Schäffer M R, Efron P A, Thornton F J, Klingel K, Gross S S, Barbul A
Department of Surgery, Sinai Hospital, Johns Hopkins Medical Institutions, Baltimore, MD 21215, USA.
J Immunol. 1997 Mar 1;158(5):2375-81.
Nitric oxide (NO) is synthesized in wounds, but its exact role and cellular source are not known. Wound fibroblasts (WF) are phenotypically characterized by increased collagen synthesis and contractility. We hypothesized that WF may be also phenotypically altered during wound healing to synthesize NO. WF were isolated from polyvinyl alcohol sponges implanted in male Lewis rats and harvested 10 days later. Proliferation in response to 10% fetal bovine serum was assessed by [3H]thymidine incorporation in a microculture system. A fibroblast-populated collagen lattice was used for assaying contractility. Collagen synthesis was determined by measuring the collagenase-sensitive fraction of protein-incorporated [3H]proline. Fibroblasts were incubated in the presence or the absence of 0.5 mM S-methyl-isothio-uronium or 0.5 mM N-monomethyl-L-arginine, both competitive inhibitors of NO synthase. WF spontaneously synthesize and release NO (4.60 +/- 0.29 nmol nitrite/microg DNA/48 h). Normal dermal fibroblasts do not synthesize NO. WF NO synthesis was limited to the first and second passages postharvest and was inhibitable by S-methyl-isothio-uronium (96%) and N-monomethyl-L-arginine (84%). In vivo iNOS expression by WF was confirmed by in situ hybridization and immunohistochemistry. Inhibition of endogenous NO synthesis had no effect on fibroblast proliferation. However, fibroblast-mediated collagen contraction was enhanced (p < 0.01), and collagen synthesis was significantly decreased (p < 0.05) by inhibiting NO synthase. The data show that WF are phenotypically altered during the healing process to synthesize NO, which, in turn, regulates their collagen synthetic and contractile activities.
一氧化氮（NO）在伤口处合成，但其确切作用和细胞来源尚不清楚。伤口成纤维细胞（WF）的表型特征是胶原蛋白合成增加和收缩能力增强。我们推测，在伤口愈合过程中，WF的表型可能也会发生改变以合成NO。从植入雄性Lewis大鼠体内的聚乙烯醇海绵中分离出WF，并在10天后收获。在微培养系统中通过[3H]胸腺嘧啶核苷掺入法评估对10%胎牛血清的增殖反应。使用成纤维细胞填充的胶原晶格来测定收缩能力。通过测量掺入蛋白质的[3H]脯氨酸的胶原酶敏感部分来确定胶原蛋白合成。将成纤维细胞在存在或不存在0.5 mM S-甲基异硫脲或0.5 mM N-单甲基-L-精氨酸（两者均为NO合酶的竞争性抑制剂）的情况下孵育。WF可自发合成并释放NO（4.60±0.29 nmol亚硝酸盐/μg DNA/48小时）。正常真皮成纤维细胞不合成NO。WF的NO合成仅限于收获后的第一代和第二代传代，并且可被S-甲基异硫脲（96%）和N-单甲基-L-精氨酸（84%）抑制。通过原位杂交和免疫组织化学证实了WF在体内的诱导型一氧化氮合酶（iNOS）表达。抑制内源性NO合成对成纤维细胞增殖没有影响。然而，通过抑制NO合酶，成纤维细胞介导的胶原收缩增强（p<0.01），胶原蛋白合成显著减少（p<0.05）。数据表明，在愈合过程中WF的表型发生改变以合成NO，而NO反过来调节它们的胶原蛋白合成和收缩活动。
