【作者】 董青；

【导师】 王洁； 董福生；

【作者基本信息】 河北医科大学 ， 口腔临床医学， 2002， 硕士

【摘要】 细胞凋亡(apoptosis)是细胞在机体内和外界环境多种因素所诱导并受基因调控的一种生理死亡，是机体维持稳态的重要机制之一。随着肿瘤发生的观念由细胞生长过度扩展到细胞不死，诱导肿瘤细胞凋亡已成为肿瘤治疗的新方向和研究热点。涎腺腺样囊性癌(salivary adenoidcysticcarcinoma，SACC)是发病率较高的涎腺恶性肿瘤，其恶性程度高，且易复发、转移。选用重组人肿瘤坏死因子α(recombined human tumor necrosis factor α，rhTNF-α)对腺样囊性癌进行凋亡的体内诱导为该肿瘤的治疗提供了一条新途径，以往的实验中我们已在体外证实TNF-α可诱导SACC细胞凋亡。本实验中，我们通过建立SACC裸鼠移植瘤模型，研究TNF-α在体内对SACC的抑瘤作用，并探讨TNF-α的作用机制及其与凋亡的关系，为临床治疗SACC提供新思路和可靠的实验依据。 材料与方法：1．材料(1)实验材料：人涎腺腺样囊性癌细胞系(SACC83)。(2)药品：重组人肿瘤坏死因子α(rhTNF-α)。(3)实验动物：12只SPF级雌性Balb／c裸小鼠，4-5周龄。2．方法：(1)动物模型建立：SACC83细胞培养于RPMI1640含新生牛血清和双抗的培养液中，37℃恒温孵育，收集细胞并调整为6×10~7／ml的细胞悬液。将SACC细胞悬液接种于裸鼠皮下，每只注射注射左前背部与右后背部2个点，每点0.2ml，待成瘤达直径6～10mm， 中文摘要分组注射1’i－J。＊）分组与给药：实验分三组，A实验组每次为 INFI 100X 10‘IU／kg，B实验组为每次 TN’F－a 10 X 10勺U／k，C对照组每次注射生理盐水，皆采用瘤体内直接给药，隔日1次，共8次，每次给药时测量体重及瘤体积。臼）标本采集与检测：处死裸鼠后测量瘤体，剥离瘤体，比较三组间移植瘤体积变化及抑瘤率，应用流式细胞仪检测三组的凋亡率及细胞周期的分布，并采用光镜、透射电镜进行形态学观察。 结果： 1．用药后瘤体增长情况：A实验组增长 0．14士0．18CC’，B实验组增长 0．09士 0．10cm’，C对照组增长 0．45 f 0．27。旷。（P＜o刀1） 2．抑瘤率：A实验组的抑瘤率为52二6％，B实验组的抑瘤率为73刀1％。 3．形态学观察：门）肉眼观察：肿瘤圆形，有包膜，表面光滑，个别瘤体表面有破溃。肿瘤切面为实性，灰白色，部分区域有黄白色豆渣样物，形似坏死组织叶2）光镜观察：肿瘤呈片状排列，部分区域可见囊腔。肿瘤细胞呈圆形或多边形，胞浆红染，胞核圆形，居中，核分裂象易见。少数区域肿瘤细胞透明，出现大量鳞状化生和角化珠，表现出肿瘤性肌上皮细胞的特征。A、B实验组中有成片的凋亡细胞，细胞体积缩小，胞浆固缩，染色质固缩。可见凋亡小体（ApoptOSIS body人 C对照组瘤细胞成片生长，仅有小灶状凋亡。＊）透射电镜：TNF－口实验组的SACC出现大量的凋亡细胞：细胞皱缩，胞体、胞核缩小，染色质固缩，呈块状或颗粒边集于核膜内侧，亦见凋亡小体，对 2 中文摘要 照组瘤细胞圆形，胞核圆形，可见切迹，瘤细胞成片排列， 偶见个别凋亡细胞。 4．流式细胞术检测三组均有特征性的凋亡峰形成八l） 凋亡率：A实验组的凋亡率为N ．sl士 l．68O，B实验组凋 亡率为 15．31士 2．230，C对照组凋亡率为 4．sl士 0．620。A、 B、C三组间凋亡率有显著性差异0＜0刀1人 组间两两比 较凋亡率亦有显著性差异（人8：＊＜0刀1；个C：＊”O刀IZ 8（：P＜0刀1人（2细胞周期：A组＊JG；期细胞数为70石3 士 1．99％，S期细胞数为 15．5士 1．5 7％，G。M期细胞数为 13．85土0．93％；B组 GofGI期细胞数为刀＊ 士5石3％，S期 细胞数为 15．3士 3．67o，GZM期细胞数为 13．59士 2．20O；C 组GJG；期细胞数为65．08士2．60O，S期细胞数为20．33 士2．42％，G。M期细胞数为 14．58士2刀0％。GyG；期细胞数 实验组较对照组增加O功刀1人S期细胞数实验组较对照 组减少（P＜0刀1八A、B组之间无显著差异，说明细胞增 殖在 G；期发生阻滞，G。M 期三组间无显著性差异 （P＞o刀1）。 结论：门）T’i－’刁能够抑制裸鼠 SACC移植瘤生长。 （2）TN’－口可诱导裸鼠体内 SACC细胞凋亡。p）本文中低 剂量的1’i－J的抑瘤效果忧于高剂量的T’i－川。更多还原

【Abstract】 Apoptosis is a physiological cell death induced by many factors in vivo and in vitro. It is controlled by genes. It is one of the important mechanisms that the body keeps stable condition. With the conception of tumor turning from excessive cell growth to no cell death, apoptosis of tumor cells has been new direction and hot area of tumor research. Salivary adenoid cystic carcinoma (SACC) is a kind of malignant tumor with high morbidity. It is highly malignant and tends to relapse and metastasis. It maybe a new way for SACC therapy to induce apoptosis by recombined human tumor necrosis factor alpha (rhTNF- a ) in vivo. In the prior research, we have demonstrated that TNF- a could induce apoptosis of SACC cells in vitro. In this study, we have made SACC animal model in nude mice and then used TNF-a to inhibit SACC . The mechanism of apoptosis was discussed. The new idea and experimental data will be provided to the clinical therapy of SACC.Materials and Methods:1. Materials: (1) cell line: human salivary adenoid cystic carcinoma cell line (SACC83) (2) drug: recombined humantumor necrosis factor alpha (rhTNF- a ) (3) animal: 12 SPF grade 4-5 weeks old female Balb/c nude mice.2. Methods:(1) animal model: SACC83 has been growing in RPMI Medium 1640 supplemented with newborn calf serum and two antibiotics in an incubator at 37. The cells were collected and regulated to 6107 per milliliter. Every mouse was injected 0.2ml of SACC cell liquid subcutaneously in the left anterior back and the right posterior back. (2) groups and reagents: When the tumors on animal model were measured approximately 6-10 mm, the mice were divided into three groups: experimental group A was given 100xl04IU/kg TNF- a , experimental group B was given 10xl04IU/kg TNF-a , group C was given normal saline and used as normal control. The reagents were injected directly into tumor mass once two days and 8 times together. (3) Samples collection and detection: The mice were killed and their tumors were measured and shelled out. The tumor volume and its inhibition ratio of each group were compared. The transplanted tumors from animal models were observed under both light microscope and electron microscope. The percentage of apoptotic cells and cell cycle distribution were detected by the flow cytometer.Results:1. The increase of tumor volume after reagents given: group A increased 0.14+0.18 cm3, group B increased0.09+0.10 cm3, and group C increased 0.45+0.27 cm3.2. The ratio of tumor inhibition: group A was 52.16%, group B was 73. 01%.3. Morpholigic observation:(1) Gross: The tumor was round shape with intact membrane and smooth surface. Occasionally, there was broken on the surface of one or two tumors. The section of tumor was solid and gray-white. Some yellowish-white materials like bean dregs were seen in a few sections, which looked like necrotic tissue.(2) Light microscope (LM): The result revealed that the tumors were arranged in mass. Cystic lumina were formed in some areas. The tumor cells were round or multisided. Cytoplasms of cells were stained red and some were transparent. The nuclei were round and stayed in central. Nuclear division can be observed easily. Lots of squamous cell differentiation and keratin pears were observed in tumors. Those showed the character of neoplastic myoepithelial cell. There were many apoptotic cells in group A and B. The apoptotic cell contracted. Cytoplasm and chromatin condensed. Apoptosis bodies were formed. The tumor cells in group C were arranged in mass with few apoptosis area.(3) Transmission electron microscope (TEM): The result revealed that many apoptotic cells were in group A and B. The nuclei of apoptotic cells were shrinked, chromatinpacked into masses piling against inside of nuclear membrane. Apoptosis bodies were also observed. Tumor cells in control group were round and nuclei were also round, some with wedge shape. Tumor cells were arranged in mass and few apoptotic cells更多还原