【摘要】：目的 本課題旨在建立一套適于脫落細(xì)胞檢測(cè)的激光顯微捕獲切割技術(shù)(Laser Capture Microdissection, LCM)的方法,并對(duì)該方法在脫落細(xì)胞類(lèi)檢材特別是降解的脫落細(xì)胞樣本檢驗(yàn)中的應(yīng)用進(jìn)行探索性研究。 方法 1以口腔上皮脫落細(xì)胞為模型,從染色方法、提取方法、循環(huán)參數(shù)等方面進(jìn)行比較研究,建立LCM方法。 2以酸性飲料和口腔上皮細(xì)胞混合物為研究對(duì)象,對(duì)Chelex-100法、Chelex-100與Microcon100相結(jié)合法、DNAIQTM提取法等不同方法分別與LCM方法進(jìn)行比較研究。 3以尿液中脫落細(xì)胞為研究對(duì)象,對(duì)目前法庭科學(xué)常用檢測(cè)方法與LCM方法進(jìn)行比較研究。結(jié)果 1利用本實(shí)驗(yàn)所建立的LCM方法捕獲10個(gè)口腔上皮細(xì)胞,即可獲得完整的DNA分型,重復(fù)性較好,為法醫(yī)實(shí)際案件中微量生物檢材的DNA檢驗(yàn)提供了一種參考方法。 2酸性飲料和口腔上皮細(xì)胞混合物僅采用Chelex-100法或與Microcon100相結(jié)合,難以獲得較高質(zhì)量的DNA,從而難以獲得較滿(mǎn)意的檢測(cè)結(jié)果；對(duì)于檸檬酸類(lèi)混合物,可直接采用DNA IQTM提取法提取,并可以通過(guò)減小洗脫體積,提高DNA濃度,從而提高DNA的檢出率；乳酸類(lèi)富含蛋白質(zhì)的混合物,可考慮采用LCM法,將靶細(xì)胞從復(fù)雜環(huán)境中分離,更利于獲得較好的檢測(cè)結(jié)果。 3新鮮尿液組：采用LCM技術(shù)提取的DNA在所有基因座上均獲得較滿(mǎn)意的結(jié)果,而采用Chelex-100提取法提取的DNA在各基因座上存在等位基因丟失、非特異性擴(kuò)增、峰值低等問(wèn)題；儲(chǔ)存尿液組：4℃儲(chǔ)存10天以?xún)?nèi)和室溫儲(chǔ)存4天以?xún)?nèi)的尿液采用LCM技術(shù)聯(lián)合DNA IQTM提取法的均可明確判讀12個(gè)以上基因座；而采用Chelex-100提取法未獲得有效分型。結(jié)論 LCM法為脫落細(xì)胞類(lèi)檢材,特別是降解的脫落細(xì)胞的DNA檢測(cè)提供了一種有效的方法,同時(shí)也為其他種類(lèi)的微量生物檢材的檢驗(yàn)提供了一種參考方法。
[Abstract]:Objective to establish a method of laser microcapture and cutting (Laser Capture Microdissection, LCM) for the detection of exfoliated cells. The application of this method in the detection of exfoliated cell samples, especially the samples of degraded exfoliated cells, was studied. Methods 1 the oral epithelial exfoliated cells were used as the model, and the LCM method was established by comparing the staining methods, extraction methods and circulatory parameters. 2 the mixture of acid beverage and oral epithelial cells was used as the research object. The methods of Chelex-100, Chelex-100 combined with Microcon100, DNAIQTM extraction and LCM were compared with each other. (3) taking exfoliated cells in urine as the research object, the common methods of forensic science and LCM were compared. Results 1 10 oral epithelial cells were captured by LCM method, and the complete DNA typing was obtained, which provided a reference method for the DNA test of trace biological samples in forensic cases. (2) the mixture of acid beverage and oral epithelial cells was only by Chelex-100 method or combined with Microcon100, so it was difficult to obtain high quality DNA, and obtain satisfactory results. The citric acid mixture can be extracted directly by DNA IQTM extraction, and the detection rate of DNA can be improved by reducing the volume of elution and increasing the concentration of DNA. Since lactic acid is rich in protein, LCM method can be used to isolate target cells from complex environment. (3) fresh urine group: DNA extracted by LCM technique had satisfactory results at all loci, while DNA extracted by Chelex-100 method had some problems such as allele loss, non-specific amplification and low peak value. In the urine storage group, the urine stored at 4 鈩，

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