口腔黏膜间充质干细胞存在及在口腔扁平苔藓中变化的初步研究
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摘要
口腔扁平苔藓(oral lichen planus, OLP)是一种常见的口腔黏膜病,其在口腔黏膜病中的发病率仅次于复发性口腔溃疡(recurrent aphthous ulcer, RAU)。该疾病常常迁延不愈,给病人造成巨大痛苦。由于口腔扁平苔藓病因不明,因此治疗以对症为主,经常使用免疫抑制剂,如糖皮质激素等。移植物抗宿主病(Graft-vs-host disease ,GVHD)是同种异体骨髓移植(bone marrow transplantation ,BMT)的一种常见的严重的并发症。口腔黏膜内的GVHD无论在临床还是组织学上与OLP都非常相似。虽然两者可能具有不同的抗原特异性,但是它们可能具有类似的免疫致病机理,同样的引起T淋巴细胞的密集浸润,基底细胞凋亡,上皮基底膜的破坏。因此通常认为关于其中一种疾病的病理生理学研究结果也能够给另一种疾病以提示。间充质干细胞(mesenchymal stem cells, MSC)属于成体干细胞,广泛分布于人体各个组织,不同组织来源的MSC具有异质性。MSC与胚胎干细胞相比避免了伦理学上的争议,并且由于其低免疫原型,异体移植常不引起明显的排斥反应,因此有着广阔的应用前景。人们在MSC研究的初期主要寄希望于其在组织工程以及再生医学等方面的应用,但实际上MSC第一次应用于临床是利用其免疫调节能力治疗GVHD,取得了较好的效果。MSC的免疫调节能力遂成为MSC研究的另一个热点。产生了众多的关于MSC治疗免疫相关疾病的基础和临床实验研究。在类风湿性关节炎、多发性硬化、移植物排斥以及自身免疫性心肌炎等的临床前实验动物模型中,MSC都展示出了良好的治疗效果。同时由于MSC具有免疫调节能力,其与机体的整体免疫平衡有关,其功能的异常可能与OLP发病有关。
由于GVHD与OLP之间存在密切的联系,我们猜测MSC同样也可能用于治疗OLP,并且口腔黏膜来源的MSC可能更具安全性。但是关于口腔黏膜是否存在MSC以及其生物学特性鲜见报道。MSC与OLP的发病是否相关也未见报道。
本实验分为两部分:
1.口腔黏膜内间充质干细胞的鉴定
目的:证明口腔黏膜内存在MSC,并且对其进行分离培养及生物学特性的研究。方法:利用大鼠为实验动物,采用标记滞留细胞(label retaining cells,LRC)技术,利用BrdU为标记物,检测大鼠口腔黏膜内可能存在的间充质干细胞;利用细胞贴壁法,分离培养人体正常口腔黏膜组织内的MSC,应用波形丝蛋白抗体(vimentin)、stro-1单克隆抗体、CD146(Mel-CAM)多克隆抗体、Oct3/4单克隆抗体、CD45单克隆抗体、Nestin单克隆抗体等检测所分离细胞的表面抗原表达。在体外条件下利用成脂和成骨诱导方法,研究所分离细胞的多向分化能力。结果:口腔黏膜内存在标记滞留细胞,利用细胞贴壁法能够从口腔黏膜中分离出一类细胞,抗原表达符合MSC的特征,例如Stro-1(+),CD146(+),CD45(-),同时我们分离出的细胞还表达Nestin。在成骨和成脂的诱导下,我们所分离的细胞具备向成骨细胞以及脂肪细胞分化的能力。结论:口腔黏膜内存在间充质干细胞,并且可以通过传统的细胞贴壁法进行分离及体外培养。同时口腔黏膜内的MSC与真皮中的MSC类似,可能具有向神经系统细胞分化的潜力。
2.MSC在OLP病变组织中的变化
目的:研究OLP组织内MSC的变化。方法:利用MSC的相对特异性标志Stro-1,CD146,Oct4等抗体检测OLP与正常组织内以上标志的表达改变。结果:Stro-1在正常组织以及OLP病变组织中均有表达,在正常组织中散在分布,集中于小血管周围。阳性细胞呈圆环状排列,符合血管周细胞的排列方式。Stro-1在OLP病变组织中主要集中于上皮基底膜下区,并且阳性表现强于正常组织。CD146在正常组织及OLP病变组织中都有表达。在正常组织中散在分布,而在OLP病变组织中主要分布于黏膜下层,固有层很少见阳性细胞,几乎全部为密集的淋巴细胞浸润所占据。阳性区域的整体分布较为弥散。但是在个别上皮萎缩较为严重的区域,上皮基底膜下方可见CD146的高表达。Oct4在正常组织以及OLP病变组织中均没有表达。结论:OLP组织与正常组织内存在MSC分布上的差别,损伤的基底膜下方增多的MSC可能是机体修复受损组织或调节过度免疫反应的一种代偿反应的结果。但增多MSC是否具备正常的功能还需进一步研究。
Oral lichen planus(OLP) is a common oral mucosal disease, whose incidence is the highest but recurrent aptha ulcer among oral mucosal diseases. OLP has protracted course of disease and usually brings patients great inconvenience. Because of the unknown of its causes, treatments are usually aimed at relieving the symptoms. Glucocorticoid is a common drug to treat OLP. Graft-vs-host disease(GVHD) is a common and severe complication of bone marrow transplantation(BMT). GVHD happening in oral mocusa is very like OLP both clinically and histologically. Although they may have different antigen specificity, they may share the same immunology pathogenic mechanism. They both lead to the infiltration of mass T lymphocytes, apoptosis of basal cells and the destruction of basal membrane. It is usually regarded that the research results about one of the two diseases can give light to the other disease. Mesenchymal stem cells(MSC) is a kind of adult stem cells(ASC). MSC broadly exist in organs and tissues. MSC from different parts is different. Because of the advantages of MSC, like no ethical disputes or low immunogenicity, it has broad prospect for applications. At first, MSC is expected to be used in tissure engineering and regeneration medicine, but acturally its first application in clinic is as an immunoregulant due to its immunoregulation properties and the result is encouraging. Consequently, the immunoregulation properties of MSC become a research warm spot. A lot of researches concerning the treatment applications of MSC as an immunoregulant for many immune relevant diseases come out. MSC has shown great effect in the preclinic animal models of some diseases, such as rheumatic arthritis, multiple sclerosis, graft rejection and so on. At the same time, because of the correlation between the immunoregulation properties of MSC and the balance of immune system, the malfunction of MSC may contribute to the pathogenesy of OLP.
Because GVHD and OLP are greatly associated, we suppose that MSC can be used to treat OLP too. And the MSC comes from oral mucosa itself may be safer. However, the existence of oral mucosal MSC is still unreported and whether there are some associations between MSC and the pathogenesy OLP is still unknown either.
This research can be devided into two main parts
1. Identification of the oral mucosal MSC
Objective: verify the existence of oral mucosal MSC and isolate them from oral mucosa. Cultivate them and study some basic bionomics of them. Methods: using label retaining cells(LRC) technique, label the rats’oral mucosa by BrdU and then test the LRC in the mucosa. Using the wall adhereing technique isolate the MSC from normal people’s buccal mucosa. Use antibodies such as vimentin, stro-1, CD146, Oct3/4, CD45 and Nestin to examine the antigen expression characters of the isolated cells. Use differention induction technology toward lipid and bone tissue ex vivo to test the potency of these cells for differentiating into mesenchymal tissues. Results: There are LRC in the rats’mucosa. We can isolate a kind of cells that are Stro-1(+),CD146(+)and CD45(-), which is in accordance with the antigen expression characters of MSC in other tissues. Besides, the cells also express Nestin. Under the differentiating induction conditions, the cells can be induced to differentiate into adipocytes and osteoblasts. Conclusions: There is MSC in oral mucosa and it can be easily isolated and cultivated by easy protocols. Like MSC in dermis, our cells may also be able to differentiate into cells in nervous system.
2. The changes of MSC in OLP tissue
Objective: Show the changes of MSC in OLP tissue. Method: Using the relatively specific antibodies for MSC such as Stro-1, CD146 and Oct4 to test the expression of MSC in OLP tissue and normal tissue. Results: Stro-1 is expressed in both tissues. Positive cells are dispersed and mainly reside surrounding small vessels. It is in accordance with the distribution of pericytes. Stro-1 positive cells in OLP tissue mainly concentrate beside along the basal membrane which is disrupted. CD146 is also expressed in both tissues. It is scattered in normal tissue but mainly reside in the submucous layer. But at some places just below the severely atrophic epithelium there is a high expression of CD146. Oct4 is negative in both tissues. Conlusions: The expression of MSC in normal tissue and OLP tissue is different. There are increased MSC just below the disrupted basal membrane. This may be a kind of compensation response. But whether these increased MSC has normal functions still need further investigation.
由于GVHD与OLP之间存在密切的联系,我们猜测MSC同样也可能用于治疗OLP,并且口腔黏膜来源的MSC可能更具安全性。但是关于口腔黏膜是否存在MSC以及其生物学特性鲜见报道。MSC与OLP的发病是否相关也未见报道。
本实验分为两部分:
1.口腔黏膜内间充质干细胞的鉴定
目的:证明口腔黏膜内存在MSC,并且对其进行分离培养及生物学特性的研究。方法:利用大鼠为实验动物,采用标记滞留细胞(label retaining cells,LRC)技术,利用BrdU为标记物,检测大鼠口腔黏膜内可能存在的间充质干细胞;利用细胞贴壁法,分离培养人体正常口腔黏膜组织内的MSC,应用波形丝蛋白抗体(vimentin)、stro-1单克隆抗体、CD146(Mel-CAM)多克隆抗体、Oct3/4单克隆抗体、CD45单克隆抗体、Nestin单克隆抗体等检测所分离细胞的表面抗原表达。在体外条件下利用成脂和成骨诱导方法,研究所分离细胞的多向分化能力。结果:口腔黏膜内存在标记滞留细胞,利用细胞贴壁法能够从口腔黏膜中分离出一类细胞,抗原表达符合MSC的特征,例如Stro-1(+),CD146(+),CD45(-),同时我们分离出的细胞还表达Nestin。在成骨和成脂的诱导下,我们所分离的细胞具备向成骨细胞以及脂肪细胞分化的能力。结论:口腔黏膜内存在间充质干细胞,并且可以通过传统的细胞贴壁法进行分离及体外培养。同时口腔黏膜内的MSC与真皮中的MSC类似,可能具有向神经系统细胞分化的潜力。
2.MSC在OLP病变组织中的变化
目的:研究OLP组织内MSC的变化。方法:利用MSC的相对特异性标志Stro-1,CD146,Oct4等抗体检测OLP与正常组织内以上标志的表达改变。结果:Stro-1在正常组织以及OLP病变组织中均有表达,在正常组织中散在分布,集中于小血管周围。阳性细胞呈圆环状排列,符合血管周细胞的排列方式。Stro-1在OLP病变组织中主要集中于上皮基底膜下区,并且阳性表现强于正常组织。CD146在正常组织及OLP病变组织中都有表达。在正常组织中散在分布,而在OLP病变组织中主要分布于黏膜下层,固有层很少见阳性细胞,几乎全部为密集的淋巴细胞浸润所占据。阳性区域的整体分布较为弥散。但是在个别上皮萎缩较为严重的区域,上皮基底膜下方可见CD146的高表达。Oct4在正常组织以及OLP病变组织中均没有表达。结论:OLP组织与正常组织内存在MSC分布上的差别,损伤的基底膜下方增多的MSC可能是机体修复受损组织或调节过度免疫反应的一种代偿反应的结果。但增多MSC是否具备正常的功能还需进一步研究。
Oral lichen planus(OLP) is a common oral mucosal disease, whose incidence is the highest but recurrent aptha ulcer among oral mucosal diseases. OLP has protracted course of disease and usually brings patients great inconvenience. Because of the unknown of its causes, treatments are usually aimed at relieving the symptoms. Glucocorticoid is a common drug to treat OLP. Graft-vs-host disease(GVHD) is a common and severe complication of bone marrow transplantation(BMT). GVHD happening in oral mocusa is very like OLP both clinically and histologically. Although they may have different antigen specificity, they may share the same immunology pathogenic mechanism. They both lead to the infiltration of mass T lymphocytes, apoptosis of basal cells and the destruction of basal membrane. It is usually regarded that the research results about one of the two diseases can give light to the other disease. Mesenchymal stem cells(MSC) is a kind of adult stem cells(ASC). MSC broadly exist in organs and tissues. MSC from different parts is different. Because of the advantages of MSC, like no ethical disputes or low immunogenicity, it has broad prospect for applications. At first, MSC is expected to be used in tissure engineering and regeneration medicine, but acturally its first application in clinic is as an immunoregulant due to its immunoregulation properties and the result is encouraging. Consequently, the immunoregulation properties of MSC become a research warm spot. A lot of researches concerning the treatment applications of MSC as an immunoregulant for many immune relevant diseases come out. MSC has shown great effect in the preclinic animal models of some diseases, such as rheumatic arthritis, multiple sclerosis, graft rejection and so on. At the same time, because of the correlation between the immunoregulation properties of MSC and the balance of immune system, the malfunction of MSC may contribute to the pathogenesy of OLP.
Because GVHD and OLP are greatly associated, we suppose that MSC can be used to treat OLP too. And the MSC comes from oral mucosa itself may be safer. However, the existence of oral mucosal MSC is still unreported and whether there are some associations between MSC and the pathogenesy OLP is still unknown either.
This research can be devided into two main parts
1. Identification of the oral mucosal MSC
Objective: verify the existence of oral mucosal MSC and isolate them from oral mucosa. Cultivate them and study some basic bionomics of them. Methods: using label retaining cells(LRC) technique, label the rats’oral mucosa by BrdU and then test the LRC in the mucosa. Using the wall adhereing technique isolate the MSC from normal people’s buccal mucosa. Use antibodies such as vimentin, stro-1, CD146, Oct3/4, CD45 and Nestin to examine the antigen expression characters of the isolated cells. Use differention induction technology toward lipid and bone tissue ex vivo to test the potency of these cells for differentiating into mesenchymal tissues. Results: There are LRC in the rats’mucosa. We can isolate a kind of cells that are Stro-1(+),CD146(+)and CD45(-), which is in accordance with the antigen expression characters of MSC in other tissues. Besides, the cells also express Nestin. Under the differentiating induction conditions, the cells can be induced to differentiate into adipocytes and osteoblasts. Conclusions: There is MSC in oral mucosa and it can be easily isolated and cultivated by easy protocols. Like MSC in dermis, our cells may also be able to differentiate into cells in nervous system.
2. The changes of MSC in OLP tissue
Objective: Show the changes of MSC in OLP tissue. Method: Using the relatively specific antibodies for MSC such as Stro-1, CD146 and Oct4 to test the expression of MSC in OLP tissue and normal tissue. Results: Stro-1 is expressed in both tissues. Positive cells are dispersed and mainly reside surrounding small vessels. It is in accordance with the distribution of pericytes. Stro-1 positive cells in OLP tissue mainly concentrate beside along the basal membrane which is disrupted. CD146 is also expressed in both tissues. It is scattered in normal tissue but mainly reside in the submucous layer. But at some places just below the severely atrophic epithelium there is a high expression of CD146. Oct4 is negative in both tissues. Conlusions: The expression of MSC in normal tissue and OLP tissue is different. There are increased MSC just below the disrupted basal membrane. This may be a kind of compensation response. But whether these increased MSC has normal functions still need further investigation.
引文
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