外周血单一核细胞的体外活化对成纤维细胞增殖和基质金属蛋白酶产生的影响

In vitro activation of peripheral blood mononuclear cells and its effects on the proliferation of and production of matrix metalloproteinases by cultured human fibroblasts

目的 研究不同活化剂对外周血单一核细胞（PBMC）产生基质金属蛋白酶（MMP）的影响以及PBMC活化后培养上清液［称为条件培养基（CM）］对培养的皮肤成纤维细胞增殖活性、产生MMP的影响。方法 抽取、分离健康人PBMC，分为植物血凝素（PHA）组、双抗体组、对照组，分别用活化剂PHA、CD3和CD28双抗体、含10%胎牛血清的RPMI 1640培养基处理细胞，72 h后收集三组CM，并将获得的CM按不同比例稀释后作用于培养的成纤维细胞，以不加CM作为对照组，培养48或24 h。采用噻唑蓝法检测细胞增殖能力，半定量反转录（RT）-PCR法检测细胞MMP-1、MMP-3、MMP-9 mRNA表达水平，酶联免疫吸附实验（ELISA）检测上清液中白介素（IL）-6、MMP-1、MMP-3、MMP-9蛋白含量。采用单因素方差分析、Tukey HSD检验、Games-Howell检验等方法进行统计学分析。 结果 与对照组相比，PHA组PBMC增殖活性及分泌IL-6、MMP-3水平明显增加，差异均有统计学意义（P 〈 0.05）；3组活化后上清液中均未检测到MMP-1、MMP-9蛋白。对照组、双抗体组、PHA组PBMC中MMP-1 mRNA相对表达水平（以靶基因与β肌动蛋白mRNA之比表示）0.083 ± 0.016、0.188 ± 0.030、0.714 ± 0.104，差异有统计学意义（P 〈 0.05），MMP-3、MMP-9 mRNA均无表达。不同稀释度CM刺激成纤维细胞后上清液中可检测到MMP-3蛋白，MMP-3含量以原液CM中最高，1/10 CM刺激时最低；在相同的稀释度刺激时，上清液中MMP-3含量以PHA刺激组最高，对照刺激组最低；不同CM刺激成纤维细胞后上清液中均未检测到MMP-1、MMP-9蛋白。不同处理组成纤维细胞的增殖能力及MMP-1、MMP-3 mRNA表达差异均无统计学意义（P 〉 0.05），MMP-9 mRNA无表达。 结论 PBMC活化后可表达MMP-1 mRNA并分泌MMP-3蛋白，PBMC活化后上清液不能刺激成纤维细胞MMP-1、MMP-3、MMP-9 mRNA及蛋白表达，提示炎症细胞可能通过自身产生MMP而发挥作用。

Objective To study the effects of different stimulators on the production of matrix metalloproteinases （MMPs） by peripheral blood mononuclear cells （PBMCs）, and to evaluate the effects of the culture supernatant of activated PBMCs , named conditioned media （CM）, on the proliferation of and production of MMPs by cultured human fibroblasts. Methods PBMCs were isolated from the venous blood samples of healthy volunteers and divided into three groups to be stimulated by phytohemagglutinin （PHA group）, the combination of antibodies against CD3 and CD28 （double-antibody group）, or the RPMI 1640 medium containing 10% fetal calf serum （control group）. After 72-hour stimulation, CM was collected from all the three groups, diluted to several different degrees. Cultured human fibroblasts were classified into several groups to be treated with different dilutions of CM from the three groups for 48 or 24 hours, with the fibroblasts untreated with CM serving as the control group. Methyl thiazolyl tetrazolium （MTT） assay was performed to evaluate cellular proliferative activity, semi-quantitative reverse transcription （RT）-PCR to detect the expressions of MMP-1, MMP-3 and MMP-9 mRNAs in cells, and enzyme-linked immunosorbent assay （ELISA） to measure the levels of interleukin （IL）-6, MMP-1, MMP-3 and MMP-9 proteins in the culture supernatant of cells. Statistical analysis was carried out mainly by using one-way analysis of variance （ANOVA）, Tukey HSD test, and Games-Howell test. Results Compared with the control group, the PHA group showed increased cellular proliferative activity, IL-6 and MMP-3 protein levels in the culture supernatant of activated PBMCs （all P 〈 0.05）. Significant differences were observed among the PHA group, double-antibody group and control group in the relative mRNA expression level （expressed as the ratio of target mRNA to β-actin mRNA） of MMP-1 in activated PBMCs（0.083 ± 0.016 vs. 0.188 ± 0.030 vs. 0.714 ± 0.104, F = 85.905, P 〈 0.05）, but neither MMP-3 nor MMP-9 mRNA was expressed by activated PBMCs. MMP-3 protein was detectable in the culture supernatant of fibroblasts after the treatment with CM, and the level of MMP-3 protein was highest in that of fibroblasts treated with undiluted CM, and lowest with 1/10 diluted CM; at the same dilutions, the level of MMP-3 protein was highest in the culture supernatant of fibroblasts treated with CM from the PHA group, but lowest with that from the control group. Neither MMP-1 nor MMP-9 protein was detected in the culture supernatant of activated PBMCs or treated fibroblasts. There were no significant differences in cellular proliferative activity of and mRNA expressions of MMP-1 or MMP-3 in fibroblasts among these groups （all P 〉 0.05）, and MMP-9 mRNA expression was undetected in the treated fibroblasts. Conclusions PBMCs can be induced to express MMP-1 mRNA and secret MMP-3 protein after activation. However, the culture supernatant of activated PBMCs has no capacity to stimulate the expressions of MMP-1, MMP-3 and MMP-9 mRNAs or proteins by fibroblasts, suggesting that inflammatory cells may function through self-production of MMPs.