Nerve Growth Factor NGF Plays And Neuropeptides SP Interaction Mechanism To Explore In The Hyperplasia Of Scar

BACKground:Scar is the skin’s natural repair response to wound healing, but its abnormal growths result in pathological Scar.Clinically common scar can be divided into superficial scar, hyperplastic scar, atrophic scar and keloid scar. Pathological changes within a limited by the boundaries of the injured area is called "hyperplastic scar(HS)", accounted for 91.4%. It is the most common clinical pathological types.It not only affects the appearance of the affected area and its contracture can lead to dysfunction.It broughts economic and psychological burden for patients with scar.But its formation mechanism is not clear and has no exact treatment at present.In recent years,the researchers on the mechanism of formation and fading of the HS has achieved good results.It recognized in the cell molecular mechanism is:(1) fibroblast cells is the main effect, because of its excessive proliferation and secretion of Collagen fiber directly lead to excessive scar excessive deposition of extracellular matrix, but CollagenⅠand Collagen has always maintained at a relatively constant Ⅲratio, namely 2:1;(2)the morphological structure and material level, it mainly show the collagen metabolism disorder, different shape and sizer, disordered arrangement, its corresponding performance is often different in different periods.Effect of scar into fiber cell proliferation and secretion of factors are much.In recent years, studies have shown that neural factors played important roles.The mainstream view is:(3) nerve growth factor characterized by two sides.on the one hand, low concentration of NGF plays under the influence of some factors can only be combined with Tr KA, then activate the NF- KB pathways, fibroblasts proliferation and activation.On the contrary hand, a high concentration of NGF is able to combine the P53, then through the JNK- BAX pathways promote cell apoptosis.(4) neuropeptide substance P(SP) can be combined with NK- 2 receptor activating regulating protein B-catenin.It causes fibroblast migration and differentiation of epidermal stem cells.(5) Fibroblasts and its positive feedback regulation mechanism, namely under exogenous SP autocrine SP.In the process of wound repair, the fibroblast cells is the main effect. Its abnormal expression is the key to scarring.The excessive proliferation of fibroblasts and collagen secretion directly causes cell outside too much.Deposit, It lead to excessive repair of wound healing, generally speaking,Ⅰand Ⅲtype collagen fiber proportion changes its characteristics, including hyperplasia scar is 2:1, keloid for 19:1, this is one of the important mechanism of scar formation.Scar is a variety of factors, a variety of signal molecules each other abnormalities, causing abnormal fibroblasts.In recent years, studies have shown that neural factors play an important role, someone confirmed neuropeptide receptors(SP) on fibroblast(Fb) proliferation regulation role, on the one hand, nerve endings will secrete SP effect of scar fibroblasts, lead to fibroblasts autocrine SP, which is one of the positive feedback regulation mechanism of Fb proliferation;On the other hand,It increased the SP and SP receptor in the early wound healing and to actively promote the proliferation of Fb; When the wound healing is complete, the process does not stop, so it comes with the whole process of scarring.But as neural factors in scar effect research deeply.For the function of SP in hyperplastic scar the understanding of the mechanism is not perfect.The related theory is not comprehensive and can not fully explain all the characteristics of hyperplastic scar.So it is necessary to study SP factor and the influence of the scar what plays a role in regulating mechanism to do further research.Scar research reports suggest different hyperplastic scar formation time the number of nerve fibers is different obviously.The number is increased with prolonged, but the number is lower than that of normal skin. in the process of hyperplastic scar repair, scar injury of middle-late neuroplasticity research are rarely reported, the secretion of neuropeptides(SP) and nerve growth factor(NGF) variation characteristics of understanding is not very perfect.Therefore need to further study in different periods in the hyperplastic scar between the building and its secretion of nerve factor of law.Nerve and microcirculation, refactoring is the important foundation of hyperplastic scar formation, nerve endings can adjust the secretion of SP and NGF plays collagen metabolism, activate the immune cells, but also for the repair and regeneration of the nerve fiber and capillary plays an important role.Neuropeptide substance P(SP) and vasoactive peptide(VIP), calcitonin gene related peptide(CGRP) and so on all can stimulate the proliferation of vascular endothelial cells, induced the emergence of new blood vessels.Evans AR studies confirm SP direct effects on vascular endothelial cells, leading to enhanced intracellular NO synthase, and thus increase SP on vascular endothelial cell proliferation effect of CGRP and can at the same time by combining endothelial cell specific receptors, increase vascular endothelial cells and DNA synthesis.Meanwhile Rayehaudhuri SK study proved NGF plays role in leather small vascular endothelial cells(HDMEC), make its proliferation.So presumably normal skin capillary number should be less than the capillary number in the hyperplastic scar, but the neuropeptides SP and nerve growth factor NGF plays in the hyperplastic scar at different times content difference is bigger, so for neural factors in the role of the HS microvascular still need further exploration.Part of hyperplastic scar in patients with clinical cash for local itching, burning pain, symptoms aggravated when eating spicy stimuli.Studies have shown that O ’connor TM SP under dependent on concentration can stimulate immune cells such as lymphocytes, monocytes, macrophages secrete immune factors, mediated immune response.Make the capillary after small vein plasma leakage, CGRP expand small arteries, skin irritation reaction and coordination.Danzani C confirmed that substance P(both a variety of inflammatory cells, and can make mast cell degranulation, and the release of histamine, leading to the zhou min.In recent years, some scholars confirm regenerated nerve fibers in the scar exists all the links, in the environment, local target cells in a body for synthesis of a large number of model NGF and maintain a high level, and high levels of NGF plays chemotaxis and activation of immune and inflammatory cells, and release a large number of cytokines and inflammatory medium, thus to make the body itching or pain reaction.Thus neuropeptides SP and nerve growth factor NGF plays hyperplastic scar is important material base of burning pain, in the process also play a role together.Might therefore in recent years, many scholars advocate clinically used steroids or antihistamines, neurotoxin accompanied by itching or burning pain treatment of hyperplastic scar, also obtained the certain effect, but for the effect of treatment with this kind of concrete mechanism, aiming at this kind of scar using condition, using the timing is not accurate, there is also dispute about what the deal could use safety, and so on curative effect and the use range is also reduced, thus exploring SP and NGF plays in the interaction of hyperplastic scar formation rule, mechanism of action has important clinical guidance and research significance..Although surgery methods continues to improve, such as "Z" "W" shape local reduction, or fiber keeping outer skin excision of scar, but it failed to start from the essence, surgical trauma is still possible for healing is out of control again or scarring again.The experiment specimens identified both by clinicians as hyperplastic scar, both by adopting the method of surgical resection.A large number of studies have shown that nerve toxoid, such as BTXA prevention and treatment of hyperplastic scar has obvious effect, but for its mechanism is not very clear.Also have the research of formation of the cell apoptosis and the HS, is closely related to the fading process, through regulating the fibroblast apoptosis is expected to be effective to prevent the HS new luminescent spot.Distribution of SP and NGF plays and content, to identify the biology.For the first time, the scar of SP and NGF plays the interaction mechanism and to study the influence on the formation of the HS, from the protein molecule and gene level to find out its regularity, to clarify the HS mechanism and mechanism.At the same time explore botulinum toxin A in different time, different severity of hyperplastic scar fibroblasts proliferation apoptosis and the effect of observation period, hope to find botox A botox can effectively treat early HS provides A more detailed theory basis of molecular biology.Objective 1.Nerve growth factor NGF plays and neuropeptides SP in the hyperplastic scar tissue and normal skin tissue pathology and molecular differences. 2. SP and NGF plays in the hyperplastic scar fibroblasts proliferation and activation in the process of relevance and influence on fibroblast proliferation, activation, and expound its possible mechanism from the molecular level 3.From cytology and molecular biology to explore botulinum toxin A(BTXA) on the effect of the HS Fb growth proliferation and apoptosis, and explore its possible mechanism formation, as to provide theoretical basis for clinical application of BTXA treatment.Methods 1. specimen collection, Gather specimens were from specimens taken from the hospital, the second people’s hospital of guangdong province from 2013-03 and 2015-01 30 specimens of 30 cases of patients with traumatic scar, before the trial after the second people’s hospital of guangdong province ethics committee approval, all experimental specimens in specimens when informed patient ethics committee requirements, and obtain the signature of the patients consent form.The experimental process in strict accordance with the experimental ethical standards of the People’s Republic of China.Specimens of inclusion and exclusion criteria included A, from 30 cases of specimens are identified by the clinicians as hyperplastic scar(HS).B, all patient without history and chronic skin disease, preoperative half a year without scar medication history. 2.specimen processing and grouping Organization for after freeze drying preservation, all within 4 hours to experiment.Specimen processing is divided into three parts: the first part is used for primary cell culture, the second part fixed in 10% formalin formaldehyde solution, used for morphological identification, such as immunofluorescence, eosin staining and immunohistochemical sappanwood;The third part put in liquid nitrogen refrigeration spare(extraction of protein and RNA in subsequent experiments, or make up for the front part of the experiment error) 3.The hyperplasia of scar HE staining and Masson dyeing for morphological observation, three color more normal tissues and in different periods, the differences of different severity of hyperplastic scar tissue4.SP, NGF plays, Collagen Ⅰ, Collagen Ⅲimmunohistochemical study, analyzing the characteristics of its distribution within the scar tissue and expression. Five particles, organization method of the trainees to develop fiber cells, summarize and improve the method of scar fibroblasts cultured and to observe the growth rule of hyperplastic scar fibroblasts and growth characteristics, and to prepare for subsequent experiments. 6.the application of PCR detection hyperplasia scar organization endothelial collagen I, III collagen, SP, model NGF gene expression, and compared with normal line organization, analyze the differences, and application of WB technology test I and the expression of collagen type III in scar tissue, application emerges from SP and NGF plays in expression in the scar tissue 7.Using flow cytometry instrument two-color method to detect different concentration of toxin A type of hyperplasia scar fibroblast apoptosis effect;Determined by MTT method were used to detect different concentrations of botulinum toxin A fibroblast proliferation, plot of inhibition rate, the analysis of differences between groups.Semi-quantitative rt-pcr detection cell Collagen Ⅰwith Collagen Ⅲm RNA expression.Results 1.scar fibroblasts morphology observation and identification(1) the organization of the HS particles after a week, see a few cells from the tissue around the climb out, it is some vortex, some radiated(FIG. 2-1 a).The original generation of Fb mostly fusiform or irregular triangle, protruding from the cytoplasm, length is differ, vortex, radial.Some cells overlap, in mass.(2) the growth curve showed that the HS FB vaccination after 1 week or so after the incubation period which began to enter the logarithmic phase of rapid growth, about 12 days into stagnation.(FIG. 2-1 b).2.HE staining results observation of scar tissue(1) normal skin structure hierarchy clear, skin thin, shape change and the distortion, thickness change is bigger.Gradually upward from the base layer to the stratum corneum cells in each layer flat, each layer structure complete, shape is normal, neatly, corneous layer is thinner, base layer visible normal amount of melanin cells, skin more nail foot, and deep dermal tissue.Leather visible border with skin parts in papillary layer and net layer underneath, and papillary layer contains more components, reticulocyte layer is given priority to with collagen fibers, collagen fiber thickness uniformity, spiraling, visible and nerves, blood vessels, such as hair follicles, sebaceous glands, sweat gland adnexal skin peptide(FIG. 1-2 a).(2) The HS skin is thinner, less than six months early than normal skin overall thickness more uniform, little change, but the epidermal cells from down to up in each layer gradient flat, has lost its normal form, the composition is not complete, skin nail Angle disappeared, part of the cell or tissue, such as base layer the melanin cell, granular layer and stratum spinosum, reduced or disappeared, and in the dermis showed a lot of enlargement disorder of collagen fibers(FIG. 1-2 b). Mid 7 to 18 months,(3) the HS skin thickening, obviously higher than that of normal, overall from down to up gradually flat cells, cell composition is not complete, has lost due to normal morphology, dermal papillary layer and net layer, but the papillary layer of the nipple amount to less than the normal skin, the tiny blood vessels and tactile corpuscle hyperplasia of its visible(sensory nerve endings structure), its net woven layer of collagen fiber is bulky, arranged disorderly(FIG. 1-2- c);(4) In the late 18 months above HS horny layer by thin as compared to normal skin thickening and basic returned to normal, but significantly more than the normal skin base layer the melanin cells, its net woven layer of collagen fiber and mixed and disorderly, thinning, hair follicles, sweat glands, sebaceous glands small number(FIG.1-2 d). 3.Masson trichromatic dyeing observation: hyperplastic scar tissue in the dermis a large number of collagen fibers, the thickness is differ, show the dermal papilla layer attachments vascular proliferation, collagen fiber, disorder of collagen arrangement(FIG. 1-3 a);Dermal collagen fiber network layer is arranged closely, near the skin disorder, nearly subcutaneous arrange rule), the capillary number is more, and a squeeze tendency reduction(FIG. 1-3 b), part of the collagen fiber cross collagen nodule formation, nearly papillary layer significantly(FIG. 1-3 c). 4.IHC results:(1) Collagen Ⅰwith Collagen Ⅲimmunohistochemical study show that type I Collagen in the HS main distribution in reticular dermis layer, bulky, most are interwoven nodular(FIG. 1-6 a).Scattered collagen type III in dermal cell mesenchyme, papillary layer color is deeper, more fine, loosely arranged, interweave line direction is different from each other.In fibroblasts, tiny blood vessels, nerve receptors are around the distribution of type I and III collagen fiber(FIG. 1-6 b).(2)SP immunohistochemical staining showed that the HS of SP distribution around the capillaries, nerve endings, mast cells, multi-core white blood cells, fibroblasts and accessories surrounding skin.SP expression in the normal skin mainly in mast cells and nerve endings, sweat glands and around the capillaries, fibroblasts of distribution is not obvious.Mid-term deepest dyeing, the microscopic direct view differences between early and late is not obvious, but still higher than normal;By image analyzer quantitative analysis shows that: the HS in early and mid content increased with the extension of time and SP, but late gradually decreased, but still higher than normal skin(figure, table 1-5). 5. RT- q PCR results showed that the HS in the dermis and normal skin can be detected in Collagen Ⅰ, Collagen Ⅲ, SP, gene expression of NGF plays.Comparedwith normal skin, Collagen within the HS Ⅰ, Collagen Ⅲ, SP, higher gene expression of NGF plays(figure and table2-4). 6.Western Blot showed that samples of scar dermal tissue and normal skin tissues can be detected with Collagen III Collagen I, and significantly higher in the HS(figure2-5). 7.Emerges from display: HS compared with normal skin tissue to, in the control group did not join the exogenous SP samples, SP, the expression of NGF plays significantly higher than the normal skin tissue;And after adding exogenous SP HS, SP and the expression of NGF plays all have increased, and significantly higher than the control group(figure2-6). 8.Botulinum toxin A function before and after the fibroblasts form no obvious change;BTXA on in vitro culture FB growth proliferation has obvious inhibitory effect.Group compared with control group concentration significantly inhibit the growth of fibroblasts, in combination with inhibition rate curve, botox effect on inhibition of scar fibroblasts in A dose dependent(FIG.3-1) 9.flow cytometry instrument Annexin V/PI double staining method to detect FB apoptosis situation shows: join BTXA scar fibroblast apoptosis rate was 12.213 + /-0.89667, no set of scar fibroblast apoptosis rate was 2.032 + /- 0.95673, compared to both the statistically significant(figure, table 3-2).Conclusion 1.This study by the original generation of scar fibroblasts culture, can be observed in hyperplastic scar fibroblasts with no significant difference on fine form normal fibroblasts 2.In the hyperplastic scar tissue Collagen with Collagen increased significantly, Ⅰ ⅢSP, model NGF content is also higher than normal tissue. 3.The hyperplastic scar fibroblasts can under the action of exogenous SP autocrine SP, as one of the positive feedback regulation mechanism of fiber cell proliferation.Andexogenous SP in the hyperplastic scar can regulate the production of NGF plays, and create synergies by NF- KB pathways, can promote the excessive proliferation of fibroblasts, pathological collagen I, III excessive metabolism. 4.Botulinum toxin A is by inhibiting the formation of the SP material, and then through the inhibition of NF- KB pathways inhibit fibroblast proliferation.