| Cell proliferation is the fundamental features of life,and it is not only invovled in individual development, body repair and other physiological processes, but also in the refractory, scar formation, tumorigenesis and other pathological processes. Therefore, a reasonable regulation of cell proliferation will promote tissue repair, improve the repair quality and inhibit the formation of scar and tumorigenesis, causing important significance especially in wound healing for its high incidence. Currently, problems with wound healing can manifest as either delayed healing or excessive healing such as the formation of hypertrophic scars. In addition, several contradictions exist between the treatment of wound healing and scar formation. For example, enhanced growth factor activity increases the rate of wound healing but may also pose a risk for increasing scar formation. Current scar treatments are empirical, unreliable, and some have side effects like delaying wound healing.In addition,the treatment targeted to receptor pathway is hard to receive ideal cure effects because of the reduced levels of active growth factors in the wound environment, mutation of the receptors of growth factors, excessive proteinase activity, imbalance between proteinases and their inhibitors. Thus, it is very important to study the mechanism of normal wound healing and seek new proliferation-promoting factors of non-receptor pathway.c-Ski is cellular homolog of v-ski gene and highly conserved across various species,distributes widely in many kinds of tissues or cells and calls Ski in human. Ski is transcriptional co-repressors that inhibit transforming growth factor-beta(TGF-beta) signaling through interaction with Smad3,Smad2 protein, and also a versatile regulator of transcription that has been shown to stimulate transcription of NFI(the nuclear factor I)and represse transcription of Rb. Ski is involved in many physiological and pathological processes which include the development of nervous system, neural axis formation, hematopoietic cell proliferation and differentiation, tumorigenesis and reorganization. All these suggest that Ski a proliferation-promoting factor. Yet, we previously found that ski is a new tissue repair–related gene expressed in fibroblasts after wounding. Furthermore, we explored the regulatory role of Ski in wound healing and scar formation through in vivo gene transfection methods, and Ski plays dual roles by both promoting wound healing and alleviating scar formation in rat skin and rabbit ear models. We demonstrated that the potential therapeutic effects of Ski are better than those of existing measures to promote wound healing and inhibit scar formation, and the possibility of simultaneously achieving these seemingly contradictory effects. Ski holds promise for future molecular and clinical applications to treat non-healing or abnormal wounds and hypertrophic scars.However, it's mechanism in cell proliferation, wound healing and scar formation is not clear. In particular, as a proto-oncogene, Ski was significantly expressed in a variety of human malignant tumors, and how the role is in infinite proliferation of the pathological process. All these are not only affect the elucidation of its biological effects, but also severely restricted the good prospects for the treatment of trauma. Moreover, studies have shown that Smad3 can inhibit cell proliferation, and NIH group firstly reported that the Smad3-null mice paradoxically show accelerated cutaneous wound healing compared with wild-type mice in 1999.These results suggest that reduction of Smad3 could promote proliferation and significantly accelerates wound healing; In addition, Smad2 and Smad3, are main downstream signalling molecules of TGF-beta( called as TGF-β1/Smad dependent pathway), have been showen to play a important role in scar formation in past few years. Thus, inhibition of Smad3/2 has become the new way to reduce scar formation. Moreover, TGF-β1/Smad dependent pathway not only inhibits cell proliferation, but also has anti-tumor effect. And the loss of its function is considered to cause the loss of its anti-tumor effect of TGF-β1.All these suggest that Ski, as corepressor of Smad3 and Smad2, maybe promoting proliferation, wound healing, scar formation and tumorigenesis through Smad3/2 pathway. To date,whether it is true need to be further confirmed.Based on the previous studies, the present study is divided into three parts: (1) The study on in the role of Ski in cell proliferation and cell proliferation regulated by TGF-β1: Different proliferation of the primary fibroblasts (FB) and fibrosarcoma cells (L929) with TGF-β1 stimulation was validated in vitro, and the proliferation role of Ski was verified by c-Ski RNAi interfered with TGF-β1 stimulation. Then, the results were verified in vivo and used to discusse whether the mechanism of promoting proliferation of Ski was involved inhibition Smad3/2 signaling; (2) The study on mechanism in the dual role of Ski in wound healing: first, we investigated a dual-role in both promoting wound healing and alleviating scar formation of Ski in cut wound of rat skin and rabbit hypertrophic scar model. Then we confirmed the dual role of ski using Ski RNA interference (RNAi) methods in a full-thickness excisional rat model, and furthermore, we investigated relationship between Ski and TGF-β1/Smad dependent pathway in wound healing in rat model using naked c-ski plasmid treatment; (3) The study on the role of Ski in hypertrophic scar formation: To investigate the role on collagen secretion of fibroblast of Ski using gene transfer techniques in vitro and in vivo, and then, to investigate Ski expression in human hypertrophic scar and the relationship between Ski and TGF-β1/Smad dependent pathway using collected scar specimens of human.The main results and conclusions of the study are as followed:1. We demonstrated that Ski is the modulator in bidirectional regulation of TGF-β1–mediated proliferation in rat skin fibroblasts in previous experiment.Then, we founded that the persistently high expression of c-Ski in L929 cells involved in proliferative effect switching of TGF-bate1 from a dual role in fibroblast to one-directional role in L929 cells on this study, and confirmed that the high expression of c-Ski was to promote sustained growth of xenograft tumor in animal experiment; In addition, the TGF-β1 concentration continuously increased in L929 cells and xenograft tumor compared with FB. These results suggest that c-Ski is an important regulator of cell proliferation, and the continuously high expression of L929 cells may be an important reason for infinite proliferation, while high concentrations of secrete TGF-β1 in L929 cells is the basis of their infinite proliferation.2. We founded that enhanced Smad signaling by low expression of c-Ski and inhibited Smad signaling by high expression of c-Ski were the reasons of different roles in cell proliferation by high and low dose of TGF-β1 respectively in previous experiment. Then, we founded that although the high expression of c-Ski didn't change the phosphorylation levels of Smad2/3 in vitro, significantly inhibited the P21 expression of the downstream signaling factor of Smad in this study, suggesting that high expression of c-Ski involved in one-directional regulation of TGF-β1-mediated proliferation in L929 cells by adjusting the Smad signaling pathway. However, the low concentrations of TGF-β1 increased the expression of c-Ski and no significantly changed the expression of P21 after 24 hours of TGF-β1 treatment, while cell proliferation remained elevated, suggesting that high expression c-Ski may adjust the non-Smad pathway to promote proliferation of L929 cells.But, the speculation needed further experiment. These results not only show that the molecular mechanism of regulating proliferation by c-Ski is related with inhibition of Smad signaling system, but also lay the foundation for wound healing treatment through the regulation of cell proliferation by c-Ski.3. We demonstrated that Ski not only promotes cell proliferation but also inhibites the transformation of myofibroblasts and the secretion of collagen in FB in previous experiment.Then, we founded the relatively low expression of Ski and the high expression of TGF-β1/Smad signaling system, which not only lay the foundation to determine the role of Ski in hypertrophic scar but also further clarify the role of TGF-β1/Smad signal system in promoting pathological scar formation. According to the previous results, Ski transfection could reverse the collagen-promoting role of Smad3 in FB and inhibited Smad signal and scar tissue formation in vivo, our dates indicate decreased inhibition of TGF-β1/Smad signaling systems by low expression of the Ski is one of the reasons of pathological scar formation, which provides a useful clue to further study the role and mechanism of Ski in scar formation.4. Ski has the dual role of promoting healing and inhibiting scar formation in rat wound model by gene transfer, and also achieved the same result in the rabbit ear hypertrophic scar model in previous experiment.Then, we founded that Ski RNAi not only significantly decreased local Ski expression levels within the wound in rat model but also slowed healing and increased scar area, which confirmed the dual role of Ski Specificity created by its own. Our data not only strongly indicate that it is possible to achieve these seemingly contradictory effects with a single treatment but also provide a new way to intervention treatment for wound healing and scar.5. The pathology and immunohistochemistry analysis of wound healing in rats model showed that the expression of c-Ski increased in the new epithelium,and ski gene transfer further improved the expression of c-Ski, which significantly increased new epithelial and made epithelial crawled farther; Ski gene transfer increased the expressions of Cyclin D and Col l in the granulation tissue and their expressions consistented with the c-Ski, leading to promote proliferation of granulation tissue; In addition, CD11b (on behalf of granulocytes and macrophages and other inflammatory cell) positive cells decreased in ski gene transfer side. These results suggest that ski gene transfer significantly accelerated wound healing by relieving inflammation, accelerating re-epithelialization and increasing granulation tissue formation, and the proliferation regulated by c-Ski may play an important role in this process.6. The pathology and immunohistochemistry analysis of scar in rats model showed that collagen on the treated side was sparsely distributed, more mature and arranged in a net-like shape similar to the adjacent unwounded tissue, andα-SMA positive cells and the expression of Col l decreased.Our dates suggest that Ski improves the quality of scar formation by increasing levels of mature collagen and remodeling the tissue, which lay the foundation for further exploring its role in hypertrophic scar.7. The molecular analysis of wound healing in rat model showed ski gene transfer inhibited the expression levels of Smad2/3 phosphorylation and its downstream signal factor P21, suggesting that the molecular mechanism of the dual role of Ski was related with Smad dependent pathway; On the other hand, the expression levels of Smad2/3 and their's phosphorylation manly increased in the late healing and collagen synthesis increased in the early healing, suggesting the molecular mechanism of the dual role of Ski may be involved in non-Smad dependent pathway, which is also the focus of our further research and key to further elucidate the molecular mechanism of dual role of Ski.
|
PDF Full Text Request