A Preliminary Study Of The Establishment Of The The Red Fluorescence Orthotopic Model Of Bladder Carcinoma In Nude Mice And Instillation Therapy With Mitomycin C

Background:bladder carcinoma is the most commonly seen malignant tumor of urinary system, whose pathogenesis has not been completely determined. In order to cure bladder carcinoma and discover therapy medicine, it is necessary to establish a ideal animal model. Because orthotopic transplantation model can simulate the development of human bladder carcinoma on animals, nowadays, most researchers are focusing on orthotopic model among other various models. There are a great many ways to establish a orthotopic model of bladder carcinoma. Usually the model is established by infusion of tumor cells after breaking the bladder mucosa in different ways or by planting the tumor cells directly to the bladder walls after laparotomy. Amongst the above two methods, the latter one is advantageous in that the tumorigenesis rate is higher while taking less time, which has been widely adopted. However, it has disadvantages such as causing greater damage to the animals and causing infections more frequently. It has been a hot study topic to establish a model more easily and with less damage. Cutting the bladder mucosa via urethra using our self-made tool is more efficient, less harmful to animals and more selective.Conventionally, animals with tumors are to be killed at a few particular time points over a period of time to be observed. As the high-speed development of molecular imaging, non-invasive observation has become possible when conducting animal models. In particular, the in-vivo imaging system, which is not dependant on radioactive agent and has high sensitivity, has promoted the development of animal models. So far, the fluorescence models of orthotopic tumors of a variety of tumors such as bladder carcinoma, prostate carcinoma, breast cancer, lung cancer, colon cancer and ovarian cancer have been established based on green fluorescence protein as reporting gene. However, at present, it is not commonly seen that tumor models are established using red fluorescence protein. We use lentivirus vector containing red fluorescence to mark EJ cells of human bladder cancer, and use these cells to build up orthotopic bladder cancer model, and then observe the growth process of tumors non-invasively using Roper in-vivo imaging system.Treating bladder cancer with only surgical resection can lead to a high recurrence rate. A general way is bladder instillation which combines surgical resection and the use of anti-cancer drugs. Mitomycin C (MMC) is one of the chemotherapeutic drugs which is clinically used after bladder cancer surgery. The function principle of MMC is that it can make DNA of bladder cancer cells disaggregate and prevent DNA replication in order to suppress the division of tumor cells to fight the tumors. After we successfully establish the model of the transplantation tumor of bladder carcinoma, we use MMC to perform instillation therapy on nude mice. Medication treatment group and comparison group observe the curative effect of MMC instillation by statistic analysis.Part One Lentivirus mediated red fluorescence protein reporter gene labeling EJ cells of human bladder cancerObjective:we use lentivirus mediated red fluorescence protein to label EJ cells of bladder cancer to explore whether there will any significant change in cytobiological characteristic of EJ cells before and after they are labeled and whether red fluorescence protein has a lasting and stable expression, preparing for the experiment in part two where we establish the model of the red fluorescent transplantation tumor of bladder carcinoma in nude mice.Methodology:EJ cells of human bladder cancer are conventionally cultured, passage, and divided into two groups. At logarithmic phase in their best condition, we perform RFP lentivirus infection with different multiplicity of infection of virus. The first group is infected by adding virus fluid directly into complete medium. The second group is infected when polybrene is added to and observed under fluorescence microscope on a daily basis. And then RFP fluorescence cell counting is conducted to calculate the infection rate under different MOI. We continue to culture and passage cells until the third generation after we attain EJ cells with lasting RFP positive expression. Growth curve is drawn by continuous cell counting for eight days. The ability of migration of cells is tested by wound healing test.Results:EJ cells are successfully infected by RFP lentivirus, can demonstrate quite strong fluorescence under fluorescence microscope and has stable expression and passage in vitro. Cryopreservation after two months, transfection efficiency shows no obvious change. The growth curve and wound healing test show that, after transfection, there is no significant change in the EJ cells’speed of multiplication and EJ cells maintain good migration ability.Conclusion:EJ cells labeled by RFP lentivirus have long-standing and stable in vitro expression. Their characteristics do not change obviously after being labeled. Thus they are available for establishing models in the next part. Part two:The establishment of the model of the red fluorescent transplantation tumor of bladder carcinoma in nude mice and instillation therapy with Mitomycin CObjective:Using EJ cells of human bladder cancer as experimental cells, adopting the methods of tumor cell seeding from abdomen under bladder wall and the methods of cutting bladder mucosa from urethra with self-made negative-pressure bladder mucosa cutter, to establish the model of transplantation tumor of bladder cancer in nude mice; using EJ cells of human bladder cancer labeled by red fluorescent protein as experimental cells, adopting the methods of tumor cell seeding from abdomen under bladder wall to establish the model of transplantation tumor of bladder cancer in nude mice and then perform instillation therapy with Mitomycin C to observe and analyze curative effect.Methodology:1. We use injection of cell mass suspension under bladder wall to construct the model of bladder cancer in nude mice and confirm the occurrence of bladder cancer through pathological section.2. We adopt the methods of cutting bladder mucosa with self-made negative-pressure bladder mucosa cutter, confirm the damage to bladder mucosa by pathological section and construct the model of bladder cancer in nude mice conduct instillation therapy with EJ cells of human bladder cancer. Pathological section can confirm the occurrence of bladder cancer.3. We carefully choose60female dude mice, aged6weeks, weighed18g±1.5g, randomly divided into three groups, namely blank group, comparison group and medication treated group with20mice in each group. Blank group is treated with instillation therapy with normal saline while comparison group and medication treated group are treated with injection of cell mass suspension under bladder wall to construct the model of bladder cancer and nude mice are observed under via in-vivo imaging system. After four weeks, the medication treated group is treated with instillation therapy with Mitomycin C for four times, once a week, one hour each time while the comparison group is treated with instillation therapy with normal saline. At the end of week four and week eight, we randomly choose two mice from comparison group and medication treated group to execute and perform pathological bladder examination. All mice are executed at the first day of the ninth week. We use the ratio of the absolute weight of nude mice’s bladders to nude mice’s weight as parameter of the growth of bladder cancer to perform statistic analysis.Results:1. The success ratio of establishing the model of subcutaneous red fluorescence in nude mice and the model of the red fluorescent transplantation of bladder carcinoma is100%when using the injection of cell mass suspension under bladder wall, and60%when using self-made bladder mucosa cutter. Pathological section can prove that self-made bladder mucosa cutter can damage bladder mucosa and can be used to construct the model of transplantation tumor of bladder carcinoma. Nude mice’s bladders emit red fluorescence when observed through in-vivo imaging system.2. By statistic analysis using the relative weight of bladders as growth parameter of bladder tumor, we prove that Mitomycin C instillation therapy is effective in curing bladder cancer in nude mice.Conclusion:The use of RFP/EJ to establish the model of the red fluorescent transplantation tumor of bladder carcinoma in nude mice and in-vivo imaging system to observe the growth of tumor is a innovative and convenient method which provide some new thoughts to the study of tumor model. The fact that Mitomycin C, the most clinically used medication in instillation therapy, is effective in curing bladder cancer in nude mice can be used as reference of clinical usage of Mitomycin C. In addition, self-made bladder mucosa cutter’s utilization can be more widespread in the future research of bladder cancer model as it is a new type of equipment that does minimum damage to bladder mucosa of nude mice.