[Reconstruction of Hematopoietic Function of Human Placental Hematopoietic Stem Cells in Non-Lethally Radiated Mice].

OBJECTIVE: To investigate the effect of human placental hematopoietic stem cells (PHSCs) on hematopoietic reconstruction in non-lethally irradiated mice. METHODS: Human placental HSCs were extracted by mechanical method combined with zymolysis and were identified by flow cytometry and colony formationtest. Twenty-five NOG mice were divided randomly into 4 groups: the blank control group (n=5), the irradiated group (n=4), the low dose PHSC group (n=8) and the high dose PHSC group (n=8). The mice in the irradiated, the low dose and the high dose PHSC groups were irradiated with X-rays at dose 1 Gy (100 cGy/min) under sterile condition. The mice in the low dose PHSC group and the high dose PHSC group were injected intravenously with 0.1 ml human placental HSC in dose of 2×106 and 1×107, respectively. The mice in the blank control and the irradiated group were injected with the same volume of saline. The mice were weighed weekly, and the changes of body weight were calculated. The peripheral blood was collected from each group at 4, 8 and 12 week for flow cytometrytic detection of human CD45+ and myeloid and lymphoid cells. RESULTS: The flow cytometry and cell-colong formation test showed that the human placental HSC accounted for more than 0.75% of total placental mononuclear cells, moreover possess the differentiation ability. Compared with the blank control group, the relative weight gain in the irradiated, the low dose PHSC, and the high dose PHSC groups decreased significantly, and the relative weight gain in the low dose PHSC and the high dose PHSC group increased significantly as compared with the irradiated group. Flow cytometry showed that at the tine-point of 12 weeks after transplantation, the human blood immune system in the high-dose PHSC mice began long-term reconstruction, while the ratio of human CD45+ cells in the low-dose PHSC mice was very low. CONCLUSION: After transplantation of human PHSC the non-lethally irradiated mice can obtain short-term and long-term reconstruetion of human blood cells, which demonstrated that human placental HSC can differentiate and reconstruct hematopoietic function in vivo of irradiated mice.

[Construction and immunological analysis of a combined multi-epitope vaccine against EGFR/HER2].

A recombinant plasmid pET28a-HBcAg-delta n was constructed, in which three mimic B-epitopes of HER family were inserted into the truncated HBc vector. The fusion protein expressed was purified and used to immunize BALB/c mice to induce antibody against the epitopes. Three mimic epitope genes were inserted into the sequences of amino acid residues 78 and 79 of HBcAg by overlap PCR. The PCR product was then cloned into pET28a to construct recombinant expression plasmid which was transformed to E. coli BL21 (DE3) and induced by IPTG. After purification, the fused protein designed HBHE was used to immunize BALB/c mice to detect humoral immunoresponse. The recombinant plasmid was successfully constructed by DNA sequencing analysis. A fusion protein with correct molecular mass was expressed and confirmed by SDS-PAGE. High titre antibody was elicited in the mice immunized with HBHE by indirect ELISA and Western blotting. The HBc particle vector containing three B-epitopes of HER family had been successfully prepared, purified and high titre antibody against HBHE was detected. All these data are helpful in further research of the broad-spectrum anti-tumour effect of combine polypeptide epi-position vaccine of EGFR and HER2.