Development Of A Novel TiO 2 High Performance Nanofiber Photosensitizer And Its Regulation On MPP8 Gene In HeLa Cells

Objective 1. To produce and determine the characteristics of the new type Ti O2 composite nanofibers. To evaluate their effects on the cervical cancer cells He La and investigate whether these new nanofibers might be possible candidate of photosensitizer for photodynamic therapy(PDT). 2. To find out the target genes MPP8 screened from the gene expression profile microarray of cervical cancer patients post-PDT biopsy and investigate the underlying mechanisms of the new type Ti O2 composite nanofibers in photodynamic therapy of cervical cancer. 3. To detect the difference of MPP8 levels in He La cells after the photodynamic treatment using the new nanofibers, further discuss the relative mechanisms.Methods 1. Different crystal forms of Ti O2 composite nanofibers were produced by calcination and acidity hydrothermal reaction, and analyze their characteristics by XRD, SEM and TEM. 2. Nanofibers as described above were administrated to He La cells, and the morphological changes were observed by fluorescence miscroscopy. MTT assay was used to detect the proliferation of He La cells with PDT; Flow cytometry was used to identify the effects of the nanofiber as the photosensitizer on the cell apoptosis. 3. M-phase phosphoprotein 8(MPP8) was chosen for the further experiments after screening the gene expression profile microarray of clinical cervical cancer patients with PDT biopsy. Difference of MPP8 expression in the tumor tissues between patient with PDT and without PDT was evaluated by immunohistochemistry and quantitative real-time PCR. 4. The stable He La cells with MPP8 knock down were constructed. The role of MPP8 in He La cells,proliferation and apoptosis was evaluated by MTT test, clone formation assay and flow cytometry5. MPP8 expression was determined by western blot and quantitative real-time PCR in He La cells after the PDT with the new type Ti O2 composite nanofiber. The underlying mechanisms were further discussed.Results 1. Four kinds of crystal forms of Ti O2 composite nanofiber were obtained by calcination and acidity hydrothermal reaction.By XRD, SEM and TEM, they were classified as: monocrystal Ti O2(B)( T(B)), monocrystal anatase(T(A)), mixed-phase anatase/Ti O2(B)(T(AB)_T), mixed-phase anatase/Ti O2(B)( T(AB)_ H). 2. The tendency of the nanofiber photosensitizer to gather in the He La cells after the coculture indicated a perfect biocompatibility. MMT assay revealed that four types of nanofiber incubation had no effect on the proliferation of the He La cells without the photodynamic therapy, however, inhibited the cell viability with photodynamic therepy, among which T(AB)_ H had the strongest effects. Consistent with MTT assay, flow cytometry revealed that four types of nanofibers induced the He La cells, apoptosis remarkably. 3. The gene expression profile microarray of cervical cancer patients with-PDT biopsy showed that MPP8 was higher in cancer tissue than in para-carcinoma tissue. This was further supported by the results of immunichemistry and real-time PCR confirmed that MPP8 levels were down-regulated in tumor tissue of cervical cancer patients with PDT compared to those without PDT. 4. Lentiviral vector was used to infect He La cells,among which more than 70% were infected successfully under the fluorescence microscope. The stable He La cells resistant to puromycin with MPP8 knock down were successfully established. Quantitative real-time PCR and western blots confirmed that MPP8 levels were about 65~70% lower compared to the control group. 5. The viability of He La cells with MPP8 knockdown was decreased significantly. The number of cell colony and cloning efficiency of the He La cells were decreased remarkably. Flow cytometry results showed that MPP8 knockdown resulted in the cell cycle retardant, indicating MPP8 decreased the proliferation ofHe La cells by slowing down their cell cycle. He La cells with MPP8 knockdown showed an elevated cell apoptosis, indicating that MPP8 might promote the proliferation of cervical cancer cells by inhibiting cell apoptosis.Conclusions 1. We produced the new type Ti O2 composite nanofiber photosensitizer which had the application potential and the preferable biocompatibility and could induced He La cells apoptosis in photodynamic effect. 2. We discovered that MPP8 might be a candidate for h the potential brand new cervical tumor marker and the target spot of PDT, and provide the new theoretical foundation of PDT mechanisms. 3. We built up the human cervical cancer cell lines-He La cells with the MPP8 gene knockdown which resulted in the He La cells cycle retardant and the apoptosis accelerating. Thus we concluded that MPP8 gene might play an important role in the malignant proliferation of cervical cancer.