【作者】 田毅；

【导师】 杨波；

【作者基本信息】 郑州大学 ， 外科学， 2010， 硕士

【摘要】 背景与目的：肿瘤干细胞学说这一概念的提出距今已有30多年的历史,该学说认为肿瘤组织中存在极少数具有自我更新和分化潜能的肿瘤细胞,并且是肿瘤增殖、生长、转移和复发的根源。1992年Reynolds对小鼠脑细胞的研究提出了神经干细胞(Neural stem cells, NSCs)概念,1997年Mckay在Science上发表NSCs的文章得到了公认。而2003年Singh从不同病理类型的人脑肿瘤中培养、鉴定出了一种拥有增殖、自我更新和分化能力并且表达神经干细胞表面标记物CD133的脑肿瘤干细胞(Brain Tumor Stem Cells, BTSCs)。近年来,大量研究证实,BTSCs是脑肿瘤的起源细胞,在肿瘤的发生、发展过程中起决定性作用,也是脑肿瘤对放射治疗和化学治疗产生耐受的根源所在,使得BTSCs成为治疗脑肿瘤的新靶点。而BTSCs相关分子标记物及其致瘤机制是神经肿瘤研究的热点领域,也为未来脑肿瘤的研究和临床治疗指明了方向。人们证明,移植后的NSCs具有向脑肿瘤定向迁徙的能力,能在肿瘤周边增殖并包绕脑肿瘤,从而降低脑肿瘤的侵袭性。然而宿主对NSCs移植的排斥和NSCs移植后潜在的致瘤性以及组织来源的伦理问题使得人们把目光投向了间充质干细胞(Mesenchymal Stromal Cells, MSCs)。MSCs是来源于中胚层的多能干细胞,具有高度自我更新能力和多向分化潜能,在适宜的体内体外环境下可以分化为造血细胞以及多种组织特别是中胚层组织细胞的能力,甚至还具有跨胚层分化为神经外胚层起源组织细胞的潜能,因而具有高度的可塑性,是细胞移植和组织工程的新型种子细胞,具有广阔的应用前景。以往研究的重点放在骨髓MSCs的研究上,而其它组织中也存在有MSCs,包括动员的外周血、脐血、乳牙、脂肪、胎盘等,然而,从这些组织中得到的细胞数量有限,从而限制了其研究发展。人脐带Wharton’s jelly间充质干细胞(Mesenchymal Stromal Cells derived from Wharton’s jelly cells, WJCs)满足了国际细胞治疗协会规定的MSCs的特点：如贴壁生长,自我更新,表达MSCs共有的表面标记物,能够向骨、软骨、脂肪、肌肉、神经细胞诱导分化,能够支持其它干细胞的扩增,对免疫系统有良好的耐受性,对肿瘤有定向迁徙的特性。WJCs通过其营养支持和免疫调节作用对很多临床前期的人类疾病的动物模型有治疗作用,如神经退行性疾病,肿瘤,心脏病等。而且脐带组织来源广泛,不存在伦理学问题。因此,本实验主要研究WJCs的生物学特性,以及WJCs和BTSCs在体外共培养后,WJCs是否能够影响BTSCs的生物学特性,并为细胞治疗提供理论依据。方法：WJCs的分离、培养：取长约10cm脐带,剥离脐带外膜并将两条脐动脉和一条脐静脉分离去除,保留Wharton’s jelly,并用PBS缓冲液充分冲洗并将其剪碎至2mm×2mm×2mm大小组织块,置于T25培养瓶中,加入含有20μg/L的bFGF和体积分数为10%的FBS的DMEM/F12培养基5ml培养。WJCs免疫表型鉴定：流式细胞仪检CD29、CD44、HLA-ABC、HLA-DR、CD34、CD45。WJCs免疫荧光鉴定：GFAP和Nestin。比较脐带Wharton’s jelly不同时间产出原代WJCs的免疫表型变化：流式细胞仪检CD29、CD44、HLA-ABC。CCK-8法比较Wharton’s jelly不同时间产出原代WJCs增殖能力变化：绘制7天细胞生长曲线。BTSCs的分离、培养：将新鲜的肿瘤组织用PBS缓冲液充分冲洗并去除外周坏死组织和毛细血管,在80目滤网中研碎过滤后加入2.5g/L胰酶和1.0g/L胶原酶Ⅳ37℃消化30分钟,将消化液过200滤网后制成单细胞悬液。计数活细胞,以2×105／ml终浓度接种于无血清培养基(Serum-free medium, SFM)中,置于T75培养瓶中,添加培养基至10ml培养。BTSCs免疫表型鉴定：流式细胞仪检CD133BTSCs免疫荧光鉴定：GFAP和NestinCCK-8法检测BTSCs增殖率：绘制7天细胞生长曲线。WJCs与BTSCs共培养实验：应用不添加任何生长因子的无血清培养基将两种细胞在24孔板中进行直接共培养。第3、7天时分别离心收集孔中悬浮细胞,应用流式细胞术检测BTSCs CD133表达；贴壁细胞应用免疫荧光法检测神经干细胞标记物巢蛋白(Nestin)和神经胶质细胞标志物胶质纤维酸性蛋白(GFAP)表达；将第3天离心所得的共培养上清液(Co-culture supernatant, CCS)重悬第3代BTSCs并与正常培养悬浮的第3代BTSCs置入96孔板中,应用酶标仪检测两组细胞生长曲线的差异。结果：WJCs的原代培养和扩增：组织块贴壁3-5天后可零星见到单个的梭形或三角形的贴壁细胞,1周后可见贴壁组织块周围有细胞向外游出,细胞由多层逐渐变为单层,细胞由密集变为松散,体型由小到大,细胞形态多为饱满的长梭形或扁平形的成纤维样细胞,核仁明显。两周后,细胞形态变为均一的纺锤形,形态类似成纤维细胞,呈平行排列生长或旋涡状生长。流式细胞仪检测第3代WJCs显示：高表达CD29(91.50%)；低表达CD44(36.92%)、HLA-ABC(28.10%)；不表达CD34(0.72%)、CD45(0.11%)和HLA-DR(0.80%)对WJCs进行Nestin和GFAP免疫荧光染色后显示：未经诱导的WJCs在荧光显微镜下不发出荧光,即不表达Nestin和GFAP。比较脐带Wharton’s jelly不同时间产出原代WJCs的免疫表型变：流式细胞仪检测CD29,CD44,HLA-ABC变化无统计学意思。CCK-8法比较Wharton’s jelly不同时间产出原代WJCs增殖能力变化：不同时间Wharton’s jelly产生原代WJCs的增殖曲线无差别。BTSCs的原代培养和扩增：接种细胞1天后,倒置显微镜下观察可见大量悬浮生长、形态多样的肿瘤细胞团和一些悬浮生长的单细胞,折光性不明显。7天后可见由2-5个细胞组成大小不等的悬浮细胞球。10天后见细胞球明显增多,体积增大,形状规则且折光性强。将细胞球吹散后传代培养,大部分1天后形成新的细胞球。BTSCs巢蛋白Nestin和GFAP免疫荧光鉴定：未经诱导的BTSCs表达Nestin而不表达GFAP。WJCs与BTSCs共培养：倒置显微镜下可见：单独孵育WJCs 24h后用PBS冲洗24孔板,去除未贴壁的细胞,板内可见大部分呈梭形,胞核呈卵圆形,散在均匀分布生长的典型WJCs。共培养1天后,可见两种现象：一种是BTSCs能够形成典型的肿瘤干细胞球,球体圆形或椭圆形,大小不等,折光性强,每个视野能见到3-5克隆团且与贴壁的WJCs相粘连,而WJCs形态数目与WJCs阴性对照几乎无差别；第二种是BTSCs很难形成肿瘤干细胞球,多为单细胞均匀分散,漂浮在培养液里,覆盖在WJCs之上,而WJCs与WJCs阴性对照相比增殖迅速,形成很多细胞集落,胞体伸长形成细胞间连接,方向不整。BTSCs阴性对照开始形成典型的肿瘤干细胞球漂浮在培养基中并且可以滚动。共培养3天后,可见两种现象：一种是与WJCs相连的BTSCs球体发生贴壁现象,肿瘤细胞从球体周围向外游出,形态多为星形,梭形,形成细胞突触呈放射状,并且与周围增生的WJCs形成连接,细胞体积比WJCs小很多,球体周围有一些单个的肿瘤细胞脱落漂浮在培养基中,WJCS增生明显尤其是与BTSCs连接处的WJCs形成细胞集落较多,细胞体积开始变大包体伸长；第二种是BTSCs依然分散漂浮在培养基中,形态与共培养1天时无差别,而WJCs增生明显占据视野的70%,细胞扁平宽大伸长。BTSCs阴性对照依然是典型的肿瘤干细胞球,WJCs阴性对照细胞数目变化不大,形态增大伸长,但依然是梭形。共培养7天后,可见两种现象：一种是肿瘤干细胞球贴壁分化的细胞增多,整个球体紧贴在孔底,BTSCs与WJCs之间的链接更加紧密,WJCs与WJCs阴性对照相比包体较大扁平；第二种是BTSCs散落在WJCs之间,有的单细胞紧紧贴在孔底上,多为星形。通过检测共培养的BTSCs的免疫表型可以得到：脑肿瘤级别高的组织分离得到的BTSCs表达CD133较高,与WJCs共培养后可以看到随着共培养天数增加,BTSCs表达CD133的量逐渐减少。第3天离心所得的共培养上清液重悬第3代BTSCs与正常培养基培养的第3代BTSCs生长曲线的对比,在共培养上清液中培养的BTSCs比在正常培养基中培养的BTSCs的生长曲线要平。结论：1.成功使用组织块法在体外培养出WJCs,并能将脐带组织块充分利用获取更多间充质干细胞,以及采用酶消化法从胶质瘤组织中成功分离出BTSCs,通过换液法尽可能到纯化干细胞。2. WJCs与BTSCs共培养后在倒置显微镜下可见脑肿瘤干细胞球随着培养时间的增加出现分解、贴壁、分化现象；贴壁的脑肿瘤干细胞免疫荧光染色GFAP和Nestin均阳性,WJCs能促使BTSCs分化。3.恶性程度高的脑肿瘤组织培养的BTSCs表达CD133量越高,而与WJCs共培养后随着时间的变化均出现CD133表达量降低,WJCs与BTSCs体外共培养后可降低BTSCs表面标记物CD133的阳性率表达。4.共培养上清液培养的BTSCs与正常培养基培养的BTSCs相比,增殖能力明显受到抑制,WJCs能够分泌抑制性因子产生对BTSCs的抑制作用。更多还原

【Abstract】 Background and Objective:The doctrine of cancer stem cells, which reckon that a small minority of tumor cells with the ability of self-renewal and potentially differentiation in tumor tissue are responsible for proliferation, growth, metastasis and recurrence of tumor, has been put forward more than 30 years ago. In 1992, Reynolds put forward the concept of neural stem cells (NSCs) based on the research on mice.5 years later, the article, wrote by Mckay concerned about NSCs, published in Science has been recognized. In 2003, Singh cultured and identified brain tumor stem cells (BTSCs) from different pathological types of human brain tumor. BTSCs has the capability of proliferation, self-renewal, differentiation and expression of neural stem cell surface marker CD133, which makes it become a new target for treatment of brain tumors. In recent years, numerous studies confirm that BTSCs is the origin of the brain tumor cells, play a decisive role in the tumor occurrence and development, and is also the roots of radiation therapy and chemotherapy tolerance.The BTSCs-related molecular markers and tumorigenic mechanism is a hot research field, but also for the future of brain tumor research and clinical treatment.It is proven that once NSCs has been transplanted into the brain tumor, it could acquire the ability of directional migration, proliferation surrounding the brain tumor, thereby reducing the invasiveness of brain tumors.However, graft-versus-host disease and the potential tumorigenicity after transplantation and ethical issues of sources of tissue make people pay more attention to the mesenchymal stem cells (MSCs).Mesenchymal stem cells (MSCs) is derived from the number of embryos in stem cells, with a high degree of self-renewal capacity and more potential to divide, in appropriate in vivo or in vitro environment, MSCs not only differentiate into hematopoietic cells, Has divided hematopoiesis outside for a variety of organizations, especially in the cells of the embryo’s ability, and even cross-layer differentiation of embryonic nerve cells of the tumor of origin potential, so they have a high degree of plasticity, is a new type of seed cells in cell transplantation and tissue engineering that have broad application prospects. Previous studies have focused on the study of bone marrow MSCs, but there are also other organizations that MSCs, including the mobilization of peripheral blood, umbilical cord blood, adipose tissue, placenta tissue and deciduous, however, these organizations have a limited number of cell restrictions on the research and development.The study found that in recent years people from the AMT, the umbilical cord, amniotic fluid can also be extracted from a good proliferation of MSCs, which for better access to MSCs to provide a new way, and with the in-depth study found that MSCs, MSCs Can also be induced to differentiate into cardiac cells, liver cells and neurons and other cells, MSCs a variety of differentiation potential of the research staff to be more and more attention. A clear case can be made for WJCs as a stromal population since they display the characteristics of MSCs as defined by the International Society for Cellular Therapy; for example, they grow as adherent cells with mesenchymal morphology, they are self-renewing, they express cell surface markers displayed by MSCs, and they may be differentiated into bone, cartilage, adipose, muscle, and neural cells. Like other stromal cells, WJCs support the expansion of other stem cells, such as hematopoietic stem cells, are well-tolerated by the immune system, and they have the ability to home to tumors. WJCs are therapeutic in several different pre-clinical animal models of human disease such as neurodegenerative disease, cancer, heart disease, etc. Whereas the source of umbilical cord is quite rich, the use of it is without ethics problem.This study is to be concentrated mainly on the research of the biological characteristics of WJCs, whether it can influence the biological characteristics of BTSCs after the two type cells co-cultured in vitro, and then provide a theoretical basis for cell therapy.Methods:The isolation and cultivation of WJCs:Taking about 10cm umbilical cord being removed the umbilical cord membrane, the two umbilical arteries and one umbilical vein were, and retained Wharton’s jelly. Wharton’s jelly is fully washed by PBS buffer and cut into pieces to the size of 2mm×2mm×2mm.WJCs immunophenotype identified:flow cytometry CD29, CD44, HLA-ABC, HLA-DR, CD34, CD45.WJCs immunofluorescence identified:GFAP and Nestin.Comparison of immune phenotype of WJCs came out from Wharton’s jelly at different times:flow cytometry CD29, CD44, HLA-ABC.Comparison of Proliferation ability of WJCs came out from Wharton’s jelly at different times by CCK-8:mapping the cell growth curve of 7 days.The isolation and cultivation of BTSCs:wash fresh tumor tissue with PBS buffer and then remove the necrotic tissue and capillary from it. Filter the tissue with 80 mesh and then digest in the 2.5 g/L trypsin and 1.0 g/L collagenaseⅣat 37℃for 30 minutes. After digestion, filter the digestive juice with 200 mesh then remained single-cell suspension. Counting of living cells in a final concentration of 2×105/ml, then inoculate it in serum-free medium (SFM) and placed in T75 culture flask added 10ml culture medium into.BTSCs immunophenotype identified:flow cytometry CD133.BTSCs immunofluorescence identified:GFAP and Nestin.CCK-8 assay of proliferation of BTSCs:mapping the cell growth curve of 7 days.WJCs co-cultured experiments with BTSCs:Co-culture of two kinds of cells in 24-well plates in serum-free medium without any growth factor. Collect the centrifuged suspension cells, and then analysis expression of CD133 by flow cytometry and at day 3 and 7 respectively. Analysis adherent cells expression of Nestin and GFAP by immunofluorescence assay at day 3 and 7 respectively. Co-culture supernatant (CCS) acquired at day 3 resuspend BTSCs. Compare the differences of cell growth curve between cultured in CCS and in normal suspension by Microplate Reader.Results:The isolation and cultivation of WJCs:It can be seen that a single spindle or triangular adherent cells come out from tissue being adherent after 3 to 5 days. After 1 week, adherent cells are swimming out of tissue. Cells gradually become from the multi-layer to single-layer, loose to intensive, and small to big. Cell morphology is mostly full of long spindle-shaped or flat fibroblast-like cells with prominent nucleoli. Two weeks later, it turns out to be uniform spindle cell morphology similar to fibroblasts, showing parallel growth or vortex growth.WJCs immunophenotype identified:high expression of CD29 (91.50%), low expression of CD44 (36.92%), HLA-ABC (28.10%), not express CD34 (0.72%), CD45 (0.11%) and HLA-DR (0.80%)WJCs immunofluorescence identified:None-induced WJCs does not express Nestin and GFAP.Comparison of immune phenotype of WJCs came out from Wharton’s jelly at different times:no difference in expression of CD29, CD44, HLA-ABC.Comparison of Proliferation ability of MSCs came out from WJCs at different times by CCK-8:no difference in cell growth curve.The isolation and cultivation of BTSCs:1 days after inoculation, it is observed under the inverted microscope that a large number of suspended growth and diversity of tumor cell clusters and some single-cell suspension with not obvious refraction.7 days later it can be seen suspension bulk of cells are composed by 2 to 5 the size of cells. After 10 days bulk of cell volume significantly increased with the regular shape and strong refraction. Disperse then subculture the cells, the majority of cells formed news bulk of cells 1 day later.BTSCs immunofluorescence identified:Without induced BTSCs express Nestin but GFAP.WJCs co-cultured experiments with BTSCs:After 24h separately incubation of WJCs in the 24-well plate, then wash plate with PBS to remove non adherent cells. It can be seen most of the spindle, homogeneous scattered growth of the typical WJCs in the bottom of the plate. After 1 day co-cultivation, it is shown two phenomena. One is that BTSCs can form a typical sphere of cells, it can be viewed 3 to 5 clone and connected with adherent WJCs, while the number and form of WJCs is almost no difference with negative control; another is that BTSCs can hardly form a typical sphere of cells, they are more dispersed as single cells, floating in the culture medium covering WJCs. Compared with negative control, WJCs rapidly proliferate and form many cell colony with the elongation of cell body connected with each other. Negative control of BTSCs began to form typical sphere of cells floating and rolling in the medium.After 3 day co-cultivation, it is shown two phenomena. One phenomenon is that sphere of BTSCs connected with WJCs begin adherent to the plate. Tumor cells are climbing out from Sphere of BTSCs with the astral, spindle morphological, and format the radial synapse and connection with proliferation of WJCs around. Adherent BTSCs is much smaller than the WJCs. Another is that BTSCs is still scattered floating in the medium without morphology changing compared to the first day. But WJCs is proliferating obviously, becoming wider and bigger and occupying the range of 70%of the vision.After 7 day co-cultivation, there is shown two phenomena. One phenomenon is that most of sphere of BTSCs connected with WJCs are adherent to the plate. Another is that BTSCs is scattered floating in the medium or adherent to the plate. Negative control of BTSCs and WJCs has normal morphology.We can see the results that the higher degree of malignant brain tumor tissue used in culturing BTSCs was, the higher expression of CD133 in BTSCs was and CD133+in BTSCs declined when co-cultured with WJCs.Growth curve of brain tumor stem cells cultured in CCS compared with in SFM at day 3, which indicates that the proliferation of BTSCs inhibited obviously.Conclusions:1. WJCs cultured by situ cultivation and BTSCs used enzyme digestion way respectively, and gathering the 3rd passage of WJCs though subculturing as well as BTSCs. The original WJCs came out from Wharton’s jelly at different times express stable positive immunophenotype and have stable growth curve. That indicates more WJCs can be gained and used.2. With the cocultivation days increasing, the phenomenon that tumor sphere cells began to be decomposed, adherent and differentiated observed by an inverted microscope. BTSCs in the co-cultured group expressed GFAP and Nestin when adherent and differentiated.3. The higher degree of malignant brain tumor tissue used in culturing BTSCs was, the higher expression of CD133 in BTSCs was. CD133+in BTSCs declined when co-cultured with WJCs.4. Growth curve of brain tumor stem cells cultured in CCS compared with in SFM at day 3, which indicates that the proliferation of BTSCs inhibited obviously. Results indicated that CD133+expression and proliferative capacity of BTSCs went down and BTSCs underwent differentiation during the co-culture in vitro.更多还原