琥珀散对人MenSCs植入式子宫内膜异位症裸鼠异位病灶及Beclin1、ZEB1表达的影响

Effects of Hupo Powder on ectopic lesions,Beclin1 and ZEB1 expression in endometriotic nude mice implanted with human MenSCs

目的探究琥珀散对人经血源间充质干细胞(MenSCs)植入式子宫内膜异位症(EMT)裸鼠的异位病灶及Bcl-2同源结构域蛋白抗体(Beclin1)、锌指E盒同源结合蛋白1(ZEB1)表达的影响。方法将裸鼠随机分为对照组、模型组、地诺孕素(0.001 g·kg^(-1))组和琥珀散(29 g·kg^(-1))组,每组10只。从EMT患者及健康女性的月经血中提取MenSCs,并借助成脂、成骨诱导分化鉴定其干细胞属性。将EMT MenSCs注射至裸鼠腹部皮下,制备EMT裸鼠模型,对照组植入正常人MenSCs。至第7天每组随机抽取1只裸鼠,开腹观察异位病灶生长情况,并借助HE染色和人类白细胞抗原A(HLAA)免疫荧光法评价模型。自造模成功连续给药21 d,末次给药后第2日取材,观测EMT异位病灶体积及血管分布;切取异位病灶组织,应用实时荧光定量PCR(qRT-PCR)、Western blotting、免疫组织化学及免疫荧光技术分别测定组织中Beclin1与ZEB1在mRNA及蛋白水平的表达。结果EMT源及正常MenSCs为长梭形,呈辐射状向周边集落扩散,且经成脂、成骨诱导分化后均有脂滴、钙结节形成。造模7 d后,对照组未见异位病灶形成,其余各组均有囊泡样病灶形成,且周围血管形成丰富,病理切片均可见子宫内膜腺体和间质,HLAA均阳性表达。与模型组比较,琥珀散能显著缩小EMT裸鼠异位病灶体积(P<0.001),改善周围血管分布。在mRNA水平,与模型组比较,地诺孕素组和琥珀散组ZEB1表达显著降低(P<0.001),Beclin1表达显著升高(P<0.001)。在蛋白水平,与模型组比较,Westernblotting结果显示,地诺孕素组和琥珀散组ZEB1表达显著降低(P<0.05),Beclin1表达显著升高(P<0.01、0.001);免疫组化结果显示,琥珀散组Beclin1大量分布在异位内膜、腺腔以及腺上皮细胞与间质细胞的胞核及胞浆中,Beclin1表达显著增加(P<0.01);免疫荧光结果显示,琥珀散组ZEB1蛋白在异位内膜、腺腔、腺上皮细胞及周围间质细胞的胞核与胞浆中的分布均明显减少,地诺孕素组、琥珀散组ZEB1蛋白表达阳性率显著降低(P<0.001)。结论琥珀散可能通过上调Beclin1、下调ZEB1表达,缩小EMT裸鼠异位病灶,改善周围血管分布。

Objective To explore the influence of Hupo Powder on the ectopic lesions and the expression of Beclin1 and ZEB1 in EMT nude mice implanted with human menstrual blood stromal cells(MenSCs).Methods Nude mice were randomly divided into control group,model group,dienogest(0.001 g·kg^(-1))group and Hupo Powder(29 g·kg^(-1))group,10 mice in each group.MenSCs were extracted from menstrual blood of EMT patients and healthy women,respectively,and identified by adipogenic and osteogenic differentiation.EMT MenSCs were sc into the abdomen of nude mice to prepare EMT nude mice model,and the control group was implanted with healthy MenSCs.On the 7th day,one nude mouse was randomly selected from each group,and the growth of ectopic lesions was observed.We then evaluated model by HE staining and human leukocyte antigen A(HLAA)immunofluorescence.Each group was administered continuously for 21 days since model had been prepared,and the ectopic lesions were collected on the 2nd day after the last administration.We observed the volume and blood vessel distribution of EMT ectopic lesions in each group.Beclin1 and ZEB1 mRNA and protein levels were detected by real-time quantitative PCR(qRT-PCR),Western blotting,immunohistochemistry and immunofluorescence.Results EMT-derived and normal MenSCs were long-fusiform,radially spreading to the surrounding colonies,and lipid droplets and calcium nodules were formed.After seven days of modeling,no ectopic lesion was found in the control group,while the other groups had vesicle-like lesions and abundant peripheral blood vessels.Endometrial glands and stroma were found in pathological sections,and HLAA was positively expressed.Compared with the model group,Hupo Powder could significantly reduce the volume of ectopic lesions in EMT nude mice(P<0.001)and improve the distribution of peripheral blood vessels.At the mRNA level,the expression of ZEB1 was decreased in the dienogest group and the Hupo Powder group(P<0.001),while the expression of Beclin1 was increased(P<0.001).At the protein level,compared with model group,Western blotting results showed that the expression of ZEB1 in dinogestrel group and Hupo Powder group was significantly decreased(P<0.05),while Beclin1 expression was significantly increased(P<0.01,0.001).Immunohistochemical results showed that Beclin1 in Hupo Powder group was distributed in ectopic intima,glandular cavity,nucleus and cytoplasm of glandular epithelial cells and stromal cells,and Beclin1 expression was significantly increased(P<0.01).Immunofluorescence results showed that ZEB1 protein distribution in ectopic endometrium,glandular cavity,glandular epithelial cells and the cytoplasm of surrounding stromal cells was significantly decreased in the Hupo Powder group,and the positive rate of ZEB1 protein expression was significantly decreased in the dinorgestrel and Hupo Powder groups(P<0.001).Conclusion Hupo Powder may reduce the ectopic lesions of EMT nude mice and improve the distribution of peripheral blood vessels by up-regulating Beclin1 and down-regulating the expression of ZEB1.