1.Application of Flow Cytometry Combined Fluorescence in Situ Hybridization to Indentify the Lymphocyte Subtypies with Epstein-Barr Virus Infection.
Hong-Yu SU ; Yi SHU ; Guo FU ; Zi-Yang LIU ; Dan ZHU ; La-Mei ZENG ; De-Yu MA ; Lin ZOU
Journal of Experimental Hematology 2022;30(3):897-907
OBJECTIVE:
To establish the technique that take the advantages of flow cytometry combined fluorescence in situ hybridization (Flow-FISH) to identify the Epstein-Barr virus(EBV) infected lymphocyte subtypies in patients' peripheral blood sample.
METHODS:
Peripheral Blood monocyte from 9 patients with EBV infection enrolled at Children's Hospital in Chongqing Medical University were isolated by Ficoll-paque centrifugal separation. The expressions of EBER1, EBER2 in cell were detected by qRT-PCR. The surface markers of cell were detected by Flow cytometry after staining with their antibodies. The cell was treated Fix-Permeabilization Buffer before hybridization with fluorescent labeled probe at 37 ℃ overnight. The cell status, surface markers and targeted mRNA are detected by flow cytometry and fluorescence microscope.
RESULTS:
It was optimized that the Fix-Permeabilization Buffer and recipe with 0.2% Tween-20 were picked out as providing a good cell integrity and high resolution of surface markers. Hybridization with 20% formamide and 7% dextran sulfate at 37 ℃ overnight is the optimal hybridization condition as a good hybridization effect, a detectable cell integrity and a high resolution of cell markers under flow cytometry detection. Finally, upon the established Flow-FISH method, lymphocyte subpopulations of the EBV+ cells from cell lines and blood samples of patients were identified successfully.
CONCLUSION
A Flow-FISH technology is established, which can be applied in the identification of EBV infected cell subtypes. This research provides a foundmental for its application in clinical test in EBV+ related proliferative diseases.
Epstein-Barr Virus Infections
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Flow Cytometry/methods*
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Herpesvirus 4, Human
;
Humans
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In Situ Hybridization, Fluorescence/methods*
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Lymphocyte Subsets
2.The Effect of KRAS on Proliferation and Apoptosis of T-ALL Cell Lines.
Zi-Yang LIU ; Yi SHU ; Guo FU ; Hong-Yu SU ; Dan ZHU ; La-Mei ZENG ; De-Yu MA ; Lin ZOU
Journal of Experimental Hematology 2022;30(4):1040-1048
OBJECTIVE:
To investigate the function of RAS protein on the progression of the T-ALL cell lines in vitro.
METHODS:
The DNA of the T-ALL cells was purified then amplified the coding regions of three RAS genes (KRAS, NRAS, HRAS) by PCR reaction. After T-A cloning, the coding regions of KRAS, NRAS and HRAS were sequenced by Sanger Sequencing. The siRNA oligonucleotides were cloned into the pSEH-361 vector, which were then packaged into retroviral together with pAMPHO and pVSVG in the HEK-293T cells. The T-ALL cells were infected with the retrovirus. The gene expressions were detected by qRT-PCR and Western blot. The T-ALL cells were stained with Annexin V-PE/7-AAD and the apoptotic cells were detected by flow cytometry. The T-ALL cells were stained with Hoechst 33258, and the cell cycle distribution was determined by flow cytometry. The expression of cleaved-Caspase 3 was stained with antibody and observed with fluorescence microscope.
RESULTS:
For RAS genes, beside the Loucy and the P12-ICH cells harbored KRAS c.6187G>A (p.KRASG12D) homozygous mutant, no missense mutation of RAS was found in other T-ALL cells genome. The pan RAS inhibitor compound 3144 showed toxicity to all tested T-ALL cells, except PEER (IC50=47.916 μmol/L). Similarly, Tipifarnib induced apoptosis of multiple T-ALL cell lines except for the PEER cells (IC50=94.2265 μmol/L). After KRAS knock-down, the T-ALL cells showed significant apoptosis and an arrested cell cycle.
CONCLUSION
The KRAS protein is vital for the progression of the T-ALL cells in vitro, it is a potential therapeutic target for T-ALL patients.
Apoptosis
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Cell Line
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Cell Proliferation
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Humans
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Mutation
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Precursor T-Cell Lymphoblastic Leukemia-Lymphoma
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Proto-Oncogene Proteins p21(ras)/genetics*