【作者】 王璐；

【导师】 徐强；

【作者基本信息】 南京大学 ， 药理学， 2011， 博士

【摘要】 本文第一章发现中药姜黄中的有效单体姜黄素可以选择性的抑制脂肪肉瘤细胞的生存率而并不杀伤正常脂肪来源细胞,这种选择性对于临床治疗脂肪肉瘤具有重要的意义。姜黄素诱导了脂肪肉瘤细胞发生了Caspase 8/3通路依赖性的凋亡,而并不影响线粒体凋亡通路。进一步证实姜黄素系通过直接结合并抑制脂肪肉瘤特异性高表达的肌浆网钙蛋白(SERCA2b)的活性,破坏了细胞内钙离子的稳态,从而引发了脂肪肉瘤细胞内质网应激。脂肪肉瘤细胞发生内质网应激的过程中,上调的CHOP蛋白作为转录因子转录出TRAIL-R2(DR5),继而活化其下游的Caspase 8,引发上述的凋亡。SERCA家族是一种跨膜10次的阳离子转运ATP酶,其结构为四聚体,氨基端和羧基端均位于胞浆内,似球型结构,能主动把胞浆钙泵入肌浆网内。SERCA家族不同的亚型在不同组织中均有表达。在本研究中,我们首次发现相比较人脂肪来源细胞,脂肪肉瘤细胞高表达SERCA2b,66.7%的人脂肪肉瘤临床样本中检测到SERCA2b的阳性表达,且与脂肪肉瘤的恶性程度呈正相关。脂肪肉瘤是来源于恶化的间叶组织细胞,其恶性程度与分化程度密切相关,因此在本文的第二章,进一步探讨了SERCA2b在脂肪肉瘤细胞分化方面的功能。发现脂肪肉瘤细胞系和原代脂肪肉瘤细胞都可以在体内发生分化,尽管无法分化出成熟的脂滴,但很多分化marker的表达量如正常脂肪细胞一样可以出现上调的现象。相比较稳定转染空载体的脂肪肉瘤细胞,稳定表达的SERCA2b可以导致细胞内ROS积累,从而激活MEK/ERK通路,促进ERK的磷酸化,过度磷酸化的ERK在分化过程中磷酸化了脂肪细胞分化关键蛋白PPAR-y,从而抑制了PPAR-y的转录功能,导致下游的脂肪分化相关基因无法正常转录,最终导致了细胞对于分化的抵抗。我们发现SERCA2b对于ERK磷酸化的影响并不通过其酶活来实现,与之前报道相一致,SERCA2b的酶活抑制剂同样可以促进ERK磷酸化,因此SERCA2b本身的表达对于ERK磷酸化的上调应该是其酶活非依赖性的上调,也为揭示肌浆网钙蛋白的新功能做了一个铺垫。由于脂肪肉瘤却有着极高的复发率,某些亚型五年复发率达到100%,反复的手术切除给病人带来极大的痛苦。我们猜测脂肪肉瘤中很可能存在着一小群干细胞样细胞亚群,有很少的数量,就可导致疾病的复发。在第三章中,首先建立了以脂肪肉瘤复发为背景的体内模型,从低度恶性的脂肪肉瘤细胞系中,筛选出一株高恶性程度亚系,经过各种表面marker的筛选和鉴定,发现整合素α6在该亚细胞系上表达量显著上升。以整合素α6为标志筛选出高表达亚群具有极高的体内增殖能力,体外克隆能力和自我更新能力,复发性脂肪肉瘤样本中也可检测到整合素α6阳性细胞亚群,提示整合素α6可以作为人脂肪肉瘤的干细胞样细胞的鉴别marker之一。而作为脂肪来源的恶性肿瘤,我们又检测了脂肪干细胞的鉴定marker在该高恶性细胞亚系上的表达,发现氨基肽酶N(CD13)和整合素α6存在着共同高表达,双阳性亚群的bc12表达量显著提高。而CD13的抑制剂可以选择性的抑制整合素α6高表达的细胞亚群,导致细胞线粒体通路的凋亡,提示CD13协助整合素α6高表达细胞亚群抵抗了凋亡。以上结果首次发现能治疗人脂肪肉瘤的小分子化合物姜黄素及其作用的靶分子SERCA2b,在此基础上研究了SERCA2b在抵抗脂肪肉瘤分化的作用,进一步发现并鉴定出脂肪肉瘤中存在的干细胞样细胞亚群及其特征。这些发现对于阐明脂肪肉瘤分化及复发的机理、研制抗脂肪肉瘤治疗药物具有指导意义。更多还原

【Abstract】 In Chapter One, we discovered that curcumin could selectively inhibit the survival rate of human liposarcoma cells but not normal adipose derived cells, which was quite different from the classical chemotherapeutic drugs used in clinical treatment. Curcumin induced Capsase8/3 dependent apoptosis in human liposarcoma SW872 cells but not Caspase9. Curcumin binds directly with and inhibits the activity of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase2(SERCA2), which is highly expressed in human liposarcoma. The disfunction of SERCA2b made by curcumin disrupted the balance of Ca2+, leading to ER stress in liposarcoma cells. CHOP as a transcription factor of TRAIL-R2(DR5) increases during cells undergo ER stress, the enrichment of DR5 on the surface of cells will induce Caspase 8 dependent apoptosis. Different SERCA family members expression in different tissues, in our studies, we discovered for the first time that 66.7% of 24 human liposarcoma samples expression high level of SERCA2, which is also correlative with the malignant degree of these samples. For liposarcoma roots in transformed normal stromal tissue, and the malignant degree of liposarcoma has close relationship with its differentiation degree, In Chapter Two, we tried to identify the possible function of SERC2b In the differentiation of liposarcoma cells. As we know, liposarcoma cell line could also differentiate in vivo as normal adipose cells. Although it is hard to induce liposarcoma cells to differentiate into mature adipose cells, some differentiation makers will also enhance during differentiation as normal adipose cells. Stably transfected SERCA2b could induce the accumulation of ROS in SW872 cells, activating MEK/ERK pathway. Activated ERK then phosphorylated PPAR-y, which prevented PPAR-y entering into nulei to transcript downstream genes, leading to the resistance of liposarcoma cells differentiation. Interestingly, we found that SERCA2b induced ERK phosphorylation in a kinase activity independent way. Consistent with previous studies, the inhibitor of SERCA2b could also induce ERK phosphorylation. All these results indicated that SERCA2b induced ROS dependent activation of ERK in a Ca2+ pump independent way. For the low metastasis ability of liposarcoma, the main clinical therapy for curing liposarcoma is surgery. However, since liposarcoma has a very high relapse rate, continual surgery will bring great pain to patients. Moreover, liposarcoma is non-sensitive to chemotherapeutic drugs treatment or radiotherapy. All these results indicated the possibility of the existence of a small group of stem cell-like cells in human liposarcoma, which be the initiator of relapse. In Chapter Three, we firstly established a high malignant sub cell line (SW872-S) from human liposarcoma cell line SW872 by re-transplantation in nude mice. We identified that integrin α6 was high expressed in SW872-S cells. Integrin a6high population of cells revealed extremely high growth ability in vitro and in vivo, as well as the ability of colony formation and self-renew. We could also find integrin a6 positive cells in liposarcoma samples from patients with relapse, but not in well-differentiated liposarcoma samples or normal adipose tissues. Our result indicated that integrin a6 could be used as important marker for identifying stem cell-like cells in human liposarcoma. For liposarcoma developed from adipose tissue, we then examined the reported stem cell markers expressing on the surface of adipose stem cells. We detected that CD 13 co-overexpression with integrin cc6, the double positive cell group exhibited highest level of BCL2, which might mediate the anti-apoptosis ability of integrin a6high cells. Consistently, CD 13 siRNA as well as its specific inhibitor selectively inhibited the survival rate of integrin α6high cells, which may contribute the therapeutic therapy targeting integrin α6high sub-population cells.It is the first time to discover the effect of curcumin on treating human liposarcoma, as well as SERCA2b, the direct interatction protein of curcumin in human liposarcoma cells. Further study proved the role played by SERCA2b in the dedifferentiation effect of liposarcoma cells. Moreover, we identified and characterized a small population of stem cell-like cells in human liposarcoma. These findings illustrate the mechanism of recurrence and dedifferentiation of human liposarcoma, and also guide significance for development of anti-liposarcoma drugs.更多还原