| Objective: HCC is one of the common malignant cancer , and insensitive to chemotherapy and radiotherapy, only 15% can effect a radical cure. The rate of recurrence and metastasis is high. Immuno-gene therapy application in prophylaxis and therapies has made a big progress in recent years. Fractalkine (FK) is a CX3C chemokine that can chemoattract T lymphocytes, monocytes and NK cells. In our study, we investigated the induction of antitumor response and its immune mechanism by FK gene-modified murine hepatocellular cells. These were groundworks for exploring immunity arosing by co-transfectant as vaccine to treat established tumors and their metastases Methods: 1.Expression of FK in MM45T.Li tumor cells: Plasmids were transfected into MM45T.Li cells with Lipofectamine 2000. Expression of FK was selected in the presence of G418 selection media .Protein expressed in cells was detected by RT-PCR and immunocytochemical staining. The effect of the FK gene modification on the in vitro growth of MM45T.Li cells was evaluated by 3H-TdR incorporation. Chemotaxis was detected by Transwell chamber; 2. Immune response induced by FK gene modified tumor vaccine:Female mice were inoculated s.c. with MM45T.Li- FK or control cells. The other group of mice were vaccinated with MM45T.Li- FK cells and were rechallenged s.c. parental MM45T.Li cells 4 weeks later. Anti-tumor effect was evaluated by tumor size. And the expression of FK in tumor tissues were detected by immunohistochemical staining; 3. Antitumor immune mechanism of FK gene modified tumor vaccine: Infiltration of lymphocytes in tumor tissues were analysied by immunohistochemical staining .T cell subsets in murine peripheral blood were detected by FACS. The CTL activity was determined by a standard 4 hr 51Cr-release assay. Splenocytes derived from immunized mice were stimulated by MM45T.Li inactivated by ultrasound,IL-4,IFN-γand IL-2 in supernatant were assayed by ELISA. Results: 1.FK expression in HCC cells: Specific bands could be amplified from total RNA of MM45T.Li-FK cells , no bands from MM45T.Li and MM45T.Li- mock cells. Immunohistochemical staining for FK was positive in MM45T.Li- FK cells. The proliferation of MM45T.Li -FK cells remained unchanged compared to that of parental MM45T.Li and MM45T.Li- mock cells,indicating that FK gene transfer itself does not affect the in vitro growth of MM45T.Li cells. The supernatant of FK gene modified cells can chemoattract lymphocytes, indicating that FK has chemotaxis in vitro. 2. Immune response induced by FK gene modified tumor vaccine: When 5×105 MM45T.Li, MM45T.Li- mock and MM45T.Li -FK cells were inoculated for each mouse, 100% of the mice developed progressively growing tumors in MM45T.Li and MM45T.Li- mock groups. Tumor growth of the MM45T.Li-FK mice were inhibited and the survival time was prolonged. Four weeks after the injection with 1×105 MM45T.Li-FK cells ,the mice were challenged s.c. with 5×105 parental MM45T.Li cells, 50% mice developed tumor. The tumor size was smaller, and the the survival time was prolonged, indicating that the protecting effect waseffectively induced by vaccination with MM45T.Li- FK . Immunohistochemical staining for FK was positive in MM45T.Li- FK tumor tissue. 3. Antitumor immune mechanism of FK gene modified tumor vaccine: 3.1 Obvious infiltrations of CD8+T cells, CD4+T cells were observed in the tumor sites; 3.2 The ratios of CD8+T cells and CD4+T cells in MM45T.Li -FK group were 50.02±4.46% and 21.70±1.75%,obviously higher than those of MM45T.Li and MM45T.Li-mock groups, which were 35.56±6.32%,41.59±7.91% and 12.51±3.06%,13.54±1.90% respectively(p<0.05); 3.3 CTL activity(%) of these groups was 16.456±0.008,18.835±0.01 and 19.696±0.017 respectively at E/T=25:1, there were no difference within these groups(p>0.05);However, it was 13.823±0.061,16±0.02 and 46.53±0.02 respectively at E/T=50:1,there were difference within these groups , MM45T.Li-FK group was increased (p<0.05); 3.4 There was no IL-4 detected in supernatant of splenocytes derived from...
|
PDF Full Text Request