1.Selection,soluble expression and identification of full human scFvs against IL-13
Dengmei ZHANG ; Siji NIAN ; Yingchun YE ; Yan YANG ; Hong YU ; Qing YUAN
Chinese Journal of Immunology 2016;(1):65-68
Objective:To select specific and neutralizing scFv against IL-13 and soluble expression and identification them.Methods:In our previous study the scFv library were constructed,and here the scFv library was enriched and the positive scFv were screened from the enriched scFv library for three round.The specific positive scFvs with better affinity were ligated with expression vector for expression and identification.Results:After three rounds of enrichment,30%of clones were positive.The two specific scFvs with better affinity and neutralization were selected from almost 500 clones and then ligated with expression vector LZ16 for soluble ex-pression.The expressed scFvs were identified by western blot and biomolecular interaction analysis.Conclusion: The specific scFvs against IL-13 with better affinity and neutralization had been selected successfully.
2. Effect of endothelial cell-targeted soluble Notch ligand hD1R protein on the proliferation of acute myeloid leukemia cells
Dengmei TIAN ; Yingmin LIANG ; Yongqing ZHANG
Chinese Journal of Hematology 2018;39(10):845-850
Objective:
To evaluate the effects of endothelial cell-targeted soluble Notch ligand hD1R protein on the proliferation of acute myeloid leukemia (AML) cells.
Methods:
The expression levels of Notch1, Notch2, Notch3, Notch4, Hes1 in bone marrow CD34+ cells from 24 cases of untreated AML (AML group) and 9 healthy controls (control group) were determined by real time quantitative polymerase chain reaction (PCR) . Recombinant hD1R protein was first induced and purified. Bone marrow CD34+ cells were co-cultured on human umbilical vein endothelial cells (HUVEC) supplemented with a cocktail containing 5 types of human cytokines (5GF) and soluble hD1R. The cultured cells were tested under different culture conditions including PBS group (PBS replaces HUVEC) , hD1R group, 5GF group, GSI group (hD1R plus GSI) . Proliferation and apoptosis of cultured cells were also analyzed. Real time quantitative PCR was used to test the expression levels of Hes1 and Bcl-2 in cultured cells.
Results:
The expression levels of Notch1 and Hes1 in primary AML patients were significantly lower, and Notch4 expression was higher compared to the control group (
3.Effect of endothelial cell-targeted soluble Notch ligand hD1R protein on expansion and engraftment of cord blood hematopoietic stem/progenitor cells.
Dengmei TIAN ; Yingmin LIANG ; Hua HAN ; Yongqing ZHANG
Chinese Journal of Hematology 2014;35(10):885-890
OBJECTIVETo evaluate the effects of endothelial cell- targeted soluble Notch ligand hD1R protein on expansion and engraftment of cord blood hematopoietic stem/progenitor cell (CB HSPCs).
METHODSRecombinant hD1R protein was first induced and purified. Human cord blood CD34⁺ cells were co-cultured on human umbilical vein endothelial cells (HUVECs) supplemented with a cocktail containing 5 types of human cytokines including TPO, SCF, FL, IL-6, IL-3 (5GF) and soluble hD1R. The expansion of CD34⁺ cells was tested under different culture conditions including PBS group (PBS replaces HUVEC), hD1R group, sup group (HUVEC supernatant replaces HUVEC), fix group (fixed HUVEC replaces HUVEC), Day 0 group (Control). Cell cycle and apoptosis of cultured cells were also analyzed. Their progeny expanded in PBS or hD1R group were transplanted into sublethally irradiated NOD/SCID mice. The percentages of human CD45⁺ (hCD45⁺) cells in the marrow of recipient mice were determined by FACS 12 weeks later.
RESULTShD1R induced more expansion in the total number of CD34⁺ cells cocultured with HUVECs plus 5GF, which was 87.50-fold increase compared to the Day 0 group, and 7.98-fold increase than that of PBS group. FACS analysis also showed that the percentage of CD34⁺ cells was 77.0% in G0/G1 phase in the hD1R group, which indicated that hD1R enhanced HSPCs expansion and inhibited apoptosis. Moreover, hD1R significantly promoted human HSPC engraftment after BM transplantation in irradiated mice.
CONCLUSIONThe Notch-mediated ex vivo expansion system has been established and hD1R promoted expansion and engraftment of human CB HSPCs, which provided the evidence for further clinical application.
Animals ; Antigens, CD34 ; Cells, Cultured ; Coculture Techniques ; Endothelial Cells ; immunology ; Fetal Blood ; Hematopoietic Stem Cells ; immunology ; Humans ; Membrane Proteins ; immunology ; Mice