小鼠胚胎毒性试验方法的建立及在评价中药早期胚胎毒性中的应用

Establishment of Mouse Embryo Toxicity Test Method and Its Application in Evaluation of Early Embryo Toxicity of Traditional Chinese Medicine

目的建立一种快速、早期的中药胚胎毒性的快速检测技术,用于中药的早期胚胎毒性筛选。方法 (1)血清制备:SD大鼠随机分为阳性对照组4只,正常组4只,雄黄组、朱砂组各3只。阳性对照组给予环磷酰胺10 mg/kg,正常组用0.5%CMC-Na灌胃给药,雄黄组和朱砂组分别将雄黄和朱砂(用0.5%CMC-Na稀释)按1.0 ml/100 g灌胃给药。末次给药后1 h,无菌取血并取上清血清;(2)细胞采集:4～6周雌性C57小鼠,用妊娠孕马血清(PMSG)腹腔注射,48 h后腹腔注射人绒毛膜促性腺激素(h CG),合笼交配。第2天检查阴栓,用M2培养液将2细胞胚胎从输卵管中冲出,用M16培养液进行培养;(3)毒性试验:在胚胎2细胞阶段加入正常组、阳性对照组、雄黄组及朱砂组不同稀释比血清,观察囊胚成活率。结果在1∶4、1∶16、1∶32的稀释比下,正常组的囊胚率为60.00%、73.33%、93.33%,高于阳性对照组的46.67%、60.00%、66.67%;朱砂组血清各个稀释比的囊胚率均未达到80%;除1∶512这个稀释比外,雄黄组血清各个稀释比2细胞胚胎的囊胚率均未达到80%。结论 (1)朱砂和雄黄的大鼠含药血清在一定浓度时,对小鼠2细胞胚胎的生长发育有一定的影响,呈现中药的胚胎毒性;(2)小鼠胚胎2细胞用于中药的早期胚胎毒性筛选方法是可行的。

Objective To establish a rapid and early rapid detection technique for the toxicity of traditional Chinese medicine embryos for the early embryotoxicity screening of traditional Chinese medicine.Method ① Serum preparation: SD rats were randomly divided into 4 positive control groups, 4 normal groups, and 3 rats in the realgar group and cinnabar group. The positive control group was given cyclophosphamide 10 mg/kg, the normal group was administered with 0.5% CMC-Na, and the realgar and cinnabar groups were respectively diluted with realgar and cinnabar (diluted with 0.5% CMC-Na) at 1.0 ml/100 g. Stomach administration. One hour after the last administration, the blood was taken aseptically and the supernatant serum was taken;②Cell collection: 4 to 6 weeks of female C57 mice were injected intraperitoneally with pregnant pregnant horse serum (PMSG). After 48 hours, human chorionic gonadotropin (hCG) was injected intraperitoneally and the cage was mated. On the second day, the sputum was examined, and the 2 cell embryos were washed out from the fallopian tube with M2 medium, and cultured with M16 medium;③Toxicity test: normal group, positive control group, realgar group and cinnabar group were added at the embryonic 2 cell stage. The blastocyst survival rate was observed with different dilution ratios of serum.Results At the dilution ratio of 1∶4, 1∶16, 1∶32, the blastocyst rate of the normal group was 60.00%, 73.33%, and 93.33%, which was higher than that of the positive control group (46.67%, 60.00%, and 66.67%). The blastocyst rate of each dilution ratio of serum did not reach 80%;Except 1∶512 dilution ratio, the blastocyst rate of each dilution ratio of 2-cell embryos in the realgar group was less than 80%.Conclusion ①The serum containing cinnabar and realgar in rats has certain effect on the growth and development of mouse 2 cell embryos at a certain concentration, showing the embryotoxicity of traditional Chinese medicine;②mouse embryo 2 cells used for early embryotoxicity screening of traditional Chinese medicine The method is feasible.