Experiment Study On Targeted Magnetic 5-Fluorouracil Nanocapsules Treating Implanted Tumor Of Human Cholangiocarcinoma In Nude Mice

Objectives:Previous studies of Shengquan Zou'group long-term gone in for the study of pathogenesis and targeted medicine of cholangiocarcinoma, and gained systemic and penetrating achievement, especially in molecular biology of cholangiocarcinoma. Based on those performance record, Prof. Zou winned the financial assistance of National Hi-Tech Study and Progress Project (863 Project)"Experiment Study on targeted Magnetic 5-Fluorouracil Nanocapsules Treating Cholangiocarcinoma"(No: 2002AA214061). The key contents include: to aim directly at intermediate and advanced stage cholangiocarcinoma, employ magnetic stent and magnetic 5-Fluorouracil nanocapsules built by hi-tech to targeted chemotherapy cholangiocarcinoma at the same time of Supporting drainage the bile duct, witch alter the traditionary method and carry out innovate and breakthrough in treating cholangiocarcinoma.This study aims at detect the effect that TM5-FuNC associating with magnetic bile duct stent treat cholangiocarcinoma in vitro and in vivo, especially to implanted tumor of human cholangiocarcinoma in nude mice, explore bio-distribution characteristic of TM5-FuNC, provide experimental data to support the concept that TM5-FuNC Hi-targeted treat cholangiocarcinoma based on the internal magnetic fields.Methods:The targeted effection and its probalble mechanisms of TM5-FuNC in treating cholangiocarcinoma were studied, concentrating on the experimental study on TM5-FuNC to treat cholangiocarcinoma based on internal magnetic field, and the role of study on targeting distribution of TM5-FuNC on nude mouse bearing Cholangiocarcinom by MRI, trying to refer an experimental basis for further study.Mtt, DNA ladder and flow cytometry were employed respectively to detect the inhibition phenomenon or apoptosis expression. Bax and Bcl-2 genes were mesured by reverse transcription-polymerase chain reaction respectively (RT-PCR).Established allotopic transplantation model of human cholangiocarcinoma in nude mice, which provid steady and reproducibility animal mode.,and then, evaluate the effect of TM5-FuNCtargeting to treat cholangiocarcinoma in different groups.High Performance Liquid Chromatography (HPLC) method for the determination of 5-FU in tissues was established and applied to determine 5-FU in mouse tissue samples. A Flame atomic absorption spectrometer (AAS) was used to detect the iron concentration in mouse tissues; MRI was used to detecet the distribution of targeting magnetic nanoparticles in vivo.SP immunohistochemistry was used to detect the expression of caspase-3, bcl-2/bax protein in 38 cases cholangiocarcinoma; Caspase-3, Bcl-2/Bax protein were detected by RT-PCR and Western-Blot allotopic transplantation tumor.Results:The effect of TM5-FuNC inhibited proliferation in human Cholangiocarcinoma QBC939 Cell line was depended on relation of time and dosage, which was more powerful than 5-Fu group. Cholangiocarcinoma QBC939 cells presented some typical feasures of apoptosis: nucler shrinkage,chromatin condensation and DNA fragmentation. agarose gel electrophoresis appeared marked DNA ladder. The apoptosis rate of QBC939 cells treated by TM5-FuNC was 23.77%, which was bigger than 5-Fu group. The level of bax mRNA was up-regulated, the level of bcl-2 mRNA was down-regulated.Tumorigenic rate of 40 nude mice were 100%; average tumorigenic period was one week; average life span of nude mice bearing cholangiocarcinoma was fifteen weeks. Tumor volume shows that the difference among 40 nude mice have no significant. The study of pathology and electron microscope were in accordience with the biological characteristics of human cholangiocarcinoma. In following experiment, the group of TM5-FuNC and magnetic stent, shows significantly therapeutic efficacy to TM5-FuNC and external magnetic fields.The concentration of both 5-FU and Fe in tumor tissues increased significantly in the group of TM5-FuNC plus magnetic field detected by HPLC and AAS respectively, and the signal intensity of tumor tissue is lower than it in control group detected by MRI.The level of Caspase-3 and Bax mRNA were up-regulated in experimental groups detected by RT-PCR and Western-Blot (P<0.05), but the level of bcl-2 mRNA had no change.ConclusionAll the above study could be sumed up as follows:1. TM5-FuNC can inhibit proliferation human Cholangiocarcinoma QBC939 Cells, induce apoptosis of human Cholangiocarcinoma QBC939 Cells, which apoptosis could relate to the up-regulating of bax gene.2. The animal model is established simply and well ; There is no obvious difference in tumor volume and histology ; Biological characteristics of cholangiocarcinoma is kept .this kind of allotopic transplantation model of human cholangiocarcinoma can be used to provid steady and reproducibility animal model for animal experiment.3. Magnetic drug targeting based on internal magnetic field can inhibit the growth of tumor tissue, and its therapeutic efficacy is better than the traditional method based on external magnetic fields.4. TM5-FuNC possesses targeting distribution when treating nude mouse bearing cholangiocarcinoma based on the inducing of magnetic field. MRI is the effective method to detected biologic distribution of TM5-FuNC iv vivo.5. To some extent, caspase-3, bcl-2 and bax protein can predict the prognosis of cholangiocarcinoma. Molecular targets of caspase-3, bcl-2 and bax may also be important factors to the pathogenesis of cholangiocardinomaThis part of medical experimental studys was based on the prophase work, including preparing successedly magnetic bile duct stent and TM5-FuNC, and corresponding toxicology, pharmacology. After those systematical and in-depth studys, some innovations can be concluded as follows:1. On the background of the era of DTS, utilizing new materials and nanotechnique, we haved prepared magnetic bile duct stent and TM5-FuNC possessed well biocompatibility, and developing systemtical studys in depth, supplying a new road for cholangiocarcianoma in mid-advanced stage. 2. Makeing use of the concept of internal magnetic fields to experimental research. The magnetic stent can not only play a part in support bile duct, but also provide internal magnetic fields in the same time. Which overcome the defect of external magnetic fields, such as, locating error, hard controling the time of operation and the strength of magnetic fields, hard aggregating in targeted tissue, et al.3. Exploying the new method by utilizing MRI techquine to detect biodistribution of TM5-FuNC in vivo in little animal model, which can provide a realtime and visualized medium to study the characteristic of TM5-FuNC in pharmacokinetics.Whatmore, we study preliminarily the potential molecule target point of TM5-FuNC acting cholangiocarcianoma, which provide fundament for further study in molecular biology.