Vaccine Generared By HMME Mediated Photodynamic Therapy In Murine Hepatoma MM45T-Li Cells

Photodynamic therapy (PDT) is a novel treatment for cancer and certain non-cancerous conditions that are generally characterized by overgrowth of unwanted or abnormal cells. PDT can not only derectly kill tumor cells, but also augment the host antitumor immune response. Recent studies have established that tumor cells treted by PDT can be used for potent antitumor vaccines. Our present study used murine hepatoma MM45T-Li cells as study model and investigate the antitumor efficiency of vaccine gerated by PDT, which may provide a new strategy for hepatocellular carcinoma.objective1. To investigate the killing effects of HMME-based photodynamic therapy on murine hepatoma MM45T-Li cells in vitro, so as to search appropriate parameters of PDT for preparing vaccines.2. To analyze the protective and therapeutic effects of vaccine generated by PDT on murie hepatocellular carcinoma in vivo.3. To study the effects of vaccine generated by PDT on antitumor immune response.Methods1. The uptake of HMME in cells was observed by fluorescent microscopy. Fluorescent intensity of HMME with various incubation time was assayed by multi microplates reader. Subcellular localization of HMME in cells was observed by confocal microscopy. The photodynamic therapy treatment on MM45T-Li cells was peformed by using HMME as photosensitizer and630nm laser as light source. The proliferation activity of cells was evaluated by MTT assay after being treated by photodynamic therapy. The effect of PDT on apoptosis was observed by Hoechst fluorescent staining analysis and AnnexinV-FITC/PI flow cytometry technology.2. MM45T-Li cells were treated by PDT with defferent light dose in the condition of power desity as15mw/cm2and after24h the lysate was used as vaccine. Normal saline (NS) was used as control and vaccine prepared by freezing and thrawing (F/T) was used as positive control. Hepatocellular carinoma animal model were prepared after immunization. The mouse was killed When tumor grew for16days. The weight and volume of tumor were mearured and compared. Pathological changes were observed by HE staining.3. Detect T cell subsets in blood by flow flow cytometry technology after immunization. Spleen lymphocytes were extracted by gradient density centrifugation. MTT assay was used to detect the specific cytotoxicity of spleen lymphocyte.4. Detect the vaccine therapeutic effects:the mouse model of hepatocellular carcinoma was established and then immunized. The observation period was60days, recording tumor volume and the tumor-bearing mouse survival time.Resuts1. The absorption of HMME in cells increased with the increase of concentration and the extension of incubating time Incubating time of4h and concentration of20μg/ml were selected as optimal parameters for PDT. HMME located in the mitochondria. MTT assay showed that the killing rate of cells raised with the increase of energy density and photosensitizer concentration. When using20μg/ml as HMME concentration and2.7J/cm2as light dose the killing rate was30.21%; when the light dose raised to7.2J/cm2the killing rate can reach90.65%. Chromatin condensation, nuclear pyknosis and karyorrhexis were observed after PDT by Hoechst fluorescent staining analysis. The apoptosis increased with the increase of energy density within the rang of0.9to4.5J/cm2. However when energy density raised to7.2J/cm2, the apoptosis decreased to4.1±2.7%and necrosis was the main cell death way.2. According to the killing effects of PDT,2.7and7.2J/cm2were selected as light dose for preparing vaccines. The results in vaccine protective role experiment demonstrated the tumor grew more slowly in2.7J/cm2PDT group. Compared with the control group, tumor volume and weight decreased obviously. In2.7J/cm PDT group and the inhibition rate has reached59.8%. While the vaccine generated by the dose of7.2J/cm2PDT had not showed significant deference comparing with control. Histopathology showed no difference in each group.3. Compared with the control,2.7J/cm PDT and7.2J/cm2PDT groups can improve the immune function of mouse, reverse T cell subsets CD4+/CD8+ratio in blood, increase NK cell ratios, inhance the spleen lymphocyte specific cytotoxicity.4. The results in vaccine therapeutic role experiment showed that2.7J/cm2PDT vaccine inhibited tumor growth in vivo. Tumors in2.7J/cm2PDT group grew slowly and even subside and the surval rate of tumor bearing mouse increased (p<0.05) when comparing with control group.Conclusion1. HMME mediated photodynamic therapy can be effective in killing MM45T-Li cells. The apoptosis induces by PDT closely related with light dose.2. Tumor cell vaccine preparaed by PDT significantly inhibited tumor growth, which has the protective and therapeutic effects on tumor bearing mouse. Vaccine preparaed by small dose of2.7J/cm2PDT has a better antitumor effect than that of a large dose of7.2J/cm2PDT3. Tumor cell vaccine preparaed by PDT in MM45T-Li cells can effectively enhance the antitumor immune response.