發(fā)布時間：2018-10-18 17:18

【摘要】：目的采用Opti Prep密度梯度離心結(jié)合免疫磁珠負向篩選法富集循環(huán)腫瘤細胞(Circulating Tumor Cell,CTC),并聯(lián)合多色免疫熒光標記和熒光原位雜交技術(shù)檢測CTC,建立一種簡易高效的富集和鑒定癌癥患者外周血CTC的方法,有效應(yīng)用于臨床腫瘤患者外周血的CTC計數(shù)檢測。在此基礎(chǔ)上,進一步探索建立單細胞捕獲技術(shù)平臺,對特定CTC細胞或者CTC細胞群進行單細胞組學(xué)的研究。顯微鏡觀察到CTC后進行顯微切割和顯微吸取以獲取單細胞進行分子鑒定,這一研究將促進CTC的分子鑒定,開發(fā)新的治療靶點,并產(chǎn)生對癌癥患者更有效的治療方案。方法1.利用不同密度細胞的沉降系數(shù)不同,配比不同濃度的Opti Prep溶液梯度離心分離外周血各細胞成分,驗證單核細胞聚集細胞層。分別選取QGY-7701等多種腫瘤細胞混入外周血,模擬體內(nèi)循環(huán)腫瘤細胞狀態(tài)。比較Opti Prep液與Ficoll液富集腫瘤細胞的效率、回收率、活性率。密度梯度離心結(jié)合免疫磁珠建立最優(yōu)的循環(huán)腫瘤細胞富集體系。2.選取5種腫瘤細胞株:肝癌細胞(QGY-7701、Huh-7、SMMC-7721)、卵巢癌細胞HO-8910、肺癌細胞A549分別進行CD45和CK18兩種抗體的細胞免疫熒光染色,CIK細胞作對照。FISH標記CIK細胞染色體數(shù)目。將腫瘤細胞混入正常人外周血模擬外周血循環(huán)腫瘤細胞進行富集,免疫熒光技術(shù)結(jié)合FISH檢測,準確識別腫瘤細胞。3.聯(lián)合富集和檢測方法精確檢測癌癥患者的外周血循環(huán)腫瘤細胞,進行方法學(xué)驗證評估。并根據(jù)患者CTC檢測數(shù)目和臨床預(yù)后數(shù)據(jù)分析我們建立的CTC檢測對于腫瘤患者預(yù)后判斷的作用。4.嘗試顯微切割和顯微吸取提取單個靶細胞及較純的目的細胞群,應(yīng)用Carl Zeiss PALM Combi System和Cell Ector Plus兩大系統(tǒng)捕獲單個細胞,并進行RT-PCR檢測腫瘤細胞的基因表達情況。結(jié)果1.Opti Prep液能有效分離單核細胞,將QGY-7701腫瘤細胞富集于白膜層。肝癌細胞(QGY-7701、Huh-7、SMMC-7721)、卵巢癌細胞HO-8910、肺癌細胞A549在Opti Prep中腫瘤細胞的富集效率均高于Ficoll,兩組間差異均有統(tǒng)計學(xué)意義(P0.05)。單細胞操作取50個左右腫瘤細胞入血模擬循環(huán)腫瘤細胞,比較Opti Prep與Ficoll的回收率較一致。2.免疫熒光結(jié)果顯示,僅CIK細胞觀察到CD45紅色熒光,而CK18則是僅CIK細胞無綠色熒光。熒光原位雜交結(jié)果表明能顯著標記細胞染色體,以此識別染色體異常細胞。3.通過對33例入組患者外周血的檢驗驗證以上富集和檢測循環(huán)腫瘤細胞的方法,均發(fā)現(xiàn)有循環(huán)腫瘤細胞的存在,說明建立的這一方法能有效檢出癌癥患者血液中的CTC。實驗結(jié)果顯示,CTC數(shù)量較多對應(yīng)較短生存期,較差預(yù)后。4.本研究根據(jù)實驗需要,涉及兩種可識別熒光標記的單細胞獲取技術(shù),結(jié)果顯示均能迅速準確的得到單個目的細胞,解決細胞異質(zhì)性問題,獲取純凈靶細胞,進而為少量CTC的下一步分子鑒定提供了機會,顯示了良好的應(yīng)用前景。結(jié)論本研究成功建立了Opti Prep密度梯度離心結(jié)合免疫磁珠負向篩選法富集CTC,并聯(lián)合多色免疫熒光標記結(jié)合熒光原位雜交技術(shù)檢測CTC,有效應(yīng)用于臨床腫瘤患者外周血的CTC鑒定。另外,嘗試單細胞捕獲新技術(shù)獲取稀有循環(huán)腫瘤細胞,為后續(xù)單細胞目的基因表達鑒定和進一步的基因測序研究探尋方向,為癌癥的預(yù)后及預(yù)測信息指明方向、為個體化指導(dǎo)治療和用藥的合理選擇提供參考。
[Abstract]:Objective To enrich circulating tumor cells (CTC) with Opti Prep density gradient centrifugation combined with immunomagnetic beads, and to establish a simple and efficient method for enriching and identifying peripheral blood CTC in cancer patients by combining multi-color immunofluorescence labeling and fluorescence in situ hybridization technique. The effect was used in the detection of CTC counts in peripheral blood of patients with clinical tumors. On the basis of this, a single cell acquisition technology platform was established to study the single cell group study of specific CTC cells or CTC cells. Microdissection and microscopic aspiration after the CTC were observed to obtain single cells for molecular identification, which would facilitate molecular identification of CTCs, develop new therapeutic targets, and produce a more effective treatment regimen for cancer patients. Method 1. The cell components of peripheral blood were obtained by gradient centrifugation of Opti Prep solution with different densities and different density cells. Multiple tumor cells, such as QGY-7701, were selected to be mixed with peripheral blood to simulate the state of circulating tumor cells in vivo. The efficiency, recovery and activity of Opti Prep liquid and Ficoll liquid enriched tumor cells were compared. The optimal circulating tumor cell enrichment system was established by density gradient centrifugation combined with immunomagnetic beads. Five tumor cell lines were selected: liver cancer cell (QGY-7701, Huh-7, NCI-7721), ovarian cancer cell HO-8910, lung cancer cell A549, and cell immunofluorescent staining of CD45 and CK18 antibodies respectively. CIK cells were used as control. FISH markers CIK cell chromosome number. the tumor cells are mixed with normal human peripheral blood to simulate peripheral blood circulating tumor cells to enrich, the immunofluorescence technique is combined with FISH detection, and the tumor cells are accurately identified. Combined enrichment and detection methods accurately detect peripheral blood circulating tumor cells in cancer patients and conduct methodological verification evaluation. Based on the patient's CTC test number and clinical outcome data, we established the effect of CTC detection on the prognosis of patients with tumor. Single target cell and pure target cell population were extracted by microdissection and microdissection. The single cell was captured by Carl Zeiss PALM Combi System and Cell Ector Plus, and the gene expression of tumor cells was detected by RT-PCR. Results 1. Opti Prep liquid could effectively separate monocytes and enrich QGY-7701 tumor cells in white membrane. The concentration of tumor cells in Oti Prep was higher than that of Ficoll (P0.05). In single cell operation, 50 or so tumor cells were injected into the blood-simulated circulating tumor cells, and the recovery rates of Opti Prep and Ficoll were more consistent. Immunofluorescence showed that only CIK cells observed CD45 red fluorescence, while CK18 was only CIK cells without green fluorescence. The results of fluorescence in situ hybridization showed that the cell chromosome could be marked by fluorescence in situ hybridization. By examining the methods of enriching and detecting circulating tumor cells in the peripheral blood of 33 patients enrolled, the existence of circulating tumor cells was found, which indicated that the established method could effectively detect the CTC in the blood of cancer patients. The results showed that the number of CTC corresponded to shorter survival and poor prognosis. According to the experimental requirements, the study relates to two single cell acquisition technologies capable of identifying fluorescent labels, and the results show that the single cell can be rapidly and accurately obtained, the problem of cell heterogeneity is solved, pure target cells are obtained, and the next step molecular identification of a small amount of CTCs is provided with the opportunity, and a good application prospect is displayed. Conclusion This study successfully established Opti Prep density gradient centrifugation combined with immunomagnetic beads to enrich CTC, and combined with fluorescent in situ hybridization technique to detect CTC, and has the effect of CTC identification in peripheral blood of clinical tumor patients. in addition, attempt to acquire rare circulating tumor cells by single cell capture novel technology, identify and further gene sequencing research search directions for subsequent single cell target gene expression identification and further gene sequencing, indicate that prognosis and prediction information of cancer refer to a bright direction, Provide reference for the reasonable choice of individualized instruction therapy and medication.
【學(xué)位授予單位】：浙江理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R730

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