消炎痛抑制肝细胞肝癌生长转移的研究

Objective To study the effects of indomethacin on proliferation and invasion of hepatocellular carcinoma (HCC) cell line MHCC97L with metastatic potential and the effect of indomethacin on the growth and metastasis of HCC. Methods (1) In vitro; Proliferation, Transwell invasion assay, cell motility assay, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) protein activity were evaluated after cells were treated with 0. 2 mmol/L indomethacin. (2)In vivo: Mice bearing xenografts in the liver were randomly divided into control and indomethacin groups. At the end of sixth week, the mice were killed and tumor volume, inhibitory rate, immunohistochemistry assay (IHA) and metastasis were evaluated. Results (1)In vitro; 0. 2 mmol/L indomethacin could inhibit the proliferation of MHCC97L cells markedly (P ＜0. 01). The average amount of invading cells per field in cell invasion assay and motility assay was 2. 2 ± 1. 3 and 4.4 ± 1. 1 respectively in indomethacin group, significantly less than in control group ( 11. 4 ± 1. 9 and 12. 8 ± 1. 8 respectively, P ＜0. 01). The expression of VEGF and MMP-2 in cells treated with indomethacin was significantly lower than in control group (P ＜0. 01). (2)In vivo; Tumor volume, incidence and number of lung metastases in control and indomethacin groups were (1700 ±422) mm3 and (1170 ± 585) mm3 (P ＜ 0. 05), 75% and 50% ( P ＞ 0.05), 2. 92 ± 2. 07 and 1.33 ±1.56 (P＜0. 05) , respectively. Inhibition rate in indomethacin group was 31.2%. IHA showed that the expression of VEGF, MMP-2, and cyclooxygenase-2 ( COX-2) was down-regulated in indomethacin group (P ＜0.01). Conclusion Indomethacin could inhibit the growth and metastasis of HCC, which was in part mediated by down-regulation of VEGF and MMP-2.     

消炎痛抑制肝细胞肝癌生长转移的研究

Objective To study the effects of indomethacin on proliferation and invasion of hepatocellular carcinoma (HCC) cell line MHCC97L with metastatic potential and the effect of indomethacin on the growth and metastasis of HCC. Methods (1) In vitro; Proliferation, Transwell invasion assay, cell motility assay, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) protein activity were evaluated after cells were treated with 0. 2 mmol/L indomethacin. (2)In vivo: Mice bearing xenografts in the liver were randomly divided into control and indomethacin groups. At the end of sixth week, the mice were killed and tumor volume, inhibitory rate, immunohistochemistry assay (IHA) and metastasis were evaluated. Results (1)In vitro; 0. 2 mmol/L indomethacin could inhibit the proliferation of MHCC97L cells markedly (P ＜0. 01). The average amount of invading cells per field in cell invasion assay and motility assay was 2. 2 ± 1. 3 and 4.4 ± 1. 1 respectively in indomethacin group, significantly less than in control group ( 11. 4 ± 1. 9 and 12. 8 ± 1. 8 respectively, P ＜0. 01). The expression of VEGF and MMP-2 in cells treated with indomethacin was significantly lower than in control group (P ＜0. 01). (2)In vivo; Tumor volume, incidence and number of lung metastases in control and indomethacin groups were (1700 ±422) mm3 and (1170 ± 585) mm3 (P ＜ 0. 05), 75% and 50% ( P ＞ 0.05), 2. 92 ± 2. 07 and 1.33 ±1.56 (P＜0. 05) , respectively. Inhibition rate in indomethacin group was 31.2%. IHA showed that the expression of VEGF, MMP-2, and cyclooxygenase-2 ( COX-2) was down-regulated in indomethacin group (P ＜0.01). Conclusion Indomethacin could inhibit the growth and metastasis of HCC, which was in part mediated by down-regulation of VEGF and MMP-2.     

绿脓杆菌制剂对人肝癌MHCC97L细胞增殖和侵袭能力的影响

Objective To investigate the effects of Pseudomonas aeruginosa vaccine (PA) on proliferation and invasiveness of the hepatocellular carcinoma cell line MHCC97L with metastatic potential. Methods Proliferation, growth curve, plate efficiency, flow cytometry, transwell invasion assay, cell motility assay, scarification test, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP2) protein activity were evaluated after cells were treated with PA at various concentrations. Results PA can inhibit the proliferation and plate efficiency of MHCC97L cell markedly in a dose-dependent manner. The IC50 of cells treated with PA for 48 h and 72 h was 3.1 ×108/ml and 1.9 × 108/ml, respectively. The doubling time increased and plate efficiency decreased gradually when cells treated with 0.5 × 108/ml, 1 × 108/ml and 2 × 108/ml PA (P＜0.01). PA could induce cell cycle arrest at the G1 phase in a dose-dependent manner by flow cytometric analysis. The average amount of invading cell per field in cell invasion assay and motility assay were 4. 8 ± 1.3 and 8. 8±2.2 when cells treated with 1× 108/ml PA, which was significantly lower than that of control group (8. 6±2. 1 and 15. 6±1.2 ) (P＜0.01) Scarification test showed that the metastatic ability of cells treated with 1 × 108/ml PA significantly lower than that in the control group. Comparison between cells treated with 1 × 108/ml PA and control group, no remarkable difference was found regarding expression of VEGF and MMP2 in supernatant of cell culture. Conclusion PA can inhibit proliferation and plate efficiency of HCC cell line MHCC97L, which is in part mediated by the cell cycle arrest at the G1 phase. PA could inhibit invasiveness of HCC cell line MHCC97L, which is unrelated to the VEGF and MMP2 protein activity.     

绿脓杆菌制剂对人肝癌MHCC97L细胞增殖和侵袭能力的影响

Objective To investigate the effects of Pseudomonas aeruginosa vaccine (PA) on proliferation and invasiveness of the hepatocellular carcinoma cell line MHCC97L with metastatic potential. Methods Proliferation, growth curve, plate efficiency, flow cytometry, transwell invasion assay, cell motility assay, scarification test, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP2) protein activity were evaluated after cells were treated with PA at various concentrations. Results PA can inhibit the proliferation and plate efficiency of MHCC97L cell markedly in a dose-dependent manner. The IC50 of cells treated with PA for 48 h and 72 h was 3.1 ×108/ml and 1.9 × 108/ml, respectively. The doubling time increased and plate efficiency decreased gradually when cells treated with 0.5 × 108/ml, 1 × 108/ml and 2 × 108/ml PA (P＜0.01). PA could induce cell cycle arrest at the G1 phase in a dose-dependent manner by flow cytometric analysis. The average amount of invading cell per field in cell invasion assay and motility assay were 4. 8 ± 1.3 and 8. 8±2.2 when cells treated with 1× 108/ml PA, which was significantly lower than that of control group (8. 6±2. 1 and 15. 6±1.2 ) (P＜0.01) Scarification test showed that the metastatic ability of cells treated with 1 × 108/ml PA significantly lower than that in the control group. Comparison between cells treated with 1 × 108/ml PA and control group, no remarkable difference was found regarding expression of VEGF and MMP2 in supernatant of cell culture. Conclusion PA can inhibit proliferation and plate efficiency of HCC cell line MHCC97L, which is in part mediated by the cell cycle arrest at the G1 phase. PA could inhibit invasiveness of HCC cell line MHCC97L, which is unrelated to the VEGF and MMP2 protein activity.     

抗肝肠钙粘连蛋白单克隆抗体对肝癌细胞生物学行为的影响

目的 观察抗肝肠钙粘连蛋白(CDH17)单克隆抗体Lic5对肝癌细胞生物学行为的影响.方法 Western blot和实时定量聚合酶链反应(PCR)检测细胞株MHCC97H、MHCC97L、PLC/PRF/5及MIHA中CDH17的表达.噻唑蓝(MTT)比色法、细胞划痕法、Transwell法及平板克隆法检测Lic5对肝癌细胞生物学行为的影响.结果 CDH17仅在细胞株MHCC97H、MHCC97L中表达,Lic5可结合肝癌细胞表面的CDH17,并抑制CDH17表达.Lic5 50mg/L组、100mg/L组、小鼠IgG组4 d细胞增殖抑制率在MHCC97H为26.1%、43.6%、6.4%,MHCC97L为26.0%、40.7%、7.7%;Lic5100mg/L组、小鼠IgG组、磷酸盐缓冲液(PBS)组48h细胞迁移抑制率在MHCC97H为36.7%、8.4%、5.6%,MHCC97L为42.3%、10.2%、7.4%(P＜0.05);穿膜细胞数在MHCC97H为(39.20±9.56)、(106.50±7.56)、(96.60±13.02)个,MHCC97L为(26.00±8.61)、(86.00±10.26)、(90.40±12.04)(P＜0.05);克隆形成数在MHCC97H为(59.30±11.68)、(141.70±19.40)、(150.30±14.64),MHCC97L为(57.20±10.21)、(132.50±9.07)、(121.70±11.93)(P＜0.01).Lic5对PLC/PRF/5及MIHA细胞的生物学行为无明显影响.结论 单克隆抗体Lic5能够下调肝癌细胞CDH17表达,抑制肝癌细胞的增殖、迁移、侵袭和克隆形成能力.

Abstract：

Objective To investigate the effect of monoclonal antibody against liver-intestine cadherin (CDH17) on the cell proliferation, migration, invasion and colony formation of hepatocellular carcinoma cell lines. Methods Hepatocellular carcinoma cell lines MHCC97H, MHCC97L, PLC/PRF/5 and MIHA were examined for CDH17 expression by Western blotting and quantitative real-time polymerase chain reaction (PGR). The combination capacity between bepatocellular carcinoma cell lines and monoclonal antibody Lic5 was detected by the way of immunofluorescence staining. The cell lines were treated with Lic5, PBS and mouse IgG respectively. Methyl thiazol tetrazolium (MTT) assay, wound healing assay, Transwell invasion assay and colony formation assay were used to study the changes in cell proliferation, migration, invasion and colony formation of hepatocellular carcinoma cell lines. Results High expression level of CDH17 was detected in MHCC97H and MHCC97L cell lines. CDH17 protein level was down-regulated but there was no significant effect on CDH17 mRNA after treatment with Lie5 in MHCC97H and MHCC97L. Cellular growth rate of MHCC97H in Lic5 (50 mg/L), Lic5 ( 100 mg/L) and mouse IgG groups was decreased by 26. 1%, 43.6% and 6. 4%, and by 26. 0%, 40. 7% and 7. 7% in MHCC97L on the 4th day respectively (P ＜0. 05 ). The inhibition rate of cell migration at 48 h was 36. 7%, 8. 4% and 5.6% in Lic5 ( 100 mg/L), mouse IgG and PBS groups in MHCC97H, and 42. 3%, 10. 2% and 7. 4% in MHCC97L respectively ( P ＜ 0. 05 ). The number of invasion cells was ( 39. 20 t 9. 56),(106.50±7.56) and (96.60±13.02) in MHCC97H, and (26.00±8.61), (86.00±10.26) and (90.40±12.04) in MHCC97L in Lic5 (50 mg/L), Lic5 (100 mg/L) and mouse IgG groups, respectively (P ＜ 0. 05 ). The number of colony formation was ( 59. 30 ± 11.68 ), ( 141.70 ± 19. 40 ) and (150.30 ±14.64) in MHCC97H, and (57.20 ± 10.21), (132.50 ±9.07) and (121.70 ±11.93) in MHCC97L in Lie5 (50 mg/L), Lic5 (100 mg/L) and mouse IgG groups, respectively (P＜ 0. 01 ).There was no significant difference between Lic5 treatment groups and controls in PLC/PRF/5 and MIHA cell lines. Conclusion The anti-CDH17 monoclonal antibody Lic5 can down-regulate CDH17 expression and inhibit the cell proliferation, migration, invasion and colony formation in hepatocellular carcinoma cell lines.     

The expression of β-adrenoceptors in human pancreatic cancer cell lines and their roles in cell invasiveness

Objective To investigate the effect and mechanism of β-adrenoceptors on norepi-nephrine-induced invasiveness of pancreatic cancer cell lines. Methods The expression of β-adrenocep-tors mRNA in human pancreatic cancer cell lines MiaPaCa-2 and BxPC3 was detected by using RT-PCR. The cells were randomly divided into control group.10 mol/L NE intervention group, 1 mol/L propranolol intervention group and NE + propranolol intervention group. After 48 h , transwell invasiveness test was used to examine the changes in invasive ability of MiaPaCa-2. The expression of MMP-2, MMP-9 and VEGF mRNA was measured by semi-quantitative RT-PCR. The levels of MMP-2 , MMP-9 and VECF proteins were assayed by immunocytochemistry. Results Both MiaPaCa-2 and BxPC3 expressed β1-and β2-adrenocep-tors. The absorbance ( A) values of invasive cells in NE, NE + propranolol, propranolol and control groups were 0.78±0.02 ,0.32±0.03 ,0.26±0.01 and 0.28±0.02 , respectively, and those in NE intervention group were significantly higher than in control and NE + propranolol groups ( P <0.05) . There was no sig-nificant difference in the number of invasive cells between propranolol and control groups ( P > 0. 05) . In NE group , the expression index of MMP-2 , MMP-9 and VEGF mRNA was 0. 87±0.02 , 1.04±0.02 and 0. 92±0. 01 , and the gray value of the protein expression was 131.20±2.34,105.32±7.21 and 115.60 ±5. 03 , respectively, which were higher than those in control and NE + propranolol groups ( P<0.05). There was no significant difference in the expression levels of MMP-2 , MMP-9 and VEGF mRNA and pro-tein between propranolol and control groups ( P>0.05 ) . Conclusion β-adrenoceptors play an important role in the process of norepinephrine-induced invasiveness of pancreatic cancer cells. NE can promote the invasiveness of MiaPaCa-2 through up-regulating the expression of MMP-2 , MMP-9 and VEGF via β-adre-noceptors.     

The expression of β-adrenoceptors in human pancreatic cancer cell lines and their roles in cell invasiveness

Objective To investigate the effect and mechanism of β-adrenoceptors on norepi-nephrine-induced invasiveness of pancreatic cancer cell lines. Methods The expression of β-adrenocep-tors mRNA in human pancreatic cancer cell lines MiaPaCa-2 and BxPC3 was detected by using RT-PCR. The cells were randomly divided into control group.10 mol/L NE intervention group, 1 mol/L propranolol intervention group and NE + propranolol intervention group. After 48 h , transwell invasiveness test was used to examine the changes in invasive ability of MiaPaCa-2. The expression of MMP-2, MMP-9 and VEGF mRNA was measured by semi-quantitative RT-PCR. The levels of MMP-2 , MMP-9 and VECF proteins were assayed by immunocytochemistry. Results Both MiaPaCa-2 and BxPC3 expressed β1-and β2-adrenocep-tors. The absorbance ( A) values of invasive cells in NE, NE + propranolol, propranolol and control groups were 0.78±0.02 ,0.32±0.03 ,0.26±0.01 and 0.28±0.02 , respectively, and those in NE intervention group were significantly higher than in control and NE + propranolol groups ( P <0.05) . There was no sig-nificant difference in the number of invasive cells between propranolol and control groups ( P > 0. 05) . In NE group , the expression index of MMP-2 , MMP-9 and VEGF mRNA was 0. 87±0.02 , 1.04±0.02 and 0. 92±0. 01 , and the gray value of the protein expression was 131.20±2.34,105.32±7.21 and 115.60 ±5. 03 , respectively, which were higher than those in control and NE + propranolol groups ( P<0.05). There was no significant difference in the expression levels of MMP-2 , MMP-9 and VEGF mRNA and pro-tein between propranolol and control groups ( P>0.05 ) . Conclusion β-adrenoceptors play an important role in the process of norepinephrine-induced invasiveness of pancreatic cancer cells. NE can promote the invasiveness of MiaPaCa-2 through up-regulating the expression of MMP-2 , MMP-9 and VEGF via β-adre-noceptors.     

The expression of β-adrenoceptors in human pancreatic cancer cell lines and their roles in cell invasiveness

Objective To investigate the effect and mechanism of β-adrenoceptors on norepi-nephrine-induced invasiveness of pancreatic cancer cell lines. Methods The expression of β-adrenocep-tors mRNA in human pancreatic cancer cell lines MiaPaCa-2 and BxPC3 was detected by using RT-PCR. The cells were randomly divided into control group.10 mol/L NE intervention group, 1 mol/L propranolol intervention group and NE + propranolol intervention group. After 48 h , transwell invasiveness test was used to examine the changes in invasive ability of MiaPaCa-2. The expression of MMP-2, MMP-9 and VEGF mRNA was measured by semi-quantitative RT-PCR. The levels of MMP-2 , MMP-9 and VECF proteins were assayed by immunocytochemistry. Results Both MiaPaCa-2 and BxPC3 expressed β1-and β2-adrenocep-tors. The absorbance ( A) values of invasive cells in NE, NE + propranolol, propranolol and control groups were 0.78±0.02 ,0.32±0.03 ,0.26±0.01 and 0.28±0.02 , respectively, and those in NE intervention group were significantly higher than in control and NE + propranolol groups ( P <0.05) . There was no sig-nificant difference in the number of invasive cells between propranolol and control groups ( P > 0. 05) . In NE group , the expression index of MMP-2 , MMP-9 and VEGF mRNA was 0. 87±0.02 , 1.04±0.02 and 0. 92±0. 01 , and the gray value of the protein expression was 131.20±2.34,105.32±7.21 and 115.60 ±5. 03 , respectively, which were higher than those in control and NE + propranolol groups ( P<0.05). There was no significant difference in the expression levels of MMP-2 , MMP-9 and VEGF mRNA and pro-tein between propranolol and control groups ( P>0.05 ) . Conclusion β-adrenoceptors play an important role in the process of norepinephrine-induced invasiveness of pancreatic cancer cells. NE can promote the invasiveness of MiaPaCa-2 through up-regulating the expression of MMP-2 , MMP-9 and VEGF via β-adre-noceptors.     

The expression of β-adrenoceptors in human pancreatic cancer cell lines and their roles in cell invasiveness

Objective To investigate the effect and mechanism of β-adrenoceptors on norepi-nephrine-induced invasiveness of pancreatic cancer cell lines. Methods The expression of β-adrenocep-tors mRNA in human pancreatic cancer cell lines MiaPaCa-2 and BxPC3 was detected by using RT-PCR. The cells were randomly divided into control group.10 mol/L NE intervention group, 1 mol/L propranolol intervention group and NE + propranolol intervention group. After 48 h , transwell invasiveness test was used to examine the changes in invasive ability of MiaPaCa-2. The expression of MMP-2, MMP-9 and VEGF mRNA was measured by semi-quantitative RT-PCR. The levels of MMP-2 , MMP-9 and VECF proteins were assayed by immunocytochemistry. Results Both MiaPaCa-2 and BxPC3 expressed β1-and β2-adrenocep-tors. The absorbance ( A) values of invasive cells in NE, NE + propranolol, propranolol and control groups were 0.78±0.02 ,0.32±0.03 ,0.26±0.01 and 0.28±0.02 , respectively, and those in NE intervention group were significantly higher than in control and NE + propranolol groups ( P <0.05) . There was no sig-nificant difference in the number of invasive cells between propranolol and control groups ( P > 0. 05) . In NE group , the expression index of MMP-2 , MMP-9 and VEGF mRNA was 0. 87±0.02 , 1.04±0.02 and 0. 92±0. 01 , and the gray value of the protein expression was 131.20±2.34,105.32±7.21 and 115.60 ±5. 03 , respectively, which were higher than those in control and NE + propranolol groups ( P<0.05). There was no significant difference in the expression levels of MMP-2 , MMP-9 and VEGF mRNA and pro-tein between propranolol and control groups ( P>0.05 ) . Conclusion β-adrenoceptors play an important role in the process of norepinephrine-induced invasiveness of pancreatic cancer cells. NE can promote the invasiveness of MiaPaCa-2 through up-regulating the expression of MMP-2 , MMP-9 and VEGF via β-adre-noceptors.     

肝癌体外培养三维模型的建立

目的 构建能模拟癌细胞从黏附、侵袭到肝内癌巢形成过程的肝癌肝侵袭转移体外实验模型.方法 将肝癌细胞MHCC97H与肝组织片层置于RWV生物反应器进行三维混合培养,收集不同培养时间的混合类组织标本,检测其病理改变及侵袭转移关联基因表达.结果 转移关联基因的表达随病理侵袭进程的不同(癌细胞黏附、侵袭、癌巢形成)而呈不同模式[初期:基质金属蛋白酶(MMP)-9,t=-5.847,P＜0.01;钙黏蛋白(CDH1),t=3.199,P＜0.05;,MMP-7和CD44,相对表达均＞2.中期:MMP-2,t=-10.295,P＜0.01;趋化因子受体(CXCR4)和骨桥蛋白(SPP1),相对表达＞2和＞1.7.后期:CXCL12,和=-4.412,P＜0.05].结论 该模型较好地模拟了肝癌肝侵袭转移的病理全过程,有助于侵袭转移病理阶段的认识及"过程"关键蛋白筛查.

Abstract：

Objective To develop a 3D experimental model of HCC intrahepatic invasion and metastasis in vitro for understanding the mechanism of invasion and metastasis. Methods The highly metastatic MHCC97H cells and a liver tissue fragment were seeded into the RWV bioreactor for 3D rotating coculture. The spherical cocultures at different time points were collected respectively for pathological morphology analysis and metastasis associated genes analysis. Results The patterns of HCC metastasis associated genes in the cocultures were diverse in parallel with different pathological states of tumor cells invasion [Early stage of invasion: matrix metalloproteinase (MMP) -9, t = - 5. 847, P ＜ 0. 01; CDH1, t = 3. 199,P ＜ 0. 05; MMP-7 and CD44, fold increase ＞ 2. Middle stage: MMP-2, t = - 10. 295, P ＜ 0. 01; CXCR4 and Secreted phosphoprotein 1 (SPP1), fold increase ＞ 2 and ＞ 1.7. kate stage: CXCL1 2,t = -4. 412,P ＜ 0. 05]. Conclusion The established experimental model better mirrors the pathological process of HCC intrahepatic invasion in vitro, which will be helpful for discovery of many metastasis "process" associated proteins.     

小剂量阿司匹林协同干扰素-α抑制肝细胞肝癌生长转移的实验研究

目的 探讨小剂量阿司匹林协同干扰素-α(IFN-α)抑制肝细胞癌(hepatocellular carcinoma,HCC)生长转移的作用及机制.方法 培养具有肺转移潜能的人MHCC97L肝癌细胞.将MHCC97L肝癌组织块种植于BALB/c nu/nu雄性裸鼠肝脏,建立人肝癌裸鼠原位模型.以不同剂量组合的阿司匹林和IFN-α作用于荷瘤裸鼠,测量肿瘤体积,计算肺转移灶数目及肺转移率.用MTT及明胶酶谱实验检测阿司匹林对MHCC97L细胞增殖及金属蛋白酶2(matrix metalloproteinases 2,MMP-2)活性的影响.用Western Blot及ELISA检测细胞及血清MMP-2及血管内皮生长因子(vascular endothelial growth factor,VEGF)蛋白水平.结果 对照组肿瘤体积为(3.12±0.85)cm3,肺转移率为66.7％.大剂量阿司匹林[45 mg/(kg· d)]治疗组肿瘤体积为(1.89±0.88)cm3 (P＞0.05),肺转移率为58.3％ (P＞0.05).而大剂量IFN-α[1.5×107/(kg·d)]治疗组、大剂量IFN-α+大剂量阿司匹林治疗组、小剂量IFN-α [7.5×106/(kg·d)]+小剂量阿司匹林15 mg/(kg· d)]治疗组肿瘤体积分别为(0.69±0.40)cm3、(0.55±0.31)cm3、(0.40±0.43)cm3,均显著低于对照组(P＜0.05),肺转移率均为0(P＜0.05).2 mmol/L阿司匹林对MHCC97L细胞增殖无显著影响(P＞0.05),但可抑制其MMP-2的活性及VEGF的水平.小剂量IFN-α+小剂量阿司匹林治疗组裸鼠血清MMP-2及VEGF显著降低(P＜0.05).结论 小剂量阿司匹林可协同IFN-α抑制HCC生长转移,抑制MMP-2和VEGF的活性和表达是其重要作用机制之一.     

绿脓杆菌制剂对人肝癌MHCC97L细胞增殖和侵袭能力的影响

目的 探讨绿脓杆菌制剂(pseudomonas aeruginosa vaccine,PA)对有转移潜能的人肝癌MHCC97L细胞增殖和侵袭能力的影响.方法 采用不同浓度的PA分别作用于MHCC97L细胞,观察细胞增殖、生长曲线、克隆形成率、流式细胞术分析(FCM)、侵袭、运动、迁移、VEGF和MMP2蛋白表达(ELISA法).结果 PA明显抑制MHCC97L细胞增殖和克隆形成,呈良好的剂量-效应关系.PA 48 h和72 h的IC50分别为3.1×108/ml和1. 9×108/ml.PA浓度为0.5×108/ml、1×108/ml和2×108/ml时,其细胞倍增时间依次增加,克隆形成率依次降低(P均＜0.01);FCM显示,G1期细胞比例随PA浓度增加而增加,S+G2期细胞比例随PA浓度增加而降低(P均＜0.01).PA浓度为1×108/ml时,穿过人工基底膜(侵袭实验)和上室底膜(运动实验)的细胞数(分别为4.8±1.3和8.8±2.2)明显低于对照组(8.6±2.1和15.6±1.2)(P均＜0.01);细胞经72 h培养后,对照组划痕逐渐愈合,1×108/ml的PA组细胞划痕依然明显.ELISA法检测发现,1×108/ml的PA组其VEGF蛋白和MMP2蛋白含量和对照组相比均无明显差异(P均＞0.05).结论 在一定条件下,绿脓杆菌制剂可抑制人肝癌MHCC97L细胞增殖和克隆形成,其作用部分是通过使细胞周期阻滞在G1期实现的;绿脓杆菌制剂可以明显抑制MHCC97L细胞的侵袭、运动和迁移能力,其作用和VEGF、MMP2蛋白分泌关系不明显.     

膜型基质金属蛋白酶-1在浸润性肝细胞癌组织的表达及其意义

目的 探讨在浸润性肝细胞肝癌(HCC)中,膜型基质金属蛋白酶-1(MT1-MMP)的表达和意义.方法 随机选取2004年1月至2006年12月间根治性手术切除的HCC标本123例,通过对肿瘤的个数、包膜是否完整、门静脉有无癌栓、有无肝外转移等标准的划分,将其分为浸润转移组(74)及无浸润组(49).术前所有病例均采用酶联免疫吸附试验(ELISA)测定血清血管内皮生长因子(VEGF)水平;并通过免疫组织化学法(ABC),测定两组术后标本癌组织的MT1-MMP蛋白及肿瘤微血管密度(MVD)的表达;定量聚合酶链反应(PCR)测定癌组织MT1-MMP mRNA的表达.结果 浸润组术前血清VEGF:(1 33.89±68.56) μg/L;无浸润组:(100.64±81.37) μg/L,差异有统计学意义(P＜0.01).MT1-MMP主要定位表达在肝癌细胞的胞膜和间质.在浸润组中,MT1-MMP蛋白及mRNA表达量明显高于无浸润组(P＜0.05);浸润组MVD:11.24±1.49,无浸润组:8.11±2.51,两者比较浸润组MVD的形成量明显增多(P＜0.01).结论 在浸润性肝细胞肝癌中,MT1-MMP的表达明显增高,并伴随血清VEGF的表达上调、癌组织内新生肿瘤微血管的形成增多.MT1-MMP高表达可作为浸润性肝癌的判定指标,并提示临床预后较差.     

表达IL-24基因的溶瘤腺病毒对肝癌细胞的抑制作用

目的 研究携带IL-24基因的溶瘤腺病毒对肝癌细胞抑制生长和转移的作用.方法 构建Ad.HS4.AFP.E1A/IL-24,病毒的复制由人甲胎蛋白启动子控制,携带IL-24基因.检测病毒在不同细胞系中的选择性复制,以及对高转移潜能人肝癌细胞系MHCC97-H的生长抑制、诱导凋亡和抑制转移能力(侵袭、运动、黏附)的作用.结果 Ad.HS4.AFP.E1A/IL-24在肝癌细胞SMMC-7721、Hep3B和MHCC97-H中选择性表达,而不影响正常肝细胞L02(P＜0.05).Ad.HS4.AFP.E1A/IL-24可明显抑制MHCC97-H细胞的增殖,诱导其凋亡,并抑制其体外运动、侵袭和黏附能力(P＜0.01).RT-PCR和明胶酶谱显示其抑制肝癌作用与抑制MMP-2的表达有关.结论 携带IL-24基因的溶瘤腺病毒能够选择性抑制肝癌细胞的增殖并抑制其转移.

Abstract：

Objective To investigate the selective oncolytic role and antitumor action of a novel recombinant adenovirus containing E1A and IL-24 on hepatocellular carcinoma cell(HCC). Methods The recombinant adenovirus expressing IL-24 (Ad. HS4. AFP. E1A/IL-24) was constructed by using modified human alpha-fetoprotein (HS4-AFP) promoter to drive adenovirus E1A gene and II-24 gene.Cell Counting Kit-8 were performed to test the selective cytotoxicity of the virus in hepatocellular carcinoma cell lines SMMC-7721, Hep3B, MHCC97-H and hepatocyte cell line L02 . The mRNA and protein expression of IL-24 gene were detected by RT-PCR and western blot. Cell growth curves and Annexin V/PI assay were used to study cell proliferation and apoptosis of MHCC97-H. The anti-metastatic effects of the recombinant adenovirus were evaluated in cell adhesion, migration, and cell motion. Matrix metalloproteinase-2 (MMP-2) expression was examined by RT-PCR and zymography.Results Selective replications of Ad. HS4. AFP. E1A/IL-24 adenovirus were observed in over expression AFP cell line MHCC97-H, a highly metastatic potential HCC cell line but not in hepatocyte cell line L02. The mRNA and protein of IL-24 were also over expressed in MHCC97-H. This recombinant adenovirus also showed the significant oncolytic action on MHCC97-H but not on L02 (P＜0. 05). Besides, the recombinant adenovirus significantly inhibited MHCC97-H metastatic potential such as cell adhesion, migration and invasion as well(P＜0.01). Conclusion The selective oncolytic adenovirus expressing E1A and II-24 has a selective antitumor effect and play an inhibitory role in metastasis of HCC.     

活化血管内皮生长因子受体1诱导肝癌细胞株MHCC97-H细胞上皮-间叶表型转化的分子机制

目的 探讨活化血管内皮生长因子受体1(VEGFR-1)诱导肝癌细胞株MHCC97-H细胞上皮-间叶表型转化(EMT)的分子机制.方法 将MHCC97-H细胞分为对照组(1％胎牛血清的DMEM培养)、PP2组(10 μmol/L PP2培养)、PBS组(10 μmol/L PBS培养)、VEGF-B组(50 μg/L的VEGF-B培养)、PP2+ VEGF-B组(10 μmol/L PP2和50 μg/L的VEGF-B培养)、PBS+ VEGF-B组(10 μmol/L PBS和50 μg/L VEGF-B培养).Westem blot法检测各组上皮标志物E-钙黏蛋白、α-连环蛋白和间叶标志物波形蛋白、N-钙黏蛋白的表达水平;细胞免疫荧光检测上述蛋白的表达部位;细胞侵袭和迁移试验检测各组MHCC 97-H细胞的侵袭和运动能力.组间比较采用t检验.结果 对照组、PP2组、PBS组、VEGF-B组、PP2+ VEGF-B组、PBS+ VEGF-B组的MHCC97-H细胞上皮标志物E-钙黏蛋白的表达分别为3.23±0.76、4.18±0.32、2.83 +0.65、2.06±0.15、6.12±0.08、1.36±0.54;α-连环蛋白的表达分别为3.01 +0.25、3.29+0.11、3.03±0.27、2.84+0.76、5.45±0.37、1.26±0.45;波形蛋白的表达分别为3.01±0.22、4.85±0.36、1.37±0.24、5.79±0.38、3.36±0.42、4.05±0.17;N-钙黏蛋白的表达分别为2.63±0.40、3.02±0.52、2.98±0.36、5.54±0.28、3.26±0.13、1.05±0.33.PP2组、PP2+ VEGF-B组的MHCC97-H细胞E-钙黏蛋白和α-连环蛋白表达明显上调,PP2+ VEGF-B组与VEGF-B组比较,差异有统计学意义(t=7.625,9.931,P＜0.05).PP2+ VEGF-B组的波形蛋白和N-钙黏蛋白的表达显著低于VEGF-B组(t=12.001,11.910,P＜0.05).VEGF-B处理6h后,VEGF-B组、PP2+ VEGF-B组、PBS+ VEGF-B组的MHCC97-H细胞迁移数量分别为19±l、5±2和16±1,VEGF-B组MHCC97-H细胞迁移数量显著多于PP2+ VEGF-B组(t=13.566,P＜0.05),PP2+ VEGF-B组中MHCC97-H细胞穿过Matrigel包被的改良侵袭小室的数量为4±2,显著少于VEGF-B组的16±l(t=12.350,P＜0.05).结论 VEGFR-1活化诱导MHCC97-H细胞发生EMT是由c-Src激酶信号转导通路介导的,c-Src作为该信号通路的关键因子之一可能是干预肝细胞癌侵袭和转移的有效靶点.     

二氢杨梅素对肝癌细胞黏附、侵袭及迁移的抑制作用及机制

目的：研究二氢杨梅素（DHM）对肝癌细胞黏附、侵袭和迁移能力的影响及其可能机制。 方法：用不同浓度DHM处理肝癌MHCC97L细胞后,分别检测细胞的黏附能力、迁移与侵袭能力,以及E-cadherin、MMP-2、MMP-9和VEGF蛋白表达。 结果：与空白对照细胞比较,DHM处理后的MHCC97L细胞黏附力明显降低、侵袭与迁移力明显减弱（均P<0.05）;E-cadherin表达明显上调,而MMP-9、VEGF蛋白表达明显下调的水平（均P<0.05）,但MMP-2蛋白的表达无明显改变（均P>0.05）。 结论：DHM可能通过调控E-cadherin、MMP-9和VEGF蛋白的表达抑制肝癌细胞的黏附、迁移和侵袭。