| Cancers are the first killer for threatening human healthes. Curing or cancelling tumors is a big problem on the world. While how to cure midbrain system disease and tumors in midbrain such as neuroblastoma and Parkinesons have become as the hottest projects to attack. It's very important to attack nerve system tumor for leveling up the quality of the sick and extend human life. The operations, chemo-treatment, treatment by Chinese herbs, gene therapy, immunization theropy are used on the patients. Though have some effects but the toxic side effect and expensive curing fees can't be bearable by the patients and their families. Some patients even give up treatments. To find no toxic side effects and effective new ways for curing cancer is most researchers'wishes. So to study biology factors Chinese herb Kanglaite injection (KLT) influence on the midbrain tissue. Then to study the mechanism of antitumor cooperation of Oncolytic virus New castle disease virus (NDV), KLT and secondary metabolite of Rhizoctonia leguminicola swaisonie (SW). Both researches are what our project want to do.This research we built S37 models,, H.E staining TH staining,NeuN staining and GFAP staining technical were used in our study for getting more antitumor mechanism informations from threemedicines. The results as follows:1. The midbrain cultures were successfully built from 14-day old mouse embryos. From 1-5 day the midbrain cultures were maintained in the basic medium and the cell bodies grew bigger and bigger. There are some short processes appeared. From 5-10 days, the midbrain culture medium was changed by N4 medium. Many long processes were sprayed out from the cell bodies and the second processes can be found. And the processes were formed very big network in the cultures. All these mean that midbrain tissue could be grown well outside body.2. It's the first time that KLT were used in the midbrain cultures to detect if there is some cytotoxixity on the midbrain cultures. The different dilutions of KLT were added. The results showed that the concentration of 0.001mg/ml could stimulate neurons growing faster and the number of the neurons increased 13.3%. While the number of the neurons in other KLT solutions had some little decrease. All means that higher concentrations of KLT do some harms to midbrain cultures. However the lower concentrations could well stimulate the growth of the neurons.3. The 200μM MPP+ were used to build Parkinsonismus mould. Different concentrations of KLT were used to do some treatments on the hurted dopaminergic neurons. The numbers of the dopaminergic neurons were counted and statistics and the morphology changes can be seen on the dopaminergic neurons. The concentration of 0.001mg/ml leveled up the number of the dopaminergic neurons 88% in contrast to the control. And the processes of the dopaminergic neurons became more longer and the neutrites possess more to the control. The results showed that KLT could be used to cure the Parkinsonismus.4. The N18TG2 neuroblastoma cells was the first time to grow in the midbrain culture medium N4 medium to detect if the N18TG2 can be growed in the N4 medium. The morphology changes and MTT assay showed that the N18TG2 can grow very well inside the N4 medium. Many big primary processes showed up and many second processes on the primary. The bodies of the N18TG2 become more bigger and bigger. MTT results suggested that the N18TG2 cells grew more quickly than the N18TG2 inside NBT medium in 12h,24h and 48h. Especially the number of N18TG2 cell in N4 medium is triple to the NBT medium. So the N18TG2 cells can grow in the midbrain medium.5. It's the first time that N18TG2 neuroblastoma cells were cocultured with midbrain tissue. The seeding densities of the N18TG2 cells are 2.5×104,5.0×104 and 105 cells/ml and were seeded in the midbrain culture. Take pictures at 12h,24h,36h and 48h separately. The numbers of the tumor cells were counted and statistics and the morphology changes could be found. The results showed that the N18TG2 could grow in the midbrain culture in all seeding densities but grew very slowly. Lower seeding densities N18TG2 cells can't do cytotoxicity on neurons in midbrain culture. While higher seeding densities made the neuron swell and the processes became shorter and the second processes disappeared. The number of the neurons was decreased.6. To detect the antitumor effects of KLT on N18TG2 neuroblastoma in N4 medium and NBT medium. The different concentrations of KLT treatments on N18TG2 neuroblastoma were added. The antitumor effect values were detected by MTT assay. The results showed that the KLT treatments have significant antitumor effects contrast to the control either in N4 medium or in NBT medium. Plenty of fragments and many apoptotic vesicles can be found in the culture suspension especially in the higher KLT concentrations. The results suggest that KLT treatments on N18TG2 neuroblastoma are dose dependent on the antitumor effects. And higher KLT solutions can make cancer cells depressed and lower KLT solutions induce apoptosis on N18TG2 neuroblastoma. The inhibition rate of 1.0mg/ml KLT is 48.7%.7. KLT was used in the coculture for studying the antitumor effects on the N18TG2 neuroblastoma cells. The different KLT solutions were added into the coculture separately for another 48h. The numbers of the tumor cells were counted and statistics and the morphology changes could be found. The results showed that 0.001mg/ml KLT solution possessed significant lowest tumor number in all the tests in different periods to the control. The inhibition rate is 36%. The morphology showed that the primary processes and second processes didn't been hurted and the networks made by processes were very clear. In other KLT solution treatment, the number of the tumor cells was decreased but not significantly to the control. Many round, no processes neuron could be found in other KLT solutions. These suggest that 0.001mg/ml KLT solution possess antitumor effect in the coculture of midbrain neurons and N18TG2 neuroblastoma.8.After different solutions of KLT treatments on the coculture of neuroblastoma cells and midbrain cells, the numbers of the dopaminergic neurons, all neurons and astrocytes were counted and statistics and the morphology changes could be found. The morphology showed that the primary processes and second processes didn't been hurted and the networks made by processes were very clear. The numbers of the dopaminergic neurons, all neurons and astrocytes were increased quickly and the bodies and the processes are very smooth. The decrease of the dopaminergic neurons, all neurons and astrocytes in the control was easy to find. Many apoptotic vesicle can be found in the bodies and the processes. KLT could level up the numbers of the dopaminergic neurons, all neurons and astrocytes and improve the lives of the neurons.9 After building the cancer models, 48 cancer models were divided into three groups. Those are control group, Kanglaite injection group and NDV+Kanglaite group. The results suggested that the tumors grew very slowly, the tumor cells were dissolved and karyopyknosis when time expand. Plenty of lymphocytes could be found in the depression area. All treatment groups markedly do effects on the cancer models. The best group is NDV+Kanglaite group, then KLT, last NDV. The antitumor mechanism of these three medcines may be that they could level up the functions of immune system and nervous system. Those can inhibit or weak the cancer cell growth.10. After building the cancer models, 48 cancer models were divided into three groups. Those are NDV group, SW group, NDV+Kanglaite group and control group. The results suggested that the tumors were necrosis, the tumor cells were dissolved and karyopyknosis when time expand. Plenty of lymphocytes could be found in the depression area. All treatment groups markedly do effects on the cancer models. The best group is SW+Kanglaite group, then NDV, last SW. The antitumor mechanism of these three medcines may be that they could level up the functions of immune system and nervous system. Those can inhibit or weak the cancer cell growth rate, make apoptosis or necrosis on tumor cells. So they have effects on the tumors. |
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