Study On The Expression Of Substance P By Fibroblasts In Vitro And Its Effect Of Collagen Synthesis In Human Hypertrophic Scar

Hypertrophic scar is the over hyperplasia of fibrous tissue limited by the area of adustum after its healing and common in the area of burn wound with deepⅡandⅢ, the pathognomonic feature of hypertrophic scar are abnormal hyperplasy of fibroblast, synthesis multiplication and over deposite of collagen. The pathogenesis has been unkown at present, it has some close relationship between the disfunction of main cell of collagen-fibroblast and some factors and protein of its autocrine. For example, transforming growth factor-α,β,TGF-a,βand IGF-binding proteins by the autocrine of fibroblast in hypertrophic scar, the control mode of which is different of normal skin, so the collagen synthesis is effected.At present, substance P is the earliest neuropeptide and its function is complicate, it exists both in central and peripheral nervous system. Not only is the acceptor of SP in immunocell (monocaryon/macrophage, mast cell et al.), fibroblast, endothelial but also synthetize and secrete SP. Autocrine SP in fibroblast is controlled by extracellular SP. Such as SP in sensory ending was exhausted by large dose of capsaicin, and then vulnerated the rat skin, and the SP of fibroblast in granulation tissue of injuried skin was weakened than no capsaicin obviously; SP of 10-8mol/L added in the culture fluid of fibroblast in normal skin for 1-3h, and the expression of SP and protein in fibroblast increased sharply. In recent study discovered SP is the important factor in the control of tissue repair. SP in sensory ending can not only promote inflammatory reaction from nerves but induce high expression of cell factors such as TGF-β,EGF,TNF in fibroblast under injuried skin, thus can promote fibroblast multiplication.The investigate indicated the content of SP was obviously higher than the nomal one in hypertrophic scar. But what's the feature of the self-expression of fibroblast, what's the influence of extracellular SP and what's the effect on collagen synthesis have not been reported at present. Study on the problems above directly will affluence the heterogeneity theory of fibroblast in hypertrophic scar,and offer a new way of healing hypertrophic scar.To investigate the feature of autocrine SP in hypertrophic scar fibroblas(tHSF)and the effect of collagen synthesis,hypertrophic scar fibroblast cultured in vitro was the target, and normal skin fibroblast(NSF) was the contrast group, and detect the expression feature of SP in the two kinds of fibroblasts from mRNA and protein with the technology of RT-PCR and ELISA. The NSF or HSF were stimulated by different doses of 10-7mol/L or 10-5mol/L SP to survey the secretion of SP in the two kinds of fibroblasts in order to analyze the effect of extracellular SP in hypertrophic scar. To investigate the effect of collagen synthesis by SP, NK-1R (Neurokinin 1 receptor) blocking agent (WIN62577) was added into the cell culture fluid of HSF and NSF, and to block SP effect on fibroblast in hypertrophic scar and then analyze the change of collagen synthesis with the technology of radioactive tracing to detect the incorporation of 3H-proline and immunohistochemistry by adding NK-1R blocking agent and no doing it.The main results and conclusion of the study are as follows:1.The assessment of hypertrophic scar fibroblast cultured in vitro and expression of SP(1) Hypertrophic scar fibroblast cultured in vitro showed long fusiform, vimentin(-),α-SMA(-). it's determined the fibroblast for the experiment.(2) The expression of SP mRNAin hypertrophic scar fibroblast was obviously higher than the nomal one, and the concentration in HSF was 0.132±0.026nmol/L, and 0.056±0.022nmol/L in NSF, there were significant difference between them(P<0.05).2. The effectof exogenous SP in hypertrophic scar fibroblast HSF cultrued in vitro and NSF were irritated by different doses of SP, and then the expression of SP mRNA in fibroblast and protein were both up-regulations obviously. But the phase to peak, amplitude and peak value were different of NSF obviously.(1) The expression of SP mRNA in fibroblastThe expression of SP mRNA both in HSF and NSF were up-regulation after adding 10-7mol/L SP, and achived to peak in 6h or 3h, and then decreased slowly. The expression of SP mRNA both in HSF and NSF were up-regulation after adding 10-5mol/L SP, and achived to peak in 3h or 6h, and the peak value were higher than NSF, and the two groups were higher than pre-stimulate 24h later. (2) The expression of SP protein in fibroblastThe expression of 10-7mol/L SP protein in HSF achived to peak in 6h, it's up to 0.367±0.026nmol/L, and step up 225%, and then decreased slowly. It's up to 0.224±0.014 nmol/L 24h later but still higher than before, both of the two groups were different significant(P<0.01). The expression of SP protein in NSF achived to peak in 3h, it's up to 0.489±0.022nmol/L, and step up 823%. It's up to 0.163±0.015nmol/L 24h later, but still higher than before(P<0.01).The expression of 10-5mol/L SP protein in HSF achived to peak in 3h, it's up to 1.476±0.068nmol/L, and step up 834%, and then decreased sharply. It's up to 0.248±0.031 nmol/L 24h later but still higher than before(P<0.01). The expression of SP protein in NSF achived to peak in 6h, it's up to 1.257±0.086nmol/L, and step up 1805%. It's up to 0.196±0.024 nmol/L 24h later, but still higher than before(P<0.01).(3) Compared with NSF, the peak value and phase between the expression of SP mRNA and protein in HSF under adding 10-7mol/L or 10-5mol/L SP had different significantly (P<0.05,or P<0.01), and the expression of SP in HSF were higher than normal one 24h later.3. the effect of collagen synthesis with SP in human hypertrophic scar(1) The wink point number of the incorporation of 3H-proline in HSF was 82.13±9.23cpm/1000cells, higher than that of NSF (35.7±9.8cpm/1000cells) , there is significant different between them(P<0.01).The wink point number of the incorporation of 3H-proline in HSF was 36.75±5.83 cpm/1000cells after adding NK-1R blocking agent, and lower than the group of contrast, and the block rate was up to 55.25%, there is significant different between them(P<0.01).The wink point number of the incorporation of 3H-proline in NSF was 24.48±3.48 cpm/1000cells after adding NK-1R blocking agent, and lower than the group of contrast, there is significant different between them(P<0.01), and the block rate was up to 31.43%.(2) The average gray scale of precollagen I was 102.18±15.26 after adding NK-1R blocking agent, and the average gray scale of precollagen III was 111.53±14.79, which were all lower than the contrast one(P<0.01). The block rates were up to 33.35%, 21.67%, respectively. The conclusions above suggest:1. The expression of SP mRNA and protein in HSF are higher than the normal one obviously, and it is probably one of the reasons that the content of SP was obviously high in HSF.2. The effect of the expression of SP in HSF by exogenous SP is different of NSF. The peak value and phase between the expression of SP mRNA and protein in HSF under adding 10-7mol/L or 10-5mol/L SP had different significantly, and the expression of SP in HSF were higher than normal one 24h later. It is suggested that the control mechanism of SP and protein in HSF are different from the normal one obviously, and the control point of balance of autocrine SP was up-regulation, which is one of the reasons that the expression of SP in HSF was high.3. SP is the important factor to promote collagen synthesis of fibroblast. The high expression of SP in HSF is the important factor to increase the intake of hydroxyproline and collagen synthesis.