本文選題：脂肪瘤 + 基因芯片�。� 參考：《第二軍醫(yī)大學》2012年博士論文

【摘要】：研究背景 脂肪瘤(lipoma)是由成熟脂肪組織增生而形成的良性腫瘤，既有的大多數(shù)教科書認為此病可發(fā)生于任何年齡，但多見于40～60歲的成年人。由于絕大多數(shù)的脂肪瘤不會惡變，無特殊不適癥狀和并發(fā)癥，易于診斷，治療手段單一（目前多以手術切除為主），因此，對脂肪瘤的全面研究未能引起足夠的重視，其研究進展也相對較慢。在臨床工作中，大多數(shù)醫(yī)生會注意到，對于單發(fā)脂肪瘤，手術基本可以治愈，預后良好，而對于皮下多發(fā)脂肪瘤患者，此病雖不至于危及生命，但會使患者終日憂心于腫瘤的不斷進展和苦于找不到徹底根治的方法和手段，從而嚴重影響患者的身心健康和生活質量。更為重要的是，隨著社會飛速進步帶來的壓力增大、不良生活習慣增加及飲食結構的變化，皮下多發(fā)脂肪瘤的發(fā)病率異常增高，門診患者中此類疾病幾乎每日可見，而且大多數(shù)此病患者都具有父代或子代皆有發(fā)病的家族傾向，這就使得針對此病的研究顯得頗為重要和十分必要。 20世紀90年代中后期，國外學者在軟組織腫瘤的細胞和分子遺傳學研究中取得了突破性進展，包括針對脂肪瘤的分子遺傳學研究，基本證實脂肪瘤細胞發(fā)生了染色體易位、重排或融合，這些遺傳學異常導致了相應基因的突變和擴增。研究結果顯示：55~57%的脂肪瘤病例存在染色體異常，主要涉及12q13-15，少數(shù)涉及6p21-23，或丟失13q中的一些成分。位于12q15區(qū)帶上的HMGIC基因（high mobility group IC gen，腫瘤相關基因高遷移率蛋白IC）重排，現(xiàn)認為在脂肪瘤的發(fā)生過程中起了主要作用。研究顯示，t(3:12)(q27-28;q13-15)導致位于12q15上的HMGIC基因與位于3q27-28上的LPP基因融合形成HMGIC-LPP融合基因，斷裂點分別為HMGIC基因為3號內顯子，LPP基因為8號內顯子。除t(3;12)(p27-28;q13-15)外，文獻上還報道了1例t(12;13)(q13-15;q12)，使HMGIC基因與LHFP基因發(fā)生融合。遺憾的是，這些染色體遺傳學改變后引起哪些相應的基因發(fā)生變化，這些變化基因的下游基因表達如何，其報道很少。從流行病學角度來看，目前，比較公認的與脂肪瘤發(fā)生、發(fā)展密切相關的因素主要有如下幾種：1.遺傳因素，臨床中可見父代單發(fā)脂肪瘤，子代出現(xiàn)多發(fā)脂肪瘤或父代多發(fā)，而子代單發(fā)或多發(fā)的情況，這與上述染色體改變學說剛好能夠互為呼應；2.生活習慣不良，如過度飲酒、高脂飲食、熬夜等；3.生活或工作壓力過大。從文獻檢索來看，近年來，國內外關于脂肪異常增生性疾病的研究更多傾向于多發(fā)性對稱性脂肪瘤病以及肥胖的研究，從這些研究中也可以給我們一定的借鑒經(jīng)驗，對于染色體異常所導致的下游基因改變的研究具有相當大的參考價值。通過網(wǎng)絡信息檢索，，國內也有言論稱脂肪瘤致瘤因子是脂肪瘤形成的真正原因。提出這一理論的人員推測在脂肪瘤患者體細胞內存在一種致瘤因子，在正常情況下，這種致瘤因子處于一種失活狀態(tài)（無活性狀態(tài)），不會發(fā)病，但在機體內環(huán)境改變時，由于體內的淋巴細胞、單核吞噬細胞等免疫細胞對致瘤因子的監(jiān)控能力下降，加之慢性炎癥刺激、全身脂肪代謝異常等誘因條件下，脂肪瘤致瘤因子活性進一步增強與細胞內的某些基因片斷結合，形成基因異常突變，導致脂肪組織沉積，最終形成脂肪瘤（http://www.clsbio.com）。筆者雖經(jīng)多方檢索，但遺憾的是未能找到針對此項論斷的專業(yè)文獻報道。但不可否認的是，脂肪瘤和其它腫瘤一樣，是正常細胞通過一系列的基因改變而轉變?yōu)槟[瘤細胞，這種改變可以是遺傳性或后天獲得性，在受到環(huán)境、飲食、輻射和病毒等因素影響引起染色體的變化，從而使mRNA轉錄發(fā)生異常，產(chǎn)生過量腫瘤相關蛋白或結構異常的蛋白，導致細胞分裂和分化失控，通過多階段、多步驟轉變?yōu)槟[瘤細胞。因此，目前我們已知的是，脂肪瘤的發(fā)病是多因素相互作用的結果，那么這些因素最終是如何導致脂肪瘤的發(fā)生？在脂肪瘤形成過程中到底是哪些基因發(fā)生了改變？又為什么只是瘤變而多不惡變哪？這些問題的答案還未可知。 針對上述問題，本課題擬通過基因芯片技術篩選出脂肪瘤與正常脂肪組織之間存在的相關差異基因，以期闡釋皮下多發(fā)脂肪瘤的發(fā)病機制，并為臨床治療提供可能的理論借鑒。 研究目的 本研究旨在通過對皮下多發(fā)脂肪瘤患者的瘤體與周圍正常脂肪組織的基因差異分析，探索皮下多發(fā)脂肪瘤的相關基因表達，為臨床預防和治療提供可能的理論借鑒。 研究方法 一.樣本采集：來源為長海醫(yī)院整形外科門診收治的男性皮下多發(fā)脂肪瘤患者，排除全身系統(tǒng)性疾病，平素體健，年齡≤60歲，全身瘤體數(shù)量≥5個，所有瘤體直徑≤3cm，發(fā)病時間≤2年，目的是為了便于捕捉脂肪瘤瘤變早期的基因表達差異。同時采集瘤體周圍正常脂肪組織作為自體對照樣本，排除個體差異所造成的基因差異，縮小篩選范圍。 二.實驗分組：1.基因芯片檢測樣本采集自3個患者，共6個，包括脂肪瘤樣本3個（實驗組，n=3）和脂肪瘤周圍正常脂肪組織樣本3個（對照組，n=3）；2.人群散發(fā)皮下多發(fā)脂肪瘤樣本和自體對照正常脂肪樣本各3個（n=3）。 三.組織病理學分析：通過大體觀察、組織病理學切片HE染色、脂肪組織特殊染色、脂肪瘤內血管及神經(jīng)分布，觀察脂肪瘤與正常脂肪組織之間的形態(tài)學共性和個性。 四.基因芯片差異基因篩選：應用Affymetrix Human U133Plus2.0芯片（人類全基因組芯片）對實驗組和對照組共6個樣本進行基因檢測，所得檢測結果通過SBC生物芯片分析系統(tǒng)進行差異基因篩選，取差異倍數(shù)（foldchange）2、p0.05的基因為差異基因。將6張基因芯片篩選出的差異基因按照實驗組和對照組進行聚類分析，并根據(jù)分析結果對其功能進行綜合分析，初步篩選出6個可能與脂肪瘤發(fā)生、增殖、信號傳導等功能改變相關基因作為重要差異基因。 五.相關基因不同樣本驗證：將兩次取材的共6例實驗組樣本及6例對照組樣本進行6個重要差異基因的RT-PCR驗證，排除及證實基因芯片結果的可靠性，并綜合基因芯片及RT-PCR兩種檢測方法所得6個基因表達量的差異和分子生物學功能，初步探討這些基因在脂肪瘤發(fā)生及發(fā)展中可能的作用及機制。 研究結果 第一部分皮下多發(fā)脂肪瘤的組織病理學分析 本課題取材的多發(fā)脂肪瘤均具有完整包膜，包膜細薄，可見少量血管分布，瘤體色黃，有一定韌性，剖面見脂肪組織質地較均一，結締組織間隔較少，將瘤體分隔為大小不一的小葉。瘤體周圍正常脂肪組織無包膜，被纖維間隔分隔成豆大的小葉狀，單位體積內間隔成分較脂肪瘤組織明顯增多。鏡下見脂肪瘤組織內主要由分化成熟的脂肪細胞構成，瘤體外周有薄的纖維組織間隔，纖維間隔向內伸展，將瘤體分成各個大小不一的分葉，不同小葉間細胞大小也具有差異，細胞擠壓呈圓形或多邊形，細胞內含有大量脂滴脫失后形成的空泡，細胞核被擠壓偏位呈扁圓或新月狀。間隔內有豐富的供血血管和其它類型細胞成分分布，小葉間排列緊密，小葉內的脂肪細胞大小不一。正常脂肪組織纖維間隔豐富，組織松散，脂肪細胞形態(tài)大小較脂肪瘤組織更為均一相近，間隔成分主要為纖維結締組織。特殊染色顯示兩組的細胞內脂滴油紅染色陽性。 第二部分皮下多發(fā)脂肪瘤致病相關基因的基因組學分析 6例樣本的基因探針結合總數(shù)為54614個，脂肪瘤組和正常脂肪組間進行統(tǒng)計學分析，結果顯示兩組差異結合探針總數(shù)共1776個（p0.05），其中差異倍數(shù)大于兩倍的差異探針結合數(shù)共374個。經(jīng)聚類分析，初篩p0.05，F(xiàn)oldchange＞2的差異基因共260個。與細胞增殖相關的結構基因差異數(shù)為36個，其中上調基因30個，下調基因6個；2個凋亡抑制基因ERBB4和NPY5R下調7倍和4.425倍；具有凋亡雙向調節(jié)功能的SOX4上調2.3118倍，具有抗凋亡作用的COMP和HGF分別上調5.9558和3.366倍，具有凋亡誘導作用的PERP下調2.387倍；脂類結合基因10個，其中上調基因5個，下調基因5個；脂類儲存正性調節(jié)基因1個，即載脂蛋白B基因（APOB）下調7倍（p=0.0044）；脂類儲存負向調節(jié)基因ABCG1上調2.5倍。另外，與腫瘤細胞增殖及調控相關的幾個基因ESM1、SOX11及HOXD10等較正常脂肪組的表達量分別增高32.81倍、31.01倍及13.99倍。結合各個基因的生物學功能和表達差異，篩選ESM1、SOX11、HOXD10、ERBB4、NPY5R及APOB進行散發(fā)人群驗證。 第三部分差異基因的散發(fā)人群驗證 應用RT-PCR驗證樣本的脂肪瘤組ESM1、SOX11、HOXD10、ERBB4、NPY5R及APOB的表達與基因組學分析結果基本一致，結果顯示六個基因的表達差異與基因芯片結果一致，并且每個樣本的變化特點與基因芯片的信號強度變化完全符合，應用RT-PCR檢測此六個基因的差異表達結果分別為：ESM1上調18.51倍，SOX11上調19.18倍，HOXD10上調20.55倍，ERBB4下調16.68倍，NPY5R下調4.99倍，APOB下調15.05倍。散發(fā)人群的PCR結果同樣顯示了與基因芯片相一致的變化規(guī)律，ESM1上調290.86倍，SOX11上調6.13倍，HOXD10上調11.2倍，ERBB4下調5.09倍， NPY5R下調7.19倍，APOB下調15.05倍。既驗證了基因芯片結果的可靠性和穩(wěn)定性，也驗證了這六個差異基因在散發(fā)人群中的表達差異是確實存在的。 研究結論 本課題應用基因芯片對皮下多發(fā)脂肪瘤瘤體與自體正常脂肪之間的差異基因進行初步篩選，并從中挑選出兩組差異倍數(shù)較大的致瘤相關基因3個（ESM1、SOX11、HOXD10）、細胞凋亡抑制基因2個（ERBB4和NPY5R）及脂類儲存調解基因1個（APOB），通過熒光定量PCR方法在散發(fā)人群中進行驗證，初步證實皮下多發(fā)脂肪瘤的發(fā)生是由于致瘤基因的異常高表達導致細胞增殖能力增強，而細胞死亡和凋亡基因的上調以及細胞凋亡抑制基因的低表達能夠促進增殖細胞的正常凋亡，防止細胞惡變，另外，脂類儲存和代謝的異常造成脂質在胞漿內的沉積所造成，初步證實脂肪瘤的個體發(fā)生是局部脂肪細胞增殖造成細胞數(shù)量增多而非原有脂肪細胞局部膨大式增長形成，為揭示脂肪瘤的發(fā)病機制提供了一定的實驗依據(jù)，并為指導臨床治療提供借鑒。
[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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R739.5

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