The Study Of Hypertrophic Scar Transforming Growth Factor-β1/Smad Signal Transduction Pathway

BackgroundHyperplastic scar is one of the most common complications after wound healing. It is a kind of skin fiber proliferative diseases, which is an excessive body’s response to trauma healing, leading to abnormal proliferation of fibroblasts, large number of collagen synthesizing and excessive deposition of collagen. According to statistics, the incidence of hyperplastic scar after burn is91.4%in china. It reaches as high as74.67%if the burn wound healing time more than two weeks, which far higher than38.00%of the foreign coverage. Once the scarring, it affected the person’s appearance, and accompanied by itching pain, and produced contracture can lead to different degrees of dysfunction, such as tendon contracture, joint dislocation, motor dysfunction, professional psychological obstacles, etc. It reduced the patient’s quality of life and brought heavy economic and psychological burden. Hyperplastic scar prevention research is a both old and new topic. Due to the pathogenesis has not yet fully understood, the treatment is still quite difficult clinical problem at present.For many years scholars at home and abroad took multi-angle, multi-level of and in-depth studies on the the hypertrophic scar occurrence, development and subsided mechanism. But until now there is no definite conclusion for the mechanism, and also there is no silver bullet for the prevention. A consistent opinion is:(1) the main effector cell of hyperplastic scar is fibroblast cells, and Its main feature is cell excessive proliferation and extracellular matrix excessive deposit;(2) the collagen metabolism disorder is the main performance at biological level;(3) TGF-β1/smad signaling pathways is closely related to fibroblast proliferation, differentiation, migration, apoptosis, collagen metabolism and many kinds of physiological and pathological process and so on. According to different sub-type, Smads has a two-way role of controling fibroblast collagen metabolism.In recent years, the ability to study the effect of mechanical stress stimuli in living vitro cells, and to observe cell morphology, structural, functional status, proliferation, differentiation and apoptosis change after mechanical stress stimuli was very rapid developments in the field of cell biology research study. It has proved that mechanical stress plays an important role in some physiological and pathological processes. The pressure treatment of hyperplastic scar has been reported as early as400years ago. Then multicenter, large sample, randomized controlled clinical trials taken by many scholars have confirmed that pressure play an important role in the burn (injury) scar prevention and control. Since the70s, pressure therapy is widely accepted by the doctor, and has been popularized and applied. The pressure treatment has become the standard and preferred method of hyperplastic scar treatment. But it is not based on mechanism but clinical experience. The pressure treatment of hyperplastic scar mechanism is not very clear currently. Research suggested that one of the main reasons for scarring is fibroblastic collagen metabolism and catabolic imbalance, and the ebb and flow of the two factor perhaps decided to wound healing and tissue reconstruction. Analyzing the causes of wound not long scaring or scaring atrophy after pressure treatment, it can be summarized as follows:the stimulative factor of scarring leveled down and the suppression leveled increase. The control of signal path is divided into three levels:the extracellular signal molecules stimulate; corresponding conduction signal molecule transfer; target gene transcription and translation. Over the years, many scholars’ research results confirmed that fibroblasts biological behavior, especially the change of collagen metabolism plays a main role in the process in the pressure treatment of hyperplastic scar. Previous studies always focused on the change of blood supply on hyperplastic scar. It believe that compressive stress make scar tissue blood flow deposition, ischemia or gore, the oxygen partial pressure decrease, thus inhibiting scar from continuing to grow. However, the mechanisms how the compressive stress affect the fibroblasts biological behavior and collagen metabolism was poorly understood.Previous studies have confirmed that TGF-β1can impact the metabolism of collagen-induced MMP-1and TIMP-1expression on the transcriptional and translational levels. Previous research showed that TGF-β1in hypertrophic scar tissue tended to increase, smad7lower compared with normal skin tissue content, and significantly increased as hyperplastic scar mature. Based on these studies, we can assume that the mechanism of pressure treatment of hypertrophic scars may associated with TGF-β1/Smad signaling pathway, which inhibition in smad3activation or raised smad7, thereby regulating the expression of MMPs on collagen synthesis and degradation. Therefore, if our study take the scarring effector cells-fibroblasts as the research object, proceed from the cell proliferation and collagen metabolism, observed intermediate molecules along the mainline of an important signaling pathway TGF-β1/smad and study the control mechanisms of the pathway to biological behavior of fibroblasts and collagen metabolism balance, there may reveal the key point how the compression therapy prompted the development of hypertrophic scars quickly to atrophic scars.There is little information about the molecular mechanism of the pressure intervention how to improve the expression of cytokines to regulate the fibroblast collagen metabolism balance in literature. It is the first time to use pressure simulation technology acting on vitro hypertrophic scar fibroblasts to find the pressure effector molecules in pathway intermediate molecules, which can affect collagen metabolism. Then blocking key intermediate molecules smad3by RNAi, the pathway intermediate molecules and collagen metabolism changes was observed to determine the effect of the pressure effector molecules in the signaling pathway to hypertrophic scar fibroblast collagen metabolism. Through the experiment, we hope to get the action point of pressure signal to balance regulation of collagen metabolism and to provide valuable experimental data for part of the mechanism of the pressure treatment of hypertrophic scars. Thereby, it preliminarily can clarify the cell reconstitution mechanism and the mechanical effect of the regulation of the signal transduction pathway, and rich relevant theory, and open up a new exploration methods for how to reduce scar or attain no scar after wound healing.Objective:1. To provide valuable experimental data for part of the mechanism of the pressure treatment of hypertrophic scars and to clarify preliminarily the molecular mechanism of mechanical compression treating hyperplastic scar via TGF-β1pathway.2. To get the pressure signal effector molecule in TGF-β1pathway, to explore therapeutic targets for the prevention and reduction of hypertrophic scars and to open up a new exploration methods for how to reduce scar or attain no scar after wound healing.Methods:1、Specimen collection, handlingThe12hyperplastic scar and3normal skin specimens, age20to60years, was from the Second People’s Hospital of Guangdong Province and Guangzhou Red Cross Hospitalfrom2011.06to2011.12. Access to specimens with the patient’s informed consent, and signed informed consent. Inclusion and exclusion criteria of Specimen:1、the clinician identification of scar tissue;2、without the pituitary, adrenal diseases, infectious diseases, skin diseases and autoimmune diseases, local infection, ulcer;3、receiving nothing treatment for the scar.Obtaining of specimens had the patient’s informed consent, and signed the Informed Consent Form;4、skin graft surgery at the same time. Tissue was cut under aseptic conditions, then was placed in a sterile container. The experiments are performed within2hours.2、Build hypertrophic scar fibroblasts cell lines.Hypertrophic scar tissue cut in the sterile operating room was brought to the sterile workplace immediately. Primary culture of hypertrophic scar fibroblasts used the method of tissue particles paste petri dish wall, under the conditions of37℃,5%CO2. It replace the liquid twice a week and the cell growth was observed under the microscope. On the6th-8th day, the primary cultured cells showed aggregation growth after changing the solution, and gradually fused and passaged, followed by in vitro cell culture for3to4passages. Cells were identified by a-SMA staining immunohistochemical:The cells were cultured in the clean sterilized coverslip. After conventional fixation, dehydration, antigen retrieval, endogenous enzyme inactivated by the hydrogen peroxide,10%goat serum for30min,and treated dropwise with a1:100dilution of an anti a-SMA (1:100diluted), incubated at4°C overnight. After washing three times, dropwise addition of biotinylated goat anti-mouse IgG and DAB color development. Instead of primary antibody with PBS as negative control. The percentage of positive cells was observed under the microscope.3、Mechanical pressure interventionHypertrophic scar fibroblasts samples (n=12) were randomly divided into a control group(n=6) and pressure stimulation group(n=6).First, the sample was added to the sample hole, and then, following the instrument instructions of the FX-4000C, installed compressed tablet, and loaded in compression clamping device. Finally, we set the parameters of the system:35mmHg. The hypertrophic scar fibroblasts samples overnight under the conditions of37℃,5%CO2.4. It use GenBank to find out TGF-β receptor, Smad3, Smad7, MMP1, MMP2, MMP9, MMP12, Collagen I and collagen III gene sequences and use the software Primer-Express5.0to design primer and probe.5、The mRNA expression of TGF-β receptors, Smad3,smad7, collagen I, collagen III of control group and pressure stimulation group was detected by Sygreen realtime fluores-cence quantitative PCR and the difference was compared.The total RNA was extracted by Trizol reagent, then was identified on the concentration, purity and integrity. It added1μg total RNA to12μl sterile distilled water and mixed, the mixture was incubated for5min at65°C, and then immediately placed on ice; The configured denaturation annealing reaction solution was added to the total RNA-containing solution, then mixed. PCR was performed in the PCR machine. The resulting cDNA placed at-20℃. Taking cDNA as a template, it begin to cDNA PCR reaction and quantitative PCR. We set cDNA PCR reaction conditions as follws:initial denaturation for3min at95°C, denaturation at95°C for40s, annealing57°C for50s, extension72℃for50s, extends at72℃for10min. Fluorescent Quantitative PCR reaction conditions were:at93°C for3minutes, then at93°C for30seconds, at55°C for45seconds, at72°C for45seconds, a total of40cycles.6、Gene expression of Collagen Ⅰ, Collagen Ⅲ,, SMAD3, SMAD7within the dermis was detected by Sygreen realtime fluores-cence quantitative PCR and the difference is compared between HS and the normal tissue. 7、Hypertrophic scar fibroblasts samples (n=12) were randomly divided into blank control group (n=6) and smad3of blocking group (n=6) samples. Lipofectamine2000mediated100nmol smad3si RNA gene transfected into hypertrophic scar fibroblasts to block smad3gene. The gene expression of MMP1, MMP2, MMP9, MMP12, Collagen Ⅰ, collagen Ⅲ was detected by RT-PCR and was compared between blank control group and blocking group.Results:1、the different expression of Blank control group and pressure experimental group TGF-β receptors, Smad3, Smad7and collagen Ⅱ, Ⅲ.According to RT-PCR results, the expression level of TGF-β receptor Smad3, Smad7, Collagen Ⅰ of the experimental group with the intervention of mechanical pressure was lower than the control group, and the difference was statistically significant (P<0.05); The two groups expression of collagen Ⅲ showed no significant difference (P>0.05).2、The different expression of MMP2, MMP9, MMP12and collagen Ⅰ, Ⅲ in the Experimental group and the control group after the intervention of si-smad3.The expression level of MMP2, MMP9, MMP12of the experimental group with the intervention of si-smad3was higher than the control group, while the collagen Ⅰ was lower, and the difference was statistically significant (P<0.05); The two groups expression of MMP1collagen Ⅲshowed no significant difference (P>0.05).Conclusion1、The mechanical pressure in the treatment of hypertrophic scars can inhibit smad3gene, so as to promote the degradation of collagen Ⅰ expression, by TGF-β1/Smad signal transduction pathway. Smad3may be one of the critical pressure effector molecules.2、Smad3can inhibit the synthesis of MMP2, MMP9, MMP12in the hypertrophic scar fibroblasts and promote the expression of type I collagen,; Smad3is the key fibrogenic factor of the TGF-β1/Smad signal transduction pathway in the hypertrophic scar fibroblasts.3、Smad3might become a biological target for the treatment of hypertrophic scars...