本文選題：卵巢上皮性癌　切入點：血管生成擬態(tài)　出處：《河北醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:血管生成擬態(tài)(vasculogenic mimicry,VM)是存在于惡性侵襲性腫瘤中的特殊血液供應(yīng)方式。本實驗通過無血清懸浮培養(yǎng)方法從人卵巢上皮性癌(簡稱卵巢癌)細(xì)胞株HO8910中分選腫瘤干細(xì)胞樣細(xì)胞,并通過流式細(xì)胞術(shù)、平板克隆形成實驗和裸鼠體內(nèi)成瘤實驗驗證分選細(xì)胞的腫瘤干細(xì)胞樣特性。比較分選細(xì)胞與親代細(xì)胞形成VM的能力,并檢測二者中基質(zhì)金屬蛋白酶2、9(matrix metalloproteinases-2,9;MMP-2,MMP-9)基因的表達(dá)水平。方法:1無血清懸浮培養(yǎng):通過無血清富含多種細(xì)胞生長因子的干細(xì)胞培養(yǎng)液懸浮培養(yǎng)親代HO8910細(xì)胞,并連續(xù)傳代,觀察懸浮細(xì)胞球隨著傳代的變化,拍照記錄,并收集一、三、五、七代懸浮細(xì)胞球進(jìn)行后續(xù)試驗。2流式細(xì)胞儀檢測第一、三、五、七代懸浮細(xì)胞中CD133+細(xì)胞的表達(dá)情況:使用CD133抗體標(biāo)記待檢測的各代細(xì)胞,通過流式細(xì)胞儀檢測,熒光信號強表達(dá)的為CD133陽性(CD133+)細(xì)胞,熒光信號表達(dá)弱的為CD133陰性(CD133-)細(xì)胞,并計算各代中CD133陽性率,實驗重復(fù)三次。3體外平板克隆形成實驗:比較親代HO8910細(xì)胞與第七代懸浮細(xì)胞的克隆形成能力,制備兩種細(xì)胞的單細(xì)胞懸液,以相同濃度的細(xì)胞分別接種在培養(yǎng)皿上,培養(yǎng)箱中培養(yǎng)1-2周,顯微鏡下觀察大于50個細(xì)胞的克隆數(shù),并計算克隆形成率(CFE),實驗重復(fù)三次。4裸鼠體內(nèi)成瘤實驗:將親代細(xì)胞和第七代懸浮細(xì)胞球制備成單細(xì)胞懸液,細(xì)胞計數(shù)分別為1×105、1×103個。重懸于0.2ml的PBS中,親代細(xì)胞接種于裸鼠的左側(cè)大腿皮下,第七代懸浮細(xì)胞接種于裸鼠的右側(cè)大腿皮下,共6只雌性裸鼠,觀察裸鼠每側(cè)皮下成瘤情況。5三維立體細(xì)胞培養(yǎng)并觀察VM的形成情況:將親代細(xì)胞和第七代懸浮細(xì)胞球制備成單細(xì)胞懸液,分別接種于Matrigel基質(zhì)膠三維立體培養(yǎng)模型中,以相同間隔的時間在倒置相差顯微鏡下觀察VM的形成情況,隨機取上、下、左、右、中五個視野拍照記錄,實驗重復(fù)3次。6實時熒光定量聚合酶鏈?zhǔn)椒磻?yīng)(real-time PCR,RT-PCR):利用RT-PCR檢測親代細(xì)胞和第七代懸浮細(xì)胞球中MMP-2、MMP-9基因的表達(dá)情況,實驗重復(fù)三次。7統(tǒng)計方法:實驗數(shù)據(jù)采用SPSS 17.0統(tǒng)計軟件進(jìn)行處理,計量資料以均數(shù)±標(biāo)準(zhǔn)差((?)±s)表示,兩組數(shù)據(jù)比較采用t檢驗;以α=0.05為檢驗水準(zhǔn),當(dāng)P0.05,差異具有統(tǒng)計學(xué)意義,當(dāng)P0.05,差異不具有統(tǒng)計學(xué)意義。結(jié)果:1親代HO8910細(xì)胞的懸浮培養(yǎng)將親代HO8910細(xì)胞置于無血清干細(xì)胞培養(yǎng)液中懸浮培養(yǎng),24小時后,絕大部分細(xì)胞呈貼壁狀態(tài),僅有一小部分細(xì)胞呈懸浮狀態(tài),繼續(xù)培養(yǎng)24小時后發(fā)現(xiàn)貼壁細(xì)胞逐漸死亡,而懸浮狀態(tài)的細(xì)胞增多,表現(xiàn)為幾個細(xì)胞聚集在一起的懸浮球。培養(yǎng)4-6天時,貼壁細(xì)胞已基本分解凋亡,懸浮細(xì)胞球隨著培養(yǎng)不斷增大、增多,且能連續(xù)傳至第七代。觀察發(fā)現(xiàn)第一代懸浮細(xì)胞球僅有幾個細(xì)胞組成,大小一致,具有一定的折光性,細(xì)胞間連接緊密,但尚能分清細(xì)胞間界限。傳至第七代時,懸浮球由幾十個細(xì)胞組成,細(xì)胞間連接緊密,界限不清,折光性增強,立體感飽滿,但細(xì)胞大小和形態(tài)仍然保持和初代一致,此結(jié)果說明懸浮細(xì)胞具有很好的自我更新能力。2流式細(xì)胞檢測結(jié)果流式細(xì)胞儀檢測第一、三、五、七代懸浮細(xì)胞球,發(fā)現(xiàn)CD133陽性細(xì)胞的表達(dá)水平隨著傳代次數(shù)呈上升趨勢,其陽性表達(dá)率分別為0.23%、31.74%、79.25%和88.18%。3體外平板克隆形成率平板克隆形成實驗結(jié)果:第七代懸浮細(xì)胞球的克隆形成率為45.67±1.53%,而親代HO8910細(xì)胞的克隆形成率僅為11.00±2.00%,前者較后者約增加了4倍,差異具有統(tǒng)計學(xué)意義。(t=23.859,P0.00)。4裸鼠體內(nèi)成瘤能力的分析將1×10~3個第七代懸浮細(xì)胞接種于雌性裸鼠右側(cè)大腿皮下,持續(xù)觀察6周后,6只裸鼠全部出現(xiàn)肉眼可見的移植瘤(6/6)。將1×10~5個親代HO8910細(xì)胞接種于雌性裸鼠左側(cè)大腿皮下,持續(xù)觀察6周后,未見裸鼠左側(cè)出現(xiàn)肉眼可見的移植瘤(0/6)。5三維立體細(xì)胞培養(yǎng)模型中VM的形成能力在Matrigel基質(zhì)膠三維立體培養(yǎng)條件下,第七代懸浮細(xì)胞于2h可見細(xì)胞間產(chǎn)生連接,4h即可形成典型的VM網(wǎng)格狀結(jié)構(gòu),此時細(xì)胞間聯(lián)系緊密,縱橫交錯,48h典型的結(jié)構(gòu)方開始分散凋亡。親代細(xì)胞4h方見細(xì)胞間產(chǎn)生聯(lián)系,但連接松散不規(guī)則,8h細(xì)胞間聯(lián)系最多,但典型的VM網(wǎng)格樣結(jié)構(gòu)較少,24h連接松散,開始凋落。6 RT-PCR方法檢測MMP-2、MMP-9基因的表達(dá)情況實時熒光定量PCR結(jié)果顯示:第七代懸浮細(xì)胞中MMP-2基因水平較親代HO8910細(xì)胞提高了6.0倍,其差異具有統(tǒng)計學(xué)意義(t=-4.544,P=0.045);第七代懸浮細(xì)胞中MMP-9基因表達(dá)較親代細(xì)胞提高了6.7倍,其差異也具有統(tǒng)計學(xué)意義(t=-4.019,P=0.014)。結(jié)論:1親代HO8910細(xì)胞在無血清富含細(xì)胞生長因子的培養(yǎng)基中懸浮培養(yǎng)可得到懸浮細(xì)胞球,并可連續(xù)傳代,經(jīng)驗證第七代懸浮細(xì)胞球可高表達(dá)腫瘤干細(xì)胞標(biāo)記物CD133、有很強的克隆形成能力及裸鼠體內(nèi)成瘤能力,說明第七代懸浮細(xì)胞球具有腫瘤干細(xì)胞樣特性。2第七代懸浮細(xì)胞球較親代細(xì)胞有更強的VM形成能力,且高表達(dá)MMP-2、MMP-9。推測腫瘤干細(xì)胞樣細(xì)胞可能通過高表達(dá)MMPs,誘導(dǎo)腫瘤細(xì)胞自身及細(xì)胞外基質(zhì)的重塑去促進(jìn)VM的形成,為腫瘤的生長、轉(zhuǎn)移提供血液支持。此研究有助于我們深入認(rèn)識卵巢癌生長、轉(zhuǎn)移及復(fù)發(fā)的機制。
[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 浜蹭唬緇嗚優(yōu)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祴鑲跨槫騫茬粏鑳?yōu)鏍肪l嗚優(yōu)鍙兘閫氳繃楂樿〃杈綧MPs,璇卞鑲跨槫緇嗚優(yōu)鑷韓鍙婄粏鑳?yōu)澶栧燌櫞ㄧ殑閲嶅鍘讳績杩沄M鐨勫艦鎴，

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