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Reinforced Thermosensitivity Effect after Transporting of pCMV-p53 Vector into C6 Cell and Laser Interstitial Thermotherapy for Rat C6 Glioma Models

Author: ShiJian
Tutor: ZhaoHongYang
School: Huazhong University of Science and Technology
Course: Surgery
Keywords: Hyperthermia Laser Gene therapy Glioma p53 Magnetic resonance Electron microscopy Apoptosis Animal models
CLC: R739.4
Type: PhD thesis
Year: 2006
Downloads: 133
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Abstract


First Chapter.Transfection wt p53 gene into C6 gliomal cell and Research the change of Biological Behavior of C6 Glioma Cell.Objective To study the method of stable transinfecting wild type p53 gene to C6 glioma cell line, and to study the biological behavior of C6 cell and the effect of wt p53 gene in tumor cells after hyperthermia. Method The recombinated eukaryon expressive vector—pCMV-p53 plasmids were extracted and identificated, then the best transfection concentration of plasmid to liposome was confirmed by application of red fluorescin. This utility eukaryotic plasmids were transmitted to C6 cells by stable transfection. The successful transfection was determined by the expression of neo gene. After stable transfection into C6 cells, the biological behaviors of the p53 transfected C6 cells which were named as C6/p53(+) cells, hyperthermia treated C6 cells, hyperthermia treated C6/p53(+) cells, thermotolerance C6 cells which had been heated repeatedly and control group cell were observed. Results The wt p53 gene segments were extracted and correctly identificated by restriction enzyme (Hind III and EcoR I). The best transfection concentration of plasmid to liposome was 1:6, neo gene expressed stablely in C6 cells transfected with positive and blank plasmid. Thermotolerance C6 cell growth was normal in vitro, growth of C6/p53(+) cells demonstrated inhibited, growth of hyperthermia treated C6 cells was decelerated obviously after 12th hour, cellproliferation activity of hyperthermia treated C6/p53(+) cells was suppressed significantly after 6th hour. Obvious apoptosis was observed in morphology in the last three experimentive groups. By flow cytometry, the apoptosis ratio of hyperthermia treated C6/p53(+) cells was most obviously raised and reached 30 times compared to control groups. Subsequent study in nude mouse model demonstrated lower succeeding rate in groups of C6/p53(+) cells (57%), hyperthermia treated C6 cells (75%) and hyperthermia treated C6/p53(+) cells (20%), compared with control group, the difference was significant(P<0.05). Conclusion p53 as targeting gene could stably realize its expression in C6 cells by eukaryon expressive vector transmiting and inhibit rat C6 glioma cells growth both in vivo and in vitro. Raised expression of wt p53 gene could reinforce the sensibitity of hyperthermia to glioma cell. This provides a theory basis to research laser interstistial themotherapy for intracranial glioma models and reinforced effect of exogenous gene in hyperthermia.Second Chapter.Establishment of C6 brain glioma models, evaluation of their growth and research of applying infrared thermograph to measure temperature in thermotherapyI Establishment of C6 brain glioma models and evaluation of their growth.Objective SD C6 brain glioma models were established with stereotactic technique and evaluated the growth of these models. Methods The C6 cells cultured in vitro were stereotaxically implanted into the right caudate nucleus of SD rat brain. The concentration of C6 cells was 1×1011L-1 free serum DMEM(the volume of injection 20μl for rats). The following step was to judge the neurological deficit scoring (NDS) on everyday from 1st day to 20th day. MRI scan and histopathology were used to evaluate the growth of implanted C6 rat glioma on different days. Tumor was confirmed with HE and staining of GFAP and S-100 immunohistochemistry. Correlation between evaluation methods and days or diameter of tumor was studied. Results Inoculated with optimized stereotactic technique, rat C6 gliomas resembled histopathological features of human glioma. This kind of model was a more reliant and reproducible one, with 100% yield of intracranial tumor as well as no extracranial growth extension. NDS correlate to the growth processes or diameter of tumor. Conclusion A rat C6 brain glioma model resembles histopathological features of human glioma, which can be used as a perfect model to study the etiology of glioma. NDS can be considered for an indirect evaluation method. II Applying infrared thermograph to measure temperature in thermotherapy research of glioma modelsObjective To study the significance of infrared thermograph measuring temperature in laser interstitial thermotherapy (LITT) research to C6 rat intracranial glioma models. Methods The C6 cell suspensions were implanted into the right caudate nucleus of SD rat brain, then intracranial glioma models were built. After MR scanning and correction of tumor location, the models were divided into groups according treating time and laser power from 2 to 10W. Semiconductor laser optical fibers were inserted in tumors for LITT, simultaneously cortex’s temperature conducted from center target was measured by ThermaCAM S65 type infrared thermograph, and (or) deep tissue’s temperature around target was measured by thermocouple. Fake operation control group was established. The changes of target tissue temperature in different groups were observed and recorded Results The target tissue temperature in every LITT groups was higher than fake operative group (P<0.05). The difference between cortex temperature conducted from center target and deep tissue temperature around target had no statistical significance (P>0.05). Conclusion There is a great significance for applying infrared thermograph technique to measure temperature in LITT research. It could measure temperature conveniently, sensitively, effectually, non invasive and the data could be treated by software. Combining thermocouple to measure deep tissue temperature, it would have a better effect.Third Chapter.Research ultramicro morphology of rat C6 intracranial gliomas post LITT by electron microscope, reinforced effect of wt p53 gene and its mechanism in vivo I. Observation Ultramicro Pathologic Structure of Rat C6 Intracranial Gliomas and Evaluation Changed Structure of Blood Brain Barrier in LITT.Objective To observe ultramicro pathologic change of rat glioma in the tumorcentral damaged tissue and tumor peripheral tissue after LITT, to evaluate the changedstructure of Blood Brain Barrier (BBB) in peripheral tissue. Methods 12 SD rats whichwere build intracranial glioma model successfully were randomly selected into LITT ratsgroup, semiconductor surgical laser was applied to prectice stereotastic LITT for gliomamodels; orther 24 nomal SD rats were randomly distributed as mannitol perfusion groupand fake operation group. The ultramicro structures in glioma central thermodamagedtissue were observed with transmission electro microscope at different periods. Inperipheral tissue, ultramicro morphologic changes of glioma cell, tumor vessel and BBBwere evaluated. Results The glioma center connected the tip of laser fibre and turn intothermodamage tissue. The main structure changes were tumor cytoclasis, damnification ofcell membrum, swelling of cell organelle such as mitochondrion, endoplasmic reticulum,disappearance of mitochondrion and sparseness of cytoplasm. Heat energy conducted totumor peripheral tissue, some cells occured apoptosis in different period. Within 5d afterLITT, contracted capillary vessel, oncreted red cell, swell endothelium cell, broken basemembrum, wide around clearance and destroyed aperture structure were observed. Theopening time of BBB in tumor peripheral tissue was longer than mannital perfusion group(P<0.05) . Conclusion Cytoclasis in tumor cemtral thermodamage tissue could beenovserved obviously by semiconductor laser treatment, cell membrance structures andchondriosome were damaged, cell organelles were destroyed, apoptosis could be found intumor peripheral tissue, BBB could be opened in a considerable period.II. Effect of interstitial hyperthermia for rat C6 intracranial gliomas by semiconductor laser and mechanism of reinforced thermosensitivity of wt p53 gene. Objective To evaluate thermotherapy effect in SD rats with different C6 intracranial glioma and nude mouse with different endermic transplanted glioma. To evaluate the relation between p53 gene expression and effect of thermotherapy. To research the mechanism of reinforced thermosensitivity. Methods 38 SD rats were treated as intracranial models and they were randomly distributed group A: LITT, group B: gene injection, group C: combined treatment of gene and hyperthermia, group D: fake operation control rats. Randomly select nude mouse with transplanted glioma to accepte LITT, named as group E: thermotreated C6/p53(+) nude mouse, group F: thermotreated C6 nude mouse, group G: thermotolerance C6 nude mouse and group H: fake operation control nude mouse. LITT was practiced in glioma models by application of semiconductor laser, pCMV-p53 plasmids were injected into local tumor of group B and group C with temperature sensitive liposome, to transfer objective gene. TUNEL pigmentations were used to check apoptosis in tumor tissue. Tumor suppressive effects, accout of apoptosis cell and growth curves were compared between different groups, wt p53 content in different groups were compared by RT-PCR, expression of HSP90 and Bcl-2 were checked by immunohistochemistry (S-P Staining). Results The tumor suppresive effects in group E was the best, next were groups C, A, F, B, G. and effects in these groups were evidently better than control groups. Positive apoptosis cells whose nucleus were contracted could be found by TUNEL pigmentation in glioma tissue after LITT. Conclusion The reinforced thermosensitivity effect of p53 gene was relative to its content in glioma tissues, and its mechanism possibly associated with decreased expression of HSP90 and increased expression of Bax, wt p53 gene injection could combined semiconductor laser interstitial thermotherapy and treated glioma minimal invasively, effectively and sensitively.

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