【作者】 石键；

【导师】 赵洪洋；

【作者基本信息】 华中科技大学 ， 外科学， 2006， 博士

【摘要】 第一部分利用重组真核表达载体pCMV-p53转染wt p53基因及对大鼠C6胶质瘤细胞的热增敏效应目的研究C6胶质瘤细胞内稳定转入野生型p53基因（wild type p53，wt p53）的方法，并探讨C6细胞在加热后的生物学行为改变及wt p53基因对肿瘤细胞加热的影响。方法提取与鉴定真核表达载体pCMV-p53，利用红色荧光蛋白确定转染质粒和脂质体的最佳比例。将此即用型的wt p53真核表达质粒稳定转染至大鼠胶质瘤细胞系C6，通过neo基因表达的检测确定目的基因转移的成功性。设置C6／p53（+）细胞组、C6细胞加热组、C6／p53（+）细胞加热组、反复加热后生长的热耐受C6细胞组和对照组，然后对转染wt p53基因及加热培养后肿瘤细胞的生物学行为进行观察。结果wt p53基因片断经限制性核酸内切酶（HindⅢand EcoRⅠ）正确鉴定和提取，转染质粒和脂质体的最佳转染比例为1:6。neo基因稳定表达于转染了阳性质粒的C6细胞中。体外实验证实热耐受C6细胞的生长未受影响；将阳性质粒转染入的C6／p53（+）细胞组生长速度减慢；加热组在加热12h后细胞增殖明显受影响；而C6／p53（+）细胞加热组的细胞增殖活性从6h起受到显著抑制；在后三个实验组中都可以观察到细胞凋亡形态学改变，证实凋亡发生，流式细胞技术证实C6／p53（+）细胞加热组凋亡率增加最为明显，达对照组的30倍，与对照组相比差异有显著性（p＜0.01）。体内实验证实C6细胞加热组、C6／p53（+）细胞组、C6／p53（+）细胞加热组的裸鼠成瘤率均有下降，分别为75％、57％、20％，而热耐受C6细胞组为100％。结论目的基因可通过真核载体基因转移技术实现在C6细胞中稳定表达，并能在体内体外抑制大鼠C6胶质瘤细胞的生长，目的基因表达可增加肿瘤细胞对加热的敏感性。为进一步研究胶质瘤热疗及导入外源基因的热增敏效应提供了理论指导。第二部分立体定向脑胶质瘤模型建立、生长评估及激光间质热疗测温技术研究1、立体定向脑胶质瘤模型建立与生长评估目的建立SD大鼠C6脑胶质瘤动物模型，比较生长评估方法。方法采用调整的立体定向技术，将体外培养的大鼠C6胶质瘤细胞浓缩悬置，调制为浓度1×10¹¹L^-1的无血清DMEM培养液（每只大鼠注入20μl），将悬液接种于SD大鼠右侧尾状核区。接种后分时段观察与评估实验鼠的神经功能评分及肿瘤的生长特性及MRI检查；做组织病理学和胶质纤维酸性蛋白（GFAP）和S-100蛋白免疫组化检查。利用笔者发明的多功能便携式大鼠神经行为学试验装置进行旷场试验与爬坡试验，比较各种评估方法与实验天数及肿瘤直径间的相关性。结果优化的立体定向接种技术使本组大鼠脑胶质瘤动物模型具有颅内生长稳定，成瘤率高，未见颅外转移病灶，实验周期短，可重复性好，适于进一步的热疗光纤插入或给药，并在组织学上接近人类特征。神经功能评分与荷瘤鼠的脑胶质瘤大小及生长进程具有相关性。结论可成功建立SD大鼠脑尾状核C6胶质瘤模型，其肿瘤MRI影像及病理特征与人脑胶质瘤相似，神经功能评分可作为大鼠脑胶质瘤生长的间接评估方法。2、红外测温技术在大鼠胶质瘤激光热疗实验研究中的应用目的探讨红外热像测温技术应用于大鼠C6胶质瘤模型激光间质热疗（Laser Interstitial Thermotherapy，LITT）研究的可行性及其意义。方法将C6胶质瘤细胞悬液接种于SD大鼠右侧尾状核形成脑内胶质瘤模型，经MR扫描监测，校正肿瘤定位，按2-10W不同功率和热疗时间分组，插入半导体激光光纤进行间质热疗，同时使用ThermaCAM S65型红外热像仪测量肿瘤的中心靶点皮层温度和（或）热电偶仪间质测量靶区周边的深部温度，设立假手术对照组。观察记录靶区内温度在各组的改变，比较组间和组内的差异性。结果LITT各组靶区温度高于假手术组（P＜0.05）；在同一治疗组内中心靶点皮层温度和靶区周边的深部温度之间有符合性，差异无统计学意义（P＞0.05）。结论红外热像测温技术在大鼠实验性LITT研究中的应用具有可行性，有测温方便、易重复、无创、无污染、高效等优点，并可行后期软件处理，对热疗研究有较大意义，联合热电偶深部测温技术有更好的应用前景。第三部分C6胶质瘤LITT后超微形态学研究与wt p53基因对在体胶质瘤模型热疗的热增敏效应及机制1、大鼠C6脑内胶质瘤热疗的超微病理改变与血脑屏障开放性目的观察大鼠脑胶质瘤LITT后肿瘤中心毁损灶与肿瘤周边的超微病理改变，评估LITT后瘤周血脑屏障（Blood Brain Barrier,BBB）的开放性。方法造膜成功的SD大鼠经随机筛选，12只荷瘤大鼠入间质热疗组，应用半导体激光对脑胶质瘤模型实行立体定向问质热疗，健康SD大鼠24只随机分为甘露醇灌注组和假手术对照组。透射电镜观察LITT后不同时间的脑胶质瘤中心热疗毁损灶的超微结构改变，热疗对肿瘤周边的细胞、肿瘤血管及BBB的超微形态学的改变。结果胶质瘤中心接触激光光纤前端成为热疗毁损灶，以细胞坏死为主，肿瘤细胞膜结构损伤，线粒体、内质网等细胞器膨胀，线粒体嵴消失，胞浆稀疏等。形态学改变明显于对照组。热能传导至肿瘤周边，可见部分瘤细胞发生不同时期的凋亡改变，并可在电镜下见到毛细血管收缩、红细胞凝固、内皮细胞肿胀；血管基膜模糊断裂、周围间隙增宽、缝隙结构破坏。瘤周BBB开放的时程较甘露醇灌注组长（P＜0.05）。结论半导体激光热疗治疗脑胶质瘤毁损灶内可见明显肿瘤细胞坏死，损伤肿瘤细胞膜结构和线粒体，细胞器破坏，瘤周可见细胞凋亡，血脑屏障长时程地开放。2、半导体激光间质热疗C6细胞胶质瘤的作用及wt p53基因热增敏机制的研究目的评价LITT对脑内胶质瘤荷瘤SD大鼠与不同皮下荷瘤裸鼠的热疗效果，研究肿瘤组织p53基因的表达与热增敏效应的关系及其机制。方法造膜成功的SD大鼠随机分为A LITT组、B基因注射组、C基因加热疗组、D假手术对照组；随机筛选出成瘤的裸鼠接受LITT试验，定为E C6／p53（+）裸鼠热疗组、F C6裸鼠热疗组和G热耐受C6裸鼠组，另设H裸鼠假手术对照组。应用半导体激光对各组模型实行LITT，pCMV-p53质粒应用热敏脂质体转染法对B组和C组实行肿瘤灶局部注射，转移目的基因wt p53。TUNEL染色法检测肿瘤组织的凋亡。比较各组抑瘤效果，凋亡细胞计数和生长抑制曲线。RT-PCR比较各组瘤组织wt p53含量，免疫组化S-P法检测HSP70和Bax的表达。结果 C组的抑瘤效果最佳，其次为E组、A组、B组、F组、G组。疗效均显著优于对照组。LITT后的瘤组织TUNEL染色可见核固缩的阳性细胞，基因加热疗组的计数最高。RT-PCR电泳条带显示C6／p53（+）细胞荷瘤鼠瘤组织内p53基因表达量最高，热耐受C6瘤组织内最低。免疫组化染色证实P53高表达的瘤组织内伴有HSP70和Bax基因表达。结论 联合外源wt p53基因导入的LITT治疗有明显的增效效应，p53基因的热增敏效果与瘤组织wt p53含量有联系，可能与上调Bax基因的表达诱导瘤细胞凋亡有关，而热耐受C6瘤组织内HSP70基因高表达。wt p53基因辅助半导体激光LITT治疗脑胶质瘤是安全、有效、敏感的。更多还原

【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.更多还原