正常与硬化肝组织基因表达差异的初步分析

摘要

目的: 了解肝硬化的基因表达概况, 寻找在肝硬化及正常肝组织中差异表达基因.

方法: 以各24例肝硬化及正常肝组织的总RNA混合定量后反转录合成含有α-³²P dATP的cDNA为探针, 与Atlas微阵列表达分析膜杂交.

结果: 放射自显影结果经AtlasImage^TM软件分析显示: 在所分析的588种已知基因中, Integrin beta 7、collagen type XVIII等17个基因在肝硬化组织中表达上调, TFDP2、BAK、ABL等98个表达下调, 参与细胞增生、凋亡、分化、细胞间相互作用、与细胞侵袭相关的基因表达水平发生了明显改变.

结论: 通过Atlas微阵列系统分析发现的这些基因的表达改变组成了一个肝硬化特异的基因表达谱. 系统地研究肝硬化的基因表达改变, 与肝硬化发生相关基因的差异变化可为肝硬化诊断和治疗提供线索.

关键词: N/A

Gene expression profiles in liver cirrhosis and normal liver tissues

Lian-Xin Liu, Zhi-Hong Chen, Lin-Feng Wu, Hong-Wei Li, Zhi-Hua Liu, Hong-Chi Jiang, Xiu-Qin Wang, Min Wu

Lian-Xin Liu, Lin-Feng Wu, Hong-Chi Jiang, Department of Surgery, First Clinical College, Harbin Medical University, Harbin 150001, Heilongjiang Province, China

Zhi-Hong Chen, Hong-Wei Li, Department of General Surgery, The Fourth Hospital of Harbin, Harbin 150028, Heilongjiang Province, China

Zhi-Hua Liu, Xiu-Qin Wang, Min Wu, National Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100021, China

Supported by: the Natural Science Foundation of Heilongjiang, No. QC01C11.

Correspondence to: Dr. Lian-Xin Liu, Department of Surgery, First Clinical College, Harbin Medical University, 23 Youzheng Street, Nangang District, Harbin 150001, Heilongjiang Province, China. liulianxin@sohu.com

Received: July 4, 2003
Revised: September 10, 2003
Accepted: October 7, 2003
Published online: February 15, 2004

Abstract

AIM: To describe liver specific gene expression profiles and to identify genes with differential expression between liver cirrhotic tissues and normal liver tissues.

METHODS: The cDNA probes which were labeled with α-³²P dATP were synthesized from total RNAs of liver cirrhosis and normal liver tissues and hybridized to two identical Atlas human cDNA expression arrays membranes containing 588 known genes respectively.

RESULTS: Autoradiographic results were analyzed by specific AtlasImage^TM (version1.01a) software. Among the 588 genes analyzed, 17 genes were found up-regulated in cirrhosis, including integrin beta 7 and collagen type XVIII, and 98 genes were down-regulated in cirrhosis, including TFDP2, BAK and ABL. Expression of the genes was associated with the regulation of cell proliferation, apoptosis, differen-tiation, cell-cell interaction, invasion regulators and cytokines altered.

CONCLUSION: The results obtained from Atlas microarray provide a comprehensive liver cirrhosis specific expression profile. These results may be helpful for identification of target genes for diagnosis and designing rational therapeutic strategies.

Key Words: N/A

0 引言

肝硬化是常见的消化系统疾病之一, 在我国主要是由于肝炎病毒感染后形成的, 其次是由于血吸虫感染形成, 部分是由于过量饮酒形成的酒精性肝硬化[1-5]. 目前对肝硬化的研究多集中在肝炎病毒或某些原因在肝硬化的发生、发展、诊断、治疗及预后的作用上, 这样就很难揭示肝硬化演进的全貌[6-18]. 很少有研究着眼于肝硬化发展的过程和在此过程中那些基因发生了显著的变化？这些变化的基因都起了什么样的作用？如何通过这些变化的基因来寻找逆转肝硬化的方法？Atlas微阵列表达分析膜利用微阵列(Microarray) 及差异杂交技术, 可快速了解我们感兴趣的许多基因的表达状况[19]. 为了解肝硬化特异的基因表达谱, 寻找在肝硬化与正常肝组织中的差异表达基因, 我们采用该技术分析了588种已知基因在肝硬化中的表达概况, 为揭示肝硬化的分子遗传学变化, 阐明肝硬化的发生机制, 以及肝硬化的早期诊断和逆转治疗提供线索.

1 材料和方法

1.1 材料

各24例肝硬化及正常肝组织取自哈尔滨医科大学第一临床医学院普通外科. 肝硬化标本取自因门静脉高压症行脾切除贲门周围血管离断术患者肝脏; 正常肝脏标本取自因肝脏外伤行剖腹探查术患者的肝脏, 肉眼下于MEM培养液中行显微剥离后置于液氮中保存.

1.2 试剂

Atlas人类cDNA表达分析微阵列(atlas human cDNA expression array)购自Clontech公司. Trizol^TM RNA提取试剂盒及第一链cDNA合成扩增试剂盒 (SuperScript^TM preamplication system for first strand cDNA synthesis)均购自 Gibco BRL公司. DNA酶(DNase), RNA酶抑制剂(RNasin)购自Promega公司. 放射性同位素α-³²P dATP, dCTP购自NEN Du-Pont公司.

1.3 方法

将各24例的肝硬化和正常肝组织分别混合后, 提取组织的总RNA, 再各取5 μg, 按照Clontech公司Atlas试剂盒操作手册, 分别反转录合成cDNA第一条链, 并掺入放射性同位素α-³²P dATP作探针, 与一套两张已经预杂交2 h的相同的Atlas微阵列膜于68℃杂交16 h. 洗膜条件为2×SSC、1% SDS 68 ℃ 15 min两次, 0.5×SSC、0.5% SDS 68 ℃ 20 min 2次, 应用富士胶片(Fuji Film)于-70 ℃放射自显影36-48 h.

1.4 结果分析

放射自显影的X光片采用Bio-Rad公司Fluor-S^TM MultiImager成像仪扫描生成相应格式的灰度文件. 再由Clontech公司专用的 AtlasImageTM (Version1.01a) 软件进行分析, 生成差异图像及表格.

2 结果

两张放射自显影的X光片(见图1)经Fluor-STM MultiImager成像仪扫描后, 由AtlasImageTM软件分析后得到对比图像. 是以正常肝脏为对照, 以肝硬化相对正常肝脏的变化比率(Ratio)和二者之间的密度值差异(Difference)来反映肝硬化的变化. 为准确反映基因的变化差异, 我们以看家基因泛素(Ubiquitin)来校正二者之间的信号差异. 在所分析的588个基因中, 二者之间的差异大于50%或密度值差异大于10 000单位的共有115个, 其中在肝硬化中表达上调的有17个, 表达下调的有98个(见表1, 其中Ratio>1为在肝硬化组织中表达上调; Ratio<1为在肝硬化组织中表达下调). 许多与细胞周期调控, 细胞增生与分化, 细胞间相互作用, 细胞间信号传递, 细胞凋亡有关的基因以及癌基因和抑癌基因等均有明显的改变.

表1 肝硬化与正常肝组织基因表达差异情况.

位置	比例	差值	基因	位置	比例	差值	基因
D7f		-46 583	placenta growth factor	D3a		-13 878	versican isoforms
F5l		-45 246	IL-inducible peptide	B1h		-13 644	BAK
F5e	0.170	-41 164	IL-13	A5b	0.204	-12 751	MEK1
C4b		-40 337	Wnt-5a	B3l	0.153	-12 644	DAP kinase 1
C7l		-39 092	RAR gamma	D7i	0.150	-12 490	GLG1
E2f	0.290	-36 259	emmprin	E6b	0.422	-12 411	catenin alpha-2
C3i	0.228	-35 930	Notch4	F6k		-12 372	TDGF1, 2, 3
A3i	0.080	-33 464	p19INK4d	D2c		-12 339	collagen type XI
C7m		-32 988	retinoid X receptor	A3j		-12 219	WEE1HU
D5f		-32 437	CD9	B1l		-11 664	FLAME-1
B2g	0.254	-32 317	TRIP	C2d		-11 434	ERCC5
C4k	0.146	-31 082	DVL	A7n		-11 310	desmin
B3j		-30 455	caspase-10	A5l		-11 230	TFDP2
C2j		-30 318	Rad	D2m		-11 077	tenascin-C
F2g	0.388	-30 177	endothelin ET2	F2f		-10 796	EGF-like growth factor
E4j	0.097	-30 058	CDC42	D4f		-10 779	GPIIIA; integrin beta3
D5k	0.085	-28114	ninjurin-1	A7g		-10 686	K4; CK4
B7j	0.250	-27 838	ABL2; ABLL	B2j		-10 436	C-IAP2; IAP1; MIHC
E7n		-27 258	HCK	A1c	0.203	-10 422	CDPK3
E2a		-26 725	MMP-19	A1e		-10 334	kinase PSSALRE
C6n		-26 592	sex gene	F3j	0.791	-10 331	EGR-1; KROX24; AT225
E2d	0.164	-26 409	TIMP-3;	B6d		-10 284	p55-c-fgr protein
E5m		-26 206	H-cadherin	C4n		-10 111	smoothened
D7g		-25 952	FLT3/FLK2	B7l	0.265	-9 485	STAT1
F5k		-25 326	IFN-gamma	B7k	0.444	-9 301	ZAP-70; ZAP70
D5g	0.170	-25 132	cytovillin 2	E4n	0.330	-8 337	P115
D6c	0.281	-23 356	semaphorin E	B4c	0.262	-8 173	WSL-LR; Apo-3
B3h	0.465	-23 309	FLICE; CAP4; CASP8; MCH5	D7n	0.353	-7 249	homeobox HOX 4A
C2n	0.087	-23 014	replication factor C	A3m	0.292	-7 008	NEDD5 protein homolog
D4b	0.136	-22 957	integrin alpha8	C3e	0.458	-6 939	RAD23
B2i	0.122	-21 022	CRAF1	A2e	0.351	-6 736	PCTAIRE-3
E4e		-20 262	P21-RAC2	A5a	0.367	-6 249	MAPKK 5;
B3i	0.536	-20 089	ICE-LAP6	E1k	0.449	-5 473	MMP-14 (MT1-MMP)
D5e	0.159	-19 823	cytohesin-1	A1i	0.455	-5 260	P23; P25; P35
F3k	0.137	-19 389	hepatocyte growth factor	C3b	0.352	-5 111	superoxide dismutase 1
E1m	0.095	-19 179	MMP-16 (MT3-MMP)	A4n	0.464	-4 929	JNK3; JNK3; P49
D6f	-18 894	semaphorin-1	A7a	0.352		-4 649	K16; CK 16;
E2b	0.636	-18 636	TIMP-1; EPA	C1m	0.380	-4 399	DNA TOPO I
C3j		-18 316	Jagged 1	B4d	0.473	-4 221	Akt1; c-Akt
C1a		-18 130	DNA-PK; XRCC7	A2f	0.449	-4 152	PITALRE
C6m		-17 970	CCK4; PTK7	D2e	2.162	10 343	collagen type XVIII
B2n	0.100	-17 919	TRAIL receptor	A5e		1 0747	PCNA; cyclin
D6h		-17 735	LAR	E1e	1.583	11 610	MMP-8; collagenase-2
A7d		-17 478	K1; CK 1	E3e	2.141	1 1693	NDK A; NM23-H1
C2c		-17 212	XRCC1	D3f		13 084	fibronectin
E1j		-17 010	MMP-13; collagenase-3	E6d		14 470	plakoglobin;
F6b		-16 980	NT-3	E3l		14 883	rhoB
B1k	0.644	-16 741	NIK	D4j	1.702	16 207	integrin beta7
B3f		-16 474	LICE2	A7m		16 433	vimentin
C5f		-15 882	eps15	D1b		16 589	byglycan
D6e		-15 220	semaphorin V	F4k		16 800	IL-5
B7g		-15 072	SKY	D1a	1.842	1 8757	CSPCP; aggrecan 1;
A6f		-14 898	b-raf	A6i		1 9497	c-myc binding protein
B5l	0.670	-14 354	rhoA	B5c		19 662	CD27BP (Siva)
E4i	0.181	-14 319	p160ROCK	A3e		28 403	mda-6; CAP20
F5d		-13 972	IL-12	F5n		32 402	MIF
C1i		-13 927	DNA polymerase	B5b		36 045	GST homolog
A4j	0.216	-13 888	ERK6; ERK5

图1 正常肝组织与肝硬化的放射自显影照片. A: 正常肝组织; B: 肝硬化.

3 讨论

自1995年Brown实验室首次报道了微阵列技术(Microarray)以来, 因其信息量大, 操作简单、可靠, 可重复性强且可以反复利用, 迅速被用于基因表达检测、DNA测序、寻找新基因、突变体和多态性的检测、药物筛选、疾病诊断和基因文库作图等方面[20]. Atlas微阵列膜是微阵列系统的一种, 也称为DNA芯片(DNA chip). 本研究结果表明: 在肝硬化中, 对细胞增生起促进作用的基因表达明显下调, 对细胞增生起抑制作用的基因表达明显上调; 与细胞间相互作用、细胞粘连、生长因子受体和细胞因子有关的基因也发生了明显改变. 这些改变构成了肝硬化不同于正常肝脏特异的基因表达谱.

在对细胞增生周期调控的基因中, 转录因子E2F家族中的配体DP2(TFDP2)在肝硬化中有明显的下降. TFDP2是E2F家族的重要配体, 虽然E2F家族有1-5五个基因, 但其配体却只有DP1和DP2两个, 尤以DP2的功能更为重要. 其是通过与E2F组分形成DNA结合复合体而发挥作用, 在细胞周期的调节和分化中发挥重要作用, 可以促进S期的进程[21]. TFDP2还可以通过调节E2F而在细胞周期中发挥作用, 其在肝硬化组织中的表达下降, 说明肝硬化时细胞的倍增时间延长. 促分裂原活化激酶激酶(mitogen-actived protein kinase kinase, MAP kinase kinase 1, MAPKK1)是胞外信号调节激酶的亚类, 其可被丝氨酸磷酸化所激活, 上游的激活因子c-raf, ras和MEK激酶均可以调节其活性[22]. 在肝硬化中表达明显下降的基因还有LAR, LAR是跨膜蛋白酪氨酸磷酸酯酶的前体, 有调节细胞与细胞粘连分子之间的作用 [23], 其在肝硬化中表达下降亦可能起同样的作用.

与细胞凋亡有关的基因在肝硬化中表达下调, BAK可以直接刺激细胞凋亡, 他通过BH3结构域和Bcl-2结合, 抑制Bcl-2的功能, 进而发挥促进凋亡作用[24], 其在肝硬化组织中的表达明显下降. DNA-PK由催化亚单位和由G22P1基因编码的自身免疫性抗原Ku构成, 催化亚单位的活性是依赖于Ku的活性来调节, 许多DNA结合蛋白, 包括许多转录因子等都是DNA-PK的底物[25]. DNA-PK还可以控制转录、凋亡和染色体端粒的长度, 他的催化亚基是Caspase家族Caspase-3介导凋亡通路上的一个重要目标蛋白[26]. DNA-PK还参与双股DNA断裂(DNA double strand breaks, DSBs)的修复和V(D)J重组, 其活性的调节可通过自身磷酸化的过程进行[25]. DNA拓扑异构酶(DNA TOPI)催化超螺旋DNA的解链, 并且在DNA的复制、转录和重组中发挥重要作用, 同时还可以改变DNA的立体构象[27]. 真核生物的DNA TOPI是高度保守的, 他的功能受抑制时, 将导致DNA双链断裂、堆积, 进而造成细胞死亡.

此外, 在肝硬化中表达上调的基因有整合蛋白β7(integrin beta7), 他调节细胞与细胞, 细胞与基质之间的相互作用, 同时还可以将各种刺激转变为细胞内信号[28]. XVIII型胶原(collagen type XVIII)属于胶原超家族中的一类, Endostatin是他的20 kDa的C末端, 而且是表皮细胞增生和血管化的一种特异的抑制剂, 同时可以使上皮细胞停止于G1期[29]. 他们在肝硬化中的表达上调是我们首次发现.

总之, 肝硬化的基因表达改变是非常复杂的, 肝硬化的发生、发展及演进正是这些基因改变共同作用的结果[30-32]. 应用Atlas微阵列膜可以同时分析上千个基因的变化, 可以提供特殊疾病的基因表达谱; 可以为人们提供研究正常细胞和疾病细胞分子解剖学的第一手资料[33]. 本研究发现的在肝硬化中较之正常肝组织有变化的许多基因大多数尚未见诸肝硬化研究的报道. 这些基因改变的发现必将为人类研究肝硬化提供新的思路；为肝硬化逆转的基因治疗提供新的可能的靶点.

备注

编辑: N/A

参考文献

1.	Reshetnyak VI, Sharafanova TI, Ilchenko LU, Golovanova EV, Poroshenko GG. Peripheral blood lymphocytes DNA in patients with chronic liver diseases. World J Gastroenterol. 2001;7:235-237.  [PubMed]  [DOI]

2.	Shen LJ, Zhang HX, Zhang ZJ, Li JY, Chen MQ, Yang WB, Huang R. Detection of HBV, PCNA and GST-pi in hepatocellular carcinoma and chronic liver diseases. World J Gastroenterol. 2003;9:459-462.  [PubMed]  [DOI]

3.	Dai WJ, Jiang HC. Advances in gene therapy of liver cirrhosis: a review. World J Gastroenterol. 2001;7:1-8.  [PubMed]  [DOI]

4.	Zheng M, Cai WM, Weng HL, Liu RH. ROC curves in evaluation of serum fibrosis indices for hepatic fibrosis. World J Gastroenterol. 2002;8:1073-1076.  [PubMed]  [DOI]

5.	Wang JY, Guo JS, Yang CQ. Expression of exogenous rat collagenase in vitro and in a rat model of liver fibrosis. World J Gastroenterol. 2002;8:901-907.  [PubMed]  [DOI]

6.	Cheng XD, Jiang XC, Liu YB, Peng CH, Xu B, Peng SY. Effect of ischemic preconditioning on P-selectin expression in hepatocytes of rats with cirrhotic ischemia-reperfusion injury. World J Gastroenterol. 2003;9:2289-2292.  [PubMed]  [DOI]

7.	Jiang XH, Zhong RQ, Yu SQ, Hu Y, Li WW, Kong XT. Construction and expression of a humanized M2 autoantigen trimer and its application in the diagnosis of primary biliary cirrhosis. World J Gastroenterol. 2003;9:1352-1355.  [PubMed]  [DOI]

8.	Wang XZ, Chen ZX, Zhang LJ, Chen YX, Li D, Chen FL, Huang YH. Expression of insulin-like growth factor 1 and insulin-like growth factor 1 receptor and its intervention by interleukin-10 in experimental hepatic fibrosis. World J Gastroenterol. 2003;9:1287-1291.  [PubMed]  [DOI]

9.	Wang HT, Chen S, Wang J, Ou QJ, Liu C, Zheng SS, Deng MH, Liu XP. Expression of growth hormone receptor and its mRNA in hepatic cirrhosis. World J Gastroenterol. 2003;9:765-770.  [PubMed]  [DOI]

10.	Wang XZ, Zhang LJ, Li D, Huang YH, Chen ZX, Li B. Effects of transmitters and interleukin-10 on rat hepatic fibrosis induced by CCl4. World J Gastroenterol. 2003;9:539-543.  [PubMed]  [DOI]

11.	Liu YK, Shen W. Inhibitive effect of cordyceps sinensis on experimental hepatic fibrosis and its possible mechanism. World J Gastroenterol. 2003;9:529-533.  [PubMed]  [DOI]

12.	Wang X, Zhong YX, Zhang ZY, Lu J, Lan M, Miao JY, Guo XG, Shi YQ, Zhao YQ, Ding J. Effect of L-NAME on nitric oxide and gastrointestinal motility alterations in cirrhotic rats. World J Gastroenterol. 2002;8:328-332.  [PubMed]  [DOI]

13.	Nie QH, Cheng YQ, Xie YM, Zhou YX, Bai XG, Cao YZ. Methodologic research on TIMP-1, TIMP-2 detection as a new diagnostic index for hepatic fibrosis and its significance. World J Gastroenterol. 2002;8:282-287.  [PubMed]  [DOI]

14.	Nie QH, Cheng YQ, Xie YM, Zhou YX, Cao YZ. Inhibiting effect of antisense oligonucleotides phosphorthioate on gene expression of TIMP-1 in rat liver fibrosis. World J Gastroenterol. 2001;7:363-369.  [PubMed]  [DOI]

15.	Weng HL, Cai WM, Liu RH. Animal experiment and clinical study of effect of gamma-interferon on hepatic fibrosis. World J Gastroenterol. 2001;7:42-48.  [PubMed]  [DOI]

16.	Huang X, Li DG, Wang ZR, Wei HS, Cheng JL, Zhan YT, Zhou X, Xu QF, Li X, Lu HM. Expression changes of activin A in the development of hepatic fibrosis. World J Gastroenterol. 2001;7:37-41.  [PubMed]  [DOI]

17.	Assy N, Paizi M, Gaitini D, Baruch Y, Spira G. Clinical implication of VEGF serum levels in cirrhotic patients with or without portal hypertension. World J Gastroenterol. 1999;5:296-300.  [PubMed]  [DOI]

18.	Wang X, Chen YX, Xu CF, Zhao GN, Huang YX, Wang QL. Relationship between tumor necrosis factor-alphaand liver fibrosis. World J Gastroenterol. 1998;4:18.  [PubMed]  [DOI]

19.

Sehgal A, Boynton AL, Young RF, Vermeulen SS, Yonemura KS, Kohler EP, Aldape HC, Simrell CR, Murphy GP. Application of the differential hybridization of Atlas Human expression arrays technique in the identification of differentially expressed genes in human glioblastoma multiforme tumor tissue. J Surg Oncol. 1998;67:234-241.  [PubMed]  [DOI]

20.	Schena M, Shalon D, Davis RW, Brown PO. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science. 1995;270:467-470.  [PubMed]  [DOI]

21.	Zhang Y, Venkatraj VS, Fischer SG, Warburton D, Chellappan SP. Genomic cloning and chromosomal assignment of the E2F dimerization partner TFDP gene family. Genomics. 1997;39:95-98.  [PubMed]  [DOI]

22.	Zheng CF, Guan KL. Activation of MEK family kinases requires phosphorylation of two conserved Ser/Thr residues. EMBO J. 1994;13:1123-1131.  [PubMed]  [DOI]

23.	Aicher B, Lerch MM, Müller T, Schilling J, Ullrich A. Cellular redistribution of protein tyrosine phosphatases LAR and PTPsigma by inducible proteolytic processing. J Cell Biol. 1997;138:681-696.  [PubMed]  [DOI]

24.	Chittenden T, Harrington EA, O'Connor R, Flemington C, Lutz RJ, Evan GI, Guild BC. Induction of apoptosis by the Bcl-2 homologue Bak. Nature. 1995;374:733-736.  [PubMed]  [DOI]

25.	Jin S, Inoue S, Weaver DT. Functions of the DNA dependent protein kinase. Cancer Surv. 1997;29:221-261.  [PubMed]  [DOI]

26.	Teraoka H, Yumoto Y, Watanabe F, Tsukada K, Suwa A, Enari M, Nagata S. CPP32/Yama/apopain cleaves the catalytic component of DNA-dependent protein kinase in the holoenzyme. FEBS Lett. 1996;393:1-6.  [PubMed]  [DOI]

27.	Pommier Y. Eukaryotic DNA topoisomerase I: genome gatekeeper and its intruders, camptothecins. Semin Oncol. 1996;23:3-10.  [PubMed]  [DOI]

28.	Yano Y, Geibel J, Sumpio BE. Cyclic strain induces reorganization of integrin alpha 5 beta 1 and alpha 2 beta 1 in human umbilical vein endothelial cells. J Cell Biochem. 1997;64:505-513.  [PubMed]  [DOI]

29.	Dhanabal M, Volk R, Ramchandran R, Simons M, Sukhatme VP. Cloning, expression, and in vitro activity of human endostatin. Biochem Biophys Res Commun. 1999;258:345-352.  [PubMed]  [DOI]

30.	Iizuka N, Oka M, Yamada-Okabe H, Mori N, Tamesa T, Okada T, Takemoto N, Hashimoto K, Tangoku A, Hamada K. Differential gene expression in distinct virologic types of hepatocellular carcinoma: association with liver cirrhosis. Oncogene. 2003;22:3007-3014.  [PubMed]  [DOI]

31.	Anders RA, Yerian LM, Tretiakova M, Davison JM, Quigg RJ, Domer PH, Hoberg J, Hart J. cDNA microarray analysis of macroregenerative and dysplastic nodules in end-stage hepatitis C virus-induced cirrhosis. Am J Pathol. 2003;162:991-1000.  [PubMed]  [DOI]

32.	Chen L, Goryachev A, Sun J, Kim P, Zhang H, Phillips MJ, Macgregor P, Lebel S, Edwards AM, Cao Q. Altered expression of genes involved in hepatic morphogenesis and fibrogenesis are identified by cDNA microarray analysis in biliary atresia. Hepatology. 2003;38:567-576.  [PubMed]  [DOI]

33.	Cole KA, Krizman DB, Emmert-Buck MR. The genetics of cancer--a 3D model. Nat Genet. 1999;21:38-41.  [PubMed]  [DOI]