To figure out the change of miR-25 expression of NSCLC H446 lung cancer in vivo, the membrane-type PBLs were administered into ICR mice through injection using established methods. Littermate neonatal rats were used as controls. At 12 h, 24 h, 36 h, and 48 h after injection, lung tissues and BALF were evaluated to determine the expression of miR-25. As shown in Figs. 1 and 2, for BALF the miR-25 levels were significantly increased after PBLs stimulation in PBLs groups. These results suggested that the expression of miR-25 increased with extension of time after injection of PBLs.
Pro-inflammatory cytokine production in mice of induced lung cancer
Inflammatory cytokines TNF-γ and IL-2 (Fig. 5) levels in the BALF increased with PBLs stimulation. Induction of PBLs resulted in a significant increase in TNF-γ production compared with the control group, moreover, IL-2 significantly increase by PBLs induction in a dose-dependent manner, the difference was statistically significant (P < 0.05). Consistently, inflammatory cytokines TNF-γ and IL-2 (Fig. 6) protein levels production in lung tissues were all increased in ICR mice. The more doses of PBLs, the higher TNF-γ and IL-2 level, the difference was statistically significant (P < 0.05).
Histological examinations of lung tissue
We also performed the Hematoxylin and eosin stain to examine lung pathological changes. As shown in Figs. 7–10, PBLs induced lung injury was promoted in experiment groups, as manifested by increase infiltration of neutrophils, increased thickening of interstitial alveolar regions, and minimal structural damage compared with that seen in the control group.
Effects of PBLs on enhancement of splenocytes proliferation and cytotoxicity
After inoculation for one week, the spleen cells were taken for proliferation experiment. Under the conditions of adding specific antigenic peptide stimulation and without specific antigenic peptide stimulation, flow cytometry was used to detect the mice splenocyte proliferation index of each experimental group. The results (see Fig. 11) showed that the PBLs group had significant difference (P < 0.05) comparing the saline control group and the pcDNA group with specific antigenic peptide stimulation. Also, the mice splenocyte proliferation index of the PBLs group without specific antigenic peptide stimulation did not have significant difference comparing the other two groups. The ratios of proliferation index obtained by adding specific antigenic peptide stimulation and without specific antigenic peptide stimulation for the three groups were 0.97, 0.99, and 1.62, respectively. This result indicates that the larger the ratio is, the higher the activity of spleen cell proliferation is under antigen stimulation.
The killing activity experiment of spleen cells further confirmed the specific killing activity of T cells. The specific killing function of PBLs group was stronger than pcDNA group (P < 0.05) and saline control group (P < 0.01). The killing rate was 32%, 23%, and 14%, respectively.
Two-color analysis of the expression of PBLs on mouse spleen B cells was shown in Fig. 11. ICR mice splenocytes were simultaneously stained with anti-mouse IgM[a] mAb DS-1 and purified anti-mouse PBLs mAb 1D3. The staining of primary antibody was detected with FITC-conjugated goat anti-mouse lg.
In vivo expression of HSP70 and PBLs in mice muscle tissues after 12 h, 24 h, 36 h, and 48 h were shown in Fig. 12. Western Blot used denaturing polyacrylamide gel electrophoresis (SDS-PAGE) for separation of protein mixture, the proteins were transferred to solid supports after separation and solid phase carrier absorb protein with non covalent interaction, which maintains the invariant properties of antigen protein or polypeptide onto membrane. Solid carrier protein or polypeptide behave as antigen and react with its corresponding antibody and enzyme labeled antibody to detect the expression of specific proteins through chromogenic substrate.
Synergistic effect of PBLs with HSP70
So far, we have demonstrated that transfection of PBLs into tumor-bearing mice significantly inhibited the growth of tumor. Furthermore, the growth was further inhibited when PBLs and HSP70 were simultaneously transfected. After inoculation for two weeks, of the average weight of tumor was only 0.42 g, significantly lower than that of PBLs transfected mice (0.84 g) or HSP70 transfected mice (1.57 g) and normal saline group (1.61 g). The experimental group treated with combined PBLs and HSP70 has significant difference (P < 0.05), suggesting that PBLs and HSP70 have stronger synergistic antitumor effect.