本文選題：胃癌 + HER2��； 參考：《中國人民解放軍醫(yī)學院》2017年博士論文

【摘要】：背景胃癌在世界范圍內一直是一個嚴重威脅人類健康的疾病,其發(fā)病率在所有惡性腫瘤中占第二位,死亡率占全部癌性致死性疾病的第二位。在消化道惡性腫瘤中占首位。雖然近年來內鏡、手術、化療、靶向等診療手段均有了一定的提高,但目前胃癌患者5年生存率仍低于30%。既往研究關注基因組學、表觀遺傳學、蛋白質組學等方面的變化。如原癌基因的激活或(和)抑癌基因的失活、蛋白相互作用,信號傳導通路,調控網(wǎng)絡等。有些研究有些成果已用于臨床,如基因HER2靶向治療在乳腺癌中的應用,為部分患者帶來益處。HER2的檢測在乳腺癌中已成常規(guī),但在胃癌尚不是,多需專門提出,但準確測定并監(jiān)測胃癌患者體內HER2狀態(tài),對于合理應用HER2靶向治療及評估預后均至關重要。目前其檢測手段是熒光原位雜交分析(FISH)和免疫組織化學,在臨床應用中有一定的局限性,比如第一,兩種方法有13%左右的不一致率,第二,免疫組化是半定量檢測,主觀性較強,而FISH檢測耗時且昂貴。第三,血清中HER2的情況可以在曲妥珠單抗治療過程中發(fā)生變化,而FISH及免疫組織化學都不能實時監(jiān)測,這些均不完善,成為本課題第一部分產生的初衷。腫瘤分子生物學的研究,除了對只占全部基因組2%的編碼基因的研究之外,還有很多未知領域,新一代基因測序技術及高通量篩選方法發(fā)展,使大量非編碼RNA被發(fā)現(xiàn),非編碼RNA主要包括長鏈非編碼RNA(long non-codingRNA,lncRNA)和微小RNA(microRNA,miRNA)。最初認為非編碼RNA無功能,隨著全部人類基因組測序的完成,人類的疾病并沒有隨之迎刃而解。所以人們開始關注數(shù)量眾多卻被忽視的非編碼RNA的研究。近些年miRNA在腫瘤生物學的調控作用已有廣泛研究。然而,lncRNA的數(shù)量眾多,功能多樣,可在表觀遺傳學、轉錄及轉錄后等水平參與基因表達的調控。相關研究尚較少,特別是在胃癌的研究上更是剛剛開始,本研究通過對胃癌及癌旁組織異常表達的lncRNA研究,有望發(fā)現(xiàn)新的與胃癌相關的lncRNA,作為胃癌診斷治療新靶點,從而形成了論文的第二部分。第一部分胃癌患者血清HER2水平檢測與組織HER2表達相關性及臨床意義目的:探索血清(胞外域)HER2水平與組織內HER2水平的相關性,探索血清HER2水平對胃癌患者的作用。方法:收集解放軍總醫(yī)院2012年12月-2013年6月期間239例胃癌庫中患者資料。應用化學熒光法檢測血清HER2水平,應用熒光原位雜交分析(FISH)及免疫組織化學的方法,檢測組織內HER2水平。畫出受試者工作特征曲線(ROC),確定出預測胃癌患者生存的血清HER2水平的最佳截斷值。結果:血清HER2水平與組織內HER2水平、腫瘤體積、腸型胃癌等均顯著相關,血清HER2水平在組織內HER2陽性的患者與組織內HER2陰性患者間有顯著的差異。得出曲線下最大面積為0.79(95%CI: 0.71-0.87, P 0.001)。敏感度及特異度分別為0.54 (95%CI:0.37-0.70)和 0.93 (95%CI:0.88-0.96)。此時的截斷值為 24.75ng/mL。高的血清 HER2水平對胃癌患者總體生存率呈負相關關系。(hazard ratio: 1.93,95%CI: 1.32-4.38, P =0.006)。結論:血清HER2水平可以作為一個能替代組織內HER2表達情況的特異性較高的標志物,HER2水平24.75ng/mL提示預后不佳。第二部分過表達lncRNA-p2819對胃癌細胞BGC-823增殖、凋亡和侵襲的影響目的:為了明確胃癌及癌旁組織中有哪些差異表達的lncRNA,這些差異表達的lncRNA有無臨床意義,干擾這些lncRNA對胃癌腫瘤生物學行為有何影響。方法:(1 )收集2014年6月-2015年10月期間解放軍總醫(yī)院胃癌患者標本90例,利用人類lncRNA芯片,取5對胃癌與癌旁組織標本篩查差異表達的lncRNA,按照變化倍數(shù)、可靠性p值、可能的靶基因信息分析,是否已有文獻報道等篩選原則選定候選lncRNA,然后擴大樣本量13例,對候選的4個lncRNA進行qRT-PCR驗證,從中選出具有潛在臨床意義的目標lncRNA。擴大樣本量到72例,行qRT-PCR檢測,并分析lncRNA表達量與臨床參數(shù)如腫瘤部位、大小、淋巴結轉移,分化程度等的相關性。(2)針對目標lncRNA做功能試驗,通過基因重組,構建真核表達載體,將重組質粒轉染BGC-823胃癌細胞系,通過MTT, AnnexinV/PI, Transwell檢測過表達LncRNA對細胞的增殖,凋亡以及侵襲的影響。結果:(1)芯片篩查發(fā)現(xiàn)5445差異表達的lncRNA,其中差異倍數(shù)超過2倍,可靠性p值0.05的lncRNA為19個上調,56個下調,候選 4 個 lncRNAp2819 (下調)、P17382 (上調)、P24084 (上調)、P27873 (上調)擴大到13對獨立胃癌及癌旁標本行qRT-PCR驗證。驗證結果均與芯片結果一致,4個候選lncRNA在癌與癌旁正常組織的表達差異均具有統(tǒng)計學意義(分別為P2819:2-△△ct, 0.012 VS0.401; P=0. 01; P17382: 2-△△Ct, 0.572 VS0.061; P=0.01; P24084:2-△△Ct, 0.243 VS0.081; P=0.03; P2819: 2-△△Ct,0.312 VS0.091; P=0.03;)。最終選出差異倍數(shù)最大的lncRNAp2819,對72例胃癌標本行qRT-PCR檢測,發(fā)現(xiàn)其表達量與腫瘤大小,部位,TNM分期顯著相關,與年齡、性病無顯著性相關。(2)構建pcDNA3.1(+)-p2819質粒,成功通過驗證,腫瘤細胞BGC-823轉染后過表達lncRNAp2819,通過MTT檢測發(fā)現(xiàn)p2819可明顯抑制細胞增殖。通過流式檢測p2819過表達可以明顯促進腫瘤細胞凋亡。對照組細胞總凋亡0.9%;轉染組總凋亡62.8%;(p0.01 )。Transwell實驗檢測p2819對細胞的侵襲的影響,見轉染LncRNA-p2819組對細胞侵襲有一定抑制作用。結論:芯片篩查出多個胃癌相關lncRNA,與胃癌腫瘤特征具有相關性,其中LncRNA-p2819過表達后對BGC-823細胞產生抑制增殖,促進凋亡,抑制侵襲的作用,說明其具有抑癌基因的特性。其作用機理及潛在臨床應用價值尚待進一步研究。
[Abstract]:Background gastric cancer has been a serious threat to human health worldwide. Its incidence is second of all malignant tumors, and the mortality rate accounts for second of all cancer fatal diseases. It is the first in the digestive tract malignant tumors. Although endoscopy, surgery, chemotherapy, and targeting treatment have been improved in recent years, But at present, the 5 year survival rate of gastric cancer patients is still lower than that of 30%. previously concerned with genomics, epigenetics, proteomics, such as the activation of proto oncogenes or (and and) inactivation of tumor suppressor genes, protein interaction, signal transduction pathway, regulatory network and so on. Some research results have been used in clinical, such as gene HER2 targeting treatment. The application of treatment in breast cancer, the detection of benefits for some patients with.HER2 has become routine in breast cancer, but it is not yet necessary for gastric cancer to be specially proposed, but it is very important to accurately determine and monitor the HER2 status in the patients with gastric cancer, which is very important for the rational use of HER2 targeting therapy and evaluation of prognosis. Analysis (FISH) and immunohistochemistry have some limitations in clinical application, such as the inconsistency rate of about 13% of the first, second methods, second, the immunohistochemistry is the semi quantitative detection, the subjectivity is strong, and the FISH detection is time-consuming and expensive. Third, the serum HER2 can be changed during the treatment of trastuzumab, and FISH The research on molecular biology of cancer, in addition to the study of the coding genes that only accounted for 2% of the whole genome, has a lot of unknown fields. The new generation of gene sequencing and high throughput screening methods have made a large number of non coding. The code RNA has been found that non coded RNA mainly includes long chain non coded RNA (long non-codingRNA, lncRNA) and tiny RNA (microRNA, miRNA). Initially, non coded RNA is unfunctional, and with the completion of all human genome sequencing, human diseases are not readily solved. MiRNA has been widely studied in recent years in the regulatory role of tumor biology. However, lncRNA has a large number of functions and can be involved in the regulation of gene expression at epigenetic, transcriptional and post transcriptional levels. The lncRNA study of abnormal expression of tissue is expected to discover new lncRNA related to gastric cancer, as a new target for diagnosis and treatment of gastric cancer, thus forming the second part of the paper. The correlation and clinical significance of serum HER2 level detection and tissue HER2 expression in the first part of gastric cancer patients: to explore the level of HER2 in serum (extracellular domain) and the level of HER2 in the tissues To explore the effect of serum HER2 level on patients with gastric cancer. Methods: to collect the data of 239 cases of gastric cancer patients during the period of June December 2012 in the General Hospital of the Liberation Army (General Hospital of the Liberation Army). The level of serum HER2 was detected by chemical fluorescence method, and the methods of fluorescence in situ hybridization (FISH) and immunohistochemical staining were used to detect the level of HER2 in the tissue. The work feature curve (ROC) was used to determine the best truncation value of the serum HER2 level for predicting the survival of gastric cancer patients. Results: serum HER2 level was significantly correlated with the level of HER2 in the tissue, tumor volume, intestinal type of gastric cancer, and the serum HER2 level was significantly different between the patients with HER2 positive in the tissues and the HER2 negative patients in the tissue. The maximum area under the line was 0.79 (95%CI: 0.71-0.87, P 0.001). The sensitivity and specificity were 0.54 (95%CI:0.37-0.70) and 0.93 (95%CI:0.88-0.96). The serum HER2 level with a truncated value of 24.75ng/mL. was negatively correlated with the overall survival rate of gastric cancer patients. (hazard ratio: 1.93,95%CI: 1.32-4.38, P). ER2 level can be used as a highly specific marker that can replace the expression of HER2 in the tissue. HER2 level 24.75ng/mL suggests poor prognosis. Second the effect of lncRNA-p2819 on the proliferation, apoptosis and invasion of BGC-823 in gastric cancer cells is to determine what differentially expressed lncRNA in gastric and paracancerous tissues. The differential expression of lncRNA has no clinical significance, interfering with the effect of these lncRNA on the biological behavior of gastric cancer. Methods: (1) to collect 90 cases of gastric cancer specimens from the General Hospital of PLA during the period of June 2014 -2015, and use human lncRNA chip to obtain 5 differential expressions of lncRNA for gastric cancer and paracancerous tissue specimens, according to the multiplier number of changes, Depending on the p value, the possible target gene information analysis, whether the candidate lncRNA was selected for the selection principle of the literature, 13 samples were enlarged, and the candidate 4 lncRNA were verified by qRT-PCR, from which the potential clinical significance target lncRNA. was enlarged to 72 cases, the qRT-PCR detection was performed, and the lncRNA expression and clinical reference were analyzed. The correlation of tumor location, size, lymph node metastasis, differentiation degree and so on. (2) to do functional tests on target lncRNA, construct eukaryotic expression vector by gene recombination, transfect the recombinant plasmid to BGC-823 gastric cancer cell line, and detect the effect of LncRNA on cell proliferation, apoptosis and invasion through MTT, AnnexinV/PI and Transwell. Fruit: (1) chip screening found 5445 differentially expressed lncRNA, of which the difference multiplier was more than 2 times, the lncRNA of the reliability p value 0.05 was 19 up-regulated, 56 down-regulation, 4 lncRNAp2819 (down regulation), P17382 (up), P24084 (up), and P27873 (up) expanded to 13 pairs of independent gastric and paracancerous markers for qRT-PCR validation. The results were all with chips. The differences in the expression of the 4 candidate lncRNA were statistically significant (P2819:2- delta delta CT, 0.012 VS0.401, P=0. 01, P17382: 2- Delta Ct, 0.572 VS0.061, P=0.01, P24084:2- Delta Ct, 0.243, 0.312;). Large lncRNAp2819, qRT-PCR detection of 72 cases of gastric cancer specimens, found that the expression of tumor size, location, TNM staging is significantly related to age, no significant correlation with age, STD. (2) construction of pcDNA3.1 (+) -p2819 plasmid, the successful pass verification, tumor cells BGC-823 transfected lncRNAp2819, through MTT detection p2819 can obviously inhibit fine. Cell proliferation by flow cytometry could significantly promote apoptosis of tumor cells. The total apoptosis in the control group was 0.9%, the total apoptosis of the transfected group was 62.8%, and the effect of p2819 on the invasion of the cells in the transfected group was detected by.Transwell test, and the transfection of LncRNA-p2819 group had a certain inhibitory effect on the invasion of the cells. Conclusion: the chip was used to screen multiple gastric cancer related lnc. RNA is related to the tumor characteristics of gastric cancer, in which the overexpression of LncRNA-p2819 inhibits the proliferation of BGC-823 cells, promotes apoptosis, inhibits the effect of invasion, and indicates the characteristics of the tumor suppressor gene. Its mechanism and potential clinical value need to be further studied.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R735.2

【參考文獻】

相關期刊論文 前5條

1 Wei-Jun Cao;Hai-Lu Wu;Bang-Shun He;Yu-Shu Zhang;Zhen-Yu Zhang;;Analysis of long non-coding RNA expression profiles in gastric cancer[J];World Journal of Gastroenterology;2013年23期

2 劉峰;楊富;張鈴;孫樹漢;;長鏈非編碼RNA與腫瘤診斷[J];分子診斷與治療雜志;2013年03期

3 夏天;肖丙秀;郭俊明;;長鏈非編碼RNA的作用機制及其研究方法[J];遺傳;2013年03期

4 左君波;朱阿考;夏加增;;長鏈非編碼RNA與腫瘤的研究進展[J];中華醫(yī)學雜志;2011年47期

5 孫甜甜;房靜遠;;長非編碼RNA與其他表觀遺傳機制在腫瘤中的相互調控[J];上海交通大學學報(醫(yī)學版);2011年07期

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