[關(guān)鍵詞]
[摘要]
目的 從體內(nèi)外水平探究白頭翁皂苷B4(AB4)與5-氟尿嘧啶(5-Fu)聯(lián)合用藥對小鼠4T1乳腺癌細胞的作用及其分子機制。方法 體外分別采用細胞增殖抑制實驗、細胞創(chuàng)傷愈合實驗、Transwell小室侵襲實驗、流式細胞分析以及實時熒光定量PCR法(qRT-PCR)檢測AB4與5-Fu聯(lián)合對4T1細胞功能和凋亡的影響;體內(nèi)借助4T1乳腺癌荷瘤小鼠模型,觀察AB4與5-Fu聯(lián)合對移植瘤生長的影響;通過蘇木精-伊紅(HE)染色法分析聯(lián)合用藥對腫瘤組織形態(tài)學(xué)和微血管密度的影響;流式細胞術(shù)檢測AB4聯(lián)合5-Fu對骨髓源性細胞(BMDCs)動員的影響;最后,通過qRT-PCR和Western blotting技術(shù)分析對腫瘤組織中相關(guān)因子mRNA及蛋白表達的影響。結(jié)果 體外實驗證實AB4聯(lián)合5-Fu顯著降低4T1乳腺癌細胞活力,并且抑制其遷移和侵襲能力,同時可誘導(dǎo)其凋亡(P<0.01);體內(nèi)實驗表明AB4聯(lián)合5-Fu顯著抑制4T1移植瘤的生長(P<0.01),減少腫瘤組織內(nèi)微血管密度以及外周循環(huán)血液中促血管新生BMDCs的細胞計數(shù)(P<0.01),還能顯著上調(diào)腫瘤組織中促凋亡因子的mRNA水平及降低抗凋亡因子的mRNA水平(P<0.01),同時抑制腫瘤血管新生相關(guān)因子蛋白的表達(P<0.01)。結(jié)論 AB4與5-Fu聯(lián)合使用可以協(xié)同抑制4T1腫瘤細胞的生長。機制可能與其對腫瘤細胞功能的直接抑制作用有關(guān),同時也與其對BMDCs動員的抑制相關(guān),這間接減少了腫瘤血管新生。
[Key word]
[Abstract]
Objective To examine the combined impact of Anemoside B4 (AB4) and 5-fluorouracil (5-Fu) on murine 4T1 breast cancer cells, both in vitro and in vivo, while also investigating the underlying molecular mechanisms. Methods A series of experiments, including cell proliferation, wound healing, matrigel invasion assays, and flow cytometry, were conducted to assess how the combination of AB 4 and 5-Fu influences 4T1 cell function and apoptosis in vitro. In vivo, a tumor model was created by subcutaneously injecting 4T1 cells into BALB/c mice to observe the combined effects of AB4 and 5-Fu on tumor growth. HE staining was used to examine changes in tumor tissue microstructure, while flow cytometry analyzed the impact of AB4 and 5-Fu on the mobilization of bone marrow-derived cells (BMDCs). Additionally, qRT-PCR and Western blotting were employed to measure mRNA levels of apoptosis-related factors and the protein expression of angiogenesis-related factors in tumors. Results The combination of AB 4 and 5-Fu significantly reduced the viability and the migration and invasion capacity of 4T1 breast cancer cells (P < 0.01). Moreover, the proportion of cells undergoing apoptosis was significantly increased when treated with AB4 and 5-Fu together (P < 0.01). Besides, the combined treatment of AB4 and 5-Fu led to a more significant tumor growth inhibition in 4T1 xenograft mouse model (P < 0.01). The intratumoral microvascular density in 4T1 xenograft was dramatically reduced according to the HE staining results (P < 0.01) and the cell count of BMDCs in peripheral blood of tumor-bearing mice was also reduced (P < 0.01). qRT-PCR analysis shown that AB4 combined with 5-Fu significantly increased the mRNA expression of pro-apoptotic factors and decreased the mRNA levels of anti-apoptotic factors in tumor tissues (P < 0.01), and the protein expression of tumor angiogenesis-related factors was inhibited. Conclusions The combined use of AB4 and 5-Fu effectively inhibited the growth of 4T1 tumor cells in a synergistic manner. This effect is likely due to both the direct inhibition of tumor cell function and the suppression of BMDC mobilization, which in turn reduces tumor angiogenesis indirectly.
[中圖分類號]
R285.5
[基金項目]
江蘇醫(yī)藥職業(yè)學(xué)院自然科學(xué)基金研究一般項目(20214107);江蘇高校哲學(xué)社會科學(xué)研究一般項目資助(2022SJYB2075);江蘇省鹽城市衛(wèi)生健康委員會2023年度醫(yī)學(xué)科研立項項目(YK2023032,YK2023097)