人胆囊癌裸鼠原位模型的建立、单克隆细胞株的分离及双自杀基因体内外杀伤作用的研究
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摘要
第一部分:
人胆囊癌裸鼠原位移植瘤模型的建立
目的 利用组织块法建立技术较为成熟的人胆囊癌裸鼠原位移植瘤模型,了解其生长特点、镜下形态、超微结构、染色体分析及肿瘤标志物的表达等生物学特性,同时利用免疫组化法检测瘤组织中一些与侵袭和转移相关的蛋白表达。
材料和方法 人胆囊癌细胞GBC-SD培养于加入10%新生牛血清的RPMI1640培养液中,胰酶消化后将约2×10~6(0.2ml)个GBC-SD细胞注射于裸鼠皮下成瘤,取所成肿瘤组织剪成小块接种于裸鼠皮下,建立组织块法皮下移植瘤模型;同时将瘤组织块接种于胆囊,胆囊原位成瘤后取出瘤组织,同法接种于下一只裸鼠的胆囊,如此反复传代,建立原位移植瘤模型:裸鼠衰竭时处死尸解,观察肿瘤生长和转移情况,并取瘤组织做常规病理切片显微镜下观察,做超薄切片透射电镜观察,SP免疫组化法检测与转移及侵袭相关的蛋白表达,实体瘤直接法行染色体分析,取原位瘤、皮下瘤模型及正常对照裸鼠的血清,酶联免疫法检测相关的肿瘤标志物。
结果 人胆囊癌裸鼠组织块法皮下移植瘤模型的成瘤率为22%,原位移植瘤模型总的成瘤率为83%,其中第一代成瘤率57%,第二代成瘤率67%,第三代成瘤率83%,第四代、第五代及第六代成瘤率100%;原位荷瘤裸鼠平均生存期为10周,皮下瘤为17周;初步观察比较两种模型,肿瘤的生长形态、常规病理切片观察无明显差异,但原位移植瘤可以稳定传代,侵袭性较强,87%的裸鼠发
Part one:Establishment of the orthotopic transplantation tumor model of humangallbladder carcinoma in nude miceObjective To use histologically intact specimens to establish a novel orthotopic transplantation tumor model of human gallbladder carcinoma in nude mice and to observe its some biological characteristics which can improve our understanding of cancer metastasis and help us to develop therapeutic strategies for advanced gallbladder cancer.Material and methods About 2×10~6(0.2ml cell suspension) GBC-SD cells were harvested and injected subcutaneously to form the transplantation tumor in nude mice. Fresh specimens from the transplantation tumor were cut into pieces of 1—2 mm3. Some of them were sewn into the gallbladder to form the orthotopic transplantation tumor model of human gallbladder carcinoma and some specimens were transplanted subcutaneously to form the subcutaneous transplantation tumor model of human gallbladder carcinoma with intact tissue in nude mice. The fresh specimens from orthotopic tumor of the last generation were transplanted into the gallbladder of some other nude mice to form the new generation. Some growing
characteristic and biological characteristic were compared between the two models. The nude mice were autopsied when they were dying. The volume of the orthotopic tumor was measured. The liver, the other organs and all the abdominal lymph nodes were detected. The fresh specimens were fixed in 10% formalin and embedded in paraffin wax for histological analysis. The tissue was cut at 4 μm and stained with hematoxylin and eosin. Immunohistochemical staining (SP procedure) was used to check the expression of factors relevant to invasiveness and metastasis. After ultramicrotomy, the section was observed with transmission electron microscopy. Futhermore quantifying CEA, AFP, CA19-9 and CA-242 in serum were measured and chromosome analysis was studied.Result The overall incidence of subcutaneous transplantation tumor was 22% and that of the orthotopic transplantation tumor was 83%. Among them that of the first generation was 57%, the second was 67%, the third was 83%, all of the next three generations were 100%. The average life-span of the nude mice with orthotopic transplantation tumor was about 10 weeks and that of subcutaneous transplantation tumor was 17 weeks. No difference was found in morphology or pathology between the two model. But the orthotopic transplantation tumor had higher local invasiveness. Local metastasis in liver was found in 87% nude mice of the orthotopic model and distant metastasis was found in 17% nude mice of the orthotopic model. Furthermore the orthotopic transplantation tumor model can propagated stably. The immunohistochemical staining showed that there was different extent positive express among different factors relevant to invasiveness and metastasis. From the transmission electron microscopy the characteristics with high invasiveness was found in orthotopic tumor. The content of tumor marker of the orthotopic transplantation tumor model is higher than that of subcutaneous model and normal nude mice in serum. Chromosome analysis the orthotopic tumor had the similar karyotype to human tumor cells. The number of chromosome ranged from 59 to 101, which demonstrated as supertriploid or subtetraploid mainly.Conclusion The orthotopic transplantation tumor model in nude mice was successfully established. The tumor in orthotopic model had the high capability of
invasiveness and metastasis. It could simulate the clinic gallbladder cancer and provided a good instrument for the study of human gallbladder carcinoma. Part two:Isolation of human gallbladder carcinoma cell clone with high invasiveness and inhibition of invasiveness of the cell clone by heparanase antisenseoligodeoxynucleotideObjective To isolate a human gallbladder carcinoma cell clone with high invasiveness derived from human gallbladder caicinoma cell line GBC-SD and to observe its some biological characteristics. To evaluate the inhibitory effect of heparanase antisense oligodeoxynucleotide(ASODN) on the invasiveness of the cell clone with high invasion potential.Material and methods Cell clone culture was conducted on the different cell clones of the source cell line GBC-SD using limited dilution method. Some biological characteristics of the different cell clones were studied by light microscope, cell growth analysis, cloning assay. Electrophoresis migration rates of different cell clones were tested to study charge properties and density of their surface. MTT assay was used for adhesion tests and the modified Boyden chamber model(Transwell chamber) was used for quantitative invasion and chemotaxis motility assay. The cell clone with highly invasiveness was selected by analyzing these results. Karyotyping was employed to characterize the target cell clone. Scanning electron microscope was used to observe the surface of the target cell clone. Flow cytometry and immunohistochemistry were used to detect the expressions of CD54 and matrix metalloproteinases respectively. The ASODN complementary to the start codon region of heparanase mRNA and its control, scrambled nonsense oligodeoxynucleotide(NSODN) were designed and synthesized and phosphorothioated. The ODNs were embedded in cationic liposome Lipofectin and transfected into the target cells. The total RNAs were extracted from the cells in 48 hours after transfection and then semi-quantitative RT-PCR and immunohistchemsitry
were performed to evalutate the heparanase gene and protein expression levels respectively. The invasiveness of transfected cells was measured quantitatively by Matrigel invasion assays(Transwell chamber).Results In all 21 cell clones were successfully isolated from the source cell line GBC-SD. No obvious difference among the 21 cell clones was observed by light microscope. Comparing the 21 cell clones, the doubling time varied from 15.38 hours to 24.65 hours, the clonogenesis from 26% to 36.4%, electrophoresis migration rates from 2.44um/s / V/cm to 6.14um/s / V/cm and the rates of adhesion from 22.6% to 45.6%. The number of cells that penetrated the filter was from 46.3 to 81.7 in quantitative invasion assay and from 81.1 to 126.4 in chemotaxis motility assay. The No.2 clone was obtained as the high invasiveness cell clone by analyzing these results and named GBC-SDHj. When GBC-SDHj was compared with GBC-SD, the number of chromosome was 59~92 versus 42~136, and fluorescent intensity and percent of positive cells of CD54 were 561.39 and 15.60% versus 34.12 and 3.17%. There were much more microvilli and filopodia, with crotches in distal end, on surface of GBC-SDHi cells than that of GBC-SD cells under scanning electron microscope. The immunohistochemistry showed that the expression levels of MMP-2 and MMP-9 in GBC-SDH, cells were higher than that of GBC-SD cells. The heparanase gene and protein expression and invasiveness of GBC-SDHj cells treated with ASODN of diferent final concentrations were significantly decreased compared with that of the controls (PO.01). Furthermore the inhibitory effects were significantly different among the cells treated with ASODN of different concentration (PO.05). The invasiveness inhibition rates were 37%, 51%, 63% and 66% at the cells treated with ASODN of the final concentrations of 0.2umol/L, 0.4umol/L, 0.8umol/L and 1.6umol/L respectivelyConclusion 1 .GBC-SDHj cell was a cell clone with high invasiveness. 2.Some biological characteristics were different between GBC-SDHj cells and GBC-SD cells. 3.Heparanase ASODN complementary to the start condon region of heparanase mRNA has a significant inhibitory effect on the invasiveness of human gallbladder carcinoma cell line in a dose-dependent manner.
Part three:Killing effects of combination of cytosine deaminase and HSV-tk genes ongallbladder cancer mediated by retroviral vectorObjective To investigate the different cell-killing and bystander effects on GBC-SD cells in vitro and vivo with double suicide genes system(CD/5-FC and HSV-tk/GCV) transferred by retroviral vector and to find a more efficient and low-toxicity suicide gene therapy for gallbladder cancer.Material and methods The PA317 cells with CD and HSV-tk double suicide genes were cultured after G418 selected and the viral supernatant was harvested. The GBC-SD cells were infected with the viral supernatant. After G418 selection, the GBC-SD/CD+tk cells in culture were picked and the expression of double suicide genes was confirmed by RT-PCR. After administration of 5-FC and/or GCV the morphological changes of those cells were observed through optical microscope and scanning electron microscope. The cell cycle and apoptosis rates were analyzed by flow cytometry (FCM). The cytoxicity efficacy of 5-FC and/or GCV on GBC-SD/CD+tk cells was evaluated by MTT method. Furthermore the GBC-SD/CD+tk and GBC-SD cells at varying mixing ratio were treated by the different concentration of 5-FC and GCV to explore the bystander effects. In vivo experiment, 36 Balb/c nude mice of four to six weeks old were divide into 6 groups at random and the cell line GBC-SD/CD+tk or/and GBC-SD were inoculated subcutaneously into the nude mice respectively. When the tumor were palpable, retroviral supernatant containing CD and HSV-tk genes were directly injected into tumors of the Group GBC-SD. For the 4 experiment groups and the GBC-SD contral group GCV and 5-FC were administrated intraperitoneally and the other contral group was given normal saline(NS). 21 days later animals were sacrificed and the sizes of the specimens were measured and processed for histopathological analysis.Results The virus supernatant containing double suicide gene was obstained
人胆囊癌裸鼠原位移植瘤模型的建立
目的 利用组织块法建立技术较为成熟的人胆囊癌裸鼠原位移植瘤模型,了解其生长特点、镜下形态、超微结构、染色体分析及肿瘤标志物的表达等生物学特性,同时利用免疫组化法检测瘤组织中一些与侵袭和转移相关的蛋白表达。
材料和方法 人胆囊癌细胞GBC-SD培养于加入10%新生牛血清的RPMI1640培养液中,胰酶消化后将约2×10~6(0.2ml)个GBC-SD细胞注射于裸鼠皮下成瘤,取所成肿瘤组织剪成小块接种于裸鼠皮下,建立组织块法皮下移植瘤模型;同时将瘤组织块接种于胆囊,胆囊原位成瘤后取出瘤组织,同法接种于下一只裸鼠的胆囊,如此反复传代,建立原位移植瘤模型:裸鼠衰竭时处死尸解,观察肿瘤生长和转移情况,并取瘤组织做常规病理切片显微镜下观察,做超薄切片透射电镜观察,SP免疫组化法检测与转移及侵袭相关的蛋白表达,实体瘤直接法行染色体分析,取原位瘤、皮下瘤模型及正常对照裸鼠的血清,酶联免疫法检测相关的肿瘤标志物。
结果 人胆囊癌裸鼠组织块法皮下移植瘤模型的成瘤率为22%,原位移植瘤模型总的成瘤率为83%,其中第一代成瘤率57%,第二代成瘤率67%,第三代成瘤率83%,第四代、第五代及第六代成瘤率100%;原位荷瘤裸鼠平均生存期为10周,皮下瘤为17周;初步观察比较两种模型,肿瘤的生长形态、常规病理切片观察无明显差异,但原位移植瘤可以稳定传代,侵袭性较强,87%的裸鼠发
Part one:Establishment of the orthotopic transplantation tumor model of humangallbladder carcinoma in nude miceObjective To use histologically intact specimens to establish a novel orthotopic transplantation tumor model of human gallbladder carcinoma in nude mice and to observe its some biological characteristics which can improve our understanding of cancer metastasis and help us to develop therapeutic strategies for advanced gallbladder cancer.Material and methods About 2×10~6(0.2ml cell suspension) GBC-SD cells were harvested and injected subcutaneously to form the transplantation tumor in nude mice. Fresh specimens from the transplantation tumor were cut into pieces of 1—2 mm3. Some of them were sewn into the gallbladder to form the orthotopic transplantation tumor model of human gallbladder carcinoma and some specimens were transplanted subcutaneously to form the subcutaneous transplantation tumor model of human gallbladder carcinoma with intact tissue in nude mice. The fresh specimens from orthotopic tumor of the last generation were transplanted into the gallbladder of some other nude mice to form the new generation. Some growing
characteristic and biological characteristic were compared between the two models. The nude mice were autopsied when they were dying. The volume of the orthotopic tumor was measured. The liver, the other organs and all the abdominal lymph nodes were detected. The fresh specimens were fixed in 10% formalin and embedded in paraffin wax for histological analysis. The tissue was cut at 4 μm and stained with hematoxylin and eosin. Immunohistochemical staining (SP procedure) was used to check the expression of factors relevant to invasiveness and metastasis. After ultramicrotomy, the section was observed with transmission electron microscopy. Futhermore quantifying CEA, AFP, CA19-9 and CA-242 in serum were measured and chromosome analysis was studied.Result The overall incidence of subcutaneous transplantation tumor was 22% and that of the orthotopic transplantation tumor was 83%. Among them that of the first generation was 57%, the second was 67%, the third was 83%, all of the next three generations were 100%. The average life-span of the nude mice with orthotopic transplantation tumor was about 10 weeks and that of subcutaneous transplantation tumor was 17 weeks. No difference was found in morphology or pathology between the two model. But the orthotopic transplantation tumor had higher local invasiveness. Local metastasis in liver was found in 87% nude mice of the orthotopic model and distant metastasis was found in 17% nude mice of the orthotopic model. Furthermore the orthotopic transplantation tumor model can propagated stably. The immunohistochemical staining showed that there was different extent positive express among different factors relevant to invasiveness and metastasis. From the transmission electron microscopy the characteristics with high invasiveness was found in orthotopic tumor. The content of tumor marker of the orthotopic transplantation tumor model is higher than that of subcutaneous model and normal nude mice in serum. Chromosome analysis the orthotopic tumor had the similar karyotype to human tumor cells. The number of chromosome ranged from 59 to 101, which demonstrated as supertriploid or subtetraploid mainly.Conclusion The orthotopic transplantation tumor model in nude mice was successfully established. The tumor in orthotopic model had the high capability of
invasiveness and metastasis. It could simulate the clinic gallbladder cancer and provided a good instrument for the study of human gallbladder carcinoma. Part two:Isolation of human gallbladder carcinoma cell clone with high invasiveness and inhibition of invasiveness of the cell clone by heparanase antisenseoligodeoxynucleotideObjective To isolate a human gallbladder carcinoma cell clone with high invasiveness derived from human gallbladder caicinoma cell line GBC-SD and to observe its some biological characteristics. To evaluate the inhibitory effect of heparanase antisense oligodeoxynucleotide(ASODN) on the invasiveness of the cell clone with high invasion potential.Material and methods Cell clone culture was conducted on the different cell clones of the source cell line GBC-SD using limited dilution method. Some biological characteristics of the different cell clones were studied by light microscope, cell growth analysis, cloning assay. Electrophoresis migration rates of different cell clones were tested to study charge properties and density of their surface. MTT assay was used for adhesion tests and the modified Boyden chamber model(Transwell chamber) was used for quantitative invasion and chemotaxis motility assay. The cell clone with highly invasiveness was selected by analyzing these results. Karyotyping was employed to characterize the target cell clone. Scanning electron microscope was used to observe the surface of the target cell clone. Flow cytometry and immunohistochemistry were used to detect the expressions of CD54 and matrix metalloproteinases respectively. The ASODN complementary to the start codon region of heparanase mRNA and its control, scrambled nonsense oligodeoxynucleotide(NSODN) were designed and synthesized and phosphorothioated. The ODNs were embedded in cationic liposome Lipofectin and transfected into the target cells. The total RNAs were extracted from the cells in 48 hours after transfection and then semi-quantitative RT-PCR and immunohistchemsitry
were performed to evalutate the heparanase gene and protein expression levels respectively. The invasiveness of transfected cells was measured quantitatively by Matrigel invasion assays(Transwell chamber).Results In all 21 cell clones were successfully isolated from the source cell line GBC-SD. No obvious difference among the 21 cell clones was observed by light microscope. Comparing the 21 cell clones, the doubling time varied from 15.38 hours to 24.65 hours, the clonogenesis from 26% to 36.4%, electrophoresis migration rates from 2.44um/s / V/cm to 6.14um/s / V/cm and the rates of adhesion from 22.6% to 45.6%. The number of cells that penetrated the filter was from 46.3 to 81.7 in quantitative invasion assay and from 81.1 to 126.4 in chemotaxis motility assay. The No.2 clone was obtained as the high invasiveness cell clone by analyzing these results and named GBC-SDHj. When GBC-SDHj was compared with GBC-SD, the number of chromosome was 59~92 versus 42~136, and fluorescent intensity and percent of positive cells of CD54 were 561.39 and 15.60% versus 34.12 and 3.17%. There were much more microvilli and filopodia, with crotches in distal end, on surface of GBC-SDHi cells than that of GBC-SD cells under scanning electron microscope. The immunohistochemistry showed that the expression levels of MMP-2 and MMP-9 in GBC-SDH, cells were higher than that of GBC-SD cells. The heparanase gene and protein expression and invasiveness of GBC-SDHj cells treated with ASODN of diferent final concentrations were significantly decreased compared with that of the controls (PO.01). Furthermore the inhibitory effects were significantly different among the cells treated with ASODN of different concentration (PO.05). The invasiveness inhibition rates were 37%, 51%, 63% and 66% at the cells treated with ASODN of the final concentrations of 0.2umol/L, 0.4umol/L, 0.8umol/L and 1.6umol/L respectivelyConclusion 1 .GBC-SDHj cell was a cell clone with high invasiveness. 2.Some biological characteristics were different between GBC-SDHj cells and GBC-SD cells. 3.Heparanase ASODN complementary to the start condon region of heparanase mRNA has a significant inhibitory effect on the invasiveness of human gallbladder carcinoma cell line in a dose-dependent manner.
Part three:Killing effects of combination of cytosine deaminase and HSV-tk genes ongallbladder cancer mediated by retroviral vectorObjective To investigate the different cell-killing and bystander effects on GBC-SD cells in vitro and vivo with double suicide genes system(CD/5-FC and HSV-tk/GCV) transferred by retroviral vector and to find a more efficient and low-toxicity suicide gene therapy for gallbladder cancer.Material and methods The PA317 cells with CD and HSV-tk double suicide genes were cultured after G418 selected and the viral supernatant was harvested. The GBC-SD cells were infected with the viral supernatant. After G418 selection, the GBC-SD/CD+tk cells in culture were picked and the expression of double suicide genes was confirmed by RT-PCR. After administration of 5-FC and/or GCV the morphological changes of those cells were observed through optical microscope and scanning electron microscope. The cell cycle and apoptosis rates were analyzed by flow cytometry (FCM). The cytoxicity efficacy of 5-FC and/or GCV on GBC-SD/CD+tk cells was evaluated by MTT method. Furthermore the GBC-SD/CD+tk and GBC-SD cells at varying mixing ratio were treated by the different concentration of 5-FC and GCV to explore the bystander effects. In vivo experiment, 36 Balb/c nude mice of four to six weeks old were divide into 6 groups at random and the cell line GBC-SD/CD+tk or/and GBC-SD were inoculated subcutaneously into the nude mice respectively. When the tumor were palpable, retroviral supernatant containing CD and HSV-tk genes were directly injected into tumors of the Group GBC-SD. For the 4 experiment groups and the GBC-SD contral group GCV and 5-FC were administrated intraperitoneally and the other contral group was given normal saline(NS). 21 days later animals were sacrificed and the sizes of the specimens were measured and processed for histopathological analysis.Results The virus supernatant containing double suicide gene was obstained
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