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Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation
Published on: November 10, 2017
[Effect of Nucleolin on Lymphoma Proliferation by Regulating Thymidine Kinase 1]
Xu-Qiao Mei1, Jian-Da Hu2, Ting Yang2
1Department of Clinical Laboratory, Zhangzhou Affiliated Hospital of Fujian Medical University, Zhangzhou 363000, Fujian Province, China.
Objective:
To investigate the mechanism of nucleolin (NCL) involved in lymphoma proliferation by regulating thymidine kinase 1 (TK1).
Methods:
Twenty-three patients with diffuse large B-cell lymphoma (DLBCL) were selected and divided into initial treatment group (14 cases) and relapsed/refractory group (9 cases). Serum TK1 and C23 protein in peripheral blood mononuclear cells were detected. Cell models of CA46-NCL-KD (CA46-NCL-knockdown) and CA46-NCL-KNC (CA46-NCL-knockdown negative control) were established by lentivirus vector mediated transfection in Burkitt lymphoma cell line CA46. The half maximal inhibitory concentration (IC50) of CA46-NCL-KD, CA46-NCL-KNC, and CA46 to adriamycin were detected by cell proliferation assay (MTS). The expression of NCL mRNA and protein in CA46-NCL-KD and CA46-NCL-KNC cells were dectected by Q-PCR and Western blot, respectively. The cell cycle of CA46-NCL-KD, CA46-NCL-KNC, and CA46 cells were detected by flow cytometry. The expression of TK1 protein in CA46-NCL-KD and CA46-NCL-KNC cells was detected by an enhanced chemiluminescence (ECL) dot blot assay.
Results:
The level of serum TK1 in the initial treatment group was 0.43(0-30-1.01) pmol/L, which was lower than 10.56(2.19-14.99) pmol/L in the relapsed/refractory group (P<0-01), and the relative expression level of NCL protein in peripheral blood was also significantly lower. The IC50 of CA46-C23-KD cells to adriamycin was (0.147±0.02) μg/ml, which was significantly lower than (0.301±0.04) μg/ml of CA46-C23-KNC cells and (0.338±0.05) μg/ml of CA46 cells (P<0.05). Compared with CA46-NCL-KNC cells, the expression of NCL mRNA and protein, TK1 protein decreased in CA46-NCL-KD cells, and the proportion of S phase and G2/M phase also decreased, while G0/G1 phase increased in cell cycle.
Conclusion:
The increased expression of NCL in DLBCL and CA46 cells indicates low sensitivity to drug. NCL may participate in regulation of lymphoma proliferation by affecting TK1 expression, thereby affecting the drug sensitivity.
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