不同转移潜能人肝癌细胞模型的分子细胞遗传学研究
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摘要
肝细胞癌是最常见的肿瘤之一，而转移复发则成为提高肝癌病人预后的主要障碍。为探索究竟何种细胞遗传改变的积累导致转移这一恶性表型的形成，复旦大学肝癌研究所在建立人肝癌转移模型(LCI-D20)基础上，成功建成了世界上首个高转移潜能的人肝癌细胞系MHCC97，并成功分离出具有不同转移潜能的细胞克隆MHCC97-H和MHCC97-L；这两种细胞经皮下接种、扩增成瘤、原位肝脏移植后肺转移率分别为100％和40％。最近又建成了具有更高转移力的细胞系HCCLM3，其细胞皮下接种后就发生了100％肺转移。这些细胞系和克隆为研究肝癌转移相关的细胞遗传学异常和分子标记提供了工具。
我所既往利用比较基因组杂交技术(CGH)在肝癌临床标本和对应转移灶中发现了8p缺失与肝癌转移有关；对高低转移模型LCI-D20和LCI-D35以及MHCC97-H细胞系的CGH分析进一步证实了这一发现。在此基础上，对22例临床标本杂和性缺失(loss of heterozygosity, LOH)的研究将缺失区域进一步缩小到8p23.3区和8p11.2区。
为查明我所新建立的这些具有同一遗传背景而又在肝癌的转移潜能上存在明显差异的细胞是否也具有8p的缺失，并弄清它们在其它染色体及其区段的DNA水平上有着怎样的相似和不同，这些相似与不同又有着怎样的意义，本课题联合应用G显带技术、比较基因组杂交技术(CGH)、多重荧光原位杂交技术(M-FISH)和位点或臂特异荧光原位杂交技术，对上述细胞进行细胞及分子遗传学研究，以期了解其分子细胞遗传学异常的特征、找出转移相关的遗传学标记，为探讨肝癌转移机制提供材料。
第一部分
以荧光原位杂交为基础的分子细胞遗传学技术平台的建立及其初步运用
荧光原位杂交(Fluorescence in situ hybridization, FISH)及其相关分子细胞遗传学技术，国内仅少数实验室做的比较理想。本部分旨在我所建立以FISH为基础的分子遗传学技术平台。
首先以双色荧光原位杂交对高转移潜能细胞克隆MHCC97-H进行间接法FISH分析。采用商业途径获得的8号着丝粒探针和自己合成的来自8q23.1区的
不同转移潜能人肝癌细胞模型的分子细胞遗传学研究
博士论文摘要
BAC探针进行FISH杂交，结果两种探针的穿透性、特异性和检测时的强度均较
好。初步发现MHCC97一H克隆8号染色体呈三体性改变和其长臂信号向短臂区
域易位及向B组染色体(4号染色体)的非交互性易位。
据此结果，利用购买的8号染色体特异性涂染探针(Whole chromosome
painting，WCp)，对MHCC97一H克隆进行杂交，不但优化了直接法探针的各项技
术要点，还验证了双色FlsH的发现。
在双色FISH的基础上，我们建立了比较基因组杂交(Co哪arative genomic
hybridization，CGH)技术，并对低转移潜能或不转移的人肝癌细胞系SMMC一7721
进行分析。CGH各项技术指标如目标片染色体长度，分裂像指数以及信号强度
和均一性都达到默认设置。在SMMC一7721细胞系中检测出了扩增的区域有IP31，
lq25，3P22一Pter， SP，6P21.3一Pter， 7P13一Pter， 8P23，SPll一q12，9q22一qter， 10P12一q21，
11pter一qZI，llplZ一ql4，14ql3一qter， 15qls一qter， 17p，Xp14一pter;而缺失区域有
4q31一35，gPZI一3，1 3qZI一31，18q，YqO
最后，我们探索了多重荧光原位杂交(Multiplex fluoreseenee in situ
hybridization，M一FISH)技术，并对HCCLM3细胞系进行分析。结果表明混合
探针的特异性和亮度均可，发生了易位的标志染色体有der(Y)t( Y;18)，
der(3)t(3:20)，der(4)(4;8)，der(9)t(9;13)，der(14)t(14:22)，der(15)t(15:21)，此外还观察
到了X，1号和8号染色体内部的结构异常。
双色FISH、染色体涂染FISH、间接法CGH和MFISH技术的建立，为运用
这些技术手段对不同转移潜能的细胞株和临床标本进行分子遗传学的分析及比
较研究提供了条件。
第二部分
不同转移潜能的人肝癌细胞克隆的分子细胞遗传学分析
我们联合应用G显带技术、CGH、MFISH以及染色体臂和/或区域特异性
荧光原位杂交技术(FISH)，对从MHCC97分离出的不同转移能力的细胞克隆
MHCC97一H、MHCC97一L和HCCLM3进行分子细胞遗传学研究。发现:
1 .1(X)(qlo)，der(Y)t(y;18)(qlZ;Pll)，+der(3)t(3:20)(P25:ql3)，
der(4)t(4;8)(q31:q22)，i(8)(qlo)，der(14)t(14:22)印13:ql3)等染色体异常在
三种细胞系中均出现，是其标志染色体，证实了它们的共同起源。
2.CGH发现三者各有其特异的遗传学改变特征，如+gqlZ一21，17q24一qter，
18q22一23，一13ql3，一13q22一31只出现在MHCC97一H中发生;而+Zq，+llq，
尹
不同转移潜能人肝癌细胞模型的分子细胞遗传学研究
博士论文摘要
+9q33一ter只出现于MHCC97一L;+3qZI一24，+3q13.3一qter，+9p12一qter，
+lopls，+12p，+17plZ一qlZ，+18pll.3一pter，+19p，一2p23一pter，等是HCCLM3
特有的改变。三种细胞中均存在8p23的缺失，而低转移或不转移的
sMMc一7721细胞8P区没有缺失;此外，我们利用改良的CGH方法，
发现在高转移的MHCC97一H细胞?
Hepatocelluar carcinoma (HCC) is one of the most common human cancers worldwide, and metastatic recurrence is the major obstacle to improve the prognosis of HCC patients. In order to known how the accumulation of genetic changes give rise to this aggressive phenotype, consistent efforts have been made by us to get the ideal cell model for in vitro studies on mechanisms underlying the carcinogenesis and tumor progression of HCC. Recently, two cell clones (MHCC97-H and MHCC97-L) with different metastatic potentials were successfully subcloned from the metastatic human HCC cell line MHCC97 in our institute, and another cell line designated HCCLM3 which was able to produce more extensive metastases via both subcutaneous and orthotopic inoculation in athymic nude mice was successively established. These cells provide us valuable tools for identifying cytogenetic aberrations and molecular markers associated with the metastasis of HCC.
In our previous studies, through CGH analysis of both clinical tumor samples and the animal models, 8p deletion was found to be one of the most obvious aberrations in HCC, and might be associated with HCC metastasis. In most recently, we had used more precise genome-wide scan methods such as microsatellite analysis to detected about 22 HCC tumor and their matched metastasis focal, and the 8p23.3 and 8p11.2 were found to be related to progression and metastasis of HCC.
To identify whether our previous founding were exist in the series of our newly established cell cultures with the same genetic background and different metastatic potentials, and to make clear what are congenerous and various traits in the chromosomal or DMA sequence levels between these cells, in this study, a combination of conventional G-banding, comparative genomic hybridization (CGH), multiplex fluorescence in situ hybridization (M-FISH) and arm or locus-specific fluorescence in situ hybridization (FISH) were used to comprehensively characterize molecular cytogenetics aberrations of the above cell cultures. This will provide clues to the molecular mechanisms involved in the HCC metastasis.
Part one
Establishment of the FISH-based molecular cytogenetic techniques and its primary application
The major objectives of this part were to establish the novel methods based on fluorescence in situ hybridization (FISH) techniques such as comparative genomic hybridization (CGH), multiplex fluorescence in situ hybridization (M-FISH) and whole chromosome painting (WCP) in our institute.
First, indirect two-color FISH was performed on the metaphase of MHCC97-H cells. The alpha satellite pericentromeric probe specific to chromosome 8 (D8Z2) was obtained from Oncor (Qbiogene, Cedex, France), and the DMA of BAG clone RP11-328M16 mapping at 8q23.1 was extracted and labeled by ourselves. The results showed that the penetrating ability, specificity and signal strength of the both probes were acceptable. The trisomy of chromosome 8 and the non-reciprocal translocation of partial 8q to one chromosome in group B (Chromosome 4) were found.
Second, the directly labeled whole chromosome painting (WCP) probes were purchased from Vysis Company (IL, USA, the MFISH probes also came from this incorporation), and the steps of the hybridization (almost the same as MFISH) were successfully optimized. The findings of numerical and structural changes of chromosome 8 in MHCC97-H were confirmed.
Based on the experience of two-color FISH, we analyzed human hepatocellular carcinoma (HCC) cell line SMMC-7721 with low or none metastatic potential by Indirect CGH. The key parameters of CGH, such as the signal granulation, signal intensity, chromosome length/width and separated chromosomes were all reached the default value. The gains of 1p31, 1q25, 3p22-pter, 5p, 6p21.3-pter, 7p13-pter, 8p23, 8p11-q12, 9q22-qter, 10p12-q21, 11pter-q21, 11p12-q14, 14q13-qter, 15q15-qter, 17p, Xp14-pter and the losses of 4q31-35, 9p21-3, 13q21-31, 18q,Yq were detected in this cell line. These changes may serve as the control for our subsequent research on mechanism o
我所既往利用比较基因组杂交技术(CGH)在肝癌临床标本和对应转移灶中发现了8p缺失与肝癌转移有关；对高低转移模型LCI-D20和LCI-D35以及MHCC97-H细胞系的CGH分析进一步证实了这一发现。在此基础上，对22例临床标本杂和性缺失(loss of heterozygosity, LOH)的研究将缺失区域进一步缩小到8p23.3区和8p11.2区。
为查明我所新建立的这些具有同一遗传背景而又在肝癌的转移潜能上存在明显差异的细胞是否也具有8p的缺失，并弄清它们在其它染色体及其区段的DNA水平上有着怎样的相似和不同，这些相似与不同又有着怎样的意义，本课题联合应用G显带技术、比较基因组杂交技术(CGH)、多重荧光原位杂交技术(M-FISH)和位点或臂特异荧光原位杂交技术，对上述细胞进行细胞及分子遗传学研究，以期了解其分子细胞遗传学异常的特征、找出转移相关的遗传学标记，为探讨肝癌转移机制提供材料。
第一部分
以荧光原位杂交为基础的分子细胞遗传学技术平台的建立及其初步运用
荧光原位杂交(Fluorescence in situ hybridization, FISH)及其相关分子细胞遗传学技术，国内仅少数实验室做的比较理想。本部分旨在我所建立以FISH为基础的分子遗传学技术平台。
首先以双色荧光原位杂交对高转移潜能细胞克隆MHCC97-H进行间接法FISH分析。采用商业途径获得的8号着丝粒探针和自己合成的来自8q23.1区的
不同转移潜能人肝癌细胞模型的分子细胞遗传学研究
博士论文摘要
BAC探针进行FISH杂交，结果两种探针的穿透性、特异性和检测时的强度均较
好。初步发现MHCC97一H克隆8号染色体呈三体性改变和其长臂信号向短臂区
域易位及向B组染色体(4号染色体)的非交互性易位。
据此结果，利用购买的8号染色体特异性涂染探针(Whole chromosome
painting，WCp)，对MHCC97一H克隆进行杂交，不但优化了直接法探针的各项技
术要点，还验证了双色FlsH的发现。
在双色FISH的基础上，我们建立了比较基因组杂交(Co哪arative genomic
hybridization，CGH)技术，并对低转移潜能或不转移的人肝癌细胞系SMMC一7721
进行分析。CGH各项技术指标如目标片染色体长度，分裂像指数以及信号强度
和均一性都达到默认设置。在SMMC一7721细胞系中检测出了扩增的区域有IP31，
lq25，3P22一Pter， SP，6P21.3一Pter， 7P13一Pter， 8P23，SPll一q12，9q22一qter， 10P12一q21，
11pter一qZI，llplZ一ql4，14ql3一qter， 15qls一qter， 17p，Xp14一pter;而缺失区域有
4q31一35，gPZI一3，1 3qZI一31，18q，YqO
最后，我们探索了多重荧光原位杂交(Multiplex fluoreseenee in situ
hybridization，M一FISH)技术，并对HCCLM3细胞系进行分析。结果表明混合
探针的特异性和亮度均可，发生了易位的标志染色体有der(Y)t( Y;18)，
der(3)t(3:20)，der(4)(4;8)，der(9)t(9;13)，der(14)t(14:22)，der(15)t(15:21)，此外还观察
到了X，1号和8号染色体内部的结构异常。
双色FISH、染色体涂染FISH、间接法CGH和MFISH技术的建立，为运用
这些技术手段对不同转移潜能的细胞株和临床标本进行分子遗传学的分析及比
较研究提供了条件。
第二部分
不同转移潜能的人肝癌细胞克隆的分子细胞遗传学分析
我们联合应用G显带技术、CGH、MFISH以及染色体臂和/或区域特异性
荧光原位杂交技术(FISH)，对从MHCC97分离出的不同转移能力的细胞克隆
MHCC97一H、MHCC97一L和HCCLM3进行分子细胞遗传学研究。发现:
1 .1(X)(qlo)，der(Y)t(y;18)(qlZ;Pll)，+der(3)t(3:20)(P25:ql3)，
der(4)t(4;8)(q31:q22)，i(8)(qlo)，der(14)t(14:22)印13:ql3)等染色体异常在
三种细胞系中均出现，是其标志染色体，证实了它们的共同起源。
2.CGH发现三者各有其特异的遗传学改变特征，如+gqlZ一21，17q24一qter，
18q22一23，一13ql3，一13q22一31只出现在MHCC97一H中发生;而+Zq，+llq，
尹
不同转移潜能人肝癌细胞模型的分子细胞遗传学研究
博士论文摘要
+9q33一ter只出现于MHCC97一L;+3qZI一24，+3q13.3一qter，+9p12一qter，
+lopls，+12p，+17plZ一qlZ，+18pll.3一pter，+19p，一2p23一pter，等是HCCLM3
特有的改变。三种细胞中均存在8p23的缺失，而低转移或不转移的
sMMc一7721细胞8P区没有缺失;此外，我们利用改良的CGH方法，
发现在高转移的MHCC97一H细胞?
Hepatocelluar carcinoma (HCC) is one of the most common human cancers worldwide, and metastatic recurrence is the major obstacle to improve the prognosis of HCC patients. In order to known how the accumulation of genetic changes give rise to this aggressive phenotype, consistent efforts have been made by us to get the ideal cell model for in vitro studies on mechanisms underlying the carcinogenesis and tumor progression of HCC. Recently, two cell clones (MHCC97-H and MHCC97-L) with different metastatic potentials were successfully subcloned from the metastatic human HCC cell line MHCC97 in our institute, and another cell line designated HCCLM3 which was able to produce more extensive metastases via both subcutaneous and orthotopic inoculation in athymic nude mice was successively established. These cells provide us valuable tools for identifying cytogenetic aberrations and molecular markers associated with the metastasis of HCC.
In our previous studies, through CGH analysis of both clinical tumor samples and the animal models, 8p deletion was found to be one of the most obvious aberrations in HCC, and might be associated with HCC metastasis. In most recently, we had used more precise genome-wide scan methods such as microsatellite analysis to detected about 22 HCC tumor and their matched metastasis focal, and the 8p23.3 and 8p11.2 were found to be related to progression and metastasis of HCC.
To identify whether our previous founding were exist in the series of our newly established cell cultures with the same genetic background and different metastatic potentials, and to make clear what are congenerous and various traits in the chromosomal or DMA sequence levels between these cells, in this study, a combination of conventional G-banding, comparative genomic hybridization (CGH), multiplex fluorescence in situ hybridization (M-FISH) and arm or locus-specific fluorescence in situ hybridization (FISH) were used to comprehensively characterize molecular cytogenetics aberrations of the above cell cultures. This will provide clues to the molecular mechanisms involved in the HCC metastasis.
Part one
Establishment of the FISH-based molecular cytogenetic techniques and its primary application
The major objectives of this part were to establish the novel methods based on fluorescence in situ hybridization (FISH) techniques such as comparative genomic hybridization (CGH), multiplex fluorescence in situ hybridization (M-FISH) and whole chromosome painting (WCP) in our institute.
First, indirect two-color FISH was performed on the metaphase of MHCC97-H cells. The alpha satellite pericentromeric probe specific to chromosome 8 (D8Z2) was obtained from Oncor (Qbiogene, Cedex, France), and the DMA of BAG clone RP11-328M16 mapping at 8q23.1 was extracted and labeled by ourselves. The results showed that the penetrating ability, specificity and signal strength of the both probes were acceptable. The trisomy of chromosome 8 and the non-reciprocal translocation of partial 8q to one chromosome in group B (Chromosome 4) were found.
Second, the directly labeled whole chromosome painting (WCP) probes were purchased from Vysis Company (IL, USA, the MFISH probes also came from this incorporation), and the steps of the hybridization (almost the same as MFISH) were successfully optimized. The findings of numerical and structural changes of chromosome 8 in MHCC97-H were confirmed.
Based on the experience of two-color FISH, we analyzed human hepatocellular carcinoma (HCC) cell line SMMC-7721 with low or none metastatic potential by Indirect CGH. The key parameters of CGH, such as the signal granulation, signal intensity, chromosome length/width and separated chromosomes were all reached the default value. The gains of 1p31, 1q25, 3p22-pter, 5p, 6p21.3-pter, 7p13-pter, 8p23, 8p11-q12, 9q22-qter, 10p12-q21, 11pter-q21, 11p12-q14, 14q13-qter, 15q15-qter, 17p, Xp14-pter and the losses of 4q31-35, 9p21-3, 13q21-31, 18q,Yq were detected in this cell line. These changes may serve as the control for our subsequent research on mechanism o
引文
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