皮下多发脂肪瘤致病相关基因分析

发布时间：2018-06-22 16:12

本文选题：脂肪瘤 + 基因芯片　；参考：《第二军医大学》2012年博士论文

【摘要】：研究背景脂肪瘤(lipoma)是由成熟脂肪组织增生而形成的良性肿瘤，既有的大多数教科书认为此病可发生于任何年龄，但多见于40～60岁的成年人。由于绝大多数的脂肪瘤不会恶变，无特殊不适症状和并发症，易于诊断，治疗手段单一（目前多以手术切除为主），因此，对脂肪瘤的全面研究未能引起足够的重视，其研究进展也相对较慢。在临床工作中，大多数医生会注意到，对于单发脂肪瘤，手术基本可以治愈，预后良好，而对于皮下多发脂肪瘤患者，此病虽不至于危及生命，但会使患者终日忧心于肿瘤的不断进展和苦于找不到彻底根治的方法和手段，从而严重影响患者的身心健康和生活质量。更为重要的是，随着社会飞速进步带来的压力增大、不良生活习惯增加及饮食结构的变化，皮下多发脂肪瘤的发病率异常增高，门诊患者中此类疾病几乎每日可见，而且大多数此病患者都具有父代或子代皆有发病的家族倾向，这就使得针对此病的研究显得颇为重要和十分必要。 20世纪90年代中后期，国外学者在软组织肿瘤的细胞和分子遗传学研究中取得了突破性进展，包括针对脂肪瘤的分子遗传学研究，基本证实脂肪瘤细胞发生了染色体易位、重排或融合，这些遗传学异常导致了相应基因的突变和扩增。研究结果显示：55~57%的脂肪瘤病例存在染色体异常，主要涉及12q13-15，少数涉及6p21-23，或丢失13q中的一些成分。位于12q15区带上的HMGIC基因（high mobility group IC gen，肿瘤相关基因高迁移率蛋白IC）重排，现认为在脂肪瘤的发生过程中起了主要作用。研究显示，t(3:12)(q27-28;q13-15)导致位于12q15上的HMGIC基因与位于3q27-28上的LPP基因融合形成HMGIC-LPP融合基因，断裂点分别为HMGIC基因为3号内显子，LPP基因为8号内显子。除t(3;12)(p27-28;q13-15)外，文献上还报道了1例t(12;13)(q13-15;q12)，使HMGIC基因与LHFP基因发生融合。遗憾的是，这些染色体遗传学改变后引起哪些相应的基因发生变化，这些变化基因的下游基因表达如何，其报道很少。从流行病学角度来看，目前，比较公认的与脂肪瘤发生、发展密切相关的因素主要有如下几种：1.遗传因素，临床中可见父代单发脂肪瘤，子代出现多发脂肪瘤或父代多发，而子代单发或多发的情况，这与上述染色体改变学说刚好能够互为呼应；2.生活习惯不良，如过度饮酒、高脂饮食、熬夜等；3.生活或工作压力过大。从文献检索来看，近年来，国内外关于脂肪异常增生性疾病的研究更多倾向于多发性对称性脂肪瘤病以及肥胖的研究，从这些研究中也可以给我们一定的借鉴经验，对于染色体异常所导致的下游基因改变的研究具有相当大的参考价值。通过网络信息检索，，国内也有言论称脂肪瘤致瘤因子是脂肪瘤形成的真正原因。提出这一理论的人员推测在脂肪瘤患者体细胞内存在一种致瘤因子，在正常情况下，这种致瘤因子处于一种失活状态（无活性状态），不会发病，但在机体内环境改变时，由于体内的淋巴细胞、单核吞噬细胞等免疫细胞对致瘤因子的监控能力下降，加之慢性炎症刺激、全身脂肪代谢异常等诱因条件下，脂肪瘤致瘤因子活性进一步增强与细胞内的某些基因片断结合，形成基因异常突变，导致脂肪组织沉积，最终形成脂肪瘤（http://www.clsbio.com）。笔者虽经多方检索，但遗憾的是未能找到针对此项论断的专业文献报道。但不可否认的是，脂肪瘤和其它肿瘤一样，是正常细胞通过一系列的基因改变而转变为肿瘤细胞，这种改变可以是遗传性或后天获得性，在受到环境、饮食、辐射和病毒等因素影响引起染色体的变化，从而使mRNA转录发生异常，产生过量肿瘤相关蛋白或结构异常的蛋白，导致细胞分裂和分化失控，通过多阶段、多步骤转变为肿瘤细胞。因此，目前我们已知的是，脂肪瘤的发病是多因素相互作用的结果，那么这些因素最终是如何导致脂肪瘤的发生？在脂肪瘤形成过程中到底是哪些基因发生了改变？又为什么只是瘤变而多不恶变哪？这些问题的答案还未可知。针对上述问题，本课题拟通过基因芯片技术筛选出脂肪瘤与正常脂肪组织之间存在的相关差异基因，以期阐释皮下多发脂肪瘤的发病机制，并为临床治疗提供可能的理论借鉴。研究目的本研究旨在通过对皮下多发脂肪瘤患者的瘤体与周围正常脂肪组织的基因差异分析，探索皮下多发脂肪瘤的相关基因表达，为临床预防和治疗提供可能的理论借鉴。研究方法一.样本采集：来源为长海医院整形外科门诊收治的男性皮下多发脂肪瘤患者，排除全身系统性疾病，平素体健，年龄≤60岁，全身瘤体数量≥5个，所有瘤体直径≤3cm，发病时间≤2年，目的是为了便于捕捉脂肪瘤瘤变早期的基因表达差异。同时采集瘤体周围正常脂肪组织作为自体对照样本，排除个体差异所造成的基因差异，缩小筛选范围。二.实验分组：1.基因芯片检测样本采集自3个患者，共6个，包括脂肪瘤样本3个（实验组，n=3）和脂肪瘤周围正常脂肪组织样本3个（对照组，n=3）；2.人群散发皮下多发脂肪瘤样本和自体对照正常脂肪样本各3个（n=3）。三.组织病理学分析：通过大体观察、组织病理学切片HE染色、脂肪组织特殊染色、脂肪瘤内血管及神经分布，观察脂肪瘤与正常脂肪组织之间的形态学共性和个性。四.基因芯片差异基因筛选：应用Affymetrix Human U133Plus2.0芯片（人类全基因组芯片）对实验组和对照组共6个样本进行基因检测，所得检测结果通过SBC生物芯片分析系统进行差异基因筛选，取差异倍数（foldchange）2、p0.05的基因为差异基因。将6张基因芯片筛选出的差异基因按照实验组和对照组进行聚类分析，并根据分析结果对其功能进行综合分析，初步筛选出6个可能与脂肪瘤发生、增殖、信号传导等功能改变相关基因作为重要差异基因。五.相关基因不同样本验证：将两次取材的共6例实验组样本及6例对照组样本进行6个重要差异基因的RT-PCR验证，排除及证实基因芯片结果的可靠性，并综合基因芯片及RT-PCR两种检测方法所得6个基因表达量的差异和分子生物学功能，初步探讨这些基因在脂肪瘤发生及发展中可能的作用及机制。研究结果第一部分皮下多发脂肪瘤的组织病理学分析本课题取材的多发脂肪瘤均具有完整包膜，包膜细薄，可见少量血管分布，瘤体色黄，有一定韧性，剖面见脂肪组织质地较均一，结缔组织间隔较少，将瘤体分隔为大小不一的小叶。瘤体周围正常脂肪组织无包膜，被纤维间隔分隔成豆大的小叶状，单位体积内间隔成分较脂肪瘤组织明显增多。镜下见脂肪瘤组织内主要由分化成熟的脂肪细胞构成，瘤体外周有薄的纤维组织间隔，纤维间隔向内伸展，将瘤体分成各个大小不一的分叶，不同小叶间细胞大小也具有差异，细胞挤压呈圆形或多边形，细胞内含有大量脂滴脱失后形成的空泡，细胞核被挤压偏位呈扁圆或新月状。间隔内有丰富的供血血管和其它类型细胞成分分布，小叶间排列紧密，小叶内的脂肪细胞大小不一。正常脂肪组织纤维间隔丰富，组织松散，脂肪细胞形态大小较脂肪瘤组织更为均一相近，间隔成分主要为纤维结缔组织。特殊染色显示两组的细胞内脂滴油红染色阳性。第二部分皮下多发脂肪瘤致病相关基因的基因组学分析 6例样本的基因探针结合总数为54614个，脂肪瘤组和正常脂肪组间进行统计学分析，结果显示两组差异结合探针总数共1776个（p0.05），其中差异倍数大于两倍的差异探针结合数共374个。经聚类分析，初筛p0.05，Foldchange＞2的差异基因共260个。与细胞增殖相关的结构基因差异数为36个，其中上调基因30个，下调基因6个；2个凋亡抑制基因ERBB4和NPY5R下调7倍和4.425倍；具有凋亡双向调节功能的SOX4上调2.3118倍，具有抗凋亡作用的COMP和HGF分别上调5.9558和3.366倍，具有凋亡诱导作用的PERP下调2.387倍；脂类结合基因10个，其中上调基因5个，下调基因5个；脂类储存正性调节基因1个，即载脂蛋白B基因（APOB）下调7倍（p=0.0044）；脂类储存负向调节基因ABCG1上调2.5倍。另外，与肿瘤细胞增殖及调控相关的几个基因ESM1、SOX11及HOXD10等较正常脂肪组的表达量分别增高32.81倍、31.01倍及13.99倍。结合各个基因的生物学功能和表达差异，筛选ESM1、SOX11、HOXD10、ERBB4、NPY5R及APOB进行散发人群验证。第三部分差异基因的散发人群验证应用RT-PCR验证样本的脂肪瘤组ESM1、SOX11、HOXD10、ERBB4、NPY5R及APOB的表达与基因组学分析结果基本一致，结果显示六个基因的表达差异与基因芯片结果一致，并且每个样本的变化特点与基因芯片的信号强度变化完全符合，应用RT-PCR检测此六个基因的差异表达结果分别为：ESM1上调18.51倍，SOX11上调19.18倍，HOXD10上调20.55倍，ERBB4下调16.68倍，NPY5R下调4.99倍，APOB下调15.05倍。散发人群的PCR结果同样显示了与基因芯片相一致的变化规律，ESM1上调290.86倍，SOX11上调6.13倍，HOXD10上调11.2倍，ERBB4下调5.09倍， NPY5R下调7.19倍，APOB下调15.05倍。既验证了基因芯片结果的可靠性和稳定性，也验证了这六个差异基因在散发人群中的表达差异是确实存在的。研究结论本课题应用基因芯片对皮下多发脂肪瘤瘤体与自体正常脂肪之间的差异基因进行初步筛选，并从中挑选出两组差异倍数较大的致瘤相关基因3个（ESM1、SOX11、HOXD10）、细胞凋亡抑制基因2个（ERBB4和NPY5R）及脂类储存调解基因1个（APOB），通过荧光定量PCR方法在散发人群中进行验证，初步证实皮下多发脂肪瘤的发生是由于致瘤基因的异常高表达导致细胞增殖能力增强，而细胞死亡和凋亡基因的上调以及细胞凋亡抑制基因的低表达能够促进增殖细胞的正常凋亡，防止细胞恶变，另外，脂类储存和代谢的异常造成脂质在胞浆内的沉积所造成，初步证实脂肪瘤的个体发生是局部脂肪细胞增殖造成细胞数量增多而非原有脂肪细胞局部膨大式增长形成，为揭示脂肪瘤的发病机制提供了一定的实验依据，并为指导临床治疗提供借鉴。
[Abstract]:Research background
Lipoma (lipoma) is a benign tumor formed by the proliferation of mature adipose tissue. Most textbooks believe that this disease can occur at any age, but most of them are at the age of 40~60. Therefore, the comprehensive study of lipoma has not been paid enough attention to, and the research progress is relatively slow. In clinical work, most doctors will notice that the operation is basically cured and the prognosis is good for the single lipoma, and for the patients with multiple lipoma, the disease will not endanger the life, but it will cause the patient. All day long is worried about the progress of the tumor and the methods and means that can not be completely cured, which seriously affects the physical and mental health and the quality of life of the patients. More importantly, the incidence of subcutaneous lipoma increases with the increasing pressure of the rapid progress of the society, the increase of the bad habits and the change of the diet structure. In the outpatient, the disease is almost daily, and most of the patients have family tendencies of the parent or offspring, which makes it very important and necessary to study the disease.
In the mid and late 1990s, foreign scholars have made breakthroughs in the study of cell and molecular genetics of soft tissue tumors, including the molecular genetics of lipoma, which basically confirmed that the lipoma cells have chromosomal translocation, rearrangement or fusion. These genetic abnormalities lead to the mutation and amplification of the corresponding genes. The results showed that there were chromosomal abnormalities in the cases of 55~57% lipoma, mainly involving 12q13-15, a few involving 6p21-23, or some components in the loss of 13q. The HMGIC gene located in the zone of 12q15 (high mobility group IC gen, tumor related gene high mobility protein IC) rearranged, which is now considered to play a major role in the occurrence of lipoma. The study showed that t (3:12) (3:12) (q27-28; q13-15) resulted in the fusion of HMGIC gene located on 12q15 and the LPP gene located on 3q27-28 to form a HMGIC-LPP fusion gene. The breakpoints were HMGIC gene 3 and LPP gene 8. Besides t (3; 12), 1 cases (12; 13) were reported in the literature. HFP genes are fused. Unfortunately, what corresponding genes are caused by the genetic changes of these chromosomes, and how the downstream genes are expressed in these genes is rarely reported. From an epidemiological point of view, the most commonly recognized factors that are closely related to the occurrence of lipoma are as follows: 1. heredity. Factors, in the clinic, we can see the single lipoma of the father generation, the multiple lipoma of the progeny or the multiple hair of the father generation, and the single or multiple generation of the progeny, which can correspond to the above chromosome change theory. 2. the living habits, such as excessive drinking, high fat diet, stay up late, and so on; 3. life or work pressure too much. Over the years, the research on adipose hyperplastic diseases at home and abroad is more likely to study on multiple symmetric lipomatosis and obesity. From these studies, we can also give us some reference experience and have considerable reference value for the study of the downstream gene changes caused by chromosomal abnormalities. It is also said that lipoma is a real reason for lipoma formation. People who put forward this theory speculate that in the body cells of the lipoma, there is a tumor factor in the body cells of the lipoma. In normal circumstances, the tumor factor is in a inactive state (inactive state) and will not occur, but in the body when the body changes in the body, it is due to the body. The ability of lymphocyte, mononuclear phagocyte and other immune cells to monitor the tumor inducing factor is decreased, in addition to chronic inflammatory stimuli and abnormal body fat metabolism, the activity of the lipoma is further enhanced with some gene fragments in the cell, forming an abnormal mutation, resulting in the formation of adipose tissue and final formation. Lipoma (http://www.clsbio.com). Although the author has been retrieved in many ways, it is regrettable to have failed to find a professional literature report aimed at this argument. But it is undeniable that the lipoma, like other tumors, is a normal cell transformed into a tumor cell through a series of gene changes, which can be inherited or acquired acquired, Factors such as environment, diet, radiation and viruses cause changes in chromosomes, causing abnormal transcription of mRNA, producing excessive tumor related proteins or proteins with abnormal structure, causing cell division and differentiation out of control and transforming into tumor cells through multistage and multistep steps. Therefore, we are now known to be the disease of lipoma. How do these factors ultimately lead to lipoma, and what genes have changed in the course of lipoma? And why does the tumor change and do not change much? The answers to these questions are still unknown.
In order to solve the above problems, we should use gene chip technology to screen out the related differential genes between lipoma and normal adipose tissue, in order to explain the pathogenesis of subcutaneous multiple lipoma and provide possible theoretical reference for clinical treatment.
research objective
The purpose of this study is to explore the gene expression of subcutaneous multiple lipoma by analyzing the gene difference between the tumor body and the normal adipose tissue in the patients with subcutaneous lipoma, and to provide a possible theoretical reference for clinical prevention and treatment.
research method
1. Sample collection: the male subcutaneous multiple lipoma patients were treated in the orthopedics outpatient department of Changhai Hospital, excluding systemic diseases, plain body health, age less than 60 years old, total body number more than 5, all tumor diameter less than 3cm, the onset time was less than 2 years, the aim is to facilitate the capture of the early gene expression difference in lipoma. At the same time, the normal adipose tissue around the tumor was collected as an autologous control sample to exclude the genetic differences caused by individual differences and to narrow the scope of screening.
Two. Experimental group: 1. gene chip detection samples were collected from 3 patients, including 6 samples, including 3 lipoma samples (experimental group, n=3) and 3 normal aliphatic tissue samples (control group, n=3); 2. people distributed subcutaneous lipoma samples and 3 samples of autologous normal fat (n=3).
Three. Histopathological analysis: by gross observation, histopathological section HE staining, special staining of adipose tissue, blood vessels and nerve distribution in lipoma, and observation of morphological generality and personality between lipoma and normal adipose tissue.
Four. Gene chip differential gene screening: Affymetrix Human U133Plus2.0 chip (human genome chip) was used to detect 6 samples of the experimental group and the control group. The results were screened by the SBC biochip analysis system, the difference multiplier (foldchange) 2, and the P0.05 gene were the difference genes. 6 The differential genes selected by Zhang Jiyin chip were cluster analyzed according to the experimental group and the control group. According to the analysis results, the functions were analyzed synthetically, and 6 genes related to lipoma, proliferation and signal transduction were screened out as important differentially genes.
Five. Different samples of related genes were verified: 6 samples of experimental group and 6 cases of control group were tested by RT-PCR to exclude and verify the reliability of gene chip results, and the difference of expression and molecular biological function of 6 bases obtained by integrated gene chip and two detection methods of RT-PCR were synthesized. Objective to explore the possible roles and mechanisms of these genes in the occurrence and development of lipoma.
Research results
The first part is a histopathological analysis of multiple cutaneous lipoma.
The multiple lipoma of this topic have complete capsule, thin film and thin film, a small amount of blood vessel distribution, the tumor is yellow and has a certain toughness. The section shows that the fat tissue is relatively uniform, the connective tissue is less spaced, and the tumor is divided into small and large lobules. The internal septum in the unit volume is more than that of the lipoma. It is found that the lipoma is mainly composed of the mature adipocytes in the lipoma tissue. The tumor is divided into thin fibrous septum in vitro, and the fibrous septum extends inward. The tumor is divided into different lobules of different sizes, and the size of the cells in different lobules is different and the cells are squeezed. The cell contains a large number of circular or polygons. The cell contains a large number of vacuoles formed after the loss of fat drops. The nucleus is compressed and flattened or crescent. There are abundant blood vessels and other types of cell components in the interval. The interlobular arrangement is close, the size of the fat cells in the lobule is different. The normal fat tissue is rich in fiber and loose tissue. The shape and size of adipocytes were more similar than those of lipoma, and the interval components were mainly fibrous connective tissue. Special staining showed that the lipid droplets in the two groups were positive.
The second part is a genomics analysis of the pathogenic genes associated with multiple cutaneous lipoma.
The total number of gene probes in 6 samples was 54614, the lipoma group and the normal fat group were analyzed statistically. The results showed that the total number of differences combined with the total number of probes was 1776 (P0.05), and the difference multiplier was more than two times the number of differential probe binding 374. The difference of the primary screening P0.05 and Foldchange > 2 were 260. The number of structural genes related to cell proliferation was 36, of which 30 were up-regulated and 6 were down regulated; 2 apoptosis suppressor genes, ERBB4 and NPY5R, were down regulated by 7 times and 4.425 times; the SOX4 with bidirectional regulation of apoptosis was 2.3118 times higher than that of COMP and HGF with anti apoptotic effect, and PE with apoptosis inducing effect, respectively. RP was down 2.387 times, and there were 10 lipid binding genes, including 5 up-regulated and 5 down regulated genes, 1 lipid storage positive regulation genes, 7 times down regulation of apolipoprotein B gene (APOB), and 2.5 times up regulation of lipid storage negative regulation gene ABCG1. In addition, several genes related to tumor cell proliferation and regulation, ESM1, SOX11 and HOXD10, etc. The expression of the normal fat group increased 32.81 times, 31.01 times and 13.99 times, respectively. ESM1, SOX11, HOXD10, ERBB4, NPY5R and APOB were screened for sporadic population according to the biological function and expression difference of each gene.
Third parts of the sporadic population of differential genes
The expression of ESM1, SOX11, HOXD10, ERBB4, NPY5R and APOB in the lipoma group with RT-PCR samples was basically consistent with the results of genomic analysis. The results showed that the differences of the expression of six genes were consistent with the gene chip results, and the change characteristics of each sample were in full conformity with the signal intensity changes of the gene chip, and RT-PCR was used to detect this six. The difference expression results were as follows: ESM1 up 18.51 times, SOX11 up 19.18 times up, HOXD10 up 20.55 times, ERBB4 down 16.68 times, NPY5R down 4.99 times, APOB down 15.05 times. PCR results in the sporadic population also showed the same change with gene chip, ESM1 up 290.86 times, SOX11 up 6.13 times up, HOXD10 up 11.2 times up, E RBB4 was down 5.09 times, NPY5R was down 7.19 times, and APOB was down 15.05 times. It not only verified the reliability and stability of the gene chip results, but also proved that the difference in the expression of these six genes in the sporadic population was true.
research conclusion
The gene chip was used to screen the differentially genes between the subcutaneous multiple lipoma tumors and the autologous normal fat, and two groups of two groups, 3 (ESM1, SOX11, HOXD10), and 2 (ERBB4 and NPY5R) and 1 lipid storage mediating genes (APOB), were selected. The quantitative PCR method was validated in sporadic population. It was preliminarily confirmed that the occurrence of multiple cutaneous lipoma was caused by abnormal high expression of tumorigenic gene.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R739.5

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