【作者】 黎江；

【导师】 钱其军；

【作者基本信息】 第二军医大学 ， 肿瘤学， 2012， 博士

【摘要】 肿瘤干细胞的存在已经被越来越多的人所认同，但由于分离到的CSCs细胞数量太少，难以支持后续实验的开展。如何在体外有效的分离，培养，扩增肿瘤干细胞，并以此为研究对象是我们思考的方向。国外学者在长期研究中发现，加入EGF和bFGF的无血清培养液有利于正常成体干细胞的体外扩增，并维持干细胞多向分化潜能。目前已经在乳腺癌、恶性神经系统肿瘤、结肠癌等恶性肿瘤中分离培养得到了类似肿瘤干细胞的一群细胞，他们是用含有EGF和bFGF的无血清培养体系维持增殖、分化潜能。我们将以此为基础进行肝癌干细胞的培养。由于现阶段分离肝癌干细胞还缺乏广谱的公认的标志物，且绝大多数分离工作均局限于肝癌细胞系，直接从人癌组织分离并培养肝癌干细胞尚无报道，严重阻碍了对肝癌干细胞的深入研究。本实验采用直接无血清培养的方法，从肝癌细胞株以及人原代肝癌组织中分离出肝癌干细胞样细胞并连续传代并尝试建立一种可有效富集肝癌干细胞样细胞的体外无血清悬浮培养技术，以期建立一种从人肝癌组织中分离培养获得肿瘤干细胞样细胞的新方法。并对培养出的球状细胞进行体外侵袭性，细胞周期的变化，细胞耐药性，克隆细胞的体外分化能力以及成瘤能力等方面验证我们获得的细胞具有肿瘤干细胞特性；进一步进行RNA深度测序和生物信息学分析，检测肝癌干细胞与肝癌细胞之间的差异。1、人肝癌干细胞样细胞的新型培养基的筛选及肿瘤细胞系的建立为更好的培养扩增肝癌干细胞，我们对培养体系进行优化，经过实验室长期的实验探索，筛选出7种有效的培养体系。培养成分包括DMEM-F12，非必需氨基酸，血清替代物，N2，B27，B27-minus VA，BSA，EGF，bFGF以及我们自己筛选的2个细胞因子IGF-1和FGF-10。通过长期的筛选实验证实C3号培养配方是我们的最佳培养体系。这个配方包含了B27-minus VA，EGF和我们筛选的IGF-1和FGF-10。利用无血清C3培养基对肝癌细胞系、原代细胞系及异种移植技术得到的肿瘤组织等多种来源的细胞进行培养，并获得了球状细胞。从肝癌细胞系Hep3B和Huh7中培养出来的球状细胞Hep3B-C、Huh7-C克隆形态越来越明显，并且能够稳定传代培养。从高侵袭性的细胞株HCCLM3和MHCC97-H及低侵袭能力的细胞株MHCC97-L分别培养得到了HCCLM3-C、MHCC97-H-C和MHCC97-L-C。在肝癌细胞系中成功得到球状生长的细胞之后，我们取病人的肿瘤组织直接进行消化培养，同样获得了球状细胞EHBH-HCSC-1和EHBH-HCSC-2细胞。同时，采用了异种移植技术，将病人的肿瘤组织直接包埋于小鼠皮下，一段时间后取小鼠的皮下肿瘤组织消化培养，分别利用常规的DMEM-F12+10%FBS和C3无血清培养体系进行培养，得到了一株呈上皮状生长的肿瘤细胞系EHBH-3和球状细胞EHBH-HCSC-3。2、球状细胞生物学特性分析经C3无血清培养体系获得了球状细胞后，进一步对这些细胞进行了生物学特性的分析：1）利用实时荧光定量PCR检测Wnt-1，CD90，Ep-CAM，NOTCH1，NOTCH2，NOTCH3在Hep3B、Huh7、MHCC97-L、HCCLM3、Hep3B-C、Huh7-C、MHCC97-L-C、HCCLM3-C中的表达情况。结果表明CD90在球状细胞中表达量都高于其相对应的贴壁肝癌细胞株。其中，Huh7-C中CD90的表达量是Huh7中表达量的10倍。球状细胞与相对应的细胞株相比NOTCH的mRNA表达均下降，而在Wnt-1和Ep-CAM的mRNA表达水平上出现了不一致的现象。2）对球状细胞进行糖原染色以及ICG的摄取实验，证实了这些细胞确实是肝癌细胞来源的细胞，而不是其他细胞污染。3）利用体外Matrigel侵袭实验直接比较Huh7细胞与Huh7-C细胞的侵袭能力。结果发现球状细胞Huh7-C与对应的Huh7细胞都具有较强的侵袭特性。定量分析结果表明，球状细胞Huh7-C的侵袭程度比Huh7高接近3倍。4）通过细胞周期的分析发现球状细胞Hep3B-C和Huh7-C主要处于G0-G1期，所占的百分比分别为74.09%和59.20%，与肝癌细胞株Hep3B和Huh7具有显著差异。5）流式细胞仪和RT-PCR检测发现在球状细胞中CD90的表达量均有不同程度的提高，两次实验结果趋势一致。在Hep3B-C细胞中，其提高倍数达10倍以上，这与之前的研究者所获得的肝癌干细胞的表型一致。同时检测了细胞的表面抗原Ep-CAM的表达情况，结果发现两种球状细胞的表达量都是下降的，这与以前的研究是相悖的。6）在Hep3B-C和Huh7-C细胞对阿霉素的摄取检测实验中发现，与相对应的Hep3B和Huh7相比较，在相同时间里面，球状细胞摄入的阿霉素的量少于普通的贴壁肝癌细胞，这是细胞产生耐药性的一个重要的条件。进一步检测细胞在不同的药物浓度下的存活率实验中证实，球状细胞Huh7-C相对于贴壁的肝癌细胞Huh7具有良好的耐药性。球状细胞对临床证实的第一个针对肝癌靶向性的药物索拉非尼的耐药性实验中也证实了这一点：应用无血清培养基培养出来的球状细胞在40umol/L的sorafinib的浓度下48小时依然有46%的细胞存活，而与此相对照的贴壁肝癌细胞Huh7基本没有细胞存活。耐药性实验证明，通过无血清条件培养获得的球状细胞与贴壁细胞相比具有良好的耐药性。7） ELISA定量检测结果说明无论是贴壁的肝癌干细胞还是球状的肝癌细胞均可以大量表达VEGF，而不同的细胞所形成的球状的细胞中其表达趋势并不是一致的，这可能与很多因素有关。8）对获得的球状细胞的进一步进行体外诱导分化。分别选取肝癌细胞的标记CK8、CK18；胆管细胞的标记CK19；血管细胞的标记Tie2。Huh7表达CK8和CK18，但不表达CK19和Tie2，而Huh7-C细胞对这4个标记表达很弱或不表达。将Huh7-C诱导后细胞均表达这4个标记因子。这个结果提示我们可能球状细胞具有多向分化的潜能，可以形成各种不同类型的细胞，这需要我们找到诱导的最佳条件，从而真正能够了解这些悬浮、球状生长的细胞。9）本课题通过给NOD/SCID鼠背部皮下注射不同细胞数量的肝癌贴壁细胞与球状生长的细胞，比较球状细胞与贴壁细胞的成瘤能力。异种移植成瘤试验的结果显示：接种100个MHCC97-L-C细胞到NOD/SCID小鼠中可以100%形成肿瘤；1000个HCCLM3-C细胞接种成瘤率高达100％；而对应的贴壁细胞成瘤率却很低。在MHCC97-H-C细胞株成瘤实验中发现100个细胞就可以在NOD/SCID鼠皮下成瘤，10000个MHCC97-H-C细胞在小鼠中成瘤率可达到100%，而相同数量的贴壁细胞MHCC97-H却不能形成肿瘤。小鼠皮下注射105个Huh7细胞都不能形成肿瘤，但是105个球状细胞Huh7-C能100%形成肿瘤。通过以上实验结果证实经C3无血清培养体系获得的球状细胞具有肿瘤干细胞的特性，可以作为肝癌干细胞研究的对象。3、肝癌干细胞样细胞的深度测序以及生物信息学分析采用Illumina HiSeq2000测序技术分别对3株肝癌干细胞株样品Huh7-C，Hep3B-C，MHCC97-H-C以及对应的3株肝癌细胞株样品Huh7，Hep3B，MHCC97-H进行RNA深度测序，采用RPKM算法对原始RNA测序数据进行预处理，消除系统误差；采用倍数法（FoldChange）筛选差异表达基因，通过文本挖掘进一步筛选到44个肝癌干细胞相关的关键基因，采用Onto-Express软件将筛选到的基因映射到Gene Ontology数据库中，进行功能富集性分析。并进一步在通路水平上进行的富集性分析，将筛选后得到的基因分别映射到GeneGO数据库和KEGG数据库中，基于KEGG数据库的分析结果仅筛选到了1条关键通路，满足统计学检测指标p-value<0.05，基于GeneGO数据的分析中则筛选到了17肝癌干细胞相关的通路（p-value<0.05），其中补体通路被2个数据库同时筛选到，可能在肝癌干细胞的分子过程中起着关键的作用。另外，本文分别对2株肝癌细胞系Hep3B，Huh7和2株肝癌干细胞系Hep3B-C，Huh7-C进行了sRNA深度测序，采用SOAP软件将sRNA定位到基因组上，分析sRNA在基因组上的表达和分布情况；将4个样品中的小RNA片段和已知的miRNA、重复序列、Genbank、Rfam、外显子和内含子、piRNA信息进行比对和注释；采用倍数法筛选在肝癌干细胞系与肝癌细胞系中表达发生变化的sRNA，利用泊松分布计算p-value值，并用Benjamini多重假设检验校正p-value值；共筛选到4个repeat片段在Hep3B和Hep3B-C、Huh7和Huh7-C中均差异表达，37个高表达、9个低表达的piRNA以及18个在Hep3B-C/Hep3B，Huh7-C/Huh7中表达同增同减的miRNA,其中高表达的miRNA有9个，低表达的有9个，具有统计学意义（p-value<0.01，q-value<0.01）。通过生物信息学分析我们得到了很多差异表达的基因，接下来的任务任重而道远。我们需要通过进一步的实验进行验证这些基因在肝癌干细胞与肝癌细胞之间的功能差异，明确肝癌干细胞其独特的标记以及特性，从而最终找到能够特异性靶向肝癌干细胞的制剂，为最终能够彻底治愈肝癌提供基础。更多还原

【Abstract】 It is commonly suggested tumors exist cancer stem cells, leading to its formation,persistence, recurrence and metastasis. But still, the isolated CSCs are too rare to supportthe subsequent experiment, emphasizing the culture and expansion CSCs in vitro isimportant. Using culture conditions that support the growth of undifferentiated CSCsmay help to devise novel diagnostic and therapeutic procedures. Recent studies havedocumented that serum-free medium with EGF and b FGF can expansion adult stem cellsand maintain pluripotency. This reminds us to find new culture medium for liver cancerstem cells. As far as we know, it is already available to separate and cultureCSC-like-cells from breast cancer, glioma, colon cancer and other malignancies. And it ispossible to maintain the potential of proliferation and differentiation in the serum-freeculture medium with EGF and b FGF. What we are striving for is optimal culturemedium for liver cancer stem cells.1. Optimized the culture medium for CSCs and expansion of human liver cancerstem cell-like cells from different sourcesIn order to successfully enrich liver CSCs, we optimized our culture system firstly.After long time exploration and screening,7effective culture systems were selected.These systems contain ingredients such as DMEM/F12, Neurobasal-A medium, KO-SR,N2, B27, B27without VA, BSA, EGF, bFGF and2cytokines as FGF-10and IGF-1. Weconfirmed C3culture formula as the ultimate culture system, which contains B27withoutVA, EGF and the cytokines FGF-10and IGF-1.By using C3culture formula, human liver cancer stem cell-like cells from differentsources were cultured and expansion.We first cultured hepatoma cell lines includingHep3B and Huh7in our optimized culture medium. The results showed that Hep3B-Cand Huh7-C displayed clone-like forms and maintained the same state after3months cellpassage. Next we cultured CSC-like cells derived from cells lines with different invasionability. HCCLM3and MHCC97-H were high-invasive cell lines and MHCC97-L waslow-invasive one. All of these cell lines were able to form clone and be stable for cellpassage. We called them as HCCLM3-C, MHCC97-H-C and MHCC97-L-C.We also got cells from tumor tissue in patients. After surgical resection, liver tumortissues were dissociated into single cells. EHBH-HCSC-1and EHBH-HCSC-2cells grewwell for more than2months in vitro as undifferentiated tumor spheres in serum-freemedium. Another way to gain cells was to engraft patient’s tumor tissues and gave rise to subcutaneous tumors in severe combined immuno-deficient mice. We took thesubcutaneous tumor for digestion and culture. These tumor cells were cultured in2different culture mediums—one was in DMEM with10%FBS and the other one was inour optimized culture system. EHBH-3display epithelial-like growth tumor cells whilethe EHBH-HCSC-3show clone-like growth.2. Biological characteristics assayWe assayed biological characteristics of these cells when we got the CSC-like cells.(1) Fluorescent quantitative PCR was used to detect the expression of Wnt-1,Wnt-1,CD90,Ep-CAM, NOTCH1, NOTCH2, NOTCH3in hepatocellularcarcinoma cells, such as: Hep3B、Huh7、MHCC97-L、HCCLM3and CSC-likecells, such as Hep3B-C、Huh7-C、MHCC97-L-C、HCCLM3-C; CD90expressedhigher in clonal cells than the corresponding adherent hepatocellular carcinomacells. Among them, CD90expression in Huh7-C was10times higher than Huh7cells. NOTCH mRNA expression level were decreased in clonal cells comparedto corresponding cell lines, but Wnt-1and Ep-CAM mRNA expression levelappeared inconsistent phenomenon.(2) We evaluated the cell source of these CSC-like cells using glycogen staining andICG uptake experiments, the results showed that these cells are indeed fromhepatocellular carcinoma cells,but not due to other cell contamination.(3) Furthermore, we performed in vitro Matrigel invasion assay to detect invasionability of Huh7-C and corresponding Huh7cells, the results showed that both ofthem had the strong invasiveness. Through the quantitative analysis we can seethat Huh7-C like cells invasion extent were nearly3times higher than Huh7.(4) Through the cell cycle analysis, the CSC-like cells Hep3B-C and Huh7-C weremainly in phase G0-G1, with percentage at74.09%and59.20%respectively,which were significantly different from cancer cell lines Hep3B and Huh7.(5) Based on both the RT-PCR and FCM analysis, expression of CD90in CSC-likecells were upregulated in different degrees. Consistent with previous study onCSC phenotype, CD90expression level in Hep3B-C was10times higher thanHep3B. Simultaneously, we detected the expression of cell surface antigenEp-CAM, and inconsistent with previous research, both two CSC-like cellsweakened their expression.(6) After surveying uptake of doxorubicin, Hep3B-C and Huh7-C took in lessdoxorubicin compared with corresponding normal HCC cell lines, and this was thought to be a critical condition. Further detection of cell survival in differentdrug concentrations verified Huh7-C wass more drug resistant than Huh7cellline. CSC-like cells show resistant difference even to sorafenib, the firstclinically confirmed liver cancer targeted drug. We applied Huh7-C cellscultured in serum-free medium for in vitro drug resistant experiment, finding46%cell survival after incubation in40μmol/L sorafinib for48hours, while thisconcentration makes adherent Huh7cells no survival. Taken together, theCSC-like cells cultured in serum-free medium showed better drug resistancethan corresponding adherent HCC cell lines.(7) ELISA quantitative detection results in both the adherent HCC cells andCSC-like cells showed great expression of VEGF, and the expression level wasinconsistent in different CSC-like cells from different HCC cell lines, whichmay be related to many factors.(8) Next we detected the differentiation capacity of clone-like cells in vitro. Wechoose CK8, CK18keratins whose presence is essentially restricted to Liver,while CK19expressed in biliary ducts and vascular cell marker Tie2. We foundthat Huh7expressed CK8and CK18but no CK19and Tie2, while Huh7-Cshowed weak or no expression of these4markers. Interestingly, differentiatedcells express all of these4markers. The result reminded us the clone cell had thepluripotency. If we can find the optimal condition to induced, we can learn morefrom the clone cells.(9) Tumorigentic potential of liver CSC-like cells were evaluated in vivo. Theinjection of1,000Huh7-C cells generated visible tumors after4-5weeks fromthe transplant while100,000live cancer cells Huh7did not induce tumorformation; We injected100MHCC97-L-C cells into NOD/SCID mices,the ratioof tumor formation is100%; For HCCLM3-C cell line, we injected5NOD/SCID mices,1,000cells per mice,the tumor formation rate is100%,whilethe tumor formation rate of corresponding adherent cancer cells is very low. Wefound100MHCC97-H-C cells could generate tumors, and when we injected10,000cells per mice, all of them could generate tumors, while the samequantity adherent MHCC97-H cells could not induce tumor formation. All thedata indicating that the CSC-like cells showed significant higher tumorogenicitythan corresponding adherent cancer cell lines.From the results above we concluded that we got a subset of cells which have the ability of CSCs, we can use them to do more research about the liver cancer stem cells.3. Deep sequencing-based expression profiling analysis of liver CSC-like cellsDeep sequencing were carried out an in-depth analysis of the expression of mRNAsin three hepatoma cells(Hep3B,Huh7and MHCC97-H) and three liver cancer stem celllines(Hep3B-C,Huh7-C and MHCC97-H-C).We identified1012differentially expressedgenes overlapped among Huh7-C vs Huh7, MHCC97-H-C vs MHCC97-H and Hep3B-Cvs Hep3B groups via foldchange method. The406of1012genes whose fold change>2were up or down regulated consistently among these samples.90genes wereup-regulated and316genes were down-regulated. In order to identify the functionalclassification of these406genes, we performed functional enrichment analysis inGeneOntology database. In addition, to identify differential genes enriched in which kindof biological pathways, pathway enrichment analysis was performed in GeneGOdatabase via MetaCore software. We showed top10significant pathways whosecorrected p value was less than0.05. We chose hypergeometric distribution to calculatethe significance values (p values) and the FDR method to correct the p value.In addition，we applied HiSeq deep sequencing to carry out an in-depth analysis ofthe expression of sRNAs in two hepatoma cells(Hep3B,Huh7) and two liver cancer stemcell lines(Hep3B-C,Huh7-C).We first mapped the small RNA tags to genome by SOAPto analyze their expression and distribution on the genome.The small RNA tags wasannotated with known miRNA, repeat, piRNA, exons, introns, rRNA, scRNA, snoRNA,snRNA and tRNA from Genbank, Rfam and miRBase. Then we used Foldchange methodto find out the differentially expressed sRNAs between hepatoma and hepatoma stemcells. Poisson distribution was used to calculate the p value, and Benjamini multiplehypothesis testing was used to correct the p value. Results revealed4repeats,37highexpressed and9low expressed piRNAs,9high expressed and9low expressed miRNAsthat are significantly diffenrentially expressed between hepatoma and liver cancer stemcells with p-value<0.01and q-value<0.01.Through bioinformatics analysis we get a lot of differences betweengene-expression. We need to verify the different function of these genes in liver cancerstem cells and tumor cells by further experiments. Also we need to find the uniquemarker and characteristic of liver cancer stem cells. In the end to find specific targetedliver cancer stem cells drugs and cured liver cancer completely.更多还原