胚胎植入期二硫化碳暴露对孕鼠脾脏淋巴细胞DNA损伤的影响

DNA damage of splenic lymphocytes in pregnant mice exposed to carbon disulfide in implantation phase

目的研究胚胎植入期二硫化碳（CS2）暴露对孕鼠脾脏淋巴细胞DNA损伤的影响，从免疫损伤角度探讨CS2致胚胎植入障碍的机制。方法分别建立不同暴露剂量和不同暴露时间2个动物模型：模型1为不同CS2暴露剂量，即在小鼠受孕第4天（GD4），分别给予4组小鼠一次性腹腔注射低（0．1LD50，157．8mg／kg）、中（0．2LD50，315．7mg／kg）、高（0．4LD50，631．4mg／kg）剂量的CS2和橄榄油对照组；模型2为不同CS2暴露时间，即4组小鼠分别在GD3、GD4、GD5和GD6一次性腹腔注射CS2（0．4LD50，631．4mg／kg），各组设平行对照。两模型对照组均注射等体积橄榄油。实验终点，制备胚胎植入期小鼠脾脏淋巴细胞单细胞悬液，用台盼蓝法检测细胞活性，并进行碱性单细胞凝胶电泳（SCGE）试验，检测孕鼠脾脏淋巴细胞DNA损伤状况。结果（1）中、高剂量组的DNA损伤程度与对照组相比差异均有统计学意义（P〈0．01）；反映DNA损伤程度的各指标与暴露剂量之间均存在回归关系，且差异有统计学意义（P〈0．01）。（2）GD3、GD4、GD5和GD6暴露组的DNA损伤程度与对照组比较，差异均有统计学意义（P〈0．01），且GD4暴露组DNA损伤程度最明显。结论胚胎植入期CS2暴露可导致孕鼠脾脏淋巴细胞的DNA损伤，并且呈明显的剂量反应关系；GD4可能是CS2暴露致孕鼠脾脏淋巴细胞DNA损伤的敏感时间点。

Objective To investigate the I^NA damage of splenic lymphocytes in pregnant mice exposed to carbon disulfide （CS2） in the implantation phase and to explore the mechanism of abnormal implantation induced by CS2 from the perspective of immune injury. Methods Mice were exposed to CS2 at different doses or at different time points in the implantation phase to establish model 1 and model 2. For model 1, mice were assigned to four groups to receive a single intraperitoneal injection of low-dose CS2 （0.1 LD50, 157.8 mg/kg）, middle-dose CS2 （0.2 LD50, 315.7 mg/kg）, and high-dose CS2 （0.4 LD50 631.4 mg/kg） as well as an equal volume of olive oil （control） on gestational day （GD） 4. For model 2, mice were assigned to four groups to receive a single intraperitoneal injection of CS2 （0.4 LD50, 631.4 mg/kg） or an equal volume of olive oil （control） on GD3, GD4, GD5, and GD6. At the end, single cell suspension of splenic lymphocytes was prepared. Cell viability was measured by trypan blue staining, and the DNA damage of splenic lymphocytes was evaluated by alkaline single cell gel electrophoresis assay. Results The middle-dose and high-dose exposure groups showed significantly more DNA damage of splenic lymphocytes than the control group （P〈0.01）; there was significant regression relationship between indicators of DNA damage and exposure doses （P〈0.01）. The GD3, GD4, GD5, and GD 6 exposure groups showed significantly more DNA damage of splenic lymphocytes than the control group （P〈0.01）, and the GD 4 exposure group had the most DNA damage. Conclusion Exposure to CS2 in the implantation phase can induce DNA damage of splenic lymphocytes in pregnant mice, and the DNA damage was aggravated with the increase in CS2 concentration. GD4 may be the sensitive time point for DNA damage of splenic lymphocytes induced by CS2 in pregnant mice.