大鼠C6脑胶质瘤模型建立及MR灌注成像研究
摘要
目的
肿瘤的微血管密度与肿瘤的分级和预后相关，肿瘤血管生成状况可作为评价肿瘤治疗疗效和预后的重要指标，研究肿瘤血管生成是目前肿瘤防治的新领域。如何在活体监测肿瘤血管生成状况(血管生成成像)便成了医学影像学面临的新课题。MR PWI是用来反映组织的微血管分布及血流灌注情况的一种检查技术，可以提供血液动力学方面的信息，因而引起了广泛的关注。脑胶质瘤是神经系统最常见的肿瘤，建立供医学影像学研究用的脑胶质瘤动物模型，必将促进脑胶质瘤的影像学基础研究进一步发展。本研究旨在建立大鼠C6脑胶质瘤模型，探讨其用于医学影像学研究的可行性，进而对其MR PWI的可行性及有关技术作一初步探讨。
材料和方法
研究的第一部分将16只SD大鼠随机分成两组，每组8只，用立体定向技术将C6细胞接种在大鼠的右侧尾状核，接种细胞量为1.0×10~6个(第一组)，1.5×10~6个(第二组)。观察大鼠术后的一般情况、体重变化、生存期和MR表现。测量肿瘤的最大径，计算肿瘤体积。记录肿瘤最大径达3mm时的瘤龄，并计算此时距大鼠死亡之间的时间(即相应的观察时间窗)。在大鼠濒死前或死亡后立即获取肿瘤标本，做病理学检查。
研究的第二部分将15只荷瘤大鼠随机分成三组，每组5只，对三组大鼠作MR PWI扫描，对比剂剂量分别为0.2mmol·kg~(-1)(第一组)、0.4mmol·kg~(-1)(第二组)和0.6mmol·kg~(-1)(第三组)。根据PWI时间-信号曲线计算rCBV、SRR_(max)、Q_(rCBV)和Q_(SRRmax)值。分析比较正常脑组织和肿瘤组织的rCBV和SRR_(max)值，以及三组大
安徽医科大学硕士学位论文
鼠的Q汇BV和QSRRm盼值。
所有数据采用 SPSS 10刀统计软件包进行正态性检验、方差分析和 t检验等处
理。
结果
在研究一中，两组大鼠皆成功接种肿瘤。肿瘤组织免疫组化 GFAP和 Sl
蛋白阳性，未见头皮下种植和肝肺等远处转移，1例腹水。荷瘤大鼠的早期症状包
括运动减少、攻击性减弱和精神萎靡等，晚期则有偏瘫、眶周出血、癫痈和恶液
质等表现，此时大鼠体重亦明显进行性减轻。MRI检查见肿瘤呈圆形或类圆形，
呈长TI低信号和长TZ高信号，大多呈环状强化。第一组大鼠平均生存期为23．50
士3．93 d，明显大于第h组 17．75士2．38 d（P＜0．of人肿瘤最大径呈直线关系随瘤
龄变化，濒死前肿瘤最大径为 9＊ 土二．36mm（第一组）和 9l 土0．96nun（第二组），
两组之间无显著性差异（P＞0刀5），但第二组的均一性较第一组好。肿瘤体积呈指
＠数关系随瘤龄变化，濒死前肿瘤体积为 515石6士303．33mm‘（第一组人 504．gi士
176．49ffiffi’（第二组人两组之间无显著性差异（p＞0．05＊第一组大鼠肿瘤最大
径达3mm时的瘤龄为门＊0土0．93 d，大于第二组8．88上1．25 d叩＜队01），相应的
观察时间窗第一组为12刀0土3．sld，大于第二组8．88上1．73d（Pwto刀5）。
在研究二中，三组大鼠皆成功进行了 MR PWI检查。三组大鼠肿瘤组织 rCBV
和 SRRm。值皆大于正常脑组织0＜0刀1人三组大鼠＊ 队分别为 1．77士0，12（第
一组L 2刀7土0．17（第二组）和1．36士0．10（第三组L三组之间有显著性差异0
＜0．m），第二组大于第一组（P＜O．m），第三组小于第一组（P＜0．m）。三组大鼠
QSRRm皿分另为 l．48土 0．25（第一组）、l．69土 0．18（第 l组）和 l二 18 i 0．06（第三组），
三组之间有显著性差异（P＜0．of），第三组小于第一组（P t 0刀5），然而第二组与
第一组之间无显著性差异p＞0刀5人
．
结论
本研究成功建立了大鼠C6脑胶质瘤模型，观察时间窗取决于接种细胞量，
3
安徽医科大学硕士学位论文
接种的细胞量越大观察时间窗越窄，根据具体的研究计划可作不同的选择。该模
型可用作 MR PWI 等医学影像学研究，MR PWI 研究中对比剂剂量以
0．Zmmolkg’－0．4tnmolkg’为宜。
Microvessel density in tumor is correlated with histologic grading and prognosis, while tumor angiogenesis is an important indicator for evaluating therapeutic effect and prognosis. Therefore angiogenesis investigation now becomes a new field in the prevention and therapy of tumors. Accordingly, to monitor tumor angiogenesis in vivo is a challenge to imaging medicine. At present, more attention is paid to MR PWI which can study distribution of microvessel and blood perfusion of tissue. Consequently, hemodynamics information is obtained by this way. However, there was little animal experimental study on imaging tumor angiogenesis. Certainly, to establish a technological platform for evaluating tumor angiogenesis in vivo, such as animal model of brain glioma, the most common tumor in central nervous system, will promote the basic research of imaging medicine and other clinical medicine. The purpose
of this study is to establish a rat C6 brain glioma model, to investigate its feasibility in imaging medicine research, especially in MR PWI research, and to initially study MR PWI technique of the model.
Materials and methods
In part one of the study, 16 rats were divided into 2 groups randomly, each group contained 8 rats. C6 cells were inoculated into the right caudate nucleus of the rats by stereotactic procedure. 1.0 X106 C6 cells were inoculated in group one, while 1.5 X 106 C6 cells in group two. On the following days, common condition, body weight, surviving time and MR features of the rats were observed. The maximum diameter of tumor was measured, and volume was calculated. Tumor age was recorded when the maximum diameter reached 3mm, and time window, the time from such tumor age to the death of rat, was counted. The specimens of tumors for pathological study were collected in the dying time or after the death of rats immediately.
In part two of the study, 15 rats with C6 brain glioma were divided into 3 groups randomly, each group contained 5 rats, then MR PWI scanning was performed. The dosage of contrast medium was 0.2mmol'kg-1 in group one, 0.4mmol kg-1 in group two and 0.6mmolkg~' in group three. According to time-signal curve of PWI, rCBV, SRRmax, QrCBv and QsRRmax were computed. The rCBV and SRRmax of normal brain were compared to those of tumor, and the QK;BV and QsRRmax of rats of 3 groups were analyzed.
In data analysis, SPSS 10.0 statistical package was employed to perform test of normality, ANOVA, and t test, et al.
Results
In part one of the study, C6 brain glioma was developed in all of the rats. The immunohistochemistry examination of tumor tissue showed the expression of GFAP and S-100 proteins. There was no implantation under scalp and no metastasis in lung
and liver, however, ascites was found in one rat. In earlier period, the rats manifestated as psychiatric depression, the power of attack weakened , and the activity decreased. In later period, the weight-loss of rats was obvious, while the symptoms includs hemiplegia, hemorrhage around orbits, epilepsy, dyscrasia, et al. On MR imaging, the tumors appeared as long TI , long Ta signal and annular enhancement with round shape or the similar. The surviving time of rats were 23.50 ± 3.93 d in group one, longer than 17.75 ± 2.38 d in group two (P<0.01). With increasing of tumor age, the maximum diameter of tumor enlarged in a linear manner. The maximum diameter in the dying time was 9.19 ± 2.36mm (group one) and 9.19±0.96mm (group two), there was no significant difference between the two groups (P>0.05), however, the homogeneity of maxmium diameters is better in group two than in group one. The volume of tumor enlarged in an exponential manner. The volume in the dying time was 515.66 ± 303.33mm3 (group one) and 5
04.91 ±176.49mm3 (group two), there was no significant difference between the two groups (P>0.05). The tumor age was 11.50 ± 0.93 d (group one) and 8.88 ± 1.25 d (group two) as tumor maximum diameter reached 3mm, the age in group one was older than that in group two (P<0.01). The time window to be used for obse
肿瘤的微血管密度与肿瘤的分级和预后相关，肿瘤血管生成状况可作为评价肿瘤治疗疗效和预后的重要指标，研究肿瘤血管生成是目前肿瘤防治的新领域。如何在活体监测肿瘤血管生成状况(血管生成成像)便成了医学影像学面临的新课题。MR PWI是用来反映组织的微血管分布及血流灌注情况的一种检查技术，可以提供血液动力学方面的信息，因而引起了广泛的关注。脑胶质瘤是神经系统最常见的肿瘤，建立供医学影像学研究用的脑胶质瘤动物模型，必将促进脑胶质瘤的影像学基础研究进一步发展。本研究旨在建立大鼠C6脑胶质瘤模型，探讨其用于医学影像学研究的可行性，进而对其MR PWI的可行性及有关技术作一初步探讨。
材料和方法
研究的第一部分将16只SD大鼠随机分成两组，每组8只，用立体定向技术将C6细胞接种在大鼠的右侧尾状核，接种细胞量为1.0×10~6个(第一组)，1.5×10~6个(第二组)。观察大鼠术后的一般情况、体重变化、生存期和MR表现。测量肿瘤的最大径，计算肿瘤体积。记录肿瘤最大径达3mm时的瘤龄，并计算此时距大鼠死亡之间的时间(即相应的观察时间窗)。在大鼠濒死前或死亡后立即获取肿瘤标本，做病理学检查。
研究的第二部分将15只荷瘤大鼠随机分成三组，每组5只，对三组大鼠作MR PWI扫描，对比剂剂量分别为0.2mmol·kg~(-1)(第一组)、0.4mmol·kg~(-1)(第二组)和0.6mmol·kg~(-1)(第三组)。根据PWI时间-信号曲线计算rCBV、SRR_(max)、Q_(rCBV)和Q_(SRRmax)值。分析比较正常脑组织和肿瘤组织的rCBV和SRR_(max)值，以及三组大
安徽医科大学硕士学位论文
鼠的Q汇BV和QSRRm盼值。
所有数据采用 SPSS 10刀统计软件包进行正态性检验、方差分析和 t检验等处
理。
结果
在研究一中，两组大鼠皆成功接种肿瘤。肿瘤组织免疫组化 GFAP和 Sl
蛋白阳性，未见头皮下种植和肝肺等远处转移，1例腹水。荷瘤大鼠的早期症状包
括运动减少、攻击性减弱和精神萎靡等，晚期则有偏瘫、眶周出血、癫痈和恶液
质等表现，此时大鼠体重亦明显进行性减轻。MRI检查见肿瘤呈圆形或类圆形，
呈长TI低信号和长TZ高信号，大多呈环状强化。第一组大鼠平均生存期为23．50
士3．93 d，明显大于第h组 17．75士2．38 d（P＜0．of人肿瘤最大径呈直线关系随瘤
龄变化，濒死前肿瘤最大径为 9＊ 土二．36mm（第一组）和 9l 土0．96nun（第二组），
两组之间无显著性差异（P＞0刀5），但第二组的均一性较第一组好。肿瘤体积呈指
＠数关系随瘤龄变化，濒死前肿瘤体积为 515石6士303．33mm‘（第一组人 504．gi士
176．49ffiffi’（第二组人两组之间无显著性差异（p＞0．05＊第一组大鼠肿瘤最大
径达3mm时的瘤龄为门＊0土0．93 d，大于第二组8．88上1．25 d叩＜队01），相应的
观察时间窗第一组为12刀0土3．sld，大于第二组8．88上1．73d（Pwto刀5）。
在研究二中，三组大鼠皆成功进行了 MR PWI检查。三组大鼠肿瘤组织 rCBV
和 SRRm。值皆大于正常脑组织0＜0刀1人三组大鼠＊ 队分别为 1．77士0，12（第
一组L 2刀7土0．17（第二组）和1．36士0．10（第三组L三组之间有显著性差异0
＜0．m），第二组大于第一组（P＜O．m），第三组小于第一组（P＜0．m）。三组大鼠
QSRRm皿分另为 l．48土 0．25（第一组）、l．69土 0．18（第 l组）和 l二 18 i 0．06（第三组），
三组之间有显著性差异（P＜0．of），第三组小于第一组（P t 0刀5），然而第二组与
第一组之间无显著性差异p＞0刀5人
．
结论
本研究成功建立了大鼠C6脑胶质瘤模型，观察时间窗取决于接种细胞量，
3
安徽医科大学硕士学位论文
接种的细胞量越大观察时间窗越窄，根据具体的研究计划可作不同的选择。该模
型可用作 MR PWI 等医学影像学研究，MR PWI 研究中对比剂剂量以
0．Zmmolkg’－0．4tnmolkg’为宜。
Microvessel density in tumor is correlated with histologic grading and prognosis, while tumor angiogenesis is an important indicator for evaluating therapeutic effect and prognosis. Therefore angiogenesis investigation now becomes a new field in the prevention and therapy of tumors. Accordingly, to monitor tumor angiogenesis in vivo is a challenge to imaging medicine. At present, more attention is paid to MR PWI which can study distribution of microvessel and blood perfusion of tissue. Consequently, hemodynamics information is obtained by this way. However, there was little animal experimental study on imaging tumor angiogenesis. Certainly, to establish a technological platform for evaluating tumor angiogenesis in vivo, such as animal model of brain glioma, the most common tumor in central nervous system, will promote the basic research of imaging medicine and other clinical medicine. The purpose
of this study is to establish a rat C6 brain glioma model, to investigate its feasibility in imaging medicine research, especially in MR PWI research, and to initially study MR PWI technique of the model.
Materials and methods
In part one of the study, 16 rats were divided into 2 groups randomly, each group contained 8 rats. C6 cells were inoculated into the right caudate nucleus of the rats by stereotactic procedure. 1.0 X106 C6 cells were inoculated in group one, while 1.5 X 106 C6 cells in group two. On the following days, common condition, body weight, surviving time and MR features of the rats were observed. The maximum diameter of tumor was measured, and volume was calculated. Tumor age was recorded when the maximum diameter reached 3mm, and time window, the time from such tumor age to the death of rat, was counted. The specimens of tumors for pathological study were collected in the dying time or after the death of rats immediately.
In part two of the study, 15 rats with C6 brain glioma were divided into 3 groups randomly, each group contained 5 rats, then MR PWI scanning was performed. The dosage of contrast medium was 0.2mmol'kg-1 in group one, 0.4mmol kg-1 in group two and 0.6mmolkg~' in group three. According to time-signal curve of PWI, rCBV, SRRmax, QrCBv and QsRRmax were computed. The rCBV and SRRmax of normal brain were compared to those of tumor, and the QK;BV and QsRRmax of rats of 3 groups were analyzed.
In data analysis, SPSS 10.0 statistical package was employed to perform test of normality, ANOVA, and t test, et al.
Results
In part one of the study, C6 brain glioma was developed in all of the rats. The immunohistochemistry examination of tumor tissue showed the expression of GFAP and S-100 proteins. There was no implantation under scalp and no metastasis in lung
and liver, however, ascites was found in one rat. In earlier period, the rats manifestated as psychiatric depression, the power of attack weakened , and the activity decreased. In later period, the weight-loss of rats was obvious, while the symptoms includs hemiplegia, hemorrhage around orbits, epilepsy, dyscrasia, et al. On MR imaging, the tumors appeared as long TI , long Ta signal and annular enhancement with round shape or the similar. The surviving time of rats were 23.50 ± 3.93 d in group one, longer than 17.75 ± 2.38 d in group two (P<0.01). With increasing of tumor age, the maximum diameter of tumor enlarged in a linear manner. The maximum diameter in the dying time was 9.19 ± 2.36mm (group one) and 9.19±0.96mm (group two), there was no significant difference between the two groups (P>0.05), however, the homogeneity of maxmium diameters is better in group two than in group one. The volume of tumor enlarged in an exponential manner. The volume in the dying time was 515.66 ± 303.33mm3 (group one) and 5
04.91 ±176.49mm3 (group two), there was no significant difference between the two groups (P>0.05). The tumor age was 11.50 ± 0.93 d (group one) and 8.88 ± 1.25 d (group two) as tumor maximum diameter reached 3mm, the age in group one was older than that in group two (P<0.01). The time window to be used for obse
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34．王建利，谢敬霞．对比增强磁共振脑血流灌注成像．临床放射学杂志，2000；19(3)：184～188．
35. Edelman RR, Mattle HP, Atkinson DJ, et al. Cerebral blood flow: Assessment with dynamic contrast-enhanced T_2~*-weighted MR imaging at 1.5T. Radiology, 1990; 176(1): 211～220.
36. Guckel F, Brix G, Rempp K, et al. Assessment of cerebral blood volume with dynamic susceptibility contrast enhanced gradient-echo imaging. J Comput Assist Tomogr, 1994; 18(3):344～351.
37. Kucharczyk J, Vexler ZS, Roberts T, et al. Echo-planar perfusion-sensitive MR imaging of acute cerebral ischemia. Radiology, 1993; 188(3): 711～717.
38. Tsuchiya K, Inaoka S, Mizutani Y, et al. Echo-planar perfusion MR of moyamoya disease. AJNR, 1998; 19(2): 211～216.
39. Bruening R, Kwong KK, Vevea MJ, et al. Echo-planar MR determination of relative cerebral blood volume in human brain tumors: T_1 versus T_2 weighting. AJNR, 1996; 17(5): 831～840.
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43. Villringer A, Rosen BR, Belliveau JW, et al. Dynamic imaging with lanthanide chelates in normal brain: Contrast due to magnetic susceptibility effects. MRM, 1988; 6(2): 164～174.
44．殷信道，冯晓源，符荣，等．超急性脑梗死及再灌注功能磁共振成像的实验模型制作．中国医学计算机成像杂志，2001；7(6)：367～369．
45. Rudin M, Sauter A. Noninvasive determination of regional cerebral blood flow in rats using dynamic imaging with Gd-DTPA. MRM, 1991; 22(1): 32～46.
46. Nighoghossian N, Berthezene Y, Philippon B, et al. Hemodynamic parameter assessment with dynamic susceptibility contrast magnetic resonance imaging in unilateral symptomatic internal carotid artery occlusion. Stroke, 1996; 27(3): 474～479.
47. Wong JC, Provenzale JM, Petrella JR. Perfusion MR imaging of brain neoplasms. AJR, 2000; 174(4): 1147～1157.
48. Rempp KA, Brix G, Wenz F, et al. Quantification of regional cerebral blood flow and volume with dynamic susceptibility contrast-enhanced MR imaging. Radiology, 1994; 193(3): 637～641.
49. Siegal T, Rubinstein R, Tzuk-shina T, et al. Utility of relative cerebral blood volume mapping derived from perfusion magnetic resonance imaging in the routine follow up of brain tumors. J Neurosurg, 1997; 86(1): 22～27.
50．余永强，郑斐群，柏亚，等．脑胶质瘤灌注成像与病理对照研究．中国医学计算机成像杂志，2001；7(3)：157～159．