体外侵袭实验分离高侵袭性和低侵袭性肾癌细胞
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摘要
肾细胞癌,是泌尿系统常见肿瘤,浸润生长和远处转移是晚期肾癌的重要特征,近1/3的肾癌患者首诊时已属晚期,预后极差。肾透明细胞癌是肾细胞癌中最常见的病理类型,约占70%～80%,因此筛选和鉴定肾透明细胞癌转移相关基因,寻找一与转移相关的生物学标志物,用于早期发现肾癌转移,对降低肾癌死亡率具有重要意义。而建立具有相同遗传背景的高、低转移性细胞株是寻找和筛选转移相关基因的基础和关键。肿瘤转移异质性理论认为,在肿瘤组织中存在着具有不同侵袭、转移能力的细胞亚群,而只有那些具有完成侵袭转移全过程能力如粘附能力、蛋白水解能力、细胞游走能力的瘤细胞才能最终形成转移灶。因此可依据某些侵袭、转移有关因素的表达与否筛选出具有不同侵袭转移能力的瘤细胞。
Matrigel胶是一种从EHS(Engelbreth-Holm-Swarm)小鼠肉瘤中提取的可溶性基底膜成分。主要由层粘连蛋白、Ⅳ型胶原蛋白、硫酸乙酞肝素糖蛋白(类肝素蛋白多糖)、纤维连接蛋白等组成。室温下Matrigel胶聚合成一种具有生物学活性的基质材料,在聚碳酸滤膜上形成一胶冻状物,封闭聚碳酸滤膜上的小孔,作用与哺乳动物细胞基底膜类似,只有那些具有蛋白水解能力的肿瘤细胞才能穿过聚碳酸滤膜上的小孔,无蛋白水解能力的非侵袭性细胞不能穿过。因此Matrigel胶可用于体外分离不同侵袭性肿瘤细胞。本研究拟构建侵袭小室,用于体外分离高侵袭性和低侵袭性肾癌细胞。分别从高侵袭性肾癌细胞和低侵袭性肾癌细胞中提取总RNA,并利用SMART技术将RNA转化成cDNA,为进一步抑制性消减杂交打下基础。
目的:筛选相同背景来源的高侵袭性和低侵袭性肾细胞癌细胞,对比二者差异。提取高、低侵袭性肾癌细胞的总RNA,并转化为cDNA,为研究肾细胞癌转移相关基因打下基础。
方法:对肾癌细胞株ACHN进行传代培养。在预实验确定铺胶浓度、消化回收时间的基础上,采用涂有Matrigel胶的Transwell对传代培养的肾癌细胞进行体外侵袭实验,分离回收高侵袭性和低侵袭性肾癌细胞。分别从高侵袭性肾癌细胞与低侵袭性肾癌细胞中提取总KNA,并利用SMART技术将RNA转化成cDNA。
结果:以浓度为1.17mg/ml、20μl/孔铺胶在Transwell,固定细胞悬液密度(5×10~6/ml),第48小时消化回收细胞较为理想。非侵袭性细胞呈散在生长,侵袭性细胞多呈聚集样生长,成功分离高侵袭性和低侵袭性肾癌细胞。从高侵袭性肾癌细胞与低侵袭性肾癌细胞提取的RNA经变性甲醛/琼脂糖凝胶电泳分析表明完整性和纯度良好,SMART技术可以利用微量的RNA合成cDNA。
结论:Transwell可体外快速分离及回收、培养不同侵袭性的肾癌细胞。用肾癌细胞株ACHN进行体外侵袭实验代表性好,且传代稳定性好。成功提取了高、低侵袭性肾癌细胞的总RNA,并转化为cDNA,为研究肾细胞癌转移相关基因奠定了坚实的基础。
Renal Cell Carcinoma occurs more commonly in urinary system, which is associated with more aggressive and extensive metastatic characteristics. One third patients have been in advanced stages when being detected for the first time,and forty percent patients reoccur or metastasize after operatiaons with worse prognosis. Renal Clear Cell Carcinoma is first most common subtype of Renal Cell Carcinoma. Therefore,developing a series of metastatic associated biological marks will be of great significance in detecting RCC effectively with the potential to reduce the death rate. It is critical step for screening and identifying metastatic associated biological mark that Renal Cell Carcinoma cell lines with different metastatic potential is established in the same genetic background.According to tumor heterogeneity theory:neoplasms contain subpoppulatiopn of cells having different metastatic potential,only those tumor cells can establish a metastasis which are capable of completing the entire process:dissociating from the primary mass,migrating through the interstitial extracellular matrix,invading from the basement membrance underlying the vascular endothelial cell layer and basement membrance to enter the interstitial matrix and establish a metastasis.Therefor,invasive and metastatic associated factors can be used as basis for isolating tumor cells with different invasive, metastatic potentials.
Malignant cells must traverse basement membrances during their migration to sites distant from the primary tumor.Matrigel is a solubulized basement membrance preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma,a tumor rish in ECM proteins.Its major component is laminin,followed by IV collagen,heparin sulfate proteoglycans,and entactin.At room temperature, Matrigel polymerizes to produce biologically active matrix material resembling the mammalian cellular basement membrane ,which are thin continuous sheets applied over filters in transwell insert,blocking non-invasive cells from migrating through the membrane.In contrast, invasive tumor cells are able to detach themselves from and invade through the Matrigel and the 8 micron membrane pores.Therefor,it is well suited for selecting differently invasive cell phenotypes in response to a chemoattractant. Then we extract total RNA from high invasive and low invasive renal cell carcinoma cells respectively and analyze them.At last we use SMART to synthesis cDNA.
Objective : To isolate the high invasive and low invasive renal cell carcinoma cells from human renal cell carcinoma (RCC) cell line ACHN in vitro, extract total RNA from high invasive and low invasive renal cell carcinoma cells respectively and synthesis cDNA.
Methods: The renal cell carcinoma cell line ACHN was serial subcultivation in vitro. Then in vitro invasion assay using the Transwells coating Matrigel were performed for separation and recovery of high invasive and low invasive cells from the serial subculture of cells. The concentration of Matrigel, trypsinization and recovery time were subsequently optimized.Then we extract total RNA from high invasive and low invasive renal cell carcinoma cells respectively and analyze them, then we use SMART to synthesis cDNA.
Results: Matrigel (diluted into 1.17mg/ml) was coated onto the filter of the Transwell; cell suspension was at a concentration of 5×10~5/ml and invasive cells were recovered on the 48h of culture. When all above were prepared well, the recovery of invasive cells was performed ideally. The growth of non-invasive cells was sporadic, while that of the invasive cells was accumulative. Total RNA extracted from high invasive and low invasive renal cell carcinoma cells are good. SMART can be used to synthesis cDNA with a small quantity of RNA.
Conclusions: In vitro the Transwell is able to quickly separate, recover and culture the highly invasive RCC cells. At the same time, the cell line ACHN had a nice representation,and it can easily go down to the future generation. we successfully extract total RNA from high invasive and low invasive renal cell carcinoma cells respectively and synthesis cDNA, which lay a substantial fundament for researching metastatic associated genes.
Matrigel胶是一种从EHS(Engelbreth-Holm-Swarm)小鼠肉瘤中提取的可溶性基底膜成分。主要由层粘连蛋白、Ⅳ型胶原蛋白、硫酸乙酞肝素糖蛋白(类肝素蛋白多糖)、纤维连接蛋白等组成。室温下Matrigel胶聚合成一种具有生物学活性的基质材料,在聚碳酸滤膜上形成一胶冻状物,封闭聚碳酸滤膜上的小孔,作用与哺乳动物细胞基底膜类似,只有那些具有蛋白水解能力的肿瘤细胞才能穿过聚碳酸滤膜上的小孔,无蛋白水解能力的非侵袭性细胞不能穿过。因此Matrigel胶可用于体外分离不同侵袭性肿瘤细胞。本研究拟构建侵袭小室,用于体外分离高侵袭性和低侵袭性肾癌细胞。分别从高侵袭性肾癌细胞和低侵袭性肾癌细胞中提取总RNA,并利用SMART技术将RNA转化成cDNA,为进一步抑制性消减杂交打下基础。
目的:筛选相同背景来源的高侵袭性和低侵袭性肾细胞癌细胞,对比二者差异。提取高、低侵袭性肾癌细胞的总RNA,并转化为cDNA,为研究肾细胞癌转移相关基因打下基础。
方法:对肾癌细胞株ACHN进行传代培养。在预实验确定铺胶浓度、消化回收时间的基础上,采用涂有Matrigel胶的Transwell对传代培养的肾癌细胞进行体外侵袭实验,分离回收高侵袭性和低侵袭性肾癌细胞。分别从高侵袭性肾癌细胞与低侵袭性肾癌细胞中提取总KNA,并利用SMART技术将RNA转化成cDNA。
结果:以浓度为1.17mg/ml、20μl/孔铺胶在Transwell,固定细胞悬液密度(5×10~6/ml),第48小时消化回收细胞较为理想。非侵袭性细胞呈散在生长,侵袭性细胞多呈聚集样生长,成功分离高侵袭性和低侵袭性肾癌细胞。从高侵袭性肾癌细胞与低侵袭性肾癌细胞提取的RNA经变性甲醛/琼脂糖凝胶电泳分析表明完整性和纯度良好,SMART技术可以利用微量的RNA合成cDNA。
结论:Transwell可体外快速分离及回收、培养不同侵袭性的肾癌细胞。用肾癌细胞株ACHN进行体外侵袭实验代表性好,且传代稳定性好。成功提取了高、低侵袭性肾癌细胞的总RNA,并转化为cDNA,为研究肾细胞癌转移相关基因奠定了坚实的基础。
Renal Cell Carcinoma occurs more commonly in urinary system, which is associated with more aggressive and extensive metastatic characteristics. One third patients have been in advanced stages when being detected for the first time,and forty percent patients reoccur or metastasize after operatiaons with worse prognosis. Renal Clear Cell Carcinoma is first most common subtype of Renal Cell Carcinoma. Therefore,developing a series of metastatic associated biological marks will be of great significance in detecting RCC effectively with the potential to reduce the death rate. It is critical step for screening and identifying metastatic associated biological mark that Renal Cell Carcinoma cell lines with different metastatic potential is established in the same genetic background.According to tumor heterogeneity theory:neoplasms contain subpoppulatiopn of cells having different metastatic potential,only those tumor cells can establish a metastasis which are capable of completing the entire process:dissociating from the primary mass,migrating through the interstitial extracellular matrix,invading from the basement membrance underlying the vascular endothelial cell layer and basement membrance to enter the interstitial matrix and establish a metastasis.Therefor,invasive and metastatic associated factors can be used as basis for isolating tumor cells with different invasive, metastatic potentials.
Malignant cells must traverse basement membrances during their migration to sites distant from the primary tumor.Matrigel is a solubulized basement membrance preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma,a tumor rish in ECM proteins.Its major component is laminin,followed by IV collagen,heparin sulfate proteoglycans,and entactin.At room temperature, Matrigel polymerizes to produce biologically active matrix material resembling the mammalian cellular basement membrane ,which are thin continuous sheets applied over filters in transwell insert,blocking non-invasive cells from migrating through the membrane.In contrast, invasive tumor cells are able to detach themselves from and invade through the Matrigel and the 8 micron membrane pores.Therefor,it is well suited for selecting differently invasive cell phenotypes in response to a chemoattractant. Then we extract total RNA from high invasive and low invasive renal cell carcinoma cells respectively and analyze them.At last we use SMART to synthesis cDNA.
Objective : To isolate the high invasive and low invasive renal cell carcinoma cells from human renal cell carcinoma (RCC) cell line ACHN in vitro, extract total RNA from high invasive and low invasive renal cell carcinoma cells respectively and synthesis cDNA.
Methods: The renal cell carcinoma cell line ACHN was serial subcultivation in vitro. Then in vitro invasion assay using the Transwells coating Matrigel were performed for separation and recovery of high invasive and low invasive cells from the serial subculture of cells. The concentration of Matrigel, trypsinization and recovery time were subsequently optimized.Then we extract total RNA from high invasive and low invasive renal cell carcinoma cells respectively and analyze them, then we use SMART to synthesis cDNA.
Results: Matrigel (diluted into 1.17mg/ml) was coated onto the filter of the Transwell; cell suspension was at a concentration of 5×10~5/ml and invasive cells were recovered on the 48h of culture. When all above were prepared well, the recovery of invasive cells was performed ideally. The growth of non-invasive cells was sporadic, while that of the invasive cells was accumulative. Total RNA extracted from high invasive and low invasive renal cell carcinoma cells are good. SMART can be used to synthesis cDNA with a small quantity of RNA.
Conclusions: In vitro the Transwell is able to quickly separate, recover and culture the highly invasive RCC cells. At the same time, the cell line ACHN had a nice representation,and it can easily go down to the future generation. we successfully extract total RNA from high invasive and low invasive renal cell carcinoma cells respectively and synthesis cDNA, which lay a substantial fundament for researching metastatic associated genes.
引文
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1.Pasco S,Ramont L,Maquart FX,et al.Control of melanoma progression by various matrikines from basement membrane macromolecules.Crit Rev Oncol Hematol.2004,49(3):221-33.Review.,
2.Carmeliet P,Jain RK.Angiogenesis in cancer and other diseases.Nature.2000;407(6801):249-57.Review.
3.Jho D,Mehta D,Ahmmed G,et al.Angiopoietin-1 opposes VEGF-induced increase in endothelial permeability by inhibiting TRPCI-dependent Ca2 influx.Cire Res,2005,96(12):1282-1290.
4.Lyden D,Hattori K,Dias S,et al.Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth.Nat Med.2001;7(11):1194-201.
5.Tian Y,Ding RY,Zhi YH,et al.Analysis of p53 and vascular endothelial growth factor expression in human gallbladder carcinoma for the determination of tumor vascularity.World J Gastroenterol.2006 21;12(3):415-9
6.Hemmerlein B,Johanns U,Halbfass J,et al.The balance between MMP-2/-9 and TIMP-1/-2 is shifted towards MMP in renal cell carcinomas and can be further disturbed by hydrogen peroxide.Int J Oncol,2004;24(5):1069-1076
7.Nelson AR,Fingleton B,Rothenberg ML,et al.Matrix metalloproteinases:biologic activity and clinical implications.J Clin Oncol.2000;18(5):1135-49..
8.朱峰-基质金属蛋白酶及其组织抑制物与肿瘤侵袭转移。国外医学:临床生物化学与检验医学分册,2001,22(5):229-231
9.Libby P,Aikawa M.Stabilization of atheroselerotic plaques;new mechanisms and clinical targets.Nat Med,2002,8:1257,
10.BariteA,CovicL.AgarwalA,et al.PART is a matrix metalloprotease-1 receptor that promotes invasion and tomorigenesis of breast cancer c ells.Cell,2005,120(3):303.
11. Evans TR.Pentheroudakis G .Paul J ,et al.Aphase I and pharmacokinetic study of capecitabine in combination, with epirubicin and cisplatinin patients with inoperable oesophag -ogastric adenocarcinoma.Ann Oncol.2002,13 (9); 14 69 .
12. Masai D .Naldini AC.Carraro F ,et al.Experssion of portease- activated receptors land 2 in melanocytic nevi and malignant melanoma.Hum Pathol,2005,36(6)s676
13. ouatasT , Salerno M , PalmieriD , etal.Basic and Translational advances In Cancer metastasis:Nm23.J Bioenerg Biomembr, 2003.35:73-9
14. Dong JT, Suzuki H, Pins, etal.Down-reguation Of the KAll Metastasis Suppressor Gene During The Progression Of Human Prostatic Cancer Infrequenily Involves Gene Mutation orallelic loss .CancerRes, 1996, 56:4387-90.
15. Shevde LA , Welch DR, Metastasis suppressor Pathways-an Evolving paradigm. Cancer Lett 2003, 198:1-20.
16. Noordzij MA, van stenbruge CJ, Schreder FH. et al.Decreased expression of cd44 in metastatic prostate cancer.Int J Cancer, 1999, 84:478-83.
17. Li H, Liu J , Hofmann M, et al.Diferential CD44 expression patterns in primary brain tumour and brain metastases.Br J Cancer, 1995, 72:160-3.
18. TengDH, Perry WL3rd, HoganJK, et al.Human mitogen-activated protein kinase kinase4 as a candidate tumor suppressor.Cancer Res, 197, 57:4177-82.
19. Kim HL, vander Griend DJ, Yang X, etal. Mitogen-activated Protein kinase kinase4 metastasis suppressor gene expression is inversely related to histological patternin advancing human prostatic cancers.Cancer Res, 2001, 61:2833-7.
20. Hori A, Honda S, Asada M, etal. Metastin suppresses the motility and growth of CHo cells transfected with its receptor .Biochem Biophys Res Commun, 2001, 256:958-63.
21. Shirasaki F, Takata M, Hatta N, et al.Loss of expression Of the metastasis suppressor gene KSSI during melanoma progression and its association with L0H of chromosome 6ql6.3-q23 .Cancer Res, 2001, 61:7422-5.
22. Rotblat B, Yizhar O, Haklai R, et al. Ras and its signals diffuse through the cell on randomly moving nanoparticles. Cancer Res. 2006;66(4):1974-81.。
23. Roger L, Gadea G, Roux P. Control of cell migration: a tumour suppressor function for p53.Biol Cell. 2006 ;98(3):141-52. Review.
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1.Pasco S,Ramont L,Maquart FX,et al.Control of melanoma progression by various matrikines from basement membrane macromolecules.Crit Rev Oncol Hematol.2004,49(3):221-33.Review.,
2.Carmeliet P,Jain RK.Angiogenesis in cancer and other diseases.Nature.2000;407(6801):249-57.Review.
3.Jho D,Mehta D,Ahmmed G,et al.Angiopoietin-1 opposes VEGF-induced increase in endothelial permeability by inhibiting TRPCI-dependent Ca2 influx.Cire Res,2005,96(12):1282-1290.
4.Lyden D,Hattori K,Dias S,et al.Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth.Nat Med.2001;7(11):1194-201.
5.Tian Y,Ding RY,Zhi YH,et al.Analysis of p53 and vascular endothelial growth factor expression in human gallbladder carcinoma for the determination of tumor vascularity.World J Gastroenterol.2006 21;12(3):415-9
6.Hemmerlein B,Johanns U,Halbfass J,et al.The balance between MMP-2/-9 and TIMP-1/-2 is shifted towards MMP in renal cell carcinomas and can be further disturbed by hydrogen peroxide.Int J Oncol,2004;24(5):1069-1076
7.Nelson AR,Fingleton B,Rothenberg ML,et al.Matrix metalloproteinases:biologic activity and clinical implications.J Clin Oncol.2000;18(5):1135-49..
8.朱峰-基质金属蛋白酶及其组织抑制物与肿瘤侵袭转移。国外医学:临床生物化学与检验医学分册,2001,22(5):229-231
9.Libby P,Aikawa M.Stabilization of atheroselerotic plaques;new mechanisms and clinical targets.Nat Med,2002,8:1257,
10.BariteA,CovicL.AgarwalA,et al.PART is a matrix metalloprotease-1 receptor that promotes invasion and tomorigenesis of breast cancer c ells.Cell,2005,120(3):303.
11. Evans TR.Pentheroudakis G .Paul J ,et al.Aphase I and pharmacokinetic study of capecitabine in combination, with epirubicin and cisplatinin patients with inoperable oesophag -ogastric adenocarcinoma.Ann Oncol.2002,13 (9); 14 69 .
12. Masai D .Naldini AC.Carraro F ,et al.Experssion of portease- activated receptors land 2 in melanocytic nevi and malignant melanoma.Hum Pathol,2005,36(6)s676
13. ouatasT , Salerno M , PalmieriD , etal.Basic and Translational advances In Cancer metastasis:Nm23.J Bioenerg Biomembr, 2003.35:73-9
14. Dong JT, Suzuki H, Pins, etal.Down-reguation Of the KAll Metastasis Suppressor Gene During The Progression Of Human Prostatic Cancer Infrequenily Involves Gene Mutation orallelic loss .CancerRes, 1996, 56:4387-90.
15. Shevde LA , Welch DR, Metastasis suppressor Pathways-an Evolving paradigm. Cancer Lett 2003, 198:1-20.
16. Noordzij MA, van stenbruge CJ, Schreder FH. et al.Decreased expression of cd44 in metastatic prostate cancer.Int J Cancer, 1999, 84:478-83.
17. Li H, Liu J , Hofmann M, et al.Diferential CD44 expression patterns in primary brain tumour and brain metastases.Br J Cancer, 1995, 72:160-3.
18. TengDH, Perry WL3rd, HoganJK, et al.Human mitogen-activated protein kinase kinase4 as a candidate tumor suppressor.Cancer Res, 197, 57:4177-82.
19. Kim HL, vander Griend DJ, Yang X, etal. Mitogen-activated Protein kinase kinase4 metastasis suppressor gene expression is inversely related to histological patternin advancing human prostatic cancers.Cancer Res, 2001, 61:2833-7.
20. Hori A, Honda S, Asada M, etal. Metastin suppresses the motility and growth of CHo cells transfected with its receptor .Biochem Biophys Res Commun, 2001, 256:958-63.
21. Shirasaki F, Takata M, Hatta N, et al.Loss of expression Of the metastasis suppressor gene KSSI during melanoma progression and its association with L0H of chromosome 6ql6.3-q23 .Cancer Res, 2001, 61:7422-5.
22. Rotblat B, Yizhar O, Haklai R, et al. Ras and its signals diffuse through the cell on randomly moving nanoparticles. Cancer Res. 2006;66(4):1974-81.。
23. Roger L, Gadea G, Roux P. Control of cell migration: a tumour suppressor function for p53.Biol Cell. 2006 ;98(3):141-52. Review.
24. Sirotkovic-Skerlev M, Krizanac S, Kapitanovic S, et al .Expression of c-myc, erbB-2, p53 and nm23-H1 gene product in benign and malignant breast lesions: coexpression and correlation with clinicopathologic parameters. Exp Mol Pathol. 2005;79(1):42-50. Epub 2005 Apr 7.