【作者】 李明；

【导师】 张世忠；

【作者基本信息】 南方医科大学 ， 神经外科， 2011， 博士

【摘要】 研究背景氩氦冷冻治疗技术是近年源自美国氩氦冷冻治疗设备而兴起的一种低温冷冻技术。目前,此项治疗技术发展迅速,因为它操作简单,靶向性强,适应症广,加之治疗过程中靶区可以适时监测,对病人损伤小,消融效果确切。已经成为无法外科根治性切除的实体肿瘤患者治疗的最佳手段之一,极大的推动了临床肿瘤治疗学的进步,明显改善了肿瘤患者的治疗条件、生存状况和预后。随着近年来氩氦冷冻在全世界范围内的开展,它可对多种良恶性肿瘤施行精确冷冻治疗,尤其在肝癌、肺癌、胰腺癌、前列腺癌、肾肿瘤、乳腺癌等治疗领域已广泛应用于临床。资料显示冷冻治疗效果不仅在于超低温直接杀伤肿瘤细胞,更重要的是激活机体抗肿瘤免疫系统。在颅内胶质瘤治疗中,对于那些对化学治疗和放射治疗无效或不适宜手术治疗的病人,氩氦冷冻治疗为这些患者提供了一个有效的方法,但这一技术在颅内胶质瘤手术中的应用还不够成熟,临床上有少量的病例报道认为其对胶质瘤的疗效较好,但尚缺乏必要的基础研究和动物实验。本研究拟建立荷C6胶质瘤的Wistar大鼠动物模型,并进行氩氦冷冻治疗,观察肿瘤细胞病理、影像以及大鼠的免疫变化情况,为其临床进一步应用提供理论依据。第一章氩氦冷冻治疗荷脑胶质瘤大鼠影像和病理检测目的①：观察氩氦冷冻治疗荷脑胶质瘤鼠后胶质瘤细胞形态变化,其死亡方式是坏死或凋亡②：观察氩氦冷冻胶质瘤大鼠后磁共振影像学变化。方法①：C6脑胶质瘤细胞的冻存和复苏；种植C6胶质瘤细胞于Wistar大鼠鼠背；荷脑胶质瘤Wistar大鼠模型的建立；动物模型的分组(空白对照组、假手术组、手术切除组、氩氦冷冻治疗组)。②：氩氦冷冻治疗脑胶质瘤大鼠的形态和影像学变化,先行各种干预手段处理背部荷脑胶质瘤大鼠,分别在各种干预手段治疗前、术后7天时,观察荷脑胶质瘤大鼠的MRI变化；HE染色观察冷冻消融的肿瘤组织的形态；分别在干预治疗前、术后6小时、12小时、24小时Tunel染色观察氩氦冷冻凋亡细胞的形态并计数；游标卡尺测量并计算、分析比较各组肿瘤的体积变化。结果①：MRI检查显示各组在干预前肿瘤组织T1WI呈低信号、T2WI呈高信号。经冷冻治疗后,T1WI信号增强,T2WI信号减低。外科手术成功切除外科手术组鼠背的胶质瘤。肿瘤的体积分析显示空白对照组、假手术组肿瘤增大,氩氦冷冻的荷瘤大鼠体积减小②：HE染色示荷C6胶质瘤大鼠经冷冻治疗后,瘤细胞破裂,细胞核固缩,呈凝固性坏死改变。冷冻后Tunel染色显示凋亡细胞主要集中分布在冰球周围损伤区带,细胞出现了凋亡特有的形态学变化,表现为细胞核着棕黄色,核固缩,染色质靠核膜周边呈蚕豆或新月形浓染,冷冻后12个小时达到凋亡的高峰,平均在200倍的每个视野达到53.818±5.018。氩氦冷冻组与空白对照组比较有统计学意义(F=28.965,P=0.000),氩氦冷冻组与假手术组比较有统计学意义(F=28.376,P=0.000)。空白对照组及假手术组的肿瘤组织中少量凋亡细胞散在分布,两组之间无显著差异(F=0.590,P=0.568)。结论采用Wistar大鼠背部种植肿瘤细胞构建荷脑胶质瘤模型,该方法简单、经济、直视下接种准确,成瘤率高,种植肿瘤细胞2～3周,肿瘤体积便可达2.5-3.5cm3,背部便于消融。同时避免因为大鼠颅腔较小,消融后脑水肿造成动物死亡。因此,鼠背荷胶质瘤是氩氦冷冻治疗脑胶质瘤的理想动物模型之一。影像学提示氩氦冷冻可以造成肿瘤组织坏死。HE染色及Tunel染色显示氩氦冷冻治疗通过坏死和凋亡两种形式达到有效的消融。第二章氩氦冷冻治疗荷脑胶质瘤大鼠的免疫学检测目的评定氩氦冷冻治疗组并与空白对照组、假手术组、手术切除组比较荷脑胶质瘤大鼠的免疫状态的改变。方法①：C6脑胶质瘤细胞的冻存和复苏；种植C6胶质瘤细胞于Wistar大鼠鼠背；荷脑胶质瘤Wistar大鼠模型的建立；动物模型的分组(空白对照组、假手术组、手术切除组、氩氦冷冻治疗组)②：免疫组化检测干预后14天肿瘤组织内CD4+、CD8+的表达情况并计数,免疫荧光检测各组干预后14天F480的表达情况并计数.③：流式细胞仪测量各组大鼠在各种干预手段治疗前、干预后7天、干预后14天、干预后21天外周血中CD3+、CD4+、CD8+、CD14+、CD16+56阳性细胞的比例.④：流式细胞仪测量各组荷瘤大鼠在各种干预手段治疗前、干预后7天、干预后14天、干预后21天脾脏细胞中的IFN-γ及IL-4的含量,并计算出Th1/Th2细胞比例.⑤：酶联免疫吸附实验检测荷脑胶质瘤鼠在各种干预手段治疗前、干预后7天、干预后14天、干预后21天血清中IL-4、IL-10、IL-12、IFN-γ细胞因子的变化。结果①：免疫组化显示氩氦冷冻治疗组CD8+细胞染色、F480荧光染色氩氦冷冻组较空白对照组及假手术组明显密集(P<0.05),但是空白对照组与假手术组之间没有差异(P>0.05)②：流式细胞仪检测外周血的T细胞亚群显示：干预治疗前、干预治疗后7、14、21天,各组血清CD3+、CD4+、CD14+、CD16+56细胞水平：氩氦冷冻组>手术切除组>假手术组,氩氦冷冻组>手术切除组>空白对照组,但是空白对照组和假手术组之间没有明显的统计学差异(P>0.05)。空白对照组、假手术组血清CD8+细胞水平随肿瘤负荷增加而不断增高,但是组内比较均无显著差异。手术治疗组随肿瘤负荷的降低,血清CD8+细胞水平有所上升,冷冻治疗组术后血清CD8+细胞水平无显著改善。但是综合分析各组在干预治疗前、干预治疗后,血清CD8+细胞相同,无显著差异(P>0.05)。③：流式细胞仪检测大鼠脾脏细胞中IFN-y和IL-4的含量,显示术后7天、14天、21天对照组和假手术组大鼠脾脏细胞中IFN-y差异没有统计学意义(P>0.05)。外科切除组的IFN-y在术后7、14、21天3个时间点均有明显升高,差异有统计学意义(F=3.442,P=0.044)。冷冻治疗组的IFN-y在术后7天、14天、21天3个时间点均有明显升高,差异有统计学意义(F=7.319,P=0.043)。四组脾脏细胞中IL-4水平术后随时间都有升高,但各组之间在同一时间点上没有差异(P>0.05)。综合分析：Th1/Th2比值向Th1转移,抗肿瘤免疫增强。④：酶联免疫吸附实验显示空白对照组,假手术组血清IL-4水平随肿瘤负荷增加而不断升高。手术治疗组随肿瘤负荷的降低,血清IL-4水平于术后7天明显下降,随后趋于稳定状态,术后14天、21天无明显变化。冷冻治疗组,血清IL-4水平于术后7天明显下降,随后趋于稳定状态,术后14天、21天无明显变化。各组之间比较,术后各时间点,手术治疗组、冷冻治疗组IL-4水平均较对照组明显降低(P<0.05)。酶联免疫吸附实验检测空白对照组、假手术组血清IL-10水平随肿瘤负荷增加略有升高,但各时间点比较无统计学差异；手术治疗组与术前比较,术后血清IL-10水平略有降低,但各时间点比较无统计学差异。冷冻治疗组术后血清IL-10水平略有降低,但各时间点比较无统计学差异。各组之间比较,术后7天、14天、21天,各组之间无统计学差异(P>0.05)。酶联免疫吸附实验检测空白对照组、假手术组血清IL-12水平随肿瘤负荷增加略有升高,但各时间点比较有统计学差异；手术治疗组与术前比较,术后血清IL-12水平略有降低,但各时间点比较无统计学差异。冷冻治疗组术后血清IL-12水平略有降低,但各时间点比较无统计学差异。组间比较显示术后7天各组之间无统计学差异(F=3.238,P=0.033)。术后14天各组之间有统计学差异(F=18.539,P=0.001),其中氩氦冷冻水平最低,达到6.646±0.758,术后21天,各组之间有统计学差异(F=19.235,P=0.000),其中氩氦冷冻组水平最低,达到6.318±1.073。酶联免疫吸附实验检测空白对照组、假手术组血清IFN-γ水平随肿瘤负荷增加而逐渐降低,手术治疗组随肿瘤负荷的降低,血清IFN-γ水平有所上升,至术后14天达到相对平衡状态,冷冻治疗组术后血清IFN-γ水平明显上升,于术后14天达到最高峰。干预治疗前各组血清IFN-γ相同,干预治疗7天、14天、21天血清IFN-γ水平：氩氦冷冻组>手术切除组>假手术组,氩氦冷冻组>手术切除组>空白对照组,各组之间比较有统计学差异(P<0.01),但是空白对照组和假手术组之间没有明显的统计学差异(F=0.443,P=0.582)。结论采用氩氦冷冻消融背部荷C6脑胶质瘤Wistar大鼠模型,运用免疫组化、流式细胞仪、酶联免疫吸附试验等多角度多手段显示可有效诱导、增强机体抗肿瘤的细胞免疫,但是这种抗肿瘤免疫术后第3周开始就有降低的趋势,因此此种抗肿瘤免疫可能只作用于机体一段时间,如何维持和促进氩氦冷冻免疫反应以及冷冻免疫的具体机制方面将是我们进一步要探讨的问题。但是本实验为氩氦冷冻技术治疗脑胶质瘤乃至中枢神经系统肿瘤术后提高免疫提供重要的参考数据,为氩氦冷冻的临床应用奠定基础。更多还原

【Abstract】 BackgroundArgon-helium cryoablation technology rise from the United States in recent years, At present, the technology developed rapidly, because it is simple, target specificly, broad indications, combined with timely treatment can be monitored in the target area, the small damage, exact ablation. That make this technology become one of the best means in treatment of solid tumors, greatly promoted the progress of clinical oncology therapeutics, significantly improved the condition of cancer patients, survival status and prognosis. With the recent advance out worldwide, this technique allows precise cryoablation of a wide variety of neoplasms, and has been used in the clinical treatment of cancers in liver cancer, lung cancer, pancreatic cancer, prostate cancer, kidney cancer, breast cancer. All these data suggest that argon-helium cryosurgery causes not only tumor tissue destructions but also enhancement of the host immune function through several possible mechanisms.The brain glioma patients that fail to respond to or are ineligible for chemotherapy or radiotherapy often present difficult clinical cases, for which argon-helium cryosurgery provide a valuable therapeutic option. Nevertheless, the application of this technique in the treatment of gliomas still remains preliminary, and reports of only a handful cases were available to demonstrate its good therapeutic effect in the management of gliomas without support by sufficient evidences from basic research and animal experiments. In this study, we performed argon-helium cryosurgeries in Wistar rat models bearing C6 gliomas, and observed the pathology of tumor cells、image and cellular immunity changes. we aimed to explore the mechanism of angon-helium cryosurgery in relation to the cellular immunity and provide experimental evidences for the clinical application of this technique in glioma treatment.Part I The MRI and pathology change after cryoablation on rat model bearing C6 gliomasObjective①In this study, we performed argon-helium cryosurgeries in Wistar rat models bearing C6 gliomas, and observed the postoperative tumor cell apoptosis or necrosis.②To observe the MRI change after cryoablation on wistar rat models bearing C6 gliomas.Materials and methods①we established Wistar rat models bearing subcutaneous C6 glioma and divided the rats into the normal control, sham-operated, surgical resection, and cryosurgery groups for corresponding treatments.②The postoperative changes in the findings by magnetic resonance imaging (MRI) and tumor cell morphology were observed, the cell apoptosis at the tumor foci was assessed with TUNEL assay after 6 hours、12 hours、24 hours, and analyze the tumor size by computing the volume.Results①MRI scanning displayed low signal intensity of the tumor tissues on T1-weighted images (T1WI) and high signal intensity on T2WI. After cryoablation, the tumors showed enhanced T1 signals and attenuated T2 signals, suggesting tumor necrosis following cryoablation. MRI demonstrated radical tumor resection in the surgical resection group without abnormal T1 or T2 signals in the tumor foci. In the cryosurgery group, the tumor volume tended to gradually decrease after the operation.②HE staining showed tumor cell rupture, nuclear condensation and coagulative necrosis of the C6 glioma following cryoablation. One week after the cryablation, obvious congestion and bleeding occurred with formation of granulation tissues on the margin of the ablated area. TUNEL assay reached the highest that was 53.818±5.018 under×200 visual field 12 hours after the cryoablation showed that the apoptotic cells occurred mostly around the ablated foci, presenting with typical apoptotic morphologies including yellowish brown nuclei, nuclear condensation and chromatin margination. It is significant difference in the cryosurgical group comparing with the normal control group (F=28.965,P=0.000), and significant difference in the cryosurgical group comparing with shamed-operationed group (F=28.376, P=0.000), In the normal control group and shamed-operationed group, only occasional apoptotic cells were found in the tumor foci, between which was no statistical significance (F=0.590,P=0.568)ConclusionsIn summary, Wistar rat models bearing C6 gliomas is simple、economical, accurate and inoculated under direct vision, into the tumor rate, growing 2 to 3 weeks of, tumor volume will reach 2.5-3.5cm3,Ablation on the rat back avoid the small rat cranial cavity which can easily result animal deaths caused by ablation of brain edema. Therefore, Rat bearing C6 glioma on back is an ideal animal glioma model is one. HE staining and Tunel staining show argon-helium cryoablation result to cell death by necrosis and apoptosis.PartⅡThe immunity change after cryoablation on rat model bearing C6 gliomasObjectiveTo evaluate the cellular immunity change after cryoablation on wistar rat models bearing C6 gliomas and provide experimental evidences for the clinical application of this technique in glioma treatment.Materials and methods①we established Wistar rat models bearing subcutaneous C6 glioma and divided the rats into the normal control, sham-operated, surgical resection, and cryosurgery groups for corresponding treatments.②The CD4+、CD8+、F480 positive cells were detected by immunohistochemistry after 14 days.③Flow cytometry was performed for analysis of the T lymphocyte subset、NK lymphocyte subset changes.④the ratio of Th1/Th2 were assessed with flow cytometry at 7 days、14 days、21 days following the cryosurgery.⑤Elisa was performed to analyse the serum IL-4、IL-10、IL-12、IFN-γat 7 days、14 days、21 days following the cryosurgery.Results①The CD4+、CD8+、F480 positive cells were were found to obviously increase on days 7、14 and 21 after the operation by immunohistochemistry after 14 days, showing significant differences from those in the other 2 groups (P<0.05). but, no significant differences between normal control and shamed operation group (P>0.05)②Compared with the preoperative levels, the percentages of CD3+、CD4+、CD14+、CD16+56 cells in the surgical resection group decreased significantly 7 days after the operation followed by gradual increase, but recovered the preoperative level till 14 days after the operation (P＜0.05). In the cryosurgery group, the CD3+、CD4+、CD14+、CD16+56 cells were found to obviously increase on days 7、14 and 21 after the operation, showing significant differences from those in the other 3 groups (P＜0.05). The CD3+、CD4+、CD14+、CD16+56 cells showed no significant differences between the normal control and sham-operated groups at 7、14 and 21 days after the operation. In all the 4 groups, CD8+cells increased with time after the operation, but no significant differences were found between the 4 groups at each of the time points for measurement (P＜0.001).③Compared with the preoperative levels, the percentages of IFN-y positive spleen cells in the surgical resection group decreased significantly 7 days after the operation followed by gradual increase, but recovered the preoperative level till 21 days after the operation (P<0.05). In the cryosurgery group, the IFN-y positive spleen cells by flow cytometer were found to obviously increase on days 7、14 and 21 after the operation, showing significant differences from those in the other 3 groups (P<0.05). The IFN-y positive cells showed no significant differences between the normal control and sham-operated groups at 7、14 and 21 days after the operation. n all the 4 groups, IL-4 positive cells increased with time after the operation, but no significant differences were found between the 4 groups at each of the time points for measurement.④In normal control group and shamed-operationed group, the serum IL-4 increased with time measured by enzyme-linked immunosorbent assay, however, the serum IL-4 decreased with time 7 days after the surgical resection, that come to a stable state, then no significant differences were found at each of the time points days 14 and days 21 for measurement (P>0.05). the serum IL-4 decreased with time 7 days after the cryosurgical treatment, that come to a stable state, then no significant differences were found at each of the time points days 14 and days 21 for measurement (P>0.05). and no significant differences were found at each of the time points days 14 and days 21 for measurement in the 4 groups (P>0.05). In normal control group and shamed-operationed group, the serum IL-10 increased with time measured by enzyme-linked immunosorbent assay, however, no significant differences were found at each of the time points days 14 and days 21 for measurement (P>0.05).the serum IL-10 decreased with time 7 days after the surgical resection, that come to a stable state, then no significant differences were found at each of the time points days 14 and days 21 for measurement (P>0.05). the serum IL-10 decreased with time 7 days after the cryosurgical treatment, that come to a stable state, then no significant differences were found at each of the time points days 14 and days 21 for measurement (P>0.05). and no significant differences were found at each of the time points days 14 and days 21 for measurement in the 4 groups (P>0.05).In normal control group and shamed-operationed group, the serum IL-12 increased with time measured by enzyme-linked immunosorbent assay, however, it is significant difference at each of the time points days 14 and days 21 for measurement (P>0.05).the serum IL-12 decreased with time 7 days after the surgical resection, however, no significant differences were found at each of the time points days 14 and days 21 for measurement (P>0.05). the serum IL-12 decreased with time 7 days after the cryosurgical treatment, that come to a stable state, then no significant differences were found at each of the time points days 14 and days 21 for measurement in the cryosurgical group(P>0.05). Multiple comparison showed no significant differences were found at each of the time points days 7 in the 4 groups(F=3.238, P=0.033), it is significant differences at the time points days 14 (F=18.539, P= 0.001) and days 21 for measurement in the 4 groups (F=19.235, P=0.000), the lowest is 6.318±1.073 in the cryosurgical group.In normal control group and shamed-operationed group, the serum IFN-y decreased with time measured by enzyme-linked immunosorbent assay, the serum IFN-y increased with time after the surgical resection, that come to a stable state after 14 days, the serum IFN-y increased with time after the cryosurgical treatment, the highest is at days 14 after cryosurgery. Multiple comparison showed no significant differences were found before operation, Cryosurgical group>Surgical resection group>shamed operation group, Cryosurgical group>Surgical resection group>Normal control group, it is significant differences at each time points days in the 4 groups. but, no significant differences were found in the normal control group and shamed-operationed group(F=0.443, P= 0.582). ConclusionsUsing argon-helium cryoablation Wistar rats bearing C6 glioma model, using immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assay, and other means can effectively induce, enhance the tumor immunity against tumor cells, But the anti-tumor immunity after 3 weeks have decreased, so that such anti-tumor immunity may only act on the body for some time, how to maintain and promote the immune response to argon-helium refrigeration and frozen the specific immune mechanism is our furthe issue. But this experiment provide important reference data in treatment of glioma patients and even the central nervous system tumors for argon-helium cryosurgery in clinical management of gliomas.更多还原