粘膜炎的疗效，及叶酸对人T淋巴细胞白血病细胞株CEM的体外作用。方法 将50只5周龄120～150gWistar大鼠（雌雄不限）随机分为3组，A组为正常对照组，B组为MTX对照组，C组为叶酸预治疗组。MTX注射后第1、3、5、7天杀鼠，观察各组死亡率、小肠组织形态学及超微结构变化。体外细胞培养，应用流式细胞术及分子生物学等方法观察不同浓度的叶酸对EEM增殖及HDMTX疗效的影响。结果 在体内叶酸预治疗可明显降低小肠组织病理学积分，使实验鼠的死亡率下降。与MTX对照组比较差异有显著性（P〈0．01）。在体外叶酸对CEM细胞的增殖有明显抑制作用，在浓度为0．4×10^-4～3×10^-4μg／L时抑制作用最明显；流式细胞术分析，叶酸浓度为3．0×10^-4μg／L时，诱导凋亡作用最强，凋亡率为6．19％；细胞表面凋亡相关蛋白bcl-2、C—myc的表达率均明显下降。结论 叶酸不但可以减轻HDMTX诱发的小肠粘膜炎的严重程度，缩短病程，还可通过下调癌基因bcl-2和C—myc的表达而诱导CEM细胞凋亡；叶酸可以安全地用于儿童急性淋巴细胞白血病HDMTX的化疗，而不必担心其余促进肿瘤细胞的增殖及影响HDMTX的疗效。

Objective To study the therapeutic effect of folic acid on high-dose Methotrexate （HDMTX） induced intestinal mucositis in Wistar rats and the proliferative effect of folic acid on CEM leukemia cell line in vivo. Methods Fifty wistar rats with five-week old and 120-150 gram weight were randomly divided into three groups. Normal control was assigned to be group A （n = I0）, MTX control as group B （n = 20）, Wistar rats being pretreated with folic acid before MTX injection as group C （n = 20）. The rats were killed at the first, third, fifth and seventh day after MTX intraperitoneal injection. The morphological changes and super microscopic architecture of the small intestines in different groups were observed. The proliferation of CEM cell line of the rats co-cultured with different levels of folic acid was observed in vivo with flow cytometry and molecular biological methods. And the influence of different levels of folic acid on the anticancer effect of MTX was assayed. Results In comparison with group B, significant increase in the survival rate, the length of small intestinal villus, and the ratio of villus/ crypt was recognized in group C （P〈0.01）. Folic acid had inhibitory effect on proliferation of CEM leukemia cell in vivo. The strongest inhibitory effect of folic acid on proliferation of CEM leukemia cell at the level from 0.4 × 10^-4 -0.3 × 10^-4μg/ L was found after co-cultured with CEM cell at 24/ 48/ 72 hours. The study of flow cytometry showed the strongest effect of folic acid in inducing apoptosis at the level of 0.3 × 10^-4 μg/L （the apoptostic rate was 6.19%）. And the expression of the cell＇ s surface apoptosis-related protein, bcl-2 and C-myc was significantly decreased. Conclusions Folic acid not only can mitigate the severity, but also can shorten the duration of intestinal mucositis induced with HDMTX in rat. Besides,it can also induce apoptosis of CEM cell by down-regulating the expression of gene bcl-2 and C-myc. So,folic acid should be combined with HDMTX chemotherapy in treating childhood acute lymphocytic leukemia safely without worrying about its effect on proliferation of leukemia cells and its impact on chemotherapy.