光敏剂TMPYP4光动力治疗宫颈癌的动物实验研究
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摘要
研究背景：
宫颈癌是常见的妇科恶性肿瘤,仅次于乳腺癌,位于妇女恶性肿瘤的第二位,90年代初,WHO宣布人乳头瘤病毒(human papilloma viruses, HPV)是引起宫颈癌变的首要因素,因此,宫颈液基细胞学和HPV病毒联合检测技术明显提高了宫颈癌筛查的准确率,尤其是对早期宫颈病变的发现有很大的帮助,同时也发现宫颈癌或宫颈上皮内瘤变患者的年龄趋于年轻化,对生育和性生活质量的要求较高,传统的治疗方法包括手术、放化疗等对生殖器官功能的保留不太理想,光动力疗法可能成为一种新的能保留生育功能的治疗方法。另外,对于年龄较大的晚期宫颈癌患者,一般情况差难以耐受化疗及放疗的不良反应时,光动力治疗也成为可选择的治疗方式之一。
光动力疗法(PDT)是基于病变细胞选择性的吸收光敏剂并在特定波长激光的激发下产生的光化学反应,对病变细胞或者组织产生杀伤效应。PDT现广泛应用于皮肤科、呼吸、消化、妇科等临床科室,成为近年研究的热点,但在发现PDT治疗作用的同时,也发现其副反应有时是严重的,如皮肤发生红斑、水泡等光敏性皮炎、皮肤溃疡等。
我们本次实验选用的光敏剂是四甲基吡啶卟啉(5,10,15,20-tetra-(N-methyl-4-pyridyl)porphyrin, TMPyP4),属于卟啉衍生物,是一种新型的水溶性光敏剂,可以结合并稳定人类端粒G-四链体的形成,抑制端粒酶的功能,而目前研究发现恶性肿瘤增殖失控的关键在于端粒酶的激活,因此,TMPyP4通过稳定G-四链体的结构,从而降低端粒酶活性,将其作用于肿瘤细胞可导致肿瘤细胞生长停止,诱导肿瘤细胞的凋亡。但近年有研究发现,端粒酶活性在一些正常细胞中也被检测到,如外周血、骨髓细胞和皮肤的基底层等组织。因此,卟啉类光敏剂和端粒酶的相互作用可能会影响机体的正常细胞,在正常组织产生副作用。另外,TMPyP4本身是一种光敏剂,在适当波长的激光照射下可发生光敏反应,对肿瘤细胞起到杀伤作用,因此从理论基础上分析,TMPyP4是一种对恶性肿瘤细胞杀伤作用比较强的药物,但它有肿瘤选择特异性较差的缺点,在发挥治疗作用的同时也可能发生相关的光敏副反应,如皮肤溃疡、脏器损伤等,因此为了避免全身副反应的发生,我们选择在肿瘤局部用药方案。
Survivin和Caspase-3是参与调节细胞凋亡的两个重要分子,Survivin是目前发现的最强的凋亡抑制因子。有研究发现,Survivin可能通过某种机制间接抑(?)Caspase-3的活性而抑制细胞凋亡,而Caspase-3是Caspase家族成员中执行细胞凋亡的重要成员之一,在细胞凋亡过程中起关键作用,大多数促进细胞凋亡的因子,最终都通过激活Caspase-3而导致细胞凋亡。目前,已有TMPyP4抑制端粒酶活性的报道,但TMPyP4-PDT对促进恶性肿瘤细胞凋亡及对凋亡相关蛋白Survivin和Caspase-3的表达影响研究较少,本研究中我们通过观察TMPyP4-PDT对裸鼠Hela细胞移植瘤的作用来探讨其对宫颈癌细胞的促凋亡机制。
根据我们前期细胞实验研究结果：TMPyP4-PDT对宫颈癌hela、siha细胞均有显著杀伤作用,而宫颈癌hela、siha细胞对TMPyP4-PDT的敏感性不同,宫颈癌hela细胞较敏感。因此,我们动物实验选择宫颈癌hela细胞接种雌性BALB/c裸小鼠,观察TMPyP4-PDT对裸鼠移植瘤的抑制作用。
因为PDT杀伤肿瘤细胞的效果取决于光敏剂浓度和光的能量密度,我们曾用光敏剂TMPyP4的较大浓度(20mg/kg)对裸鼠宫颈癌移植瘤行瘤周注射,发现在没有激光照射的情况下就发生了皮肤溃烂,持续时间较长(四周)。预实验时我们也曾选择经尾静脉注射光敏剂给动物全身用药(10mg/kg),发现光敏剂在尾静脉中长时间聚集,避光四周,然后移至自然光线下放置2天发现裸鼠尾巴部分出现干性坏死(见图1-2),而肿瘤增大明显,生长未被抑制,裸鼠消瘦,提示全身用药治疗效果不理想。
综上所述,为了较大限度的降低TMPyP4-PDT副反应,同时又能达到治疗宫颈癌的效果,我们设计多次低剂量TMPyP4-PDT的治疗方案,通过局部瘤体内用药方法来探讨TMPyP4-PDT对宫颈癌的治疗作用,并探讨其促进宫颈癌细胞凋亡的机制。
第一部分：光敏剂TMPyP4多次低剂量光动力疗法治疗宫颈癌的动物实验研究
研究目的：光动力疗法(PDT)是一种近年研究发现的适合治疗宫颈癌的新方法,但光动力疗法的副作用有时是严重的,为了减少光敏疗法副反应的发生,又能在较大程度上达到治疗恶性肿瘤的效果,我们设计本实验来研究观察TMPyP4多次低剂量光动力疗法对裸鼠宫颈癌Hela细胞移植瘤的治疗效果及副反应。
材料与方法：雌性BALB/c裸小鼠(5-6周,称重18-20克)20只,SPF级条件饲养,裸鼠皮下注射宫颈癌Hela细胞(1×107细胞/200μL),瘤细胞接种10天后,达到标准的荷瘤小鼠为18只,标准成瘤率为90%,将荷瘤小鼠随机分为三组,每组6只(n=6),A组为对照组,无任何治疗；B组为一次用药光疗组,用TMPyP4(10mg/kg)瘤内注射和照射蓝色激光(108J／cm2)一次,药物注射与激光照射间隔时间为6小时；C组为三次用药光疗组,用TMPyP4(10mg/kg)瘤内注射和照射蓝色激光(108J/cm2)三次,每次治疗间隔时间2天。开始治疗后,监测肿瘤体积,在C组治疗结束后2周,比较各组肿瘤体积大小,同时观察裸鼠生活状态以及皮肤和组织器官的损害情况及血液中血常规和肝肾功能的变化情况。
结果：C组移植瘤体积与A组和B组比较明显受到抑制(P<0.05)；而实验结束时治疗组裸鼠体重增加,生活状态良好,对照组体重下降,生活状态较差；另外治疗组裸鼠皮肤和内脏器官未见明显损伤,血常规和肝肾功能与对照组相比无明显变化,同时肿瘤切片HE染色发现C组移植瘤细胞凋亡明显增多。
结论：我们的研究结果表明,多次低剂量TMPyP4光动力治疗可显著抑制裸鼠宫颈癌移植瘤的生长、促进肿瘤细胞凋亡,治疗组裸鼠生活状态良好,未见消瘦,血常规和肝肾功能无明显变化,同时皮肤及内脏器官未见明显副反应发生。
第二部分：TMPyP4-PDT对宫颈癌裸鼠移植瘤细胞凋亡及Survivin和Caspase-3表达影响的研究
研究目的：细胞凋亡又称细胞程序性死亡是指为维持内环境稳定,由一系列酶参与、多种基因控制的细胞自主有序的死亡过程。细胞凋亡与肿瘤的发生发展关系密切,目前细胞凋亡的确切机制仍不清楚,但细胞往往会激活相对立的两个过程,凋亡和抗凋亡,由两种信号通路的强弱对比决定最后的结局。所以,细胞凋亡一直是恶性肿瘤研究的热点,也是药物治疗研究的靶点。有研究发现,低剂量光敏剂光动力治疗导致细胞凋亡,而高剂量光动力疗法主要引起细胞坏死。我们采用原位末端标记法(TUNEL)研究观察TMPyP4-PDT治疗组宫颈癌细胞的凋亡情况,TUNEL法的原理是生物素或荧光素等标记物被标记在DNA缺口末端的3’-OH上,在TdT催化下,通过标记物显色系统在组织细胞原位检测凋亡细胞,对完整的单个凋亡细胞或凋亡小体进行原位染色,与普通HE染色方法相比,能更准确的反映细胞凋亡的形态特征并进行组间统计学分析对比。生存素(Survivin)和半胱氨酸天冬氨酸蛋白酶3(Caspase-3)在细胞凋亡的发生机制中是两个比较关键的因子,本研究联合检钡Survivin和Caspase-3在裸鼠宫颈癌移植瘤中的表达,探讨TMPyP4-PDT促进宫颈癌细胞凋亡的机制。
材料与方法：按照实验第一部分分组情况,取各组移植瘤石蜡切片,用TUNEL法检测肿瘤细胞的凋亡情况,用全自动荧光显微镜进行观察和结果分析。TUNEL检测方法为每例标本随机取10个高倍视野,检测1000个细胞中凋亡细胞所占百分比计数为凋亡指数(AI)。survivin和caspase-3蛋白的检测采用免疫组化SP法,免疫组化阳性为细胞浆中棕黄色颗粒,采用全自动显微镜和图像采集分析系统,每只裸鼠选3张切片进行分析,每张切片连续读取10张高倍视野,计算每视野阳性染色的光密度(0D)值,最后计算每组裸鼠的平均光密度值进行统计学分析。
结果：荧光强度检测结果显示, TUNEL阳性细胞以绿色荧光表示。TMPyP4-PDT三次治疗组的TUNEL阳性(绿色荧光)细胞较单次治疗组和对照组明显增多,凋亡指数高于另外两组,差异有显著性意义；survivin和caspase-3蛋白免疫组化检测结果显示在三次治疗组凋亡抑制因子survivin蛋白表达降低,凋亡关键因子caspase-3表达升高。
结论：多次低剂量TMPyP4-PDT疗法对裸鼠移植瘤宫颈癌细胞有明显的促凋亡作用,其机制可能与survivin蛋白表达降低和caspase-3表达升高有关。
Backgrounds:
Cervical cancer is a common gynecological tumor, second only to breast cancer in women. At the beginning of the90's, WHO announced that human papilloma virus (HPV) is the leading cause of cervical carcinogenesis.Therefore, combined detection of liquid based cytology and HPV can significantly improve the accuracy of cervical cancer screening, especially of great help for early cervical lesions, at the same time we found that cervical cancer or cervical intraepithelial neoplasia in patients with younger age, higher requirements on the reproduction and quality of life. The traditional treatment methods including operation, radiotherapy and chemotherapy, which keep on reproductive organ function is not very ideal, photodynamic therapy may become a new method can preserve fertility treatment. In addition, for older patients with advanced cervical cancer, poor general condition of adverse reactions to tolerate the chemotherapy and radiotherapy, photodynamic therapy has become one of the alternative treatment.
Photodynamic therapy (PDT) is a photochemical reaction of which diseased cells selected absorption of photosensitizer and excited at a particular wavelength laser, to product the killing effect on cells or tissues. PDT is now widely used in the Department of dermatology, respiratory, digestive, gynecological clinical departments, become a research hot spot in recent years.At the same time,we also found that the side reaction is sometimes serious, such as photosensitive dermatitis, skin ulcer.
We used four methyl pyridine porphyrin photosensitizer (5,10,15,20-tetra-(N-methyl-4-pyridyl)porphyrin, TMPyP4) in this research,which is a new type of water-soluble photosensitizers. It can combine the human telomeric G-four chain bodies, to inhibit telomerase function. Some study found that the key to control the proliferation of malignant tumors is the activation of telomerase, therefore, the structure of G-four chain bodies and TMPyP4, which reduced the telomerase activity, to lead to tumor cells growth arrest, apoptosis. But recent studies have found, telomerase activity was also detected in some normal cells, such as peripheral blood, bone marrow cells and the basal layer of the skin and other tissues. Therefore, the interaction of porphyrin photosensitizer and telomerase may affect the normal cells in the body, to cause side effects in normal tissues. In addition, the TMPyP4itself is a kind of photosensitizer, has the photosensitized reaction of the corresponding laser irradiation, the killing effect on tumor cells.Therefore, based on theoretical analysis, drug cytotoxicity of TMPyP4act on tumor cells is strong, but it has shortcomings of the poor tumor specific, may also occur photosensitive side effects associated with treatment function, such as the skin ulcer, organ damage, so in order to avoid systemic adverse reactions, we chose the local tumor therapy.
Survivin and Caspase-3are two important molecules involved in the regulation of apoptosis, Survivin is most of apoptosis inhibition factor which was found now. Studies have found that, survivin may inhibit Caspase-3indirectly through a mechanism of activity and inhibition of apoptosis. Caspase-3is one of the important members of the executive apoptotic caspase family members, plays a key role in the process of apoptosis promoting factor, most cell apoptosis through activation of Caspase-3, ultimately leading to apoptosis. At present, TMPyP4inhibit telomerase activity had been reported, but the TMPyP4-PDT to promote apoptosis of malignant tumor cells and effect on the expression of apoptosis related protein Survivin and Caspase-3, in this study we observed the effects of TMPyP4-PDT on Hela cells in nude mice to investigate the impact of cervical cancer cell apoptosis mechanism.
According to our previous experimental results: TMPyP4-PDT had a significant killing effect on cells of cervical cancer HeLa and SiHa cells, but the sensitivity of cervical cancer HeLa and SiHa cells to TMPyP4-PDT were different, HeLa cells was more sensitive. Therefore, we choose the animal experimental inoculation of cervical cancer HeLa cell of female BALB/c nude mice, to observe the inhibitory effect of TMPyP4-PDT on transplanted tumor in nude mice.
Because the effect of PDT killing tumor cell depended on the photosensitizer concentration and light energy density, we had higher concentration of photosensitizer TMPyP4(20mg/kg) on cervical carcinoma in nude mice for peritumoral injection, we found that the skin ulceration occured in no case of laser irradiation, and continued longer time(four weeks).(Figure1-1). In preliminary experiments we have chosen animal systemic administration (10mg/kg) by tail vein injection of photosensitizer, found that the photosensitizer aggregation long time in tail vein, evades the light for four weeks, and then moved to the natural light for2days,the nude mice tail appeared partly dry necrosis (Figure1-2), and tumor growth was not inhibited, nude mice weight loss, indicating that the treatment effect of systemic administration was not ideal.
By way of summary, in order to maximize the side effects of TMPyP4-PDT, and achieve the effect of treatment of cervical cancer, we designed the therapy of multiple low dose TMPyP4-PDT, to explore the therapeutic effect of TMPyP4-PDT on the cervical cancer by local intratumoral administration method, and to explore the mechanism of promoting apoptosis of cervical cancer cells.
Part1:Animal Experimental Study of photosensitizer TMPyP4multiple low dose of photodynamic therapy for the treatment of cervical cancer
Objective: photodynamic therapy (PDT) is a new method of suitable for the treatment of cervical cancer recentlly, but the side effect of photodynamic therapy is sometimes serious, in order to reduce the side effects of the photodynamic therapy, and can achieve the effect of treatment of malignant tumor in the great degree, we design this experiment to study the treatment and the side effects of multiple low dose of photodynamic therapy of TMPyP4on transplanted tumor of cervical cancer Hela cells in nude mice.
Materials and methods:20female BALB/c nude mice (5-6weeks, weighing18-20g) had been breeded on SPF conditions. Subcutaneous injection of cervical cancer Hela cells (1×107cells/200μL).10days after tumor cell inoculation,18mice reached standard, standard tumor formation rate is90%, The mice bearing tumor were randomly divided into three groups,6rats in each group (n=6). Group A was the control group, no treatment; group B for once drug therapy group, Using TMPyP4(10mg/kg) was injected intratumoral and irradiated by the blue laser(108J/cm2). The time interval of drug injection and laser irradiation is6hours; Group C for three-time PDT therapy, TMPyP4(10mg/kg) was injected intratumoral and irradiated by blue laser (108J/cm2) for three times, interval of each treatment was2days. After treatment, the tumor volume was monitored, we compared the size of tumor in nude mice, and observed the state of life, skin, tissue and organ damage.
Results:the tumor volume of C group was significantly inhibited which compared with A group and B group (P<0.05). At the end of the experiment, the weight of nude mice in the treatment group increased, lived in good condition, the body weight of the control group decreased, poor living condition; On the other hand,the nude mouse skin and internal organs of treatment groups were not obviously damaged, and apoptosis of cervical cell, in C group was found significantly increased by HE.
Conclusion:Our results showed that multiple low dose TMPyP4photodynamic therapy can significantly inhibit the cervical cancer xenograft growth in nude mice, promote the apoptosis of tumor cells in nude mice, the treatment group living in good condition, adverse reactions on the skin and internal organs were not happened.
Part2:The effect of TMPyP4-PDT on the expression of cervical cancer cell apoptosis、Survivin and Caspase-3in nude mice
Objective: apoptosis is also called programmed cell death which refers to the maintenance of the homeostasis and the death process including multi gene controlled by a series of enzymes. The development of tumor Closely related with cell apoptosis, the exact mechanism is still not clear, but the cells tend to activate the two process opposite, apoptosis and anti apoptosis, the strength of two kinds of signal transduction pathway determines the final outcome. Therefore, cell apoptosis has been a hot research on malignant tumor, and drug treatment target. Some studies found that low doses photosensitizer photodynamic therapy induced cell apoptosis, and the high dose of photodynamic therapy mainly caused the cell necrosis. We used in situ end labeling (TUNEL) study the cervical carcinoma cell apoptosis treated by TMPyP4-PDT. The principle of TUNEL method is that the biotin or fluorescence marker was labeled in the DNA nick end3'-OH, catalyzed by TdT, the color system was markered in the detection of apoptotic cells tissue cells in situ, in situ staining of intact single apoptotic cells and apoptotic bodies. Compared with common HE staining method, morphological characteristics of apoptosis can be reflected more accurately and analyzed statistically. Survivin and cysteine aspartic proteinase3(Caspase-3) in the mechanism of apoptosis are the two key factors, the expression of the combined detection of Survivin and Caspase-3in cervical cancer xenograft in nude mice, may investigate the mechanisms of TMPyP4-PDT inducing apoptosis of cervical cancer cells.
Materials and methods: according to the first part of the experiment and the tumor paraffin section, TUNEL was used to detect the apoptosis of tumor cells, results were investigated by automatic fluorescence microscopy. TUNEL detection method is that10HPF of each case were randomly selected, apoptotic cells were detected in1000cell,percentage count of apoptosis cell is apoptosis index (AI). Detection of Survivin and caspase-3protein is immunohistochemical SP method, brown granules is thoughted the positive cell by immunohistochemical staining of cytoplasm using automatic microscope and image analysis system, each mouse selected3sections of each section, read for10high power field, to calculate the positive staining value,that is optical density(OD), the average optical density was analyzed statistically each group of nude mice.
Results:the testing results of fluorescence intensity display is that TUNEL positive cells expressed green fluorescence. The TUNEL positive cells of C group were more than the single treatment group and the control group,the apoptosis index was higher than the other two groups, which was significant difference; Survivin and caspase-3protein by immunohistochemical detection showed that the expression of the survivin protein was decreased in the three-time treatment groups,but the expression of the caspase-3protein increased.
Conclusion:multiple low dose TMPyP4-PDT therapy could induce apoptosis of cervical cancer cells in nude mice, and that may be related to the expression of survivin decreased and expression of Caspase-3increased.
宫颈癌是常见的妇科恶性肿瘤,仅次于乳腺癌,位于妇女恶性肿瘤的第二位,90年代初,WHO宣布人乳头瘤病毒(human papilloma viruses, HPV)是引起宫颈癌变的首要因素,因此,宫颈液基细胞学和HPV病毒联合检测技术明显提高了宫颈癌筛查的准确率,尤其是对早期宫颈病变的发现有很大的帮助,同时也发现宫颈癌或宫颈上皮内瘤变患者的年龄趋于年轻化,对生育和性生活质量的要求较高,传统的治疗方法包括手术、放化疗等对生殖器官功能的保留不太理想,光动力疗法可能成为一种新的能保留生育功能的治疗方法。另外,对于年龄较大的晚期宫颈癌患者,一般情况差难以耐受化疗及放疗的不良反应时,光动力治疗也成为可选择的治疗方式之一。
光动力疗法(PDT)是基于病变细胞选择性的吸收光敏剂并在特定波长激光的激发下产生的光化学反应,对病变细胞或者组织产生杀伤效应。PDT现广泛应用于皮肤科、呼吸、消化、妇科等临床科室,成为近年研究的热点,但在发现PDT治疗作用的同时,也发现其副反应有时是严重的,如皮肤发生红斑、水泡等光敏性皮炎、皮肤溃疡等。
我们本次实验选用的光敏剂是四甲基吡啶卟啉(5,10,15,20-tetra-(N-methyl-4-pyridyl)porphyrin, TMPyP4),属于卟啉衍生物,是一种新型的水溶性光敏剂,可以结合并稳定人类端粒G-四链体的形成,抑制端粒酶的功能,而目前研究发现恶性肿瘤增殖失控的关键在于端粒酶的激活,因此,TMPyP4通过稳定G-四链体的结构,从而降低端粒酶活性,将其作用于肿瘤细胞可导致肿瘤细胞生长停止,诱导肿瘤细胞的凋亡。但近年有研究发现,端粒酶活性在一些正常细胞中也被检测到,如外周血、骨髓细胞和皮肤的基底层等组织。因此,卟啉类光敏剂和端粒酶的相互作用可能会影响机体的正常细胞,在正常组织产生副作用。另外,TMPyP4本身是一种光敏剂,在适当波长的激光照射下可发生光敏反应,对肿瘤细胞起到杀伤作用,因此从理论基础上分析,TMPyP4是一种对恶性肿瘤细胞杀伤作用比较强的药物,但它有肿瘤选择特异性较差的缺点,在发挥治疗作用的同时也可能发生相关的光敏副反应,如皮肤溃疡、脏器损伤等,因此为了避免全身副反应的发生,我们选择在肿瘤局部用药方案。
Survivin和Caspase-3是参与调节细胞凋亡的两个重要分子,Survivin是目前发现的最强的凋亡抑制因子。有研究发现,Survivin可能通过某种机制间接抑(?)Caspase-3的活性而抑制细胞凋亡,而Caspase-3是Caspase家族成员中执行细胞凋亡的重要成员之一,在细胞凋亡过程中起关键作用,大多数促进细胞凋亡的因子,最终都通过激活Caspase-3而导致细胞凋亡。目前,已有TMPyP4抑制端粒酶活性的报道,但TMPyP4-PDT对促进恶性肿瘤细胞凋亡及对凋亡相关蛋白Survivin和Caspase-3的表达影响研究较少,本研究中我们通过观察TMPyP4-PDT对裸鼠Hela细胞移植瘤的作用来探讨其对宫颈癌细胞的促凋亡机制。
根据我们前期细胞实验研究结果：TMPyP4-PDT对宫颈癌hela、siha细胞均有显著杀伤作用,而宫颈癌hela、siha细胞对TMPyP4-PDT的敏感性不同,宫颈癌hela细胞较敏感。因此,我们动物实验选择宫颈癌hela细胞接种雌性BALB/c裸小鼠,观察TMPyP4-PDT对裸鼠移植瘤的抑制作用。
因为PDT杀伤肿瘤细胞的效果取决于光敏剂浓度和光的能量密度,我们曾用光敏剂TMPyP4的较大浓度(20mg/kg)对裸鼠宫颈癌移植瘤行瘤周注射,发现在没有激光照射的情况下就发生了皮肤溃烂,持续时间较长(四周)。预实验时我们也曾选择经尾静脉注射光敏剂给动物全身用药(10mg/kg),发现光敏剂在尾静脉中长时间聚集,避光四周,然后移至自然光线下放置2天发现裸鼠尾巴部分出现干性坏死(见图1-2),而肿瘤增大明显,生长未被抑制,裸鼠消瘦,提示全身用药治疗效果不理想。
综上所述,为了较大限度的降低TMPyP4-PDT副反应,同时又能达到治疗宫颈癌的效果,我们设计多次低剂量TMPyP4-PDT的治疗方案,通过局部瘤体内用药方法来探讨TMPyP4-PDT对宫颈癌的治疗作用,并探讨其促进宫颈癌细胞凋亡的机制。
第一部分：光敏剂TMPyP4多次低剂量光动力疗法治疗宫颈癌的动物实验研究
研究目的：光动力疗法(PDT)是一种近年研究发现的适合治疗宫颈癌的新方法,但光动力疗法的副作用有时是严重的,为了减少光敏疗法副反应的发生,又能在较大程度上达到治疗恶性肿瘤的效果,我们设计本实验来研究观察TMPyP4多次低剂量光动力疗法对裸鼠宫颈癌Hela细胞移植瘤的治疗效果及副反应。
材料与方法：雌性BALB/c裸小鼠(5-6周,称重18-20克)20只,SPF级条件饲养,裸鼠皮下注射宫颈癌Hela细胞(1×107细胞/200μL),瘤细胞接种10天后,达到标准的荷瘤小鼠为18只,标准成瘤率为90%,将荷瘤小鼠随机分为三组,每组6只(n=6),A组为对照组,无任何治疗；B组为一次用药光疗组,用TMPyP4(10mg/kg)瘤内注射和照射蓝色激光(108J／cm2)一次,药物注射与激光照射间隔时间为6小时；C组为三次用药光疗组,用TMPyP4(10mg/kg)瘤内注射和照射蓝色激光(108J/cm2)三次,每次治疗间隔时间2天。开始治疗后,监测肿瘤体积,在C组治疗结束后2周,比较各组肿瘤体积大小,同时观察裸鼠生活状态以及皮肤和组织器官的损害情况及血液中血常规和肝肾功能的变化情况。
结果：C组移植瘤体积与A组和B组比较明显受到抑制(P<0.05)；而实验结束时治疗组裸鼠体重增加,生活状态良好,对照组体重下降,生活状态较差；另外治疗组裸鼠皮肤和内脏器官未见明显损伤,血常规和肝肾功能与对照组相比无明显变化,同时肿瘤切片HE染色发现C组移植瘤细胞凋亡明显增多。
结论：我们的研究结果表明,多次低剂量TMPyP4光动力治疗可显著抑制裸鼠宫颈癌移植瘤的生长、促进肿瘤细胞凋亡,治疗组裸鼠生活状态良好,未见消瘦,血常规和肝肾功能无明显变化,同时皮肤及内脏器官未见明显副反应发生。
第二部分：TMPyP4-PDT对宫颈癌裸鼠移植瘤细胞凋亡及Survivin和Caspase-3表达影响的研究
研究目的：细胞凋亡又称细胞程序性死亡是指为维持内环境稳定,由一系列酶参与、多种基因控制的细胞自主有序的死亡过程。细胞凋亡与肿瘤的发生发展关系密切,目前细胞凋亡的确切机制仍不清楚,但细胞往往会激活相对立的两个过程,凋亡和抗凋亡,由两种信号通路的强弱对比决定最后的结局。所以,细胞凋亡一直是恶性肿瘤研究的热点,也是药物治疗研究的靶点。有研究发现,低剂量光敏剂光动力治疗导致细胞凋亡,而高剂量光动力疗法主要引起细胞坏死。我们采用原位末端标记法(TUNEL)研究观察TMPyP4-PDT治疗组宫颈癌细胞的凋亡情况,TUNEL法的原理是生物素或荧光素等标记物被标记在DNA缺口末端的3’-OH上,在TdT催化下,通过标记物显色系统在组织细胞原位检测凋亡细胞,对完整的单个凋亡细胞或凋亡小体进行原位染色,与普通HE染色方法相比,能更准确的反映细胞凋亡的形态特征并进行组间统计学分析对比。生存素(Survivin)和半胱氨酸天冬氨酸蛋白酶3(Caspase-3)在细胞凋亡的发生机制中是两个比较关键的因子,本研究联合检钡Survivin和Caspase-3在裸鼠宫颈癌移植瘤中的表达,探讨TMPyP4-PDT促进宫颈癌细胞凋亡的机制。
材料与方法：按照实验第一部分分组情况,取各组移植瘤石蜡切片,用TUNEL法检测肿瘤细胞的凋亡情况,用全自动荧光显微镜进行观察和结果分析。TUNEL检测方法为每例标本随机取10个高倍视野,检测1000个细胞中凋亡细胞所占百分比计数为凋亡指数(AI)。survivin和caspase-3蛋白的检测采用免疫组化SP法,免疫组化阳性为细胞浆中棕黄色颗粒,采用全自动显微镜和图像采集分析系统,每只裸鼠选3张切片进行分析,每张切片连续读取10张高倍视野,计算每视野阳性染色的光密度(0D)值,最后计算每组裸鼠的平均光密度值进行统计学分析。
结果：荧光强度检测结果显示, TUNEL阳性细胞以绿色荧光表示。TMPyP4-PDT三次治疗组的TUNEL阳性(绿色荧光)细胞较单次治疗组和对照组明显增多,凋亡指数高于另外两组,差异有显著性意义；survivin和caspase-3蛋白免疫组化检测结果显示在三次治疗组凋亡抑制因子survivin蛋白表达降低,凋亡关键因子caspase-3表达升高。
结论：多次低剂量TMPyP4-PDT疗法对裸鼠移植瘤宫颈癌细胞有明显的促凋亡作用,其机制可能与survivin蛋白表达降低和caspase-3表达升高有关。
Backgrounds:
Cervical cancer is a common gynecological tumor, second only to breast cancer in women. At the beginning of the90's, WHO announced that human papilloma virus (HPV) is the leading cause of cervical carcinogenesis.Therefore, combined detection of liquid based cytology and HPV can significantly improve the accuracy of cervical cancer screening, especially of great help for early cervical lesions, at the same time we found that cervical cancer or cervical intraepithelial neoplasia in patients with younger age, higher requirements on the reproduction and quality of life. The traditional treatment methods including operation, radiotherapy and chemotherapy, which keep on reproductive organ function is not very ideal, photodynamic therapy may become a new method can preserve fertility treatment. In addition, for older patients with advanced cervical cancer, poor general condition of adverse reactions to tolerate the chemotherapy and radiotherapy, photodynamic therapy has become one of the alternative treatment.
Photodynamic therapy (PDT) is a photochemical reaction of which diseased cells selected absorption of photosensitizer and excited at a particular wavelength laser, to product the killing effect on cells or tissues. PDT is now widely used in the Department of dermatology, respiratory, digestive, gynecological clinical departments, become a research hot spot in recent years.At the same time,we also found that the side reaction is sometimes serious, such as photosensitive dermatitis, skin ulcer.
We used four methyl pyridine porphyrin photosensitizer (5,10,15,20-tetra-(N-methyl-4-pyridyl)porphyrin, TMPyP4) in this research,which is a new type of water-soluble photosensitizers. It can combine the human telomeric G-four chain bodies, to inhibit telomerase function. Some study found that the key to control the proliferation of malignant tumors is the activation of telomerase, therefore, the structure of G-four chain bodies and TMPyP4, which reduced the telomerase activity, to lead to tumor cells growth arrest, apoptosis. But recent studies have found, telomerase activity was also detected in some normal cells, such as peripheral blood, bone marrow cells and the basal layer of the skin and other tissues. Therefore, the interaction of porphyrin photosensitizer and telomerase may affect the normal cells in the body, to cause side effects in normal tissues. In addition, the TMPyP4itself is a kind of photosensitizer, has the photosensitized reaction of the corresponding laser irradiation, the killing effect on tumor cells.Therefore, based on theoretical analysis, drug cytotoxicity of TMPyP4act on tumor cells is strong, but it has shortcomings of the poor tumor specific, may also occur photosensitive side effects associated with treatment function, such as the skin ulcer, organ damage, so in order to avoid systemic adverse reactions, we chose the local tumor therapy.
Survivin and Caspase-3are two important molecules involved in the regulation of apoptosis, Survivin is most of apoptosis inhibition factor which was found now. Studies have found that, survivin may inhibit Caspase-3indirectly through a mechanism of activity and inhibition of apoptosis. Caspase-3is one of the important members of the executive apoptotic caspase family members, plays a key role in the process of apoptosis promoting factor, most cell apoptosis through activation of Caspase-3, ultimately leading to apoptosis. At present, TMPyP4inhibit telomerase activity had been reported, but the TMPyP4-PDT to promote apoptosis of malignant tumor cells and effect on the expression of apoptosis related protein Survivin and Caspase-3, in this study we observed the effects of TMPyP4-PDT on Hela cells in nude mice to investigate the impact of cervical cancer cell apoptosis mechanism.
According to our previous experimental results: TMPyP4-PDT had a significant killing effect on cells of cervical cancer HeLa and SiHa cells, but the sensitivity of cervical cancer HeLa and SiHa cells to TMPyP4-PDT were different, HeLa cells was more sensitive. Therefore, we choose the animal experimental inoculation of cervical cancer HeLa cell of female BALB/c nude mice, to observe the inhibitory effect of TMPyP4-PDT on transplanted tumor in nude mice.
Because the effect of PDT killing tumor cell depended on the photosensitizer concentration and light energy density, we had higher concentration of photosensitizer TMPyP4(20mg/kg) on cervical carcinoma in nude mice for peritumoral injection, we found that the skin ulceration occured in no case of laser irradiation, and continued longer time(four weeks).(Figure1-1). In preliminary experiments we have chosen animal systemic administration (10mg/kg) by tail vein injection of photosensitizer, found that the photosensitizer aggregation long time in tail vein, evades the light for four weeks, and then moved to the natural light for2days,the nude mice tail appeared partly dry necrosis (Figure1-2), and tumor growth was not inhibited, nude mice weight loss, indicating that the treatment effect of systemic administration was not ideal.
By way of summary, in order to maximize the side effects of TMPyP4-PDT, and achieve the effect of treatment of cervical cancer, we designed the therapy of multiple low dose TMPyP4-PDT, to explore the therapeutic effect of TMPyP4-PDT on the cervical cancer by local intratumoral administration method, and to explore the mechanism of promoting apoptosis of cervical cancer cells.
Part1:Animal Experimental Study of photosensitizer TMPyP4multiple low dose of photodynamic therapy for the treatment of cervical cancer
Objective: photodynamic therapy (PDT) is a new method of suitable for the treatment of cervical cancer recentlly, but the side effect of photodynamic therapy is sometimes serious, in order to reduce the side effects of the photodynamic therapy, and can achieve the effect of treatment of malignant tumor in the great degree, we design this experiment to study the treatment and the side effects of multiple low dose of photodynamic therapy of TMPyP4on transplanted tumor of cervical cancer Hela cells in nude mice.
Materials and methods:20female BALB/c nude mice (5-6weeks, weighing18-20g) had been breeded on SPF conditions. Subcutaneous injection of cervical cancer Hela cells (1×107cells/200μL).10days after tumor cell inoculation,18mice reached standard, standard tumor formation rate is90%, The mice bearing tumor were randomly divided into three groups,6rats in each group (n=6). Group A was the control group, no treatment; group B for once drug therapy group, Using TMPyP4(10mg/kg) was injected intratumoral and irradiated by the blue laser(108J/cm2). The time interval of drug injection and laser irradiation is6hours; Group C for three-time PDT therapy, TMPyP4(10mg/kg) was injected intratumoral and irradiated by blue laser (108J/cm2) for three times, interval of each treatment was2days. After treatment, the tumor volume was monitored, we compared the size of tumor in nude mice, and observed the state of life, skin, tissue and organ damage.
Results:the tumor volume of C group was significantly inhibited which compared with A group and B group (P<0.05). At the end of the experiment, the weight of nude mice in the treatment group increased, lived in good condition, the body weight of the control group decreased, poor living condition; On the other hand,the nude mouse skin and internal organs of treatment groups were not obviously damaged, and apoptosis of cervical cell, in C group was found significantly increased by HE.
Conclusion:Our results showed that multiple low dose TMPyP4photodynamic therapy can significantly inhibit the cervical cancer xenograft growth in nude mice, promote the apoptosis of tumor cells in nude mice, the treatment group living in good condition, adverse reactions on the skin and internal organs were not happened.
Part2:The effect of TMPyP4-PDT on the expression of cervical cancer cell apoptosis、Survivin and Caspase-3in nude mice
Objective: apoptosis is also called programmed cell death which refers to the maintenance of the homeostasis and the death process including multi gene controlled by a series of enzymes. The development of tumor Closely related with cell apoptosis, the exact mechanism is still not clear, but the cells tend to activate the two process opposite, apoptosis and anti apoptosis, the strength of two kinds of signal transduction pathway determines the final outcome. Therefore, cell apoptosis has been a hot research on malignant tumor, and drug treatment target. Some studies found that low doses photosensitizer photodynamic therapy induced cell apoptosis, and the high dose of photodynamic therapy mainly caused the cell necrosis. We used in situ end labeling (TUNEL) study the cervical carcinoma cell apoptosis treated by TMPyP4-PDT. The principle of TUNEL method is that the biotin or fluorescence marker was labeled in the DNA nick end3'-OH, catalyzed by TdT, the color system was markered in the detection of apoptotic cells tissue cells in situ, in situ staining of intact single apoptotic cells and apoptotic bodies. Compared with common HE staining method, morphological characteristics of apoptosis can be reflected more accurately and analyzed statistically. Survivin and cysteine aspartic proteinase3(Caspase-3) in the mechanism of apoptosis are the two key factors, the expression of the combined detection of Survivin and Caspase-3in cervical cancer xenograft in nude mice, may investigate the mechanisms of TMPyP4-PDT inducing apoptosis of cervical cancer cells.
Materials and methods: according to the first part of the experiment and the tumor paraffin section, TUNEL was used to detect the apoptosis of tumor cells, results were investigated by automatic fluorescence microscopy. TUNEL detection method is that10HPF of each case were randomly selected, apoptotic cells were detected in1000cell,percentage count of apoptosis cell is apoptosis index (AI). Detection of Survivin and caspase-3protein is immunohistochemical SP method, brown granules is thoughted the positive cell by immunohistochemical staining of cytoplasm using automatic microscope and image analysis system, each mouse selected3sections of each section, read for10high power field, to calculate the positive staining value,that is optical density(OD), the average optical density was analyzed statistically each group of nude mice.
Results:the testing results of fluorescence intensity display is that TUNEL positive cells expressed green fluorescence. The TUNEL positive cells of C group were more than the single treatment group and the control group,the apoptosis index was higher than the other two groups, which was significant difference; Survivin and caspase-3protein by immunohistochemical detection showed that the expression of the survivin protein was decreased in the three-time treatment groups,but the expression of the caspase-3protein increased.
Conclusion:multiple low dose TMPyP4-PDT therapy could induce apoptosis of cervical cancer cells in nude mice, and that may be related to the expression of survivin decreased and expression of Caspase-3increased.
引文
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