As_2O_3抗肝癌侵袭转移作用及As_2O_3药物微球制备的实验研究
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摘要
第一部分:三氧化二砷对人高转移肝癌细胞株MHCC97H侵袭迁移能力影响
目的:了解三氧化二砷对MHCC97H肝癌细胞的侵袭迁移能力影响。方法:MHCC97H细胞与不同浓度三氧化二砷共孵育,细胞形态学观察,描记生长曲线,划痕实验细胞运动能力观察,Transwell小室了解三氧化二砷对细胞侵袭力的影响,RT—PCR检测三氧化二砷对MMP—9、Timp—1基因表达的影响。结果:培养细胞加药后细胞形态发生明显改变,悬浮细胞增多,贴壁减少,部分细胞变形、体积变小,空泡变性,部分细胞核固缩,胞浆内颗粒样物增多,这些表现随着药物作用时间的延长和药物浓度的增加而明显。三氧化二砷对MHCC97H有生长抑制作用,此抑制作用与药物浓度和作用时间均有依赖关系,生长抑制率随着药物浓度的增加、作用时间的延长而升高,2μmol/L三氧化二砷对MHCC97H细胞生长即表现出抑制作用,这种抑制作用在72h后明显加强。随着三氧化二砷浓度的增高MHCC97H细胞运动能力减弱,细胞变圆,伸展性变差,运动能力减弱。与空白对照组相比,2μmol/L浓度的三氧化二砷对97H细胞的运动能力就发生影响,8μmol/L浓度时明显抑制。Transwell实验中穿膜的细胞数也随着三氧化二砷浓度增加而减少,说明细胞侵袭能力下降。RT—PCR检测显示药物处理后MHCC97H细胞的MMP—9、Timp—1基因表达随三氧化二砷药物浓度的增加而减弱。结论:三氧化二砷能抑制MHCC97H细胞的生长,使细胞运动能力减弱,并抑制了肿瘤细胞的侵袭能力。
Invasion and metastasis is the most prominent obstacles to further improvement of clinical treatment efficacy in hepatocellular carcinoma (HCC). As_20_3 has apoptosis-inducing effect in many solid tumor cell lines, and MHCC97H is a highly metastatic human hepatocellular carcinoma cell line. Purpose: TO assess the anti-invasion and migration effect of As_20_3 on MHCC97H cell line. Methods: A wide range concentration of arsenic trioxide were cultured with MHCC97H with different time. We observed the cells change. Cell growth curve, scratch test, Transwell permeable supports were used to assess the anti-invasion and migration effect of As_2O_3 on MHCC97H cell line. RT-PCR test the express of MMP-9 and Timp-1 gene in MHCC97H which treated by different concentration of arsenic trioxide. Result: After culturing with As_20_3, MHCC97H cell present paste wall cells decrease. Float, vacuolation, deformation, granulation and chromatin condensation were observed. Those appearance were increased with the prolong of time and the concentration of arsenic trioxide increasing. Scratch test show that the cells which can creep out of the line were decreased after cultured with arsenic trioxide. MHCC97H cells were cultured with different concentration of arsenic trioxide in transwell permeable supports, shown the cells which can pass through the pores decreased with the concentration increase. PT-PCR test showed the express of MMP-9 and Timp-1 gene in MHCC97H were decreased with concentration of arsenic trioxide increasing. Conclusion: As_20_3 can inhabit the invasion and migration of the MHCC97H cell line.
Purpose: To investigate the inhibitory effect of As_203 on angiogenesis , growth and lung metastasis of LCI-D20 hepatocellular carcinoma in nude mice. Method: LCI-D20 tumor tissues were implanted into the livers of nude mice to establish models in present study. 20 nude mice were randomly divided into a treatment group and a control group, and then treated with As203 at a dose of 3mg/kg daily injection through the tail vein, or 0.9% sodium chloride solution (0. lml) as control. Therapy began on the 7 day after operation and continued for 10 consecutive days. A week later the nude mice were were killed. The liver tumor were weighed, microvessel density (MVD) was analyzed in CD34-stained tumor sections, lung metastatic lesions were searched. Results: LCI-D20 tumor implanted into liver successfully. On day 27 after implantation .average tumor volume was 0. 99±0. 45g in the As203 treated group; whereas it was 2.33 ±0. 78g in the control. The difference between two groups was statistically significant. A dramatically decreased microvessel density in tumor tissues was revealed in the As203 -treated mice compared with control tumor tissues 18. 87±3. 02 versus 48. 33±6. 37, P < 0.01). Conclusion: As203 is able to inhibit angiogenesis , growth and lung metastasis of LCID20 hepatocellular carcinoma in nude mice. The growth inhibitory mechanisms of As2O3 may be attributed to antiangiogenesis in this experiment.
Purpose: To optimize the preparation of sustained release microspheres of As203 using the biodegradable materials PLGA for interventional radiology therapy. Methods: The orthogonal test design was used to optimize the technology of preparation with good reproducibility. The surface morphology of the microspheres was observed by scanning electron microscope. The mean diameter and the size distribution of microspheres, the drug loading, the incorporation efficiency, the reappearances of pharmaceutical technology, drug release in vitro were examined. Result: the As2O3 PLGA mocrospheres were regular in their morphology. Drug was enveloped in microspheres but not physically mixed with PLGA. The average particle size was 47. 27±0. 836 u m with 91.25% of the microspheres being in the range of 20-100y m; the drug loading was 14. 60±0. 30%. The reappearance of pharmaceutical technology was good. The microspheres were stable for three months at 4"C and room temperature. The in vitro release properties could be expressed by the Weibull quation: InIn(l/(l-F(t)))=1.0214Int-5.1881 r=0.97. Conclusion: The technology of preparation was successful and As2O3-PLGA microspheres showed sighificant sustained release and suitable for interventional radiology therapy.
目的:了解三氧化二砷对MHCC97H肝癌细胞的侵袭迁移能力影响。方法:MHCC97H细胞与不同浓度三氧化二砷共孵育,细胞形态学观察,描记生长曲线,划痕实验细胞运动能力观察,Transwell小室了解三氧化二砷对细胞侵袭力的影响,RT—PCR检测三氧化二砷对MMP—9、Timp—1基因表达的影响。结果:培养细胞加药后细胞形态发生明显改变,悬浮细胞增多,贴壁减少,部分细胞变形、体积变小,空泡变性,部分细胞核固缩,胞浆内颗粒样物增多,这些表现随着药物作用时间的延长和药物浓度的增加而明显。三氧化二砷对MHCC97H有生长抑制作用,此抑制作用与药物浓度和作用时间均有依赖关系,生长抑制率随着药物浓度的增加、作用时间的延长而升高,2μmol/L三氧化二砷对MHCC97H细胞生长即表现出抑制作用,这种抑制作用在72h后明显加强。随着三氧化二砷浓度的增高MHCC97H细胞运动能力减弱,细胞变圆,伸展性变差,运动能力减弱。与空白对照组相比,2μmol/L浓度的三氧化二砷对97H细胞的运动能力就发生影响,8μmol/L浓度时明显抑制。Transwell实验中穿膜的细胞数也随着三氧化二砷浓度增加而减少,说明细胞侵袭能力下降。RT—PCR检测显示药物处理后MHCC97H细胞的MMP—9、Timp—1基因表达随三氧化二砷药物浓度的增加而减弱。结论:三氧化二砷能抑制MHCC97H细胞的生长,使细胞运动能力减弱,并抑制了肿瘤细胞的侵袭能力。
Invasion and metastasis is the most prominent obstacles to further improvement of clinical treatment efficacy in hepatocellular carcinoma (HCC). As_20_3 has apoptosis-inducing effect in many solid tumor cell lines, and MHCC97H is a highly metastatic human hepatocellular carcinoma cell line. Purpose: TO assess the anti-invasion and migration effect of As_20_3 on MHCC97H cell line. Methods: A wide range concentration of arsenic trioxide were cultured with MHCC97H with different time. We observed the cells change. Cell growth curve, scratch test, Transwell permeable supports were used to assess the anti-invasion and migration effect of As_2O_3 on MHCC97H cell line. RT-PCR test the express of MMP-9 and Timp-1 gene in MHCC97H which treated by different concentration of arsenic trioxide. Result: After culturing with As_20_3, MHCC97H cell present paste wall cells decrease. Float, vacuolation, deformation, granulation and chromatin condensation were observed. Those appearance were increased with the prolong of time and the concentration of arsenic trioxide increasing. Scratch test show that the cells which can creep out of the line were decreased after cultured with arsenic trioxide. MHCC97H cells were cultured with different concentration of arsenic trioxide in transwell permeable supports, shown the cells which can pass through the pores decreased with the concentration increase. PT-PCR test showed the express of MMP-9 and Timp-1 gene in MHCC97H were decreased with concentration of arsenic trioxide increasing. Conclusion: As_20_3 can inhabit the invasion and migration of the MHCC97H cell line.
Purpose: To investigate the inhibitory effect of As_203 on angiogenesis , growth and lung metastasis of LCI-D20 hepatocellular carcinoma in nude mice. Method: LCI-D20 tumor tissues were implanted into the livers of nude mice to establish models in present study. 20 nude mice were randomly divided into a treatment group and a control group, and then treated with As203 at a dose of 3mg/kg daily injection through the tail vein, or 0.9% sodium chloride solution (0. lml) as control. Therapy began on the 7 day after operation and continued for 10 consecutive days. A week later the nude mice were were killed. The liver tumor were weighed, microvessel density (MVD) was analyzed in CD34-stained tumor sections, lung metastatic lesions were searched. Results: LCI-D20 tumor implanted into liver successfully. On day 27 after implantation .average tumor volume was 0. 99±0. 45g in the As203 treated group; whereas it was 2.33 ±0. 78g in the control. The difference between two groups was statistically significant. A dramatically decreased microvessel density in tumor tissues was revealed in the As203 -treated mice compared with control tumor tissues 18. 87±3. 02 versus 48. 33±6. 37, P < 0.01). Conclusion: As203 is able to inhibit angiogenesis , growth and lung metastasis of LCID20 hepatocellular carcinoma in nude mice. The growth inhibitory mechanisms of As2O3 may be attributed to antiangiogenesis in this experiment.
Purpose: To optimize the preparation of sustained release microspheres of As203 using the biodegradable materials PLGA for interventional radiology therapy. Methods: The orthogonal test design was used to optimize the technology of preparation with good reproducibility. The surface morphology of the microspheres was observed by scanning electron microscope. The mean diameter and the size distribution of microspheres, the drug loading, the incorporation efficiency, the reappearances of pharmaceutical technology, drug release in vitro were examined. Result: the As2O3 PLGA mocrospheres were regular in their morphology. Drug was enveloped in microspheres but not physically mixed with PLGA. The average particle size was 47. 27±0. 836 u m with 91.25% of the microspheres being in the range of 20-100y m; the drug loading was 14. 60±0. 30%. The reappearance of pharmaceutical technology was good. The microspheres were stable for three months at 4"C and room temperature. The in vitro release properties could be expressed by the Weibull quation: InIn(l/(l-F(t)))=1.0214Int-5.1881 r=0.97. Conclusion: The technology of preparation was successful and As2O3-PLGA microspheres showed sighificant sustained release and suitable for interventional radiology therapy.
引文
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2. Engers R, Gabbert HE. Mechanism of cancer metastasis: cell biological aspects and clinical implication. J Cancer Res Clin Oncol. 2000; 126:682-692.
3. Mira E, Manes S, Lacalle RA, Marquez AC. Insulin-like growth factor I-trigger cell migration and invasion are mediated by matrix metalloproteinase-9. Endocrinlology 140:1657-1664.
4. Kondapaka SB, Fridman R, Reddy KB Epidermal growth factor and amphiregulin up-regulate matrix metalloproteinase-9(MMP-9) in human breast cancer cells. Int J Cancer. 1997; 70:722-726.
5. Reddy KB, Krueger JS, Kondapaka SB, Diglio CA Mitogen-activated protein kinase(MAPK) regulates the expression of progelatinase B(MMP-9) in breast epithelial cells. Int J Cancer. 1999; 82:268-273.
6. Gianelli G, Falk-Marzillier J, Schiraldi O, Stetler-Stevenson WG, Quaranta V Introduction of cell migration by matrix metalloproteinase-2 cleavage of laminin-5. Science. 1997; 277:225-228.
7. Grignon DJ, et al. High levels of tissue inhibitor of metalloproteinase-2 (TIMP-2) expression are associated with poor outcome in invasive bladder. cancer. Cancer Res. 1996; 56:1654-1659.
8. Hayakawa T, Yamashita K, et al. Cell growth-promoting activity of tissue inhibitor of metalloproteinase-2(TIMP-2). 1994, J Cell Sci 107(pt 9):2373-2379.
9.汤钊猷复发与转移——原发性肝癌研究的一个重点,中华肝胆外科杂志1999,5:3-5.
10. Folkman J. Role of angiogenesis in tumor growth and metastasis. Semin Oncol 2002; 29(6 Suppl 16):15-18
11. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell, 1996; 86:353-364.
12.赵万洲 韩锐肿瘤血管生成抑制剂研究策略及进展 中华肿瘤杂志2000:22 (2):93～95.
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