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The Establishment and Clinical Significance of a Quantitative Method for Detection of Circulating Colorectal Tumor Cells in Peripheral Blood

Author: WangBinZhe
Tutor: HuWeiMin
School: Guangzhou Medical College
Course: Oncology
Keywords: Colorectal cancer Circulating tumor cells Human telomerase reverse transcriptase Immune sorting magnetization technology Real-time quantitative reverse transcriptase polymerase chain reaction
CLC: R735.3
Type: Master's thesis
Year: 2010
Downloads: 49
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Abstract

Objective: To establish the immunosuppressive magnetization sorting technology and real-time quantitative reverse transcription polymerase chain reaction method of quantitative detection of circulating tumor cells, and to explore the clinical significance of quantitative detection of colorectal cancer in peripheral blood of patients with tumor cells. Methods: In vitro cultured colon cancer HCT-116 cells, will be divided into 101-106 of six different quantity levels, peripheral venous blood mixed with 5 ml of health, by density gradient centrifugation of peripheral blood mononuclear cells. Separated cells were divided into immune Enrichment group (A) and non-enriched group (B), another set of negative control group. Group A and the corresponding negative control group and package monoclonal antibody to CD326 immunomagnetic bead separation and enrichment of the role magnetic activated cell sorting device, extraction of total RNA enriched sorting specimens. Group B and the corresponding negative control group without immune enrichment processing, direct extraction of total RNA. A and B samples of total RNA using real-time quantitative RT-PCR was used to detect which human telomerase reverse transcriptase (hTERT) mRNA expression levels of each group were carried out 10 experiments, statistical analysis of test results, control experiments research and establish a higher sensitivity of the detection of circulating blood tumor cells. Extract 22 previously untreated patients with colorectal cancer patients and 22 healthy human peripheral venous blood by density gradient centrifugation of peripheral blood mononuclear cells. Peripheral blood mononuclear cells of 22 patients were divided into immune Enrichment group (C group) and non-enriched group (D), 22 healthy peripheral blood mononuclear cell samples is set to E group. Extracted total RNA by RT-PCR was used to detect the hTERT-mRNA expression levels of the three groups of test results to explore the immunosuppressive the magnetization sorting technology and real-time fluorescent quantitative reverse transcription polymerase chain reaction quantitative detection of colorectal cancer in peripheral blood The clinical significance of tumor cells. Results: The PCR amplification of all the negative control group (1) in cell experiments the curve showed irregular wavy lines, not the typical S-type amplification curve, and that all the negative control group specimens were not specific PCR amplification; ( 2) A group and B group 10 experiments 101-106 orders of magnitude cell group PCR amplification mean Ct values ??are shown in Table 5; relative quantitative comparison of 10 experiments 101-106 orders of magnitude cell group hTERT-mRNA expression levels of , A group was significantly higher than in group B, the results were statistically significant (P lt; 0.05), see Table 6; (3) 22 patients with colorectal cancer in group C and group D PCR amplification Ct values ??are shown in Table VII ; relative quantitative comparison of two groups of 22 specimens of hTERT-mRNA expression levels, C group was significantly higher than in group D, the results were statistically significant (P lt; 0.05), see Table 8; (4) the relative quantitative comparison colorectal cancer patients and healthy persons hTERT-mRNA expression levels of C was significantly higher than in group E, the results were statistically significant (P lt; 0.05), are shown in Table 9; (5) C group, 22 patients with colorectal cancer hTERT-mRNA of the expression level of sex, age, tumor size, location and tumor depth of invasion was no significant correlation was not statistically significant (P gt; 0.05). Patients with lymph node metastasis and TNM staging was statistically significant results (P lt; 0.05), are shown in Table 10. Conclusion: The experimental study established the immunosuppressive magnetization sorting technique and RT-PCR detection method, the detection sensitivity is better than simply using RT-PCR detection method for the quantitative detection of tumor cells in peripheral blood by measuring the expression levels of hTERT-mRNA of . Colorectal cancer in peripheral blood of patients with hTERT-mRNA expression correlated with lymph node metastasis and TNM stage was significantly associated micrometastases in patients with colorectal cancer provide new ideas for early clinical. Further research, diagnosis, treatment and prognosis of patients with colorectal cancer have a greater clinical value.

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