人骨髓来源间充质干细胞对肝癌细胞增殖和侵袭能力的影响
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摘要
第一部分骨髓间充质干细胞对肝癌的增殖和侵袭转移能力的影响
目的:确定人骨髓来源间充质干细胞(MSC)对肝癌细胞体内外增殖和侵袭转移能力的影响,从而为进一步研究MSC在肝癌发生发展中的作用提供理论依据。
方法:培养MSC并进行表型鉴定,选取5-8代细胞用于以下研究。(1)体外试验:提取MSC的条件培养基(MSC-CM),在高转移潜能人肝细胞癌细胞系MHCC97-H中加入不同浓度的MSC-CM,采用CyQUANT细胞增殖试验和Transwell细胞侵袭试验,比较MSC-CM对MHCC97-H细胞的增殖和侵袭能力的影响。(2)体内试验:分别皮下注射MHCC97-H和MHCC97-L细胞建立肝癌的裸鼠转移皮下瘤模型,并建立裸鼠MHCC97-H肝内原位移植瘤模型(Xenograft model)。在不同模型中注射MSC细胞并以PBS作为对照,观察动物的成瘤和肿瘤生长情况,动物饲养35天后切去肿瘤组织,测量体重和肿瘤质量;另外切取小鼠肝肺组织,固定包埋后间断50μm病理切片,显微镜下观察肺转移的情况,主要比较各组标本平均肺转移细胞数,肺转移灶的个数以及肺转移率。
结果:(1)体外增殖实验结果显示:MHCC97-H细胞与0,25%、50%的MSC-CM共培养后,其OD值分别为236.24±57.15和283.59±62.16,明显高于对照组(211.65±54.72,P=0.02),提示MSC-CM可明显促进肝癌细胞的增殖。(2)体外侵袭实验结果显示:经与MSC-CM共培养后MHCC97-H的穿膜细胞数目减少(11.37±3.54 vs 20.00±5.35,P=0.0021,提示MSC-CM抑制肝癌细胞的侵袭能力。(3)MSC对肝癌体内生长的影响:在MHCC97-H皮下瘤模型中注射MSC,35天后肿瘤体积显著大于PBS对照组(3080.51±1234.78 vs 2223.75±1000.60 mm~3,P=0.046),平均每天的肿瘤增长量也高于对照组(102.65±63.66 vs 72.07±52.13 mm~3/day,P=0.013),这种生长速度的差别主要位于成瘤及注射后的前20天内,随着时间增加差别逐渐缩小。在MHCC97-L皮下瘤模型中注射MSC45天后,MSC注射组与对照组的肿瘤体积无显著性差别(1913.67±1336.59 vs 1989.63±466.08,P=0.898),平均每天的增长量(62.95±.30.45 vs 63.04±50.57,P=0.99)。在Xenograft模型中MSC注射组较对照组有更高的瘤体比值(0.15±0.51 vs 0.13±0.04,P=0.03)。为进一步检验MSC是否对肿瘤生长的影响具有时效性,我们于Xenograft模型(n=10)建立15天后再注射MSC细胞,结果表明,晚期阶段MSC注射对肿瘤的生长无明显作用。(4)MSC对肝癌转移模型肺转移的影响:肺组织切片统计结果显示,在MHCC97-H皮下瘤模型中,MSC注射组的平均肺转移细胞数目显著低于对照组(49.75±53.35 Vs 227.22±224.00,P=0.046);在Xenograft模型中,同期和后期MSC注射组肺转移率均低于对照组(50%vs100%,P=0.024;20%vs 100%,P=0.015),与MSC的注射时间无关。
结论:注射MSC可促进肝癌细胞的体外增殖和体内生长,同时受肿瘤细胞类型(不同转移潜能的肝癌细胞)和肿瘤生长阶段的影响;但MSC可明显抑制肝癌的体外侵袭和体内肺转移。MSC可能为抗肝癌转移的一种新方法。
第二部分骨髓间充质干细胞在裸鼠人肝癌转移模型体内的分布
目的:观察MSC静脉注射后,在裸鼠体内的归巢和分布情况,为进一步研究MSC的作用机制提供理论指导。
方法:利用目前国内外现有的多种细胞标记方法的优缺点,分别采用转染绿色荧光蛋白GFP基因和红色荧光蛋白RFP基因的方法标记人高转移肝癌细胞MHCC97-H和MSC细胞,并筛选出稳定的转染细胞株,另外采用DAPI,Brdu标记MSC。为观察MSC在荷瘤鼠体内的分布,将DAPI标记的MSC细胞注入MHCC97-H荷瘤鼠体内,4天后,切取肿瘤组织,肝脏,肺脏,脾脏,心脏,快速冰冻切片后荧光显微镜下观察DAPI-MSC在器官组织中的分布,骨髓细胞涂片后观察DAPI-MSC在骨髓中的分布。为观察MSC在肿瘤组织中的分布,采用Brdu标记MSC,并注入Xenograft模型鼠体内,2周后应用Brdu单克隆抗体进行肿瘤组织的免疫组化检测。为观察MSC与肝癌细胞体外增殖的影响,将GFP标记的MHCC97-H细胞与RFP标记的MSC细胞进行体外共培养。
结果:肝癌组织中MSC数明显高于非肿瘤组织中。在骨髓、脾脏、血管内皮组织中也存在相对较多的MSC。MSC在肿瘤组织中主要定位于基质,MSC注射组较非注射组有更多间质构成。MSC较高的迁徙归巢性能与MSC存在较强的基底膜降解能力有关,及肿瘤细胞分泌物的趋化作用有关。MSC能在体外与MHCC97-H细胞共生长,且存在少量细胞融合现象。
结论:MSC具有向肿瘤组织归巢的性能,并参与了肿瘤基质构建。结合第一部分结果说明,MSC通过向肿瘤组织的归巢和向间质成分分化从而可以改变肿瘤微环境并影响肿瘤的生长和转移。MSC与肿瘤细胞体外可以发生融合,也可能是影响肿瘤细胞生物学机制的可能途径。
第三部分骨髓间充质干细胞对肝癌侵袭和侵袭转移影响的机制研究
目的:本部分主要研究骨髓间充质干细胞对肝癌增殖和侵袭转移能力影响的机制。
方法:将肝癌细胞系MHCC97-H与不同比例的MSC-CM共培养后于不同时间点提取肝癌细胞系的总蛋白及总mRNA;提取动物实验中肿瘤组织标本的总蛋白及总RNA,采用Real-time PCR,Western blot,免疫化学,ELISA,明胶酶谱分析的方法,比较MSC处理和未处理肿瘤细胞和组织中PCNA,Ki67,MMP2,MMP9,VEGF,MVD等与增殖侵袭密切相关指标的变化。第一,二部分试验结果MSC对肿瘤生成的促进作用和对肿瘤转移的抑制作用,并可成为肿瘤基质的重要来源,提示MSC在肿瘤与肿瘤微环境中发挥了双重作用,结合TGFβ/Smad信号通路的作用特点,选择其分子作为重要研究对象。检测MSC对肝癌细胞TGFβ/Smad信号通路分子表达的影响。并采用抗体和小干扰RNA阻断TGFβ的方法,进一步观察MSC对肿瘤生长侵袭作用的影响。
结果:
1)MSC对肝癌细胞和组织与增殖侵袭密切相关指标表达的影响
(1)MSC对增殖指标PCNA和Ki67表达的影响:MSC-CM显著的升高了MHCC97-H细胞的PCNA和Ki67表达水平,在MHCC97-H细胞培养液中加入50%的MSC-CM后,PCNA和ki67的mRNA表达水平升高了1.8和7倍。
(2)MHCC97细胞对MSC体外表型变化的影响:MHCC97-H的条件培养基(CM)能显著地升高MSC细胞中Flk1的表达,在MSC细胞培养液中加入MHCC97-H—CM后,Flk1的mRNA表达水平升高了2.64倍,同时降低了OCT-4的表达水平。
(3)MSC对MMP2和MMP9表达的影响:体外MSC-CM可显著的降低MMP2的mRNA和蛋白表达水平,但对MMP9无明显影响。体内实验对于组织标本的明胶酶谱分析显示MSC可显著的降低MMP2的表达水平和活性。在MSC注射组与非注射组MMP2 mRNA水平为(1.13±0.55 vs 1.61±1.52,P=0.05),且MMP2的表达水平与肿瘤的转移相关(非转移组与转移组mRNA水平为:0.86±0.30 vs.1.46±0.84,P=0.032)。
2)MSC对肝癌细胞和组织与增殖侵袭的影响与TGFβ/Smad信号通路有关
(1)MSC对TGFβ信号通路分子TGFβ,Smad2,Smad7表达的影响及对肝癌生长转移的相关性:体外实验:采用western blot检测发现,MSC-CM与肿瘤细胞MHCC97-H共培养后,TGFβ的表达显著的下调。体内实验:在MHCC97-H皮下瘤模型中,MSC注射后TGFβ的表达水平显著的降低(1.34±1.01 vs 2.42±2.03,P=0.047)。且肿瘤的大小和转移与TGFβ的表达水平存在相关性。将TGFβ的表达水平按中位值分为高低两组后,发现低水平组有较大的肿瘤体积(3076.97±1416.52 vs2272.87±783.36mm~3,P=0.044);非转移组较转移组有较低的TGFβ水平(1.19±0.80 vs 2.31±1.70,P=0.05);Smad7的表达水平也显著下调(1.532±1.256vs 2.56±1.99,P=0.036)。在Xenograft模型中,MSC显著的上调了TGFβ的表达水平(1.30±0.83 vs.0.67±0.52,P=0.037),但TGFβ的表达水平与肿瘤的大小无相关性(回归系数R=0.162,P=0.534),与肿瘤转移也无相关性(TGFβ在转移与非转移组织标本:1.09±0.77 vs 0.58±0.53,P=0.24)。MSC还显著的下调Smad2(1.01±0.14 vs 1.25±0.38,P=0.043)和Smad7(0.76±0.29 vs 1.408±0.503,P=0.006)的mRNA水平。而Smad2,Smad7的mRNA水平与肿瘤转移均有相关性(Smad2在非转移组与转移组的表达水平是1.01±0.17 vs 1.25±0.34,P=0.024;Smad7在非转移组与转移组的表达水平是1.02±0.23,2.13±0.85,P=0.021)。Smad7还与肿瘤的大小呈负相关(回归系数R=0.494,P=0.044)。这一结果说明,MSC通过改变TGFβ/Smad信号通路中多个分子的表达水平影响肿瘤的增殖和侵袭能力。
(2)TGFβ与MMP2表达相关分析:实验中还发现TGFβ与MMP2表达之间存在相关性。依据每组模型鼠中TGFβ,Smad2和Smad7表达水平的中位数将其分为高低表达两组,在MHCC97-H模型中,TGFβ低表达组MMP2的表达水平较低(1.25±1.01 vs1.82±1.95 P=0.047);在Xenograft模型中,Smad2低表达组MMP2的mRNA表达水平较低(0.99±0.28 vs 1.32±0.45,P=0.041)。体外实验在MHCC97-H的培养液中同时添加MSC-CM后,随着TGFβ表达水平的下调,MMP2的表达水平也下调,且两指标的波动变化方向一致。
(3)TGFβ抗体可以阻断MSC的促增殖作用:MHCC97-H培养液加入MSC-CM并培养48hrs后细胞增殖能力明显增加(16.45±4.73 vs 22.45±4.84,P=0.045)但加入TGFβ抗体后,MSC-CM的促细胞增殖能力不明显(16.45±4.73 vs 18.02±3.68,P=0.586)。
(4)MSC促进肿瘤血管生成并与TGFβ表达有关:VEGF及MVD是衡量肿瘤血管生成的重要指标,通过对组织标本裂解液VEGF的ELISA检测,显示MSC显著的升高了MHCC97-H模型鼠肿瘤组织中的VEGF浓度(OD值:0.34±0.08 vs 0.28±0.07,P=0.04),但VEGF和肿瘤的大小以及转移无相关性。体外实验采用VEGF受体(Flk1)抗体阻断VEGF的作用,对肿瘤增殖性的影响也无显著性。在Xenograft模型中,通过对微血管密度(MVD)的检测,发现MSC显著的增加了肿瘤MVD值(28±9.20 vs18.11±3.30,P=0.009),且MVD与肿瘤组织中VEGF的浓度无相关性,但与肿瘤TGFβ的表达水平有相关性(25.5±8.9 VS 19.6±8.3,P=0.038),说明TGFβ参与了肿瘤的血管生成。
(5)TGFβ的表达水平与肝癌细胞自身特性和肿瘤的微环境均相关:因为MSC在肝癌皮下瘤模型(MHCC97-H)和肝内移植瘤模型(Xenograft)中对TGFβ的表达显示了不同的调节方向,为探讨影响TGFβ/Smad信号通路分子表达的因素,选取前期不同肝癌模型标本(均未注射MSC)进行检测。TGFβ的表达水平与肝癌细胞自身特性有关:TGFβ和Smad2在MHCC97H和MHCC97L细胞系中存在显著差别(0.18±0.15vs 0.40±0.19,P=0.011;0.99±0.17 vs 2.56±0.66,P=0.017),TGFβ在MHCC97-H模型中表达水平也低于MHCC97-L模型(1.27±1.05 vs 2.72±1.63,P=0.001)。TGFβ,Smad2,Smad7的表达还与肿瘤的器官微环境密切相关:当将MHCC97-H和MHCC97-L细胞种植于裸鼠皮下时,TGFβ的表达水平均显著的增加(MHCC97-H:0.18±0.15 vs1.27±1.05,P=0.002;MHCC97-L:0.40±0.19 vs 2.72±1.63,P＜0.001),但是当把MHCC97-H的皮下瘤移植于肝脏组织后,TGFβ表达水平降低并接近细胞系水平(1.27±1.05 vs 0.32±0.25;P=0.009)。Smad2在MHCC97-H细胞系和MHCC97-H模型中的表达水平也低于在肝移植瘤模型中的表达水平(0.99±0.17 vs 2.13±0.64P=0.016;1.19±0.76 vs 2.13±0.64 P=0.003),但在MHCC97-L细胞系的表达水平高于MHCC97-L模型(2.56±0.66 vs 1.44±0.55,P=0.023)。Smad7的表达水平在细胞注入皮下后也明显的降低(MHCC97-H:12.36±1.62 vs 1.18±0.62,P=0.034;MHCC97-L:46.98±30.39 vs 1.64±0.89,P＜0.001)。TGFβ的表达水平还与肿瘤质量呈线性负相关的关系,(相关系数R=0.387,P=0.042),而Smad2的表达水平与肿瘤质量呈线性正相关(R=0.427,P=0.023),Smad2的表达水平还与肿瘤的转移密切相关,(转移组和非转移组Smad2的表达水平:1.71±0.78 vs 1.08±0.61,P=0.054)
(6)TGFβsiRNA对肝癌增殖侵袭性的影响:为进一步验证TGFβ在MSC对肿瘤增殖转移影响中的作用,采用TGFβsiRNA分别干扰MHCC97-H细胞和MSC细胞的TGFβ的表达。发现干扰后,TGFβ和MMP2的表达水平均明显下调,且肿瘤的增殖性和侵袭性均降低,干扰MSC细胞后,其MSC-CM对肿瘤的增殖侵袭性的影响以及对MMP2表达的影响均消失。
结论:TGFβ在肝癌的增殖侵袭转移中发挥了重要的作用,同时在肿瘤于微环境的相互作用中也发挥重要作用,骨髓间充质干细胞主要通过改变TGFβ中多个信号分子的表达来影响该信号通路,并最终影响肝癌的侵袭转移。同时TGFβ表达与MMP2的表达密切相关,TGFβ还参与了肿瘤的血管生成。
为了探讨骨髓间充质干细胞对肝癌增殖侵袭的影响,在本课题第一部分中通过体外细胞共培养,以及在裸鼠人肝癌模型中进行MSC注射的方法,发现了MSC在肝癌生长中的促进作用和对肝癌转移的抑制作用。为了探讨MSC的作用机制,第二部分采用多种方法对MSC细胞进行了标记,发现静脉注射MSC后在肿瘤组织中会形成相对较高的分布,归巢的MSC在肿瘤中主要参与了肿瘤基质的构建,说明骨髓间充质干细胞主要通过改变肿瘤的基质微环境而发挥其对肿瘤生长转移的影响。本课题第三部分探讨了MSC作用的分子机制,首先检测了与肝癌增殖转移密切相关的分子标记物PCNA,Ki67,MMP2,MMP9,及VEGF的变化,发现MSC升高了肝癌细胞PCNA,Ki67的表达水平,抑制了MMP2的表达和酶活性,并且MMP2的表达与肿瘤的转移密切相关。另外,结合MSC对肿瘤的双向性作用特点,重点选择了TGFβ/Smad信号通路中的多个分子进行检测,发现TGFβ/Smad信号通路中多个分子的变化与MSC作用的发挥密切相关,尤其是TGFβ和Smad7的下调不仅与MSC的促肿瘤生长有关而且也同时与MSC的抑制肿瘤转移作用相关,采用抗体及小干扰RNA阻断的方法也验证了TGFβ在MSC对肝癌生长转移的影响中发挥了重要作用,TGFβ/Smad信号通路还明显的影响MMP2的表达水平,并与MSC的促进肿瘤血管生成作用有关,通过这些结果,我们推断MSC对肝癌细胞增殖侵袭性的影响主要是通过TGFβ/Smad信号通路。由于在不同动物模型中MSC对TGFβ的表达的调控呈现不一致现象,为此比较了不同动物模型肿瘤中TGFβ表达水平的差别,我们发现TGFβ的表达不仅与肿瘤细胞本身特性有关,而且与肿瘤的器官微环境相关,这一结果强调应在器官微环境背景下来进行TGFβ/Smad信号通路的研究。
本研究的应用价值
1.确立了骨髓间充质干细胞在肝癌生长中的促进作用,以及对肝癌转移的抑制作用,这一实验结果为进一步的临床研究提供了依据。
2.归巢的骨髓间充质干细胞在肿瘤中主要参与了肿瘤基质的构建,说明骨髓间充质干细胞主要通过改变肿瘤的基质微环境而发挥其对肿瘤生长转移的影响,这一现象的发现为通过调控肿瘤微环境来治疗肿瘤转移提供了一种新的手段。
3.TGFβ/Smad信号通路中多个分子的变化与骨髓间充质干细胞的作用的发挥密切相关,为进一步研究骨髓间充质干细胞对肿瘤影响的双向作用机制提供了新思路
创新点
1.首次发现了MSC对肝癌生长的促进作用和对肝癌转移的抑制作用。
2发现MSC注射后主要参与肿瘤基质构建并改变肿瘤基质微环境。并发现了MSC体外可与肝癌细胞发生细胞融合现象。
3.首次发现了MSC对肝癌生长转移的作用机制主要与TGFβ/Smad信号通路的调控密切相关。
尚待解决的问题
1.如何有效的抑制MSC的促进生长作用将是首要的问题,实验中发现MSC的促进肿瘤增殖的作用存在时效性特点,进一步完善机制将是有益的。
2.骨髓间充质干细胞对肿瘤增殖侵袭性影响作用的发挥与多种因素有关,包括细胞因子的作用,细胞间的相互作用,以及肝脏干细胞反应等,全面深入的探讨这些因素有助于进一步完善机制。
Effect of Human Mesenchymal Stem Cell on the Proliferation and Metastasis of Hepatocellular Carcinoma
Hepatocellular carcinoma(HCC) is one of the most common cancers in the world,with important mortality in sub-Saharan Africa and eastern Asia. The extremely poor prognosis of HCC is largely due to the high rate of tumor recurrence after surgery or intra-hepatic metastases that develop through invasion of the portal vein or spread to other parts of the liver. Therefore,exploring the mechanisms involved in invasion and metastasis of HCC is the key issue for the further prolonging the survival of patients with HCC,which could provide new therapeutic targets for HCC metastasis Recently,accumulated evidences indicate that stem cells derived from bone marrow contribute to malignant transformation,tumor vascularization and cancer cell migration.VEGFR1-position haemtopoietic bone marrow progenitors can initiate the pre-metastastie niche for incoming tumor cells .These suggest that recognizing the roles of stem cells from marrow in the development and progression of cancers have important values in the diagnosis and treatment of human cancers.Especially,it is worth note that liver disease strongly relate with bone marrow cells.Oval cells/hepatocytes could be derived from circulating bone marrow cells,and damaged hepatocytes can alter the lineage commitment of bone marrow stem cells toward that of hepatocytes.Moreover,the liver stem cell responses and hepatocarcinogenesis are indirectly influenced by bone marrow cell. However,the exact significance of bone marrow-derived cells to liver cancer is far from fully elucidated.
Mesenchymal stem cells(MSC) from adult bone marrow can be induced both in vitro and in vivo to differentiate into a variety of mesenchymal tissues, including bone,cartilage,tendon,fat,bone marrow stroma,and muscle .A rare cells within human bone marrow MSC,which called Multipotent adult progenitor cell(MAPC),could differentiate not only into mesenchymal lineage cells but also endothelium and endoderm.When implant MSC in the utero of sheep,MSC can demonstrate a site specific differentiation.In addition,MSC seemed to have unique immunologic characteristics that allow persistence in a xenogeneic environment.Therefore,MSC present an intriguing model for examining the differentiation of stem cells,and also for cell and gene therapy.
However,a little is known about the precise functional contribution of MSC to the growth and progression of tumor including HCC,and the results from a few reports are controversial.Studeny and Fierro demonstrated that MSC could promote tumor growth,whereas Ohisson found that MSC could inhibit the proliferation of colon cancer cell line H1D1.
Therefore,in this study,we aimed to determine the role of MSC in the growth and metastasis of human HCC,and explore its possible mechanisms involved.
PARTⅠ.Study about the in vivo and in vitro Effect of Mesenchymal Stem Cells on the Proliferation and Metastasis of Liver Cancer Cell Line
Aim:
To explore the possible role of MSC in the progression of HCC.
Methods:
The fifth to eigth passage cells were assessed by immunocytochemistry and RT-PCR to asurres the phenotype of MSC.And then,MSC cells were amplified in vitro and used in the following experiment.The HCC cells(MHCC97-H) were co-cultured with MSC-CM(MSC-conditioned medium),and the effect of MSC on the in vitro proliferation and metastasis of HCC cells were investigated. To investigate the in vivo effect of MSC on HCC growth and metastasis, MHCC97-H and MHCC97-L cells were subcutaneously inoculated in nude mice (named MHCC97H and MHCC97-L model),and MHCC97-H subcutaneous tumor tissues were orthotopically implanted in the liver of nude mice(named Xenograft model).MSC cells were begun to inject at different time of tumor progression. The tumor growth and pulmonary matastasis were analyzed.
Result:
1.MSC can enhance cancer cells growth but inhibit invasion in vitro. Co-culture with MSC-CM could significantly promote the proliferation of MHCC97-H cells,as compared with those cells cultured alone.Detected by CyQUANT proliferation assay,the OD value of MHCC97H cells cultured with 0, 25%,and 50%MSC-CM were 211.65±54.72,236.24±57.15and 283.59±62.16 respectively,P=0.02).MHCC97-H cells treated with MSC-CM have a lower capacity of invasion;the number of invaded cells(11.37±3.54) was much less than that of the controls.(20.00±5.35,t=3.80,P=0.002) by using Transwell assay test.
2.MSC can enhance tumor growth but inhibit metastasis in vivo.MSC could also enhance the in vivo growth of MHCC97-H cells.In the MHCC97-H models, the tumor volume in MSC-treated group was much bigger than that of the controls(3080.51±1234.78 vs 2223.75±1000.60mm~3,P=0.045),and also the average tumor growth rate of the MSC-treated group was much increased than that of the controls(102.65±63.66 vs 72.07±52.13mm~3/day,P=0.013).In Xenograft model,the median ratio of tumor to body weight in the MSC-treated mice was much greater than that in the controls(0.15 vs 0.13,P=0.03). However,in MHCC97-L models,the difference of tumor size between two groups were no significant(1913.67±1336.59 vs 1989.63±466.08,P=0.898). Similarly,in Xenograft models,when injected MSC in later phase,the difference between two groups were no significant(2.63±0.65 vs 3.1±0.47, P=0.23).MSC could significantly decrease the number of metastatic tumor cells in the lungs(in MHCC97-H model:49.75±53.35 vs 227.22±224.00 for MSC-injected mice and controls,respectively,P=0.046);In MHCC97-L models MSC decreased the pulmonary metastatic foci(1.33±1.97 vs 4.50±2.81, P=0.047);In Xenograft model,whether injected in earlier or later phase, MSC could statistically decrease the pulmonary metastasis rate(50%vs 100%, P=0.015;20%vs 100%,P=0.024).
Conclusion:
MSC could enhance the HCC growth,but significantly inhibit the pulmonary metastasis of HCC,These suggest that MSC transfusion might be a new and helpful strategy in the control of metastatic and recurrence of HCC after operation.On the other hand,because of the enhancement effect of MSC on the proliferation of HCC,the safety of MSC injection should also be pay attention to.The detail mechanism of the effect of MSC on HCC metastasis deserves further investigation
PARTⅡ.The Distribution of MSC in Mice Body Bearing HCC
Aim:
To explore the distribution of MSC in mice body after injection,and to provide evidence for further studies about mechanism.
Methods:
1.To detect the in vivo distribution of MSC,MSC labeled with DAPI were injected into the nude mice bearing tumor,four days later,the tumors and organs,such as lung,liver,spleen and heart were removed,freezly sliced and were observed under fluroscense microscopy.MSC labeled with Brdu were also injected into the nude mice,and the tumors were removed and immnostained with anti-Brdu antibody 14 days after injection.
2.To observe the in vitro interaction between HCC cells and MSC,GFP-MHCC97H cells which were stably tansfected with a GFP expression plasmid vector were co-cultured with DAPI-MSCs or MSC cells(transfected with RFP expression plasmid vector) at a ratio of 5:1.The culture was observed in different times.
Result:
1.MSC can well distribute in tumor tissues.4 days after DAPI-MSC injection, more DAPI-MSC cells presented in tumor site than in normal liver.DAPI-MSC cells could also distribute into bone marrow and spleen.
2.MSC can participated in tumor stroma formation.Brdu-MSC mainly existed in tumor stroma.The MSC was found to be merged into the tumor tissues of HCC after intravenous injection into nude mice model bearing human HCC.As compared with the controls,the tumor composed more mesenchymal components than controls
3.Fusion can be found between GFP-MSC and MHCC97-H tumor cells.After 4 days cultured in vitro,fusion can be found between GFP-MSC and MHCC97-H, but this phenomenon was not obviously increased in the following day.
Conclusion:
These results suggest that MSC is one of important derivation of tumor stroma, and MSC may play an important role between tumor cells and tumor stroma cells. And combined with the results from part one,these results suggest MSC can change microenvironment of HCC and then to reverse the metastasis of liver cancer cells.
PARTⅢ.Mechanism of the Effect of MSC on the Proliferation and Metastasis of HCC
Aim:
To investigate the mechanism of the effect of MSC on the proliferation and metastasis of HCC cells.
Methods:
MHCC97-H was cultured with 0%,25%and 50%of MSC-CM for 24hrs,48hrs and 72hrs respectively.Total RNA and total protein of HCC cells were extracted from MHCC97-H cells and from HCC tissues of nude mice models respectively; Real-time PCR,western blot,gelatin zymography and ELISA were performed to identify the express levels of PCNA,Ki67,MMP2,MMP9,VEGF,TGFβ,Smad2 and Smad7,respectively.TGF-βantibody and TGF-βsiRNA were added into MHCC97-H medium,and the proliferative and matastatic capabilities of cells were evalulated.
Rusults
1.The effect of MSC on the proliferative and invasive markers of HCC
1) MSC-CM enhanced the expression of PCNAand Ki67.Compared with culture alone,the PCNA and Ki67 expression of HCC cells co-cultured with 50%MSC-CM were significantly increased for 1.8 and 7 times.
2) MSC down regulated the expression of MMP2.MMP2 levels in MHCC97-Hcells cultured with MSC-CM were lower than in cells cultured alone.And both the mRNA levels and the activity of MMP2 were reduced in MSC-injected mice than that of control(1.13±0.55 vs 1.61±1.52,P=0.05).Moreover,the downregulated MMP2 were correlated with tumor metastasis(0.86±0.30 vs. 1.46±0.84,P=0.032).However,after co-cultured with MSC-CM,MMP9 level didn't change according Western blotting analysis.The expression of MMP9 between MSC injection group and NO-MSC injection group was also no significantly different by immunohistochemistry staining.
2.MSC influence proliferation and invasion of HCC via TGF-β/Smad pathway
1).MSC can effect on the expression of components of TGF-β/Smad pathway. In vitro study,MSC-CM significantly decreases the expression of TGF-β1 in 48hrs by western blot analysis.In MHCC97-H model,compared with no MSC injection samples,the mRNA expression level was 1.81-fold decreased for TGF-β1(1.34±1.01 vs 2.42±2.03,P=0.047),and 1.67-fold for Smad7 (1.532±1.256vs2.561±1.985P=0.036).Moreover,the lower expression level of TGFβcorrelated with larger tumor size(3076.97±1416.52 vs 2272.869±783.36mm~3,P=0.044).No metastasis group had lower expression of TGF-β1 than in metastasis group(1.19±0.8 vs 2.31±1.7,P=0.05).In Xenograft model tumor,TGFβin MSC injection group was 1.95-fold higher than controls(1.30±0.83 vs 0.67±0.52 P=0.037),but TGFβlevels did not correlate with tumor size and tumor metastasis.While compared with controls, Smad2 and Smad7 in MSC injection group were 1.25-fold(1.01±0.14 vs 1.25±0.38,P=0.043) and 1.85-fold(0.76±0.29 vs 1.41±0.50,P=0.006) decreased.Both Smad2 and Smad7 levels were significantly correlated with tumor size and metastasis.
2).The relation between TGFβ/Smad pathway and MMP2.To assess whether there exist relation between TGFβ/Smad pathway and MMP2,All mice model samples were divided into 2 groups according the median of TGFβ,Smad2 and Smad7 expression level,and we found samples with lower TGFβlevel in MHCC97-H model as well as samples with lower Smad2 levels in Xenograft models correlated with lower expression level of MMP2(1.25±1.01 vs 1.82±1.95, P=0.047 and 1.25±1.009 vs 1.82±1.95,P=0.047,respectivly).
3).Enhancement of VEGF and MVD by MSC.MSC could significantly up-regulate the expression of VEGF in MHCC97H models(OD Value in MSC-injection group and no MSC-injection group were 0.34±0.08 vs 0.28±0.07,P=0.04),But the expression of VEGF didn't correlate with tumor size and metastasis.Compared with control,MSC significantly increased MVD of tumor tissues in Xenograft models(28.0±9.2 vs 18.1±3.3,P=0.009) Moreover,when divided all samples into two groups according the median of VEGF and TGFβlevel,no relation between VEGF and MVD were found(23.7±8.7 vs 19.6±6.5,P=0.375),but the relation between TGFβand MVD were found to be existed(25.5±8.9 vs 19.6±8.3,P=0.038).
4).The expression of TGFβ/Smads correlated with hallmark of HCC cells and its microenvironment.TGFβ,Smad2 in MHCC97-H cell line was lower than that of MHCC97-L cell line(0.18±0.15 vs 0.40±0.19,P=0.011;0.99±0.17 vs 2.56±0.66,P=0.017),and TGFβin MHCC97-H model was also lower than that of MHCC97-L models(1.27±1.05 vs 2.72±1.63,P=0.001).
When MHCC97-H or MHCC97-L cells were inoculated subcutaneously in mice bodies, the TGFβlevels were statistically increased(MHCC97-H:0.18±0.15 vs. 1.27±1.05,P=0.002;MHCC97-L:0.40±0.19 vs 2.72±1.63,P＜0.001),but after implanting MHCC97-H subcutaneous tumor in mice liver,the levels were significantly decreased(1.27±1.05 vs 0.32±0.25,P=0.O09).Smad2 in MHCC97-H cell line and MHCC97-H models was lower than in Xenograft model (0.99±0.17 vs 2.13±0.64,P=0.016;1.19±0.76 vs 2.13±0.64,P=0.003),but it was higher in MHCC97-L cell line than in MHCC97-L models(2.56±0.66 vs 1.44±0.55,P=0.023).After inoculated subcutaneously,Smad7 levels were statistically decreased(MHCC97-H model:12.36±1.62 vs 1.18±0.62,P=0.034; MHCC97-L model:46.98±30.39 vs 1.64±0.89,P＜0.001)these mean Organ microenvironment can effect on the expression of TGFβand Smads.In addition, TGFβlevels linearly and negatively correlatedwith tumor weight.(R=0.387, P=0.042),Smad2 levels linearly and positively correlated with tumor weight(R=0.427,P=0.023).Moreover,metastasis samples had a higher Smad2 level than no metastasis samples.(1.71±0.78 vs 1.08±0.61,P=0.054)
5).siRNA TGF can influence the proliferation and metastasis of HCC.After interfered by TGFβsiRNA,the proliferative and metastasis ability of both MSC and MHCC97-H cell were significantly decreased.The effect of MSC-CM on the MHCC97-H can also be significantly blocked.And with the TGFβlevel down regulated,the MMP2 levels were down regulated at same time.
Conclusion:
1.Taken together,our results indicated that MSC have an enhanced ability in promoting tumor growth and inhibiting tumor invasion,which might be partially due to down regulation of TGFβ/Smad pathway.
2.Close correlation between the expressions of TGFβand MMP2 was also found.
3.The effect of TGFβand TGFβ/Smad pathway play a crucial role between HCC and its microenvironment.
目的:确定人骨髓来源间充质干细胞(MSC)对肝癌细胞体内外增殖和侵袭转移能力的影响,从而为进一步研究MSC在肝癌发生发展中的作用提供理论依据。
方法:培养MSC并进行表型鉴定,选取5-8代细胞用于以下研究。(1)体外试验:提取MSC的条件培养基(MSC-CM),在高转移潜能人肝细胞癌细胞系MHCC97-H中加入不同浓度的MSC-CM,采用CyQUANT细胞增殖试验和Transwell细胞侵袭试验,比较MSC-CM对MHCC97-H细胞的增殖和侵袭能力的影响。(2)体内试验:分别皮下注射MHCC97-H和MHCC97-L细胞建立肝癌的裸鼠转移皮下瘤模型,并建立裸鼠MHCC97-H肝内原位移植瘤模型(Xenograft model)。在不同模型中注射MSC细胞并以PBS作为对照,观察动物的成瘤和肿瘤生长情况,动物饲养35天后切去肿瘤组织,测量体重和肿瘤质量;另外切取小鼠肝肺组织,固定包埋后间断50μm病理切片,显微镜下观察肺转移的情况,主要比较各组标本平均肺转移细胞数,肺转移灶的个数以及肺转移率。
结果:(1)体外增殖实验结果显示:MHCC97-H细胞与0,25%、50%的MSC-CM共培养后,其OD值分别为236.24±57.15和283.59±62.16,明显高于对照组(211.65±54.72,P=0.02),提示MSC-CM可明显促进肝癌细胞的增殖。(2)体外侵袭实验结果显示:经与MSC-CM共培养后MHCC97-H的穿膜细胞数目减少(11.37±3.54 vs 20.00±5.35,P=0.0021,提示MSC-CM抑制肝癌细胞的侵袭能力。(3)MSC对肝癌体内生长的影响:在MHCC97-H皮下瘤模型中注射MSC,35天后肿瘤体积显著大于PBS对照组(3080.51±1234.78 vs 2223.75±1000.60 mm~3,P=0.046),平均每天的肿瘤增长量也高于对照组(102.65±63.66 vs 72.07±52.13 mm~3/day,P=0.013),这种生长速度的差别主要位于成瘤及注射后的前20天内,随着时间增加差别逐渐缩小。在MHCC97-L皮下瘤模型中注射MSC45天后,MSC注射组与对照组的肿瘤体积无显著性差别(1913.67±1336.59 vs 1989.63±466.08,P=0.898),平均每天的增长量(62.95±.30.45 vs 63.04±50.57,P=0.99)。在Xenograft模型中MSC注射组较对照组有更高的瘤体比值(0.15±0.51 vs 0.13±0.04,P=0.03)。为进一步检验MSC是否对肿瘤生长的影响具有时效性,我们于Xenograft模型(n=10)建立15天后再注射MSC细胞,结果表明,晚期阶段MSC注射对肿瘤的生长无明显作用。(4)MSC对肝癌转移模型肺转移的影响:肺组织切片统计结果显示,在MHCC97-H皮下瘤模型中,MSC注射组的平均肺转移细胞数目显著低于对照组(49.75±53.35 Vs 227.22±224.00,P=0.046);在Xenograft模型中,同期和后期MSC注射组肺转移率均低于对照组(50%vs100%,P=0.024;20%vs 100%,P=0.015),与MSC的注射时间无关。
结论:注射MSC可促进肝癌细胞的体外增殖和体内生长,同时受肿瘤细胞类型(不同转移潜能的肝癌细胞)和肿瘤生长阶段的影响;但MSC可明显抑制肝癌的体外侵袭和体内肺转移。MSC可能为抗肝癌转移的一种新方法。
第二部分骨髓间充质干细胞在裸鼠人肝癌转移模型体内的分布
目的:观察MSC静脉注射后,在裸鼠体内的归巢和分布情况,为进一步研究MSC的作用机制提供理论指导。
方法:利用目前国内外现有的多种细胞标记方法的优缺点,分别采用转染绿色荧光蛋白GFP基因和红色荧光蛋白RFP基因的方法标记人高转移肝癌细胞MHCC97-H和MSC细胞,并筛选出稳定的转染细胞株,另外采用DAPI,Brdu标记MSC。为观察MSC在荷瘤鼠体内的分布,将DAPI标记的MSC细胞注入MHCC97-H荷瘤鼠体内,4天后,切取肿瘤组织,肝脏,肺脏,脾脏,心脏,快速冰冻切片后荧光显微镜下观察DAPI-MSC在器官组织中的分布,骨髓细胞涂片后观察DAPI-MSC在骨髓中的分布。为观察MSC在肿瘤组织中的分布,采用Brdu标记MSC,并注入Xenograft模型鼠体内,2周后应用Brdu单克隆抗体进行肿瘤组织的免疫组化检测。为观察MSC与肝癌细胞体外增殖的影响,将GFP标记的MHCC97-H细胞与RFP标记的MSC细胞进行体外共培养。
结果:肝癌组织中MSC数明显高于非肿瘤组织中。在骨髓、脾脏、血管内皮组织中也存在相对较多的MSC。MSC在肿瘤组织中主要定位于基质,MSC注射组较非注射组有更多间质构成。MSC较高的迁徙归巢性能与MSC存在较强的基底膜降解能力有关,及肿瘤细胞分泌物的趋化作用有关。MSC能在体外与MHCC97-H细胞共生长,且存在少量细胞融合现象。
结论:MSC具有向肿瘤组织归巢的性能,并参与了肿瘤基质构建。结合第一部分结果说明,MSC通过向肿瘤组织的归巢和向间质成分分化从而可以改变肿瘤微环境并影响肿瘤的生长和转移。MSC与肿瘤细胞体外可以发生融合,也可能是影响肿瘤细胞生物学机制的可能途径。
第三部分骨髓间充质干细胞对肝癌侵袭和侵袭转移影响的机制研究
目的:本部分主要研究骨髓间充质干细胞对肝癌增殖和侵袭转移能力影响的机制。
方法:将肝癌细胞系MHCC97-H与不同比例的MSC-CM共培养后于不同时间点提取肝癌细胞系的总蛋白及总mRNA;提取动物实验中肿瘤组织标本的总蛋白及总RNA,采用Real-time PCR,Western blot,免疫化学,ELISA,明胶酶谱分析的方法,比较MSC处理和未处理肿瘤细胞和组织中PCNA,Ki67,MMP2,MMP9,VEGF,MVD等与增殖侵袭密切相关指标的变化。第一,二部分试验结果MSC对肿瘤生成的促进作用和对肿瘤转移的抑制作用,并可成为肿瘤基质的重要来源,提示MSC在肿瘤与肿瘤微环境中发挥了双重作用,结合TGFβ/Smad信号通路的作用特点,选择其分子作为重要研究对象。检测MSC对肝癌细胞TGFβ/Smad信号通路分子表达的影响。并采用抗体和小干扰RNA阻断TGFβ的方法,进一步观察MSC对肿瘤生长侵袭作用的影响。
结果:
1)MSC对肝癌细胞和组织与增殖侵袭密切相关指标表达的影响
(1)MSC对增殖指标PCNA和Ki67表达的影响:MSC-CM显著的升高了MHCC97-H细胞的PCNA和Ki67表达水平,在MHCC97-H细胞培养液中加入50%的MSC-CM后,PCNA和ki67的mRNA表达水平升高了1.8和7倍。
(2)MHCC97细胞对MSC体外表型变化的影响:MHCC97-H的条件培养基(CM)能显著地升高MSC细胞中Flk1的表达,在MSC细胞培养液中加入MHCC97-H—CM后,Flk1的mRNA表达水平升高了2.64倍,同时降低了OCT-4的表达水平。
(3)MSC对MMP2和MMP9表达的影响:体外MSC-CM可显著的降低MMP2的mRNA和蛋白表达水平,但对MMP9无明显影响。体内实验对于组织标本的明胶酶谱分析显示MSC可显著的降低MMP2的表达水平和活性。在MSC注射组与非注射组MMP2 mRNA水平为(1.13±0.55 vs 1.61±1.52,P=0.05),且MMP2的表达水平与肿瘤的转移相关(非转移组与转移组mRNA水平为:0.86±0.30 vs.1.46±0.84,P=0.032)。
2)MSC对肝癌细胞和组织与增殖侵袭的影响与TGFβ/Smad信号通路有关
(1)MSC对TGFβ信号通路分子TGFβ,Smad2,Smad7表达的影响及对肝癌生长转移的相关性:体外实验:采用western blot检测发现,MSC-CM与肿瘤细胞MHCC97-H共培养后,TGFβ的表达显著的下调。体内实验:在MHCC97-H皮下瘤模型中,MSC注射后TGFβ的表达水平显著的降低(1.34±1.01 vs 2.42±2.03,P=0.047)。且肿瘤的大小和转移与TGFβ的表达水平存在相关性。将TGFβ的表达水平按中位值分为高低两组后,发现低水平组有较大的肿瘤体积(3076.97±1416.52 vs2272.87±783.36mm~3,P=0.044);非转移组较转移组有较低的TGFβ水平(1.19±0.80 vs 2.31±1.70,P=0.05);Smad7的表达水平也显著下调(1.532±1.256vs 2.56±1.99,P=0.036)。在Xenograft模型中,MSC显著的上调了TGFβ的表达水平(1.30±0.83 vs.0.67±0.52,P=0.037),但TGFβ的表达水平与肿瘤的大小无相关性(回归系数R=0.162,P=0.534),与肿瘤转移也无相关性(TGFβ在转移与非转移组织标本:1.09±0.77 vs 0.58±0.53,P=0.24)。MSC还显著的下调Smad2(1.01±0.14 vs 1.25±0.38,P=0.043)和Smad7(0.76±0.29 vs 1.408±0.503,P=0.006)的mRNA水平。而Smad2,Smad7的mRNA水平与肿瘤转移均有相关性(Smad2在非转移组与转移组的表达水平是1.01±0.17 vs 1.25±0.34,P=0.024;Smad7在非转移组与转移组的表达水平是1.02±0.23,2.13±0.85,P=0.021)。Smad7还与肿瘤的大小呈负相关(回归系数R=0.494,P=0.044)。这一结果说明,MSC通过改变TGFβ/Smad信号通路中多个分子的表达水平影响肿瘤的增殖和侵袭能力。
(2)TGFβ与MMP2表达相关分析:实验中还发现TGFβ与MMP2表达之间存在相关性。依据每组模型鼠中TGFβ,Smad2和Smad7表达水平的中位数将其分为高低表达两组,在MHCC97-H模型中,TGFβ低表达组MMP2的表达水平较低(1.25±1.01 vs1.82±1.95 P=0.047);在Xenograft模型中,Smad2低表达组MMP2的mRNA表达水平较低(0.99±0.28 vs 1.32±0.45,P=0.041)。体外实验在MHCC97-H的培养液中同时添加MSC-CM后,随着TGFβ表达水平的下调,MMP2的表达水平也下调,且两指标的波动变化方向一致。
(3)TGFβ抗体可以阻断MSC的促增殖作用:MHCC97-H培养液加入MSC-CM并培养48hrs后细胞增殖能力明显增加(16.45±4.73 vs 22.45±4.84,P=0.045)但加入TGFβ抗体后,MSC-CM的促细胞增殖能力不明显(16.45±4.73 vs 18.02±3.68,P=0.586)。
(4)MSC促进肿瘤血管生成并与TGFβ表达有关:VEGF及MVD是衡量肿瘤血管生成的重要指标,通过对组织标本裂解液VEGF的ELISA检测,显示MSC显著的升高了MHCC97-H模型鼠肿瘤组织中的VEGF浓度(OD值:0.34±0.08 vs 0.28±0.07,P=0.04),但VEGF和肿瘤的大小以及转移无相关性。体外实验采用VEGF受体(Flk1)抗体阻断VEGF的作用,对肿瘤增殖性的影响也无显著性。在Xenograft模型中,通过对微血管密度(MVD)的检测,发现MSC显著的增加了肿瘤MVD值(28±9.20 vs18.11±3.30,P=0.009),且MVD与肿瘤组织中VEGF的浓度无相关性,但与肿瘤TGFβ的表达水平有相关性(25.5±8.9 VS 19.6±8.3,P=0.038),说明TGFβ参与了肿瘤的血管生成。
(5)TGFβ的表达水平与肝癌细胞自身特性和肿瘤的微环境均相关:因为MSC在肝癌皮下瘤模型(MHCC97-H)和肝内移植瘤模型(Xenograft)中对TGFβ的表达显示了不同的调节方向,为探讨影响TGFβ/Smad信号通路分子表达的因素,选取前期不同肝癌模型标本(均未注射MSC)进行检测。TGFβ的表达水平与肝癌细胞自身特性有关:TGFβ和Smad2在MHCC97H和MHCC97L细胞系中存在显著差别(0.18±0.15vs 0.40±0.19,P=0.011;0.99±0.17 vs 2.56±0.66,P=0.017),TGFβ在MHCC97-H模型中表达水平也低于MHCC97-L模型(1.27±1.05 vs 2.72±1.63,P=0.001)。TGFβ,Smad2,Smad7的表达还与肿瘤的器官微环境密切相关:当将MHCC97-H和MHCC97-L细胞种植于裸鼠皮下时,TGFβ的表达水平均显著的增加(MHCC97-H:0.18±0.15 vs1.27±1.05,P=0.002;MHCC97-L:0.40±0.19 vs 2.72±1.63,P＜0.001),但是当把MHCC97-H的皮下瘤移植于肝脏组织后,TGFβ表达水平降低并接近细胞系水平(1.27±1.05 vs 0.32±0.25;P=0.009)。Smad2在MHCC97-H细胞系和MHCC97-H模型中的表达水平也低于在肝移植瘤模型中的表达水平(0.99±0.17 vs 2.13±0.64P=0.016;1.19±0.76 vs 2.13±0.64 P=0.003),但在MHCC97-L细胞系的表达水平高于MHCC97-L模型(2.56±0.66 vs 1.44±0.55,P=0.023)。Smad7的表达水平在细胞注入皮下后也明显的降低(MHCC97-H:12.36±1.62 vs 1.18±0.62,P=0.034;MHCC97-L:46.98±30.39 vs 1.64±0.89,P＜0.001)。TGFβ的表达水平还与肿瘤质量呈线性负相关的关系,(相关系数R=0.387,P=0.042),而Smad2的表达水平与肿瘤质量呈线性正相关(R=0.427,P=0.023),Smad2的表达水平还与肿瘤的转移密切相关,(转移组和非转移组Smad2的表达水平:1.71±0.78 vs 1.08±0.61,P=0.054)
(6)TGFβsiRNA对肝癌增殖侵袭性的影响:为进一步验证TGFβ在MSC对肿瘤增殖转移影响中的作用,采用TGFβsiRNA分别干扰MHCC97-H细胞和MSC细胞的TGFβ的表达。发现干扰后,TGFβ和MMP2的表达水平均明显下调,且肿瘤的增殖性和侵袭性均降低,干扰MSC细胞后,其MSC-CM对肿瘤的增殖侵袭性的影响以及对MMP2表达的影响均消失。
结论:TGFβ在肝癌的增殖侵袭转移中发挥了重要的作用,同时在肿瘤于微环境的相互作用中也发挥重要作用,骨髓间充质干细胞主要通过改变TGFβ中多个信号分子的表达来影响该信号通路,并最终影响肝癌的侵袭转移。同时TGFβ表达与MMP2的表达密切相关,TGFβ还参与了肿瘤的血管生成。
为了探讨骨髓间充质干细胞对肝癌增殖侵袭的影响,在本课题第一部分中通过体外细胞共培养,以及在裸鼠人肝癌模型中进行MSC注射的方法,发现了MSC在肝癌生长中的促进作用和对肝癌转移的抑制作用。为了探讨MSC的作用机制,第二部分采用多种方法对MSC细胞进行了标记,发现静脉注射MSC后在肿瘤组织中会形成相对较高的分布,归巢的MSC在肿瘤中主要参与了肿瘤基质的构建,说明骨髓间充质干细胞主要通过改变肿瘤的基质微环境而发挥其对肿瘤生长转移的影响。本课题第三部分探讨了MSC作用的分子机制,首先检测了与肝癌增殖转移密切相关的分子标记物PCNA,Ki67,MMP2,MMP9,及VEGF的变化,发现MSC升高了肝癌细胞PCNA,Ki67的表达水平,抑制了MMP2的表达和酶活性,并且MMP2的表达与肿瘤的转移密切相关。另外,结合MSC对肿瘤的双向性作用特点,重点选择了TGFβ/Smad信号通路中的多个分子进行检测,发现TGFβ/Smad信号通路中多个分子的变化与MSC作用的发挥密切相关,尤其是TGFβ和Smad7的下调不仅与MSC的促肿瘤生长有关而且也同时与MSC的抑制肿瘤转移作用相关,采用抗体及小干扰RNA阻断的方法也验证了TGFβ在MSC对肝癌生长转移的影响中发挥了重要作用,TGFβ/Smad信号通路还明显的影响MMP2的表达水平,并与MSC的促进肿瘤血管生成作用有关,通过这些结果,我们推断MSC对肝癌细胞增殖侵袭性的影响主要是通过TGFβ/Smad信号通路。由于在不同动物模型中MSC对TGFβ的表达的调控呈现不一致现象,为此比较了不同动物模型肿瘤中TGFβ表达水平的差别,我们发现TGFβ的表达不仅与肿瘤细胞本身特性有关,而且与肿瘤的器官微环境相关,这一结果强调应在器官微环境背景下来进行TGFβ/Smad信号通路的研究。
本研究的应用价值
1.确立了骨髓间充质干细胞在肝癌生长中的促进作用,以及对肝癌转移的抑制作用,这一实验结果为进一步的临床研究提供了依据。
2.归巢的骨髓间充质干细胞在肿瘤中主要参与了肿瘤基质的构建,说明骨髓间充质干细胞主要通过改变肿瘤的基质微环境而发挥其对肿瘤生长转移的影响,这一现象的发现为通过调控肿瘤微环境来治疗肿瘤转移提供了一种新的手段。
3.TGFβ/Smad信号通路中多个分子的变化与骨髓间充质干细胞的作用的发挥密切相关,为进一步研究骨髓间充质干细胞对肿瘤影响的双向作用机制提供了新思路
创新点
1.首次发现了MSC对肝癌生长的促进作用和对肝癌转移的抑制作用。
2发现MSC注射后主要参与肿瘤基质构建并改变肿瘤基质微环境。并发现了MSC体外可与肝癌细胞发生细胞融合现象。
3.首次发现了MSC对肝癌生长转移的作用机制主要与TGFβ/Smad信号通路的调控密切相关。
尚待解决的问题
1.如何有效的抑制MSC的促进生长作用将是首要的问题,实验中发现MSC的促进肿瘤增殖的作用存在时效性特点,进一步完善机制将是有益的。
2.骨髓间充质干细胞对肿瘤增殖侵袭性影响作用的发挥与多种因素有关,包括细胞因子的作用,细胞间的相互作用,以及肝脏干细胞反应等,全面深入的探讨这些因素有助于进一步完善机制。
Effect of Human Mesenchymal Stem Cell on the Proliferation and Metastasis of Hepatocellular Carcinoma
Hepatocellular carcinoma(HCC) is one of the most common cancers in the world,with important mortality in sub-Saharan Africa and eastern Asia. The extremely poor prognosis of HCC is largely due to the high rate of tumor recurrence after surgery or intra-hepatic metastases that develop through invasion of the portal vein or spread to other parts of the liver. Therefore,exploring the mechanisms involved in invasion and metastasis of HCC is the key issue for the further prolonging the survival of patients with HCC,which could provide new therapeutic targets for HCC metastasis Recently,accumulated evidences indicate that stem cells derived from bone marrow contribute to malignant transformation,tumor vascularization and cancer cell migration.VEGFR1-position haemtopoietic bone marrow progenitors can initiate the pre-metastastie niche for incoming tumor cells .These suggest that recognizing the roles of stem cells from marrow in the development and progression of cancers have important values in the diagnosis and treatment of human cancers.Especially,it is worth note that liver disease strongly relate with bone marrow cells.Oval cells/hepatocytes could be derived from circulating bone marrow cells,and damaged hepatocytes can alter the lineage commitment of bone marrow stem cells toward that of hepatocytes.Moreover,the liver stem cell responses and hepatocarcinogenesis are indirectly influenced by bone marrow cell. However,the exact significance of bone marrow-derived cells to liver cancer is far from fully elucidated.
Mesenchymal stem cells(MSC) from adult bone marrow can be induced both in vitro and in vivo to differentiate into a variety of mesenchymal tissues, including bone,cartilage,tendon,fat,bone marrow stroma,and muscle .A rare cells within human bone marrow MSC,which called Multipotent adult progenitor cell(MAPC),could differentiate not only into mesenchymal lineage cells but also endothelium and endoderm.When implant MSC in the utero of sheep,MSC can demonstrate a site specific differentiation.In addition,MSC seemed to have unique immunologic characteristics that allow persistence in a xenogeneic environment.Therefore,MSC present an intriguing model for examining the differentiation of stem cells,and also for cell and gene therapy.
However,a little is known about the precise functional contribution of MSC to the growth and progression of tumor including HCC,and the results from a few reports are controversial.Studeny and Fierro demonstrated that MSC could promote tumor growth,whereas Ohisson found that MSC could inhibit the proliferation of colon cancer cell line H1D1.
Therefore,in this study,we aimed to determine the role of MSC in the growth and metastasis of human HCC,and explore its possible mechanisms involved.
PARTⅠ.Study about the in vivo and in vitro Effect of Mesenchymal Stem Cells on the Proliferation and Metastasis of Liver Cancer Cell Line
Aim:
To explore the possible role of MSC in the progression of HCC.
Methods:
The fifth to eigth passage cells were assessed by immunocytochemistry and RT-PCR to asurres the phenotype of MSC.And then,MSC cells were amplified in vitro and used in the following experiment.The HCC cells(MHCC97-H) were co-cultured with MSC-CM(MSC-conditioned medium),and the effect of MSC on the in vitro proliferation and metastasis of HCC cells were investigated. To investigate the in vivo effect of MSC on HCC growth and metastasis, MHCC97-H and MHCC97-L cells were subcutaneously inoculated in nude mice (named MHCC97H and MHCC97-L model),and MHCC97-H subcutaneous tumor tissues were orthotopically implanted in the liver of nude mice(named Xenograft model).MSC cells were begun to inject at different time of tumor progression. The tumor growth and pulmonary matastasis were analyzed.
Result:
1.MSC can enhance cancer cells growth but inhibit invasion in vitro. Co-culture with MSC-CM could significantly promote the proliferation of MHCC97-H cells,as compared with those cells cultured alone.Detected by CyQUANT proliferation assay,the OD value of MHCC97H cells cultured with 0, 25%,and 50%MSC-CM were 211.65±54.72,236.24±57.15and 283.59±62.16 respectively,P=0.02).MHCC97-H cells treated with MSC-CM have a lower capacity of invasion;the number of invaded cells(11.37±3.54) was much less than that of the controls.(20.00±5.35,t=3.80,P=0.002) by using Transwell assay test.
2.MSC can enhance tumor growth but inhibit metastasis in vivo.MSC could also enhance the in vivo growth of MHCC97-H cells.In the MHCC97-H models, the tumor volume in MSC-treated group was much bigger than that of the controls(3080.51±1234.78 vs 2223.75±1000.60mm~3,P=0.045),and also the average tumor growth rate of the MSC-treated group was much increased than that of the controls(102.65±63.66 vs 72.07±52.13mm~3/day,P=0.013).In Xenograft model,the median ratio of tumor to body weight in the MSC-treated mice was much greater than that in the controls(0.15 vs 0.13,P=0.03). However,in MHCC97-L models,the difference of tumor size between two groups were no significant(1913.67±1336.59 vs 1989.63±466.08,P=0.898). Similarly,in Xenograft models,when injected MSC in later phase,the difference between two groups were no significant(2.63±0.65 vs 3.1±0.47, P=0.23).MSC could significantly decrease the number of metastatic tumor cells in the lungs(in MHCC97-H model:49.75±53.35 vs 227.22±224.00 for MSC-injected mice and controls,respectively,P=0.046);In MHCC97-L models MSC decreased the pulmonary metastatic foci(1.33±1.97 vs 4.50±2.81, P=0.047);In Xenograft model,whether injected in earlier or later phase, MSC could statistically decrease the pulmonary metastasis rate(50%vs 100%, P=0.015;20%vs 100%,P=0.024).
Conclusion:
MSC could enhance the HCC growth,but significantly inhibit the pulmonary metastasis of HCC,These suggest that MSC transfusion might be a new and helpful strategy in the control of metastatic and recurrence of HCC after operation.On the other hand,because of the enhancement effect of MSC on the proliferation of HCC,the safety of MSC injection should also be pay attention to.The detail mechanism of the effect of MSC on HCC metastasis deserves further investigation
PARTⅡ.The Distribution of MSC in Mice Body Bearing HCC
Aim:
To explore the distribution of MSC in mice body after injection,and to provide evidence for further studies about mechanism.
Methods:
1.To detect the in vivo distribution of MSC,MSC labeled with DAPI were injected into the nude mice bearing tumor,four days later,the tumors and organs,such as lung,liver,spleen and heart were removed,freezly sliced and were observed under fluroscense microscopy.MSC labeled with Brdu were also injected into the nude mice,and the tumors were removed and immnostained with anti-Brdu antibody 14 days after injection.
2.To observe the in vitro interaction between HCC cells and MSC,GFP-MHCC97H cells which were stably tansfected with a GFP expression plasmid vector were co-cultured with DAPI-MSCs or MSC cells(transfected with RFP expression plasmid vector) at a ratio of 5:1.The culture was observed in different times.
Result:
1.MSC can well distribute in tumor tissues.4 days after DAPI-MSC injection, more DAPI-MSC cells presented in tumor site than in normal liver.DAPI-MSC cells could also distribute into bone marrow and spleen.
2.MSC can participated in tumor stroma formation.Brdu-MSC mainly existed in tumor stroma.The MSC was found to be merged into the tumor tissues of HCC after intravenous injection into nude mice model bearing human HCC.As compared with the controls,the tumor composed more mesenchymal components than controls
3.Fusion can be found between GFP-MSC and MHCC97-H tumor cells.After 4 days cultured in vitro,fusion can be found between GFP-MSC and MHCC97-H, but this phenomenon was not obviously increased in the following day.
Conclusion:
These results suggest that MSC is one of important derivation of tumor stroma, and MSC may play an important role between tumor cells and tumor stroma cells. And combined with the results from part one,these results suggest MSC can change microenvironment of HCC and then to reverse the metastasis of liver cancer cells.
PARTⅢ.Mechanism of the Effect of MSC on the Proliferation and Metastasis of HCC
Aim:
To investigate the mechanism of the effect of MSC on the proliferation and metastasis of HCC cells.
Methods:
MHCC97-H was cultured with 0%,25%and 50%of MSC-CM for 24hrs,48hrs and 72hrs respectively.Total RNA and total protein of HCC cells were extracted from MHCC97-H cells and from HCC tissues of nude mice models respectively; Real-time PCR,western blot,gelatin zymography and ELISA were performed to identify the express levels of PCNA,Ki67,MMP2,MMP9,VEGF,TGFβ,Smad2 and Smad7,respectively.TGF-βantibody and TGF-βsiRNA were added into MHCC97-H medium,and the proliferative and matastatic capabilities of cells were evalulated.
Rusults
1.The effect of MSC on the proliferative and invasive markers of HCC
1) MSC-CM enhanced the expression of PCNAand Ki67.Compared with culture alone,the PCNA and Ki67 expression of HCC cells co-cultured with 50%MSC-CM were significantly increased for 1.8 and 7 times.
2) MSC down regulated the expression of MMP2.MMP2 levels in MHCC97-Hcells cultured with MSC-CM were lower than in cells cultured alone.And both the mRNA levels and the activity of MMP2 were reduced in MSC-injected mice than that of control(1.13±0.55 vs 1.61±1.52,P=0.05).Moreover,the downregulated MMP2 were correlated with tumor metastasis(0.86±0.30 vs. 1.46±0.84,P=0.032).However,after co-cultured with MSC-CM,MMP9 level didn't change according Western blotting analysis.The expression of MMP9 between MSC injection group and NO-MSC injection group was also no significantly different by immunohistochemistry staining.
2.MSC influence proliferation and invasion of HCC via TGF-β/Smad pathway
1).MSC can effect on the expression of components of TGF-β/Smad pathway. In vitro study,MSC-CM significantly decreases the expression of TGF-β1 in 48hrs by western blot analysis.In MHCC97-H model,compared with no MSC injection samples,the mRNA expression level was 1.81-fold decreased for TGF-β1(1.34±1.01 vs 2.42±2.03,P=0.047),and 1.67-fold for Smad7 (1.532±1.256vs2.561±1.985P=0.036).Moreover,the lower expression level of TGFβcorrelated with larger tumor size(3076.97±1416.52 vs 2272.869±783.36mm~3,P=0.044).No metastasis group had lower expression of TGF-β1 than in metastasis group(1.19±0.8 vs 2.31±1.7,P=0.05).In Xenograft model tumor,TGFβin MSC injection group was 1.95-fold higher than controls(1.30±0.83 vs 0.67±0.52 P=0.037),but TGFβlevels did not correlate with tumor size and tumor metastasis.While compared with controls, Smad2 and Smad7 in MSC injection group were 1.25-fold(1.01±0.14 vs 1.25±0.38,P=0.043) and 1.85-fold(0.76±0.29 vs 1.41±0.50,P=0.006) decreased.Both Smad2 and Smad7 levels were significantly correlated with tumor size and metastasis.
2).The relation between TGFβ/Smad pathway and MMP2.To assess whether there exist relation between TGFβ/Smad pathway and MMP2,All mice model samples were divided into 2 groups according the median of TGFβ,Smad2 and Smad7 expression level,and we found samples with lower TGFβlevel in MHCC97-H model as well as samples with lower Smad2 levels in Xenograft models correlated with lower expression level of MMP2(1.25±1.01 vs 1.82±1.95, P=0.047 and 1.25±1.009 vs 1.82±1.95,P=0.047,respectivly).
3).Enhancement of VEGF and MVD by MSC.MSC could significantly up-regulate the expression of VEGF in MHCC97H models(OD Value in MSC-injection group and no MSC-injection group were 0.34±0.08 vs 0.28±0.07,P=0.04),But the expression of VEGF didn't correlate with tumor size and metastasis.Compared with control,MSC significantly increased MVD of tumor tissues in Xenograft models(28.0±9.2 vs 18.1±3.3,P=0.009) Moreover,when divided all samples into two groups according the median of VEGF and TGFβlevel,no relation between VEGF and MVD were found(23.7±8.7 vs 19.6±6.5,P=0.375),but the relation between TGFβand MVD were found to be existed(25.5±8.9 vs 19.6±8.3,P=0.038).
4).The expression of TGFβ/Smads correlated with hallmark of HCC cells and its microenvironment.TGFβ,Smad2 in MHCC97-H cell line was lower than that of MHCC97-L cell line(0.18±0.15 vs 0.40±0.19,P=0.011;0.99±0.17 vs 2.56±0.66,P=0.017),and TGFβin MHCC97-H model was also lower than that of MHCC97-L models(1.27±1.05 vs 2.72±1.63,P=0.001).
When MHCC97-H or MHCC97-L cells were inoculated subcutaneously in mice bodies, the TGFβlevels were statistically increased(MHCC97-H:0.18±0.15 vs. 1.27±1.05,P=0.002;MHCC97-L:0.40±0.19 vs 2.72±1.63,P＜0.001),but after implanting MHCC97-H subcutaneous tumor in mice liver,the levels were significantly decreased(1.27±1.05 vs 0.32±0.25,P=0.O09).Smad2 in MHCC97-H cell line and MHCC97-H models was lower than in Xenograft model (0.99±0.17 vs 2.13±0.64,P=0.016;1.19±0.76 vs 2.13±0.64,P=0.003),but it was higher in MHCC97-L cell line than in MHCC97-L models(2.56±0.66 vs 1.44±0.55,P=0.023).After inoculated subcutaneously,Smad7 levels were statistically decreased(MHCC97-H model:12.36±1.62 vs 1.18±0.62,P=0.034; MHCC97-L model:46.98±30.39 vs 1.64±0.89,P＜0.001)these mean Organ microenvironment can effect on the expression of TGFβand Smads.In addition, TGFβlevels linearly and negatively correlatedwith tumor weight.(R=0.387, P=0.042),Smad2 levels linearly and positively correlated with tumor weight(R=0.427,P=0.023).Moreover,metastasis samples had a higher Smad2 level than no metastasis samples.(1.71±0.78 vs 1.08±0.61,P=0.054)
5).siRNA TGF can influence the proliferation and metastasis of HCC.After interfered by TGFβsiRNA,the proliferative and metastasis ability of both MSC and MHCC97-H cell were significantly decreased.The effect of MSC-CM on the MHCC97-H can also be significantly blocked.And with the TGFβlevel down regulated,the MMP2 levels were down regulated at same time.
Conclusion:
1.Taken together,our results indicated that MSC have an enhanced ability in promoting tumor growth and inhibiting tumor invasion,which might be partially due to down regulation of TGFβ/Smad pathway.
2.Close correlation between the expressions of TGFβand MMP2 was also found.
3.The effect of TGFβand TGFβ/Smad pathway play a crucial role between HCC and its microenvironment.
引文
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