人卵巢癌HO8910细胞系中的肿瘤干细胞样细胞通过MMP促进血管生成拟态的形成

发布时间：2018-03-21 01:54

  本文选题：卵巢上皮性癌　切入点：血管生成拟态　出处：《河北医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:血管生成拟态(vasculogenic mimicry,VM)是存在于恶性侵袭性肿瘤中的特殊血液供应方式。本实验通过无血清悬浮培养方法从人卵巢上皮性癌(简称卵巢癌)细胞株HO8910中分选肿瘤干细胞样细胞,并通过流式细胞术、平板克隆形成实验和裸鼠体内成瘤实验验证分选细胞的肿瘤干细胞样特性。比较分选细胞与亲代细胞形成VM的能力,并检测二者中基质金属蛋白酶2、9(matrix metalloproteinases-2,9;MMP-2,MMP-9)基因的表达水平。方法:1无血清悬浮培养:通过无血清富含多种细胞生长因子的干细胞培养液悬浮培养亲代HO8910细胞,并连续传代,观察悬浮细胞球随着传代的变化,拍照记录,并收集一、三、五、七代悬浮细胞球进行后续试验。2流式细胞仪检测第一、三、五、七代悬浮细胞中CD133+细胞的表达情况:使用CD133抗体标记待检测的各代细胞,通过流式细胞仪检测,荧光信号强表达的为CD133阳性(CD133+)细胞,荧光信号表达弱的为CD133阴性(CD133-)细胞,并计算各代中CD133阳性率,实验重复三次。3体外平板克隆形成实验:比较亲代HO8910细胞与第七代悬浮细胞的克隆形成能力,制备两种细胞的单细胞悬液,以相同浓度的细胞分别接种在培养皿上,培养箱中培养1-2周,显微镜下观察大于50个细胞的克隆数,并计算克隆形成率(CFE),实验重复三次。4裸鼠体内成瘤实验:将亲代细胞和第七代悬浮细胞球制备成单细胞悬液,细胞计数分别为1×105、1×103个。重悬于0.2ml的PBS中,亲代细胞接种于裸鼠的左侧大腿皮下,第七代悬浮细胞接种于裸鼠的右侧大腿皮下,共6只雌性裸鼠,观察裸鼠每侧皮下成瘤情况。5三维立体细胞培养并观察VM的形成情况:将亲代细胞和第七代悬浮细胞球制备成单细胞悬液,分别接种于Matrigel基质胶三维立体培养模型中,以相同间隔的时间在倒置相差显微镜下观察VM的形成情况,随机取上、下、左、右、中五个视野拍照记录,实验重复3次。6实时荧光定量聚合酶链式反应(real-time PCR,RT-PCR):利用RT-PCR检测亲代细胞和第七代悬浮细胞球中MMP-2、MMP-9基因的表达情况,实验重复三次。7统计方法:实验数据采用SPSS 17.0统计软件进行处理,计量资料以均数±标准差((?)±s)表示,两组数据比较采用t检验;以α=0.05为检验水准,当P0.05,差异具有统计学意义,当P0.05,差异不具有统计学意义。结果:1亲代HO8910细胞的悬浮培养将亲代HO8910细胞置于无血清干细胞培养液中悬浮培养,24小时后,绝大部分细胞呈贴壁状态,仅有一小部分细胞呈悬浮状态,继续培养24小时后发现贴壁细胞逐渐死亡,而悬浮状态的细胞增多,表现为几个细胞聚集在一起的悬浮球。培养4-6天时,贴壁细胞已基本分解凋亡,悬浮细胞球随着培养不断增大、增多,且能连续传至第七代。观察发现第一代悬浮细胞球仅有几个细胞组成,大小一致,具有一定的折光性,细胞间连接紧密,但尚能分清细胞间界限。传至第七代时,悬浮球由几十个细胞组成,细胞间连接紧密,界限不清,折光性增强,立体感饱满,但细胞大小和形态仍然保持和初代一致,此结果说明悬浮细胞具有很好的自我更新能力。2流式细胞检测结果流式细胞仪检测第一、三、五、七代悬浮细胞球,发现CD133阳性细胞的表达水平随着传代次数呈上升趋势,其阳性表达率分别为0.23%、31.74%、79.25%和88.18%。3体外平板克隆形成率平板克隆形成实验结果:第七代悬浮细胞球的克隆形成率为45.67±1.53%,而亲代HO8910细胞的克隆形成率仅为11.00±2.00%,前者较后者约增加了4倍,差异具有统计学意义。(t=23.859,P0.00)。4裸鼠体内成瘤能力的分析将1×10~3个第七代悬浮细胞接种于雌性裸鼠右侧大腿皮下,持续观察6周后,6只裸鼠全部出现肉眼可见的移植瘤(6/6)。将1×10~5个亲代HO8910细胞接种于雌性裸鼠左侧大腿皮下,持续观察6周后,未见裸鼠左侧出现肉眼可见的移植瘤(0/6)。5三维立体细胞培养模型中VM的形成能力在Matrigel基质胶三维立体培养条件下,第七代悬浮细胞于2h可见细胞间产生连接,4h即可形成典型的VM网格状结构,此时细胞间联系紧密,纵横交错,48h典型的结构方开始分散凋亡。亲代细胞4h方见细胞间产生联系,但连接松散不规则,8h细胞间联系最多,但典型的VM网格样结构较少,24h连接松散,开始凋落。6 RT-PCR方法检测MMP-2、MMP-9基因的表达情况实时荧光定量PCR结果显示:第七代悬浮细胞中MMP-2基因水平较亲代HO8910细胞提高了6.0倍,其差异具有统计学意义(t=-4.544,P=0.045);第七代悬浮细胞中MMP-9基因表达较亲代细胞提高了6.7倍,其差异也具有统计学意义(t=-4.019,P=0.014)。结论:1亲代HO8910细胞在无血清富含细胞生长因子的培养基中悬浮培养可得到悬浮细胞球,并可连续传代,经验证第七代悬浮细胞球可高表达肿瘤干细胞标记物CD133、有很强的克隆形成能力及裸鼠体内成瘤能力,说明第七代悬浮细胞球具有肿瘤干细胞样特性。2第七代悬浮细胞球较亲代细胞有更强的VM形成能力,且高表达MMP-2、MMP-9。推测肿瘤干细胞样细胞可能通过高表达MMPs,诱导肿瘤细胞自身及细胞外基质的重塑去促进VM的形成,为肿瘤的生长、转移提供血液支持。此研究有助于我们深入认识卵巢癌生长、转移及复发的机制。
[Abstract]:Objective: vasculogenic mimicry (vasculogenic mimicry VM) is a special way of blood supply in malignant invasive tumors. Through this experiment, cultured from human epithelial ovarian cancer serum free suspension (EOC) cell line HO8910 in sorting cancer stem cell like cells by flow cytometry, experiment and nude mice experiment sorting cells tumor stem cell like characteristics of plate clone formation. Sorting and parental cells the ability to form VM, and the detection of matrix metalloproteinase two in 2,9 (matrix metalloproteinases-2,9; MMP-2, MMP-9) gene expression level. Methods: 1 serum free suspension culture with serum-free rich a variety of cell growth factors stem cell culture parental HO8910 cells were cultured and passaged, observe the cell suspension with ball change, passage and collect a photograph, three, five, seven Generation of ball suspension cells for subsequent experiments of.2 flow cytometry in first, third, five, the expression of CD133+ cells in suspension cells of the seven generation: the use of CD133 antibody to detect the cells by flow cytometry, fluorescence intensity expression of CD133 positive cells (CD133+), the expression of the weak fluorescence signal negative for CD133 (CD133-) cells, and calculate the positive rate of CD133 in each generation, the experiment was repeated three times in vitro.3 clone formation experiment: cloning of parental HO8910 cells and the seventh generation of suspension cells forming ability, cell suspension preparation of two kinds of single cell, with the same concentration of the cells were seeded in culture dishes, the incubator for 1-2 weeks, the number of clones under the microscope more than 50 cells, and calculate the clone formation rate (CFE), the experiment was repeated three times.4 in nude mice: the parental cells and the seventh generation of suspension cell preparation Single cell suspension, cell counts were 1 * 105,1 * 103. Resuspended in 0.2ml PBS in the left thigh subcutaneous parental cells were inoculated in nude mice, the right thigh subcutaneous seventh generation suspension cells were inoculated in nude mice, a total of 6 female nude mice, the mice were observed on each side of subcutaneous tumor.5 three-dimensional cell cultured and observed the formation of VM: the parental cells and the seventh generation of suspension cell prepared into single cell suspension were inoculated on Matrigel Matrigel three-dimensional culture model, at the same time interval under the inverted microscope observation of VM formation condition, randomly selected, under the left and right. In view of five photographs, the experiment was repeated 3 times.6 real-time fluorescence quantitative polymerase chain reaction (real-time PCR RT-PCR): the use of MMP-2 and seventh RT-PCR generation parental cell suspension cell, the expression of MMP-9 gene, the experiment was repeated three times.7 statistics Methods: the experimental data were processed by SPSS 17 statistical software, measurement data to mean + standard deviation ((?) + s) said that the two sets of data were compared with t test; when the P0.05 to test the level of a =0.05, and the difference has statistical significance, when P0.05, the difference was not statistically significant. Suspension: 1 parental HO8910 cells to parental HO8910 cells on cells cultured in suspension in serum, 24 hours later, most cells adherent state, only a small part of the cells were suspended and incubated for 24 hours after the discovery of adherent cells gradually died, and suspension cells increased, performance for several ball suspended cells together. When cultured for 4-6 days, the adherent cells have basic decomposition apoptosis, cell suspension culture with the ball is increasing, increased, and can be continuously passaged for seventh generations. The first generation of suspension cell was observed only a few fine The cellular composition, the same size, with certain refractive index, cells connected closely, but it is able to distinguish between the cells. At the seventh passage, a ball suspended by dozens of cells, cells are closely connected, is unclear, refractive index enhancement, stereo sense is full, but the cell size and shape remain the first, the cell suspension has good self-renewal ability of.2 flow cytometry results of flow cytometry in first, third, five, seven generation of suspension cell, found that CD133 positive cells expression level with the passage number increased, the positive rate was 0.23%, 31.74%, 79.25% and 88.18%.3 in vitro the plate clone formation rate of clone formation experiment results: the seventh generation of the suspension cell clone formation rate was 45.67 + 1.53%, and the clone formation rate of parental HO8910 cells was 11 + 2%, compared with the increase of about 4 Times, the difference was statistically significant. (t=23.859, P0.00).4 analysis of nude mice will be 1 * 10~3 seventh generation cell suspension inoculated in female nude mice right thigh subcutaneous, continuous observation after 6 weeks, 6 mice all visible tumor (6/6). The 1 * 10~5 parent HO8910 cells were inoculated on female nude mice left thigh subcutaneous, continued after 6 weeks of observation, no visible on the left side of the nude mice transplanted tumor (0/6).5 three-dimensional cell culture model of VM formation in Matrigel Matrigel three-dimensional culture condition, the seventh generation cell suspension in 2H cells generated between the connection, 4H can form VM a typical grid structure, the close connection between cells, the structure of a typical 48h arranged in a crisscross pattern, began to disperse apoptosis. Parent cell 4H see links between cells, but loosely connected irregular 8h cells between the most typical, but The VM grid like structure is less, 24h loose connection,.6 began to fall RT-PCR method to detect the MMP-2 expression of MMP-9 gene by real-time fluorescence quantitative PCR results showed that: the seventh generation of cells suspended in MMP-2 gene expression compared with parental HO8910 cells increased 6 times, the difference was statistically significant (t=-4.544, P=0.045); MMP-9 gene seventh generation suspension expression compared with parental cells increased by 6.7 times, the difference was statistically significant (t=-4.019, P=0.014). Conclusion: 1 parental HO8910 cells in serum-free medium containing cell growth factor in suspension culture can get cell suspension ball, and continuous passage, verified by the seventh generation cells suspended high expression the cancer stem cell marker CD133, clone forming ability and strong ability of tumorigenicity in the nude mice, indicating that seventh generation of suspension cell tumor stem cell like characteristics of the seventh generation of.2 cell suspension with ball 浜蹭唬缁嗚優鏈夋洿寮虹殑VM褰㈡垚鑳藉姏,涓旈珮琛ㄨ揪MMP-2,MMP-9.鎺ㄦ祴鑲跨槫骞茬粏鑳炴牱缁嗚優鍙�鑳介�氳繃楂樿〃杈綧MPs,璇卞�艰偪鐦ょ粏鑳炶嚜韬�鍙婄粏鑳炲�栧熀璐ㄧ殑閲嶅�戝幓淇冭繘VM鐨勫舰鎴，

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