北方汉族瘢痕疙瘩临床特征分析及相关基因多态性研究
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摘要
前言
瘢痕疙瘩(Keloid)是易感个体在皮肤损伤修复过程中成纤维细胞大量增殖,胶原基质大量分泌形成的良性真皮肿瘤,其临床特征为瘢痕组织超越原损伤范围持续生长,侵袭周围外观正常皮肤,手术切除后容易复发。国内外学者对其进行了大量研究,但发病机制目前仍不清楚。近年来,遗传学方面的研究已成为瘢痕疙瘩基础研究的主要方向,其中分子遗传学方面的研究已成为最新热点。Marneros等通过全基因组扫描将一个日本人瘢痕疙瘩家系易感基因定位于2q23,一个非裔美国黑人家系定位于7p11,提示瘢痕疙瘩的发生存在明显的遗传异质性。
痕痕疙瘩的人群患病率报道不一,分别从扎伊尔的16%到英国的不到1%;不同人种的患病率相差很大,深色人种与浅色人种的患病率从2:1到19:1不等。大多数瘢痕疙瘩病例呈散发状态,但也存在着家庭聚集现象。在南印度的一项1000人的流行病学调查中有家族史者占1.9%,另一项247人的研究表明瘢痕疙瘩患者中有家族史者占3.2%。有些综合症也可以合并瘢痕疙瘩,并且也表现为家族聚集现象,如Rubinstein-Taybi综合症和Goeminne综合症,但它们是与瘢痕疙瘩不同的疾病。国外流行病学调查结果表明瘢痕疙瘩患者无性别差异,发病高峰集中在10-30岁年龄段;安徽医科大学进行的南方汉族瘢痕疙瘩流行病学调查结果与国外流行病学调查结果相似,但是目前尚缺乏北方汉族瘢痕疙瘩临床流行病学资料。
单核苷酸多态性(SNP)是近年来出现的第三代遗传标记,对疾病的早期风险性评估、早期诊断与预防以及相应的治疗等诸多方面有巨大的应用价值。单核苷酸多态性,尤其是位于蛋白编码区和表达调控序列的SNP可能具有特定功能作用,并可以影响疾病发生的易感性。
基质金属蛋白酶(metal matrix proteinase,MMPs)是一个高度保守的依赖于锌离子的内切蛋白水解酶家族,是细胞外基质降解过程中最主要的蛋白水解酶,在伤口愈合、恶性肿瘤的浸润与转移等病理生理过程中发挥重要作用。MMPs家族至少发现与人类有关的17个成员,根据结构及酶与底物亲和力的不同,主要将MMPs分为4组:①胶原酶(collagenase)以MMP-1为代表,主要消化Ⅰ、Ⅱ、Ⅲ、Ⅶ、Ⅹ型胶原和蛋白多糖;②明胶酶(gelatinase)MMP-2、MMP-9,又称为明胶酶A、B,主要消化Ⅳ、Ⅴ、Ⅶ、Ⅹ型胶原和弹性纤维;③基质溶解素(stromelysin)MMP-3、MMP-7、MMP-10、MMP-11,主要作用于纤维黏连蛋白、层黏连蛋白、弹力纤维、Ⅲ、Ⅳ、Ⅵ、Ⅸ型胶原及MMP-1、MMP-8、MMP-9;④膜型金属蛋白酶(membrane-type matrix proteinase)MT1-MMP、MT2-MMP、MT3-MMP,主要作用于明胶Ⅳ型胶原、MMP-2。目前发现MMPs基因有一些多态性位点可以影响MMPs的结构与功能,且与炎症及纤维化疾病的发生发展有关。其中MMP-9基因尤其受到关注。人类MMP-9基因定位于染色体20q12-13,研究发现MMP-9有两个多态性位点,C-1562T和CA-repeat,其中C-1562T研究较多。
白细胞介素-6(Interleukin 6)是炎症起始阶段的一个重要致炎因子,具有广泛免疫调节作用,通过诱导多种细胞合成和分泌多种急性期蛋白促进感染时多形核白细胞产生、活化、促进B细胞增殖、分化及产生免疫球蛋白、促进T细胞增殖、分化等,在急性期炎症反应中扮演着十分重要的角色。1998年Daniel Fishman首先发现了启动子-174位点存在G/C多态性,发现这种多态性对于IL-6基因表达具有调控作用。对IL-6启动子区多态性的临床研究有助于从免疫基因的角度理解疾病的发生、发展规律,探索携带不同等位基因的个体对某一疾病的易患性。
本研究从流行病学调查着手,历时三年,在临床收集680瘢痕疙瘩病例,进行详细的问卷调查,从这些患者中采集120例志愿者肘静脉血2毫升,应用聚合酶链反应—限制性片段长度多态性(Polymerase chain reaction restriction fragmentlength polymorphism,PCR-RFLP)方法和测序结合对120例辽宁籍汉族瘢痕疙瘩患者和180例种族与地域相匹配的正常对照者的MMP-9 -1562C/T基因多态性位点和IL-6-174G/C基因多态性位点进行检测,以探讨这些功能性SNP位点与瘢痕疙瘩的遗传易感性的关系,为揭示瘢痕疙瘩遗传学发病机理提供重要线索。
材料和方法
一、研究对象
1、病例组
纳入流行病学调查的680例瘢痕疙瘩患者为中国医科大学附属第一医院皮肤科门诊及住院患者,均为北方汉族人,符合临床诊断标准:增生瘢痕组织超过原损伤范围,病程大于1年。其中知情同意后采血的120例纳入多态性研究,均为彼此间无血缘关系的北方籍汉族人,其中男性54例,女性66例,发病年龄12～52岁,平均年龄26.4±5.9岁。
2、正常对照组
180例,为随机抽取的健康体检志愿者,北方汉族人,其中男84例,女96例,平均年龄24.3±2.7岁,彼此间无血缘关系,排除瘢痕疙瘩病史及家族史。
二、实验材料
10%SDS、EDTA、蛋白酶K、Tris饱和酚、氯仿、醋酸钠、无水乙醇、TE缓冲液、琼脂糖、LA Taq酶。
三、实验方法
1、采用标准的调查问卷,利用SPSS 13.0软件分析流行病学调查数据。
2、应用聚合酶链反应—限制性片段长度多态性(PCR-RFLP)方法对MMP-9-1562C/T及IL-6-174G/C等位基因进行分型。
3、利用测序方法验证PCR-RFLP结果。
四、统计学处理
利用SPSS 13.0软件分析流行病学调查数据。Hardy-Weinberg平衡吻合性检验、组间基因型频率及等位基因频率的差异性比较采用x~2检验,并以优势比(oddsratio,OR)及其95%可信区间(confidence interval,CI)表示相对风险度。使用SPSS13.0软件分析数据,认为P＜0.05有统计学意义(具有显著性差异)。
结果
1、瘢痕疙瘩发病无性别差异;发病高峰集中在10～30岁;有家族史患者发病年龄提前,而且有家族史患者更容易发生多发性瘢痕疙瘩。
2、瘢痕疙瘩与MMP-9-1562C/T基因多态性的相关研究
(1)瘢痕疙瘩组和正常对照组人群MMP-9-1562C/T基因型分布符合Hardy-Weinberg平衡定律。
(2)瘢痕疙瘩组MMP-9-1562C/T等位基因的频率与正常对照组比较,差异有统计学意义(P＜0.05)。
3、瘢痕疙瘩与IL-6-174G/C基因多态性的相关研究
(1)瘢痕疙瘩组和正常对照组人群IL-6-174G/C基因型分布符合Hardy-Weinberg平衡定律。
(2)瘢痕疙瘩组IL-6-174G/C等位基因的频率与正常对照组比较,差异无统计学意义(P＞0.05)。
结论
1、流行病学调查结果显示北方汉族瘢痕疙瘩患者发病无性别差异;发病高峰集中在10～30岁;有家族史患者发病年龄提前,而且更容易发生多发性瘢痕疙瘩。
2、北方汉族瘢痕疙瘩组和正常对照组人群MMP-9-1562C/T基因频率已达到Hardy-Weinberg遗传平衡。
3、北方汉族MMP-9基因T等位基因的存在可能与瘢痕疙瘩的形成和发展有一定的相关性。
4、北方汉族瘢痕疙瘩组和正常对照组人群IL-6-174G/C基因频率已达到Hardy-Weinberg遗传平衡。
5、北方汉族IL-6-174G/C等位基因与瘢痕疙瘩的形成和发展可能不具有相关性。
Introduction
Keloids are fibrous overgrowths that develop at sites of cutaneous injury and are characterized by the deposition of excessive extracellular matrix collagen,synthesized by the increased number of fibroblasts.The excessive scar tissue proliferates beyond the boundaries of the original wound.Keloids are considered to be benign tumors and, unlike normal scars and hypertrophic scars,do not regress spontaneously,commonly recurring following excision.Although the cause of keloids is unknown,it is thought that they are due to a failure to turn off the healing process needed to repair broken skin and genetic factors influence susceptibility and modify progression.
Marneros studied two families with an autosomal dominant inheritance pattern of keloids.One African-American family showed a high degree of variability in the extent of keloid formation between family members,whereas the second family from Japan showed a pattern of full penetrance and the formation of only small keloids.They performed a genomewide linkage search for genes predisposing to keloid formation in these two families and identified linkage to chromosome 2q23 for the Japanese family. The African-American family showed evidence for a keloid susceptibility locus on chromosome 7p11.The observed locus heterogeneity in autosomal dominant keloid disease is consistent with the clinical heterogeneity of this scarring disorder.This study provides the first genetic evidence for keloid susceptibility loci and serves as a basis for the identification of responsible genes.
Keloid scar populations reported prevalence rates vary greatly all over,from Zaire to the England of 16%less than 1%,the prevalence rate of different races are also a huge difference,dark ethnic origin of patients with light-colored disease increased from 2:1 to 19:1 range.The majority of cases of keloid were distributed,but there are also gathered home situation.At a South Indian epidemiological survey of 1000 people has a family history in 1.9%,another study of 247 people shows that there is a family history of keloid patients accounted for 3.2%.Some keloid syndrome can also be the emergence of the merger,and also has a family history,such as Rubinstein-Taybi syndrome and syndrome Goeminne,but they are different diseases and keloids.Data indicate that the majority of foreign patients with no gender differences in keloid.The occurrence of keloids with age-related,usually is 10-30 years of age in China's north. There is still a lack of clinical epidemiological data Han keloid.
Single nucleotide polymorphism(SNP) in recent years are the third generation of genetic markers,early disease risk assessment,early diagnosis and prevention as well as the corresponding treatment and many other applications have enormous value.In particular,single nucleotide polymorphisms are located in protein coding region and the SNP sequence of expression and regulation may have a specific function and can affect disease susceptibility.
Matrix metalloproteinases(metal matrix proteinase,MMPs) are a family of highly conserved zinc ions dependent proteolytic enzymes.Extracellular matrix degradation are the main course of proteolytic enzymes,playing an important role in the process in cell migration,angiogenesis,wound healing,the infiltration and metastasis of malignant tumors.
MMPs family has at least 17 members,according to the structure and different substrate affinity,MMPS mainly divided into 4 groups:①collagenase,MMP1 represented,the main digestionⅠ,Ⅱ,Ⅲ,Ⅶ,Ⅹcollagen and proteoglycans;②gelatinase,MMP2,MMP9,also known as gelatinase-A,B,the main digestiveⅣ,Ⅴ,Ⅶ,Ⅹcollagen and elastic fibers;③matrilysin(stromelysin) MMP3,MMP7,MMP10, MMP11,a key role in fibronectin,laminin,elastic fiber,Ⅲ,Ⅳ,Ⅵ,Ⅸcollagen and MMP1,MMP8,MMP9;④membrane-type MMP(membrane-type matrix proteinase) MT1-MMP,MT2-MMP,MT3-MMP,a major role in collagen typeⅣgelatinase, MMP2.The majority of MMPs significantly expressed in the physiological or pathological remodeling process.At present,there is a number of MMPs gene polymorphism can affect the structure and function of MMPs,fibrosis,inflammation and the development of disease.Human MMP-9 gene located in chromosome 20q12-13,research has found MMP-9 polymorphism,C-1562T and the CA-repeat.
In this study,we adopt epidemiology study of 680 cases of Han nationality keloid cases,Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and sequencing to study MMP-9 -1562C/T and IL-6 -174G/C gene polymorphisms of 120 cases of Han nationality keloid cases in North China and 180 cases of race and geographical matched normal controls.Investigating MMPg-1562C/T and IL-6 -174G/C gene polymorphism could provide clues to the genetic susceptibility of keloid and reveal the pathogenesis of keloid.
Material and method
1.Subjects
(1).case group:Epidemiological investigations into 680 cases of patients with keloid for First Affiliated Hospital of China Medical University dermatology out-patient clinics and hospitalized patients with the clinical diagnostic criteria:scar tissue over the scope of the original injury,the course is greater than 1 year.120 keloid patients were diagnosed in Department of Dermatology,First Hospital of China Medical University,conforms to the clinical diagnosis standard:excessive scar tissue proliferates beyond the boundaries of the original wound and the course of disease is more than 1 year.All patients were unrelated North China Han person,in which male 54 examples,feminine 66 examples,age of onset is 12-52 year old,average age is 26.4(±5.9) years old.
(2).Normal control group:180 examples,a random selection of volunteers for health examination,include 84 male and 96 female.The average age is 24.3(±2.7) years old,no blood relationship between them,and the keloid medical history and family history were excluded.
2.Materials
10%SDS,EDTA,proteinase K,Tris saturated phenol,chloroform,sodium acetate, alcohol,TE buffer,agarose,8%denatured polyacrylamide solution,LA Taq enzyme.
3.Methods
(1) Polymerase chain reaction(PER)
(2) The polymerase chain reaction - restrictive fragment length polymorphism (PCR-RFLP) method to genotyping the allele type.
(3) Direct sequencing.
4.Statistical Analysis
To determine whether genotype was in Hardy-Weinberg equilibrium,aχ2 test was performed.The genotype frequency and the allele frequency of three genes compared byχ2,and odds ratio(OR) and 95%confidence interval(CI) indicate the relative risk. Statistical analyses were performed in SPSS 13.0.
Result
1.Results of epidemiology study
There is no gender differences in China north Han keloid.The peak of keloids is usually 10-30 years of age.
2.Results of MMP-9 -1562C/T gene polymorphism research
(1) Keloid group and the normal population control group MMP-9 -1562C / T genotype distribution of the balance in line with the Hardy-Weinberg law.
(2) Keloid group MMP-9-1562C / T allele frequency compared with normal control group,the difference has statistical significance(P＜0.05).
3.Results of I1-6 - 174G/C gene polymorphism research
(1) Keloid group and the normal population control group I1-6 -174 G/C genotype distribution of the balance in line with the Hardy-Weinberg law.
(2) Keloid group I1-6 -174G/C allele frequency compared with normal control group,the difference has no statistical significance(P＞0.05).
Conclusion
1.Epidemiological investigation showed that no gender differences in the incidence of keloid;incidence peak concentrated in the 10-30 years of age;have a family history of early onset of age;have a family history of multiple keloid-prone patients.
2.Keloids and normal control group,the crowd MMP-9 -1562C/T allele frequency have reached the Hardy-Weinberg genetic equilibrium.
3.MMP-9 gene T allele may be associated with the existence of the formation and development of keloids.
4.Keloids and normal control group,the crowd I1-6 -174G/C allele frequency have reached the Hardy-Weinberg genetic equilibrium.
5.I1-6 -174G/C polymorphism may not be associated with the existence of the formation and development of keloids.
瘢痕疙瘩(Keloid)是易感个体在皮肤损伤修复过程中成纤维细胞大量增殖,胶原基质大量分泌形成的良性真皮肿瘤,其临床特征为瘢痕组织超越原损伤范围持续生长,侵袭周围外观正常皮肤,手术切除后容易复发。国内外学者对其进行了大量研究,但发病机制目前仍不清楚。近年来,遗传学方面的研究已成为瘢痕疙瘩基础研究的主要方向,其中分子遗传学方面的研究已成为最新热点。Marneros等通过全基因组扫描将一个日本人瘢痕疙瘩家系易感基因定位于2q23,一个非裔美国黑人家系定位于7p11,提示瘢痕疙瘩的发生存在明显的遗传异质性。
痕痕疙瘩的人群患病率报道不一,分别从扎伊尔的16%到英国的不到1%;不同人种的患病率相差很大,深色人种与浅色人种的患病率从2:1到19:1不等。大多数瘢痕疙瘩病例呈散发状态,但也存在着家庭聚集现象。在南印度的一项1000人的流行病学调查中有家族史者占1.9%,另一项247人的研究表明瘢痕疙瘩患者中有家族史者占3.2%。有些综合症也可以合并瘢痕疙瘩,并且也表现为家族聚集现象,如Rubinstein-Taybi综合症和Goeminne综合症,但它们是与瘢痕疙瘩不同的疾病。国外流行病学调查结果表明瘢痕疙瘩患者无性别差异,发病高峰集中在10-30岁年龄段;安徽医科大学进行的南方汉族瘢痕疙瘩流行病学调查结果与国外流行病学调查结果相似,但是目前尚缺乏北方汉族瘢痕疙瘩临床流行病学资料。
单核苷酸多态性(SNP)是近年来出现的第三代遗传标记,对疾病的早期风险性评估、早期诊断与预防以及相应的治疗等诸多方面有巨大的应用价值。单核苷酸多态性,尤其是位于蛋白编码区和表达调控序列的SNP可能具有特定功能作用,并可以影响疾病发生的易感性。
基质金属蛋白酶(metal matrix proteinase,MMPs)是一个高度保守的依赖于锌离子的内切蛋白水解酶家族,是细胞外基质降解过程中最主要的蛋白水解酶,在伤口愈合、恶性肿瘤的浸润与转移等病理生理过程中发挥重要作用。MMPs家族至少发现与人类有关的17个成员,根据结构及酶与底物亲和力的不同,主要将MMPs分为4组:①胶原酶(collagenase)以MMP-1为代表,主要消化Ⅰ、Ⅱ、Ⅲ、Ⅶ、Ⅹ型胶原和蛋白多糖;②明胶酶(gelatinase)MMP-2、MMP-9,又称为明胶酶A、B,主要消化Ⅳ、Ⅴ、Ⅶ、Ⅹ型胶原和弹性纤维;③基质溶解素(stromelysin)MMP-3、MMP-7、MMP-10、MMP-11,主要作用于纤维黏连蛋白、层黏连蛋白、弹力纤维、Ⅲ、Ⅳ、Ⅵ、Ⅸ型胶原及MMP-1、MMP-8、MMP-9;④膜型金属蛋白酶(membrane-type matrix proteinase)MT1-MMP、MT2-MMP、MT3-MMP,主要作用于明胶Ⅳ型胶原、MMP-2。目前发现MMPs基因有一些多态性位点可以影响MMPs的结构与功能,且与炎症及纤维化疾病的发生发展有关。其中MMP-9基因尤其受到关注。人类MMP-9基因定位于染色体20q12-13,研究发现MMP-9有两个多态性位点,C-1562T和CA-repeat,其中C-1562T研究较多。
白细胞介素-6(Interleukin 6)是炎症起始阶段的一个重要致炎因子,具有广泛免疫调节作用,通过诱导多种细胞合成和分泌多种急性期蛋白促进感染时多形核白细胞产生、活化、促进B细胞增殖、分化及产生免疫球蛋白、促进T细胞增殖、分化等,在急性期炎症反应中扮演着十分重要的角色。1998年Daniel Fishman首先发现了启动子-174位点存在G/C多态性,发现这种多态性对于IL-6基因表达具有调控作用。对IL-6启动子区多态性的临床研究有助于从免疫基因的角度理解疾病的发生、发展规律,探索携带不同等位基因的个体对某一疾病的易患性。
本研究从流行病学调查着手,历时三年,在临床收集680瘢痕疙瘩病例,进行详细的问卷调查,从这些患者中采集120例志愿者肘静脉血2毫升,应用聚合酶链反应—限制性片段长度多态性(Polymerase chain reaction restriction fragmentlength polymorphism,PCR-RFLP)方法和测序结合对120例辽宁籍汉族瘢痕疙瘩患者和180例种族与地域相匹配的正常对照者的MMP-9 -1562C/T基因多态性位点和IL-6-174G/C基因多态性位点进行检测,以探讨这些功能性SNP位点与瘢痕疙瘩的遗传易感性的关系,为揭示瘢痕疙瘩遗传学发病机理提供重要线索。
材料和方法
一、研究对象
1、病例组
纳入流行病学调查的680例瘢痕疙瘩患者为中国医科大学附属第一医院皮肤科门诊及住院患者,均为北方汉族人,符合临床诊断标准:增生瘢痕组织超过原损伤范围,病程大于1年。其中知情同意后采血的120例纳入多态性研究,均为彼此间无血缘关系的北方籍汉族人,其中男性54例,女性66例,发病年龄12～52岁,平均年龄26.4±5.9岁。
2、正常对照组
180例,为随机抽取的健康体检志愿者,北方汉族人,其中男84例,女96例,平均年龄24.3±2.7岁,彼此间无血缘关系,排除瘢痕疙瘩病史及家族史。
二、实验材料
10%SDS、EDTA、蛋白酶K、Tris饱和酚、氯仿、醋酸钠、无水乙醇、TE缓冲液、琼脂糖、LA Taq酶。
三、实验方法
1、采用标准的调查问卷,利用SPSS 13.0软件分析流行病学调查数据。
2、应用聚合酶链反应—限制性片段长度多态性(PCR-RFLP)方法对MMP-9-1562C/T及IL-6-174G/C等位基因进行分型。
3、利用测序方法验证PCR-RFLP结果。
四、统计学处理
利用SPSS 13.0软件分析流行病学调查数据。Hardy-Weinberg平衡吻合性检验、组间基因型频率及等位基因频率的差异性比较采用x~2检验,并以优势比(oddsratio,OR)及其95%可信区间(confidence interval,CI)表示相对风险度。使用SPSS13.0软件分析数据,认为P＜0.05有统计学意义(具有显著性差异)。
结果
1、瘢痕疙瘩发病无性别差异;发病高峰集中在10～30岁;有家族史患者发病年龄提前,而且有家族史患者更容易发生多发性瘢痕疙瘩。
2、瘢痕疙瘩与MMP-9-1562C/T基因多态性的相关研究
(1)瘢痕疙瘩组和正常对照组人群MMP-9-1562C/T基因型分布符合Hardy-Weinberg平衡定律。
(2)瘢痕疙瘩组MMP-9-1562C/T等位基因的频率与正常对照组比较,差异有统计学意义(P＜0.05)。
3、瘢痕疙瘩与IL-6-174G/C基因多态性的相关研究
(1)瘢痕疙瘩组和正常对照组人群IL-6-174G/C基因型分布符合Hardy-Weinberg平衡定律。
(2)瘢痕疙瘩组IL-6-174G/C等位基因的频率与正常对照组比较,差异无统计学意义(P＞0.05)。
结论
1、流行病学调查结果显示北方汉族瘢痕疙瘩患者发病无性别差异;发病高峰集中在10～30岁;有家族史患者发病年龄提前,而且更容易发生多发性瘢痕疙瘩。
2、北方汉族瘢痕疙瘩组和正常对照组人群MMP-9-1562C/T基因频率已达到Hardy-Weinberg遗传平衡。
3、北方汉族MMP-9基因T等位基因的存在可能与瘢痕疙瘩的形成和发展有一定的相关性。
4、北方汉族瘢痕疙瘩组和正常对照组人群IL-6-174G/C基因频率已达到Hardy-Weinberg遗传平衡。
5、北方汉族IL-6-174G/C等位基因与瘢痕疙瘩的形成和发展可能不具有相关性。
Introduction
Keloids are fibrous overgrowths that develop at sites of cutaneous injury and are characterized by the deposition of excessive extracellular matrix collagen,synthesized by the increased number of fibroblasts.The excessive scar tissue proliferates beyond the boundaries of the original wound.Keloids are considered to be benign tumors and, unlike normal scars and hypertrophic scars,do not regress spontaneously,commonly recurring following excision.Although the cause of keloids is unknown,it is thought that they are due to a failure to turn off the healing process needed to repair broken skin and genetic factors influence susceptibility and modify progression.
Marneros studied two families with an autosomal dominant inheritance pattern of keloids.One African-American family showed a high degree of variability in the extent of keloid formation between family members,whereas the second family from Japan showed a pattern of full penetrance and the formation of only small keloids.They performed a genomewide linkage search for genes predisposing to keloid formation in these two families and identified linkage to chromosome 2q23 for the Japanese family. The African-American family showed evidence for a keloid susceptibility locus on chromosome 7p11.The observed locus heterogeneity in autosomal dominant keloid disease is consistent with the clinical heterogeneity of this scarring disorder.This study provides the first genetic evidence for keloid susceptibility loci and serves as a basis for the identification of responsible genes.
Keloid scar populations reported prevalence rates vary greatly all over,from Zaire to the England of 16%less than 1%,the prevalence rate of different races are also a huge difference,dark ethnic origin of patients with light-colored disease increased from 2:1 to 19:1 range.The majority of cases of keloid were distributed,but there are also gathered home situation.At a South Indian epidemiological survey of 1000 people has a family history in 1.9%,another study of 247 people shows that there is a family history of keloid patients accounted for 3.2%.Some keloid syndrome can also be the emergence of the merger,and also has a family history,such as Rubinstein-Taybi syndrome and syndrome Goeminne,but they are different diseases and keloids.Data indicate that the majority of foreign patients with no gender differences in keloid.The occurrence of keloids with age-related,usually is 10-30 years of age in China's north. There is still a lack of clinical epidemiological data Han keloid.
Single nucleotide polymorphism(SNP) in recent years are the third generation of genetic markers,early disease risk assessment,early diagnosis and prevention as well as the corresponding treatment and many other applications have enormous value.In particular,single nucleotide polymorphisms are located in protein coding region and the SNP sequence of expression and regulation may have a specific function and can affect disease susceptibility.
Matrix metalloproteinases(metal matrix proteinase,MMPs) are a family of highly conserved zinc ions dependent proteolytic enzymes.Extracellular matrix degradation are the main course of proteolytic enzymes,playing an important role in the process in cell migration,angiogenesis,wound healing,the infiltration and metastasis of malignant tumors.
MMPs family has at least 17 members,according to the structure and different substrate affinity,MMPS mainly divided into 4 groups:①collagenase,MMP1 represented,the main digestionⅠ,Ⅱ,Ⅲ,Ⅶ,Ⅹcollagen and proteoglycans;②gelatinase,MMP2,MMP9,also known as gelatinase-A,B,the main digestiveⅣ,Ⅴ,Ⅶ,Ⅹcollagen and elastic fibers;③matrilysin(stromelysin) MMP3,MMP7,MMP10, MMP11,a key role in fibronectin,laminin,elastic fiber,Ⅲ,Ⅳ,Ⅵ,Ⅸcollagen and MMP1,MMP8,MMP9;④membrane-type MMP(membrane-type matrix proteinase) MT1-MMP,MT2-MMP,MT3-MMP,a major role in collagen typeⅣgelatinase, MMP2.The majority of MMPs significantly expressed in the physiological or pathological remodeling process.At present,there is a number of MMPs gene polymorphism can affect the structure and function of MMPs,fibrosis,inflammation and the development of disease.Human MMP-9 gene located in chromosome 20q12-13,research has found MMP-9 polymorphism,C-1562T and the CA-repeat.
In this study,we adopt epidemiology study of 680 cases of Han nationality keloid cases,Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and sequencing to study MMP-9 -1562C/T and IL-6 -174G/C gene polymorphisms of 120 cases of Han nationality keloid cases in North China and 180 cases of race and geographical matched normal controls.Investigating MMPg-1562C/T and IL-6 -174G/C gene polymorphism could provide clues to the genetic susceptibility of keloid and reveal the pathogenesis of keloid.
Material and method
1.Subjects
(1).case group:Epidemiological investigations into 680 cases of patients with keloid for First Affiliated Hospital of China Medical University dermatology out-patient clinics and hospitalized patients with the clinical diagnostic criteria:scar tissue over the scope of the original injury,the course is greater than 1 year.120 keloid patients were diagnosed in Department of Dermatology,First Hospital of China Medical University,conforms to the clinical diagnosis standard:excessive scar tissue proliferates beyond the boundaries of the original wound and the course of disease is more than 1 year.All patients were unrelated North China Han person,in which male 54 examples,feminine 66 examples,age of onset is 12-52 year old,average age is 26.4(±5.9) years old.
(2).Normal control group:180 examples,a random selection of volunteers for health examination,include 84 male and 96 female.The average age is 24.3(±2.7) years old,no blood relationship between them,and the keloid medical history and family history were excluded.
2.Materials
10%SDS,EDTA,proteinase K,Tris saturated phenol,chloroform,sodium acetate, alcohol,TE buffer,agarose,8%denatured polyacrylamide solution,LA Taq enzyme.
3.Methods
(1) Polymerase chain reaction(PER)
(2) The polymerase chain reaction - restrictive fragment length polymorphism (PCR-RFLP) method to genotyping the allele type.
(3) Direct sequencing.
4.Statistical Analysis
To determine whether genotype was in Hardy-Weinberg equilibrium,aχ2 test was performed.The genotype frequency and the allele frequency of three genes compared byχ2,and odds ratio(OR) and 95%confidence interval(CI) indicate the relative risk. Statistical analyses were performed in SPSS 13.0.
Result
1.Results of epidemiology study
There is no gender differences in China north Han keloid.The peak of keloids is usually 10-30 years of age.
2.Results of MMP-9 -1562C/T gene polymorphism research
(1) Keloid group and the normal population control group MMP-9 -1562C / T genotype distribution of the balance in line with the Hardy-Weinberg law.
(2) Keloid group MMP-9-1562C / T allele frequency compared with normal control group,the difference has statistical significance(P＜0.05).
3.Results of I1-6 - 174G/C gene polymorphism research
(1) Keloid group and the normal population control group I1-6 -174 G/C genotype distribution of the balance in line with the Hardy-Weinberg law.
(2) Keloid group I1-6 -174G/C allele frequency compared with normal control group,the difference has no statistical significance(P＞0.05).
Conclusion
1.Epidemiological investigation showed that no gender differences in the incidence of keloid;incidence peak concentrated in the 10-30 years of age;have a family history of early onset of age;have a family history of multiple keloid-prone patients.
2.Keloids and normal control group,the crowd MMP-9 -1562C/T allele frequency have reached the Hardy-Weinberg genetic equilibrium.
3.MMP-9 gene T allele may be associated with the existence of the formation and development of keloids.
4.Keloids and normal control group,the crowd I1-6 -174G/C allele frequency have reached the Hardy-Weinberg genetic equilibrium.
5.I1-6 -174G/C polymorphism may not be associated with the existence of the formation and development of keloids.
引文
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10 Dan Leibovici,H.Barton Grossman,Colin P.Dinney,et al.Polymorphisms in Inflammation Genes and Bladder Cancer:From Initiation to Recurrence,Progression,and Survival.
11 Bayat A,Arscott G,Oilier WE,et al.Description of site-specific morphology of keloid phenotypes in an Afrocaribbean population.Br J Plast Surg.2004 Mar;57(2):122-133.
12 Bloom D.Heredity of keloids.NY State Med J.1956;56:511-519
13 Marneros AG,Norris JE,Watanabe S,et a/.Genome scans provide evidence for keloid susceptibility loci on chromosomes 2q23 and 7pl 1J Invest Dermatol,2004,122(5):1126-1132.
14 Lim CP,Phan TT,Lim IJ,Cao X.Cytokine profiling and Stat3 phosphorylation in epithelial-mesenchymal interactions between keloid keratinocytes and fibroblasts.J Invest Dermatol.2009 Apr;129(4):851-61.
15 Ghazizadeh M.Essential role of IL-6 signaling pathway in keloid pathogenesis.J Nippon Med Sch.2007 Feb;74(1):11-22.
16 Uitto J.IL-6 signaling pathway in keloids:a target for pharmacologic intervention? J Invest Dermatol.2007 Jan;127(1):6-8.
17 Ghazizadeh M,Tosa M,Shimizu H,et al.Functional implications of the IL-6 signaling pathway in keloid pathogenesis.J Invest Dermatol.2007 Jan;127(1):98-105.
18 Tosa M,Ghazizadeh M,Shimizu H,et al.Global gene expression analysis of keloid fibroblasts in response to electron beam irradiation reveals the involvement of interleukin-6 pathway.J Invest Dermatol.2005 Apr;124(4):704-13.
19 Giugliano G,Pasquali D,Notaro A,et al.Verapamil inhibits interleukin-6 and vascular endothelial growth factor production in primary cultures of keloid fibroblasts.Br J Plast Surg.2003 Dec;56(8):804-9.
20 Xue H,McCauley RL,Zhang W.Elevated interleukin-6 expression in keloid fibroblasts.J Surg Res.2000 Mar;89(1):74-7.
21 Xu S,Wang Z,Bao W.The responses of fibroblasts from three parts of keloids and normal skin to interleukin-1 beta and interleukin-6.Zhonghua Zheng Xing Shao Shang Wai Ke Za Zhi.1998 Jan;14(1):23-5.
22 McCauley RL,Chopra V,Li YY,et al.Altered cytokine production in black patients with keloids.J Clin Immunol.1992 Jul;12(4):300-8.
23 Fishman D,Faulds G,Jeffery R,et al.The effect of novel polymorphisms in the interleukin-6 (IL-6)gene on IL-6 transcription and plasma IL-6 levels,and an association with systemic-onset juvenile chronic arthritis.J Clin Invest.1998 Oct 1;102(7):1369-76.
24 Yucesoy B,Johnson VJ,Kissling GE,et al.Genetic susceptibility to progressive massive fibrosis in coal miners.Eur Respir J.2008 Jun;31(6):1177-82.
25 Sfrent-Cornateanu R,Mihai C,Balan S,et al.The IL-6 promoter polymorphism is associated with disease activity and disability in systemic sclerosis.J Cell Mol Med.2006Oct-Dec;10(4):955-9.
26 Analysis of IL6 and ILIA gene polymorphisms in UK and Dutch patients with sarcoidosis.Grutters JC,Sato H,Pantelidis P,Ruven HJ,McGrath DS,Wells AU,van den Bosch JM,Welsh KI,du Bois RM.Sarcoidosis Vase Diffuse Lung Dis.2003 Mar;20(1):20-7.
27 Elevated interleukin-6 plasma levels are regulated by the promoter region polymorphism of the IL6 gene in primary Sjogren's syndrome and correlate with the clinical manifestations of the disease.Hulkkonen J,Pertovaara M,Antonen J,Pasternack A,Hurme M.Rheumatology (Oxford).2001 Jun;40(6):656-61.
28 Analysis of tumor necrosis factor-alpha,lymphotoxin-alpha,tumor necrosis factor receptor Ⅱ,and interleukin-6 polymorphisms in patients with idiopathic pulmonary fibrosis.Pantelidis P,Fanning GC,Wells AU,et al.Am J Respir Crit Care Med.2001 May;163(6):1432-6.
29 de Souza AP,Trevilatto PC,Scarel-Caminaga RM,et al.Analysis of the MMP-9 (C-1562 T)and TIMP-2 (G-418C)gene promoter polymorphisms in patients with chronic periodontitis.J Clin Periodontol.2005 Feb;32(2):207-11.
30 Wan R,Mo Y,Zhang X,Chien S,et al.Matrix metalloproteinase-2 and-9 are induced differently by metal nanoparticles in human monocytes:The role of oxidative stress and protein tyrosine kinase activation.Toxicol Appl Pharmacol.2008 Dec 1;233(2):276-85.
31 Gardi C,Arezzini B,Fortino V,et al.Effect of free iron on collagen synthesis,cell proliferation and MMP-2 expression in rat hepatic stellate cells.Biochem Pharmacol.2002 OCT 1;64(7):1139-45.
32 Sakaida I,Hironaka K,Terai S,et al.Gadolinium chloride reverses dimethylnitrosamine (DMN)-induced rat liver fibrosis with increased matrix metalloproteinases (MMPs)of Kupffer cells.Life Sci.2003 Jan 10;72(8):943-59.
33 Sage EH,Reed M,Funk SE,e/ al.Cleavage of the matricellular protein SPARC by matrix metalloproteinase-3 produces polypeptides that influence angiogenesis.J Biol Chem.2003 Sep 26;278(39):37849-57.
34 Hughes WM Jr,Rodriguez WE,Rosenberger D,et al.Role of copper and homocysteine in pressure overload heart failure.Cardiovasc Toxicol.2008 Fall;8(3):137-44.
35 Tamai E,Miyata S,Tanaka H,et al.High-level expression of his-tagged clostridial collagenase in Clostridium perfringens.Appl Microbiol Biotechnol.2008 Sep;80(4):627-35.
36 Wang GY,Bergman MR,Nguyen AP,et al.Cardiac transgenic matrix metalloproteinase-2 expression directly induces impaired contractility.Cardiovasc Res.2006 Feb 15;69(3):688-96.
37 Lin SC,Chung MY,Huang JW,et al.Correlation between functional genotypes in the matrix metalloproteinases-1 promoter and risk of oral squamous cell carcinomas.J Oral Pathol Med.2004 Jul;33(6):323-6.
38 Duong HS,Zhang QZ,Le AD,et al.Elevated prolidase activity in keloids:correlation with type I collagen turnover.Br J Dermatol.2006 May;154(5):820-828.
39 Vokurka J,Klapusova L,Pantuckova P,et al.The association of MMP-9 and IL-18 gene promoter polymorphisms with gingivitis in adolescents.Arch Oral Biol.2009 Feb;54(2):172-8.
40 Lee YJ,Woo M,Nam JH,et al.Matrix metalloproteinase-9 promoter polymorphisms in Korean patients with systemic lupus erythematosus.Hum Immunol.2008 Jun;69(6):374-9.
41 Demacq C,Vasconcellos VB,Marcaccini AM,et al.Functional polymorphisms in the promoter of the matrix metalloproteinase-9 (MMP-9)gene are not linked with significant plasma MMP-9 variations in healthy subjects.Clin Chem Lab Med.2008;46(1):57-63.
42 Gürkan A,Emingil G,Saygan BH,et al.Gene polymorphisms of matrix metalloproteinase-2,-9 and-12 in periodontal health and severe chronic periodontitis.Arch Oral Biol.2008 Apr;53(4):337-45.
43 Krumsiek A,Kropf S,Gardemann A.Tumor necrosis factor-alpha G(-308)A promoter polymorphism,matrix metalloproteinase (MMP)-3 5A/6A gene variation,MMP-9 C(-1562)T promoter polymorphism and risk and extent of ischemic heart disease.Clin Chem Lab Med.2008;46(2):292-5.
44 Gurkan A,Emingil G,Saygan BH,et al.Matrix metalloproteinase-2,-9,and-12 gene polymorphisms in generalized aggressive periodontitis.J Periodontol.2007 Dec;78(12):2338-47.
45 Tang LJ,Chen XF,Zhu M,et al.Matrix metalloproteinase-1,-3,and-9 gene polymorphisms and the risk of idiopathic dilated cardiomyopathy in a Chinese Han population.Clin Biochem.2007Dec;40(18):1427-30.
46 Zivkovic M,Djuric T,Dincic E,et al.Matrix metalloproteinase-9-1562 C/T gene polymorphism in Serbian patients with multiple sclerosis.J Neuroimmunol.2007 Sep;189(l-2):147-50.
47 Tu HF,Wu CH,Kao SY,et al.Functional-1562 C-to-T polymorphism in matrix metalloproteinase-9 (MMP-9)promoter is associated with the risk for oral squamous cell carcinoma in younger male areca users.J Oral Pathol Med.2007 Aug;36(7):409-14.
48 Nasr HB,Mestiri S,Chahed K,et al.Matrix metalloproteinase-1 (-1607)1G/2G and-9 (-1562)C/T promoter polymorphisms:susceptibility and prognostic implications in nasopharyngeal carcinomas.Clin Chim Acta.2007 Sep;384(l-2):57-63.
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14 Wan R,Mo Y,Zhang X,Chien S,et al.Matrix metalloproteinase-2 and -9 are induced differently by metal nanoparticles in human monocytes:The role of oxidative stress and protein tyrosine kinase activation.Toxicol Appl Pharmacol.2008 Dec 1;233(2):276-85.
15 Gardi C,Arezzini B,Fortino V,et al.Effect of free iron on collagen synthesis,cell proliferation and MMP-2 expression in rat hepatic stellate cells.Biochem Pharmacol.2002 OCT 1;64(7):1139-45.
16 Sakaida I,Hironaka K,Terai S,et al.Gadolinium chloride reverses dimethylnitrosamine (DMN)-induced rat liver fibrosis with increased matrix metalJoproteinases (MMPs)of Kupffer cells.Life Sci.2003 Jan 10;72(8):943-59.
17 Sage EH,Reed M,Funk SE,et al.Cleavage of the matricellular protein SPARC by matrix metalloproteinase-3 produces polypeptides that influence angiogenesis.J Biol Chem.2003 Sep 26;278(39):37849-57.
18 Hughes WM Jr,Rodriguez WE,Rosenberger D,et al.Role of copper and homocysteine in pressure overload heart failure.Cardiovasc Toxicol.2008 Fall;8(3):137-44.
19 Tamai E,Miyata S,Tanaka H,et al.High-level expression of his-tagged clostridial collagenase in Clostridium perfringens.Appl Microbiol Biotechnol.2008 Sep;80(4):627-35.
20 Wang GY,Bergman MR,Nguyen AP,et al.Cardiac transgenic matrix metalloproteinase-2 expression directly induces impaired contractility.Cardiovasc Res.2006 Feb 15;69(3):688-96.
21 Lin SC,Chung MY,Huang JW,et al.Correlation between functional genotypes in the matrix metalloproteinases-1 promoter and risk of oral squamous cell carcinomas.J Oral Pathol Med.2004 Jul;33(6):323-6.
22 Duong HS,Zhang QZ,Le AD,et al.Elevated prolidase activity in keloids:correlation with type 1 collagen turnover.Br J Dermatol.2006 May;154(5):820-828.
23 Vokurka J,Klapusova L,Pantuckova P,et al.The association of MMP-9 and IL-18 gene promoter polymorphisms with gingivitis in adolescents.Arch Oral Biol.2009 Feb;54(2):172-8.
24 Lee YJ,Woo M,Nam JH,et al.Matrix metalloproteinase-9 promoter polymorphisms in Korean patients with systemic lupus erythematosus.Hum Immunol.2008 Jun;69(6):374-9.
25 Demacq C,Vasconcellos VB,Marcaccini AM,et al.Functional polymorphisms in the promoter of the matrix metalloproteinase-9 (MMP-9)gene are not linked with significant plasma MMP-9 variations in healthy subjects.Clin Chem Lab Med.2008;46(1):57-63.
26 Giirkan A,Emingil G,Saygan BH,et al.Gene polymorphisms of matrix metalloproteinase-2,-9 and-12 in periodontal health and severe chronic periodontitis.Arch Oral Biol.2008 Apr;53(4):337-45.
27 Krumsiek A,Kropf S,Gardemann A.Tumor necrosis factor-alpha G(-308)A promoter polymorphism,matrix metalloproteinase (MMP)-3 5A/6A gene variation,MMP-9 C(-1562)T promoter polymorphism and risk and extent of ischemic heart disease.Clin Chem Lab Med.2008;46(2):292-5.
28 Giirkan A,Emingil G,Saygan BH,et al.Matrix metalloproteinase-2,-9,and-12 gene polymorphisms in generalized aggressive periodontitis.J Periodontol.2007 Dec;78(12):2338-47.
29 Tang LJ,Chen XF,Zhu M,et al.Matrix metalloproteinase-1,-3,and-9 gene polymorphisms and the risk of idiopathic dilated cardiomyopathy in a Chinese Han population.Clin Biochem.2007 Dec;40(18):1427-30.
30 Zivkovic M,Djuric T,Dincic E,et al.Matrix metalloproteinase-9-1562 C/T gene polymorphism in Serbian patients with multiple sclerosis.J Neuroimmunol.2007 Sep;189(l-2):147-50.
31 Tu HF,Wu CH,Kao SY,et al.Functional-1562 C-to-T polymorphism in matrix metalloproteinase-9 (MMP-9)promoter is associated with the risk for oral squamous cell carcinoma in younger male areca users.J Oral Pathol Med.2007 Aug;36(7):409-14.
32 Nasr HB,Mestiri S,Chahed K,et al.Matrix metalloproteinase-1 (-1607)1G/2G and-9 (-1562)C/T promoter polymorphisms:susceptibility and prognostic implications in nasopharyngeal carcinomas.Clin Chim Acta.2007 Sep;384(l-2):57-63.
33 Xu E,Xia X,Lü B,et al.Association of matrix metalloproteinase-2 and-9 promoter polymorphisms with colorectal cancer in Chinese.Mol Carcinog.2007 Nov;46(l l):924-9.
34 Lu Z,Cao Y,Wang Y,et al.Polymorphisms in the matrix metalloproteinase-1,3,and 9 promoters and susceptibility to adult astrocytoma in northern China.J Neurooncol.2007 Oct;85(1):65-73.
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52 Lim CP,Phan TT,Lim IJ,Cao X.Cytokine profiling and Stat3 phosphorylation in epithelial-mesenchymal interactions between keloid keratinocytes and fibroblasts.J Invest Dermatol.2009 Apr;129(4):851-61.
53 Ghazizadeh M.Essential role of IL-6 signaling pathway in keloid pathogenesis.J Nippon Med Sch.2007 Feb;74(1):l 1-22.
54 Uitto J.IL-6 signaling pathway in keloids:a target for pharmacologic intervention?J Invest Dermatol.2007 Jan;127(1):6-8.
55 Ghazizadeh M,Tosa M,Shimizu H,et al.Functional implications of the IL-6 signaling pathway in keloid pathogenesis.J Invest Dermatol.2007 Jan;127(1):98-105.
56 Tosa M,Ghazizadeh M,Shimizu H,et al.Global gene expression analysis of keloid fibroblasts in response to electron beam irradiation reveals the involvement of interleukin-6 pathway.J Invest Dermatol.2005 Apr;124(4):704-13.
57 Giugliano G,Pasquali D,Notaro A,et al.Verapamil inhibits interleukin-6 and vascular endothelial growth factor production in primary cultures of keloid fibroblasts.Br J Plast Surg.2003 Dec;56(8):804-9.
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64 Analysis of IL6 and IL1A gene polymorphisms in UK and Dutch patients with sarcoidosis.Grutters JC,Sato H,Pantelidis P,Ruven HJ,McGrath DS,Wells AU,van den Bosch JM,Welsh KI,du Bois RM.Sarcoidosis Vase Diffuse Lung Dis.2003 Mar;20(1):20-7.
65 Elevated interleukin-6 plasma levels are regulated by the promoter region polymorphism of the IL6 gene in primary Sjogren's syndrome and correlate with the clinical manifestations of the disease.Hulkkonen J,Pertovaara M,Antonen J,Pasternack A,Hurme M.Rheumatology (Oxford).2001 Jun;40(6):656-61.
66 Analysis of tumor necrosis factor-alpha,lymphotoxin-alpha,tumor necrosis factor receptor Ⅱ,and interleukin-6 polymorphisms in patients with idiopathic pulmonary fibrosis.Pantelidis P,Fanning GC,Wells AU,et al.Am J Respir Crit Care Med.2001 May;163(6):1432-6.
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8 Omo-Dare P.Genetic studies on keloid.J Natl Med Assoc.1975,67:428-432.
9 Hui xue,Mccauleyr L,Wenru Zhang.Elevated interleukin-6 expression in keloid fibroblasts.The Journal of Surgical Research.2000,vol.89,nol,pp.74-77 (24 ref.).
10 Dan Leibovici,H.Barton Grossman,Colin P.Dinney,et al.Polymorphisms in Inflammation Genes and Bladder Cancer:From Initiation to Recurrence,Progression,and Survival.
11 Bayat A,Arscott G,Oilier WE,et al.Description of site-specific morphology of keloid phenotypes in an Afrocaribbean population.Br J Plast Surg.2004 Mar;57(2):122-133.
12 Bloom D.Heredity of keloids.NY State Med J.1956;56:511-519
13 Marneros AG,Norris JE,Watanabe S,et a/.Genome scans provide evidence for keloid susceptibility loci on chromosomes 2q23 and 7pl 1J Invest Dermatol,2004,122(5):1126-1132.
14 Lim CP,Phan TT,Lim IJ,Cao X.Cytokine profiling and Stat3 phosphorylation in epithelial-mesenchymal interactions between keloid keratinocytes and fibroblasts.J Invest Dermatol.2009 Apr;129(4):851-61.
15 Ghazizadeh M.Essential role of IL-6 signaling pathway in keloid pathogenesis.J Nippon Med Sch.2007 Feb;74(1):11-22.
16 Uitto J.IL-6 signaling pathway in keloids:a target for pharmacologic intervention? J Invest Dermatol.2007 Jan;127(1):6-8.
17 Ghazizadeh M,Tosa M,Shimizu H,et al.Functional implications of the IL-6 signaling pathway in keloid pathogenesis.J Invest Dermatol.2007 Jan;127(1):98-105.
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