卵巢上皮性癌细胞与腹膜间皮细胞相互作用对卵巢上皮性癌细胞基质金属蛋白酶表达的影响

目的探讨卵巢上皮性癌(卵巢癌)细胞与腹膜间皮细胞(HPMC)相互作用对卵巢癌细胞基质金属蛋白酶(MMP)表达的影响。方法用酶联免疫吸附实验(ELISA)检测卵巢癌细胞SKOV3条件培养液(SKOV3-CM)中的转化生长因子β1(TGF-β1)水平。用RT-PCR技术检测SKOV3-CM对HPMC纤维粘连蛋白(Fn)基因的mRNA表达的影响。以无血清培养液、SKOV3-CM、SKOV3-CM+TGF-β1中和抗体、SKOV3-CM+非特异性IgG刺激HPMC,制备成不同的HPMC-CM,并用不同的HPMC-CM、HPMC-CM1+抗Fn抗体及HPMC-CM1+IgG培育SKOV3细胞,以RT-PCR和ELISA分别检测SKOV3细胞MMP-2、MMP-9基因的mRNA及蛋白表达。结果SKOV3-CM中TGF-β1水平为(236±22)ng/L。SKOV3-CM刺激HPMC后,HPMCFn基因的mRNA表达量为2·643±0·051,高于SKOV3-CM刺激前的水平(1·328±0·025),两者比较,差异有统计学意义(P<0·05);SKOV3-CM+TGF-β1中和抗体刺激HPMC后,HPMCFn基因的mRNA表达量为1·897±0·035,低于SKOV3-CM刺激者,两者比较,差异有统计学意义(P<0·01)。用无血清培养液培育的SKOV3细胞检测不到MMP-2基因的mRNA及蛋白表达。HPMC-CM1培育SKOV3细胞后,MMP-2、MMP-9基因的mRNA表达量分别为0·226±0·012、2·66±0·07,蛋白表达量分别为(15·0±0·8)、(37·2±3·5)μg/L,均高于无血清培养液培育者,两者分别比较,差异有统计学意义(P<0·01)。HPMC-CM1+抗Fn抗体培育SKOV3细胞后,MMP-2、MMP-9基因的mRNA表达量分别为0·138±0·007、1·82±0·06,蛋白表达量分别为(8·8±0·7)、(25·8±2·5)μg/L,均低于HPMC-CM1培育者,两者分别比较,差异有统计学意义(P<0·01)。HPMC-CM2培育SKOV3细胞后,MMP-2、MMP-9基因的mRNA表达量分别为0·467±0·018、4·28±0·09,蛋白表达量分别为(39·3±3·6)、(62·0±5·3)μg/L,均高于HPMC-CM1培育者,两者分别比较,差异有统计学意义(P<0·01)。HPMC-CM3培育SKOV3细胞后,MMP-2、MMP-9基因的mRNA表达量分别为0·331±0·015、3·52±0·08,蛋白表达量分别为(27·6±1·9)、(50·0±4·1)μg/L,均低于HPMC-CM2培育者,两者分别比较,差异有统计学意义(P<0·05),但仍高于HPMC-CM1培育者,两者分别比较,差异有统计学意义(P<0·05)。结论卵巢癌细胞分泌TGF-β1可活化HPMC,上调HPMCFn表达。HPMC来源的Fn可促进卵巢癌细胞MMP-2及MMP-9基因的mRNA和蛋白表达。

Interactions of ovarian carcinoma cells and human peritoneal mesothelial cells involved in matrix metalloproteinases expressions of ovarian carcinoma cells

OBJECTIVE: To investigate the interactions of ovarian carcinoma cells and human peritoneal mesothelial cells (HPMC) involved in matrix metalloproteinases (MMP) expressions of ovarian carcinoma cells. METHODS: The conditioned medium (CM) of ovarian carcinoma cell SKOV3 was tested by enzyme-linked immunosorbent assay (ELISA) for transforming growth factor beta1 (TGF-beta1). The impact of SKOV3-CM in the presence or absence of TGF-beta1 neutralizing antibody on fibronectin (Fn) gene expression of HPMC was studied by RT-PCR. HPMC were pretreated with serum-free medium, SKOV3-CM, SKOV3-CM + TGF-beta1 neutralizing antibody, and SKOV3-CM + IgG, then the supernatant was collected as HPMC-CM(1), HPMC-CM(2), HPMC-CM(3) and HPMC-CM(4). SKOV3 were incubated with different HPMC-CM, HPMC-CM(1) + antibody against Fn or HPMC-CM(1) + IgG. MMP-2 and MMP-9 gene mRNA expressions and protein expressions of SKOV3 were detected by RT-PCR and ELISA respectively. RESULTS: TGF-beta1 in SKOV3-CM was (236 +/- 22) ng/L. Fn gene mRNA expressions of HPMC before and after stimulation by SKOV3-CM were 1.328 +/- 0.025 and 2.643 +/- 0.051, and the latter was higher than the former (P < 0.05). Fn gene mRNA expressions of HPMC stimulated by SKOV3-CM + TGF-beta1 neutralizing antibody was 1.897 +/- 0.035, which was less than that of SKOV3-CM group (P < 0.01). Before SKOV3 were incubated with HPMC-CM, MMP-9 protein and mRNA expressions were (14.5 +/- 1.6) microg/L and 1.50 +/- 0.04, but protein and mRNA expressions of MMP-2 were scarcely detected. When SKOV3 were incubated with HPMC-CM(1), mRNA expressions of MMP-2 and MMP-9 were 0.226 +/- 0.012 and 2.66 +/- 0.07, protein expressions were (15.0 +/- 0.8) and (37.2 +/- 3.5) microg/L, which were all higher than those of SKOV3 without treatment of HPMC-CM (P < 0.01). Following incubation of SKOV3 with HPMC-CM(1) + antibody against Fn, mRNA expressions of MMP-2 and MMP-9 were 0.138 +/- 0.007 and 1.82 +/- 0.06, protein expressions were (8.8 +/- 0.7) and (25.8 +/- 2.5) microg/L, which decreased compared with those of HPMC-CM(1) group (P < 0.01). Following incubation of SKOV3 with HPMC-CM(2), mRNA expressions of MMP-2 and MMP-9 were 0.467 +/- 0.018 and 4.28 +/- 0.09, protein expressions were (39.3 +/- 3.6) and (62.0 +/- 5.3) microg/L, which were higher than those of HPMC-CM(1) group (P < 0.01). Following incubation of SKOV3 with HPMC-CM(3), gene expressions of MMP-2 and MMP-9 were 0.331 +/- 0.015 and 3.52 +/- 0.08, protein expressions were (27.6 +/- 1.9) and (50.0 +/- 4.1) microg/L, which decreased compared with HPMC-CM(2) group (P < 0.05), but were still higher than those of HPMC-CM(1) group (P < 0.05). CONCLUSIONS: Ovarian carcinoma cells activate HPMC through TGF-beta1, which induces higher expressions of MMP of ovarian carcinoma cells. Up-regulating Fn expression of HPMC may be one of action mechanisms of ovarian tumor cells. Fn derived from HPMC stimulates MMP-2 and MMP-9 expressions of ovarian carcinoma cells at gene and protein levels.