HPPH光动力学治疗大鼠C6脑胶质瘤肿瘤生长实验研究的磁共振表现

Magnetic Resonance Evaluation for C6Glioma Growth after the Photodynamic Therapy with 2-[1-Hexyloxyethyl]-2-Devinyl Pyropheophorbide-Alpha(HPPH)in the C6 Glioma Model of the Rat

目的:以不同剂量新型光敏剂HPPH光动力学疗法治疗大鼠C6脑胶质细胞瘤模型,通过磁共振平扫及增强扫描来测定各组肿瘤的大小,绘出生长曲线,选择最佳的治疗剂量并与传统HPD-PDT(血卟啉光动力学疗法)作对比,并以病理、电镜检查作验证。方法:建立大鼠C6脑胶质细胞瘤模型建立,设立对照组(肿瘤未治疗组、单注射HPPH 0.45mg/kg组、单注射HPD5mg/kg组、单波长667nm激光照射组、单波长630nm激光照射组),光动力学治疗组(HPPH-PDT各组(0.15、0.30、0.45mg/kg组)、HpD-PDT 5mg/kg组)。单注射HPPH 0.45mg/kg组、HPPH-PDT组和单注射HPD 5mg/kg组、HpD-PDT组自尾静脉推注入光敏剂,光动力学组注光敏剂18h后照激光。以波长667nm的半导体激光照射HPPH-PDT组和单667nm激光照射组肿瘤,功率密度200mW/cm2,每光斑照射20min,能量密度为240J/cm2;以波长630nm的半导体激光照射HpDPDT组及单630nm激光照射组肿瘤,功率密度200mW/cm2,每光斑照射20min,能量密度为240J/cm2。肉眼观察鼠行为变化,以磁共振平扫及增强扫描测量治疗前、治疗后第7、14天或对照组肿瘤生长第7、14、21天肿瘤体积,绘制肿瘤生长曲线,于PDT后14天或对照组肿瘤生长21天鼠脑,磁共振增强扫描测量后处死鼠,取脑组织作病理、电镜检查。结果:大鼠PDT治疗后,光动力学治疗组疗效较好的鼠,未出现异常表现,而对照组及光动力学治疗疗效较差组出现大鼠消瘦、萎靡或偏瘫、视盲。所有鼠均未出现皮肤红斑、水肿等光毒性反应。磁共振增强扫描测定肿瘤生长不同时间体积,并绘出肿瘤生长曲线。光动力学治疗前及治疗后14天肿瘤体积比(P14/P0),即肿瘤生长第21天与7天比(T21/T7),HPPH 0.15、0.30、0.45mg/kg-PDT组、HPD5mg/kg-PDT组值为4.43±4.80、0.37±0.25、0.71±0.42、8.31±1.56;对照组单注药HPPH 0.45mg/kg组、单注药HPD5mg/kg组、单激光667nm照射组、单激光630nm照射组、未治疗肿瘤组为17.01±0.36、16.66±0.31、18.37±0.47、17.66±0.04、20.24±1.75。T检验治疗组与对照组有显著及非常显著差别(P值<0.050、0.001),治疗组中HPPH各组疗效优于HPD5mg/kg-PDT组,T检验有显著差异(P值<0.050)HPPH-PDT组中HPPH 0.3mg/kg-PDT组、HPPH 0.45mg/kg-PDT组疗效接近,T检验无显著差别(P值>0.050),但明显优于HPPH0.15mg/kg-PDT组,T检验有显著差异,(P值<0.050)各对照组间无明显差异(P值>0.050)。病理检查正常脑组织仅见神经元细胞及胶质细胞;而接种C6胶质细胞瘤后大鼠脑可见大片密集成巢状生长,色深、大小不一、有核分裂的肿瘤细胞,随着肿瘤生长时间的增加,肿瘤细胞的密度加大。未光动力学治疗对照组的病理表现与上相同。光动力治疗后可见肿瘤细胞变性、色变淡、细胞密度减少,脑组织水肿及毛细血管扩张,程度与光敏剂HPPH的剂量有关,HPPH 0.15mg/kg-PDT较差一些、而HPPH 0.3mg/kg-PDT、HPPH 0.45mg/kg-PDT较明显、有的只有少量的肿瘤细胞,仅可见正常脑神经元细胞及胶质细胞,而HPD-PDT后的脑肿瘤细胞变性程度较HPPH-PDT更差一些,标本中仍可见较多的肿瘤细胞。电镜变化:大鼠正常脑可见神经元细胞及有髓鞘轴突及无鞘轴突。大鼠C6脑胶质细胞移植瘤未治疗对照组:均是活的肿瘤细胞,细胞体积大,细胞膜、核膜完整,核大,有核仁,有核切迹。大鼠C6脑胶质细胞移植瘤光动力学治疗组出现不同程度细胞凋亡及凋亡坏死的表现,核固缩,染色质边集的凋亡细胞,凋亡小体,细胞质空泡变性,线粒体空泡变性,细胞膜、核膜破裂。随着剂量增加,肿瘤细胞凋亡、坏死程度增加。HPPH 0.15mg/kg-PDT早期凋亡肿瘤细胞多见,有较多有活性肿瘤细胞;0.30mg/kg及0.45mg/kg HPPH-PDT组肿瘤凋亡及坏死程度增加。程度相似,但边缘脑组织有少许神经元固缩,程度0.45mg/kg HPPH-PDT组大于0.30mg/kg HPPH-PDT组。HPD-PDT组也是以早期凋亡肿瘤细胞多见,有较多有活性的肿瘤细胞,肿瘤的凋亡程度较HPPH 0.15mg/kg-PDT差。结论:磁共振增强扫描成像分析,能在无创情况下动态观察大鼠接种C6胶质瘤脑组织内肿瘤的生长情况,测量肿瘤肿块大小。HPPH-PDT能治疗大鼠脑C6胶质瘤,疗效优于HPD-PDT,HPPH 0.3mg/kg为HPPH-PDT最佳光敏剂剂量。

ObjectiveThe objective is to evaluate the efficacy of the photodynamic therapy （PDT） for the glioma treatment, usingdifferent doses of 2-[-1-hexyloxyethyl]-2-devinyl pyropheophorbide-alpha（HPPH）as a photosensitizer, and find an optimal doseof the HPPH by observing the growth curve of the glioma with magnetic resonance imaging （MRI）, which is then comparedwith traditional hematoporphyrin derivative（HPD）-PDT. Methods：After establishing rat C6 glioma model, the animals were di-vided into nine groups： untreated glioma group, HPPH （0.45 mg/kg） group, HPD （5 mg/kg）, 667 nm laser treated group,630 nm laser treated group, HPPH-PDT groups （0. 15, 0. 30, 0. 45 mg/kg, respectively）, and HPD-PDT（5 mg/kg）group. At18 h after photosensitizers were injected via rat tail vein, while the 667 nm laser at energy density of 240 J/cm2 with a power of200 mW/cm2was used to treat the glioma for 20 min each spot in the HPPH-PDT group and the 667 nm laser treated group, theHPD-PDT group and the 630 nm laser treated group were treated with 630 nm laser. The behaviors of the rat were observed,and the growth curve of the glioma was assessed with MRI before and 7 and 14 d after the treatment. The glioma was taken forpathological and electron microscope examination in 14d post-treatment. Results： After the PDT, some animals have no abnor-mal behaviors, others exhibit reduced body weight, apathy, paralysis, or blindness, but there was no phototoxicity. The rattumor size was measured with enhanced MRI. The ratios of the volume between in P14 animals and in P0 rats were 4.43±4.80,0.37±0. 25, 0. 7±0. 42, and 8. 31±1. 56 in HPPH 0. 15 mg/kg-PDT group, HPPH 0. 30 mg/kg-PDT group, HPPH0.45 mg/kg-PDT group, and HPD 5 mg/kg-PDT group, respectively, while 17.01=[=0. 36, 16.66=[=0.31, 18.37±0.47, 17.66±0.04, and 20. 244-1.75 were noted in the HPPH 0.45 mg/kg group, HPD 5 mg/kg group, 667 nm laser group, 630 lasergroup, and untreated glioma group, respectively. There was a significant difference between the treated group and untreatedgroup. The efficacy of the HPPH treatment is significantly greater than that in the HPD treatment, and the one of HPPH0. 30 mg/kg-PDT group is similar to that of HPPH 0. 45 mg/kg-PDT, which, however, is better than that in HPPH0.15 mg/kg-PDT. Pathological results.. Normal brain has neurons and glia cells, hut after implanted in the brain, the tumor cellsgrow in nest pattern and exhibit different sizes and nuclear division. The tumor cell density was increased with the tumorgrowth. After the PDT, degenerated cells, density-reduced tumor, edema, and enlarged capillary were observed, which is relat-ed to the HPPH dose. The pathological changes were more obvious in the HPPH 0. 30 mg/kg-PDT and HPPH 0.45 mg/kg-PDT groups than that in the HPPH 0.15 mg/kg-PDT group. The tumor cell degeneration was less serious post the HPD-PDTthan the one after the HPPH-PDT, and more tumor cells were noted in the former. Electron microscope results： The neuron andits axon were observed in the normal brain. The glioma cells show big volume, intact cell and nuclear membrane, visible nueleo-li, and nuclear notch in the untreated glioma group, but after the PDT, apoptosis and necrosis were observed in the tumor cellsincluding cell shrinkage, nuclear condensation, chromatin assembly, the formation of apoptotic bodies, the vacuolar degenera-tion of cytoplasm and mitochondria, the cell and nuclear membrane breakdown. In the HPPH 0. 15 mg/kg-PDT group, most ap-optotic cells were at the early stage of the apoptosis, and there were more tumor cells, yet more apoptotic cells and fewer numberof the tumor cells were noted in the HPPH 0. 30 mg/kg-PDT and the HPPH 0. 45 mg/kg-PDT groups, in which the latter was more ob-vious than the former. In the HPD-PDT, the apoptosis of the tumor cells was less serious than the one in HPPH 0. 15 mg/kg-PDTgroup. Conclusions： MRI is an efficient technique to observe the growth of the C6 glioma in rat C6 glioma model. Also, the HPPH-PDTis more efficient than the HPD-PDT. The optimal HPPH dose is 0.30 mg/kg for the HPPH-PDT.