本文選題：α型葉酸受體 + 循環(huán)腫瘤細胞��； 參考：《南方醫(yī)科大學(xué)》2014年碩士論文

【摘要】：據(jù)報道近十年腎癌的全球發(fā)病率大約上升了2%-4%,占全世界腫瘤人數(shù)的2%。亞洲是腎癌的低發(fā)病區(qū),但是近年來隨著經(jīng)濟的發(fā)展,生活方式的西化以及診斷水平的提升,亞洲地區(qū)腎癌發(fā)病率的增長速度明顯高于歐洲。據(jù)調(diào)查我國1988～1992、1993-1997、1998-2002年3個時間段腎和泌尿系統(tǒng)其他惡性腫瘤的發(fā)病率分別為4.26/10萬、5.40/10萬及6.63/10萬人口,發(fā)病率呈逐年上升趨勢。腎癌發(fā)病率在我國泌尿生殖系統(tǒng)腫瘤中位居第二。 腎細胞癌(RCC)起源于腎實質(zhì)的腎小管或集合管上皮細胞,占腎癌的80%-90%。其惡性程度高,對放化療均不敏感,盡管影像學(xué)技術(shù)在不斷發(fā)展,仍有接近30%的腎細胞癌患者在初次就診時即已存在轉(zhuǎn)移病灶。一旦出現(xiàn)遠處轉(zhuǎn)移,則其五年生存率不足10%,所以腎細胞癌的早期診斷和治療對病人預(yù)后產(chǎn)生深遠影響。盡管有關(guān)腎細胞癌相關(guān)血漿蛋白例如紅細胞生成素(EPO),鐵蛋白,特異性烯醇化酶以及轉(zhuǎn)錄生長因子-β(TGF-β)用于根治性腎切除術(shù)后預(yù)后評價的報道,但是這些標志物在腎癌診療過程中的實際意義有待于更多證據(jù)的支持。循環(huán)腫瘤細胞(CTCs)是1869年由Ashworth首次報道,其定義是指由原發(fā)腫瘤部位脫落并進入血液循環(huán)系統(tǒng)的惡性腫瘤細胞。CTCs在許多實體腫瘤患者外周血中被發(fā)現(xiàn),并進一步證實與腫瘤臨床分期、預(yù)后密切相關(guān),美國臨床腫瘤學(xué)會(ASCO)也在2007年將CTCs推薦作為乳腺癌的一種腫瘤標志物。α-FR是一種通過糖基磷脂酰肌醇(GPI)錨定在細胞膜表面的糖蛋白,由257個氨基酸組成,分子量約38kDa。其在正常上皮細胞中表達極其有限,而在上皮來源的惡性腫瘤中過度表達；文獻報道超過75%的RCC其a-FR呈高度表達。目前有關(guān)腎細胞癌CTCs的研究相對較少,主要原因在于檢測手段的限制;例如常見的RT-PCR, CellSearch系統(tǒng)及流式細胞儀等CTCs檢測手段并非適合于RCC。 因此,本研究在文獻報道的基礎(chǔ)上先從mRNA和蛋白質(zhì)水平檢測a-FR在兩種人腎癌細胞株ACHN、7860中的表達情況；再嘗試采用基于a-FR的配體探針PCR技術(shù)來有效檢測RCC患者外周血CTCs,初步闡述RCC患者外周血CTCs檢測的臨床意義。為RCC患者檢測外周血CTCs提供了一種切實可行的技術(shù)手段,國內(nèi)外尚無相關(guān)報道。 目的： 探索基于a-FR的配體探針PCR定量檢測腎細胞癌(RCC)患者外周血循環(huán)腫瘤細胞(CTCs)的可行性,并進一步闡釋該法定量檢測RCC患者外周血CTCs的臨床意義。 方法： 一：(1)：選擇兩株人腎癌細胞株ACHN、7860,采用含10%FBS的無葉酸培養(yǎng)基RPMI-1640常規(guī)培養(yǎng),取對數(shù)生長期細胞胰酶消化收集。另取健康志愿者外周血收集白細胞做陰性對照。Trizol提取三種細胞總RNA,常規(guī)凝膠電、泳檢測RNA完整性；采用商品化試劑盒(PrimeScriptTM RT Master Mix Kit)對目的基因(a-FR)進行特異性擴增,內(nèi)參基因為GAPDH。制作溶解曲線及擴增曲線,采用AAt法行相對定量分析；并紫外燈下觀察凝膠電泳后的擴增產(chǎn)物。(2)：取106的三種細胞提取總蛋白,采用BCA法行蛋白定量檢測, Western blot法檢測蛋白樣品中a-FR的表達情況；GAPDH作內(nèi)參,采用化學(xué)發(fā)光法檢測雜交信號,分析表達差異。(3)：為驗證本實驗方法能敏感而有效地從外周血中檢測到CTCs,我們將培養(yǎng)的人腎癌細胞株ACHN進行人工摻血實驗。重懸細胞分為5/ul、50/ul、500/ul、5000/ul和50000/ul五個濃度梯度,并設(shè)置PBS陰性對照組,每份樣品設(shè)置三個復(fù)孔。摻入3ml健康志愿者新鮮外周血后按照標準檢測流程行CTCs檢測(如下文所述),采用雙盲實驗計算不同濃度梯度的CTCs檢測值與已知數(shù)量摻血腫瘤細胞的相關(guān)性。 二：(1)：3ml新鮮外周血標本首先利用紅細胞裂解液去除紅細胞,PBS重懸后利用標記有抗CD45抗體的免疫磁珠分選技術(shù)陰性富集CTCs,加入活化液用以活化和釋放腫瘤細胞表面a-FR,加入特異性識別a-FR并標記有Taqman探針的小片段單鏈DNA(數(shù)據(jù)未予公布),洗滌去除未結(jié)合的多余探針,最后將結(jié)合a-FR的探針洗脫。(2)：ABI Viia7熒光定量PCR儀對洗脫的小片段DNA進行特異性擴增,6個標準品用以制定標準曲線。反應(yīng)條件：stagel:95℃2min,40℃30sec,60℃lmin,8℃5min,95℃lmin,1cycle; stage2:95℃10sec,35℃30sec,72℃5sec,40cycle;在步驟2中35℃退火時檢測熒光信號。采用Graphpad Prism5軟件對標準品起始拷貝數(shù)的對數(shù)和所測得的Ct值進行最小二乘法回歸分析得到標準曲線。樣品原始拷貝數(shù)通過將熒光定量PCR所測得的Ct值代入標準曲線而得到。每份樣品三個復(fù)孔原始拷貝數(shù)的平均值乘以0.144轉(zhuǎn)換成拷貝數(shù)/3ml。 三：我們共收集45例初診腎細胞癌患者外周血標本共91份,其中術(shù)前1天血標本45份,術(shù)后7天血標本39份,還有3位術(shù)后病人在隨訪過程中采集血標本共7份。并且收集相關(guān)臨床資料。合并其他惡性腫瘤者未納入RCC組。另外采集了50例健康志愿者和35例腎臟良性病變患者的外周血標本共85份。RCC患者中男性19例,女性26例,年齡為29歲-87歲(55.58±14.16)；所有RCC均經(jīng)病理學(xué)確診,包括透明細胞癌31例,嫌色細胞癌4例,乳頭狀癌8例,腎集合管癌2例。參照2010年AJCC腎癌TNM分期標準,T1期21例,T2期14例,T3期8例,T4期2例,M1期4例；stageⅠ21例,stageⅡ14例,stageⅢ4例,stageIV6例。腎臟良性病變包括腎結(jié)石13例、腎積水7例、腎囊腫7例及腎錯構(gòu)瘤6例、腎脂肪瘤2例。其中男性24例,女性11例,年齡22歲-72歲(50.74±14.28)。健康志愿者中男性27例,女性23例,年齡20歲-68歲(45.80±12.82),全部無惡性腫瘤證據(jù),亦無家族性腫瘤病史,健康志愿者未進行常規(guī)螺旋CT檢查。靜脈血標本均通過常規(guī)靜脈穿刺得到,舍棄最初的2.5ml血液以減小皮膚來源上皮細胞污染的風(fēng)險。所有血標本采集后立即置于4℃冰箱暫時保存,并于采血后5h內(nèi)行外周血CTCs檢測。為防止交叉污染,腫瘤細胞摻血實驗與病人血標本CTCs檢測場所相互獨立。分析三組間CTCs水平有無差異,RCC病人的CTCs水平是否同腫瘤TNM分期、臨床分期相關(guān),手術(shù)前后CTCs水平是否發(fā)生變化等。 四：統(tǒng)計學(xué)分析：所有統(tǒng)計分析采用SPSS13.0軟件(SPSS Inc., Chicago,IL,USA)。一般線性相關(guān)分析將用于腫瘤細胞摻血實驗中計算各濃度CTCs檢測值與已知數(shù)量摻血腫瘤細胞的相關(guān)系數(shù)。為確定該法檢測的閾值我們進行了ROC曲線分析。各組間差別采用One-way ANOVA單因素方差分析,并進行簡單統(tǒng)計描述。RCC患者中,CTC檢測值在不同TNM分期、臨床分期及手術(shù)前后的差異將采用兩獨立樣本t檢驗或One-way ANOVA單因素方差分析,并對兩種術(shù)式在局限性RCC患者中手術(shù)前后CTCs檢測值的差值進行兩獨立樣本t檢驗。p值0.05認為差異有統(tǒng)計學(xué)意義。 結(jié)果： 熒光定量PCR：△△t法相對定量分析ACHN和786-0其a-FRmRNA的表達量分別是正常人WBC39.77倍和23.12倍。 Western blot:GAPDH蛋白在ACHN、786-0及正常人WBC中表達相對恒定,而a-FR在人腎癌細胞株ACHN和786-0中呈高表達,在WBC幾乎不表達。 腫瘤細胞摻血回收實驗：基于a-FR的配體探針PCR技術(shù)能在3ml外周血中檢測到5-5×104個ACHN細胞,經(jīng)直線相關(guān)分析提示CTCs檢測值與摻入腫瘤細胞數(shù)目呈正相關(guān),相關(guān)系數(shù)r=0.99(p0.01)。 CTCs檢測數(shù)據(jù)行ROC曲線分析,當臨界值設(shè)為15.4Copies/3ml時,ROC曲線下面積最大,此時檢測的敏感度和特異度分別為95.6%和98.8%。 RCC術(shù)前組外周血CTCs水平顯著高于健康對照組和腎臟良性病變組(p0.01,p0.01),并且腎細胞癌CTCs水平同腫瘤的TNM分期、臨床分期顯著相關(guān)(p0.01)。 根治性腎切除術(shù)和保留腎單位的部分腎切除術(shù)對于T1、T2期不伴轉(zhuǎn)移RCC患者術(shù)后外周血CTCs水平的影響無差異(p=0.320)。 結(jié)論： 基于a-FR的配體探針PCR能有效檢測到RCC患者外周血中的CTCs,本實驗方法定量檢測CTCs可彌補腎癌TNM分期的不足,可能在評估RCC患者治療效果和監(jiān)測術(shù)后復(fù)發(fā)方面具有一定的價值。
[Abstract]:It is reported that the global incidence of renal cancer has risen by about 2%-4% in recent ten years. 2%. Asia, which accounts for the number of tumors in the world, is a low incidence area of renal cancer. However, in recent years, with the development of the economy, the Westernization of life style and the improvement of the diagnostic level, the rate of renal cancer incidence in Asia is obviously higher than that in Europe. According to the survey of China, the incidence of renal cancer is 1988 to 1992,1. In the 3 period of 993-19971998-2002, the incidence of kidney and other malignant tumors of the urinary system was 4.26/10 million, 5.40/10 million and 6.63/10 million, and the incidence rate was increasing year by year. The incidence of renal cancer was second in the urogenital system tumor in China.
Renal cell carcinoma (RCC) originates from renal tubules or collecting tubular epithelial cells in renal parenchyma. The 80%-90%. of renal cell carcinoma is highly malignant and is not sensitive to radiotherapy and chemotherapy. Although imaging techniques are developing continuously, there are still 30% renal cell carcinoma patients with metastatic lesions at the first visit. Less than 10%, so early diagnosis and treatment of renal cell carcinoma have a profound impact on patients' prognosis. Although renal cell related plasma proteins such as erythropoietin (EPO), ferritin, specific enolase, and transcription growth factor beta (TGF- beta) are used to evaluate the prognosis after radical nephrectomy, but these markers The practical significance of the diagnosis and treatment of renal cancer remains to be supported by more evidence. Circulating tumor cells (CTCs), first reported by Ashworth in 1869, are defined as.CTCs, a malignant tumor cell that has fallen from the original tumor site and entered the blood circulation system, and is found in the peripheral blood of many solid tumor patients and further confirmed with the tumor. The clinical stage is closely related to the prognosis. The American Society of Clinical Oncology (ASCO) also recommended CTCs as a tumor marker of breast cancer in 2007. Alpha -FR is a glycoprotein anchored on the surface of the cell membrane through glycosyl phosphatidylinositol (GPI), consisting of 257 amino acids, which are approximately 38kDa. in normal epithelial cells and are extremely limited in expression. It is overexpressed in epithelial malignant tumors; the literature reports that more than 75% of the RCC a-FR is highly expressed. There are relatively few studies on CTCs of renal cell carcinoma, mainly due to the restriction of detection methods; for example, CTCs detection methods such as common RT-PCR, CellSearch system and flow cytometry are not suitable for RCC..
Therefore, on the basis of the literature report, the expression of a-FR in two human renal cancer cell lines ACHN, 7860 was detected from the mRNA and protein levels, and the a-FR based ligand probe PCR technique was used to detect the peripheral blood CTCs in the peripheral blood of RCC patients. The clinical significance of CTCs detection in peripheral blood of RCC patients was preliminarily discussed. The detection of RCC patients was for the detection of RCC patients. Peripheral blood CTCs provides a practical technique, and there is no report on it at home and abroad.
Objective:
To explore the feasibility of quantitative detection of peripheral blood circulating tumor cells (CTCs) in renal cell carcinoma (RCC) patients by a-FR based ligand probe PCR, and to further explain the clinical significance of this method for quantitative detection of CTCs in peripheral blood of patients with RCC.
Method錛，

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