目的 探讨经超声提取的防风多糖(USPS)进行硫酸酯化修饰后对人乳腺癌MCF-7细胞体外增殖的抑制作用以及对肿瘤相关巨噬细胞募集和驯化的影响。方法 ①采用氯磺酸-吡啶法对USPS进行硫酸酯化修饰得到硫酸酯化USPS(S-USPS)。②采用四甲基偶氮唑蓝(MTT)法测定USPS和S-USPS在不同给药浓度下对MCF-7细胞生长的抑制作用。③体外诱导生成THP-1来源的巨噬细胞,采用Transwell法检测S-USPS作用上清对巨噬细胞募集的影响。④采用qRT-PCR和ELISA法检测S-USPS对MCF-7细胞趋化因子表达和分泌的影响。⑤采用qRT-PCR法检测S-USPS、CCL3对巨噬细胞促炎和抗炎因子表达的影响。结果 ①与USPS相比,S-USPS体外可明显抑制MCF-7细胞的增殖活性(P<0.05),且抑制率呈剂量和时间依赖性。②S-USPS可显著促进MCF-7细胞趋化因子3(CCL3)的转录和分泌。③与空白对照和MCF-7细胞上清组相比,S-USPS可上调巨噬细胞白介素1β(IL-1β)、IL-6、肿瘤坏死因子α(TNF-α)mRNA的表达,同时下调转化生长因子β(TGF-β)、IL-10 mRNA的表达(P<0.05)。④向MCF-7条件培养基中加入CCL3中和抗体,可显著逆转S-USPS对IL-1β、TNF-α表达的促进作用(P<0.05)。结论 硫酸酯化防风多糖可通过作用于CCL3促使MCF-7细胞对巨噬细胞的募集作用,并驯化巨噬细胞向M1型分化。
Abstract
OBJECTIVE To observe the inhibition of saposhnikovan sulphate obtained by ultrasonic extraction(S-USPS) on human leukemia MCF-7 cells in vitro and the effect of S-USPS on the recruitment and re-education of macrophages by MCF-7 cells. METHODS S-USPS was obtained from saposknikovan(SPS) by the method of chlorosulfonic acid-pyridine complex. The inhibition of S-USPS and USPS on proliferation of MCF-7 cells was measured by MTT. THP-1-derived macrophages were differentiated in vitro. The effect of S-USPS on the recruitment of macrophages by MCF-7 cells was detected by transwell migration assay. The effects of S-USPS on the expression and secretion of chemokine were detected by qRT-PCR and ELISA. The effects of S-USPS and CCL3 on the expression of inflammatory cytokines were detected by qRT-PCR. RESULTS The inhibitory rate of S-USPS on the proliferation of MCF-7 was dose-dependent and time-dependent and was higher than that of USPS(P<0.05).S-USPS increased the transcription and secretion of CCL3 by MCF-7 cells. There were higher expressions of IL-1β, IL-6, and TNF-α mRNA and lower expressions of TGF-β, IL-10 mRNA of macrophages in the S-USPS group than in the control group and MCF-7 supernatant group(P<0.05). The promotion of S-USPS on the expressions of IL-1β and TNF-α was inhibited by CCL3 neutralizing antibody(P<0.05). CONCLUSION There are significant evidences that the saposhnikovan sulphate effectively promotes the recruitment of macrophages by MCF-7 cells in vitro, and re-educates their secretion profile toward M1-type.
关键词
防风多糖 /
MCF-7细胞 /
硫酸酯化 /
CCL3 /
巨噬细胞
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Key words
saposhnikovan /
MCF-7 cell /
sulfation /
CCL3 /
macrophage
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中图分类号：
R96
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脚注
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基金
江西省自然科学基金项目资助(S2017ZRMSB1204)
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