冷冻联合rhTNF-α治疗脑胶质瘤的实验研究及临床应用研究
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摘要
概述
冷冻治疗肿瘤已有数十年历史。近年来,随着冷冻设备的更新和冷冻技术的完善,在准确定位肿瘤和最大限度杀灭肿瘤组织等方面取得了长足的进步。
冷冻的作用主要就是使局部快速降温、冻结、复温,在此过程中产生一系列的病理变化,最终导致细胞受损而死亡。由细胞内、外液中因冻结而分离出的纯水形成冰晶,并由此而产生的一系列变化是冷冻致组织损伤的重要机制。冷冻治疗过程中还存在冷冻速度的区别,因此致细胞损伤的作用机制可能也存在一些差别。目前关于冷冻致细胞死亡的机制主要存在以下几种学说：①迅速的低温使细胞内外的水分形成冰晶,细胞脱水,细胞内电解质浓度增高,使脂蛋白复合体受到破坏,引起细胞膜的溶解损伤从而致细胞死亡；②冷冻还可伤害线粒体中的光磷酸化作用,使三磷酸腺苷的合成发生不可逆性的裂解,造成细胞器膜的损害,而琥珀酸脱氢酶的活性有赖于线粒体膜的完整性,溶酶体膜的破坏可导致其中酶的释放,引起细胞的自溶,内质网、细胞核的膜均可在冷冻过程中发生破裂,而细胞可因细胞器的破坏而发生死亡；③冷冻过程中由于细胞内外冰晶形成,可造成细胞内的脱水状态,体液浓缩,细胞内电解质浓缩,浓度达到有害的程度,同时酸碱度也随之改变,出现细胞酸中毒和代谢障碍,从而导致胞浆、胞膜、细胞内成分蛋白产生变性,进而导致细胞死亡；④冷冻还可使细胞膜、蛋白等细胞成分中的结构水丢失,细胞因此失去功能而死亡；⑤温度休克学说认为温度的急剧变化较绝对温度对细胞的杀伤作用更重要,这可能是由于细胞内组成成分缩胀比率不均衡以致细胞破裂的结果；⑥组织冷冻后,血流速度减慢,这在小静脉表现的尤为明显,小静脉出现血流郁滞,这种血流郁滞乃至造成微循环的阻断,另外,冷冻还可直接损伤毛细血管壁,使血小板和其他血细胞与损伤的血管壁黏附,在血管内形成血栓栓塞。毛细血管腔出现阻塞后,则血流停止,组织细胞出现缺血性梗死。
冷冻后继发的组织细胞坏死还与冷冻后所引起的一系列生化反应有关。冷冻致细胞死亡的机制可能与细胞凋亡有关。凋亡是与坏死不同的另一种细胞死亡机制,即程序性死亡,它是一种耗能的主动的促细胞消亡的方式。我们前期研究已证实冷冻治疗可引起肿瘤中心区坏死、周边肿瘤细胞凋亡。而冷冻免疫是冷冻治疗的又一重要机制。全身冷冻免疫实验表明,冷冻HCA实体瘤,除原瘤生长受抑制外,还可能产生肿瘤特异性移植免疫以及抑制再植瘤生长。Bayjoo实验显示NK细胞的细胞毒性在冷冻后增强,临床也偶见原发灶经冷冻后转移灶自行退缩现象。Joosten等在冷冻治疗小鼠皮肤肿瘤(colon26-B)实验中发现除局部肿瘤坏死外,还可诱导全身抗肿瘤反应,血浆中IL-1和TNF-α水平较直接切除组高。由此可见冷冻本身能改变机体免疫状态,为联合TNF-α治疗提供了条件。
TNF-α的众多生物学作用中,其抗瘤作用倍受人们关注。大量实验研究和初步临床应用的资料证实TNF-α对某些肿瘤细胞具有细胞抑制和杀伤作用。TNF的抗癌作用主要是由于：①TNF为激发性细胞因子,可启动广泛的免疫炎性反应,介导天然细胞毒细胞、单核细胞和巨噬细胞.对癌细胞的杀伤和溶瘤作用；②TNF可作用于血管内皮细胞,使肿瘤的血管变形受损或形成血栓,导致肿瘤发生出血性坏死或因营养缺乏而消退；③TNF可通过诱导某些肿瘤细胞凋亡来抑制癌细胞增生。由于其毒副作用和剂量依赖性,TNF-a常与放疗,化疗(VP16,5-Fu,MMC,ADM等),热疗等联合用药,以提高其抗瘤作用,减少其用量和副作用。
脑胶质瘤约占脑肿瘤总数的40%-50%,平均生存期仅为51周,多采用手术切除、放疗、化疗等治疗手段,但复发率高,预后仍不理想。若将冷冻与免疫治疗联合应用于脑胶质瘤的治疗,其疗效如何,国内外鲜见报道。本实验采用冷冻联合免疫增强剂——TNF-α治疗脑胶质瘤,探讨其杀瘤机制,评价其疗效,为临床应用提供理论依据。本课题分三个部分：
第一部分：建立大鼠皮层C6脑胶质瘤模型,为脑胶质瘤的研究提供实验基础。借助于脑立体定位技术,在雄性Wistar大鼠脑S1区接种C6细胞悬液。随机分成4个周组和1个自然死亡组后观察大鼠的生存期,并采用MRI、病理解剖、HE染色及GFAP免疫组织化学方法,动态观察各周组肿瘤生长情况。以了解模型的稳定性。
第二部分：冷冻联合rhTNF-α治疗C6大鼠脑胶质瘤。本部分通过TUNEL法、流式细胞仪、免疫组化、MRI等手段观察大鼠C6脑胶质瘤经冷冻、rhTNF—α及冷冻联合rhTNF-α治疗后,脑胶质瘤细胞凋亡的数目、p21WAF1/CIP1和PCNA的表达、肿瘤体积、荷瘤鼠生存期、及凋亡、p21WAF1/CIP、PCNA三者之间的相互关系,以探讨它们杀伤肿瘤细胞的作用和机制,为冷冻联合rhTNF-α治疗脑胶质瘤的临床应用提供理论依据。第三部分冷冻切除联合rhTNF-α治疗脑胶质瘤的临床研究,对通过MRI初步判断为脑胶质瘤患者采用冷冻切除联合rhTNF-α治疗。观察rhTNF-α毒副作用,出院前及以后每6个月复查一次MRI。了解联合治疗可否降低复发率,延长病人的生存时间,能否推广应用。
目的：为脑胶质瘤的研究提供实验基础,建立稳定可靠的动物模型。方法：借助于脑立体定位技术,在25只雄性Wistar大鼠脑S1区接种含有10g/L琼脂糖的C6细胞悬液。随机分成4个周组和1个自然死亡组后观察大鼠的生存期,并采用MRI、病理解剖、HE染色及GFAP免疫组织化学方法,动态观察各周组肿瘤生长情况。结果：在不同周组中,所有检查均显示C6脑胶质瘤模型的建立成功,GFAP阳性。4周内无死亡,自然死亡组在第5周死亡2只、余3只于第6周死亡。结论：该方法建立的S1区脑胶质瘤模型具备与人脑胶质瘤生长相似的特性,肿瘤形成时间短,生存期稳定,且无脑外转移和颅外扩散,适合各种脑胶质瘤实验治疗研究。
目的本实验通过观察大鼠C6脑胶质瘤经冷冻、免疫(rhTNF-α)及冷冻联合免疫(rhTNF-α)治疗后,脑胶质瘤细胞凋亡的数目、p21WAFI/CIP1和PCNA的表达、肿瘤体积、荷瘤鼠生存期、及凋亡、p21WAF1/C1P1、PCNA三者之间的相互关系,以探讨它们杀伤肿瘤细胞的作用和机制,为冷冻联合免疫(rhTNF-α)疗法的临床应用提供理论依据。
方法本研究包括(1)C6脑胶质瘤细胞的培养,C6细胞在含有10%新生小牛血清的DMEM培养基中,5%二氧化碳、37℃条件下培养。(2)大鼠C6脑胶质瘤动物模型的建立,选纯种Wistar雄性大鼠120只,借助于立体定位技术、将10μl含有1×106个C6细胞悬液缓慢注射入大鼠右侧大脑S1区皮层。(3)动物分组及治疗的实施：待肿瘤生长至第14天、直径约6mm时,随机将荷瘤鼠分为对照、单冷、单免(rhTNF-α)及冻免(rhTNF-α)四组,每组30只,对不同的组别进行相应的治疗。于接种后第21天,每组随机抽取20只按不同检测要求取材,各组余10只用于测量肿瘤体积和观察生存期。(4)TUNEL法和流式细胞仪检测细胞凋亡。(5)免疫组化方法观察p21WAF1/CIP1及PCNA蛋白的表达。(6)MRI测肿瘤体积。(7)直接观察各组荷瘤鼠的生存期。
结果HE染色显示颅内肿瘤形成；GFAP阳性。联合治疗(冻免组)与其他三组比较：肿瘤细胞凋亡、p21WAF1/CIP1蛋白表达显著增多,PCNA蛋白表达显著减少,肿瘤体积缩小,生存期延长。凋亡细胞数与p21WAF1/CIP1蛋白表达呈正相关；与PCNA蛋白表达之间呈显著负相关；p21WAF1/CIP1蛋白表达也与PCNA蛋白表达之间呈显著负相关。
结论凋亡是冷冻、免疫(rhTNF—α)杀伤肿瘤细胞的一个重要机制；冷冻联合免疫(rhTNF—α)治疗可产生协同效应；p21WAF1/CIP1参与了凋亡的调控；PCNA参与DNA的合成,其下调反应细胞增殖受抑制,有利于凋亡的发生,其表达受p21WAF1/CIP1负调控。
目的探讨冷冻切除联合重组人肿瘤坏死因子(rhTNF-α)治疗脑胶质瘤的效果。
方法对于18例脑胶质瘤患者采用冷冻切除联合rhTNF-α治疗。观察rhTNF-α毒副作用,出院前及以后每6个月复查一次MRI评估治疗。结果肿瘤全切除17例,1例次全切除。术前9例有病例对侧肢力减退者,均在1周内恢复。rhTNF-α常见的毒副作用为发热,局部红肿及肌肉疼痛发生率为38.9%(7/18)。术后随访6-20个月,除1例次全切者术后15个月死亡外,余17例均幸存。
结论本结果提示冷冻切除联合rhTNF-α治疗脑胶质瘤可能成为胶质瘤治疗的一种新策略。
OVERVIEW
Gliomas originate from brain glial cells and account for approximately80%of malignant primary brain tumors. The prognosis for patients with gliomas is generally poor with an average survival time of51weeks. Treatment depends on the location of the tumor, the cell type, and the grade of malignancy and often involves a combination of surgery, radiation therapy, chemotherapy, and targeted molecular therapy.
Cryosurgery is a surgical technique established as rapid freezing, slow thawing and repetition of the freeze-thaw cycles, which has been used for the treatment of skin, oral cavity, liver, bone and prostate tumors. Besides the local destruction on tumor tissues caused by ice crystal formation in the rapid freezing period, the slow thawing period also causes microcirculatory failure. Furthermore, It has been shown that cryosurgery postulates inhibition of secondary tumor growth and metastases associated with increased plasma cytokine tumor necrosis factor-alpha (TNF-a)and enhances natural killer cytotoxicity in liver tumors. Cryosurgery began to treat brain tumors in the1960s. Despite advances of imaging technologies to facilitate this application, it still poses challenges. Insufficient freezing does not induce cell death resulting in tumor recurrences. Conversely, over-freezing may damage normal brain tissues causing serious complications. It has been shown that molecular adjuvant approaches could enhance cryosurgery by reducing local recurrence and complications
TNF-a is a multifunctional cytokine that plays a key role in apoptosis and cell survival. Although systemic TNF-α administration is dose-dependent and associated with severe toxicity, TNF-a has been demonstrated to have anti-tumor effects in animal and human. More importantly, systemic TNF-a administration augments the effect of cryosurgery in prostate cancer and breast cancer by increasing cryo-sensitivity. However, it is not clear whether TNF-a enhances the cryosurgery effects in brain tumors.
This study is designed to test the hypothesis that systemic TNF-a administration and cryosurgery play a synergistic effect on the treatment of brain tumors. For this purpose we developed a more reproducible rat brain cortex glioma model to facilitate cryosurgery delivery, compared with the deep brain tumor models developed at the caudate nucleus and brainstem. By using this model, we investigated survival time, tumor size, tumor cell proliferation, and apoptosis. Additionally, we also monitored brain gliomas tumor patients treated with cryosurgery and TNF-a. Part I:We developed a rat cortical C6glioma model to provide experimental basis for the study of brain gliomas. C6cells were implanted into the male Wistar rat brain SI District using a stereotaxic technology,
Part II:Rats were followed up for90days and MRI was used to monitor tumor size in time series. Pathological anatomy examinations were performed and tissues were collected at the end of studies. DNA fragmentation was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL assay); P21WAF1/CIP1and proliferating cell nuclear antigen (PCNA) expression levels were scored using immunohistochemical staining.
The study is composed with three part:
Part III:In order to assess combination therapy the possibility of lowering the recurrence rate and prolong survival time in clinical, we followed up brain gliomas patients treated with cryosurgery and TNF-. Patients were received MRI test before discharge and every6-month after discharge to monitor tumor size. Side effects of rhTNF-a were also monitored.
Objective To establish a stable and dependable animal model as a laboratory foundation for treatment research in human brain glioma. Methods By stereotaxic technique,25male Wistar rats were inoculated with c6cell suspension containing10g.L-1agarose in their brain S1area. After being randomized to4week groups and1natural death group the rats were observed of survival period and the examinations such as MRI, patholoanatomy, HE stain and GFAP immunohistochemistry were prformed on each week group rats to dynamically observe the tumor growth
Results All examinations showed intracranial tumors existing in different week groups. Expression of GFAP was positive. The death was not detected in4weeks. However, in natural death group, two rats died in No.5week and the others died in No.6week. Conclusions The S1area brain glioma model established by this method in rats had the same characters as human brain glioma. The tumor growth period was shirt and the subsisting period was stable without extracerebral metastasis and extracranial extension. This model is suitable for laboratory and treatment study of brain gliomas.
Objective This study was performed to detect the number of apoptosis cells, the expression of p21WAF1/CIP1and PCNA, the relationship among the three factors after rat C6glioma-model treated with cryotherapy or immunotherapy(rhTNF-α) or combination cryotherapy and immunotherapy(rhTNF-α). At the same time, the tumor volume and the survival period of rat with tumor were observed. Try to investigate their effect and mechanism of killing glioma cells and provide scientific evidence for clinical application.
Methods (1) C6glioma cells were cultured. C6glioma was cultured in DMEM containing10%new born bovine serum, under condition of5%carbon dioxide and37℃.(2) Rat C6brain glioma models were established. By stereotaxic technique,120male Wistar rats were respectively inoculated with suspension containing1×106C6cells in their right brain cortex S1area..(3) The division and the performance of treatment:The rats were randomly divided into the control group (n=30),the cryotherapy group (n=30),the immunotherapy(rhTNF-α) group (n=30) and the combination group (n=30) of the later two methods. When the tumor diameter were approximate6mm at14th day after inoculation, the corresponding treatment was performed according to the different groups.20rats of each group were randomly drawed to sacrifice for different examining purpose at21st day following inoculation. The other10rats of each group was to observe tumor volume and subsisting period.(4) Glioma cell apoptosis was observed with TUNEL and flow cytometry.(5) Immunohistochemical staining was used to analyze the expression of p21WAF1/CIP1and PCNA protein.(6) The tumor volume was measured by MRI.(7) The rat survival period of each group was directly observed.
Results MR and HE stain showed intracranial tumors existing.Expression of GFAP was positive. After comparing the combination group with the other three groups, we found increase of TUNEL and p21WAF1/CIP1positive cells, reduced tumor volume and prolonged survival phase..The number of apoptotic cells was positively correlated with those of p21WAF1/CIP1. The number of PCNA positive cells were negative realationship with those of apoptotic cells and a significant negative correlation with those of p21WAF1/CIP1.
Conclusions The S1area brain glioma model had the same characters as human brain glioma. This model is suitable for laboratory and treatment study of brain gliomas. Apoptosis may be one of the most important mechanism of cryotherapy and immunotherapy(rhTNF-α) in killing the tumor cells. The combination of cryotherapy and immunotherapy(rhTNF-α) show marked synergistic effect compared with them alone. P21WAF1/CIP1took part in the reducing of apoptosis of rat glioma, and involved in controlling expression of PCNA. PCNA participated in the synthesis of DNA, and down-regulation of the expression reflected inhibited proliferation of cells Part III Clinical observationof combination therapy against brainglioma by freeze ablation and rhTNF-α
Objective To evaluate the therapeutical effect of cryosurgery combinated with recombinant human tumor necrosis factor a (rhTNF-a) on the brain gliomas.
Methods The brain gliomas were removed by microsurgery after the cryosurgery and then were treated by intramuscular injection of rhTNF-a. The poisonous and side effects of rhTNF-a were observed in all the patients. MRI was performed before and per6month after the discharge from hospital in all the patients. Results Of18patients with brain gliomas,17underwent total resection of the tumors and1subtotal. The weakened muscle power was recovered differently in degree in9patients whose muscle power was weakened before the operation. The occurrent rate of the poisonous and side effects of rhTNF-a including fever, red and swollen region, bone and muscle pain and son on was38.9%(7/18). The following up showed that of18patients,17survived for more than18months and1who received subtotal resection of the tumor died15months after the treatment.
Conclusion It is suggested that cryosurgery combinated with rhTNF-a may be a new method to treat the brain gliomas.
冷冻治疗肿瘤已有数十年历史。近年来,随着冷冻设备的更新和冷冻技术的完善,在准确定位肿瘤和最大限度杀灭肿瘤组织等方面取得了长足的进步。
冷冻的作用主要就是使局部快速降温、冻结、复温,在此过程中产生一系列的病理变化,最终导致细胞受损而死亡。由细胞内、外液中因冻结而分离出的纯水形成冰晶,并由此而产生的一系列变化是冷冻致组织损伤的重要机制。冷冻治疗过程中还存在冷冻速度的区别,因此致细胞损伤的作用机制可能也存在一些差别。目前关于冷冻致细胞死亡的机制主要存在以下几种学说：①迅速的低温使细胞内外的水分形成冰晶,细胞脱水,细胞内电解质浓度增高,使脂蛋白复合体受到破坏,引起细胞膜的溶解损伤从而致细胞死亡；②冷冻还可伤害线粒体中的光磷酸化作用,使三磷酸腺苷的合成发生不可逆性的裂解,造成细胞器膜的损害,而琥珀酸脱氢酶的活性有赖于线粒体膜的完整性,溶酶体膜的破坏可导致其中酶的释放,引起细胞的自溶,内质网、细胞核的膜均可在冷冻过程中发生破裂,而细胞可因细胞器的破坏而发生死亡；③冷冻过程中由于细胞内外冰晶形成,可造成细胞内的脱水状态,体液浓缩,细胞内电解质浓缩,浓度达到有害的程度,同时酸碱度也随之改变,出现细胞酸中毒和代谢障碍,从而导致胞浆、胞膜、细胞内成分蛋白产生变性,进而导致细胞死亡；④冷冻还可使细胞膜、蛋白等细胞成分中的结构水丢失,细胞因此失去功能而死亡；⑤温度休克学说认为温度的急剧变化较绝对温度对细胞的杀伤作用更重要,这可能是由于细胞内组成成分缩胀比率不均衡以致细胞破裂的结果；⑥组织冷冻后,血流速度减慢,这在小静脉表现的尤为明显,小静脉出现血流郁滞,这种血流郁滞乃至造成微循环的阻断,另外,冷冻还可直接损伤毛细血管壁,使血小板和其他血细胞与损伤的血管壁黏附,在血管内形成血栓栓塞。毛细血管腔出现阻塞后,则血流停止,组织细胞出现缺血性梗死。
冷冻后继发的组织细胞坏死还与冷冻后所引起的一系列生化反应有关。冷冻致细胞死亡的机制可能与细胞凋亡有关。凋亡是与坏死不同的另一种细胞死亡机制,即程序性死亡,它是一种耗能的主动的促细胞消亡的方式。我们前期研究已证实冷冻治疗可引起肿瘤中心区坏死、周边肿瘤细胞凋亡。而冷冻免疫是冷冻治疗的又一重要机制。全身冷冻免疫实验表明,冷冻HCA实体瘤,除原瘤生长受抑制外,还可能产生肿瘤特异性移植免疫以及抑制再植瘤生长。Bayjoo实验显示NK细胞的细胞毒性在冷冻后增强,临床也偶见原发灶经冷冻后转移灶自行退缩现象。Joosten等在冷冻治疗小鼠皮肤肿瘤(colon26-B)实验中发现除局部肿瘤坏死外,还可诱导全身抗肿瘤反应,血浆中IL-1和TNF-α水平较直接切除组高。由此可见冷冻本身能改变机体免疫状态,为联合TNF-α治疗提供了条件。
TNF-α的众多生物学作用中,其抗瘤作用倍受人们关注。大量实验研究和初步临床应用的资料证实TNF-α对某些肿瘤细胞具有细胞抑制和杀伤作用。TNF的抗癌作用主要是由于：①TNF为激发性细胞因子,可启动广泛的免疫炎性反应,介导天然细胞毒细胞、单核细胞和巨噬细胞.对癌细胞的杀伤和溶瘤作用；②TNF可作用于血管内皮细胞,使肿瘤的血管变形受损或形成血栓,导致肿瘤发生出血性坏死或因营养缺乏而消退；③TNF可通过诱导某些肿瘤细胞凋亡来抑制癌细胞增生。由于其毒副作用和剂量依赖性,TNF-a常与放疗,化疗(VP16,5-Fu,MMC,ADM等),热疗等联合用药,以提高其抗瘤作用,减少其用量和副作用。
脑胶质瘤约占脑肿瘤总数的40%-50%,平均生存期仅为51周,多采用手术切除、放疗、化疗等治疗手段,但复发率高,预后仍不理想。若将冷冻与免疫治疗联合应用于脑胶质瘤的治疗,其疗效如何,国内外鲜见报道。本实验采用冷冻联合免疫增强剂——TNF-α治疗脑胶质瘤,探讨其杀瘤机制,评价其疗效,为临床应用提供理论依据。本课题分三个部分：
第一部分：建立大鼠皮层C6脑胶质瘤模型,为脑胶质瘤的研究提供实验基础。借助于脑立体定位技术,在雄性Wistar大鼠脑S1区接种C6细胞悬液。随机分成4个周组和1个自然死亡组后观察大鼠的生存期,并采用MRI、病理解剖、HE染色及GFAP免疫组织化学方法,动态观察各周组肿瘤生长情况。以了解模型的稳定性。
第二部分：冷冻联合rhTNF-α治疗C6大鼠脑胶质瘤。本部分通过TUNEL法、流式细胞仪、免疫组化、MRI等手段观察大鼠C6脑胶质瘤经冷冻、rhTNF—α及冷冻联合rhTNF-α治疗后,脑胶质瘤细胞凋亡的数目、p21WAF1/CIP1和PCNA的表达、肿瘤体积、荷瘤鼠生存期、及凋亡、p21WAF1/CIP、PCNA三者之间的相互关系,以探讨它们杀伤肿瘤细胞的作用和机制,为冷冻联合rhTNF-α治疗脑胶质瘤的临床应用提供理论依据。第三部分冷冻切除联合rhTNF-α治疗脑胶质瘤的临床研究,对通过MRI初步判断为脑胶质瘤患者采用冷冻切除联合rhTNF-α治疗。观察rhTNF-α毒副作用,出院前及以后每6个月复查一次MRI。了解联合治疗可否降低复发率,延长病人的生存时间,能否推广应用。
目的：为脑胶质瘤的研究提供实验基础,建立稳定可靠的动物模型。方法：借助于脑立体定位技术,在25只雄性Wistar大鼠脑S1区接种含有10g/L琼脂糖的C6细胞悬液。随机分成4个周组和1个自然死亡组后观察大鼠的生存期,并采用MRI、病理解剖、HE染色及GFAP免疫组织化学方法,动态观察各周组肿瘤生长情况。结果：在不同周组中,所有检查均显示C6脑胶质瘤模型的建立成功,GFAP阳性。4周内无死亡,自然死亡组在第5周死亡2只、余3只于第6周死亡。结论：该方法建立的S1区脑胶质瘤模型具备与人脑胶质瘤生长相似的特性,肿瘤形成时间短,生存期稳定,且无脑外转移和颅外扩散,适合各种脑胶质瘤实验治疗研究。
目的本实验通过观察大鼠C6脑胶质瘤经冷冻、免疫(rhTNF-α)及冷冻联合免疫(rhTNF-α)治疗后,脑胶质瘤细胞凋亡的数目、p21WAFI/CIP1和PCNA的表达、肿瘤体积、荷瘤鼠生存期、及凋亡、p21WAF1/C1P1、PCNA三者之间的相互关系,以探讨它们杀伤肿瘤细胞的作用和机制,为冷冻联合免疫(rhTNF-α)疗法的临床应用提供理论依据。
方法本研究包括(1)C6脑胶质瘤细胞的培养,C6细胞在含有10%新生小牛血清的DMEM培养基中,5%二氧化碳、37℃条件下培养。(2)大鼠C6脑胶质瘤动物模型的建立,选纯种Wistar雄性大鼠120只,借助于立体定位技术、将10μl含有1×106个C6细胞悬液缓慢注射入大鼠右侧大脑S1区皮层。(3)动物分组及治疗的实施：待肿瘤生长至第14天、直径约6mm时,随机将荷瘤鼠分为对照、单冷、单免(rhTNF-α)及冻免(rhTNF-α)四组,每组30只,对不同的组别进行相应的治疗。于接种后第21天,每组随机抽取20只按不同检测要求取材,各组余10只用于测量肿瘤体积和观察生存期。(4)TUNEL法和流式细胞仪检测细胞凋亡。(5)免疫组化方法观察p21WAF1/CIP1及PCNA蛋白的表达。(6)MRI测肿瘤体积。(7)直接观察各组荷瘤鼠的生存期。
结果HE染色显示颅内肿瘤形成；GFAP阳性。联合治疗(冻免组)与其他三组比较：肿瘤细胞凋亡、p21WAF1/CIP1蛋白表达显著增多,PCNA蛋白表达显著减少,肿瘤体积缩小,生存期延长。凋亡细胞数与p21WAF1/CIP1蛋白表达呈正相关；与PCNA蛋白表达之间呈显著负相关；p21WAF1/CIP1蛋白表达也与PCNA蛋白表达之间呈显著负相关。
结论凋亡是冷冻、免疫(rhTNF—α)杀伤肿瘤细胞的一个重要机制；冷冻联合免疫(rhTNF—α)治疗可产生协同效应；p21WAF1/CIP1参与了凋亡的调控；PCNA参与DNA的合成,其下调反应细胞增殖受抑制,有利于凋亡的发生,其表达受p21WAF1/CIP1负调控。
目的探讨冷冻切除联合重组人肿瘤坏死因子(rhTNF-α)治疗脑胶质瘤的效果。
方法对于18例脑胶质瘤患者采用冷冻切除联合rhTNF-α治疗。观察rhTNF-α毒副作用,出院前及以后每6个月复查一次MRI评估治疗。结果肿瘤全切除17例,1例次全切除。术前9例有病例对侧肢力减退者,均在1周内恢复。rhTNF-α常见的毒副作用为发热,局部红肿及肌肉疼痛发生率为38.9%(7/18)。术后随访6-20个月,除1例次全切者术后15个月死亡外,余17例均幸存。
结论本结果提示冷冻切除联合rhTNF-α治疗脑胶质瘤可能成为胶质瘤治疗的一种新策略。
OVERVIEW
Gliomas originate from brain glial cells and account for approximately80%of malignant primary brain tumors. The prognosis for patients with gliomas is generally poor with an average survival time of51weeks. Treatment depends on the location of the tumor, the cell type, and the grade of malignancy and often involves a combination of surgery, radiation therapy, chemotherapy, and targeted molecular therapy.
Cryosurgery is a surgical technique established as rapid freezing, slow thawing and repetition of the freeze-thaw cycles, which has been used for the treatment of skin, oral cavity, liver, bone and prostate tumors. Besides the local destruction on tumor tissues caused by ice crystal formation in the rapid freezing period, the slow thawing period also causes microcirculatory failure. Furthermore, It has been shown that cryosurgery postulates inhibition of secondary tumor growth and metastases associated with increased plasma cytokine tumor necrosis factor-alpha (TNF-a)and enhances natural killer cytotoxicity in liver tumors. Cryosurgery began to treat brain tumors in the1960s. Despite advances of imaging technologies to facilitate this application, it still poses challenges. Insufficient freezing does not induce cell death resulting in tumor recurrences. Conversely, over-freezing may damage normal brain tissues causing serious complications. It has been shown that molecular adjuvant approaches could enhance cryosurgery by reducing local recurrence and complications
TNF-a is a multifunctional cytokine that plays a key role in apoptosis and cell survival. Although systemic TNF-α administration is dose-dependent and associated with severe toxicity, TNF-a has been demonstrated to have anti-tumor effects in animal and human. More importantly, systemic TNF-a administration augments the effect of cryosurgery in prostate cancer and breast cancer by increasing cryo-sensitivity. However, it is not clear whether TNF-a enhances the cryosurgery effects in brain tumors.
This study is designed to test the hypothesis that systemic TNF-a administration and cryosurgery play a synergistic effect on the treatment of brain tumors. For this purpose we developed a more reproducible rat brain cortex glioma model to facilitate cryosurgery delivery, compared with the deep brain tumor models developed at the caudate nucleus and brainstem. By using this model, we investigated survival time, tumor size, tumor cell proliferation, and apoptosis. Additionally, we also monitored brain gliomas tumor patients treated with cryosurgery and TNF-a. Part I:We developed a rat cortical C6glioma model to provide experimental basis for the study of brain gliomas. C6cells were implanted into the male Wistar rat brain SI District using a stereotaxic technology,
Part II:Rats were followed up for90days and MRI was used to monitor tumor size in time series. Pathological anatomy examinations were performed and tissues were collected at the end of studies. DNA fragmentation was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL assay); P21WAF1/CIP1and proliferating cell nuclear antigen (PCNA) expression levels were scored using immunohistochemical staining.
The study is composed with three part:
Part III:In order to assess combination therapy the possibility of lowering the recurrence rate and prolong survival time in clinical, we followed up brain gliomas patients treated with cryosurgery and TNF-. Patients were received MRI test before discharge and every6-month after discharge to monitor tumor size. Side effects of rhTNF-a were also monitored.
Objective To establish a stable and dependable animal model as a laboratory foundation for treatment research in human brain glioma. Methods By stereotaxic technique,25male Wistar rats were inoculated with c6cell suspension containing10g.L-1agarose in their brain S1area. After being randomized to4week groups and1natural death group the rats were observed of survival period and the examinations such as MRI, patholoanatomy, HE stain and GFAP immunohistochemistry were prformed on each week group rats to dynamically observe the tumor growth
Results All examinations showed intracranial tumors existing in different week groups. Expression of GFAP was positive. The death was not detected in4weeks. However, in natural death group, two rats died in No.5week and the others died in No.6week. Conclusions The S1area brain glioma model established by this method in rats had the same characters as human brain glioma. The tumor growth period was shirt and the subsisting period was stable without extracerebral metastasis and extracranial extension. This model is suitable for laboratory and treatment study of brain gliomas.
Objective This study was performed to detect the number of apoptosis cells, the expression of p21WAF1/CIP1and PCNA, the relationship among the three factors after rat C6glioma-model treated with cryotherapy or immunotherapy(rhTNF-α) or combination cryotherapy and immunotherapy(rhTNF-α). At the same time, the tumor volume and the survival period of rat with tumor were observed. Try to investigate their effect and mechanism of killing glioma cells and provide scientific evidence for clinical application.
Methods (1) C6glioma cells were cultured. C6glioma was cultured in DMEM containing10%new born bovine serum, under condition of5%carbon dioxide and37℃.(2) Rat C6brain glioma models were established. By stereotaxic technique,120male Wistar rats were respectively inoculated with suspension containing1×106C6cells in their right brain cortex S1area..(3) The division and the performance of treatment:The rats were randomly divided into the control group (n=30),the cryotherapy group (n=30),the immunotherapy(rhTNF-α) group (n=30) and the combination group (n=30) of the later two methods. When the tumor diameter were approximate6mm at14th day after inoculation, the corresponding treatment was performed according to the different groups.20rats of each group were randomly drawed to sacrifice for different examining purpose at21st day following inoculation. The other10rats of each group was to observe tumor volume and subsisting period.(4) Glioma cell apoptosis was observed with TUNEL and flow cytometry.(5) Immunohistochemical staining was used to analyze the expression of p21WAF1/CIP1and PCNA protein.(6) The tumor volume was measured by MRI.(7) The rat survival period of each group was directly observed.
Results MR and HE stain showed intracranial tumors existing.Expression of GFAP was positive. After comparing the combination group with the other three groups, we found increase of TUNEL and p21WAF1/CIP1positive cells, reduced tumor volume and prolonged survival phase..The number of apoptotic cells was positively correlated with those of p21WAF1/CIP1. The number of PCNA positive cells were negative realationship with those of apoptotic cells and a significant negative correlation with those of p21WAF1/CIP1.
Conclusions The S1area brain glioma model had the same characters as human brain glioma. This model is suitable for laboratory and treatment study of brain gliomas. Apoptosis may be one of the most important mechanism of cryotherapy and immunotherapy(rhTNF-α) in killing the tumor cells. The combination of cryotherapy and immunotherapy(rhTNF-α) show marked synergistic effect compared with them alone. P21WAF1/CIP1took part in the reducing of apoptosis of rat glioma, and involved in controlling expression of PCNA. PCNA participated in the synthesis of DNA, and down-regulation of the expression reflected inhibited proliferation of cells Part III Clinical observationof combination therapy against brainglioma by freeze ablation and rhTNF-α
Objective To evaluate the therapeutical effect of cryosurgery combinated with recombinant human tumor necrosis factor a (rhTNF-a) on the brain gliomas.
Methods The brain gliomas were removed by microsurgery after the cryosurgery and then were treated by intramuscular injection of rhTNF-a. The poisonous and side effects of rhTNF-a were observed in all the patients. MRI was performed before and per6month after the discharge from hospital in all the patients. Results Of18patients with brain gliomas,17underwent total resection of the tumors and1subtotal. The weakened muscle power was recovered differently in degree in9patients whose muscle power was weakened before the operation. The occurrent rate of the poisonous and side effects of rhTNF-a including fever, red and swollen region, bone and muscle pain and son on was38.9%(7/18). The following up showed that of18patients,17survived for more than18months and1who received subtotal resection of the tumor died15months after the treatment.
Conclusion It is suggested that cryosurgery combinated with rhTNF-a may be a new method to treat the brain gliomas.
引文
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