Development of anti-skin ulcer drug based on the new concept -Application of the stimulatory effect of TNF-alpha on the production of NGF in fibroblasts

(1)研究了tnf - α和其他细胞因子在刺激Swiss 3T3细胞NGF生成中的可能相互作用。tnf - α对成纤维细胞NGF产生的刺激活性与il -1 α / β、ifn - γ和BMP-2协同作用，但被tgf - β拮抗。这些发现强化了tnf - α与il -1 α / β、ifn - γ和BMP-2协同作用，通过间接机制刺激成纤维细胞中NGF的产生，在调节损伤后周围神经再生中发挥重要作用。(2)研究了两种类型的TNF受体TNF- r1和TNF- r2在调节TNF刺激成纤维细胞中NGF生成素的能力中的作用。抗TNF- r1抗体而非抗TNF- r2抗体增加了这些细胞中NGF mRNA的水平，刺激了NGF的产生，表明抗TNF- r1介导了TNF的活性，刺激了NGF基因的表达。然而，导致NGF基因表达的受体后信号通路是独特的，不涉及ERK MAP激酶、p38 MAP激酶、C激酶和A激酶的激活。(3)在自发性II型糖尿病小鼠C57BL-KsJ （ab/db JcL）背侧全层皮肤创面上应用外用tnf - α，观察其对皮肤创面修复特别是周围神经再生的影响。100 ng/cm^2 tnf - α处理的小鼠伤口消退率显著高于对照组。然而，接受1和10杯/cm^2 tnf - α治疗的小鼠伤口恶化。此外，与对照组相比，tnf - α处理（100 ng/cm^2）组周围神经的再生更为明显。这些结果表明，适当浓度的tnf - α有望加速皮肤伤口愈合，特别是在神经再生方面。

(1) A possible interaction between TNF-alpha and other cytokines in stimulating the production of NGF in Swiss 3T3 cells was investigated. TNF-alpha's stimulatory activity on fibroblasts NGF production was synergized by IL-1alpha/beta, IFN-gamma and BMP-2, but was antagonized by TGF-beta. These findings reinforce the idea that TNF-alpha, in concert with IL-1alpha/beta, IFN-gamma and BMP-2, plays an essential role in regulating the regeneration of peripheral nerve following injury through an indirect mechanism by which it stimulates NGF production in fibroblasts.(2) The role of the two types of TNF receptors, TNF-R1 and TNF-R2, in mediating the capacity of TNF to stimulate NGF productoin in fibroblasts was investigated. An agonistic anti-TNF-R1 antibody, but not an agonistic anti-TNF-R2 antibody, increased the NGF mRNA level and stimulated the NGF production in these cells, indicating that anti-TNF-R1 mediated the TNF's activity to stimulate the NGF gene expression. The post receptor signaling pathway leading to NGF gene expression, however, was unique and did not involve the activatoin of ERK MAP kinases, p38 MAP kinase, C kinase nor A kinase.(3) The effect of topical TNF-alpha on cutaneous wound repair with special reference to regeneration of peripheral nerve was investigated in C57BL-KsJ (ab/db JcL) mice that spontaneously develop type II Diabetes Melitus by applying it to full thickness skin wound in dorsal skin. Regression rate of wound was significantly higher in mice treated with 100 ng/cm^2 TNF-alpha than control. However, wound worsened in mice that received 1 and 10 mug/cm^2 TNF-alpha. Furthermore, regeneration of peripheral nerve was more apparent in the TNF-alpha-treated (100 ng/cm^2) group as compared with control. These results suggests that TNF-alpha at appropriate concentrations is expected to accelerate cutaneous wound healing, especially in terms of nerve regeneration.