| Objective:(1) To investigate the mRNA expression of kinectin in hepatocellular carcinoma(HCC) cell line BEL-7404, and evaluate the possibility of immunotherapy as a molecular marker of diagnosis, as well as a liver-specific target for treatment. (2) Kinectin-MBP,SMP-30-MBP recombinant fusion protein were expressed and purified by using genetic engineering methods in vitro respectively. Investigate the ability of pulsed DC with hepatocellular carcinoma-associated antigen kinectin-MBP, SMP-30-MBP to stimulate autologous T lymphocytes into CTL, and also detect its cytotoxicity on BEL-7404. (3) preliminarily explore the clinicial application of kinectin in HCC and its role of its biological mechanism.Methods:(1) With reverse transcripts polymerase chain reaction(RT-PCR), the kienctin mRNA level of BEL-7404 HCC cell Line, hepatocellular carcinoma tissue, corresponding adjacent non-HCC tissue and normal liver tissue were analyzed. (2) Kinectin-MBP, SMP-30-MBP fusion protein were expressed and purified by means of recombinant protein technology in vitro,and were incubated with human peripheral blood dendritic cells (Dendritic cell,DC),then to induce cytotoxic T lymphocyte (CTL) with pulsed DC.Then cytotoxicity of CTL against BEL-7404 hepatoma cell was assayed by LDH 4 hours release.(3) Detect the proliferation of T cells CD4+, CD8+T cell subset with SupervisionTM, HRP immunohistochemistry, and to determine the role of kinectin in HCC.Results:(1)The mRNA of Kinectin have a strong expression in BEL-7404 cell line and HCC tissue,low expression in corresponding adjacent non-HCC tissue and normal liver tissue.Successfully expressed and purified kinectin-MBP, SMP-30-MBP by used of recombinant protein technology in vitro, the molecular weight are consistent with the expected molecular weight by electrophoresis.(2) The results of DC culture, T lymphocyte proliferation, CTL's cytotoxicity toward target cell BEL-7404:PBMCs presented typical morphologic characteristics of DCs after induced by rhGM-CSF and rhIL-4 for 7 days.Comparing with the other two groups, the level of CIL-12 and CIFN-yraised significantly in culture medium supernatants of kinectin-MBP treated group (p<0.05); The proliferation capacity of autologous T cell cultured with the kinectin-MBP, SMP-30-MBP fusion protein-pulsed DC were significantly higher than MBP-pulsed, non-treated cultured DC group (p<0.05). The greater of the ratio of stimulated cells (DC) accounts of effector cells (T cells), the stronger the proliferation ability; higher anti-tumor activity were found by DC-induced The CTL of the BEL-7404 hepatoma cells in kinectin, SMP-30 fusion protein-pulsed groups, the highest killing rate was found when the ratio of effector:target was 25:1 (65.00±1.47%; 64.40±1.40% respectively).(3) The results of immunohistochemistry:the number of CD8 positive T-cell increased remarkably in kinectin-MBP-DC-T cell group, compared with the MBP-DC-T cell group, DC-T cells group and normal cultured T cells(p<0.05). Furthermore, there are no significant difference of the number of CD4 positive T-cells, CD4/CD8 among of these four group (P> 0.05).Conclusion:The mRNA level of kinectin (D2 alternative splicing area) showed strong positive expression in the hepatoma cell lines BEL-7404 and HCC tissues. And it can be used as molecular markers for early diagnosis and possible targets for immunotherapy of human HCC. The DCs be pulsed with kinectin, SMP-30 fusion protein can stimulate T lymphocyte proliferation effectively; the pulsed DC can also induce CTL successfully, and have strong cytotoxic activity toward the BEL-7404.The DCs be pulsed of kinectin, fusion protein have a marked anti-tumor function, they are expected to develop the new vaccines for HCC biological therapy. |
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