發(fā)布時間：2018-05-30 10:18

  本文選題：肝細胞肝癌 + 異種移植模型��； 參考：《復(fù)旦大學(xué)》2008年碩士論文

【摘要】： 研究背景:原發(fā)性肝癌是我國乃至全球的高發(fā)惡性腫瘤之一。全世界每年新診斷的原發(fā)性肝癌病例有一半在中國。盡管臨床上已有多種治療肝癌的成熟方法,比如手術(shù)切除、化療、放療等,但肝癌患者五年的復(fù)發(fā)與轉(zhuǎn)移率仍高達60%,總體生存率低于5%。這可能與肝癌發(fā)病隱匿,腫瘤易早期播散;傳統(tǒng)療法不能徹底消滅患者體內(nèi)的癌細胞;傳統(tǒng)療法存在較大的毒副作用而不能反復(fù)應(yīng)用等有關(guān)。 復(fù)旦大學(xué)肝癌研究所經(jīng)過長期研究,已建立了人肝細胞癌裸小鼠模型、高轉(zhuǎn)移人肝細胞癌裸小鼠模型、高轉(zhuǎn)移人肝細胞癌細胞系LCI-D20、MHCC97H、HCCLM3和HCCLM6,為肝癌治療、轉(zhuǎn)移復(fù)發(fā)研究提供了有效的手段,但這些模型的缺點是不能方便、連續(xù)、直觀地觀察原位腫瘤及肺和腹腔轉(zhuǎn)移灶,定量也不夠準確。因此,有必要建立一個能夠?qū)崟r方便觀察的人肝癌裸鼠模型。 表達熒光蛋白的HCC(Hepatocellular Carcinoma)細胞株和相應(yīng)的裸鼠模型為腫瘤生長轉(zhuǎn)移的實時觀察提供了簡便的方法。近年來,國外已經(jīng)建立了熒光可視的黑素瘤、前列腺癌等模型,而穩(wěn)定表達熒光的肝癌裸鼠模型尚未建立。本研究用慢病毒載體系統(tǒng)建立了穩(wěn)定表達綠色/紅色熒光蛋白的HCCLM3細胞及相應(yīng)的裸鼠模型,并且研究了其腫瘤生物學(xué)特性。 研究目的:建立穩(wěn)定表達熒光蛋白的高轉(zhuǎn)移人肝癌裸鼠模型并研究其腫瘤生物學(xué)特性,建立一個良好的肝癌動物試驗研究平臺。 一表達熒光蛋白的高轉(zhuǎn)移人肝癌細胞系的建立及其生物學(xué)特性 本研究用假慢病毒載體系統(tǒng)把增強型綠色熒光蛋白或紅色熒光蛋白(GFP/RFP)基因?qū)敫咿D(zhuǎn)移人肝癌細胞系HCCLM3,使其成為穩(wěn)定表達綠色/紅色熒光蛋白的細胞,分別將其命名為HCCLM3-G、HCCLM3-R。體外連續(xù)培養(yǎng)300天、60代,在熒光顯微鏡下觀察,細胞熒光強、表達穩(wěn)定。提取新細胞基因組DNA,用PCR方法證實GFP/RFP基因已經(jīng)整合入細胞基因組,且能夠穩(wěn)定傳代。試驗對比了轉(zhuǎn)染前后的肝癌細胞在形態(tài)、增殖、核型以及主要蛋白AFP、CK8、P16、HbsAg等表達方面的變化,發(fā)現(xiàn)轉(zhuǎn)染后的肝癌細胞的上述生物學(xué)特性無明顯改變。 本研究還利用了轉(zhuǎn)染GFP/RFP后細胞穩(wěn)定表達熒光的特點,分析了細胞熒光面積與細胞計數(shù)的相關(guān)性,確立了用計算細胞熒光面積替代細胞計數(shù)獲得細胞生長曲線的方法。該方法是一種快速、方便、連續(xù)、可重復(fù)的研究體外細胞增殖的新方法,可以應(yīng)用于體外抗肝癌藥物的篩選。 二熒光可視高轉(zhuǎn)移人肝癌裸鼠模型的建立及其腫瘤生物學(xué)特性 本研究在建立穩(wěn)定表達綠色/紅色熒光的肝癌細胞系的基礎(chǔ)上,用皮下接種及組織塊移植法分別建立了裸鼠皮下和肝臟原位腫瘤模型。定期在熒光體視顯微鏡下活體觀察皮下和原位腫瘤。原位腫瘤移植后2周,即可觀察到腫瘤熒光,腫瘤存活率100%。經(jīng)過裸鼠體內(nèi)連續(xù)傳代6次,腫瘤依然穩(wěn)定而強烈地表達綠色/紅色熒光。第6周處死裸鼠,解剖觀察后可見兩種原位腫瘤模型的肺轉(zhuǎn)移率、肝內(nèi)播散率和腹腔轉(zhuǎn)移率均分別為100%、100%、90%。綠色和紅色熒光模型在裸鼠體內(nèi)傳代6代,共9個月后,腫瘤熒光表達穩(wěn)定,而且在成瘤率、轉(zhuǎn)移率和AFP的表達方面也保持穩(wěn)定。 傳統(tǒng)的動物模型不借助病理檢驗就難以了解腫瘤的轉(zhuǎn)移情況,定量也不準確。利用穩(wěn)定表達熒光蛋白的腫瘤模型建立了一種活體定量肝臟原位腫瘤大小、離體定量肺、腹腔轉(zhuǎn)移的比較方便、可信的方法。本研究同時按常規(guī)方法計算出腫瘤體積,通過分析腫瘤體積與累積光密度(Integrated Optical Density,IOD)之間的相關(guān)性,確認了腫瘤IOD曲線可以替代腫瘤體積描述裸鼠肝臟原位腫瘤的生長情況。肺轉(zhuǎn)移是腫瘤生物學(xué)特點的另一個重要指標,過去無熒光的裸鼠肝臟原位人肝癌模型是通過肺石蠟標本連續(xù)切片后讀片,按切片最大轉(zhuǎn)移灶細胞數(shù)分級,是一種半定量的分析方法,不夠方便和經(jīng)濟。而利用表達熒光蛋白的新模型就可以比較準確方便地對肺和腹腔轉(zhuǎn)移灶進行定量。
[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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R735.7;R-332

【共引文獻】

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