Ski在细胞增殖、创伤愈合及瘢痕形成中作用与意义研究

发布时间：2018-05-03 19:25

本文选题：Ski + Smad3/2　；参考：《第三军医大学》2011年博士论文

【摘要】：细胞增殖是生命的基本特征,不但参与个体的发育、机体的修复等生理过程,而且参与难愈、瘢痕形成、肿瘤发生等病理过程。因此,合理调控细胞增殖,不仅有利于促进组织修复,而且对提高修复质量,抑制瘢痕乃至肿瘤形成具有重要意义。创伤愈合发生率极高,且缓慢的愈合过程和不良的愈合结果(例如增生性瘢痕和瘢痕疙瘩)是人类创伤愈合领域所面临的两大难题。尽管目前促进创伤愈合和抑制过度瘢痕形成的治疗方法有很多,但是,在促愈和瘢痕治疗之间还存在着一定的矛盾:如生长因子类促愈治疗的同时具有加重瘢痕形成的风险;对于瘢痕的治疗目前主要依赖经验性治疗,且治疗效果不好,有的副作用大甚至影响愈合。另外,由于存在创伤后创面受体表达容易发生缺乏及变异、创面局部蛋白酶浓度升高、蛋白酶和其抑制因子比例失衡等因素,使受体类治疗途径难以达到理想促愈效果。因此,寻求新的非受体作用途径的促增殖剂,对促进创伤愈合尤其是放创复合伤、大面积创伤、烧伤等难愈伤口有着十分重要的意义。 c-Ski作为一种鸟类癌基因v-ski的细胞内同源物蛋白,在各种动物中有较高的同源性,在人体中称之为Ski。Ski在不同类型的组织和细胞中分布广泛,不仅可以抑制Smad3/2活性,还可调节多种核因子的转录活性,如促进NFI(促核因子I)的转录、抑制癌基因Rb的转录等,从而参与神经系统发育、造血细胞的增殖和分化、肿瘤发生、组织再生等多种生理、病理过程。我们前期研究发现Ski是一创伤修复相关基因,继而,在大鼠切割伤模型上的转基因实验证实Ski具有在促愈的同时减轻瘢痕形成的双重作用,并在兔耳增生瘢痕模型上得到了验证。并且Ski表现出比现有促愈措施或抗瘢痕方法更好的效果,这不仅为同时实现促进创伤愈合和抑制瘢痕形成这看似矛盾的效应成为可能,而且提示Ski可能是一个具有治疗难愈性伤口和增生性瘢痕的临床应用前景的分子。已有文献及前期研究表明Ski可能是一个细胞增殖的调节因子,并具有促进愈合和减轻瘢痕的双重作用,但是,其在细胞增殖、创伤愈合及瘢痕形成中的作用机制还不清楚,尤其,作为原癌基因的Ski在多种人类恶性肿瘤中显著表达,在这种细胞无限增殖病理过程中作用如何所知不多,不但影响其生物学效应的阐明,也严重制约了其在创伤治疗中的应用前景。不过,已有研究表明Smad3具有抑制细胞增殖的作用,表明抑制Smad3可具有促进增殖作用;同时,1999年美国NIH小组首次报道,Smad3基因敲除小鼠的伤口愈合速率增加了2倍,这一结果证实了抑制Smad3具有促愈作用;另外,近些年研究表明TGF-beta信号系统尤其是Smad3/2信号因子(他们转导的信号系统也称为TGF-β1/Smad依赖途径)高活性是导致瘢痕形成的重要原因,因此,抑制Smad3/2成为了减轻瘢痕形成的新途径。而且,TGF-β1/Smad依赖途径抑制细胞增殖作用从而使TGF-β1具有抗肿瘤发生作用,此途径的功能障碍被认为是TGF-β1抗肿瘤作用丧失的原因。这些都提示Ski基因作为Smad3、Smad2的辅阻遏子,其可能通过调节Smad3/2而在促进增殖、伤口愈合、瘢痕形成甚至肿瘤发生等方面发挥作用,是否如此需要进一步的证实。为此,本课题在前期研究的基础上,分三个部分进行如下研究:(1)Ski在细胞增殖及TGF-β1调节增殖中的作用研究:本部分研究采用原代成纤维细胞(Fibroblast,FB)和纤维肉瘤细胞(L929),通过体外实验,在明确TGF-β1对FB和L929增殖调节作用不同的基础上,利用RNAi干扰c-Ski后结合TGF-β1刺激来验证Ski在其调节增殖中所发挥的作用,并探讨其促进增殖的机理是否与其抑制Smad3/2有关;进一步,采用纤维肉瘤细胞移植瘤模型,对TGF-β1通过调节Ski来促进肿瘤生长进行验证;(2)Ski在创伤愈合中的双重作用和机理研究:首先,采用大鼠创伤及兔耳增生性瘢痕模型来观察Ski促愈和减轻瘢痕的双重作用,并采用RNAi干扰质粒局部皮下注射来验证Ski双重作用的特异性;进一步,利用Ski裸质粒治疗大鼠创伤愈合模型,探讨Ski双重作用的分子机制是否与调节TGF-β1/Smad依赖途径有关;(3)Ski及TGF-β1/Smad依赖途径相关信号因子在人体增生性瘢痕中的表达观察:利用收集的人体瘢痕标本来检测Ski及其相关调节因子的表达规律,以此探讨Ski在人体增生性瘢痕中的表达及与TGF-β1/Smad依赖途径的关系。主要结果与结论如下: 1.在前期发现c-Ski参与TGF-β1双向调节FB细胞增殖变化作用的基础上,本研究采用体外细胞实验发现,c-Ski在L929细胞中持续高表达与TGF-β1对FB和L929的增殖调节存在着双向到单向的转变有关,并在动物移植瘤实验中证实高表达的c-Ski促进移植瘤持续生长;此外,实验发现与FB不同的是,L929细胞自分泌及移植瘤组织中的TGF-β1浓度持续增高。上述结果提示c-Ski是重要的细胞增殖调节因子,且其持续、过高表达可能是L929肿瘤细胞持续增殖的重要原因,而L929肿瘤细胞分泌高浓度TGF-β1是其发挥持续增殖作用的基础。Ski的这一重要作用可能是创伤后细胞增殖在创伤修复完成后能自动停止而肿瘤细胞不能停止的机制之一。 2.前期实验发现c-Ski的低表达增强Smad信号而c-Ski高表达抑制Smad信号是高低浓度TGF-β1不同调节细胞增殖作用不同的原因。本研究通过L929细胞体外实验发现,高表达的Ski虽然没有改变Smad2/3的磷酸化水平,但可显著抑制其下游信号因子P21的表达水平,提示高表达的c-Ski可通过调节Smad途径参与TGF-β1的单向促进L929增殖作用;不过,实验中还发现低浓度TGF-β1刺激24小时后,c-Ski表达水平增高但P21表达水平无明显变化,而细胞增殖仍旧增高,提示高表达的c-Ski促进L929增殖作用存在调节非Smad途径的可能。上述结果不仅表明c-Ski调节增殖作用的分子机制与抑制Smad信号系统有关,而且为其能通过调节细胞增殖而参与调节愈合奠定基础。 3.细胞研究发现Ski不但具有促增殖作用还有调节成纤维细胞向肌成纤维细胞转化和胶原分泌等作用。本实验通过对人体瘢痕标本的检测,发现在病理性瘢痕中Ski表达相对较低而TGF-β1/Smad信号系统高表达,这不但为确定Ski在瘢痕形成中的作用提供了线索,也进一步验证了TGF-β1/Smad信号系统在病理性瘢痕中的促进作用。结合前期转染Ski实验可逆转Smad3对成纤维细胞的促胶原分泌和抑制组织Smad信号及瘢痕形成的结果,表明低表达的Ski降低了对TGF-β1/Smad信号系统的抑制是病理性瘢痕形成的原因之一,这为今后Ski在瘢痕形成中的作用及机制研究提供了有用的线索。 4.前期通过转ski基因实验在大鼠创伤模型中发现Ski具有促愈和减轻瘢痕形成的双重作用,且在兔耳增生性瘢痕模型上也取得同样的结果。本研究采用RNAi方法在大鼠创伤模型中发现干扰不仅显著降低Ski表达水平,同时伤口愈合减慢和愈后瘢痕面积增大,这反向确认了Ski双重作用来自于其本身。这不仅使同时实现促进创伤愈合和抑制瘢痕形成这看似矛盾的效应成为可能,也为创伤愈合和瘢痕的早期干预性治疗提供了新的思路。 5.大鼠创伤模型愈合过程的病理学及免疫组化分析结果表明,新生上皮中c-Ski表达增高,且转ski基因使其表达进一步提高的同时使新生上皮明显增多、爬行更远;转ski基因侧肉芽组织增多,Cyclin D和Col l表达增高且与Ski表达增高一致;另外,转ski基因侧创面内CD11b(代表粒细胞和巨噬细胞等炎性细胞标志)阳性细胞减少。上述结果提示转ski基因除调节增殖作用外,还可可通过减轻炎症反应、加快上皮化、增加肉芽形成而达到促进愈合作用。 6.大鼠创伤模型愈后瘢痕的病理学及免疫组化分析结果表明,转ski基因侧胶原排布似网状,瘢痕胶原成熟度高更接近正常皮下胶原,而且α-SMA阳性细胞减少、Col l表达降低,提示转ski基因可通过减少胶原沉积和加快瘢痕组织重构而达到抑制瘢痕形成作用。 7.大鼠创伤模型的分子检测结果表明,转ski基因抑制了Smad3/2的磷酸化水平及其下游信号因子P21的表达水平,提示Ski双重作用的分子机制与Smad依赖途径相关;另一方面,Smad3/2表达及其磷酸化水平在愈合后期增高,且愈合早期胶原合成增高,则提示Ski双重作用的分子机制存在调节非Smad依赖途径的可能,这可能是我们今后继续研究的重点和进一步阐明Ski双重作用分子机制的关键。
[Abstract]:Cell proliferation is the basic characteristic of life. It not only participates in the physiological processes of individual development, body repair and other physiological processes, but also participates in the pathological processes such as hard healing, scar formation and tumor occurrence. Therefore, the rational regulation of cell proliferation is beneficial not only to the promotion of tissue repair, but also to the improvement of the quality of repair and the formation of scar and even the formation of tumor. The high incidence of wound healing, the slow healing process and poor healing results, such as hypertrophic scars and keloids, are the two major challenges in the field of human healing. Although there are many treatments to promote wound healing and inhibit hypertrophic scar formation, there is still a presence between healing and scar therapy. Certain contradictions: for example, growth factor therapy has the risk of aggravating scar formation while the treatment of scar is mainly dependent on empirical treatment, and the treatment effect is not good, some side effects may even affect healing. In addition, due to the lack and variation of the expression of the wound receptor, the local protease in the wound surface is easy to occur. The increase in concentration and the imbalance of the proportion of protease and its inhibitory factors make it difficult for the receptor therapy pathway to achieve the ideal healing effect. Therefore, it is of great significance to seek a new non receptor pathway to promote the healing of wound healing, especially wound healing, trauma, and burn wounds.
C-Ski, as an intracellular homologous protein of avian oncogene v-Ski, has high homology in various animals. It is called Ski.Ski in different types of tissues and cells. It not only inhibits Smad3/2 activity, but also regulates the transcriptional activity of many kinds of nuclear factors, such as promoting the transcription of NFI (nuclear factor I) and inhibiting the transcription of NFI (nuclear factor I). The transcription of the oncogene Rb and so on, thus participating in the development of the nervous system, the proliferation and differentiation of the hematopoietic cells, the occurrence of tumor and the tissue regeneration, and other physiological and pathological processes. Our previous study found that Ski is a wound repair related gene, and then the transgenic experiment on the rat model of cutting injury confirmed that Ski has the reduction of scar while reducing the scar while reducing the scar. The dual effect of the formation is verified in the rabbit ear hypertrophic scar model. And Ski shows a better effect than the existing healing measures or anti scar methods. This is not only possible to achieve the paradoxical effect of promoting wound healing and inhibiting scar formation at the same time, but also suggests that Ski may be a refractory injury. Molecular markers for the clinical application of oral and hypertrophic scars.
The previous literature and previous studies have shown that Ski may be a regulator of cell proliferation and has the dual role of promoting healing and reducing scar. However, the mechanism of its role in cell proliferation, wound healing and scar formation is not clear, especially, as the proto oncogene Ski is expressed in a variety of human malignant tumors, in this case The role of cell unlimited proliferation in the pathological process is not well known, which not only affects the explanation of its biological effects, but also seriously restricts its application in the treatment of trauma. However, some studies have shown that Smad3 has the effect of inhibiting cell proliferation, indicating that inhibition of Smad3 can promote proliferation. At the same time, the United States NIH team in 1999 was the first It is reported that the rate of wound healing in Smad3 knockout mice increased by 2 times, and this result confirmed that inhibition of Smad3 has a promoting effect. In addition, recent studies have shown that the high activity of the TGF-beta signal system, especially the Smad3/2 signal factor, which is also called the TGF- beta 1/Smad dependent pathway, is an important cause of scar formation. Therefore, inhibition of Smad3/2 has become a new way to reduce scar formation. Furthermore, the TGF- beta 1/Smad dependent pathway inhibits cell proliferation and makes TGF- beta 1 antitumor. The dysfunction of this pathway is considered to be the cause of the loss of TGF- beta 1. These suggest that the Ski gene is a repressor of Smad3, Smad2, and it can be used as a repressor. Whether Smad3/2 can play a role in promoting proliferation, wound healing, scarring and even tumor formation can be further verified.
To this end, on the basis of previous research, three parts are studied as follows: (1) the role of Ski in cell proliferation and TGF- beta 1 regulation of proliferation: this part studies the use of primary fibroblasts (Fibroblast, FB) and fibrosarcoma cells (L929). Through in vitro experiments, the regulation of TGF- beta 1 on the proliferation of FB and L929 is different. On the basis of RNAi interference c-Ski and TGF- beta 1 stimulation to verify the role of Ski in its regulation and proliferation, and to explore whether its mechanism of promoting proliferation is related to its inhibition of Smad3/2; further, the fibrosarcoma cell transplantation tumor model is used to verify the growth of the tumor by regulating Ski to promote the growth of the tumor; (2) Ski in the wound healing. The double role and mechanism of the combination: first, the double effects of Ski promoting and reducing scar were observed by rat trauma and rabbit ear hypertrophic scar model, and the specificity of Ski double action was verified by RNAi interfering plasmids. Further, Ski naked plasmids were used to treat the wound healing model of rats, and the double action of Ski was discussed. Whether the molecular mechanism is related to the regulation of TGF- beta 1/Smad dependence; (3) the expression of Ski and TGF- beta 1/Smad dependent signaling factors in human hypertrophic scars: to detect the expression of Ski and its related regulatory factors by using collected human cicatricial specimens to explore the expression of Ski in human hypertrophic scars and the expression of Ski in human hypertrophic scars. The main results and conclusions are as follows: TGF- 1/Smad dependent pathway.
1. in the early stage, we found that c-Ski participates in the two-way regulation of FB cell proliferation by TGF- beta 1.
In vitro cell test, the persistent high expression of c-Ski in L929 cells was related to the bidirectional to unidirectional transformation of TGF- beta 1 to the proliferation regulation of FB and L929. In the animal transplantation tumor experiment, the high expression of c-Ski promoted the sustained growth of the transplanted tumor. In addition, the experiment found that the L929 cell autocrine and the transplant tumor group were different from the FB. The concentration of TGF- beta 1 in the fabric continues to increase. These results suggest that c-Ski is an important cell proliferation regulator, and its persistence, high expression may be an important reason for the sustained proliferation of L929 tumor cells, and the secretion of high concentration of TGF- beta 1 from L929 tumor cells is the important role of the basal.Ski for its continuous proliferation. Cell proliferation is one of the mechanisms by which cell proliferation can stop automatically and the tumor cells can not stop after wound healing.
2. earlier experiments found that the low expression of c-Ski enhanced the Smad signal and the high expression of c-Ski was the reason for the different proliferation of TGF- beta 1 cells with high and low concentration. This study found that the high expressed Ski could inhibit the downstream signal factor P21 significantly, although the high expressed Ski did not change the phosphorylation of Smad2/3. The expression level suggested that high expression of c-Ski could contribute to the proliferation of L929 by regulating Smad pathway in TGF- beta 1. However, in the experiment, the expression level of c-Ski was increased but the expression level of P21 was not obviously changed after 24 hours of low concentration of TGF- beta 1, but the proliferation of cells was still higher, suggesting that the high expression of c-Ski promoted the effect of L929 proliferation. It is possible to regulate the non Smad pathway. These results not only indicate that the molecular mechanism of the proliferation of c-Ski is related to the inhibition of the Smad signal system, but also lays the foundation for its involvement in regulating the healing of cells by regulating cell proliferation.
The 3. cell study found that Ski not only promotes proliferation but also regulates the transformation of fibroblasts into myofibroblast and collagen secretion. This experiment shows that the expression of Ski is relatively low in pathological scars and high expression of TGF- beta 1/Smad signal system in pathological scars, which not only determines the formation of Ski in scar formation. The effect of TGF- beta 1/Smad signaling system in pathological scar was further verified. Ski experiment combined with pre transfection could reverse the collagen secretion of Smad3 and inhibit the formation of Smad signal and scar formation of tissue, indicating that low expression of Ski reduced the inhibition of TGF- beta 1/Smad signal system. It is one of the causes of pathological scar formation, which provides a useful clue for the research of the role and mechanism of Ski in scar formation in the future.
4. in the early stage of the ski gene experiment, we found that Ski had double effects on promoting and alleviating scar formation in the rat model, and the same results were obtained in the rabbit ear hypertrophic scar model. This study found that interference not only significantly reduced the level of Ski expression, but also slowed the healing of wound healing in the rat model of traumatic hypertrophic scar. The increase in scar area, which confirms that the double effect of Ski comes from itself, not only makes it possible to realize the paradoxical effect of promoting wound healing and inhibiting scar formation, but also provides new ideas for the early intervention treatment of wound healing and scar.
The pathological and immunohistochemical results of the healing process of the 5. rat models showed that the expression of c-Ski in the newborn epithelium was increased, and the expression of ski gene was increased and the expression of the new epithelium was increased, and the ski gene lateral granulation tissue was increased, the expression of Cyclin D and Col l increased and the expression of Ski was increased and the expression of Ski was higher. The positive cells of CD11b (the inflammatory cell markers such as granulocyte and macrophage) in the ski gene side were reduced. The results suggested that the transfer of ski gene, in addition to the regulation of proliferation, also accelerated the epithelialization by alleviating the inflammatory response, and increased the formation of granulation to promote the more cooperative use.
The pathological and immunohistochemical results of the healing scar of the 6. rat models showed that the reticular formation of the ski gene was similar to the normal subcutaneous collagen, and the -SMA positive cells decreased and the expression of Col l decreased. It suggested that the transfer of ski gene could be achieved by reducing the deposition of collagen and accelerating the reconstruction of scar tissue. Inhibition of scar formation.
The molecular detection results of the 7. rat models showed that the ski gene inhibited the phosphorylation level of Smad3/2 and the expression level of the downstream signal factor P21, suggesting that the molecular mechanism of the double action of Ski was related to the Smad dependent pathway; on the other hand, the expression of Smad3/2 and its phosphorylation were increased at the later stage of healing, and the collagen synthesis was increased at the early stage of healing. It is suggested that the molecular mechanism of the double action of Ski has the possibility of regulating non Smad dependence, which may be the key to further study and further clarify the mechanism of Ski double action.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

【参考文献】