發(fā)布時間：2018-06-23 01:25

  本文選題：鼻咽纖維血管瘤 + 性激素��； 參考：《復旦大學》2013年博士論文

【摘要】：鼻咽纖維血管瘤,又稱青少年鼻咽纖維血管瘤(juvenile nasopharyngeal angiofibroma, JNA),是一種幾乎只發(fā)生于青少年男性的鼻咽部良性腫瘤。JNA發(fā)病率較低,腫瘤極易出血,手術(shù)徹底切除難度較大。組織學上,JNA沒有包膜,由未成熟的內(nèi)皮細胞和成纖維細胞組成,雖然為良性腫瘤,但JNA具有局部侵犯的特征,易侵犯鼻竇、翼腭窩、顳下窩、翼板、眶上裂,甚至顱內(nèi)。 JNA特定的發(fā)病年齡和性別很早就受到學者們的關(guān)注,眾多學者期望從中找到研究JNA發(fā)病機制的相關(guān)線索。早期的研究認為JNA患者垂體-性腺軸發(fā)生混亂導致腫瘤的發(fā)生,但隨后的研究表明JNA患者體內(nèi)的性激素水平正常,且并沒有第二性征的發(fā)育異常。近期的研究則關(guān)注腫瘤本身性激素受體的表達與疾病的關(guān)系,但至今為止并沒有統(tǒng)一的結(jié)論。 本研究收集了40例JNA患者的血清標本,對血清中的睪酮,游離睪酮,雌激素,孕激素的水平進行elisa檢測,對比JNA患者的血清激素水平與對照組相比是否存在異常；將70例JNA的腫瘤組織和10例中甲組織制作成組織芯片,檢測雄激素受體(androgen receptor, AR)、雌激素受體α (estrogen receptor α, ER-α)、雌激素受體β (estrogen receptor α, ER-β)、孕激素受體(progesterone receptor, PR)及血管內(nèi)皮生長因子(vascular endothelial growth factor receptor, VEGF)在JNA中的表達及其相互關(guān)系；對5例高侵襲JNA組織、4例低侵襲JNA組織和5例正常中甲黏膜組織進行了表達譜基因芯片的研究,試圖發(fā)現(xiàn)參與JNA發(fā)病的關(guān)鍵基因及信號通路。 JNA易侵犯鄰近組織甚至顱內(nèi),術(shù)中極易出血,侵犯范圍較廣的腫瘤手術(shù)常常無法切除干凈,且存在較大風險,藥物及靶向治療一直是研究的重點。JNA腫瘤組織中性激素受體的表達以及表達譜芯片的研究為進一步探究JNA治療的新方法提供了基礎。 第一部分 鼻咽纖維血管瘤患者血清中性激素的表達 目的：鼻咽纖維血管瘤(JNA)是一種幾乎只發(fā)生于青少年男性的良性腫瘤,但至今病因不明。JNA特殊的發(fā)病年齡和性別提示性激素在腫瘤發(fā)病過程的可能起作用,本研究旨在探明JNA患者是否存在青春期性激素水平異常以及性激素水平是否與腫瘤分期相關(guān)。 方法：采集40例JNA患者(平均年齡18.3歲,均為術(shù)后病理證實)及14例正常對照組患者(平均年齡19.7歲)的血清,對照組患者為同時段、同年齡段、同性別的鼻中隔、骨瘤及視神經(jīng)減壓的患者。用Elisa法檢測JNA組與對照組患者血清中的睪酮、游離睪酮、雌二醇及孕酮的水平,比較兩組間性激素的水平以及雌雄激素的比值是否存在差異。收集40例JNA患者的腫瘤分期數(shù)據(jù),分析其與血清中性激素的水平是否存在關(guān)聯(lián)。 結(jié)果：實驗數(shù)據(jù)表明,JNA組與對照組相比,血清中的睪酮、游離睪酮、雌二醇及孕酮的水平并沒有顯著異常,且血清中睪酮與雌二醇的比值、游離睪酮和雌二醇的比值也無明顯差異。腫瘤分期與JNA患者血清中的性激素水平?jīng)]有顯著相關(guān)性。 結(jié)論：雖然JNA是一種特發(fā)于青少年男性的鼻咽部良性腫瘤,但是患者循環(huán)血清中的性激素水平以及雌雄激素的比值并無異常,性激素的水平與腫瘤分期也無相關(guān)性。這說明JNA患者本身性激素水平正常,也可能并不存在垂體-性腺軸的失平衡,腫瘤的發(fā)生可能是由于原位腫瘤組織在正常性激素水平的刺激下所產(chǎn)生的。 第二部分 鼻咽纖維血管瘤組織中性激素受體和VEGF的表達及其意義 目的：眾多學者對雄激素及雌激素受體在鼻咽纖維血管瘤(JNA)中的表達進行了研究,但目前為止并沒有統(tǒng)一的結(jié)論。本研究試圖通過高通量的免疫組織芯片技術(shù)對性激素受體及血管內(nèi)皮生長因子(VEGF)的表達及其與腫瘤預后的關(guān)系進行研究,并進行RNA水平及蛋白水平的驗證。 方法：收集70例JNA患者(平均年齡17.0歲,術(shù)后病理證實)的腫瘤組織及10例對照組患者的中甲黏膜組織(平均年齡21.5歲),制作成組織芯片。對照組患者為同時段、同年齡段、同性別的鼻中隔、骨瘤及額篩囊腫的患者。應用組織芯片分別對雄激素受體、雌激素受體α、雌激素受體β、孕激素受體及VEGF進行免疫組化染色,分析其在腫瘤組織中的表達與對照組相比是否存在差異,以及幾種性激素受體之間、性激素受體與VEGF之間是否存在表達關(guān)聯(lián)性。采集70例JNA患者的臨床病理數(shù)據(jù),隨訪無瘤生存時間,隨訪時間為2-98個月,分析JNA患者的發(fā)病年齡、腫瘤分期、術(shù)中出血、無瘤生存時間與腫瘤組織中性激素受體、VEGF表達水平之間的關(guān)系。 采用Real time PCR法檢測12例JNA腫瘤組織及4例JNA患者中甲黏膜組織中雄激素受體、雌激素受體α、雌激素受體p、孕激素受體及VEGF mRNA的表達水平,統(tǒng)計分析實驗組與對照組之間的差異,以及幾種性激素受體之間、性激素受體與VEGF之間是否存在表達關(guān)聯(lián)性。 采用Western blot檢測6例JNA腫瘤組織及3例JNA患者中甲黏膜組織中雄激素受體、雌激素受體α、雌激素受體β、孕激素受體的表達水平,驗證組織芯片及PCR的實驗結(jié)果。 結(jié)果：統(tǒng)計結(jié)果顯示,與中甲黏膜相比,雄激素受體、雌激素受體α、雌激素受體β、孕激素受體及VEGF在JNA腫瘤組織中有較高表達,統(tǒng)計學差異明顯,p值小于0.05。雄激素受體、雌激素受體a、雌激素受體β、孕激素受體表達量之間彼此存在正相關(guān),p值小于0.05。雌激素受體α、雌激素受體β與VEGF的表達量呈正相關(guān)(p值均為0.006)。雌激素受體α高表達組的無瘤生存時間較短(p值均為0.031),其余性激素受體的高表達雖然提示無瘤生存時間縮短的趨勢,但并無明顯的統(tǒng)計學差異。Real time PCR及Western blot檢測的實驗結(jié)果與組織芯片一致。 結(jié)論：JNA腫瘤組織中高表達雄激素受體、雌激素受體α、雌激素受體β、孕激素受體及VEGF。性激素本身存在彼此調(diào)節(jié)的關(guān)系,雌激素受體和血管性生長因子可能參與腫瘤的發(fā)生和發(fā)展。生存分析顯示高表達雌激素受體α提示術(shù)后易復發(fā)。 第三部分 鼻咽纖維血管瘤基因表達譜及相關(guān)基因研究 目的：表達譜芯片是近期較為成熟的分子生物學技術(shù),本研究試圖通過表達譜芯片找到參與JNA發(fā)病的主要基因和信號通路,分析差異表達基因,為下一步的研究提供前期數(shù)據(jù)。 方法：本研究應用包含30000條人類全長基因的PCR產(chǎn)物為靶基因制成的寡聚核苷酸基因芯片,檢測5例高侵襲的JNA組織、4例低侵襲的JNA組織以及5例JNA患者中甲黏膜組織的基因表達譜,分析差異表達基因,旨在通過研究JNA基因表達譜的改變,從基因水平探討JNA的發(fā)病機制。 結(jié)果：高侵襲組與低侵襲組相比,基因芯片篩選出83條上調(diào)表達基因和122條下調(diào)表達基因；高侵襲組與對照組相比,基因芯片篩選出1553條上調(diào)表達基因和1640條下調(diào)表達基因；低侵襲組與對照組相比,基因芯片共篩選出1181條上調(diào)表達基因和1239條下調(diào)表達基因。其中部分差異表達基因是與性激素結(jié)合、細胞凋亡,內(nèi)皮細胞侵襲轉(zhuǎn)移等密切相關(guān)的基因。 結(jié)論：通過基因表達譜芯片檢測及相關(guān)分析,發(fā)現(xiàn)JNA存在多個基因表達及調(diào)控機制的異常,這些差異表達基因為研究JNA的發(fā)病機制提供了線索,有望為JNA的基礎研究和臨床治療提供新的方向。
[Abstract]:Nasopharyngeal fibroangioma, also known as juvenile nasopharyngeal angiofibroma (JNA), is a relatively low incidence of.JNA in nasopharyngeal benign tumors that only occur in young men. The tumor is very easy to bleed and the operation is difficult to remove. In group learning, JNA has no capsule, from immature endothelial cells and Although fibroblast is a benign tumor, JNA is characterized by local invasion. It is easy to invade the paranasal sinuses, pterygopalatine fossa, infratemporal fossa, wing plate, supraorbital fissure, and even intracranial.
JNA's specific age and sex are very early in the attention of scholars. Many scholars expect to find a clue to study the pathogenesis of JNA. Early studies suggest that the disorder of the pituitary gonadal axis in JNA patients leads to cancer, but subsequent studies have shown that the levels of sex hormones in the JNA patients are normal and have no secondary sex. Recent studies have focused on the relationship between the expression of sex hormone receptors and diseases, but so far no conclusion has been reached.
The serum samples of 40 patients with JNA were collected, and the serum levels of testosterone, free testosterone, estrogen and progestin were detected by ELISA. The serum hormone levels in JNA patients were compared with those in the control group. 70 cases of JNA tumor tissue and 10 cases of middle thyroid tissue were made into tissue chips to detect androgen receptor (Andro). Gen receptor, AR), estrogen receptor alpha (estrogen receptor alpha, ER- alpha), estrogen receptor beta (estrogen receptor A, ER- beta), progesterone receptor (progesterone receptor, PR) and vascular endothelial growth factor (VEGF) in 5 cases of highly invasive tissue, 4 cases of low invasive JNA tissues and 5 normal middle thyroid tissues were studied by gene chip. The key genes and signaling pathways involved in the pathogenesis of JNA were found.
JNA is easy to invade adjacent tissues and even intracranial. It is very easy to bleed in the operation, and the tumor operation with a wide range of invasion is often unresectable and there is a great risk. Drug and target therapy has been the focus of research on the expression of neutral hormone receptor in.JNA tumor tissue and the study of expression spectrum chip to provide a new method for further exploring the treatment of JNA. The foundation.
Part one
Expression of serum sex hormones in patients with nasopharyngeal fibroangioma
Objective: nasopharyngeal angioma (JNA) is a benign tumor that is almost only occurring in young men. But to date, the specific age of pathogenesis of.JNA and sex suggestive hormone may play a role in the process of cancer. The purpose of this study is to determine whether there is abnormal sex hormone levels in puberty and sex hormone levels in JNA patients. It is not associated with tumor staging.
Methods: the serum of 40 patients with JNA (average age 18.3 years, confirmed by postoperative pathology) and 14 normal controls (average age 19.7 years old) were collected. The control group was at the same time, the same age, the same sex other nasal septum, the osteoma and the optic nerve decompression. The serum testosterone in JNA and the control group was detected by Elisa method. The levels of testosterone, estradiol and progesterone were compared between the two groups of sex hormones and whether there were differences in the ratio of male androsteroid. The tumor staging data of 40 patients with JNA were collected to determine whether there was a correlation with the level of serum neutral hormones.
Results: the experimental data showed that there was no significant difference in serum testosterone, free testosterone, estradiol and progesterone in serum, and there was no significant difference in the ratio of testosterone to estradiol in serum and the ratio of estradiol and estradiol in serum, and there was no significant correlation between the tumor staging and the level of sex hormone in serum of patients with JNA.
Conclusion: Although JNA is a benign tumor of the nasopharynx of adolescent male, there is no abnormal sex hormone level and the ratio of sex hormone in the circulating serum, and there is no correlation between the level of sex hormone and tumor staging. This indicates that the level of sex hormone in patients with JNA is normal and may not have the loss of the pituitary gonadal axis. The occurrence of tumor may be due to the stimulation of normal tumor hormones at normal sex hormone levels.
The second part
Expression and significance of sex hormone receptor and VEGF in nasopharyngeal fibroangioma tissue
Objective: many scholars have studied the expression of androgen and estrogen receptor in nasopharyngeal angioma (JNA), but so far there is no unified conclusion. This study attempts to express the expression of sex hormone receptor and vascular endothelial growth factor (VEGF) by high throughput immuno tissue microarray and its relationship with the prognosis of the tumor. Conduct research and verify the RNA level and protein level.
Methods: 70 cases of JNA patients (average age 17 years old, pathologically confirmed) tumor tissue and 10 cases of control group (mean age 21.5 years old) were made into tissue chips. The control group was the same age group, the same age group, the same sex other nasal septum, the osteoma and the frontal sieving cyst. The hormone receptor, estrogen receptor alpha, estrogen receptor beta, progesterone receptor and VEGF were immunohistochemical staining. The difference between the expression of the tumor tissue and the control group was analyzed, and the correlation between the sex hormone receptor and the VEGF was found. The clinicopathological data of 70 patients with JNA were collected. No tumor survival time was followed up for 2-98 months. The relationship between the onset age of JNA, tumor stage, intraoperative hemorrhage, tumor free survival time and the level of VEGF expression of neutrophilic hormone receptor and tumor tissue were analyzed.
The Real time PCR method was used to detect the expression of androgen receptor, estrogen receptor alpha, estrogen receptor P, progesterone receptor and VEGF mRNA in 12 JNA tumor tissues and 4 JNA patients. The difference between the experimental group and the control group was statistically analyzed, and there were several sex hormone receptors between the sex hormone receptors and VEGF. In the expression of relevance.
The expression levels of androgen receptor, estrogen receptor alpha, estrogen receptor beta and progesterone receptor were detected by Western blot in 6 cases of JNA tumor tissue and 3 cases of JNA patients. The experimental results of tissue microarray and PCR were verified.
Results: the results showed that androgen receptor, estrogen receptor alpha, estrogen receptor beta, progesterone receptor and VEGF were highly expressed in JNA tumor tissues compared with middle thyroid mucosa, and the statistical difference was obvious, P was less than 0.05. androgen receptor, estrogen receptor A, estrin receptor beta, and progesterone receptor expression existed in positive phase. The p value was less than 0.05. estrogen receptor alpha, the expression of estrogen receptor beta was positively correlated with the expression of VEGF (P value was 0.006). The tumor free survival time of the estrogen receptor alpha high expression group was shorter (P value was 0.031), while the high expression of the other sex hormone receptors indicated the trend of shortening the tumor free survival time, but there was no significant statistical difference of.Real time. The results of PCR and Western blot test were consistent with tissue microarray.
Conclusion: the high expression of androgen receptor, estrogen receptor alpha, estrogen receptor beta, progesterone receptor and VEGF. sex hormone itself are related to each other in JNA tumor tissue. Estrogen receptor and vascular growth factor may participate in the occurrence and development of tumor. Survival analysis shows that high expression of estrogen receptor alpha suggests recurrence after operation.
The third part
Study on gene expression profile and related genes of nasopharyngeal fibroangioma
Objective: the expression chip is a relatively mature molecular biology technique in the near future. This study attempts to find the main genes and signal pathways involved in the pathogenesis of JNA by expressing the spectral chip, and analyze the differentially expressed genes, and provide the preliminary data for the next step of research.
Methods: in this study, the oligonucleotide microarray of the PCR products containing 30000 human full-length genes was used as the target gene. The gene expression profiles of 5 highly invasive JNA tissues, 4 low invasive JNA tissues and 5 JNA patients were detected and the differentially expressed genes were analyzed. The aim of this study was to study the modification of JNA gene expression profiles. Change, explore the pathogenesis of JNA from the gene level.
Results: compared with the low invasion group, 83 up-regulated genes and 122 down regulated genes were screened by gene chip compared with the low invasion group. Compared with the control group, the high invasion group screened 1553 up-regulated genes and 1640 down regulated genes, and the low invasion group screened 1181 up-regulated genes compared with the control group. Genes and 1239 down regulated expression genes, some of which are differentially expressed genes, are closely related to sex hormone binding, apoptosis, endothelial cell invasion and metastasis.
Conclusion: through the detection and correlation analysis of gene expression spectrum, it is found that there are many abnormal genes expression and regulation mechanism in JNA. These differentially expressed genes provide clues for the study of the pathogenesis of JNA. It is expected to provide a new direction for the basic and clinical treatment of JNA.
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R739.63

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