双极雄激素治疗恩杂鲁胺抵抗前列腺癌模型的建立

An animal model of bipolar androgen treatment for enzalutamide-resistant prostate cancer

目的:探讨双极雄激素治疗(BAT)治疗的机制和探索未来联合免疫治疗的可能性。方法:以庚酸睾酮为实验药物,RM1细胞荷瘤后进行阉割和恩杂鲁胺治疗后进展的雄性C57小鼠为恩杂鲁胺抵抗的去势抵抗性前列腺癌(CRPC)动物模型,通过设立阴性对照组(对照组、去势+恩杂鲁胺组)、空白对照组(空白注射组、空植入体组)和实验组(雄激素注射组、雄激素植入体组)观察小鼠存活情况并应用酶联免疫吸附实验(ELISA)检测小鼠血清中的雄激素含量来确认安全性和释放效率。以腹腔注射法和植入体法为BAT给药方式,治疗荷瘤恩杂鲁胺抵抗小鼠模型,检测体内肿瘤的大小和重量,蛋白质印迹法(Western blot)检测肿瘤组织磷酸化组蛋白(γH2AX)的变化,流式细胞仪和免疫组织化学法检测组织中CD3+T细胞变化,GraphPad软件进行统计分析,两组间比较采用t检验,多组间比较采用单因素方差分析,多组间两两比较用LSD-t检验。结果:注射法和植入体法均是有效的给药方式,并且能够显著提高血液中雄激素水平[(31.97±2.90)、(20.70±0.92)ng/ml比(1.14±0.43)、(1.21±0.34)ng/ml,F=149.653、40.399,P<0.01]和降低小鼠荷瘤大小[(907.60±55.95)、(894.71±100.65)mm 3比(1685.82±12.37)、(1642.83±8.30)mm 3,F=3.744、2.938,P<0.01],差异有统计学意义,相较于植入体法,注射法高浓度雄激素维持时间短但降低肿瘤大小效果好,能引起更多的DNA损伤(F=14.855,P<0.01),且能够部分提高CD3+T细胞的浸润(F=9.782,P<0.01),差异有统计学意义,但安全程度欠佳。结论:植入体法与注射法给予BAT均有效,注射法效果稍好于植入体法,注射法DNA损伤程度更大且造成更多的免疫细胞浸润,未来联合免疫治疗可能具有更好的效果。

Objective Bipolar androgen therapy(BAT)can improve the prognosis of enzalutamide-resistant castration-resistant prostate cancer(CRPC)patients and its mechanism has not been elucidated.In order to explore the mechanism of BAT treatment and the possibility of combined immunotherapy in the future.Methods Using testosterone enanthate as the treatment drug,the male C57 mice castrated after bearing tumor cell line were treated with enzalutamide,and enzalutamide-resistant CRPC animal models were established.Two negative control groups(control,Castration+enzalutamide),two blank groups(blank injection,blank implant)and two experimental groups(androgen injection,androgen implant)was set up.The survival of model was observed,and enzyme linked immunosorbent assay(ELISA)was used to determine serum testosterone content.Testosterone injection and testosterone implant were used as BAT to treat enzalutamide-resistant models.The size and quality of tumors off the body were measured.TheγH2AX protein was detected by Western blotting.Flow cytometry and immunohistochemistry were used to detect CD3+T cells of the tumor tissue.GraphPad software was used for statistical analysis.Results Both the injection and the implant were effective methods of releasing testosterone,which could significantly increase blood androgen levels[(31.97±2.90),(20.70±0.92)ng/ml vs.(1.14±0.43),(1.21±0.34)ng/ml,F=149.653,40.399,P<0.05]and reduce the size of tumor[(907.60±55.95),(894.71±100.65)mm3 vs.(1685.82±12.37),(1642.83±8.30)mm3,F=3.744,2.938,P<0.01].As compared with implant method,injection method had increased concentration and testosterone level in a short time.It could cause more DNA damage(F=14.855,P<0.01),and partially increased the infiltration of CD3+T cells(F=9.782,P<0.01),but the level of security was poor.Conclusion Both the implantation method and the injection method are effective in BAT.The injection method has a slightly better effect than the implant method.The injection method has a greater degree of DNA damage and causes more immune cell infiltration,suggesting combined immunotherapy with BAT may have better effect in the future.