【作者】 宋欣燕；

【导师】 孔北华；

【作者基本信息】 山东大学 ， 妇产科学， 2008， 博士

【摘要】 【研究背景与目的】卵巢恶性肿瘤是妇科三大恶性肿瘤之一,发病率位居女性生殖系统恶性肿瘤第三位,因缺乏特异性症状及有效的早期诊断手段,多数患者确诊时已为晚期。卵巢恶性肿瘤易转移及广泛播散,易复发,预后差,5年生存率仅30%左右,死亡率居妇科恶性肿瘤的首位。关于卵巢癌发生发展的机制也有较多研究探讨,但目前还未有明确的解释。近年来,随着干细胞的研究进展,人们发现肿瘤细胞与干细胞有较多相似性,因而提出了“肿瘤干细胞”的假说。此假说认为:肿瘤的发生、发展及复发归因于肿瘤组织内存在具有无限增殖能力的肿瘤干细胞,而肿瘤干细胞有可能来自于干细胞的突变或融合。随着肿瘤干细胞假说的提出,人们发现许多肿瘤组织中都存在这样一群数目稀少的具有干细胞特点的细胞,这些细胞可以在免疫缺陷的小鼠体内形成完整的肿瘤组织,被称为边缘(sidepopulation,SP)细胞。在几株卵巢癌细胞系中也发现了这种细胞。肿瘤干细胞假说的提出为肿瘤的发生机制探讨及治疗学的研究提出了新的思路,从干细胞的角度来研究探讨肿瘤的各种生物学行为等,使肿瘤细胞的重塑成为可能。干细胞治疗是当今研究的热点,其中首推全能干细胞。全能干细胞是目前所知的最具有多向分化潜能的干细胞,包括胚胎干细胞、胚胎生殖干细胞等。由于其特殊的生物学特性,使其在细胞治疗、组织修复、发育生物学、药物学等领域有着极为广阔的应用前景,成为21世纪生物医学的热点。全能干细胞的自我更新及多向分化的潜能,在多种信号通路的调节下维持在稳定的正常的平衡状态。因而,我们猜测这种具有正常发展模式的全能干细胞是否能使肿瘤细胞的生长得到控制?全能干细胞在肿瘤研究领域的应用,多是先对其进行诱导分化,再支持进一步治疗,而这种直接研究的报道较少。但已有报道鼠胚胎干细胞可以在体外抑制胰腺癌细胞的生长。近年来又有研究表明人胚胎干细胞可以逆转黑色素瘤细胞的恶性行为。胚胎生殖干细胞是全能干细胞的一种,来源于胎儿生殖嵴的原始生殖细胞,在生物学行为上与来自内细胞团的胚胎干细胞相似,且取材方便,目前还未有其可形成畸胎瘤的报道。因而,我们通过体外共培养和体内动物模型,设计研究了人胚胎生殖干细胞对卵巢癌细胞生长的作用。第一部分人胚胎生殖干细胞的分离、培养与鉴定【研究目的】从废弃流产胎儿的生殖嵴分离原始生殖细胞,培养并使其转化为人胚胎生殖干细胞,并为后续实验奠定基础。【研究方法】1、取孕13.5天小鼠的胎鼠背部皮肤,分离培养小鼠胚胎成纤维细胞,制备小鼠胚胎成纤维细胞(mouse embryonic fibroblast,MEF)饲养层。2、取5-7周废弃的流产胎儿的生殖嵴及其周围组织,分离出人原始生殖细胞(human primary germ cells,hPGCs)。经体外分化抑制培养,获得由hPGCs转化成的人胚胎生殖干细胞(human embryonic germ cells,hEGCs),并在体外增殖传代,简单比较胰酶消化传代法与胶原酶+机械法消化传代法的差别,初步观察探讨人白血病抑制因子(leukemia inhibitory factor,LIF)的作用。3、取不同代数的部分hEGCs克隆,检测其碱性磷酸酶活性、胚胎阶段特异性抗原(stage-specific embryonic antigen,SSEA)-1、3、4及TRA-1-60、TRA-1-81、OCT-4的表达,并对体外自发分化进行初步的观察,以鉴定该细胞。【结果】1、分离并纯化培养了小鼠胚胎成纤维细胞,取其3-5代制备MEF饲养层备用。2、将消化后的5-7周流产胎儿的生殖嵴接种于MEF饲养层上,培养2天后就出现了较明显的细胞团。经过体外抑制培养,hEGCs可以传至第11代。在传代中发现胶原酶+机械法更适合hEGCs的传代生长。在培养中发现去除LIF对细胞体外分化抑制培养无明显的影响。3、免疫组化及免疫荧光显示培养的细胞具有较高的碱性磷酸酶活性,并表达SSEA-1、3、4,TRA-1-60,TRA-1-81,OCT-4。符合文献报道的hEGCs的特征。撤去条件培养液后,hEGCs在体外自发分化为神经球样结构及具有自动收缩节律的心肌样细胞。【结论】成功从废弃流产胎儿的生殖嵴及其周围组织中分离出hPGCs,并经体外分化抑制培养,获得由hPGCs转化成的hEGCs。经初步鉴定该细胞符合hEGCs的特征,为后续实验奠定了基础。第二部分人胚胎生殖干细胞在体内外对人卵巢癌细胞SKOV3生长的作用研究【研究目的】通过体外共培养研究人胚胎生殖干细胞对人卵巢癌细胞SKOV3生长的作用,并进行初步的机制探讨。利用SCID小鼠建立卵巢癌的动物模型,初步研究hEGCs在体内对卵巢癌生长的作用。【研究方法】一、体外实验1、实验分两组,实验组将两种细胞先后接种在六孔板中共培养,对照组只接种SKOV3细胞,共培养期间两组的培养液相同。共培养48h后,通过HE染色比较两组SKOV3细胞生长的情况,并通过细胞计数统计两组SKOV3细胞的生长变化。2、通过免疫组化的方法检测两组SKOV3细胞的tunel和caspase-9的表达情况,以观察细胞凋亡的变化。3、通过western blot检测两组SKOV3细胞的AKT和p-AKT蛋白的表达;并通过realtime RT-PCR检测两组SKOV3细胞的akt mRNA的表达情况,并比较其差异。二、体内实验1、将40只小鼠随机分成两组,利用SKOV3卵巢癌细胞在SCID小鼠皮下建立人卵巢癌的动物模型。2、在小鼠皮下出现可触到的瘤块时(约在接种后7天,瘤块直径约0.5cm),实验组将培养的一代或二代人胚胎生殖干细胞(约2×10~5/0.1mlPBS)注入肿瘤组织内,对照组仅注射0.1mlPBS,观察并比较两组动物的肿瘤生长情况。3、在14天、24天、35天时分别处死两组部分小鼠,将肿瘤组织固定做石蜡切片,进行tunel凋亡检测。【结果】一、体外实验1、共培养48h后,HE染色显示实验组SKOV3细胞的分布较对照组稀疏,且越靠近hEGCs克隆周围的SKOV3细胞越稀疏。细胞计数显示,共培养48h后,实验组SKOV3细胞计数为(7.8±2.4)×10~4,明显少于对照组(11.5±2.3)×10~4。2、实验组tunel染色的阳性信号明显多于对照组,且阳性信号大多聚集于干细胞克隆周围。caspase-9染色的结果与tunel染色相似的,实验组的阳性信号也多于对照组。3、western blot显示实验组p-AKT蛋白的表达明显弱于对照组,AKT蛋白表达无明显差异。realtime RT-PCR显示实验组akt mRNA的表达都少于对照组,尤以akt2 mRNA最为明显。二、体内实验1、同期观察测量发现实验组动物的肿瘤生长慢于对照组。2、Tunel凋亡染色发现,同期肿瘤组织中实验组的凋亡信号多于对照组,且随时间延长实验组凋亡信号分布逐渐弥散增多。【结论】HEGCs可以在体外共培养中抑制SKOV3细胞生长。其机制可能是通过降调p-AKT和akt mRNA的表达,激活caspase-9,诱导SKOV3细胞的凋亡。体内初步实验发现hEGCs也可以抑制卵巢癌的生长,此作用可能是通过诱导了癌细胞的凋亡引起的。第三部分微流控芯片技术在体外共培养hEGCs和SKOV3细胞中的应用【研究目的】利用微流控芯片技术共培养hEGCs和SKOV3细胞,并与孔板共培养方法进行比较。【研究方法】1、根据实验要求设计微流控芯片的模型,根据常规的步骤制备微流控芯片,消毒灭菌备用。2、实验组将hEGCs和SKOV3细胞分别接种于微流控芯片中不同的培养槽中,对照组只接种SKOV3细胞于相应的培养槽中,共培养48h,保持培养液的单向流动。3、共培养期间利用显微镜实时观察并记录实验组与对照组SKOV3细胞的生长情况。4、共培养结束后,检测两组SKOV3细胞的tunel的表达情况。5、用孔板共培养作为平行对照,比较两种培养方法。【结果】1、设计并制备了实验所需的微流控芯片,并达到细胞培养的要求。栅栏结构的设计使培养液可以缓慢单向的流动,在12h后在进液孔与出液孔之间达到平衡。2、在微流控芯片中,我们可以实时观察到SKOV3细胞的生长情况,并可以直观的比较实验组与对照组SKOV3细胞生长的差异。实验组SKOV3细胞生长变化较小,而对照组可以看到明显的SKOV3细胞的生长增殖,通过测量SKOV3细胞生长的外缘距出液孔外缘的距离可以比较两组的差别。3、微流控芯片中的SKOV3细胞的tunel结果显示实验组的凋亡信号多于对照组。实验组的凋亡信号沿着液流方向逐渐减少。4、将微流控芯片共培养与孔板共培养比较,发现微流控芯片具有较多的优点,例如可以直观实时地观察细胞的生长变化,可以控制培养液的流向和流速,可以控制微环境的变化等。【结论】微流控芯片技术优于传统的共培养模式:1)微流控芯片技术可以把两种贴壁细胞分离开来共培养;2)可以实时监控细胞生长变化,便于比较。3)可以控制液流的方向,研究单向的作用;4)设计方便,花费较小。微流控芯片更适合于共培养和研究细胞之间的作用。通过分泌细胞因子来抑制SKOV3细胞的生长有可能是hEGCs作用的途径之一。更多还原

【Abstract】 【Background】Ovarian cancer is one of the three most common malignant gynecologic tumors, whose morbidity is the third in the women’s genital cancers.Because of deficiency of special symptoms and effective earlier diagnosis,it is an advanced tumor when diagnosed.Ovarian cancer is generally characterized by poor prognosis,a high rate of recurrence and early metastases.The 5-year overall survival rate is approximately 30%.The cause of ovarian cancer is unknown.In recent years,the hypothesis of cancer stem cells has been put forward because of the similarities between the stem cells and cancer cells.It is supposed that there are cancer stem cells in the tumor which contribute to the development of cancer and cancer stem cells may come from the mutation of normal stem cells.Now some kinds of side population cells which have some characteristics resemble to stem cells have been found in several kinds of cancers including ovarian cancer.The hypothesis of cancer stem cells brings new perspectives for the treatment and plasticity of tumors.Pluripotent stem cells are the most popular one in the stem cell treatment.They demonstrate self-renewal and display the ability to differentiate into a variety of normal cell types of all three primary cell lineages based on different microenvironments.They operate normally under the control of many factors.This comparison evokes our question:do the human pluripotent stem cells,that follow this normal developmental process,have the potential to keep the growth of tumor cells under control? There is some evidence that embryonic stem(ES)cells may inhibit somatic cell growth.Lightfoot et al.reported that mouse ES cells could inhibit the growth of human pancreatic carcinoma cells in vitro.A recent study by Hendrix et al. demonstrated that metastatic melanoma cells could be reverted to a normal,skin cell-like type with the ability to form colonies similar to human embryonic stem cells (hESCs)under the microenvironment of two hESCs lines.The human embryonic germ cell(hEGC),that has similar characteristics to embryonic stem cells coming from the inner cells,belongs to the pluripotent stem cell and comes from primordial germ cells.Our current study investigated the potential effect of hEGCs on a human ovarian cancer cell line(SKOV3)both in vitro and in vivo.PART 1THE ISOLATION,CULTURE AND IDENTIFICATION OF HUMAN EMBRYONIC GERM CELLSObjective:To isolate human primary germ cells from the gonads of abortion fetuses and turn them into human embryonic germ cells through conditioned culture.Methods:1、The mouse embryonic fibroblast coming from day 13 post-coitus fetuses of KM mouse was used as feeder layer cells.2、Human primary germ cells were isolated from the gonads of abortion embryos at 5-7 weeks postconception.Human embryonic germ cells were cultured and passaged under conditioned medium.During the passage,we compared the effect of collagenase IV + mechanical digestion and trypsin.Whether LIF has effect on the culture of human embryonic germ cells was simply investigated.3、Some hEGCs clones were taken out for characterization.Alkaline phosphatase (AKP)activity and Antibodies for stage-specific embryonic antigens(SSEA-1,3, 4),OCT-4,TRA-1-60,TRA-1-81 were detected.Spontaneous differentiation was also tested.Results: 1、The mouse embryonic fibroblasts were cultured and passaged.Three to five passage were used as feeder layer cells.2、After 2-3 days’ primary culture,groups of tightly packed cells with distinct cell borders were recognized.HEGCs could be cultured and passaged to passage 11 under conditioned medium.Collagenase IV + mechanical digestion was fit for the passage of hEGCs.Little difference was observed after LIF was taken out of the conditioned medium.3、Analysis was performed on some hEG clones at different passages and high level of AP activity was shown in EG clones.In addition to AKP activity,EG clones were characterized by a range of cell surface markers including:SSEA-1,SSEA-3, SSEA-4,OCT-4,TRA-1-60,TRA-1-81.When we replaced the conditioned culture medium with DMEM/F-12 and 10%heat-inactivated fetal calf serum, hEGCs could spontaneous differentiate into nerve-sphere liked cells or cells which could contract automatically.Conclusion:We successfully isolated and cultured hEGCs which conform to the characterization reported.PART 2THE EFFECT OF HUMAN EMBRYONIC GERM CELLS ON SKOV3 CELLS IN VITRO AND IN VIVOObjective:To investigate the effect of human embryonic germ cells on SKOV3 in vitro through coculture and in vivo using a xenograft model in SCID mouse.Methods:1、In vitro experiment1)、The SKOV3 cells and hEGCs were seeded orderly onto the 6-well plates for coculture.Only SKOV3 cells were cultured in the control groups.After coculturing for 48h,HE and cell counting were performed to compare the proliferation of SKOV3 cells between the two groups.2)、Tunel apoptosis and caspase-9 activity were detected by immtmocytochemistry.3)、The expression of AKT and p-AKT were examined through western blot.At the same time,akt mRNA was measured by real-time PCR.2、In vivo experiment1)、Forty mice were randomly separated into two groups.The SKOV3 cells were subcutaneously injected into the fight flank of the SCID mice to form a xenograft model.2)、Seven days after SKOV3 cells injection,2×10~5 EG cells in 0.1ml PBS were injected into the tumors in the experiment groups,only 0.1ml PBS was injected into the tumors in control groups.Tumor volume was assessed by measuring two axes(R1,R2)and calculated using the formula:V=1/6πR12 R2.3)、The mice were sacrificed respectively at day 14,24,35.Tumor tissues were fixed in 10%formaldehyde,and embedded in paraffin wax,then cut into 6-μm-thick sections.The sections were also taken for tunel apoptosis assay.Results:1、In vitro experiment1)、After coculturing,HE staining exhibited that SKOV3 cells near the EG clones were sparser than those far off.Cell counting showed that there was a 1.5-fold growth reduction for SKOV3 cells in the coculture group.There were(7.8±2.4)×10~4 in the coculture group,while(11.5±2.3)×10~4 in the control group.2)、There were more positive signals of tunel and caspase-9 in the coculture group, especially near the clones,than the control group.3)、SKOV3 cells in the coculture group showed less p-AKT and akt mRNA than those in the control group.2、In vivo experiment1)、The time-course of tumor volume change was detected.It is noteworthy that the rumors in the EG group grew dramatically slower than those in the control group.2)、More positive signals of the tunel apoptosis were detected in the experiment group.Conclusion: The hEGCs could inhibit the growth of SKOV3 cells in vitro by inducing apoptosis by inhibiting AKT pathway and activating caspase-9.The transplantation of EG cells suppressed the growth of tumor which may be caused by inducing apoptosis of SKOV3 cells.PART 3COCULTURE HUMAN EMBRYONIC GERM CELLS WITH SKOV3 CELLS ON A MICROFLUIDIC CHIPObjective:A well-designed microfluidic device with unidirectional-perfusion has been developed to observe the effect of human embryonic germ(hEG)cells on SKOV3 cells.Methods:1、The microfluidic chip was designed according our experiment and fabricated as reported.2、The hEGCs and SKOV3 cells in the experiment group were seeded in the inlet reserviors and the outlet reserviors separately,and were cocultured for two days. In the control group,just SKOV3 cells were seeded in the outlet reservoirs.The medium was perfused unidirectionally from the inlet to the outlet reserviors.3、The growth of SKOV3 cells were observed online.4、Tunel apoptosis was detected after coculturing.5、Coculture on 6-well plate was used as a parallel group to compare the differences between the two cell culture methods.Results:1、A microfluidic chip suitable for cell culturing was designed according to the experiment.The barrier makes it possible that medium can flow slowly and come to the balance between the inlet reserviors and the outlet reservoirs after 12h. 2、Using our device,the growth of SKOV3 cells could be observed online.The growth inhibition of SKOV3 cells by hEG cells was monitored intuitionally.3、More apoptosis signals in SKOV3 cells culture area were detected in the coculture group,which decreased along flowing of the medium.4、Compared with the traditional coculture,microfluidic chip showed many advantages in the experiment.Conclusion:In conclusion,the results demonstrated that microfluidic chip might be a potential tool to invest the effect of stem cells on cancer cells with intuitionistic cell-based screens.The perfused microfluidic system could control the change of culture microenvironment better and assure uniform perfusion of the cell culture media throughout the cell culture chamber.更多还原