瘢痕疙瘩成纤维细胞Fas及P53蛋白凋亡缺陷的相关研究
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摘要
研究背景及目的:
对于瘢痕疙瘩来说,目前尚无有效的治疗手段,其综合治疗的效果也不理想,存在较高的复发率。一般认为瘢痕疙瘩成纤维细胞属于非正常细胞,与肿瘤细胞类似,虽然组织内存在大量凋亡的细胞,但是瘢痕疙瘩成纤维细胞为多克隆来源,体内存在不同表型的成纤维细胞,经体外培养后细胞变成单一表型,而且这种细胞不易凋亡。寻找瘢痕疙瘩成纤维细胞凋亡缺陷机制及有效诱导其凋亡的方法对瘢痕疙瘩治疗有重要意义。
基于以上观点,课题组致力于研究瘢痕疙瘩成纤维细胞凋亡缺陷的机制,探索瘢痕疙瘩的基因治疗,寻找有效的瘢痕疙瘩治疗方法。通过对瘢痕疙瘩成纤维细胞p53基因及fas基因系统研究后发现:1.瘢痕疙瘩病人血细胞中p53基因第72位密码子Pro等位基因频率明显高于Arg等位基因频率,与Arg/Arg及Arg/Pro基因型人群相比,Pro/Pro基因型人群易患瘢痕疙瘩。血细胞中p53基因第72位密码子多态性位点的基因型检测对判断瘢痕疙瘩高危个体具有指导意义,并开发出了可用于普通人群的p53基因第72位密码子基因多态性检测试剂盒。2.Fas单克隆抗体在ng-μg水平即可迅速诱导增生性瘢痕成纤维细胞的凋亡,而在相同浓度下,瘢痕疙瘩成纤维细胞对此却无反应。3.增生性瘢痕成纤维细胞Fas表达较低,但瘢痕疙瘩成纤维细胞Fas却呈高表达,两者间差异显著,瘢痕疙瘩细胞的凋亡异常不是由于缺乏受体所导致的。4.瘢痕疙瘩Fas介导死亡信号传导阻滞,而且阻断的位点位于上游。5.携带正常人fas基因的脂质体及重组腺病毒均能有效地将fas基因导入,并且能使目的基因在靶细胞内完整且有效的表达,瘢痕疙瘩细胞Fas通道能得以稳定重建,且通道重建后瘢痕疙瘩细胞增殖-凋亡状况的失衡得到了明显的改善。
通过以上研究结果,我们对瘢痕疙瘩的发病机制与p53基因及fas基因的关系有了比较充分的了解,但是有些问题还值得我们进一步探讨。
p53基因外显子4第72编码子基因多态性是近年研究的热点,不同的基因型与多种肿瘤发病相关,其产物对细胞凋亡有不同的影响,局部组织基因型存在相互变异的现象,而且Arg等位基因占优势的肿瘤预后明显好于Pro等位基因占优势的肿瘤。瘢痕疙瘩类似良性肿瘤,患者局部组织基因多态性有无变异,不同的基因型对蛋白的表达有无影响,与临床表型的关系如何,这些都是我们必须回答的问题。
与增生性瘢痕相比,瘢痕疙瘩成纤维细胞Fas蛋白表达强阳性,而经体外培养的瘢痕疙瘩成纤维细胞FasMcAb不能有效诱导凋亡,分析其中的原因:其一为Fas蛋白结构异常,导致不能有效诱导凋亡。另外Fas蛋白是一种糖蛋白,只有糖基化的蛋白才能有效表达于细胞膜上,蛋白糖基化的程度与功能存在明显相关。因此我们采用Western Blot法检测病理性瘢痕成纤维细胞Fas蛋白糖基化水平,PI染色、流式细胞术检测成纤维细胞FasMcAb作用后的凋亡率,分析Fas蛋白糖基化水平与FasMcAb诱导凋亡的相关性,并利用蛋白糖基化抑制剂衣霉素(Tunicamycine,TM)抑制成纤维细胞Fas蛋白糖基化,观察成纤维细胞Fas蛋白糖基化水平变化后对凋亡的影响。
目前临床上没有确切有效的瘢痕疙瘩治疗方法,转染野生型fas基因能有效诱导瘢痕疙瘩成纤维细胞凋亡,具有广阔的前景,但是尚不能直接用于病人,短期内主要还是研究药物治疗为主。在各种诱导凋亡的信号转导通路中,受体通路与线粒体通路是2条基本通路。缺氧可有效启动线粒体依赖的caspases激活途径诱导凋亡,顺铂可同时启动Fas受体途径及线粒体依赖的caspases激活途径诱导凋亡,这在冠心病发病机制的研究中及肿瘤的治疗中已进行了广泛的研究,但在瘢痕疙瘩领域,无论在临床治疗或实验研究中,除基因转染外,尚未见报道有效的诱导瘢痕疙瘩成纤维细胞凋亡的方法。因此我们利用缺氧及顺铂处理瘢痕疙瘩成纤维细胞,观察是否能有效诱导其凋亡。
本实验在课题组先前研究结果的基础上再次探讨凋亡基因p53及fas,以组织标本及体外培养成纤维细胞为研究对象,旨在阐明它们在瘢痕疙瘩不同部位成纤维细胞凋亡过程中扮演的角色以及功能缺陷的可能机制,并对顺铂及缺氧诱导瘢痕疙瘩成纤维细胞凋亡的机制进行了初步探讨,为今后这方面的治疗工作进行了初步研究。
方法:
1.以正常人为对照,收集35例瘢痕疙瘩病人血标本及组织标本,瘢痕疙瘩组织标本分中央部和周边部。提取血标本及组织标本的DNA,多聚酶链式反应(PCR)扩增p53基因外显子4,并进行基因测序;采用免疫组织化学方法检测P53蛋白的表达。
2.采用组织培养技术,以手术切除的瘢痕疙瘩及增生性瘢痕为组织来源的体外培养的成纤维细胞为研究对象,Western Blot法及流式细胞术检测衣霉素处理前后成纤维细胞Fas糖蛋白的表达及FasMcAb诱导凋亡率的变化。
3.顺铂及缺氧诱导瘢痕疙瘩成纤维细胞凋亡,流式细胞术检测检测成纤维细胞凋亡率的变化,Western Blot法检测Bcl-2和Bax蛋白表达的变化。
结果:
1.p53基因72位编码子遗传基因型有三种:CCC/CCC、CCC/CGC、CGC/CGC,其产物分别是Pro/Pro、Pro/Arg、Arg/Arg。瘢痕疙瘩组Pro/Pro、Pro/Arg、Arg/Arg三种遗传基因型分布与临床表型密切相关,大体积瘢痕疙瘩患者遗传基因型为Pro/Pro,局部瘢痕组织则存在由周边的Pro向中央Arg变异的趋势。瘢痕疙瘩P53蛋白表达位于胞浆中,阳性率35/35,与瘢痕疙瘩临床表型及部位密切相关。正常皮肤无表达。
2.增生性瘢痕成纤维细胞Fas蛋白糖基化水平高于瘢痕疙瘩成纤维细胞,两组成纤维细胞FasMcAb作用后凋亡率与Fas蛋白糖基化成正相关,衣霉素可明显降低成纤维细胞Fas蛋白糖基化水平及FasMcAb作用后凋亡率。
3.缺氧条件下培养瘢痕疙瘩成纤维细胞,其凋亡率及Bcl-2和Bax蛋白的表达无明显变化。顺铂作用后瘢痕疙瘩成纤维细胞凋亡率增加,Bcl-2的表达下调,Bax的表达增加。
结论:
1.瘢痕疙瘩临床表型与p53基因外显子4第72编码子基因多态性密切相关,遗传基因型为Pro/Pro患者一般表现为大体积、多发;瘢痕疙瘩患者局部组织基因型有Pro向Arg变异的趋势,而大体积瘢痕疙瘩患者P53蛋白由边缘部的阴性表达向中央部的阳性表达转变,基因多态性变异与蛋白表达一致;Arg基因型编码的蛋白有利于瘢痕疙瘩成纤维细胞凋亡及瘢痕疙瘩的成熟。
2.FasMcAb诱导病理性瘢痕成纤维细胞凋亡与成纤维细胞Fas蛋白糖基化水平成正相关,而衣霉素可显著降低成纤维细胞Fas蛋白糖基化水平。
3.顺铂可诱导瘢痕疙瘩成纤维细胞凋亡,其作用机制可能与Bcl-2和Bax等凋亡调控基因的表达有关。
Background
Accumulating evidence indicates that unbalance between proliferation and apoptosis of fibroblasts contributes mainly to the formation of keloid and hypertrophic scar. So far, treatment for keloid is less effective. Fibroblasts from keloid are know to be abnormal ones, their apoptosis function was impaired, thus it is necessary for us to find out the cause, in which apoptotic function was impaired.
Our previous study has demonstrated that P53 and Fas were involved in keloid formation. Detection of polymorphisms in p53 gene codon 72 was important for deciding who are susceptible to keloid. Monoclonal antibody against Fas at a dose of ng-μg induced the apoptosis in Proliferative fibroblasts derived from hypertrophic scar, not in the fibroblasts from keloid. Fas protein expression was decreased in hypertrophic scar and increased in keloid. Abnormal apoptosis occurred in keloid was not associated with the loss of Fas receptors. Fas-mediated death signals were blocked at the upstream. Transfection of normal fas gene into target cells could improve the unbalance of proliferation and apoptosis in the fibroblast.
Polymorphisms in p53 gene codon 72 were intensively investigated. Different gene types of p53 are involved in the development of tumor. The products expressed by different gene typed of P53 may affect the cell apoptosis of fibroblast. However, Polymorphisms in p53 gene and protein expression in the local tissue from keloid are unclear.
Objective
The present study was done to observe the relationship between clinical phenotype of keloid and the polymorphisms in p53 gene codon 72, and explode the role of gene variation of p53 in the keloid formation and possible mechanism of impaired apoptosis. Meanwhile, we also investigated the effects of cisplatin and hypoxia on the apoptosis of fibroblasts from keloid.
Methods
1. The tissue and blood samples were taken from patients with keloid (n=35) and healthy individuls (n=26). The diagnosis of keloid was based on clinical manifestation, histological examination. The samples from patients with keloids were divided into three groups according to clinical phenotype. DNA of the tissue and blood samples was extracted. The tissue samples of the keloid were divided into peripheral and central areas. The expression of P53 in protein levels was detected by immunostaining, and DNA sequencing was performed with sequence-based typing (SBT).
2. Fibroblasts from hypertrophic scar and keloid were cultured. Western Blot and Flow cytometry were used to detect the effect of turicamycin on Fas protein expression and FasMcAb-induce apoptosis.
3. Cisplatin and hypoxia induced the apoptosis of fibroblasts from keloid. Flow cytometry were used to detect the rate of apoptois. Western Blot was used to examine the expression of Bcl-2 and Bax.
Results
1. Immunostaining results revealed that P53 was detectable in all the keloid samples. Three types of gene variation of p53 codon 72, including Pro/Pro, Pro/Arg, Arg/Arg were observed in keloids, and associated with clinical phenotype of keloids. Arg polymorphic variants of P53 were increased from the peripheral area to center of keloids. The gene type of p53 codon 72 in normal skin were Arg/Arg in eighteen samples, Pro/Arg in six samples. P53 expression in keloids was mainly located in the cytoplasm of the fibroblasts, and showed positive staining in all samples detected in the study. P53 expression was not detectable in normal skin.
2. The glycosylation level in Fas protein was higher in hypertrophic scar than in keloid. FasMcAb induced the apoptosis in cultured fibroblast, this action positively correlated with the glycosylation level in Fas protein. Treatment with turicamycin in cultured fibroblast decreased markedly the glycosylation level of Fas protein, and FasMcAb-induced apoptosis.
3. Hypoxia did not affect the apoptosis and Bax protein expression in cultured fibroblasts derived from keloid. Treatment with cisplatin in cultured fibroblast from keloid increased the apoptosis, downregulated Bcl-2 expression and upregulated Bax expression.
Conclusion:
1. There are possible associations between the codon 72 polymorphism and the clinical phenotype of keloids, the alteration of codon 72 polymorphism might be beneficial to apoptosis of fibroblasts in keloids.
2. FasMcAb-induced apoptosis in the cultured fibroblast correlated positively with the glycosylation level in Fas protein. Turicamycin decreased markedly the glycosylation level in Fas protein.
3. Cisplatin increased the apoptosis in cultured fibroblast, which might be associated with the altered expression in Bcl-2 and Bax.
对于瘢痕疙瘩来说,目前尚无有效的治疗手段,其综合治疗的效果也不理想,存在较高的复发率。一般认为瘢痕疙瘩成纤维细胞属于非正常细胞,与肿瘤细胞类似,虽然组织内存在大量凋亡的细胞,但是瘢痕疙瘩成纤维细胞为多克隆来源,体内存在不同表型的成纤维细胞,经体外培养后细胞变成单一表型,而且这种细胞不易凋亡。寻找瘢痕疙瘩成纤维细胞凋亡缺陷机制及有效诱导其凋亡的方法对瘢痕疙瘩治疗有重要意义。
基于以上观点,课题组致力于研究瘢痕疙瘩成纤维细胞凋亡缺陷的机制,探索瘢痕疙瘩的基因治疗,寻找有效的瘢痕疙瘩治疗方法。通过对瘢痕疙瘩成纤维细胞p53基因及fas基因系统研究后发现:1.瘢痕疙瘩病人血细胞中p53基因第72位密码子Pro等位基因频率明显高于Arg等位基因频率,与Arg/Arg及Arg/Pro基因型人群相比,Pro/Pro基因型人群易患瘢痕疙瘩。血细胞中p53基因第72位密码子多态性位点的基因型检测对判断瘢痕疙瘩高危个体具有指导意义,并开发出了可用于普通人群的p53基因第72位密码子基因多态性检测试剂盒。2.Fas单克隆抗体在ng-μg水平即可迅速诱导增生性瘢痕成纤维细胞的凋亡,而在相同浓度下,瘢痕疙瘩成纤维细胞对此却无反应。3.增生性瘢痕成纤维细胞Fas表达较低,但瘢痕疙瘩成纤维细胞Fas却呈高表达,两者间差异显著,瘢痕疙瘩细胞的凋亡异常不是由于缺乏受体所导致的。4.瘢痕疙瘩Fas介导死亡信号传导阻滞,而且阻断的位点位于上游。5.携带正常人fas基因的脂质体及重组腺病毒均能有效地将fas基因导入,并且能使目的基因在靶细胞内完整且有效的表达,瘢痕疙瘩细胞Fas通道能得以稳定重建,且通道重建后瘢痕疙瘩细胞增殖-凋亡状况的失衡得到了明显的改善。
通过以上研究结果,我们对瘢痕疙瘩的发病机制与p53基因及fas基因的关系有了比较充分的了解,但是有些问题还值得我们进一步探讨。
p53基因外显子4第72编码子基因多态性是近年研究的热点,不同的基因型与多种肿瘤发病相关,其产物对细胞凋亡有不同的影响,局部组织基因型存在相互变异的现象,而且Arg等位基因占优势的肿瘤预后明显好于Pro等位基因占优势的肿瘤。瘢痕疙瘩类似良性肿瘤,患者局部组织基因多态性有无变异,不同的基因型对蛋白的表达有无影响,与临床表型的关系如何,这些都是我们必须回答的问题。
与增生性瘢痕相比,瘢痕疙瘩成纤维细胞Fas蛋白表达强阳性,而经体外培养的瘢痕疙瘩成纤维细胞FasMcAb不能有效诱导凋亡,分析其中的原因:其一为Fas蛋白结构异常,导致不能有效诱导凋亡。另外Fas蛋白是一种糖蛋白,只有糖基化的蛋白才能有效表达于细胞膜上,蛋白糖基化的程度与功能存在明显相关。因此我们采用Western Blot法检测病理性瘢痕成纤维细胞Fas蛋白糖基化水平,PI染色、流式细胞术检测成纤维细胞FasMcAb作用后的凋亡率,分析Fas蛋白糖基化水平与FasMcAb诱导凋亡的相关性,并利用蛋白糖基化抑制剂衣霉素(Tunicamycine,TM)抑制成纤维细胞Fas蛋白糖基化,观察成纤维细胞Fas蛋白糖基化水平变化后对凋亡的影响。
目前临床上没有确切有效的瘢痕疙瘩治疗方法,转染野生型fas基因能有效诱导瘢痕疙瘩成纤维细胞凋亡,具有广阔的前景,但是尚不能直接用于病人,短期内主要还是研究药物治疗为主。在各种诱导凋亡的信号转导通路中,受体通路与线粒体通路是2条基本通路。缺氧可有效启动线粒体依赖的caspases激活途径诱导凋亡,顺铂可同时启动Fas受体途径及线粒体依赖的caspases激活途径诱导凋亡,这在冠心病发病机制的研究中及肿瘤的治疗中已进行了广泛的研究,但在瘢痕疙瘩领域,无论在临床治疗或实验研究中,除基因转染外,尚未见报道有效的诱导瘢痕疙瘩成纤维细胞凋亡的方法。因此我们利用缺氧及顺铂处理瘢痕疙瘩成纤维细胞,观察是否能有效诱导其凋亡。
本实验在课题组先前研究结果的基础上再次探讨凋亡基因p53及fas,以组织标本及体外培养成纤维细胞为研究对象,旨在阐明它们在瘢痕疙瘩不同部位成纤维细胞凋亡过程中扮演的角色以及功能缺陷的可能机制,并对顺铂及缺氧诱导瘢痕疙瘩成纤维细胞凋亡的机制进行了初步探讨,为今后这方面的治疗工作进行了初步研究。
方法:
1.以正常人为对照,收集35例瘢痕疙瘩病人血标本及组织标本,瘢痕疙瘩组织标本分中央部和周边部。提取血标本及组织标本的DNA,多聚酶链式反应(PCR)扩增p53基因外显子4,并进行基因测序;采用免疫组织化学方法检测P53蛋白的表达。
2.采用组织培养技术,以手术切除的瘢痕疙瘩及增生性瘢痕为组织来源的体外培养的成纤维细胞为研究对象,Western Blot法及流式细胞术检测衣霉素处理前后成纤维细胞Fas糖蛋白的表达及FasMcAb诱导凋亡率的变化。
3.顺铂及缺氧诱导瘢痕疙瘩成纤维细胞凋亡,流式细胞术检测检测成纤维细胞凋亡率的变化,Western Blot法检测Bcl-2和Bax蛋白表达的变化。
结果:
1.p53基因72位编码子遗传基因型有三种:CCC/CCC、CCC/CGC、CGC/CGC,其产物分别是Pro/Pro、Pro/Arg、Arg/Arg。瘢痕疙瘩组Pro/Pro、Pro/Arg、Arg/Arg三种遗传基因型分布与临床表型密切相关,大体积瘢痕疙瘩患者遗传基因型为Pro/Pro,局部瘢痕组织则存在由周边的Pro向中央Arg变异的趋势。瘢痕疙瘩P53蛋白表达位于胞浆中,阳性率35/35,与瘢痕疙瘩临床表型及部位密切相关。正常皮肤无表达。
2.增生性瘢痕成纤维细胞Fas蛋白糖基化水平高于瘢痕疙瘩成纤维细胞,两组成纤维细胞FasMcAb作用后凋亡率与Fas蛋白糖基化成正相关,衣霉素可明显降低成纤维细胞Fas蛋白糖基化水平及FasMcAb作用后凋亡率。
3.缺氧条件下培养瘢痕疙瘩成纤维细胞,其凋亡率及Bcl-2和Bax蛋白的表达无明显变化。顺铂作用后瘢痕疙瘩成纤维细胞凋亡率增加,Bcl-2的表达下调,Bax的表达增加。
结论:
1.瘢痕疙瘩临床表型与p53基因外显子4第72编码子基因多态性密切相关,遗传基因型为Pro/Pro患者一般表现为大体积、多发;瘢痕疙瘩患者局部组织基因型有Pro向Arg变异的趋势,而大体积瘢痕疙瘩患者P53蛋白由边缘部的阴性表达向中央部的阳性表达转变,基因多态性变异与蛋白表达一致;Arg基因型编码的蛋白有利于瘢痕疙瘩成纤维细胞凋亡及瘢痕疙瘩的成熟。
2.FasMcAb诱导病理性瘢痕成纤维细胞凋亡与成纤维细胞Fas蛋白糖基化水平成正相关,而衣霉素可显著降低成纤维细胞Fas蛋白糖基化水平。
3.顺铂可诱导瘢痕疙瘩成纤维细胞凋亡,其作用机制可能与Bcl-2和Bax等凋亡调控基因的表达有关。
Background
Accumulating evidence indicates that unbalance between proliferation and apoptosis of fibroblasts contributes mainly to the formation of keloid and hypertrophic scar. So far, treatment for keloid is less effective. Fibroblasts from keloid are know to be abnormal ones, their apoptosis function was impaired, thus it is necessary for us to find out the cause, in which apoptotic function was impaired.
Our previous study has demonstrated that P53 and Fas were involved in keloid formation. Detection of polymorphisms in p53 gene codon 72 was important for deciding who are susceptible to keloid. Monoclonal antibody against Fas at a dose of ng-μg induced the apoptosis in Proliferative fibroblasts derived from hypertrophic scar, not in the fibroblasts from keloid. Fas protein expression was decreased in hypertrophic scar and increased in keloid. Abnormal apoptosis occurred in keloid was not associated with the loss of Fas receptors. Fas-mediated death signals were blocked at the upstream. Transfection of normal fas gene into target cells could improve the unbalance of proliferation and apoptosis in the fibroblast.
Polymorphisms in p53 gene codon 72 were intensively investigated. Different gene types of p53 are involved in the development of tumor. The products expressed by different gene typed of P53 may affect the cell apoptosis of fibroblast. However, Polymorphisms in p53 gene and protein expression in the local tissue from keloid are unclear.
Objective
The present study was done to observe the relationship between clinical phenotype of keloid and the polymorphisms in p53 gene codon 72, and explode the role of gene variation of p53 in the keloid formation and possible mechanism of impaired apoptosis. Meanwhile, we also investigated the effects of cisplatin and hypoxia on the apoptosis of fibroblasts from keloid.
Methods
1. The tissue and blood samples were taken from patients with keloid (n=35) and healthy individuls (n=26). The diagnosis of keloid was based on clinical manifestation, histological examination. The samples from patients with keloids were divided into three groups according to clinical phenotype. DNA of the tissue and blood samples was extracted. The tissue samples of the keloid were divided into peripheral and central areas. The expression of P53 in protein levels was detected by immunostaining, and DNA sequencing was performed with sequence-based typing (SBT).
2. Fibroblasts from hypertrophic scar and keloid were cultured. Western Blot and Flow cytometry were used to detect the effect of turicamycin on Fas protein expression and FasMcAb-induce apoptosis.
3. Cisplatin and hypoxia induced the apoptosis of fibroblasts from keloid. Flow cytometry were used to detect the rate of apoptois. Western Blot was used to examine the expression of Bcl-2 and Bax.
Results
1. Immunostaining results revealed that P53 was detectable in all the keloid samples. Three types of gene variation of p53 codon 72, including Pro/Pro, Pro/Arg, Arg/Arg were observed in keloids, and associated with clinical phenotype of keloids. Arg polymorphic variants of P53 were increased from the peripheral area to center of keloids. The gene type of p53 codon 72 in normal skin were Arg/Arg in eighteen samples, Pro/Arg in six samples. P53 expression in keloids was mainly located in the cytoplasm of the fibroblasts, and showed positive staining in all samples detected in the study. P53 expression was not detectable in normal skin.
2. The glycosylation level in Fas protein was higher in hypertrophic scar than in keloid. FasMcAb induced the apoptosis in cultured fibroblast, this action positively correlated with the glycosylation level in Fas protein. Treatment with turicamycin in cultured fibroblast decreased markedly the glycosylation level of Fas protein, and FasMcAb-induced apoptosis.
3. Hypoxia did not affect the apoptosis and Bax protein expression in cultured fibroblasts derived from keloid. Treatment with cisplatin in cultured fibroblast from keloid increased the apoptosis, downregulated Bcl-2 expression and upregulated Bax expression.
Conclusion:
1. There are possible associations between the codon 72 polymorphism and the clinical phenotype of keloids, the alteration of codon 72 polymorphism might be beneficial to apoptosis of fibroblasts in keloids.
2. FasMcAb-induced apoptosis in the cultured fibroblast correlated positively with the glycosylation level in Fas protein. Turicamycin decreased markedly the glycosylation level in Fas protein.
3. Cisplatin increased the apoptosis in cultured fibroblast, which might be associated with the altered expression in Bcl-2 and Bax.
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