稳定表达荧光的高转移人肝癌裸鼠模型的建立及其肿瘤生物学特性

发布时间：2018-05-30 10:18

本文选题：肝细胞肝癌 + 异种移植模型　；参考：《复旦大学》2008年硕士论文

【摘要】： 研究背景:原发性肝癌是我国乃至全球的高发恶性肿瘤之一。全世界每年新诊断的原发性肝癌病例有一半在中国。尽管临床上已有多种治疗肝癌的成熟方法,比如手术切除、化疗、放疗等,但肝癌患者五年的复发与转移率仍高达60%,总体生存率低于5%。这可能与肝癌发病隐匿,肿瘤易早期播散;传统疗法不能彻底消灭患者体内的癌细胞;传统疗法存在较大的毒副作用而不能反复应用等有关。复旦大学肝癌研究所经过长期研究,已建立了人肝细胞癌裸小鼠模型、高转移人肝细胞癌裸小鼠模型、高转移人肝细胞癌细胞系LCI-D20、MHCC97H、HCCLM3和HCCLM6,为肝癌治疗、转移复发研究提供了有效的手段,但这些模型的缺点是不能方便、连续、直观地观察原位肿瘤及肺和腹腔转移灶,定量也不够准确。因此,有必要建立一个能够实时方便观察的人肝癌裸鼠模型。表达荧光蛋白的HCC(Hepatocellular Carcinoma)细胞株和相应的裸鼠模型为肿瘤生长转移的实时观察提供了简便的方法。近年来,国外已经建立了荧光可视的黑素瘤、前列腺癌等模型,而稳定表达荧光的肝癌裸鼠模型尚未建立。本研究用慢病毒载体系统建立了稳定表达绿色/红色荧光蛋白的HCCLM3细胞及相应的裸鼠模型,并且研究了其肿瘤生物学特性。研究目的:建立稳定表达荧光蛋白的高转移人肝癌裸鼠模型并研究其肿瘤生物学特性,建立一个良好的肝癌动物试验研究平台。一表达荧光蛋白的高转移人肝癌细胞系的建立及其生物学特性本研究用假慢病毒载体系统把增强型绿色荧光蛋白或红色荧光蛋白(GFP/RFP)基因导入高转移人肝癌细胞系HCCLM3,使其成为稳定表达绿色/红色荧光蛋白的细胞,分别将其命名为HCCLM3-G、HCCLM3-R。体外连续培养300天、60代,在荧光显微镜下观察,细胞荧光强、表达稳定。提取新细胞基因组DNA,用PCR方法证实GFP/RFP基因已经整合入细胞基因组,且能够稳定传代。试验对比了转染前后的肝癌细胞在形态、增殖、核型以及主要蛋白AFP、CK8、P16、HbsAg等表达方面的变化,发现转染后的肝癌细胞的上述生物学特性无明显改变。本研究还利用了转染GFP/RFP后细胞稳定表达荧光的特点,分析了细胞荧光面积与细胞计数的相关性,确立了用计算细胞荧光面积替代细胞计数获得细胞生长曲线的方法。该方法是一种快速、方便、连续、可重复的研究体外细胞增殖的新方法,可以应用于体外抗肝癌药物的筛选。二荧光可视高转移人肝癌裸鼠模型的建立及其肿瘤生物学特性本研究在建立稳定表达绿色/红色荧光的肝癌细胞系的基础上,用皮下接种及组织块移植法分别建立了裸鼠皮下和肝脏原位肿瘤模型。定期在荧光体视显微镜下活体观察皮下和原位肿瘤。原位肿瘤移植后2周,即可观察到肿瘤荧光,肿瘤存活率100%。经过裸鼠体内连续传代6次,肿瘤依然稳定而强烈地表达绿色/红色荧光。第6周处死裸鼠,解剖观察后可见两种原位肿瘤模型的肺转移率、肝内播散率和腹腔转移率均分别为100%、100%、90%。绿色和红色荧光模型在裸鼠体内传代6代,共9个月后,肿瘤荧光表达稳定,而且在成瘤率、转移率和AFP的表达方面也保持稳定。传统的动物模型不借助病理检验就难以了解肿瘤的转移情况,定量也不准确。利用稳定表达荧光蛋白的肿瘤模型建立了一种活体定量肝脏原位肿瘤大小、离体定量肺、腹腔转移的比较方便、可信的方法。本研究同时按常规方法计算出肿瘤体积,通过分析肿瘤体积与累积光密度(Integrated Optical Density,IOD)之间的相关性,确认了肿瘤IOD曲线可以替代肿瘤体积描述裸鼠肝脏原位肿瘤的生长情况。肺转移是肿瘤生物学特点的另一个重要指标,过去无荧光的裸鼠肝脏原位人肝癌模型是通过肺石蜡标本连续切片后读片,按切片最大转移灶细胞数分级,是一种半定量的分析方法,不够方便和经济。而利用表达荧光蛋白的新模型就可以比较准确方便地对肺和腹腔转移灶进行定量。
[Abstract]:Background: primary liver cancer is one of the high incidence of malignant tumors in China and even around the world. Half of the world's newly diagnosed cases of primary liver cancer are in China every year. Although there are many methods to treat liver cancer, such as surgical resection, chemotherapy, radiotherapy and so on, the recurrence and metastasis rate of liver cancer patients in five years is still up to 60%. The survival rate is lower than 5%., which may be occult with the pathogenesis of liver cancer, and the tumor is easily disseminated early; traditional therapy can not completely eliminate the cancer cells in the patient's body; the traditional therapy has large toxic side effects and can not be used repeatedly.
After a long study, the Liver Cancer Institute of Fudan University has established a nude mouse model of human hepatocellular carcinoma, a highly metastatic HCC nude mouse model, a highly metastatic human hepatocellular carcinoma cell line LCI-D20, MHCC97H, HCCLM3 and HCCLM6, which provide effective means for the treatment of liver cancer and transfer recurrence, but the shortcomings of these models are inconvenient and continuous. It is necessary to establish a human hepatoma nude mouse model that can be observed in real time and conveniently.
The HCC (Hepatocellular Carcinoma) cell lines expressing fluorescent protein and the corresponding nude mice model provide a simple method for the real-time observation of tumor growth and metastasis. In recent years, foreign models such as fluorescent visible melanoma and prostate cancer have been established, and the nude mice model for the stable expression of fluorescent liver cancer has not been established. The HCCLM3 cells and their corresponding nude mice expressing stable green / red fluorescent protein were established, and their tumor biological characteristics were studied.
Objective: to establish a nude mouse model of high metastatic human hepatocellular carcinoma (HCC) with stable expression of fluorescent protein and to study its biological characteristics, and to establish a good experimental research platform for liver cancer.
Establishment of a highly metastatic human hepatoma cell line expressing fluorescent protein and its biological characteristics
This study used the pseudo lentivirus vector system to transfer the enhanced green fluorescent protein or red fluorescent protein (GFP/RFP) gene into the high metastatic human hepatoma cell line HCCLM3, making it a cell that stably expressed green / red fluorescent protein. It was named HCCLM3-G, and the HCCLM3-R. body was cultured for 300 days, 60 generations, and observed under the fluorescence microscope. The cell fluorescence was strong and the expression was stable. The genomic DNA of the new cells was extracted. The PCR method was used to confirm that the GFP/RFP gene had been integrated into the cell genome and could be stable. The changes in the morphology, proliferation, karyotype and the expression of the major protein AFP, CK8, P16, HbsAg, etc. were compared before and after the transfection, and the hepatoma cells after transfection were found. There was no obvious change in the biological characteristics mentioned above.
This study also made use of the characteristics of stable expression of fluorescence after transfection of GFP/RFP cells, analyzed the correlation between cell fluorescence area and cell count, and established a method to obtain cell growth curve by calculating cell fluorescence area instead of cell count. This method is a fast, convenient, continuous, and repeatable method for the study of cell proliferation in vitro. The method can be applied to the screening of anti hepatoma drugs in vitro.
Establishment of two fluorescent visible high metastatic human liver cancer nude mice model and its tumor biological characteristics
On the basis of establishing a liver cancer cell line that expresses green / red fluorescence, the tumor model of the subcutaneous and liver in nude mice was established by subcutaneous inoculation and tissue mass transplantation. The tumor in situ and in situ were observed regularly under the fluorescence microscope. The tumor fluorescence and tumor could be observed 2 weeks after the transplantation of the tumor in situ. The survival rate of 100%. was continuously passaged in nude mice for 6 times. The tumor remained stable and strongly expressed in green / red fluorescence. Sixth weeks were executed in nude mice. The pulmonary metastasis rate of two tumor model in situ was observed after sixth weeks, and the rate of intraperitoneal transmission and peritoneal metastasis were 100% and 100% respectively, and the green and red fluorescence models were passaged in the nude mice for 6 generations. After 9 months, the fluorescence expression of tumor was stable, and the tumor formation rate, metastasis rate and AFP expression remained stable.
The traditional animal model is difficult to understand the metastasis of tumor without the aid of pathological examination. It is not accurate to quantify the tumor. Using a tumor model with stable expression of fluorescent protein, a kind of living quantitative liver tumor in situ tumor size, in vitro quantitative lung, abdominal metastasis is more convenient and credible. The tumor volume, by analyzing the correlation between the tumor volume and the Integrated Optical Density (IOD), confirms that the tumor IOD curve can replace the tumor volume to describe the growth of the liver in situ tumor in nude mice. The lung metastasis is another important index of the tumor biological characteristics, in the past non fluorescent nude mice liver in situ The cancer model is read by continuous slice of paraffin specimens. It is a semi quantitative analysis method according to the number of cell number of the maximum metastases, which is not convenient and economical. A new model of the expression of fluorescent protein can be used to quantify the lung and abdominal metastases accurately and conveniently.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R735.7;R-332

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