Recognition and capture of metastatic hepatocellular carcinoma cells using aptamer-conjugated quantum dots and magnetic particles.

저자: Wang FB, Rong Y, Fang M, Yuan JP, Peng CW, Liu SP, Li Y.

저널: Biomaterials. 2013 May;34(15):3816-27.

사용한 제품: Dynabeads M-280 Streptavidin (Cat# 11205D, 11206D, 60210)

Abstract

Metastatic recurrence is the most important biological behavior of hepatocellular carcinoma (HCC) and the main cause of treatment failure. Early prediction of metastasis is currently impossible due to the lack of specific molecular probes to recognize metastatic HCC cells. Aptamers have recently emerged as promising potential molecular probes for biomedical applications. Two well-matched HCC cell lines including HCCLM9 with high metastatic potential and MHCC97-L with low metastatic potential, were used to select aptamers for HCC metastasis. With a whole-cell-SELEX strategy, in which HCCLM9 cells were used as target cells and MHCC97-L cells as subtractive cell, 6 potential aptamers had been generated. Detailed study on selected aptamer LY-1 revealed that it could bind metastatic HCC cells with high affinity and specificity, not only in cells culture and animal models of HCC metastasis, but also in clinical HCC specimens. Moreover, the aptamer LY-1 and magnetic particles conjugates could efficiently capture the HCC cells from complex mixture whole blood. These studies demonstrated that this HCC specific aptamer LY-1 could be a promising molecular probe to recognize metastatic HCC cells.

제품 사용법

Random DNA library and primers

The cell-SELEX DNA library contains a 40-base central random sequence flanked by primer sites on either side (50-ATC CAG AGT GAC GCA GCA-N40-TGG ACA CGG TGG CTT AGT-30 ). The FITC-labeled forward primer (50-FITC-ATC CAG AGT GAC GCA GCA-30) and biotin-labeled reverse primer (50- Bio-ACT AAG CCA CCG TGT CCA-30) were used in PCR to obtain the double-labeled DNA and to separate the singlestranded DNA by streptavidin-coated magnetic particles. The FITC-labeled sequences were used to monitor progress of selection by flow cytometry (Beckman Coulter, USA). All sequences were synthesized by SBS Genetech Co., Ltd and purified by reverse phase HPLC.

Capture the HCC cells in real peripheral blood samples by the aptamerconjugated magnetic particles

To evaluate the targeting capture capacity of the selected aptamer LY-1 in a complex biological environment, HCCLM9 cells (1  105) were prepared as described above and mixed with the whole blood. After lysis of red blood cells inNH4Cl solution, the remaining cells (HCCLM9 cells and WBC) were incubated with bio-labeled aptamer on ice for 30 min. After washing, the cells were then incubated with streptavidin-coated magnetic particles (Dynabeads, M-280 Streptavidin) at room temperature for 30 min. Afterwashing, the suspension of the cells-aptamer-magnetic particles was dropped on the glass slides pre-treated with poly-lysine. After being dried in roomair, the slideswere gentlywashed once in PBS andimmediately fixed for 5 min in methanol solution which was pre-cooled at  20  C. The cells were first incubated with rabbit anti-human keratin 19 and mouse anti-human AFP primary anti-bodies (R&D Systems) overnight at 4  C, then washed and incubated with FITC-conjugated anti-rabbit or anti-mouse IgG secondary antibody (dilution 1:200, Santa Cruz Biotechnology Inc) for 60 min at room temperature. In order to evaluate the capture efficiency of the aptamer-conjugated magnetic particles, different amounts HCCLM9 cells (104, 103 and 102 HCC cells) were respectively mixed with the whole blood. The other treatment steps were the same as described above.