【作者】 楼朝阳；

【导师】 冯英明； 陈志南；

【作者基本信息】 第四军医大学 ， 肿瘤学， 2004， 硕士

【摘要】 目的：原发性肝癌是目前发病率高、治疗困难、死亡率高的恶性肿瘤。迄今已建立的一系列人肝癌细胞系(cell line)和人肝癌细胞系的动物模型，为肝癌的发病机理和治疗研究奠定了良好的基础。由于每一种肝癌细胞的染色体特性和癌基因位点均有所不同，而且体外建立的人肝癌细胞系会发生转化，包括基因的丢失、基因的漂移等，和体内的实体瘤之间有显著的差异，同一种细胞系经过反复传代后也会发生转化，这样就给实验的结果带来不确定性。临床及实验资料说明现有的肝癌细胞系资源还远远不能满足研究的需要。因此建立新型人肝癌细胞系有助于研究肝癌发病、进展、转移机制的共同点和不同点。同时可以对多种肝癌细胞系进行药物敏感性实验研究，有望筛选出对某一类肝癌有效的药物，指导临床治疗。 方法： 1．人肝癌细胞系的建立 组织块培养法对活体人肝癌标本进行原代培养；采用自然纯化和反复贴壁法行单一肝癌细胞纯化；光学显微镜观察细胞生长情况；胰蛋白酶法消化传代培养细胞；采用慢冻和快融进行细胞的冻存和复苏。第四军医大学硕士学位论文2.人肝癌细胞系FHCC一98的生物学特性鉴定 细胞形态学观察采用HE染色，光镜观察;超微结构用电镜观察;胎盘蓝染色计数检测冻存后复苏存活率;细胞计数法测定贴壁率;MTT法测细胞生长期曲线;流式细胞仪分析细胞周期和DNA组成;常规制备染色体标本，拍照分析核型;软琼脂克隆形成实验检测细胞克隆形成率;ConA凝集实验检测细胞的聚集特性;Westem一blotting和免疫组织化学法分析CK、AFP和肝癌相关抗原HAb 1 SG/CD147在细胞中的表达;采用PAGE检测FHCC一98的乳酸脱氢酶同工酶谱，拍照分析;裸鼠皮下接种FHCC一98细胞悬液，接种后观察成瘤情况，计算异种移植率;透射电子显微镜鉴定细胞系的污染情况。3.药物对肝癌细胞的敏感性研究3.1 MTT法测定药物对肝癌细胞生长的抑制率 用taxotere、ADM作用于FHee一95和MHee一97一L肝癌细胞，在不同时间点和不同浓度在ELISA仪上测定吸光值，计算药物抑制率。3.2侵袭实验测定药物对肝癌细胞运动能力的抑制 应用Millieell一PCF小室置于24孔板，实验组加Taxotere和ADM，对照组不加药，小室滤膜HE染色后在100倍显微镜下观察照相，图像分析仪计数后计算抑制率。3.3 SCGE测定药物对肝癌细胞FHCC一98 DNA的损伤 采用两层制胶法，细胞裂解后在碱性条件下电泳，莹光显微镜观察Taxotere和ADM对细胞核的损伤。结果:1.细胞原代培养早期，生长不稳定，细胞传代培养时需要较高的接种密度，而且细胞倍增时间不同。到第十二代时细胞生长已非常稳定。细胞呈上皮样，大小不一，以多角形、多边形 第4页第四军医大学硕士学位论文为主，占95%以上。细胞在高密度时(>l xlo。/mL)可见重叠及堆积生长。传代时间一般为2一3天。2.FHCC一98细胞生物学特性鉴定 透射电镜下见细胞大小不等，以不规则型为主，细胞表面有丰富微绒毛，细胞间有桥粒紧密连接等，有少量连接复合体;细胞间有形成腺结构及条索结构倾向，可形成类似胆小管样结构;细胞质丰富，有大量糖原颗粒形成糖原湖样结构;线粒体、粗面内质网较发达，有丰富的游离核糖体;细胞核增大，核浆比例增大，核不规则，有多核细胞，核膜明显凹陷，核仁1一5个不等;核异型性明显，核内以常染色质为主，异染色质较少，可见有核分裂像，偶见异常核分裂。冻存后复苏存活率为95%。细胞生长曲线显示上皮样恶性肿瘤生长特性，细胞群体倍增时间为ZI.4h，sh后贴壁率已超过90%，细胞能在软琼脂中生长，克隆形成率为32%。DNA倍体分析为为四倍体，DNA周期分析:G:为75%，GZ为7%，S为18%，DNA指数为1 .8 57。染色体众数以三倍体为主。LDH同工酶谱显示四条带，缺少LDH;，LDH;和LDHS含量较高。FHCC-98细胞的肝癌相关抗原HAb 18G/C D 147和细胞角蛋白均显示强阳性，而AFP阴性。FHCC一98细胞在低浓度ConA中出现聚集。FHCC一98细胞在裸鼠中的成瘤性为100%。透射电镜检测FHCC一98细胞无支原体污染。3.抗癌药物对不同肝癌细胞的抑制率3.1经 Taxotere处理的FHCC一98细胞的增殖有明显的抑制，且随时间和浓度的增加其抑制率增加，药物在SX10一Zog/mL浓度作用FHCC一98细胞48h后其增殖抑制率达到30%以上，而对MHCC一97一L细胞无作用。ADM对两株肝癌细胞均有作用，较Taxotere的作用为强，新建细胞系FHCC一98对ADM的敏感性较MHCC一97一L强，经Prism统计软件分析不同浓度药物的 第5页第四军医大学硕士学位论文抑制率均有统计学意义(P<0.05)。3.2侵袭小室实验显示Taxotere对FHCC一98的穿膜能力有明显的抑制，且呈浓度依赖性。ADM结果不确定。3.3 SCGE显示阿霉素对FHCC一98细胞的DNA有一定程度的损伤。细胞核出现部分碎裂，细胞核电泳出现彗星现象。而泰索帝组没有出现彗星想象。结论:1.由人肝癌组织培养的FHCC一98细胞系能稳定生长，具有无限生长的能力。该细胞系保持了来源组织的一些特性，AFP和HBsAg阴性，细更多还原

【Abstract】 Aims: Hepatocellular carcinoma (HCC) is a kind of malignant disease that is difficult to treat and has a high incidence and a high mortality at present. The established cell lines and animal models of human liver cancer have settled a good foundation for study of pathogenesis and treatment of HCC. There are variations of chromosomes and genie location among HCC cell lines, and the established HCC cell lines in vitro could be transformed, include loss and drift of genes, etc, also there is obvious difference between cell lines and their original tissue. Yet the same cell line could be transformed after passaged repeatedly, so the test results are uncertainty. Clinic and laboratory data shows limit resource of cell lines can’t satisfy the needs of study. More kinds of new hum-an HCC cell lines are needed, and can be help to study the commonand different points of pathogenesis, progress and metastasis ofliver cancer. Drug sensitivity can be tested through kinds of HCCcell lines, so that some efficient drugs can be screened againstcertain liver cancer and provide instruction on clincic treatment.Methods:1.Establishment of human HCC cell lineThe sample of human HCC was primary cultured by tissue culturing. The single HCC cell were obtained by natural purification and anchoring repeatedly. The cells were passaged with trypsin. Growth status was observed under light microscope.The cells were freezed with temperature down slowly in liquid nitrogen and resuscitated quickly at 37C. 2.Characteristics of human HCC cell line FHCC-98.FHCC-98 cell morphology was observed under light and electron microscope. The survival rate was measured by counting cells stained with protamine Blue.Seeding efficiency was measured by counting cell number every 2 h. Cell growth curve was plotted with MTT assay. Studies of cell cycle and stage were performed by flow cytometry.Chromosomes was made by traditional method and analyzed by photographed. Cloning efficiency was measured by counting clone number growing in soft agar. The coagulation characteristics was detected by ConA test. Expressions of tumor markers such as AFP, CK and HAbl8G/CD147 were detected by SP immunocytochemistry or Western blotting. LDH isoenzymes were detected by polyacrylamid gel PAGE and photographed analyzed. Xenograft was performed by inoculating FHCC-98 cellsinto the flanks of the nude mice s.c and the tumors were observed, thus xenograft rate was counted. Cell contamination was detected by transmission electron microscopy.3.Study of drug sensitivity against HCC cell lines3.1 MTT assay was used to detect the inhibition rate of HCC cell growthTaxotere and ADM were used to effect on FHCC-98 and MHCC-97-L cells. OD was measured by ELISA at different time and concentration, and inhibition rate was counted.3.2 Invasion test was used to investigate the mobility inhibition of FHCC-98Millicell-PCF transwell chambers were put in 24-plate, taxotere and ADM were added to test group, and no drugs in controlled one. Transwellchamber’s membrane was HE stained and photographed. Inhibition rate was measured with imaging analyser.3.3 DNA damage of FHCC-98 cell was dectected by SCGETwo layers of gel was made, and FHCC-98 cells were el-ectrophoresised in alkaline condition after lysis, then nucleus damage by taxotere and ADM was observed under fluorescence microscope. Results:1. Cells grew instably at the early stage of primary culture and needed a high density when passaged. Population doubling time was not the same. But the cells grew very stably at 12th passage. Cells were mainly polygonal epithelial-like in various size over 95%. Cells grew in overlap and piled up in high density (>1 X 106/mL). Passaging time was 2 -3 days average.2. Transmission electron-microscopy showed cells were invarious size, mainly irregular type. There were abundant microvilli on the cell surface. Desmosomes and gap junction could be seen between cells, also with a few junctional complexs. There was a tendency to form structure of gland and strand alike. St更多还原