Huh7细胞中边缘群细胞分选及其增殖分裂能力的研究
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摘要
研究背景:
恶性肿瘤给人类的生存构成严重的威胁,现有治疗措施几乎都不能改变晚期恶性肿瘤复发并最终导致患者死亡的现状。由于肿瘤的侵袭以及长期治疗导致耐药仍然是当前攻克恶性肿瘤的主要障碍,因此,阐明肿瘤耐药的分子机制具有重大的理论意义及临床应用价值。肿瘤干细胞学说认为肿瘤组织中存在极少量干细胞样肿瘤细胞亚群,它们具有无限增殖的潜能,在启动肿瘤形成和生长中起着决定性的作用,而其余的大多数细胞,经过短暂的分化,最终死亡。肿瘤干细胞是造成肿瘤耐药的最根本原因,其对化疗药物的耐受性是肿瘤治疗的主要障碍。目前鉴定肿瘤干细胞主要有检测其特异性表面抗原标志物及检测边缘群细胞( side population, SP)两种方法。本研究试图探讨肝癌细胞株Huh7细胞中边缘群细胞的分选及其增值分裂能力,为肝癌的靶向治疗提供新的思路。
研究目的:
探讨Huh7细胞中边缘群细胞的分选及边缘群细胞在增值能力及分裂能力方面与非边缘群细胞的差异,为肝癌的治疗探索新的思路。
研究方法:
1.细胞培养: Huh7细胞常规培养于10%FBS DMEM培养基中。
2.单细胞悬液的制备:对数生长期细胞常规胰酶消化后吹打均匀,调整细胞密度为2～3×10~6/L(细胞计数板计数),部分细胞悬液先加150μmol/L维拉帕米37℃孵育20min作对照组,实验组和对照组均加入10μg/ml Hoechst33342在水浴锅中37℃避光孵育90min,每15min低速震荡摇匀一次,用10 mmol/L HEPES、10%FBS Hank’s液洗涤3次(离心力800转/min,5min/次),后置冰上,分选前经40μm孔径的滤网过滤制备成单细胞悬液,分选前5min加入P I至终浓度为2μg/ml。
3.采用流式细胞仪分选Huh7细胞中的边缘群细胞和非边缘群细胞。
4.利用荧光显微镜观察边缘群细胞和非边缘群细胞内Hoechst33342荧光染色强度差异。
5.以半定量RT-PCR测定边缘群细胞和非边缘群细胞ABCG2 mRNA表达水平,ABCG2 PCR产物送公司测序,GAPDH为内参。
6.免疫荧光染色检测边缘群细胞和非边缘群细胞ABCG2蛋白表达的差异并观察其增值能力和不对称分裂能力的差异。
研究结果:
1.Huh7细胞经流式细胞仪分选发现非维拉帕米处理组边缘群细胞比例约为2.22% ,经维拉帕米阻断后边缘群细胞比例减少至0.74%。
2.荧光显微镜下观察发现边缘群细胞中Hoechst33342的荧光强度较非边缘群细胞明显弱。
3.ABCG2 mRNA表达水平在边缘群细胞中是非边缘群细胞的4.32倍(P<0.01)。
4.边缘群细胞增值能力是非边缘群细胞的2.7倍(P<0.01)。
5.边缘群细胞能产生ABCG2(+)和ABCG2(-)子代细胞,而非边缘群细胞只能产生ABCG2(-)子代细胞。
研究结论:
Huh7细胞中存在少量边缘群细胞(2.22%),边缘群细胞增值能力较非边缘群细胞强且具有不对称分裂(自我更新和定向分化)能力,具有肿瘤干细胞的特性;因此边缘群细胞分选可以作为肿瘤干细胞分离的一种方法,边缘群细胞可能在肿瘤的形成、进展、耐药及复发转移中具有更重要作用。
Background:
The malignant tumors pose a serious threat to the survival of human beings. The existing treatment measures hardly change the status that patients with advanced cancers eventually lead to recurrence and even death. Presently, tumor invasion and drug resistance owing to long-term treatment are still major obstacles to overcome cancer. Therefore, to clarify the molecular mechanism of tumor drug resistance has both important value of basic theory and clinical applications. There are a very small number of stem cell-like tumor cells in tumor tissues based on the theory of cancer stem cells, which have unlimited potential for proliferation, and play a decisive role in tumor formation, drug resistance, and recurrence and metastasis, while the majority of the remaining cells will go to die after a short spell of division. Cancer stem cells are the most fundamental cause of drug resistance, whose tolerance to chemotherapeutic drugs is a main obstacle to cancer treatment.
Presently, the isolation of cancer stem cells is mainly based on the markers of surface antigen-specific antibody or detection of the side population cells through FACS. This study aims to explore the the side population cells in hepatocellular carcinoma cell line Huh7 and their special proliferation ability and division ability, and to provide a new way of thinking for the targeted therapy of hepatocellular carcinoma.
Objectives:
The aim of this study is to explore the isolation of side population cells in Huh7 cell line and their special proliferation ability and division ability compared to non-side population counterparts, to provide a new way of thinking for the targeted therapy of hepatocellular carcinoma.
Methods:
1. Cell Culture: Huh7 cells were cultured in 10% FBS DMEM medium regularly.
2. Preparation of single cell suspension solution: cells growing in the logarithmic phase are used for the study, and the cell concentration adjusts to 2 ~ 3×10~6 / mL. Add 150μmol/L Verapamil to part of the cell suspension solution for the control group, then the cell suspension solution are cultured at 37℃for 20min. 10μg/ml Hoechst33342are added both experimental group and control group, and are incubated in the 37℃water pot and kept in dark for 90min, vibrating them at low speed every 15 minutes. At last, they are washed three times ( 800 round / min, 5min / time) with 10 mmol/L HEPES, 10% FBS Hank's solution, and put on ice . Both experimental group and control group are filtered through 40μm sieve to get single cell suspension solution before sorting, adding PI to a final concentration of 2μg/ml before FACS.
3. Isolation of side population cells in Huh7 cell line through FACS.
4. Hoechst33342 fluorescence intensity in side population and non-side population cells are detected by fluorescence microscope.
5. ABCG2 mRNA expression levels between side population and non-side population cells are detected by semi-quantitative RT-PCR, and the product of RT-PCR is sequenced by sequencing company. GAPDH is an internal control.
6. Expression of ABCG2 protein in both side population and non-side population cells are detected by Fluorescent Immunocytochemistry assay, and their different ability of proliferation and asymmetrical division are observed.
Results:
1. The ratio of side population cells in Huh7 cell lines is about 2.22% through FACS, while in verapamil group, which decreases to 0.74%.
2. Hoechst33342 fluorescence intensity in side population cells is significantly weaker than that of non-side population cells under fluorescence microscope.
3. ABCG2 mRNA expression level in the side population cells is 4.32-fold (P <0.01) to that of non-side population cells, and the product of RT-PCR is confirmed by sequencing.
4. The proliferation ability of side population cells is 2.7-fold (P <0.01) to that of non-side population cells.
5. Side population cells can generate both ABCG2 (+) and ABCG2 (-) cells, while non-side population cells can only generate ABCG2 (-) cells.
Conclusion:
There is a small fraction of side population cells (2.22%) in Huh7 cell line, whose proliferation ability is stronger than that of non-side population cells. Side population cells have asymmetric division ability (self-renewal and directed differentiation), which is the trait of cancer stem cells. So the isolation of side population cells can be used as a method to isolate cancer stem cells, and side population cells may play an important role in tumor formation, drug resistance, even recurrence and metastasis.
恶性肿瘤给人类的生存构成严重的威胁,现有治疗措施几乎都不能改变晚期恶性肿瘤复发并最终导致患者死亡的现状。由于肿瘤的侵袭以及长期治疗导致耐药仍然是当前攻克恶性肿瘤的主要障碍,因此,阐明肿瘤耐药的分子机制具有重大的理论意义及临床应用价值。肿瘤干细胞学说认为肿瘤组织中存在极少量干细胞样肿瘤细胞亚群,它们具有无限增殖的潜能,在启动肿瘤形成和生长中起着决定性的作用,而其余的大多数细胞,经过短暂的分化,最终死亡。肿瘤干细胞是造成肿瘤耐药的最根本原因,其对化疗药物的耐受性是肿瘤治疗的主要障碍。目前鉴定肿瘤干细胞主要有检测其特异性表面抗原标志物及检测边缘群细胞( side population, SP)两种方法。本研究试图探讨肝癌细胞株Huh7细胞中边缘群细胞的分选及其增值分裂能力,为肝癌的靶向治疗提供新的思路。
研究目的:
探讨Huh7细胞中边缘群细胞的分选及边缘群细胞在增值能力及分裂能力方面与非边缘群细胞的差异,为肝癌的治疗探索新的思路。
研究方法:
1.细胞培养: Huh7细胞常规培养于10%FBS DMEM培养基中。
2.单细胞悬液的制备:对数生长期细胞常规胰酶消化后吹打均匀,调整细胞密度为2～3×10~6/L(细胞计数板计数),部分细胞悬液先加150μmol/L维拉帕米37℃孵育20min作对照组,实验组和对照组均加入10μg/ml Hoechst33342在水浴锅中37℃避光孵育90min,每15min低速震荡摇匀一次,用10 mmol/L HEPES、10%FBS Hank’s液洗涤3次(离心力800转/min,5min/次),后置冰上,分选前经40μm孔径的滤网过滤制备成单细胞悬液,分选前5min加入P I至终浓度为2μg/ml。
3.采用流式细胞仪分选Huh7细胞中的边缘群细胞和非边缘群细胞。
4.利用荧光显微镜观察边缘群细胞和非边缘群细胞内Hoechst33342荧光染色强度差异。
5.以半定量RT-PCR测定边缘群细胞和非边缘群细胞ABCG2 mRNA表达水平,ABCG2 PCR产物送公司测序,GAPDH为内参。
6.免疫荧光染色检测边缘群细胞和非边缘群细胞ABCG2蛋白表达的差异并观察其增值能力和不对称分裂能力的差异。
研究结果:
1.Huh7细胞经流式细胞仪分选发现非维拉帕米处理组边缘群细胞比例约为2.22% ,经维拉帕米阻断后边缘群细胞比例减少至0.74%。
2.荧光显微镜下观察发现边缘群细胞中Hoechst33342的荧光强度较非边缘群细胞明显弱。
3.ABCG2 mRNA表达水平在边缘群细胞中是非边缘群细胞的4.32倍(P<0.01)。
4.边缘群细胞增值能力是非边缘群细胞的2.7倍(P<0.01)。
5.边缘群细胞能产生ABCG2(+)和ABCG2(-)子代细胞,而非边缘群细胞只能产生ABCG2(-)子代细胞。
研究结论:
Huh7细胞中存在少量边缘群细胞(2.22%),边缘群细胞增值能力较非边缘群细胞强且具有不对称分裂(自我更新和定向分化)能力,具有肿瘤干细胞的特性;因此边缘群细胞分选可以作为肿瘤干细胞分离的一种方法,边缘群细胞可能在肿瘤的形成、进展、耐药及复发转移中具有更重要作用。
Background:
The malignant tumors pose a serious threat to the survival of human beings. The existing treatment measures hardly change the status that patients with advanced cancers eventually lead to recurrence and even death. Presently, tumor invasion and drug resistance owing to long-term treatment are still major obstacles to overcome cancer. Therefore, to clarify the molecular mechanism of tumor drug resistance has both important value of basic theory and clinical applications. There are a very small number of stem cell-like tumor cells in tumor tissues based on the theory of cancer stem cells, which have unlimited potential for proliferation, and play a decisive role in tumor formation, drug resistance, and recurrence and metastasis, while the majority of the remaining cells will go to die after a short spell of division. Cancer stem cells are the most fundamental cause of drug resistance, whose tolerance to chemotherapeutic drugs is a main obstacle to cancer treatment.
Presently, the isolation of cancer stem cells is mainly based on the markers of surface antigen-specific antibody or detection of the side population cells through FACS. This study aims to explore the the side population cells in hepatocellular carcinoma cell line Huh7 and their special proliferation ability and division ability, and to provide a new way of thinking for the targeted therapy of hepatocellular carcinoma.
Objectives:
The aim of this study is to explore the isolation of side population cells in Huh7 cell line and their special proliferation ability and division ability compared to non-side population counterparts, to provide a new way of thinking for the targeted therapy of hepatocellular carcinoma.
Methods:
1. Cell Culture: Huh7 cells were cultured in 10% FBS DMEM medium regularly.
2. Preparation of single cell suspension solution: cells growing in the logarithmic phase are used for the study, and the cell concentration adjusts to 2 ~ 3×10~6 / mL. Add 150μmol/L Verapamil to part of the cell suspension solution for the control group, then the cell suspension solution are cultured at 37℃for 20min. 10μg/ml Hoechst33342are added both experimental group and control group, and are incubated in the 37℃water pot and kept in dark for 90min, vibrating them at low speed every 15 minutes. At last, they are washed three times ( 800 round / min, 5min / time) with 10 mmol/L HEPES, 10% FBS Hank's solution, and put on ice . Both experimental group and control group are filtered through 40μm sieve to get single cell suspension solution before sorting, adding PI to a final concentration of 2μg/ml before FACS.
3. Isolation of side population cells in Huh7 cell line through FACS.
4. Hoechst33342 fluorescence intensity in side population and non-side population cells are detected by fluorescence microscope.
5. ABCG2 mRNA expression levels between side population and non-side population cells are detected by semi-quantitative RT-PCR, and the product of RT-PCR is sequenced by sequencing company. GAPDH is an internal control.
6. Expression of ABCG2 protein in both side population and non-side population cells are detected by Fluorescent Immunocytochemistry assay, and their different ability of proliferation and asymmetrical division are observed.
Results:
1. The ratio of side population cells in Huh7 cell lines is about 2.22% through FACS, while in verapamil group, which decreases to 0.74%.
2. Hoechst33342 fluorescence intensity in side population cells is significantly weaker than that of non-side population cells under fluorescence microscope.
3. ABCG2 mRNA expression level in the side population cells is 4.32-fold (P <0.01) to that of non-side population cells, and the product of RT-PCR is confirmed by sequencing.
4. The proliferation ability of side population cells is 2.7-fold (P <0.01) to that of non-side population cells.
5. Side population cells can generate both ABCG2 (+) and ABCG2 (-) cells, while non-side population cells can only generate ABCG2 (-) cells.
Conclusion:
There is a small fraction of side population cells (2.22%) in Huh7 cell line, whose proliferation ability is stronger than that of non-side population cells. Side population cells have asymmetric division ability (self-renewal and directed differentiation), which is the trait of cancer stem cells. So the isolation of side population cells can be used as a method to isolate cancer stem cells, and side population cells may play an important role in tumor formation, drug resistance, even recurrence and metastasis.
引文
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22. Chiba T, Zheng YW, Kita K, Yokosuka O, Saisho H, Onodera M, Miyoshi H, Nakano M, Zen Y, Nakanuma Y, Nakauchi H, Iwama A, Taniguchi H. Enhanced self-renewal capability in hepatic stem/progenitor cells drives cancer initiation. Gastroenterology. 2007;133:937-50
23. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med. 1996; 183: 1797 - 1806
24. Chiba T, Kita K, Zheng YW, et al. Side population purified from hepatocellular carcinoma cells harbors cancer stem cell -like properties. Hepatology. 2006; 44: 240 - 251
25. Hirschmann-Jax C, Foster AE, Wulf GG, Nuchtern JG, Jax TW, Gobel U, Goodell MA, Brenner MK. A distinct " side population" of cells with high drug efflux capacity in human tumor cells. Proc Natl Acad Sci USA. 2004; 101: 14228 -14233
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20. Yang ZF, Ho DW, Ng MN, Lau CK, Yu WC, Ngai P, Chu PW, Lam CT, Poon RT, Fan ST. Significance of CD90+ cancer stem cells in human liver cancer. Cancer Cell. 2008; 13: 153-166
21. Ma S, Chan KW, Hu L, Lee TK, Wo JY, Ng IO, Zheng BJ, Guan XY. Identification and characterization of tumorigenic liver cancer stem/progenitor cells. Gastroenterology. 2007; 132: 2542-2556
22. Chiba T, Zheng YW, Kita K, Yokosuka O, Saisho H, Onodera M, Miyoshi H, Nakano M, Zen Y, Nakanuma Y, Nakauchi H, Iwama A, Taniguchi H. Enhanced self-renewal capability in hepatic stem/progenitor cells drives cancer initiation. Gastroenterology. 2007;133:937-50
23. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med. 1996; 183: 1797 - 1806
24. Chiba T, Kita K, Zheng YW, et al. Side population purified from hepatocellular carcinoma cells harbors cancer stem cell -like properties. Hepatology. 2006; 44: 240 - 251
25. Hirschmann-Jax C, Foster AE, Wulf GG, Nuchtern JG, Jax TW, Gobel U, Goodell MA, Brenner MK. A distinct " side population" of cells with high drug efflux capacity in human tumor cells. Proc Natl Acad Sci USA. 2004; 101: 14228 -14233
26. Haraguchi N, Utsunomiya T, Inoue H, Tanaka F, Mimori K, Barnard GF, Mori M. Characterization of a side population of cancer cells from human gastrointestinal system. Stem Cells. 2006; 24: 506 - 513
27. Hadnagy A, Gaboury L, Beaulieu R, Balicki D. SP analysis may be used to identify cancer stem cell populations. Exp Cell Res. 2006; 312, 3701–3710
28. Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, Morris JJ, Lagutina I, Grosveld GC, Osawa M, Nakauchi H, Sorrentino BP. The ABC transporter Bcrp1 /ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side - population phenotype. Nat Med. 2001; 7: 1028 - 1034
29. Frances J Sharom . ABC multidrug transporters: structure, function and role in chemoresistance. Pharmacogenomics. 2008; 9: 105–127
30. Jordan CT, Guzman ML, Noble M. Cancer stem cells. N Engl J Med. 2006; 355: 1253-1261
31. Schofield R. The relationship between the spleen colony-forming cell and the haemopoietic cell. Blood Cell. 1978; 4: 7-25
32. Barh D, Malhotra R, Ravi B, Sindhurani P. microRNA let-7: an emerging next-generation cancer therapeutic. Curr Oncol. 2010; 17: 70-80
33. Peter ME.Regulating cancer stem cells the miR way. Cell Stem Cell. 2010; 6: 4-6
34. Liang Y, Zhong Z, Huang Y, Deng W, Cao J, Tsao G, Liu Q, Pei D, Kang T, Zeng YX. Stem-like cancer cells are inducible by increasing genomic instability in cancer cells. J Biol Chem. 2010; 285: 4931-40
[1]丁景弦,白小明,张海,汪亦品,彭韬,冷静. Huh7细胞中边缘群细胞分选及其增殖分裂能力的研究。南京医科大学学报,2010, 30: 139- 143
[2] Bai XM, Jiang H, Ding JX, Peng T, Ma J, Wang YH, Zhang L, Zhang H, Leng J . Prostaglandin E2 upregulates survivin expression via the EP1 receptor in hepatocellular carcinoma cells. Life sci. 2010, 86: 214-223