摘要
目前,肝癌的发生机制与细胞起源仍然存在着较大的争议,肿瘤干细胞理论认为癌症是一种干细胞疾病,因此,深入研究肝癌干细胞不仅能够阐明肝癌发生的机制,而且对肝癌的临床诊断、治疗及预后具有重要的意义。目前,肝癌干细胞研究中的首要问题是肝癌干细胞的鉴定方法尚未成熟和标准化,缺乏足够特异、准确的分子标记。新近研究表明CD90在肝脏发生期间的肝干／祖细胞细胞中表达,而成熟肝细胞中不表达CD90,其功能复杂,在细胞的粘附、凋亡,肿瘤的生长,转移等多种细胞活动中均有其参与,也有文献报道利用CD90分离出具有干细胞性的肝癌细胞,本文利用免疫磁珠间接标记法对人肝癌细胞系SMMC-7721进行CD90阳性细胞的分选和初步鉴定研究。
目的：分选富集人肝癌细胞系SMMC-7721中的CD90阳性细胞及CD90阴性细胞,做单克隆化培养及传代培养,初步分析比较CD90阳性细胞与阴性细胞的干细胞标志物和相关基因的表达。
方法：利用免疫磁珠间接标记法富集SMMC-7721中的CD90阳性细胞,以未分选的7721细胞和CD90阴性细胞为对照,传代培养；同时对CD90阳性细胞及CD90阴性细胞利用细胞有限稀释法进行单克隆培养及扩大培养；流式细胞仪分析每组和每代细胞的CD90、CD133、CD34、CD45等干细胞标志物表达,实时荧光定量PCR检测AFP、CCNA2、CLDN10、PTEN、SPINT、TFDP1等17个肝癌相关基因的表达,免疫细胞化学染色分析AFP、CK18、CK19等蛋白表达。
结果：四次磁珠分选实验均较为成功地富集了7721中的CD90+阳性细胞,但所得目的细胞数及纯度较低,分选前CD90在该细胞系中的表达率为0.8%～1.2%,分选后其表达率依次为7.5%、9.0%、30.7%和27.2%,细胞数分别为1.37×104、1.23×104、1.42×104和2.85×104。经过改良PBS配方(PBS+2%FBS+0.5μM EDTA)所得目的细胞纯度及细胞活性有提高至30.7%,但细胞数没有明显增加。
镜下观察有限稀释法培养的细胞,大多数克隆培养的细胞均能持续分裂增殖,最初某些克隆间存在着形态差异,但随着其生长传代及扩大培养,传代至P2时细胞即呈现相互混杂的多种形态,克隆之间细胞形态差异趋于模糊。另外,少数克隆培养的细胞生长3-4d后观察到其细胞皱缩并最终死亡；同时,在扩大培养的克隆化细胞时,即使在同一培养孔中也会有部分细胞皱缩、死亡。
流式细胞仪检测CD34、CD45、CD90/Thy-1、CD133等干细胞表面标志物在三组细胞传代过程中表达率的变化情况。结果表明CD90的表达率在P0代细胞时,CD90+组为69.9%,CD90-组为0.4%,7721组为5.2%；其在P1、P2、P3、P4代细胞中分别为3.0%、2.3%、0.7%；1.1%、0.1%、1.0%；8.1%、4.4%、8.3%；6.7%、3.7%、7.4%。CD90+组中CD34表达率0.3%～7.8%,CD45：33.1%～96.0%,CD133：2.0%～59.1%；在7721组各标志物表达率分别为0.8%-11.2%、25.7%～53.2%和3.6%～13.6%；CD90-组为0.9%～12.3%、19.5%～70.0%和2.5%-11.1%。提示随着细胞传代,这些标志物的表达率存在着差异和波动,但各组表达率也逐渐趋于接近。
免疫细胞化学染色显示分选后P1代CD90+组的AFP、GPC3与另外两组均呈阴性表达,CK18、PCNA与其它两组均阳性表达,组间无差异；CK19、Vimentin在SMMC-7721组与CD90-组中为阳性染色,而CD90+组为阴性。
采用秩和检验分析CD90+组和CD90-组基因表达差异,表明单克隆培养所得细胞CD90+组与CD90-组间PTEN基因的表达差异有统计学意义(p=0.044),而AFP等7个基因以及传代培养所得两组细胞ANGPT等17个基因表达差异均无统计学意义(p>0.05)。
结论：本研究结果初步表明,(1)免疫磁珠间接标记分选能够成功分离目的细胞,但分选时对低温、避光等环境条件要求比较严格,并且需要操作者有较丰富的经验,任何细节上的疏漏均会导致细胞回收率及纯化程度的降低,进而影响实验的进行。(2)肝癌细胞系7721中并非只有CD90阳性细胞具有自我更新及持续增殖的能力,CD90-细胞同样都具有持续增殖与多向分化能力,推测肝癌干细胞除CD90外,可能还表达其它表面标志物；(3)流式细胞仪分析、免疫细胞化学和基因表达分析提示,不同组细胞在单克隆培养及传代扩大培养过程中,部分克隆存在一定的差异,这种差异是否会引起不同克隆细胞侵袭性、致瘤性等生物学行为的改变,需要裸鼠体内移植实验的验证等更深入的研究工作。
Objective:To isolate CD90 positive cells and CD90 negative cells from human hepatocellular carcinoma cell line SMMC-7721, for it stem cells characteristic research.
Methods:Isolate CD90 positive cells by magnetic microbeads cell sorting. Dilute it to 10～15 cells per milliliter Dulbecco's Modified Eagle Media:Nutrien t Mixture F-12 for monoclonal cultivation. Other goal cells make each group t o the same density, place it to culture bottle. Detect the expression rate of ste m cell markers CD34, CD45, CD90 and CD133 of each group, Real-time fluo rescence quantitative PCR to detect the disparity of gene expression. The candi date gene include AFP、CCNA2、CLDN10、PTEN、SPINT、TFDP1 et al. Det ect the disparity of protein(AFP、CK18、CK19) expression between each group cells by immunocytochemistry. SMMC-7721 did not undergo MACs and CD9 0 negative cells which collected from cell sorting for control.
Result:Four times cell sorting we were successful in isolating the purpose cell, But the resulting purpose cell number and purity is low, CD90 expression for 0.8-1.2% before the four sorting. The number and purity of purpose cell were 1.37X 104; 7.5%、1.23×104; 9.0%、1.42×104; 30.7%、2.85×104; 27.2% respectively. Later, we added low concentration(0.5μM) of ethylene diamine tetraacetic acid and 2% fetal bovin serum in phosphate buffer solution to prevent cells agglomerate, and give a sufficient combination of antibodies and purpose cells. So the sorting purity raised to 30.7%, but, there's no apparent elevation of the purpose cells number.
There have difference of morphology among some monoclonal cultivated cells, but whith the generation cultivation, these difference get ambiguouse gradually. Majority cells have the capacity to proliferation and differentiation into cells which express other stem cell markers, only minority cells of three groups necrosis after transient proliferation. Even in the same cultivate cell, there have cells necrosis and well growth.
The result of fluorescence activated cell sorting shows that P0 generation cells expression rate of CD90 in CD90 positive Group:69.9%; CD90 negative group: 0.4%;7721 group:5.2%, and the expression rate of CD90 in three groups from P1 to P4 were 3.0%、2.3%、0.7%; 1.1%、0.1%、1.0%; 8.1%、4.4%、8.3%; 6.7%、3.7%、7.4%。. In CD90 positive group expression rate of CD34 were 0.3-7.8%; CD45: 33.1%-96.0%; CD133:2.0%～59.1%; expression rate of each marker in 7721 group were0.8%～11.2%;25.7%～53.2%; 3.6%～13.6%; and an appropriate rate of 0.9%～12.3%; 19.5%～70.0%;2.5%～11.1% in CD90 negative group. This result indicated that expression rate have variance among each group, but with cells differentiation, the expression rate of various stem cell markers returned to the level of SMMC-7721 gradually.
And the outcome of immunocytochemistry stain indicated that expression of AFP, Gpc3, CK18 and PCNA there's no difference between CD90 positive and negative group cells. But CK19 and Vimentin negative stain in CD90 positive group while positive stain in SMMC-7721 and CD90 positive group.CD90 positive cells group and CD90 negative cells group.
The gene expression testing results take rank sum test for analysis, results of analysis indicated that expression variance of gene PTEN between CD90 positive group and negative group from monoclonal cell culture are significantly. (p=0.044), CD90 negative group have a high expression of this cancer inhibit gene than CD90 positive group cells. While, seven other gene ANGPT, AFP, APC of monoclonal cultivation and E2F1, HGF, P16 et al seventeen gene of two groups from serial subcultivation expression variance have no statistical significance (p> 0.05)
Conclusion:This study indicated that, (1) indirect marked microbeads cell sorting have a strictly request of temperature and protect from light. Need an experienced manipulator, a tiny negligence can take gross influence to efficiency of sorting. (2) Not only CD90 positive cells, but also CD90 negative cells of hepatocellular carcinoma can self renew and proliferation consistently. They also have the capacity of multidifferentiation. (3) Result of Real-time fluorescence quantitative test and immunocytochemistry indicated that have variance of protein and gene expression between CD90 positive and negative group cells in different degree. So, it need a further study to varificate if these variance of different clone result in different invasity and carcinogenisis of them.
引文
[1]Nowell P D The clonal evolusion of tumor cell population [J]. Science,1976,194 (4260):23-28.
[2]颜政,方驰华人肝细胞癌细胞亚群的克隆分离及异质性机制的初步研究.[J]世界华人消化杂志.2006,14(5)：481-485
[3]Snow C. Flow cytometer electronics. [J] Cytometry A,2004,57 (2):63-69.
[4]Siegel D L. Selecting antibodies to cell-surface antigens using manetic sorting techniques. [J] Methods Mol Biol,2002,178:219-226
[5]刘胜军,方驰华流式细胞仪、免疫磁珠及亲和板结合分离法分选C-kit+肝癌细胞的比较.[J]中华消化外科杂志2007,6,6(3)：206-208
[6]Yang ZF, Ngai P, Ho DW, et al. Identification of local and circulating cancer stem cells in human liver cancer, [J] Hepatology,2008,47(3):919-928.
[7]梁宏刚,张开明免疫磁珠法分选骨髓CD34+细胞：纯度影响因素的分析[J]中国组织工程研究与临床康复2009.13(40)7825-7828
[8]Lee E S, Han E M, Kim Y S, et al. Occurrence of c-kit+ tumor cells in hepatitis B virus-associated hepatocellular carcinoma [J]. Am J Clin Pathol,2005,124(1):31-36
[9]Hope KJ, Jin L, Dick JE. Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacity [J] Nat Immunol,2004, 5:738-743
[10]Tang Y, Katuri V, Dillner A,et al. Disruption of transforming growth fact or-beta signaling in ELF beta-spectrin-deficient mice. [J] Science.2003 Jan 24; 299(5606):574-7
[11]Durnez A, Verslype C, Nevens F, et al. The clinic pathological and prog nostic relevance of cytokeratin 7 and 19 expression in hepatocellular carcinoma. A possible progenitor cell origin. [J]. Histopathology,2006,49(2):138-151.
[12]Suetsugu A, Nagaki M, Aoki H,et al. Characterization of CD133+ hepatocellular carcinoma cells ascancer stem/progenitor cells [J]. Biochem Biophys Res. Commun,2006,351(4):820-824
[13]Chiba T, Kita K, Zheng YW, et al.Side population purified from hepatoce llullar carcinoma cells harbors cancer stem cell-like properties[J].Hepatology,200 6,4;4(1):240-251
[14]Ma S, Chan KW, Hu L, Lee TK, Wo JY, Ng IO,et al. Identification and characterization of tumorigenic liver cancer stem/progenitor cells.[J] Gastroenter ology 2007; 132:2542-2556
[15]Yang ZF, Ngai P, Ho DW, et al. Identification of local and circulating c ancer stem cells in human liver cancer. [J] Hepatology.2008;47(3):919-28.
[16]Yang ZF, Ngai P, Ho DW, et al. Significance of CD90+ Cancer Stem C ells in Human Liver Cancer. [J] Cancer cell.2008:13,153-166.
[17]Shi GM, Xu Y, Fan J, Zhou J, Identification of side population cells in h uman hepatocellular carcinoma cell lines with stepwise metastatic potentials. [J] Cancer Res. Clin. Oncol.2008:134(11):1155-63.
[18]Yamashita T, Ji J, Budhu A, et al. EpCAM-positive hepatocellular carcino ma cells are tumor-initiating cells with stem/progenitor cell features. [J] Gast roenterology.2009:136(3):1012-24.
[19]Grozdanov PN, Yovchev MI, Dabeva MD. The oncofetal protein glypican-3 is a novel marker of hepatic progenitor/oval cells [J]. Lab Invest,2006,86(1 2):1272-1284
[20]Rege, T.A., and Hagood, J.S. Thy-1 as a regulator of cell-cell and cell-m atrix interactions in axon regeneration, apoptosis, adhesion, migration, cancer, a nd fibrosis.[J] FASEB.20,1045-1054.
[21]Dannis Y.Y. Riehle K.J. Lazaro C. Isolation of multipotent progenitor cell s from human fetal liver capable of differentiating into liver and mesenchymal lineages. [J] Proc.Natl. Acad. Sci.USA 103,9912-9917.
[22]Wu XZ, Chen D. Origin of hepatocellular carcinoma:role of stem cells. [J] Gastroenterol Hepatol,2006,21:1093-1098.
[23]Alison MR, Lovell MJ. Liver cancer:the role of stem cells.[J] Cell Prolif, 2005,38:407-421
[24]Tovara V, Villa nueva A, Llovet JM. Cell biology and genetics in liver c ancer, [J] Gastroenterol Hepatol,2007:30(6):360-369.
[25]Dumble ML, Croager EJ, Yeoh GC, et al. Generation and characterization of p53 null transformed hepatic progenitor cells:oval cells give rise to hepat ocellular carcinoma [J] Carcinogenesis,2002,23(3):435-445
[26]Yamamoto T, Uenishi T, Ogawa M, Ichikawa T, et al. Immunohistologic attempt to find carcinogenesis from hepatic progenitor cell in hepatocellular car cinoma. [J] Dig Surg,2005:22(5):364-370.
[27]Lee JS, Heo J, Libbrecht L, et al. A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells. [J] Nat Med.20 06;12(4):410-416.
[28]Ho JW, Pang RW, Lau C, et al. Significance of circulating endothelial pr ogenitor cells in hepatocellular carcinoma [J] Hepatology,2006:44(4):836-843.
[29]Gournay J, Auvigene I, Pichard V, et al. In vivo cell lineage analysis dur ing chemical hepatocarcinogenesis in rats using retroviral-mediated gene transfer: evidence for dedifferentiation of mature hepatocytes [J] LabInvest,2002,82(6): 781-788
[30]王阔,索金友,邓婧,等原发性肝癌不同病理组织类型中肝干细胞的起源分析[J]第三军医大学学报2006：28(2)：114-116
[31]李玉林,唐建武,第6版病理学[M]人民卫生出版社,1979：93-103
[1]Nowell P D The clonal evolusion of tumor cell population [J]. Science, 1976,194(4260):23-28.
[2]Bonnet D, Dick J E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hemotopoietic cell [J]. Nature Med, 1997,3(7):730-737.
[3]Al-Hajj M, Wicha M S, Benito Hernandez A, et al. Prospective identifica tion of tumorigenic breast cancer cells [J]. Proc Natl Acad Sci USA,2003,100 (7):3983-3988.
[4]Kondo T, Setoguchi T, Taga T. Persistence of a small subpopulation of c ancer stem-like cells in the C6 glioma cell line [J]. Pro Natl Acad Sci USA,2 004,101(3):781-786.
[5]Germain L, Noel M, Gourdeau H, Marceau N. Promotion of growth and differentiation of rat ductular oval cells in primary culture. [J]. Cancer Researc h 1998;48:368-378
[6]Fialkow PJ. Biochim Biophys Acta,1976,458:283
[7]Hope KJ, Jin L, Dick JE. Acute myeloid leukemia originates from a hier archy of leukemic stem cell classes that differ in self-renewal capacity [J]. Nat Immunol,2004,5:738-743
[8]Tang Y, Katuri V, Dillner A, et al. Disruption of transforming growth fa ctor-beta signaling in ELF beta-spectrin-deficient mice. [J]. Science.2003,4;299 (5606):574-7
[9]Kitisin K, Ganesan N, Tang Y, et al. Disruption of transforming growth f actor-beta signaling through beta-spectrin ELF leads to hepatocellular cancer thr ough cyclin D1 activation. [J]. Oncogene.2007;1;26(50):7103-10.
[10]Tang Y, Katuri V, Dillner A, Mishra B, Deng CX, Mishra L. Disruptio n of transforming growth factor-beta signaling in ELF beta-spectrin-deficient mi ce. [J]. Science 2003;299:574-577
[11]Dumble M L, Croager E J, Yeoh G C, et al. Generation and charact erization of p53 null transformed hepatic progenitor cells:oval cells give rise t o hepatocellular carcinoma [J]. Carcinogenesis,2002,23(3):435-445
[12]Tanaka S, Yamamoto T et al Potentiality of combined hepatocellular and intrahepatic cholangiocellular carcinoma originating from a hepatic precursor c ell:Immunohistochemical evidence [J]. Hepatol Res.2005 May;32(1):52-7.
[13]朱言亮,陈孝平,张万广,等大鼠实验性肝癌发生中卵圆细胞的变化[J]世界华人消化杂志,2006,14(29)：2830-2833.
[14]Ho JW, Pang RW, Lau C, et al. Significance of circulating endothelia 1 progenitor cells in hepatocellular carcinoma [J]. Hepatology,2006,44(4):836-843.
[15]Gournay J, Auvigene I, Pichard V,et al. In vivo cell lineage analysisd uring chemical hepatocarcinogenesis in rats using retroviral-mediated gene trans fer:evidence for dedifferentiation of mature hepatocytes [J]. Lab Invest,2002,82 (6):781-788
[16]Ponder K P. Analysis of liver development, regeneration, and carcinog enesis by genetic marking studies[J]. FASEB J,1996,10(7):673-682
[17]周思朗、李鹏、曹漫明、等.大鼠肝癌干细胞生物学行为研究[J].中华肝脏病杂志2006,14(5)：364-366.
[18]Durnez A, Verslype C, Nevens F, et al. The clinicopathological and pro gnostic relevance of cytokeratin 7 and 19 expression in hepatocellular carcinom a. A possible progenitor cell origin. [J]. Histopathology,2006,49(2):138-151.
[19]Suetsugu A, Nagaki M, Aoki H, et al. Characterization of CD 133+ hepa tocellular carcinoma cells ascancer stem/progenitor cells [J]. Biochem Biophys Res. Commun,2006,351(4):820-824.
[20]Yin S, Li J, Hu C, et al. CD133 positive hepatocellular carcinomacell s possess high capacity for tumorigenicity [J]. Int J Cancer,2007,120(7):1444-1 450.
[21]Ma S, Chan KW, Hu L, Lee TK, Wo JY, Ng IO, et al. Identification a nd characterization of tumorigenic liver cancer stem/progenitor cells. [J]. Gastro enterology 2007;132:2542-2556
[22]Rege, T.A. Hagood, J.S. Thy-1 as a regulator of cell-cell and cellmatrix interactions in axon regeneration, apoptosis, adhesion, migration, cancer,and fi brosis. [J]. FASEB 2006,20,1045-1054.
[23]Dennis. J.E, Esterly. K, Awadallah. A. et. al Clinical-scale Expansion of a Mixed Population of Bone Marrow Derived Stem and Progenitor Cells for Potential Use in Bone Tissue Regeneration. [J]. Stem Cells 2007,25,2575-2582.
[24]Dan. Y.Y. Riehle. K.J. Lazaro. C. et al. Isolation of multipotent progenit or cells from human fetal liver capable of differentiating into liver and mesenc hymal lineages. [J]. Proc. Natl. Acad. Sci. USA 2006,103,9912-9917.
[25]Yang ZF, Ngai P, Ho DW, et al. Identification of local and circulating c ancer stem cells in human liver cancer. [J]. Hepatology.2008;47(3):919-28
[26]Lara-Pezzi, E. Serrador, J.M. Montoya, M.C. et al. The hepatitis B virus X protein (HBx) induces a migratory phenotype in a CD44-dependent manner: Possible role of HBx in invasion and metastasis. [J]. Hepatology 2001;33,127 0-1281
[27]Yang ZF, Ngai P, Ho DW, et al. Significance of CD90+ Cancer Stem Cells in Human Liver Cancer. [J]. Cancer cell.2008:13,153-166.
[28]Yamashita T, Ji J, Budhu A, et al. EpCAM-positive hepatocellular carcin oma cells are tumor-initiating cells with stem/progenitor cell features. [J].Gastr oenterology.2009; 136(3):1012-24.
[29]Grozdanov PN, Yovchev MI, Dabeva MD. The on cofetal protein glyp i can-3 is a novel marker of hepatic progenitor/oval cells [J]. Lab Invest,2006,8 6(12):1272-1284
[30]Goodell MA, Rosenzweig M, Kim H, et al. Dye efflux studies sugest th at hematopoietic stem cells expressing low or undetectable levels of CD34 anti gen exist in multiple species. [J]. Nat Med,1997.3:1337-1345
[31]Schinkel AH, Mayer U, Wagenaar E, et al. Normal viability and altered pharmacokinetics in mice lacking mdrl-type(drag-transporting) P-glycoproteins [J].Proc Natl Acad Sci USA,1997,94:4028-4033
[32]Zhou S, Schuetz JD, Bunting KD, et al The ABC transporter Bcrpl/ABC G2 is expressed in a wide variety of stem cells and is a molecular determinati on of the side-population phenotype. [J]. Nat Med.2001,7:1028-1034
[33]Chiba T, Kita K, Zheng Y W, et al. Side population purified from hepa tocellular carcinoma cells harbors cancer stem cell like properties [J]. Hepatolo gy,2006,44(1):240-251.
[34]Zheng Y W, Taniguchi H. Diversity of hepatic stem cells in the fetal a nd adult liver. [J]. Semi Liver Dis.2003,23(4):337-348.
[35]Haracughi N, Utsunomiya T, Inoue H et al. Characterization of a side p opulation of cancer cells from human gastrointestinal system. [J]. Stem Cells, 2006,24(3):506-513
[36]Woodward WA, Chen Ms, Behbod F, et al. WNT/beta-catenin mediates a radiation resistance of mouse mamma progenitor cells. [J]. Porc Natl Acad s ci USA,2007,104(2):618-623
[37]Zhou S, Morris JJ, Barnes YX, et, al BCRP1 gene expression is require d for normal numbers of side population stem cells in mice, and confers relati ve protection to mitoxantrone in hematopoietic cells in vivo [J]. Proc Natl Aca d Sci USA,2002,9:12339-12344
[38]Wulf G G. Luo KL. Jackson KA et al. Cells of the hepatic side popula tion contribute to liver regeneration and can be replenished by bone marrow st em cells [J]. Haematologica,2003,88:368-378
[39]Smyth MJ, Krasovskis E, Sutton VR, et al. The drug efflux protein, P-glycoprotein, additionally protects drug-resistant tumor cells from multiple form of caspase-dependent apoptosis. [J]. Proc Natl Acad Sci USA.1998.95:7024-70 29