摘要
目的：探讨抗菌肽hCAP18／LL-37在卵巢癌微环境中的作用及表达调控机制。方法采用侵袭实验检测巨噬细胞对卵巢癌细胞SKOV3侵袭能力的影响。采用实时荧光定量PCR（ qRT-PCR）和Western blot检测hCAP18／LL-37和versican V1蛋白的表达。采用干扰质粒抑制 SKOV3细胞中versican V1的表达，分析巨噬细胞hCAP18／LL-37的表达及SKOV3细胞的侵袭能力。结果共培养组（SKOV3细胞与巨噬细胞共培养）的侵袭穿膜数为（112．8±17．1）个，高于SKOV3培养组[SKOV3细胞单独培养，（8．2±1．9）个]，差异有统计学意义（P＜0．05）。 hCAP18／LL-37中和抗体共培养组（SKOV3细胞、巨噬细胞和hCAP18／LL-37中和抗体共培养）的侵袭穿膜细胞数为（22．2±5．6）个，少于对照IgG共培养组[SKOV3细胞、巨噬细胞和对照IgG共培养，（100．6±25．2）个]，差异有统计学意义（P＜0．05）。与SKOV3细胞共培养后，巨噬细胞中 hCAP18／LL-37蛋白和 mRNA水平均升高，而在SKOV3细胞中无变化。与巨噬细胞共培养后，卵巢癌细胞中versican V1蛋白的表达和分泌均升高。干扰卵巢癌SKOV3细胞中versican V1的表达（ SKOV3ver－／－细胞）后，巨噬细胞中hCAP18／LL-37蛋白和mRNA水平均低于对照细胞株（ SKOV3ver＋／＋细胞）；与巨噬细胞共培养后，SKOV3ver－／－细胞的穿膜细胞数[（24．8±4．6）个]低于SKOV3ver＋／＋细胞[（104．6±16．0）个]，差异有统计学意义（P＜0．05）。结论卵巢癌微环境中，巨噬细胞表达分泌的抗菌肽hCAP18／LL-37促进卵巢癌细胞的侵袭，其表达受到肿瘤细胞分泌的versicanV1蛋白调控。
Objective To investigate the effect of antibacterial peptide hCAP18/LL-37 on ovarian cancer microenvironment and the regulatory mechanism of its expression. Methods We assessed the effect of macrophage-promoted ovarian cancer cells invasion using BioCoat Matrigel invasion chamber. The expressions of hCAP18/LL-37 and versican V1 were determined by real-time PCR and Western blot analysis. SKOV3 cells were transfected with shRNA plasmid to abrogate the expression of versican V1, and then the expression of hCAP18/LL-37 in macrophages and the invasiveness of SKOV3 cells were assayed. Results The Matrigel invasion assay showed that after co-culture with macrophages for 4 days,the number of penetrated SKOV3 cells was 112. 8 ± 17. 1/per high power field, significantly higher than that in the SKOV3 cells cultured alone （8.2±1.9/per high power field） （P＆lt;0.05）. Addition of hCAP/LL-37 neutralizing antibody into the co-cultured macrophage-SKOV3 cells markedly inhibited the macrophage-promoted SKOV3 cells invasion. The penetrated SKOV3 cells was 22.2±5.6/per high power field, significantly lower than the 100.6± 25.2/per high power field in the control macrophage-SKOV3 co-cultured cells （P〈0.05）. The expressions of hCAP18/LL-37 mRNA and protein in macrophages were remarkably enhanced upon co-culture with SKOV3 cells, but not changed in SKOV3 cells cultured alone. The expression and secretion of versican V1 in the ovarian cancer cells were also significantly increased after co-cultured with macrophages. Knockdown of versican V1 in SKOV3 cells by small interfering RNA significantly reduced the expression of hCAP18/LL-37 〈br〉 mRNA and protein in the macrophages, as well as decreased the invasiveness of SKOV3 cells （P〈0.05）. Conclusions In the cancer microenvironment, the macrophage secreted hCAP18/LL-37 promote the invasiveness of ovarian cancer cells, and the hCAP18/LL-37 expression is regulated by versican V1 protein released by ovarian cancer cells.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2015年第10期725-730,共6页
Chinese Journal of Oncology
基金
国家自然科学基金（81272603、81472179）
上海市浦江人才计划（13PJ1407300）
上海申康医院发展中心课题（SHDC22014008）
教育部留学归国人员科研启动基金
