肝门部胆管癌浸润与转移方式的实验研究
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摘要
在临床意义上，肝门部胆管癌是一种恶性程度高预后差的肿瘤。
有关它的生长、浸润和转移的机制一直困扰着广大的外科医生。从形
态学和分子生物学的角度来观察和研究胆管癌的表现，可能更深入的
揭示它的发生、发展和其恶性生物学行为的机理。基于这种思路，本
实验是将计算机技术和基因芯片技术结合到肝门部胆管癌的研究中，
为探索如何提高此病的临床治疗效果打下基础。
目的：1．为研究肝门部胆管癌的发展与转移机制提供一种新的思路。
为建立科学的临床治疗观念提供形态学和分子生物学的依据。
方法：3例手术切除的较完整肝门部胆管癌及其周围正常组织，病理
证实均为中低分化的胆管腺癌。运用石蜡包埋和连续组织切片获得二
维肝门部胆管癌大体组织断面，以体素重建法，结合插值拟合与光照
处理技术，对手术切除的肝门部胆管癌和其相关连的组织进行大块标
本三维重建；观察癌细胞的立体分布。另外，分别提取胆管癌和正常
组织的RNA，作为待测样品。用荧光染料标记制备成探针与基因芯片
进行杂交，通过检测探针分子的杂交信号强度而获取样品分子的数量
和序列信息，寻找出胆管癌组织中差异表达的基因。
一
结果：！．整体重建效果图像(图10《5)兰色的为胆管，红色的为肿
瘤细胞；从重建的效果区””“、上及不同的角度显示肿瘤的大体形态及其
在空间上与周围组织的比邻关系。
2．胆管和胆管癌的三维关系图像(图 16士0)在整体重建的基础上调
节密度梯度，隐去胆管和肿瘤细胞之外的其余组织结构；只显示胆管
和肿瘤细胞，更直观的展示了肿瘤细胞沿胆管壁和胆管周围浸润生长
的三维图像。
3．神经侵润的三维图像(图2125)兰色的为一条完整的神经束，红
色为肿瘤细胞：从三维重建的图像上及不同的角度和方位显示肿瘤细
胞沿神经鞘膜进行自上而下的浸润生长。
4．筛选出246项；这246项数据所代表的基因在与两种探针杂交时
表现出较大的差异。其中新基因102条。下调基因164条，上调基因
82条；这246条基因代表15种类别，其中癌基因17条，离子通道和
运输蛋白基因4条，细胞周期蛋白基因16条，外压反应蛋白基因l
条，细胞骨架和运动基因12条，细胞凋亡基因3条，DNA合成和修复
重组基因 4条，DNA结合、转录基因 17条，细胞受体基因 6条，兔疫
相关基因 11条，代谢基因 33条，蛋白质翻译合成基因 30条，发育
相关基因10条，细胞信号和传递蛋白基因58条，其它类基因57
条。
结论：1．成功地建立了基于微机的肝门部胆管癌病理图像三维重建。
2．真实反映了重建区域内各结构立体形态，空间位置和相互的毗邻关
系。
5
博士论文 中国人民解放军军医进修学院
一
3．可以对重建的图像进行各种操作，诸如不同方位的立体视图，肿瘤
的各种几何尺寸的测量和空间定位，不同组织的单独显示或多种组织
的重叠显示。
4．从癌细胞在胆管和神经分布模式可应用于改进根治性手术方式和评
估手术效果。
5．可应用于外科的计算机辅助教学。
6．成功地筛选出胆管癌的差异表达基因。其实际理论意义仍有待于进
一步研究。
HBDC are highly malignant tumors with poor
prognosis. The spreading mode and mechanism of invasion and
metastasis are still confusing to many clinicians. The most
important factor affecting prognosis is "radical"
resection of the tumcr. More under standing of the spread
of the cancer cells along the bile duct and nerves is
needed. According to this, we use computer technique and
DNA microarray in the study.
Objective: 1. To establish an algorithm of three-dimensional
resconstruction of HBDC.
2. To find a reconstruction technique where all HBDC
structures can be individually or globally analyzed
according to what is required.
Methods: Taken from 3 paraffin-embedded tissues HBDC
specimens were cut into serial histologic
sections. Information were inputted into computer after the
processese of scanning and recognizing. By the techniques of
reconstruction, the 3D stereo HBDC models were retrieved
from 2D digita1 data. Furthermore, we developed a cDNA
array representing 2048cDNA clusters, to profi1e the gene
expression patterns in 3 sets of c1inica1 HBDC samp1es
inc1uding paired cancer and non--tumorous biliary duct.
Results: 1. HBDC comprising severa1 components can be
ana1yzed as a who1e or each structure may be viewed
s eparat e 1 y.
2. It a1so show the re1ationships between the constituent
parts such as bi1e duct and tumor.
3. The 3D mode of spread a1ong nerve revealed that the
tumor stayed sporadica1ly in perineural space.
4. 1t was observed that 246 genes were differentia11y
expressed in paired cancer and distal non--tumorous bi1e
duct tissues. includes l64 down--regu1ated genes, 82 up-
regu1ated genes. the 246 genes represent l5
categori zations: l7 oncogenes, 4 ionic channe1 genes, 16
cyc1 in genes, l2 cytoske1eton g3nes, 3 apoptosis genes, 4 DNA
synthesis and recombination genes, l7 DNA transcription
genes, 6 ce11 recipient genes, 1l inununization corre1ated
genes, 33 metabo1 ism genes, 30 protein translate and
synthesis genes, 10 growth correlated genes, 58 cel1 signa1
and transducin genes, 57 other genes.
Conclusion: 1. The method of the microcoInPuter aided 3D
reconstruction was suc.t5sfu11y estab1ished in HBDC.
2. The reconstructed 3D image showed HBDC realistica11y.
3. A11 structures reconstructed could be disp1ayed alone, in
any group or total1y and rotated in 3D space or rotated
continuous1y at different speed.
4. It can be app1ied in se1ecting the pattern of "radica1"
resection and judge the resu1t of operation from the spread
of the cancer ce11s a1ong the bile duct and nerves.
5. Such a series images of HBDC might be app1icated as a
cornerstone of the computer aid instruction in surgery.
6. The differentia11y expressed genes was successfu11y
screened in HBDC.
有关它的生长、浸润和转移的机制一直困扰着广大的外科医生。从形
态学和分子生物学的角度来观察和研究胆管癌的表现，可能更深入的
揭示它的发生、发展和其恶性生物学行为的机理。基于这种思路，本
实验是将计算机技术和基因芯片技术结合到肝门部胆管癌的研究中，
为探索如何提高此病的临床治疗效果打下基础。
目的：1．为研究肝门部胆管癌的发展与转移机制提供一种新的思路。
为建立科学的临床治疗观念提供形态学和分子生物学的依据。
方法：3例手术切除的较完整肝门部胆管癌及其周围正常组织，病理
证实均为中低分化的胆管腺癌。运用石蜡包埋和连续组织切片获得二
维肝门部胆管癌大体组织断面，以体素重建法，结合插值拟合与光照
处理技术，对手术切除的肝门部胆管癌和其相关连的组织进行大块标
本三维重建；观察癌细胞的立体分布。另外，分别提取胆管癌和正常
组织的RNA，作为待测样品。用荧光染料标记制备成探针与基因芯片
进行杂交，通过检测探针分子的杂交信号强度而获取样品分子的数量
和序列信息，寻找出胆管癌组织中差异表达的基因。
一
结果：！．整体重建效果图像(图10《5)兰色的为胆管，红色的为肿
瘤细胞；从重建的效果区””“、上及不同的角度显示肿瘤的大体形态及其
在空间上与周围组织的比邻关系。
2．胆管和胆管癌的三维关系图像(图 16士0)在整体重建的基础上调
节密度梯度，隐去胆管和肿瘤细胞之外的其余组织结构；只显示胆管
和肿瘤细胞，更直观的展示了肿瘤细胞沿胆管壁和胆管周围浸润生长
的三维图像。
3．神经侵润的三维图像(图2125)兰色的为一条完整的神经束，红
色为肿瘤细胞：从三维重建的图像上及不同的角度和方位显示肿瘤细
胞沿神经鞘膜进行自上而下的浸润生长。
4．筛选出246项；这246项数据所代表的基因在与两种探针杂交时
表现出较大的差异。其中新基因102条。下调基因164条，上调基因
82条；这246条基因代表15种类别，其中癌基因17条，离子通道和
运输蛋白基因4条，细胞周期蛋白基因16条，外压反应蛋白基因l
条，细胞骨架和运动基因12条，细胞凋亡基因3条，DNA合成和修复
重组基因 4条，DNA结合、转录基因 17条，细胞受体基因 6条，兔疫
相关基因 11条，代谢基因 33条，蛋白质翻译合成基因 30条，发育
相关基因10条，细胞信号和传递蛋白基因58条，其它类基因57
条。
结论：1．成功地建立了基于微机的肝门部胆管癌病理图像三维重建。
2．真实反映了重建区域内各结构立体形态，空间位置和相互的毗邻关
系。
5
博士论文 中国人民解放军军医进修学院
一
3．可以对重建的图像进行各种操作，诸如不同方位的立体视图，肿瘤
的各种几何尺寸的测量和空间定位，不同组织的单独显示或多种组织
的重叠显示。
4．从癌细胞在胆管和神经分布模式可应用于改进根治性手术方式和评
估手术效果。
5．可应用于外科的计算机辅助教学。
6．成功地筛选出胆管癌的差异表达基因。其实际理论意义仍有待于进
一步研究。
HBDC are highly malignant tumors with poor
prognosis. The spreading mode and mechanism of invasion and
metastasis are still confusing to many clinicians. The most
important factor affecting prognosis is "radical"
resection of the tumcr. More under standing of the spread
of the cancer cells along the bile duct and nerves is
needed. According to this, we use computer technique and
DNA microarray in the study.
Objective: 1. To establish an algorithm of three-dimensional
resconstruction of HBDC.
2. To find a reconstruction technique where all HBDC
structures can be individually or globally analyzed
according to what is required.
Methods: Taken from 3 paraffin-embedded tissues HBDC
specimens were cut into serial histologic
sections. Information were inputted into computer after the
processese of scanning and recognizing. By the techniques of
reconstruction, the 3D stereo HBDC models were retrieved
from 2D digita1 data. Furthermore, we developed a cDNA
array representing 2048cDNA clusters, to profi1e the gene
expression patterns in 3 sets of c1inica1 HBDC samp1es
inc1uding paired cancer and non--tumorous biliary duct.
Results: 1. HBDC comprising severa1 components can be
ana1yzed as a who1e or each structure may be viewed
s eparat e 1 y.
2. It a1so show the re1ationships between the constituent
parts such as bi1e duct and tumor.
3. The 3D mode of spread a1ong nerve revealed that the
tumor stayed sporadica1ly in perineural space.
4. 1t was observed that 246 genes were differentia11y
expressed in paired cancer and distal non--tumorous bi1e
duct tissues. includes l64 down--regu1ated genes, 82 up-
regu1ated genes. the 246 genes represent l5
categori zations: l7 oncogenes, 4 ionic channe1 genes, 16
cyc1 in genes, l2 cytoske1eton g3nes, 3 apoptosis genes, 4 DNA
synthesis and recombination genes, l7 DNA transcription
genes, 6 ce11 recipient genes, 1l inununization corre1ated
genes, 33 metabo1 ism genes, 30 protein translate and
synthesis genes, 10 growth correlated genes, 58 cel1 signa1
and transducin genes, 57 other genes.
Conclusion: 1. The method of the microcoInPuter aided 3D
reconstruction was suc.t5sfu11y estab1ished in HBDC.
2. The reconstructed 3D image showed HBDC realistica11y.
3. A11 structures reconstructed could be disp1ayed alone, in
any group or total1y and rotated in 3D space or rotated
continuous1y at different speed.
4. It can be app1ied in se1ecting the pattern of "radica1"
resection and judge the resu1t of operation from the spread
of the cancer ce11s a1ong the bile duct and nerves.
5. Such a series images of HBDC might be app1icated as a
cornerstone of the computer aid instruction in surgery.
6. The differentia11y expressed genes was successfu11y
screened in HBDC.
引文
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