【作者】 后晓南；

【导师】 王智彪； 伍烽；

【作者基本信息】 重庆医科大学 ， 妇产科学， 2002， 博士

【摘要】 从发生学角度来看，肿瘤与早期胚胎在细胞的增殖、生长和分化等方面都有许多共同特点，其中早期胚胎的着床植入在形成对母体的侵袭后，囊胚迅速增殖，创建独立的血供，从母体获得营养、壮大自身的体积，就象一个恶性的肿瘤细胞在肆虐机体，唯一不同的是事件发展的结果:一个是可控的生理阶段，另一个则是不可控的病理过程。因此两者侵袭机体生物学行为上的可参照性，使得肿瘤侵袭与囊胚植入的相似性研究可以作为研究生命起源、胚胎着床与肿瘤侵袭与转移的突破口。近年来胚胎与肿瘤细胞的比较性研究已大大的促进了两门学科的发展，但是单纯对立两种生命现象进行两类不同细胞行为的比较性研究还不能解释实质性问题，只有将两种细胞形式--两种生命形式放在同一环境中，统一对立地研究相互影响相互作用后所表现的整体生物学行为的变化，从中找到可能的内在规律，才能指导科研实践。在进行胚胎干细胞的获取与培养的实验研究中，都发现这样一种现象，即将脱带后的<WP=6>小鼠囊胚放在饲养层细胞上培养时（如原代小鼠、人的子宫上皮细胞，原代小鼠胚胎成纤维细胞及STO细胞），在培养的第三天，小鼠囊胚滋养层巨细胞充分扩展并可以推开饲养层细胞直接在培养物表面生长形成一胚盘样结构。就是这种能够推开底层细胞并生长的行为，使人容易想到肿瘤细胞在侵犯周围组织时所表现出来的进攻性，那么作为都具有侵袭特征的两种类型的细胞，在共处于同一培养环境时，对环境的反应性及相互反应将会发生什么样的变化呢？据此，本课题在建立体外共培养体系的基础上，对胚胎与肿瘤细胞相互作用的生物学行为进行了观察与研究。拟从共培养对象改变的角度来建立新的思维方式，并试图最终从宏观整体来解读生命的多样性。本实验建立在囊胚体外着床模型的基础上，同样应用的是共培养体系，但在本共培养体系中我们应用生长旺盛的未经过有丝分裂抑制剂处理的肿瘤细胞作为囊胚的共培养细胞，我们没有对肿瘤细胞进行任何处理，任其在优越的生长环境中生长，因此也使我们能够从肿瘤细胞生长的角度观察囊胚对肿瘤细胞的侵袭及其自身的生长变化情况。本实验的目的不只是在于建立共培养体系，而更重要的在于我们试图通过这种共培养实验可以认识到小鼠囊胚生物学行为的高级性，并通过认识这种特殊的高级性，我们想要达到一个全新审视定义生命所蕴藏的能力的最终目的。<WP=7>目的:1. 建立小鼠囊胚与肿瘤细胞共培养体系，证明小鼠囊胚在肿瘤细胞上贴附与扩展的可能性。2.了解囊胚着床部位肿瘤细胞的生物学行为, 从全新的视角观察比较小鼠囊胚与肿瘤细胞相互作用时生物学行为的变化，解释胚胎与肿瘤细胞之间的联系, 并探讨发生变化的可能机制。意义: 1. 可以提供一个体外正常组织侵袭肿瘤细胞的极简明的模型，表明囊胚生物学本质的高级性。2. 研究的结果一方面可以补充细胞间相互作用的理论，另一方面可能补充胚胎着床和肿瘤发生的机制，从而进一步促进两门学科的交叉、渗透与发展，对生物学基础理论的研究具有重要意义及其应用前景。内容:1. 小鼠囊胚体外侵袭肿瘤细胞群的行为观察。2. 小鼠囊胚在正常细胞群上的生长行为。3. 共培养体系中，肿瘤细胞群与正常细胞群对小鼠囊胚黏附扩展能力影响的比较性研究。4. 研究在共培养体系中局部肿瘤细胞对小鼠囊胚侵袭行为的反应。5. 探讨人纤维蛋白凝胶作为小鼠囊胚体外三维培养的支持物，建立简明的小鼠囊胚体外侵袭三维模型的可能性。<WP=8>结果:一 、小鼠囊胚侵袭肿瘤细胞的生长行为将妊娠第四天的小鼠囊胚与不同来源的肿瘤细胞及正常细胞株进行体外二维共培养，（共培养的细胞包括人肺癌细胞株A549、人低分化卵巢黏液性癌细胞株3AO、人低分化卵巢浆液性囊腺癌细胞株HO-8910、人卵巢癌细胞株SKOV3、人宫颈癌细胞株Hela、人肾癌细胞株GRC-1、人膀胱移行细胞癌细胞株T24、人成骨肉瘤细胞株MG-63、人脐静脉内皮细胞株ECV-304、小鼠黑色素细胞株B16、小鼠成纤维细胞株PA317、新生鼠肺成纤维细胞原代MF1、仓鼠卵巢上皮细胞株CHO、兔成骨细胞原代培养的冻存细胞RF10代），结果发现小鼠囊胚在单层生长的肿瘤细胞及正常细胞上可以形成晚期囊胚，进而正常脱带，脱带后的小鼠囊胚可以顺利的贴附在未进行处理的单层贴壁式生长的肿瘤细胞群及正常细胞群的上表面，并在继续培养过程中出现滋养层巨细胞的进一步扩展形成胚盘样区域，与其在正常饲养层细胞上的生长行为相似，而正常增殖的肿瘤细胞不会再生长在滋养层巨细胞的上面，而是在滋养层巨细胞的外围形成肿瘤细胞的重叠生长, 与小鼠囊胚扩展出来的滋养层巨细胞有明显的界限。二、小鼠囊胚体外侵袭肿瘤细胞与正常细胞的比较性研究初步实验结果表明小鼠囊胚在体外对底物细胞的贴附与扩展行为的发生无种属特异性，而且与相作用的细胞的分化水平可能无关, 其中除<WP=9>了共培养细胞对小鼠囊胚36小时扩展能力的影响没有显著性差异，P值大于0.05外, 共培养细胞对小鼠囊胚体外黏附扩展能力都具有一定的影响，共培养细胞之间具有显著性差异，P值均小于0.001。但是这种影响与共培更多还原

【Abstract】 1. Mouse Blastocyst Invade Tumor Cells in vitro During blastocyst implantation, apoptosis is observed morphologically at the implantation site of endometrium. The objective of this study was to demonstrate biochemical evidence of apoptosis in tumor and normal cells using a mouse implantation model--co-culture model. Blastocyst from day 4 pregnant mice were cultured on tumor cell lines of human (human lung cancer cells line--A549、human ovary cancer cells line--3AO、HO-8910、SKOV3、 Henrietta Lacks strain of cancer cells--Hela 、human bladder cancer cells line--T24、human osteoblastoma cells line--MG-63 、 human kidney cancer cells line--GRC-1)、 mouse melanoma cells line--B16、<WP=12>human umbilicus vein endothelial cells line -- EUV-304、mouse embryo fibroblast--PA317、Chinese hamster ovary --CHO cells line and primary rabbit embryo osteoblast--RF10、primary newborn mouse lung fibroblast--MF1 for 96 hours. Attachment was observed after 12 hours period of culture and almost all hatched blastocysts attached by 48 hours period. Blastocyst outgrowth was initiated after 24-36 hours of co-culture and almost all attached blastocysts demonstrated outgrowth after 60 hours of incubation. Trophoblastic cells between co-cultured cells-trophoblast units dislodged co-cultured cells.2. Effect of Tumor and Normal Cells on Attachment and Outgrowth of Mouse Blastocyst in vitro Some types of co-cultured cell monolayers were significantly efficient at promoting advanced stages of blastocyst in vitro, such as attachment and outgrowth. The mouse embryo fibroblast--PA317 were selected as control for testing. When co-cultured 48 hours to 72 hours later all kinds of cells can affected the ability of blastocyst attachment, significantly different between normal cells and tumor cells (P<0.001), but there were no significantly difference between normal cells (P>0.05). At first stage when co-cultured 36 hours later there were no<WP=13>significantly difference from all kinds of cells (P>0.05). But after that times some tumor cells can affect the ability of blastocyst outgrowth. There were significantly differences between these cells. And then we took normal and tumor cells as the whole to analysis, co-cultured with tumor cells and normal cells did not affect the ability of development of mouse blastocyst in vitro (P>0.05). 3．Interactions Between Mouse Blastocyst and Tumor Cell Lines in vitroSome co-cultured cells dislodged by trophoblastic cells in tumor-trophoblast unit demonstrated morphological features of apoptosis by TUNEL and Annexin V--EGFP staining when co-cultured 48 hours days later such as A549 cell. But on the other way PCNA were observed in the detached tumor cells （A549、3AO ）around the blastocyst, or in tumor epithelial cells at the tumor-fetal interface when co-cultured 96 hours in vitro.These results suggested there were autocrine/paracrine regulation of the biological of tumor and normal cells at mouse blastocyst implantation, that blastocyst may produce specific factors that can induce apoptosis, in both tumor cells and normal cells，then can stimulate tumor cells proliferate.<WP=14>4. A Three-dimensional Model of Mouse Blastocysts Implantation in vitroThe cultured mouse blastocyst obtained on the 4th day of pregnancy on human fibrin gel and observed the invasive behavior of blastocyst to develop a new embryo invasion three-dimensional model. This study showed that: When mouse blasocyst was cultured on it, human fibrin gel did not have influenced behaviors of mouse blastocyst. The whole blastocyst retained three-dimensional structure. Trophoblastic cells did not flatten but retained a polarized removal and invasion like did in vivo. It suggested human fibrin gel could be used as substratum of three-dimensional model for investigating blastocyst invasive behavior.更多还原