| In recent years, researchers all over the world are working to discover new malignant tumor treatment strategy. Pulsed electric field(PEF), as a new biomedical engineering technique in cancer therapy, is gradually paid great attention. According to the pulse duration, PEF can be classified into millisecond, microsecond, nanosecond and picosecond. At present,researchers focus on the millisecond, microsecond and nanosecond pulse range for a more in-depth study and has made a great progress. The electrochemical therapy, based on the reversible electroporation phenomena caused by microsecond PEF, is a new effective method for the treatment of malignant tumor after surgery, radiotherapy and chemotherapy.Intracellular electromanipulation, caused by nanosecond pulse electric field,also showed its prospect in the treatment of tumor. However, the application of millisecond, microsecond or nanosecond PEF requires the use of an invasive needle which limits the clinical application of this method.Picosecond pulsed electric field can be delivered non-invasively and precisely to deep tissue with impulse pulse radiation antenna. It is able toavoid damage to normal tissue at the time of killing tumor cells, so as to realize the noninvasive tumor treatment. Therefore, picosecond pulse electric field, matching impulse pulse radiation antenna, attracts people’s great interest for its excellent properties in the field of tumor biomedical treatment.New blood vessels play an important role on tumor growth, invasion and metastasis, so inhibiting tumor angiogenesis and blocking tumor nutrition supply are the main theoretical basis for tumor anti-angiogenesis therapy. Cemazar et al, found that microsecond pulse electric field can damage endothelial cells directly, lead to the suspension of blood flow and increase the toxic effect; Chen et al, found that nanosecond pulse electric field can reduce the expression of VEGF and MVD in tumors. However,the effect of ps PEF on cervical cancer angiogenesis is not clear.Cervical cancer is known as "good" cancer, for there is a longer "window period" from precancerous lesion to early malignant lesion which provides ps PEF treatment a very suitable opportunity. In this subject,through the animal tests, the effect and mechanism of ps PEF on angiogenesis of cervical cancer xenograft tumor were explored in depth. To some extent, it can improve the mechanism for picosecond pulse electric field used in tumor therapy.Part 1 Killing and anti-angiogenesis effects of picosecond pulsed electric fields on cervical cancer xenograft modelsChapter 1 Construct xenograft models in BALB/c nude miceObjective: To construct cervical cancer xenograft models in BALB/c nude mice.Methods: Tumor bearing mice were established by injection of 5×106He La cells in 200 μl PBS solution in the right lower limb. Tumors were allowed to grow to 1.0cm3 size then were harvested and cut into multiple1.5×1.5 mm3 pieces and immediately grafted subcutaneously to construct transplanted tumors. About 3 weeks the transplanted tumors reached1.0cm3 size.Results: The cervical cancer xenograft models were successfully constructed.Chapter 2 The killing effect of picosecond pulsed electric fields on cervical cancer xenograft modelsObjective: To observe the effect of picosecond pulse electric field on the cervical cancer xenograft tumor in vitro.Methods: The xenograft models were randomly divided into four groups(six per each group): control(no treatment), 50 k V/cm, 60 k V/cm and 70 k V/cm ps PEF treatment group. The following parameters were fixed:frequency(3Hz), duration(800ps), pulse numbers(2000), and exposure time(11 minutes). Following anesthetized by intraperitoneal(i.p.) injection of 10% chloral hydrate(4.5 ml/kg), tumors were subjected to field Part 1 Killing and anti-angiogenesis effects of picosecondpulsed electric fields on cervical cancer xenograft modelsstimulation protocol. One week after ps PEF treatment the direct effect of ps PEF on tumor tissue was observed by hematoxylin and eosin(H&E)staining and transmission electron microscopy(TEM).Results: Necrotic area of tumor tissue developed along with the necrosis degree of cancer cells aggravated with an increase in ps PEF intensity. In TEM sections, with the increase in ps PEF intensity, the mitochondria and endoplasmic reticulum of cancer cells showed swelling and expansion at different degrees while the links between cancer cells were gradually reduced. In the 70 k V/cm treatment group, the structure of cancer cells as well as vascular endothelial cells could hardly be identified.Conclusion: Ps PEF exhibited dramatic killing effect in cervical cancer xenograft models by exerting direct effect on cancer cells and vascular endothelial cells.Chapter 3 The anti-angiogenesis effect of picosecond pulsed electric fields on cervical cancer xenograft modelsObjective: To identify the effect of picosecond pulse electric field on the tumor angiogenesis in vivo and vitro.Methods: The same as the previous experiment grouping and field stimulation protocol. To explore the changes of blood vessels, PA tomography was performed in vivo before and seven days after ps PEF treatment after injection of 100 μl cardiogreen(5×10-5M) in the tail veinand immunohistochemical technique(IHC) was used in vitro to detect the microvessel density(MVD).Results: The PA signal amplitude and MVD value of the subcutaneously transplanted tumors kept decreasing along with an increasing in the ps PEF electric field intensity from control group to70 k V/cm treatment group. A statistically significant decrease was observed in 60 k V/cm and 70 k V/cm treatment groups compared to the control(p<0.01).Conclusion: Ps PEF exhibited a dramatic anti-angiogensis effect in cervical cancer xenograft models.Part 2 The anti-angiogenic mechanism of picosecond pulseelectric field on cervical cancer xenograft modelsObjective: To study the anti-angiogenic mechanism of picosecond pulse electric field on cervical cancer xenograft models.Methods: The same as the previous experiment grouping and field stimulation protocol. To explore the changes of micro-environment within tumors, PA tomography was performed in vivo to detect the oxygen saturation(s O2) of tumors before and seven days after ps PEF treatment.Seven days after ps PEF treatment, the animals were anesthetized by intraperitoneal injection of 10% chloral hydrate(4.5 ml/kg). The tumors from each group were excised and snap-frozen in liquid nitrogen forprotein and m RNA preparation. Vascular endothelial growth factor(VEGF)and hypoxia-inducible transcription factors 1α(HIF1α) were detected by IHC. Their protein expressions and gene transcription levels were evaluated using western blot(WB) and quantitative reverse transcription and polymerase chain reaction(RT-PCR).Results: Hypoxic area and the degree of hypoxia were increased gradually along with the increase of the ps PEF electric field intensity.Along with the increase of ps PEF electric field intensity, protein expression and gene levels of VEGF and HIF-1α were decreased. Statistical differences were identified in 60 k V/cm and 70 k V/cm treatment groups in comparison with the control group(p<0.05).Conclusion: Ps PEF induced severe and prolonged hypoxia in the cervical cancer xenograft models and down-regulated the protein and m RNA levels of VEGF and HIF1α at the same time. |
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