Study On The Establishment Of Breast Cancer Model By Intraductal Administration Of MNU And Drug Intervention

Part one: study on the occurrence and biological behavior of breast cancer induced by intraductal injection of MNU in ratsBackground: N-methyl-N-nitrosourea(MNU)is used as a strong carcinogen to establish a chemically induced breast cancer model of rat.In the past few decades,conventional methods of drug administration through intraperitoneal and caudal vein injection of MNU,easy to operate,but it has some shortcomings in practical research:breast tumors randomly appear in 6 pairs of mammary glands in rats and the numbers of tumor are variable.Intraductal administration delivers MNU through the natural ducts of the breast for local accurate drug administration,so there is gap for improvement in the construction of the model.If MNU is injected into rat mammary gland through mammary duct,we can theoretically construct an improved rat breast cancer animal model.Methods: In experiment one,12 female SD rats with 4-week age were randomly divided into two groups.The experimental group was given a single intraductal injection of MNU(the same dose of injected intraperitoneally,50mg/kg),and the control group was injected with the solvent of MNU working solution.After the successful establishment of the model,30 female SD rats with 4-week age were randomly divided into 5 groups: solvent control group,MNU gradient dose group(0.5mg 1.0 and 2.0 mg)and intraperitoneal injection group.The latent period of tumor formation,in situ tumor formation and tumor growth rate of three groups of gradient dose groups were compared comprehensively,and the appropriate dose of intraductal injection of MNU was selected.At the same time,the tumor formation rate,histology and immunohistochemical expression of ER,HER2 and Ki-67 between i.p and i.duc the group were compared.The distant organ metastasis was evaluated at the end of the experiment.Results: In experiment one,all the tumors occurred after intraductal injection of MNU,and the fourth pair of glands outside the breast developed tumors at the same time.In experiment 2,the average latency in the gradient dose group(0.5 2.0mg 1.0and 0.5mg)was 55.3,43.7 and 44.2 days,respectively.The body weight of the control group was higher than that of the three gradient dose groups.In the gradient dose group,the tumor volume of 0.5mg group increased faster than that of the 0.5mg group.According to the one-way ANOVA,the growth of tumor volume in 1.0mg and 2.0mg groups was faster than that in 0.5mg group,and the difference was statistically significant.There was no significant difference in latency and body weight among the three groups(p>0.05).There was no difference in tumor formation rate between gradient dose group and intraperitoneal injection of MNU group.However,based on the number of mammary glands exposed by carcinogenic factors,the tumor formation rate of glands increased significantly,and the difference was statistically significant(p<0.01).The results of histological types showed that the tumors induced by the gradient dose group were malignant,mainly papillary carcinoma and ethmoid carcinoma,and benign tumors such as intraductal papilloma and fibroadenoma appeared in the intraperitoneal injection group.Immunohistochemical staining showed that ER expression was positive in both intraductal injection of MNU 1.0 mg and intraperitoneal injection of MNU.The expression of HER2 was negative and Ki-67 was low in both groups.No organ metastasis was found in all rats.Conclusion: It is feasible to establish rat model of breast cancer by intraductal injection of MNU.Compared with intraperitoneal injection of MNU,intraductal injection of 1.0mg MNU at a smaller dose can induce rat breast tumors with the same tumor formation rate and fixed location.The pathological types of tumors are mainly papillary carcinoma and cribriform carcinoma,and the proportion of malignant tumors is higher.The proportion of special types of breast cancer is larger,indicating a better prognosis.Part two: Study on the time point and effect of early intervention with intraductal administration of albumin-bound paclitaxelBackground: In the part one,we studied the feasibility of constructing a model by intraductal injection of MNU,but the responsiveness of this improved model to anti-tumor drugs has not yet been verified.Albumin-bound paclitaxel is an improved preparation of solvent-based paclitaxel,which enhances the anti-tumor effect while avoiding life-threatening allergic reactions.The current clinical studies on albumin paclitaxel in the treatment of ER-positive early breast cancer show that the therapeutic effect is also significant.However,due to the increase in the effective concentration of paclitaxel,grade 3-4 adverse reactions still occur frequently.Therefore,intraductal use of albumin paclitaxel is expected to achieve the therapeutic effect while reducing the dose and reducing systemic adverse reactions.Methods: In experiment one,15 female SD rats with 4-week age were randomly divided into 5 groups,and MNU 1.0 mg was injected into the fourth pair of mammary ducts with a single injection.From the 3-7 weeks after the injection,the rats were dissected at a fixed time every week.The mammary glands of a group of rats were sectioned to observe the ductal epithelial hyperplasia to the state of malignant transformation.After determining the onset time of ductal epithelial cell malignant transformation,in experiment two,18 female SD rats with 4-week-old age were randomly divided into 3 groups,and a single injection of MNU 1.0 mg into the fourth pair of mammary ducts was performed.Carry out the tail vein and intraductal intervention of albumin-bound paclitaxel when the early malignant transformation appears,and compare the effects of different administration methods on tumor growth rate,weight change,overall survival,histological morphology,cell proliferation,and apoptosis-related indicators.Observe the changes of mental state,blood routine and biochemical indexes of rats after administration.The end point of the experiment is to evaluate the metastasis of distant organs.Results: The rat mammary glands obtained every week in the experiment one were stained by HE staining and immunohistochemistry(α-SMA and Calponin).It was determined that the rat mammary glands could form an early duct in situ at 5-6weeks after intraductal injection of MNU.cancer.In the drug intervention experiment,the tail vein or intraductal administration of the breast was performed on the 40 th day.Compared with the control group and the tail vein injection group,the fluorescent TUNEL and Caspase-3 immunofluorescence staining results showed that the fluorescence intensity was higher in the intraductal albumin paclitaxel injection group.High,the difference is statistically significant(p<0.01).Western blot results of Cleaved Caspase-3,Bcl-2 and Bax showed a higher apoptosis rate,and the percentage of expression of Ki-67,a tumor proliferation index,significantly decreased,and the difference was statistically significant(p<0.05).Within one week after the administration of albumin-bound paclitaxel,the intravenous group had varying degrees of hair loss and decreased activity,but this phenomenon did not occur in the remaining 2 groups.No distant organ metastasis was seen in the three groups of rats.Conclusion: In the rat breast cancer model induced by intraductal injection of MNU1.0mg,the start of ductal epithelial cell malignancy is 5-6 weeks after intervention.Early chemical intervention in the MNU-induced rat breast cancer model can achieve better prevention Compared with intravenous administration of paclitaxel,intra-catheter administration of albumin paclitaxel has better curative effect and has fewer side effects.