| 1.Background and PurposeAccording to the data analysis of the American Cancer Society, malignant tumor is one of the major reasons leading to disease deaths worldwide, most of the malignant tumor derived from epithelial tissue, most tumors are easily diffused in the early stage, and more than90%of death of patients because of tumor metastasis. According to the resent study, the pattern of invasion and metastasis including local infiltration, vascular invasion, intravascular transport, intravascular aggregation, extravasation, occult metastases formation and hibernation, ultimately, angiogenesis and new metastases will appear under some stimulation. And tumor cells in the peripheral circulation is known as Circulating Tumor C ells (CTCs).In recent years, it has been found that circulating tumor cells present in different tumor patients, studies on CTCs have become the hot topic for cancer researcher. CTCs have shown its remarked clinical significance to evaluate prognosis, disease stage, disease recurrence, tumor metastasis, efficacy of therapy and cancer patients’ survival time and other aspects. There have been accumulating evidence to demonstrate that CTCs are becoming an independent clinical indicator to assess the risk of disease recurrence, guidance and detection the clinical therapy. The methods of CTCs detection currently accepted, however, is only dependent on the physical characteristics of the tumor cells and molecular markers, the sensitivity and specificity are limited. So how can we identify the very few of specific tumor cells from the hundreds of millions of peripheral circulation is a challenging topic, but with a high impact for clinical application.With the continuous development of molecular biology of tumor,scientists using gene engineering to transform and build the wild-type virus, so that the virus has the ability to specifically recognize tumor cells; because of its stability, a wide range of infections, infection efficiency,low toxicity and high safety factor, adenovirus become the focus of the virus research. Based on these viewpoints, our topic try to build a new oncolytic adenovirus through genetic engineering. Telomerase-specific replication-selective oncolytic adenovirus is an oncolytic adenovirus which can only replicate specifically in the tumour cells with a high activity of telomerase and express green fluorescent protein (GFP) as the viral crucial gene E1A expression is controlled by the promoter of telomerase. The positive CTCs are easily detected through fluorescent microscope.The current CTCs detections in clinic are wildely used breast, prostate and colon cancer, there is little information about lung cancer, while the number of lung cancer patients in China is the first of that in the world, and the incidence of the cancer is rapidly increasing year by year, the peak of the incidence we expected has not come. Therefore, our experiment will focus on the clinical significance of the Telomerase-specific replication-selective oncolytic adenovirus in lung cancer cells, and aims to provide a new spicific indicator for clinical diagnosis and prognostic analysis for lung cancer.2.MethodsThe well-characterized human lung cancer cell lines, A549and H1299were applied to this study. The two cell lines were digested and counted before adding into10%DMEM, then incubated with Adll-Tel-GFP at concentration for varied time course at37℃,stained with DAPI and observed under the fluorescent microscope, with the counting of GFP positive cells.10%DMEM without tumor cells was used as a negative control; The whole blood cells without tumor cells was used as a negative control. In order to validate its potential application in the clinic, the two cell lines were spiked into7.5ml whole blood from healthy donors at different number;after lysis of red blood cells, the cell pellet of white blood cells including spiked tumor cells were incubated with the virus at an optimal concentration under37℃, for24hours, then fixed and smeared onto slides that were mounted with DAPI mounting solution. The GFP-positive cells were counted under the fluorescent microscope. The experimental results are analyzed by SPSS17.0processing.3.Results(1)The telomerase-specific replication-selective oncolytic adenovirus developed in our lab can specifically recognize and infect the lung tumor cells, and express the GFP in these tumor cells.(2)The positive rate of telomerase-specific replication-selective oncolytic adenovirus expression in two lung cancer cell lines of A549and H1299is not statistically significant (p=0.435), suggesting the similar activity of the virus for different lung cancer cell lines.(3)The telomerase-specific replication-selective oncolytic adenovirus does not infect human leukocyte.(4)The sensitivity of the method developed in this study is more than90%for detection human lung cancer cell lines in the mocked samples.4.ConclusionThe telomerase-specific replication-selective oncolytic adenovirus developed in our lab can specifically detect lung cancer cells in the circulating blood with a very high sensitivity and specificity. The method optimized in our lab is a simple, rapid and useful approach for detection of CTC, especially for the viable CTCs, which warrants further clinical application study for human lung cancer... |
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