黑色素瘤动物模型 z-bo 2020-12-02 444

　　Abstract: Uveal melanoma (UM) cell line, when exposed to blue light in vitro, showed a significant increase in proliferation ability. To determine whether a similar effect can be seen in vivo, we studied the effect of blue light irradiation on xenograft animal models. Methods: Twenty New Zealand white rabbits were injected with 1×106 human uveal melanoma (UM) cells into the suprachoroidal space of the right eye. The animals were equally divided into two groups. The experimental group was exposed to blue light, while the control group was free from blue light. After the animals died, the eyeballs were removed and the acid pink-B method was used to evaluate the proliferation rate of re-cultured tumor cells. The cells are re-cultured in order to maintain any changes in the cells in the body. In addition, the expression of proliferating cell nuclear antigen (PCNA) protein was used to determine the difference in cell proliferation between the formalin and paraffin-embedded (FFPE) eye fixation groups. Results: Statistically, blue light irradiation caused a significant increase in the proliferation of tumor cell lines in the eye (P<0.01). Compared with the control group, the expression of PCNA in the FFPE blue light treatment group was significantly higher. Conclusion: More and more data indicate that blue light exposure may affect the progression of melanoma. Our research results support this concept and warrant further research to evaluate the ability of blue light filters to slow the progression of melanoma disease.

　　 Introduction: Uveal melanoma (UM) is the most common ocular malignant tumor in adults. The incidence of UM ranges from 4.3 to 10.9 cases per million, depending on the specific criteria used to diagnose the disease. Although it is a relatively rare malignancy, approximately 50% of patients initially diagnosed with UM will eventually develop liver metastases within 10 to 15 years. The precursors of this disease include the presence of choroidal nevi, which often occurs in an aging population. As we age, the human lens becomes more and more yellow. This process is believed that the yellowed lens can effectively filter more blue light. After cataract surgery, removal of the aging lens is also accompanied by loss of natural blue light filtering ability. Further studies have shown that blue light exposure may play a role in the malignant transformation of melanocytes, which may eventually lead to the development of melanoma. It has previously been shown that rats exposed to blue light for a long time are pathologically diagnosed as intraocular melanoma.

　　Method: Animals Twenty female New Zealand white rabbits were randomly divided into two groups, the control group and the experimental group, with an average initial weight of 3.2±0.18 kg and 3.2±0.17 kg, respectively. To avoid conflicts when using female animals, fights may occur when male animals are kept in groups. Animals receive daily subcutaneous injection of cyclosporine A (CsA) for immunosuppression and avoid rejection of human cells. The experimental CsA was used for 8 full weeks to prevent tumor regression. In the past 4 weeks of experiments, CsA used the dosage schedule recommended in previous studies: 15 mg/kg/day 3 days before cell inoculation, and 4 weeks later, followed by 10 mg/kg/day. The dose of CsA is adjusted weekly according to the animal's weight loss.

　　Cell line and cell injection procedure: The injection process and subsequent animal handling were carried out as previously described. The 92.1 human uveal melanoma cell line was kindly suggested by Dr. Antonia from the research institute. The selection of this cell line is based on the previous research in our laboratory. This cell line shows high proliferation and invasive potential in vitro. The cells were cultured at 37°C and 5% CO2. The cells were cultured in RPMI-1640 medium supplemented with 5% heat-inactivated fetal bovine serum, 1% amphotericin B (Invitrogen) and 1% penicillin streptomycin. Suspended in the medium, according to the previously described technique, 0.1 ml of culture medium containing one million (cell viability greater than 99%) cells will be injected into the suprachoroidal space of each rabbit's right eye. Ketamine (35 mg/kg;) and xylazine (5 mg/kg) were used for anesthesia during the operation.

　　 Blue light irradiation: The 20 rabbits used in this experiment were randomly divided into two groups, 10 rabbits respectively. Rabbits in the experimental group were exposed to blue light for 8 hours a day for 8 weeks. The animals are placed in a large enclosure with blue light-emitting devices. The device is a large metal cage in which twenty-four 6600K bulbs are suspended, each covered by a sheet of polycarbonate film, allowing only the blue part of the spectrum to pass through. The device is placed in the middle of the cage, and the suspended bulb reaches the ground about 6" to obtain maximum light at eye level. In addition, the cage is lined with 3'high-reflective aluminum to ensure sufficient blue light in all areas of the cage. All The lights are connected to a timer, turned on at 11 am and turned off at 7 pm. During the blue light exposure period, protective goggles were provided to all persons entering the house area. The control group was placed in an adjacent cage, made of a polycarbonate The ester film covering ensures that the blue part of the visible spectrum is blocked from entering the control cage.

　　 Fundus examination: Dilation of pupils with tropinamide and fundus examination with indirect ophthalmoscope. Exclude any existing eye diseases before cell inoculation, and record clinical intraocular tumor development after weekly cell inoculation.

　　 Euthanasia: Two weeks after the inoculation of the cells into the eyes, one animal in each group was euthanized every week in order to record the time progression of the disease, especially the development of tumor metastasis. The selection criteria are based on the appearance of the animal, CsA toxicity and veterinary recommendations. The remaining rabbit groups (n = 4) were sacrificed at the end of the experiment. The method of euthanasia is through intramuscular injection of ketamine-xylazine anesthesia followed by cardiac puncture and bleeding (35mg/kg-5 mg/kg). An autopsy was performed on each animal that was executed. Enucleated eyes and other organs that may metastasize disease, such as collecting lung, liver and kidney specimens are stored in 10% neutral formalin buffer. Formalin-fixed, paraffin-embedded section specimens were subjected to histopathological evaluation and stained with hematoxylin and eosin.

　　 Reculture of cells after euthanasia: Before treatment, the right eye of each rabbit was fixed with formaldehyde to obtain a fresh tumor sample. The cells were cultured in RPMI containing 5% fetal bovine serum and overgrown the plate before inoculation in order to carry out proliferation experiments. During euthanasia, all blood collected from cardiac puncture is processed by the Ficoll-Paque® method in order to obtain a white blood cell layer. This was done for the entire duration of the experiment to capture and record the presence of tumor cells (CMCs). CMCs can adhere to the bottom of the six-well plate, and the remaining non-adherent white blood cells will be washed away. Before the start of the inoculation experiment, CMCs overgrown the plate. All re-cultured cells (primary tumor, CMCS) are passaged once in order to maintain the possible phenotypic changes of these cells in the body.

　　Immunohistochemistry: Use this standard fully automated instrument for immunohistochemistry. The fully automated barcode labeling section processing includes section baking, solvent-free deparaffinization, and CC1 (Tris/EDTA buffer pH 8) antigen retrieval. The sections were incubated with mouse anti-human monoclonal proliferating cell nuclear antigen (PCNA) antibody (1:200 dilution) at 37°C for 30 minutes. The second is the use of biotin-labeled secondary antibody (37°C, 8 minutes) and avidin/streptavidinase co-incubation (37°C, 8 minutes). Finally, use fast red chromogenic substrate and HE staining to detect antibodies. As a positive control, human small intestine and colon specimens were tested with PCNA antibodies. The main antibody of the negative control group was omitted. PCNA nuclear expression sections in tumor specimens and surrounding eye tissues were analyzed. A total of 10 rabbit xenografts were used for analysis. The overall tumor positive rate and intensity were also evaluated.

　　 Immunocytochemistry: The cultured cells are diluted to a concentration of 250,000 cells/ml, and a 300 μl solution of this concentration is evenly placed on each plate. The immunohistochemical staining of melanosomes with anti-human monoclonal antibodies on all sections mainly uses Ventana? automatic immunostaining machine, using the standard avidin-biotin complex method. Pathologists believe that HMB-45 is an effective labeling method for identifying the presence of uveal melanoma cells. These methods are to ensure that the re-cultured cells are uveal melanoma cells.

　　Result: Fundus examination At the time point of the first week, two rabbits in the control group and 3 rabbits in the experimental group were detected with intraocular masses. By 3 weeks, the total number of tumors visible in the control and experimental groups were 4 and 5, respectively. These numbers remain unchanged until the end of the experiment.

　　 Histopathology study In the 6 animals of the control group and 4 animals of the experimental group, the intraocular material can be detected macroscopically. Histopathological evaluation of the eyeball showed that seven of the control group developed tumors and 5 of the experimental group developed tumors. No macro-metastatic disease was found in any group. The four animals in the control group and the four animals in the experimental group showed the metastatic disease in serial sections of lungs. No liver metastasis was seen. There was no statistically significant difference between the two groups.

　　 Reculture of cells after euthanasia: A total of 5 primary tumors in the control group and 4 primary tumors in the experimental group were successfully re-cultured for subsequent screening analysis and proliferation experiments. In addition, 2 CMC cultures in the control group and 1 CMC culture in the experimental group were used for subsequent screening and proliferation test analysis.

　　Immunohistochemistry All the rabbit eyes of the paraffin control group showed PCNA negative (n = 5). In the paraffin blue light treatment group, 3 rabbit eyes were strongly positive (85-100%); 2 rabbit eyes were slightly positive after staining with PCNA (n = 5).

　　Immunocytochemistry: All re-cultured samples (primary tumor, CMCS) stained positive for monoclonal mouse anti-human melanocyte markers. This specific positive indicates that all re-cultured cells in the proliferation experiment do exist in the 92.1 line of human uveal melanoma cells originally inoculated into rabbit eyes.

　　 Conclusion: In summary, our current evidence shows that blue light irradiation can affect uveal melanoma cells, further confirming the results of previous in vitro studies. Our data showed that the proliferation of uveal melanoma cells increased significantly after blue light irradiation. These data require further investigation to evaluate the efficacy of blue light filtering intraocular lens in slowing down the progression of uveal melanoma.