【作者】 陈艾保；

【导师】 丁彦青；

【作者基本信息】 第一军医大学 ， 病理学， 2000， 硕士

【摘要】 大肠癌转移是一个复杂的过程，是肿瘤和宿主相互作用的共同结果，其中肿瘤细胞在肿瘤侵袭的过程中起主导作用，研究瘤细胞生物学特性不仅对研究肿瘤的发生及其发展具有重要意义，而且对了解它们的转移特性具有重要作用。Lovo细胞是大肠癌未分化的高转移腺癌细胞，其转移期间较长，4周的转移率约为40-50％。目前国内应用的Lovo细胞由于经过多次的体外传代培养，细胞已出现退化现象，转移率有逐渐下降的趋势，转移周期偏长。本实验采用体内连续传代方法，应用免疫缺陷动物裸鼠，经脾反复接种瘤细胞，建立肝转移模型，从转移灶分离瘤细胞，经过多次重复接种，使某些高转移亚系瘤细胞逐渐适应并呈优势生长和稳定下来，同时去除不适应和不稳定亚系，获得了较稳定的具有较强转移能力的亚系，该亚系具有在某器官形成高选择转移灶特性，经该方法筛选出的Lovo细胞亚系可用于各种实验性大肠癌的实验和治疗研究。 本研究主要内容包括： 1、应用裸鼠体内连续传代方法，从脾接种建立肝转移模型，通过这种实验性转移，选择并建立高侵袭转移的Lovo细胞亚系。 2、进行Lovo细胞亚系体外相关生物学特性的检测，通过调查瘤细胞电泳泳动速率、运动能力、生长活力、细胞周期和DNA含量及体外侵袭能力等参数，综合分析Lovo亚系细胞的侵袭能力。 3、进行Lovo细胞亚系裸鼠脾脏种植的体内观察与鉴定，探讨它的遗传稳定性、对环境的适应性和转移潜能。 主要方法及结果： l、应用体内连续传代方法，把L。V。细胞接种入裸鼠脾脏，选择肝转移灶，进行瘤细胞分离培养，重复3次脾接种，筛选出高侵袭转移的L。VO细胞亚系。 二、对高侵袭转移L。V。亚系细胞进行裸鼠脾种植实验，结果显示L。VO亚系细胞有遗传很稳定、环境适应性强和转移潜能高等特点。 3、对高侵袭转移L。V。亚系细胞和母系L。V。细胞进行瘤细胞电泳泳动速率检测，研究瘤细胞表面电荷性质和密度变化，结果显示LOVO亚系细胞电泳泳动速率明显高于母系LOV。细胞。 4、应用MTT法测定高侵袭转移L。v。亚系细胞和母系L。v。细胞的活力，研究瘤细胞的生长速度，结果显示L叫。亚系细胞和母系LOVO细胞生长速度有显著差别。 5、应用琼脂糖滴法测定瘤细胞的运动能力，结果显示高侵袭转移LOVO亚系细胞移出琼脂糖滴的细胞数和距离明显高于母系L。V。细胞。 6、应用单层NIH3T3细胞法测定瘤细胞的体外侵袭能力，结果显示高侵袭转移LOvO亚系细胞侵袭能力明显高于母系L。v。细胞。 7、应用 FCM测定瘤细胞的细胞周期和 DNA含量，结果显示高侵袭转移LOV。亚系细胞SP明显高于其母系L。V。细胞，说明DNA合成增加。 本研究结果提示： L。V。亚系细胞是L。VO细胞一高侵袭转移细胞亚系。更多还原

【Abstract】 Metastasis of human colorectal carcinoma(HCC),which is the interaction result between tumor and its host, is a complicated process. Tumor cell is the main part of tumor metastasis.Study on biological feature of tumor cell is significant in the research of tumor’s genesis and development.Lovo is HCC tumor cell and undifferentiated adenocarcinoma cell.Although it’s a strong invasive cell line,its metastasis period is rather long with a high metastasis rate which is up to 40-50% in 4w’s metastasis.Lovo cell applied nowadays begins to decline because of the repeated culture of go- down-to future generation in vitro,metastasis rate tends to decrease gradually with a longer metastasis period.This experiment is to construct a model of liver metastasis by means of continous culture of go-down-to future generation in vivo using nude mouse to repeatedly inoculate tumor’s cell to its spleen and then separate tumor cell from metastasis field to make some subline tumor cells adapt gradually and show its dorminant growth and become stable after several times inoculation,Simultaneously,we remove those 4 unstable subline to obstain relatively stable ones with a comparatively strong metastasis ability.In addition,this subline possesses a high function of selecting one organ to form metastasis field,Lovo cell chosen by this way can both be cultured in vitro and grow easily in proper organ in vivo.Therefore the lovo cell can be used in various experiment and treatment study for HCC. Main contents of this research include: 1. Apply a continous go-down-to the future generation method of mouse, constructing a liver-metastasized nude mouse’s model from spleen inoculation to select strong invasive lovo cell subline through repeated experimental metastasis. 2. From its electrophoresis migration rate. its movement ability its growth curve cell cycline. quantity of DNA and metastasis in vitro to examine lovo cell and analyse its metastasis ability. Main methods and results: 1. Construction of strong invasive lovo cell subline by continous go-down-to future generation in vivo.To found a liver metastasis model by inoculating love cell in nude mouse spleen. 5 2. Make tumor cell’s eletrophoresis migration rate examination for strong invasive lovo cell subline and its mother love cell and to study charge properties and density of tumor cell’s surface.Result shows the electrophoresis migration rate of lovo cell subline is obviously higher than that of its mother lovo cell. 3. Use MTT method to examine growth curve of strong invasive lovo cell subline and its mother lovo cell in order to study cell’s growth speed.The result shows a great difference in growth speed between lovo cell subline and its mother lovo cell. 4. Apply agrose drop method to examine tumor cell’s ability to move.Result shows the cell’s amount and distance of emigrating agrose drop is obviously higher than that of its mother lovo cell.Apply NIH3T3 cell to test tumor cell’s metastasis ability in vitro.The result shows strong invasive lovo cell subline’s ability of invasing NIH3T3 cell is obviously higher than that of its mother lovo cell. 5. Apply FCM to examine更多还原