【摘要】：目的循環(huán)血液中活性腫瘤細(xì)胞的檢測(cè)被看作是液體活檢,具有動(dòng)態(tài)監(jiān)測(cè)的功能,在腫瘤早期診斷、個(gè)體化治療、療效評(píng)價(jià)、預(yù)后判斷等方面具有重要的臨床意義,然而由于其在血液中數(shù)量很少,循環(huán)腫瘤細(xì)胞(circlulating tumor cells,CTCs)的分離和鑒定成為臨床應(yīng)用的難點(diǎn)。復(fù)制選擇性溶腫瘤腺病毒Ad11-5ETel-GFP是一種通過(guò)同源重組獲得的病毒,可以選擇性的在具有端粒酶活性的細(xì)胞內(nèi)復(fù)制并表達(dá)綠色熒光蛋白(Green Fluorescent Protein,GFP)。本研究旨在利用該病毒捕獲循環(huán)腫瘤細(xì)胞,從而建立一種可重復(fù)、簡(jiǎn)單、無(wú)創(chuàng)的檢測(cè)方法,并對(duì)該檢測(cè)方法的可靠性和臨床應(yīng)用價(jià)值進(jìn)行探討。方法首先通過(guò)實(shí)驗(yàn)驗(yàn)證我們所建立的方法能夠有效捕獲到腫瘤細(xì)胞,包括:將AD11-5ETel-GFP病毒加入正常人外周血中,觀察GFP在正常白細(xì)胞的表達(dá)情況;將A549(肺癌細(xì)胞系),SKBR3(乳腺癌細(xì)胞系)分別以10、50、100個(gè)細(xì)胞三個(gè)梯度加入1ml人外周血中,建立循環(huán)腫瘤細(xì)胞的模型并檢測(cè)其回收率;使用抗人CD45抗體進(jìn)行細(xì)胞免疫染色,熒光顯微鏡下觀察結(jié)果。以上結(jié)果說(shuō)明本實(shí)驗(yàn)建立的新型檢測(cè)方法能有效的捕獲循環(huán)細(xì)胞。將該方法應(yīng)用于頭頸部惡性腫瘤的患者,收集臨床標(biāo)本48例,將標(biāo)本中紅細(xì)胞裂解,加入病毒于37℃條件下孵育24h,固定涂片后于熒光顯微鏡下觀察計(jì)數(shù)。并分析不同臨床參數(shù)頭頸部腫瘤患者術(shù)前外周血CTCs的檢出情況。結(jié)果1.復(fù)制選擇性溶腫瘤病毒不感染正常人白細(xì)胞。2.分別于正常人外周血中加入的10,50,100個(gè)A549、SKBR3細(xì)胞,其檢出率分別為77.5%,84.0%,80.25%;80.0%,82.5%,81.25%,應(yīng)用pearson卡方檢驗(yàn)比較兩組間差異,結(jié)果顯示用溶腫瘤腺病毒法檢測(cè)兩組不同的腫瘤細(xì)胞,其檢出率無(wú)顯著差異,即本實(shí)驗(yàn)所研究的方法對(duì)各腫瘤細(xì)胞系均敏感且回收率較高。并通過(guò)細(xì)胞免疫染色實(shí)驗(yàn)進(jìn)一步驗(yàn)證所捕獲的細(xì)胞為循環(huán)腫瘤細(xì)胞。3.在收集的48例頭頸部惡性腫瘤的臨床標(biāo)本中,19例患者CTCs陽(yáng)性,檢出率為39.6%,且CTCs的檢出率與性別、年齡、腫瘤分期、腫瘤的發(fā)病部位、淋巴結(jié)轉(zhuǎn)移與否等臨床參數(shù)無(wú)關(guān),與腫瘤分化程度相關(guān),腫瘤分化越差,CTCs的檢出率越高。結(jié)論本研究建立了一種新型循環(huán)腫瘤細(xì)胞的檢測(cè)方法,初步確定了溶腫瘤腺病毒檢測(cè)CTCs的標(biāo)準(zhǔn),并對(duì)該方法的可靠性及臨床應(yīng)用價(jià)值進(jìn)行了評(píng)估。該方法能夠特異性示蹤循環(huán)腫瘤細(xì)胞,具有重要的臨床應(yīng)用價(jià)值。
[Abstract]:Objective the detection of active tumor cells in circulating blood is regarded as fluid biopsy, which has the function of dynamic monitoring. It has important clinical significance in early diagnosis of tumor, individualized treatment, evaluation of curative effect and judgment of prognosis. However, the isolation and identification of circulating tumor cells (circlulating tumor cells,CTCs) has become a difficulty in clinical application due to its small number in the blood. Replication-selective adenovirus (Ad11-5ETel-GFP) is a homologous recombinant virus that can selectively replicate and express green fluorescent protein (Green Fluorescent Protein,GFP) in cells with telomerase activity. The purpose of this study was to establish a repeatable, simple and noninvasive detection method by using the virus to capture circulating tumor cells, and to discuss the reliability and clinical application value of the detection method. Methods the method was proved to be effective in capturing tumor cells, including: AD11-5ETel-GFP virus was added into the peripheral blood of normal people to observe the expression of GFP in normal white blood cells; A549 (lung cancer cell line) SKBR3 (breast cancer cell line) was added to the peripheral blood of 1ml with three gradients of 10,50100 cells, and the model of circulating tumor cells was established and the recovery rate was detected. The results were observed under fluorescence microscope. The above results show that the new detection method established in this experiment can capture circulating cells effectively. The method was applied to the patients with head and neck malignant tumor. 48 clinical specimens were collected. The red blood cells were lysed in the specimens and incubated with the virus at 37 鈩，

本文編號(hào)：2246752