[關鍵詞]
[摘要]
目的 探究琥珀散對人經(jīng)血源間充質(zhì)干細胞(MenSCs)植入式子宮內(nèi)膜異位癥(EMT)裸鼠的異位病灶及 Bcl-2 同源結構域蛋白抗體(Beclin1)、鋅指E盒同源結合蛋白1(ZEB1)表達的影響。方法 將裸鼠隨機分為對照組、模型組、地諾孕素(0.001 g·kg-1)組和琥珀散(29 g·kg-1)組,每組 10 只。從 EMT 患者及健康女性的月經(jīng)血中提取 MenSCs,并借助成脂、成骨誘導分化鑒定其干細胞屬性。將EMT MenSCs注射至裸鼠腹部皮下,制備EMT裸鼠模型,對照組植入正常人MenSCs。至第7天每組隨機抽取1只裸鼠,開腹觀察異位病灶生長情況,并借助HE染色和人類白細胞抗原A(HLAA)免疫熒光法評價模型。自造模成功連續(xù)給藥21 d,末次給藥后第2日取材,觀測EMT異位病灶體積及血管分布;切取異位病灶組織,應用實時熒光定量PCR(qRT-PCR)、Western blotting、免疫組織化學及免疫熒光技術分別測定組織中Beclin1與ZEB1在mRNA及蛋白水平的表達。結果 EMT源及正常MenSCs為長梭形,呈輻射狀向周邊集落擴散,且經(jīng)成脂、成骨誘導分化后均有脂滴、鈣結節(jié)形成。造模7 d后,對照組未見異位病灶形成,其余各組均有囊泡樣病灶形成,且周圍血管形成豐富,病理切片均可見子宮內(nèi)膜腺體和間質(zhì),HLAA 均陽性表達。與模型組比較,琥珀散能顯著縮小EMT裸鼠異位病灶體積(P<0.001),改善周圍血管分布。在 mRNA水平, 與模型組比較,地諾孕素組和琥珀散組ZEB1表達顯著降低(P<0.001),Beclin1 表達顯著升高(P<0.001)。在蛋白水平,與模型組比較,Western blotting結果顯示,地諾孕素組和琥珀散組ZEB1 表達顯著降低 (P<0.05),Beclin1表達顯著升高(P<0.01、0.001);免疫組化結果顯示,琥珀散組 Beclin1 大量分布在異位內(nèi)膜、腺腔以及腺上皮細胞與間質(zhì)細胞的胞核及胞漿中,Beclin1 表達顯著增加(P<0.01);免疫熒光結果顯示,琥珀散組 ZEB1 蛋白在異位內(nèi)膜、腺腔、腺上皮細胞及周圍間質(zhì)細胞的胞核與胞漿中的分布均明顯減少,地諾孕素組、琥珀散組 ZEB1 蛋白表達陽性率顯著降低(P<0.001)。結論 琥珀散可能通過上調(diào) Beclin1、下調(diào) ZEB1 表達,縮小 EMT 裸鼠異位病灶,改善周圍血管分布。
[Key word]
[Abstract]
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.
[中圖分類號]
R285.5
[基金項目]
國家自然科學基金面上項目(81973895);北京中醫(yī)藥大學重點攻關項目(2020-JYB-ZDGG-143-3)