Rapala K
Department of Surgery University of Turku, Finland.
Ann Chir Gynaecol Suppl. 1996;211:1-53.
The inflammatory phase in wound healing is considered to be a preparatory process for the formation of new tissue. A monocyte-derived cytokine, tumor necrosis factor-alpha (TNF-alpha), is a highly conserved molecule known to play a major role in the pathogenesis of gram-negative shock. Besides this, previous experimental studies show that TNF-alpha may have either a beneficial or detrimental role in wound healing. The purpose of the present study was to examine the effects of TNF-alpha on developing granulation tissue in rats as well as on rat and human granulation tissue cells in culture. Subcutaneously implanted cylindrical hollow sponges were used for studying the effects of locally applied TNF-alpha on granulation tissue in rats. These implants were treated either on the day of implantation or for the first 4 or 7 days after implantation with a solution containing various amounts of TNF-alpha while the control implants were treated correspondingly with the carrier solution only. The analyses of the granulation tissue were carried out 4, 7, 14 and 21 days after implantation. In the histological specimen these sponges were cut into small pieces and stained with Weigert van Gieson to visualize collagen. The amount of granulation tissue grown into the sponge was calculated from the cross section of every sponge. For the cell culture studies fibroblasts were released from human and rat granulation tissue which was cut into small pieces and digested by collagenase and DNase in Hank's balanced salt solution. The cells were exposed to 1, 10, or 100 ng/ml of TNF-alpha and the rate of collagen synthesis was measured as synthesis of protein-bound 3H-hydroxyproline. The number of cells in the culture dishes was counted with Bürger's hemocytometer after detaching the cells with trypsin treatment. As interleukin-1 (IL-1) and TNF-alpha overlap in many of their functions, the effects of lipopolysaccharide (LPS), human interleukin 1 beta (IL-1) and prostaglandin E2 (PGE2) on experimental granulation tissue in rats as well as on rat granulation tissue cells in culture were studied with the same method. After a single application of TNF-alpha into the sponge, no essential differences between the groups were detected. However, after daily applications of TNF-alpha for 4 days, an inhibitory effect on tissue repair was observed after 4 and 7 days. Collagen formation, indicated by the hydroxyproline content of the sponge, was significantly lower in the group treated with TNF-alpha than in the controls. This effect was not observed after 14 or 21 days. These findings were confirmed in the histological samples. In the cultures of rat granulation tissue fibroblasts TNF-alpha decreased 3H-hydroxyproline production to about 75% of that in the controls and it had also a decreasing effect on pro alpha 1(I) and pro alpha 1(III) collagen mRNA levels maximally by 67% and 77% of the control level, respectively. In the cultures of human granulation tissue fibroblasts a similar inhibiting effect on the production of collagen was seen. TNF-alpha decreased the production of 3H-hydroxyproline to 56% of the control value with a dose of 100 ng/ml. Similarly, IL-1 beta decreased hydroxyproline content of granulation tissue seven days postoperatively and PGE2 decreased nonsignificantly the amounts of hydroxyproline but the steady-state levels of pro alpha 1(I) and pro alpha 1(III) collagen chain mRNAs were slightly elevated. In the IL-1 beta-treated fibroblast cultures collagen production decreased by 15% compared with that of the controls. PGE2 decreased collagen production by 34% of that in the controls. This effect could be abolished with indomethacin. Indomethacin alone stimulated collagen production by 40%. In vivo IL-1 decreases the formation of normal granulation tissue. This effect may be partly due to IL-1 stimulated secretion of PGE2.
伤口愈合中的炎症阶段被认为是新组织形成的一个准备过程。单核细胞衍生的细胞因子肿瘤坏死因子-α（TNF-α）是一种高度保守的分子，已知在革兰氏阴性菌感染性休克的发病机制中起主要作用。除此之外，先前的实验研究表明，TNF-α在伤口愈合中可能具有有益或有害的作用。本研究的目的是研究TNF-α对大鼠肉芽组织发育以及对培养的大鼠和人肉芽组织细胞的影响。皮下植入的圆柱形中空海绵用于研究局部应用TNF-α对大鼠肉芽组织的影响。这些植入物在植入当天或植入后的前4天或7天用含有不同量TNF-α的溶液处理，而对照植入物仅用载体溶液进行相应处理。在植入后4、7、14和21天对肉芽组织进行分析。在组织学标本中，将这些海绵切成小块，并用魏格特·范·吉森染色法使胶原蛋白可视化。从每个海绵的横截面计算长入海绵的肉芽组织的量。对于细胞培养研究，从人及大鼠肉芽组织中分离出成纤维细胞，将其切成小块，并在汉克平衡盐溶液中用胶原酶和脱氧核糖核酸酶消化。将细胞暴露于1、10或100 ng/ml的TNF-α中，并将胶原合成速率测定为与蛋白质结合的3H-羟脯氨酸的合成。在用胰蛋白酶处理使细胞脱离后，用布尔格血细胞计数器对培养皿中的细胞进行计数。由于白细胞介素-1（IL-1）和TNF-α在许多功能上重叠，因此用相同方法研究了脂多糖（LPS）、人白细胞介素1β（IL-1）和前列腺素E2（PGE2）对大鼠实验性肉芽组织以及对培养的大鼠肉芽组织细胞的影响。在海绵中单次应用TNF-α后，未检测到各组之间有本质差异。然而，在连续4天每日应用TNF-α后，在4天和7天后观察到对组织修复的抑制作用。用海绵中的羟脯氨酸含量表示的胶原形成，在TNF-α处理组中明显低于对照组。在14天或21天后未观察到这种效应。这些发现在组织学样本中得到证实。在大鼠肉芽组织成纤维细胞培养物中，TNF-α使3H-羟脯氨酸的产生降低至对照组的约75%，并且它对原α1（I）和原α1（III）胶原mRNA水平也有降低作用，分别最大降低至对照水平的67%和77%。在人肉芽组织成纤维细胞培养物中，观察到对胶原产生有类似的抑制作用。TNF-α在剂量为100 ng/ml时使3H-羟脯氨酸的产生降低至对照值的56%。同样，IL-1β在术后7天降低肉芽组织的羟脯氨酸含量，PGE2使羟脯氨酸量无明显降低，但原α1（I）和原α1（III）胶原链mRNA的稳态水平略有升高。在IL-1β处理的成纤维细胞培养物中，胶原产生与对照组相比降低了15%。PGE2使胶原产生降低至对照组的34%。这种效应可用吲哚美辛消除。单独使用吲哚美辛可使胶原产生增加40%。在体内，IL-1减少正常肉芽组织的形成。这种效应可能部分归因于IL-1刺激PGE2的分泌。
