Establishment Of Quantitative Methodology For Rat Estrus Cycle Cells And Its Application In Toxicological Studies

Objective:1.Optimization of rat vaginal smear staining method provides a basis for accurate and rapid judgment of estrus cycle.2.Through quantitative analysis of all kinds of vaginal exfoliated cells in female rats with normal estrus cycle,established a cell quantitative method that can accurately distinguish the four stages of estrus cycle,and the accuracy of this method was verified using two animal models: estradiol benzoate(E2B)administration and continuous illumination.3.Cell quantitative method was used to evaluate whether the new drug TJ1933 had interference on the estrus cycle of rats.Method:1.Optimization of dyeing method: Vaginal smear was prepared by collecting vaginal discharge approximately the same time in the morning and three vaginal smears were prepared by the same vaginal fluid,which were used for papanicolaou stain(Pap),shorr stain(S)and giemsa stain(G),and continuous collection of vaginal secretions for 15 d.After the smear was fixed and stained,the estrus cycle was judged by ordinary optical microscope.According to the result,the sensitivity,specificity and accuracy of the three staining methods were compared,and the best staining was selected methodology.2.Establishment and verification of quantitative method: 24 rats with normal estrus cycle were collected once every day in the afternoon before modeling,and collected continuously for 15 d.from 720 smears,45 high-quality smears were selected for cell count at each stage to establish cell quantitative criteria.The E2 B model wasestablished after 15 d,12 rats were given a single subcutaneous hypodermic injection(i.h.)of 5ug/m L E2 B on the first day of emotional period;and a continuous illumination model was established in 12 rats 24 h for 7d.After the model was established,vaginal exfoliated cells were collected once a day for 15 d.The established cell quantitative methodology was used to evaluate the estrus cycle of rats in two model groups.the evaluation results were compared with the literature results to verify the accuracy and reliability of the quantitative method.3.Application in the evaluation of new drugs: healthy female rats were randomly divided into solvent control group,TJ1933 low dose group,medium dose group and high dose group,10 rats in each group according to body weight.The administration group was given 2,6,18 mg/kg of TJ1933 solution by gavage,and the control group was given 0.5% sodium carboxymethyl cellulose solution(CMC-Na).Every day at the same time to the stomach once,after the administration of vaginal smear,continuous 14 d.After fixation and shorr stain,the effect of TJ1933 on estrus cycle was evaluated by cell quantitative method.Result:1.Comparison of three staining methods: shorr stain made the cells bright in color,clear in shape and structure,and provided good color and better chromatin details,which was helpful to determine different cell types;shorr stain was more sensitive and specific in the judgment of estrus cycle than papanicolaou stain and giemsa stain;shorr stain was more accurate in the judgment of estrus cycle than papanicolaou stain and giemsa stain,reaching 97.8%;and the sensitivity and specificity of the results were compared and the accuracy was compared by comparing the staining effect of the three staining methods,the sensitivity and specificity of the result judgment.The comprehensive analysis shows that shorr stain is the best dyeing method.2.By counting 45 vaginal exfoliated cell smears at each stage of normal estrus cycle rats,the proportion of nuclear epithelial cells,enucleated cornified cells andleukocytes in the four stages of the estrus cycle was determined,and the cell quantitative standard was established.Among them,the ratio of nuclear epithelial cells,enucleated cornified cells and leukocytes is 7:4:1 in preoestrus;in estrus,the ratio of enucleated cornified cells,nucleated epithelial cells and leukocytes is 4:3:1;in metoestrus,the ratio of leukocytes,nucleated epithelial cells and enucleated cornified cells is 20:2:1;in dioestrus,the ratio of leukocytes,nucleated epithelial cells and enucleated cornified cells is 10:2:1.A cell quantitative standard was used to evaluate the estrus cycle after modeling rats with E2 B administration and continuous illumination.the results showed that the cell quantitative cell quantitative evaluation were consistent with the description of literature results,which verified the accuracy of cell quantitative methodology to evaluate the changes of estrus cycle in rats.3.There was no significant difference between the estrus cycle of rats in TJ1933 and solvent control groups,and 100% mice in the solvent control group and TJ1933 administration group were 100% successful in mating and conception,indicating that TJ1933 had no effect on the reproductive ability of female mice.Conclusion:In this study,a quantitative determination method for estrus cycle of rats has been established for the first time in GLP laboratories in China.This method can accurately and rapidly determine estrus cycle,especially play a more effective role in the judgment of transition period,which is difficult to distinguish.It breaks through the bottleneck of inaccurate and time-consuming results caused by relying only on qualitative methods to identify estrus cycle for many years,improves the level and capability of female reproductive toxicity evaluation technology,further improves the technical system for non-clinical safety evaluation of drugs,and provides an important technical guarantee for the safety evaluation of new drugs.