Journal: Cancer Science

Article Title: Dendritic cells pulsed with α‐fetoprotein and mutant P53 fused gene induce bi‐targeted cytotoxic T lymphocyte response against hepatic carcinoma

doi: 10.1111/j.1349-7006.2008.00820.x

Figure Lengend Snippet: Reconstruction of enhanced green fluorescent protein/α‐feroprotein–mutant P53 plasmid (pEGFP‐C3/AFP‐mtP53) and the expression of the AFP‐mtP53 gene in eukaryotic cells. (a and b) Gel electrophoretic patterns of reconstructed plasmid pEGFP‐C3/AFP‐mtP53. (a) Reverse transcription–polymerase chain reaction amplification results. Lanes 1 and 6, molecular weight; lane 2, 1.8 kb AFP cDNA amplified; lanes 3 and 4, other samples; lane 5, positive control. (b) Double restriction enzyme reaction results of pEGFP‐C3/AFP‐mtP53 in three samples (no. 1–3). Lanes 1 and 11, molecular weight; lanes 2–4, 1.8 kb AFP cDNAs (sample 1–3); lanes 5–7, 1.2 kb mtP53 cDNA (samples 1–3); lanes 8–10, 3.0 kb AFP‐mtP53 fusion gene in samples 1–3. (c–f) Fluorescent microscopy analysis of human embryonic kidney HEK293 cells transfected with plasmid pEGFP‐C3/AFP‐mtP53 (×200). (c) HEK293 cells transfected with pEGFP‐C3/AFP‐mtP53 emitted green fluorescence 24 h after transfection. (d) The expression of AFP by the immunocytochemical staining method showed that there were red stains in transfected HEK293 cells. (e) Immunofluorescence assay showed that transfected HEK293 cells expressed orange fluorescence, a compound of green fluorescence (fluorescein‐isothiocyanate‐conjugated AFP antibody) and red fluorescence (Cy3‐conjugated mtP53 antibody). (f) Blank HEK293 cells. (g) Construction map of pEGFP‐C3/AFP‐mtP5. The AFP gene is located between 1336 and 3122 bp, and the p53 gene is located between 3140 and 4325 bp.

Article Snippet: Plasmid vector pEGFP‐C3 was purchased from BD Biosciences (San Jose, CA) and mtP53 cDNA was donated by Dr Zhe Wang (Fourth Military Medical University, Xian, China).

Techniques: Mutagenesis, Plasmid Preparation, Expressing, Reverse Transcription, Polymerase Chain Reaction, Amplification, Molecular Weight, Positive Control, Microscopy, Transfection, Fluorescence, Staining, Immunofluorescence

Journal: Cancer Science

Article Title: Dendritic cells pulsed with α‐fetoprotein and mutant P53 fused gene induce bi‐targeted cytotoxic T lymphocyte response against hepatic carcinoma

doi: 10.1111/j.1349-7006.2008.00820.x

Figure Lengend Snippet: Photograph of dendritic cells (DCs) transfected with enhanced green fluorescent protein/α‐feroprotein–mutant P53 plasmid (pEGFP‐C3/AFP‐mtP53), reverse transcription–polymerase chain reaction (RT‐PCR) results of AFP‐P53 fused gene, and flow cytometry analyses pre‐ and post‐transfection. (a and b) Fluorescent microscopy photograph of DCs transfected with pEGFP‐C3/AFP‐mtP53 (×200). (a) DCs expressing green fluorescence, 24 h after transfection. (b) No fluorescence was observed in blank DCs. (c and d) RT‐PCR assay of mRNA of AFP‐mtP53 fused gene. (c) Total RNA extracted from transfected DCs. (d) Gel electrophoretic pattern of RT‐PCR results. Lanes 1–7: molecular weight (DNA ladder 1 kb); sample 1 (3.7 kb containing GFP, AFP, and p53); negative control; glyceraldehyde‐3‐phosphate dehydrogenase; positive control; sample 2; and molecular weight. (e and f) Flow cytometry analysis of DCs pre‐ and post‐transfection. (e) Phenotypes of DCs pre‐transfection. (f) Phenotypes of DCs post‐transfection. From left to right: selection gate, CD1a, CD11c, CD80, CD86, and HLA‐DR.

Article Snippet: Plasmid vector pEGFP‐C3 was purchased from BD Biosciences (San Jose, CA) and mtP53 cDNA was donated by Dr Zhe Wang (Fourth Military Medical University, Xian, China).

Techniques: Transfection, Mutagenesis, Plasmid Preparation, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Microscopy, Expressing, Fluorescence, Molecular Weight, Negative Control, Positive Control, Selection

Journal: Cancer Science

Article Title: Dendritic cells pulsed with α‐fetoprotein and mutant P53 fused gene induce bi‐targeted cytotoxic T lymphocyte response against hepatic carcinoma

doi: 10.1111/j.1349-7006.2008.00820.x

Figure Lengend Snippet: Cytotoxic T lymphocyte responses in different hepatic cancer cells

Article Snippet: Plasmid vector pEGFP‐C3 was purchased from BD Biosciences (San Jose, CA) and mtP53 cDNA was donated by Dr Zhe Wang (Fourth Military Medical University, Xian, China).

Techniques: Inhibition

Journal: Cancer Science

Article Title: Dendritic cells pulsed with α‐fetoprotein and mutant P53 fused gene induce bi‐targeted cytotoxic T lymphocyte response against hepatic carcinoma

doi: 10.1111/j.1349-7006.2008.00820.x

Figure Lengend Snippet: Dendritic cells (DCs) induced cytotoxic T lymphocyte responses against hepatocellular carcinoma cells. Left to right in each column, enhanced green fluorescent protein/α‐feroprotein–mutant P53 plasmid (pEGFP‐C3/AFP‐mtP53), pEGFP‐C3/AFP, pEGFP‐C3/mtP53, pEGFP‐C3, and control. The growth inhibitory rates of HepG2 cells were 51.6 ± 4.2, 48.3 ± 3.6, 32.5 ± 3.9, 28.1 ± 3.1, and 23.7 ± 2.7, respectively. The growth inhibitory rates of pEGFP‐C3/AFP‐mtP53 and pEGFP‐C3/AFP groups were higher than those of other groups with significant difference (P < 0.05). The growth inhibitory rates of SMMC7721 cells were 23.1 ± 2.7, 21.7 ± 3.1, 18.5 ± 2.6, 20.2 ± 2.4, and 15.7 ± 3.3, respectively. There was no statistical difference in any group (P > 0.05). The growth inhibitory rates of HMCC97 cells were 65.2 ± 4.8, 50.2 ± 4.2, 47.7 ± 3.8, 24.5 ± 2.6, and 26.5 ± 3.1, respectively. The rate of the pEGFP‐C3/AFP‐mtP53 group was the highest in all groups with significant difference compared with other groups (P < 0.05). The rates of the pEGFP‐C3/AFP and pEGFP‐C3/mtP53 groups were also higher than those of the pEGFP‐C3 and control groups (P < 0.05). Although the rate of the pEGFP‐C3/AFP group was higher than that of the pEGFP‐C3/mtP53 group, there was no statistical difference (P > 0.05).

Article Snippet: Plasmid vector pEGFP‐C3 was purchased from BD Biosciences (San Jose, CA) and mtP53 cDNA was donated by Dr Zhe Wang (Fourth Military Medical University, Xian, China).

Techniques: Mutagenesis, Plasmid Preparation