Research On The Inhibitory Effect Of Brachytherapy With ¹²⁵i Seeds In Treatment Of Retinoblastoma

Background:Tumor is the disease that not only with abnormal proliferation and differentiation related, but is also associated with the abnormal cell death. Molecular biology of the tumor showed that the activation of oncogene and proto-oncogene and the tumor occurrence and development have a very close relationship, the activation and expression of such genes not only directly stimulate tumor growth, but also blocked tumor apoptosis process, while which are able to induce the apoptosis gene mutations, expressed low level, the interaction between them result the degree of apoptosis of tumor cells in low performance, relatively them over-value.Therefore reducing of the apoptosis plays an important role in the accumulation of tumor cells. Studies also have found that when apoptosis is inhibited, especially with cell cycle control exist, can promote malignant transformation of cells, and affect tumor cell response against the tumor treatment. Therefore, an effective way to treat cancer is to induce tumor cell apoptosis.In 1972, Kerr firstly proposed such concept of the biological ,which is"cell apoptosis ",the concept of apoptosis is also known as programmed cell death (PCD), which reflect a adaptive harmful reaction of the non-acute death of cells to stimulate the coordination ,and also is the death process.than an energy-dependent, subject to strict regulation and control a variety of mechanisms within cells of cell suicide .And different from the cell necrosis, that cells are not accompanied by the disappearance of inflammatory reaction occurs. In recent years, with the rapidly development of the molecular biology, the study of the apoptosis deeply, the apoptosis is in pathogenesis, diagnosis and treatment of the cancer that play an increasingly important role of the importance of life sciences.One of the mechanisms of tumor treatment is interfere with the DNA synthesis and cell division, thereby inducing apoptosis of tumor cells, to inhibit or remove the primary tumor. The sensitivity of the tumor to treatment, are often treated initially by apoptosis of tumor cells out of the degree of reaction. Therefore, the determination of apoptosis, has becomede to assesse the tumor efficacy as a new indicator, in addition, the apoptosis test is also a fast and effective way.to screening the cancer drug.At present,there are many methods to detect the apoptosis in vitro, including 1, morphological method, commonly used methods are observation of ordinary light microscopy, fluorescence microscopy observation and transmission electron microscopy observation; 2, agarose gel electrophoresis method, the classical method is extracted DNA of the apoptotic cells, agarose gel for electrophoresis, observed by UV lamp , showing a of 180 - 200bp size ladder band pattern; 3, flow cytometry, including Hoechst 33342 staining, Hoechst 33342/PI double staining method, etc.Through the instrument of the living tissue imaging of apoptosis cells was developed new technology this year, which is very useful of the detection for designing of cancer treatment and research, imaging principle of the apoptotic cells in vivo is apoptosis program in activated, apoptotic cells themselves produce a series of pathophysiological changes, in the process will produce many types of identifiable, specific chemical signal, using this feature, is now widely used radioisotope the introduction of specific compounds produced in vivo and with the combination of specificity, in vitro application of imaging instrument detected the distribution of radioactivity in vivo. As radionuclide imaging of apoptotic cells to meet the security, early, non-invasive conditions, has become the most extensive body of apoptosis detection. The 99mTc labeled Annexin V is a commonly class agent of imaging drugs used radionuclide imaging. Annexin V is an endogenous physiological protein, widely distributed in various organs and tissues of eukaryotes, 99mTc-Annexin V and Annexin V have the same physical characteristics; phosphatidylserine (PS) is a component of the cell membrane, Annexin V and PS have a high affinity, normally, PS exists only inside the cell membrane,the 99mTc-Annexin V can not enter the cell membrane to combine the PS and so can not imaging, but when cell apoptosis, PS occurs re-distribution, that is, the inner surface of plasma membrane PS flip, irreversible exposed, in the presence of calcium ions, 99mTc-Annexin V and PS combination of fast and close imaging.The experiment is based on the above principle and method, we should detect the cells which were irradiated by ¹²⁵I, and observe the state that ¹²⁵I induced retinoblastoma cell apoptosis; as well as, in vitro, using the radionuclide imaging instrument to detect the effect of ¹²⁵I seeds that theat the retinoblastoma transplantation tumor ,which in order to offer the experimental basis for clinical research that in the treatment of metastatic retinoblastoma.Objective:To research the state of apoptosis induced by 125 I irradiation on Rb cells and to explore the therapeutic effect of interstitial brachytherapy with ¹²⁵I seeds in treatment of retinoblastoma tumors in mice.Methods:Cell experiments:1. Experimental group: 0.1mci 0.5mci of ¹²⁵I irradiation of retinoblastoma cells, induced apoptosis.2. The control group: with final concentration of 20umol / L, 40umol / L of curcumin to observe the effect of curcumin on the induction of cell apoptosis.3. The results of Blank control group, experimental group and control group and experimental group + control group, that used of Hoechst 33258 staining, flow cytometry methods to detect ,comparing the results to observe the effect of apoptosis that inducted by ¹²⁵I and Curcumin , meanwhile observe the synergistic effect of Curcumin for ¹²⁵I Animal experiments:1.To establish the model ofretinoblastoma in NOD-SCID mice subcutaneously.2. The treatment group: implante ¹²⁵I seed into tumor.3. Control group :Treatment of non-intervention.4. After ¹²⁵I seeds irradiation 20 days ,the treatment group and control group by comparing the tumor volume, Pathological examination and the SPECT imaging to observe the effect of the treatment of retinoblastoma with ¹²⁵I seed brachytherapy.Results:Cell experiments:1. Hoech 33258 staining showed that curcumin group, ¹²⁵I irradiation group, and (curcumin +125 I) group can induce apoptosis of retinoblastoma; curcumin with final concentration 20umol / L has no synergistic effect for ¹²⁵I(0.1mci,0.3mci), curcumin with final concentration 40umol / L has synergistic effects for ¹²⁵I.2. Apoptosis rate by flow cytometry showed that ¹²⁵I and curcumin can induce apoptosis of retinoblastoma cells, curcumin with final concentration 40umol / L has synergistic effects for ¹²⁵I.Animal experiments:1. Compared with the control group, ¹²⁵I in the treatment group was significantly inhibited tumor growth, tumor inhibition rate of up to 56%.2. HE stain pathology showed particle irradiation by ¹²⁵I, appear a large number cells of degeneration , necrosis and apoptosis.3. SPECT imaging results show that, after seed implantation, due to appear a large number of apoptotic cells, the region of tumor can be seen clearly in the imaging Conclusions:1. ¹²⁵I irradiation can induce the apoptosis of Rb cells.2. The interstitial brachytherapy with ¹²⁵I seeds in human retinoblastoma (SO-RB50 cells) had significantly larger treatment.