Abstract:

Kang Yuying*, Sun Caihong, Ju Mei，Chen Kun，Gu Heng. *Department of Dermatovenereology, Shanxi Dayi Hospital, Shanxi Academy of Medical Sciences, Taiyuan 030032, China Corresponding authors: Chen Kun, Email: kunchen181@aliyun.com; Gu Heng, Email: guheng@aliyun.com 【Abstract】 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.