| Background:Pathological scar, one of complications after skin trauma and post-operative, has a bad effect on the patient’s appearance and psychological. Its mechanism and prevention has been the focus of the medical profession.There is no clear conclusion on its mechanism, prevention and treatment.For many years, many scholars approach the problem from different perspectives on hyperplastic scar formation and regression,expect to find a breakthrough in the treatment of scar. A more consistent view are that:①hypertrophic scar is characterized by excessive cell proliferation and extracellular matrix, in which fibroblasts are the major effector cells;②The hypertrophic scar is mainly the result of collagen metabolism disorders at the biological level;③TGF-β1/smad signaling pathways are closely related with physiological and pathological procession of fibroblasts, such as proliferation, differentiation, migration, apoptosis and collagen metabolism and so on.Fibroblasts,as the major effector cells in the wound healing process, often shows excessive proliferation and active function, causing a cascade of reactions, out of control in wound repair, excessive deposition of extracellular matrix, the formation of pathological scar. Thus, the abnormal function of fibroblasts plays an important role in pathological scar hyperplasia. Lekic etal. describes fibroblasts as engineers, builders and administrators of wound repair.Since Convey etal. cultured hypertrophic scar fibroblasts in1959,it started a new stage of understanding of the mechanism of scar in vitro and the cellular level. In recent years, with the deepening basic research of pathological scar,relationship of fibroblasts and scar and mechanism of scar formation and invasive growth have become a hot topic of medical research. However, the primary culture of scar fibroblasts is still a more difficult for beginners for a high failure rate.Therefore, the establishment of efficient cultivation methods is particularly necessary for scar fibroblasts.The prevailing view is that The hypertrophic scar is mainly the result of collagen metabolism disorders at the biological level. The excessive synthesis of collagen and collagen proportion change during the scar formation process is the important reason of hypertrophic scar.Collagen metabolism disorder has a close relationship with the dynamic balance of TIMPs and MMPs and TGF-beta signaling pathway. However,the changes of collagen and its regulation in hypertrophic scar is still unknown.The dynamic balance between MMPs and TIMPs is an important factor affecting the metabolism of collagen.But there are different opinions on changes of collagenase.Ghahary et al reported that mRNA expression of MMP-1in human scar fibroblast derived fibroblast collagenase was significantly lower than normal skin fibroblasts, while there was no significant difference in the mRNA expression of TIMP-1.Zhao etal. confirmed that MMP-3mRNA expression level in the HS group was lower than the normal group,which reached the same results.Likely, in the study of abnormal skin scars,the expression of TIMP-1was significantly higher than that of normal tissue. The same thing occurred in the liver and kidney fibrosis.In addition,many opposite conclusions were reported.it is possible to provide clues for the mechanism of scar formation by exploring the changes of MMPs and TIMPs in the scar tissue and its related factors.TGF-β1/smad signaling pathways plays an important role in the collagen metabolism of hypertrophic scar.SMADs is the main transduction molecules from the receptor to the cell nucleus.Different Smad have two-way regulation on fibroblast. Numerous studies have confirmed that Smad2/3is the promoting factor, while SAMD7being the inhibitory role.However, variation and its influencing factors of Smad2/3and SAMD7is also the focus of the study because of the interaction of various signaling pathways.Lots of work have been done for the pathogenesis and treatment of the HS and fruitful results have been achieved, but there still no effective or satisfactory treatment for HS so far. Surgical resection is the most common treatment for hypertrophic scar. Despite the improvements were done, Surgical resection may still be re-trauma healing and lead to excessive scar formation. This group have adopted surgical treatment due to the dysfunction and ugly appearance.The surgical specimens were used for this experiment. It was reported that breviscapine can inhibit fibrosis progression,but it has not been reported whether it can inhibit proliferation of pathological scar fibroblast.Other studies suggest that apoptosis is closely related to the formation of HS.And the regulation of fibroblast apoptosis is expected to become a new bright spot of the effective prevention and treatment of HS.In this study,we observe the growth variation and biological identification in the establishment of efficient primary culture of scar fibroblasts, and firstly explore the impact of Breviscapine on proliferation and apoptosis of scar fibroblast, hope to find effective drugs for the treatment of hypertrophic scars. The metabolism of collagen fibers in the dermis layer of scar tissue related molecules, and tap one of the important molecular variation; to provide ideas for the clarification and treatment of mechanism of hypertrophic scar. Objective:1. Searching for an efficient method of scar fibroblasts culture in vitro.2.Detecting the morphology, molecular biology of pathological scar,to understand formation mechanism of hyperplastic scar in depth.3.To study the effects of Breviscapine on proliferation and apoptosis of cultured scar fibroblasts. It may be developed as a new drug for the treatment of pathological.Methods:1. Specimen collectionThe24Traumatic scar specimens, age18to56years, was from the Second People’s Hospital of Guangdong Province and Guangzhou Red Cross Hospital from2011.06to2011.12. 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, and a preliminary classification (hypertrophic, atrophic, keloid);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.2. Specimen handling, groupingSpecimen was saved with freeze-drying method,and was used for primary culture within4hours. The specimens were divided into three parts:1. Cells primary culture;2. Fixed in10%formaldehyde solution for morphological identification (eg, immunohistochemistry, etc.);3. Frozen in liquid nitrogen alternate (extraction of protein and RNA). three normal skin used as normal controls3.Primary culture of scar fibroblasts:Observing the scar fibroblasts growth regulation and the growth characteristics; Drawing cell growth curves by MTT assay; identifition of scar fibroblasts. 4.Observing the differences morphological characteristics between hypertrophic scar and normal tissue by HE staining and masson trichrome staining.5.Distribution characteristics and expression of Collagen Ⅰ and Collagen Ⅲ was analyzed in the scar tissue by immunohistochemical staining.6.Gene expression of Collagen Ⅰ, Collagen Ⅲ, MMP-1, TIMP-1, SMAD3, SMAD7within the dermis is detected by RT-qPCR and the difference is compared between HS and the normal tissue. Protein expression of MMP-1, of SMAD3and TIMP-1by Western Blot technology.7.Fibroblasts were cultured as an experimental model and the effect of breviscapine were studied. Cell proliferation was determined by MTT assay, and Apoptosis of cultured fibroblasts is induced by breviscapine at the IC50with Annexin Ⅴ/PI staining and flow-cytometry.Results:1. Morphology and identification of scar fibroblast(1) A small number of cells climb out from HS organization in the shape of radial or spiral between two and seven days.(Fig1-1a, b) Primary fibroblasts were in the shape of spindle-shaped or irregular triangle. Nuclei were round or oblong ina-SMA of Immunohistochemistry staining.(2) It is spherical when the fibroblast has not been adherent in HS fibroblast subculture (Fig1-1c), and then extended, showing spindle-shaped, long rectangular, polygonal or rules-shaped (Fig1-1d). Cell was in the state of the convergence at2～3d (Fig1-1e). It is arranged loosely and in larger cell gap in Low cell density (Fig1-1e) and arranged in parallel or radial and swirling arrangement in high cell density,(Fig1-1f).(3) Growth curve:After a short incubation period, Scar fibroblasts showed a gradually increasing trend growth in6to8days (Fig1-3) and into the stagnation of the number after10days.(4) The results of α-SMA of Immunohistochemical staining is shown in fig1-5. We can identify fibroblasts by positive staining cells and its morphology.2. HE staining and Masson staining:1. Fewer fibroblast and microvascular in normal skin (Fig2-3a).2. Increased fibroblast and microvascular can be seen in Scar tissue (Fig2-3e). Thick collagen in dermis is disorganized, the collagen fiber was cross-cutting,swirling, nodular, among which there are collagen fibers,vessels and fibroblast(Fig2-3b, d).3. Marked thickening of the epidermal Scar tissue is thicker than normal epidermis (Fig2-3a).3. Result of IHC:Collagen Ⅰ is mainly distributed in the reticular dermis. The distribution of Collagen Ⅲ is in the papillary dermis. Both of two fibers are found in the vessel wall.4. RT-qPCR results:collagen Ⅰ, collagen Ⅲ, MMP-1and TIMP-1, SMAD3, SMAD7gene expression in dermal tissue of the scar and normal skin can be detected. Compared with normal tissue, gene expression of collagenⅠ (t=5.85, P<0.01)、 collagenⅢ (t=4.515, P<0.05), MMP-1(t=-13.22, P<0.01), TIMP-1(t=2.52, P<0.05)、smad7(t=-2.503, P<0.05) were significant different in scar. We can see collagenⅠ、collagenⅢ、TIMP-1is increased and MMP-1is decreased. There was no significant difference in gene expression of Smad3(t=-0.339, P>0.05), and Smad7(p=0.0629>0.05).(Fig2-7and Tab2-1).5. Western blot:MMP-1and TIMP-1, of SMAD3can be detected in dermal of the scar tissue samples and normal skin.But with a lower Protein expression than normal tissue.(Fig2-9).6. In vitro, the proliferation of fibroblasts is significantly inhibited by breviscapine in the manner of dose-dependent(F=3.309,P<0.01). Compared with the control group, proliferation of fibroblasts is significantly inhibited in the concentration of80,100,150μmol·ml-1(P<0.05). Apoptosis rate of fibroblasts induced by breviscapine at the IC50is significantly increased.7. The results of Flow cytometric Annexin V/PI double dyeing show apoptosis rate of experimental group is14.2050±0.95553in IC50, Apoptosis rate of control group is4.0220±0.31412, a statistically significant difference in both (t=22.653, P<0.05). The results of RT-PCR show that with increasing of concentration, the scar fibroblasts collagen Ⅰ and collagen Ⅲ mRNA are influenced obviously, there is a statistically significant difference,(fig3-4, tab3-1)Conclusion:1. Establishing scar fibroblast cell lines and naming them, and complementing primary culture theory of scar fibroblast.2. A better understanding of different fibroblasts between HS and normal skin is observed by observing the morphological changes during the growth prosess.3. Collagen Ⅰ and collagen Ⅲ in the hypertrophic scar tissue increased significantly. The formation of HS is closely related with the reduced expression of MMPs, and increased expression of TIMPs.4. The formation process of scar is a complex process of multiple signal paths, multi-factor interactions.5. Breviscapine can significantly inhibit the proliferation and induce the apoptosis of cultured scar fibroblasts. It may be developed as a new drug for the treatment of pathological scar. |
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