本文選題：膠質(zhì)母細胞瘤 + U87��； 參考：《南方醫(yī)科大學(xué)》2014年碩士論文

【摘要】：研究背景 神經(jīng)膠質(zhì)瘤,簡稱膠質(zhì)瘤,由于發(fā)生于神經(jīng)外胚層,故又稱作神經(jīng)外胚層腫瘤或神經(jīng)上皮腫瘤。神經(jīng)膠質(zhì)瘤是顱內(nèi)原發(fā)惡性腫瘤中最常見的一種,根據(jù)WTO分級,可將神經(jīng)膠質(zhì)瘤分為4個等級,多形膠質(zhì)母細胞瘤(glioblastoma multiforme GBM)屬于Ⅳ級膠質(zhì)瘤,在現(xiàn)有的治療手段下,患者確診GBM后的生存期平均為1-2年。目前對它的治療主要是以手術(shù)治療為主的綜合治療。GBM本身病死率高,又極易復(fù)發(fā),癌細胞存在一定的化療耐藥性及放射抵抗,預(yù)后欠佳,為社會和家庭帶來了巨大的經(jīng)濟和精神負擔(dān)。開展膠質(zhì)瘤相關(guān)的基礎(chǔ)與臨床研究對于尋找對其更為有效的治療方法意義重大。 U251細胞和U87細胞為膠質(zhì)瘤體外實驗兩種常用的工具細胞,兩種細胞系從GBM中分離建立而來,具有很強的代表性。U251細胞來源于一位老年男性GBM患者,而U87來源于一位女性GBM患者,均由Ponten.J教授及其同事建立。自兩細胞系建立以來,以該兩種細胞為對象的大量研究使得GBM的增殖,侵襲,遷移和凋亡等生物學(xué)習(xí)性越來越清晰。從目前的研究觀察到,盡管兩者均來源于GBM患者,但U251和U87細胞系在細胞生物學(xué)特征并不相同,表現(xiàn)為在正常培養(yǎng)的情況下,顯微鏡下觀察到的U251細胞跟U87細胞的細胞形態(tài)并不一致,U251細胞胞體成呈裙擺形,樹突軸突等突起較為短小,U87細胞的胞體成梭形,有多個較長軸突,相對于U251而言,U87細胞具有更強的增殖,遷移和侵襲能力,而對TMZ的敏感性比U251強,與實驗中觀察到的結(jié)果為相同,由此可見,U251細胞和U87細胞雖同來源于GBM,但生物學(xué)性狀并不完全一致,尤其是侵襲能力、成瘤能力差異顯著。既然U251和U87侵襲性、成瘤能力差異顯著,那么,以二者為工具,探討其分子基礎(chǔ),將有助于揭示GBM侵襲的分子機制,為GBM治療提供潛在分子靶點,但是,經(jīng)文獻檢索檢索發(fā)現(xiàn),U251細胞和U87細胞究竟存在哪些蛋白表達差異,并不清楚。 因此,本研究以U251細胞和U87細胞為研究對象,用蛋白組學(xué)的方法蛋白雙向電泳及同位素相對標(biāo)記與絕對定量技術(shù)(isobaric tags forrelative and absolute quantitation, iTRAQ)方法對比分析兩細胞蛋白表達譜表達情況,探討這兩種細胞系來源是否一致,并檢測兩細胞差異蛋白的表達,通過對差異蛋白進行GO及pathway富集分析等生物信息學(xué)分析,解析兩細胞細胞形態(tài),增殖,遷移和侵襲能力及藥敏性等細胞生物學(xué)差異的原因,從篩選得到的差異蛋白中選出部分蛋白質(zhì),驗證其蛋白表達水平,干擾其U251細胞和U87細胞中表達,觀察干擾后U251細胞和U87細胞生物學(xué)特征的變化。 研究方法 一、U251細胞和U87細胞培養(yǎng),使用MTT檢測兩細胞系的增殖能力及對不同濃度TMZ的藥敏性,使用劃痕實驗檢測其遷移能力。 二、蛋白雙向凝膠電泳(two-dimensional gel electrophoresis2-DE)檢測U251和U87細胞系蛋白表達譜,驗證其組織學(xué)來源是否一致。 三、iTRAQ比較U251細胞和U87細胞系差異蛋白表達譜,結(jié)果使用行基質(zhì)輔助激光解析電離-飛行時間質(zhì)譜分析(MALDI-TOF-MS)找差異蛋白點,并使用G0富集分析及Pathway富集分析等對兩細胞差異蛋白進行分析。 四、用qPCR技術(shù)驗證U251細胞和U87細胞系10對基因TPM4, FLNC, C10orf58, PDLIM1, MYH10, PSIP1, YA61, SYNM, SLC9A3R2and BCAM mRNAs表達差異。 五、使用siRNA干擾兩種細胞系TPM4,PDLIM1, PSIP13種基因表達,qPCR技術(shù)檢測干擾效率。劃痕實驗分別檢測siRNA干擾后U251和U87細胞各處理組細胞的遷移能力。 六、統(tǒng)計學(xué)處理：采用SPSS19.0軟件進行統(tǒng)計學(xué)處理。計量資料用x±s表示,組間比較采用單因素方差分析,P0.05定義為有統(tǒng)計學(xué)意義。 研究結(jié)果 一、同等培養(yǎng)條件下,U87比U251細胞增殖更快,侵襲能力更強,同時,前者比后者對TMZ更為敏感。 二、Image Master檢測U251和U87細胞系的蛋白質(zhì)表達譜之間的差異點,發(fā)現(xiàn)兩者蛋白表達譜基本一致,提示U251和U87細胞系組織學(xué)來源一致,,與ATCC數(shù)據(jù)庫描述一樣,而存在著少數(shù)差異蛋白,提示其生物學(xué)性質(zhì)存在差異。 三、同位素相對標(biāo)記與絕對定量技術(shù)分析兩細胞系蛋白組,總蛋白數(shù)量3660種,根據(jù)蛋白質(zhì)豐度水平,當(dāng)差異倍數(shù)達到1.5倍以上,且經(jīng)統(tǒng)計檢驗其p-value值小于0.05時,視為差異蛋白,507種蛋白在表達量上存在明顯的差異,其中U251細胞相對U87細胞上調(diào)蛋白數(shù)量為244種,下調(diào)蛋白數(shù)量為263種。 四、qPCR檢測結(jié)果提示,在10個基因當(dāng)中,C10orf58, FLNC, PDLIM1,YA61,TPM4在U251細胞中的表達比在U87細胞中表達的高,MYH10, PSIP1, SYNM, SLC9A3R2and BCAM,在U251細胞中的表達比在U87細胞中表達的低,差異均超過了4倍,比較iTRAQ檢測結(jié)果,提示以上10種差異蛋白的基因表達水平在兩細胞上下調(diào)方向一致。 五、siRNA干擾TPM4,PDLIM1,PSIP1表達后劃痕試驗提示U251中干擾TPM4表達組細胞(以下簡稱T組)遷移最快,干擾PSIP1表達組細胞次之(以下簡稱PS組),干擾PDLIM1表達組細胞(以下簡稱PD組)與陰性對照組(NC組)細胞遷移最慢,后兩組細胞遷移速度并沒有明顯的差異。 研究結(jié)論 U251細胞和U87細胞系來源于膠質(zhì)母細胞瘤,它們的組織學(xué)來源是一致的,兩者蛋白表達譜有差異,差異表達蛋白總共達到了507種,其中PDLIM1, TPM4基因和相對應(yīng)蛋白表達在U251細胞中的表達比在U87細胞中表達的高,PSIP1基因和相對應(yīng)蛋白表達在U251細胞中的表達比在U87細胞中表達的低,PDLIM1,TPM4對U87遷移有負向調(diào)節(jié)作用,即可抑制U87細胞的遷移,PSIP1基因表達量在兩細胞中較低,對兩者遷移能力影響不明顯。 本研究意義 一、為基于U251細胞和U87細胞系的研究提供蛋白水平差異的數(shù)據(jù)資料。 二、由于兩種GBM生物學(xué)特性并不完全一致,進一步闡明差異蛋白的作用,有助于GBM特定功能的解析。
[Abstract]:Research background
Glioma, called glioma, is also called neuroectodermal tumor or neuroepithelial tumor because it occurs in the neuroectoderm. Glioma is the most common type of primary intracranial malignant tumor. According to WTO classification, glioma can be divided into 4 grades, and the glioblastoma multiforme GBM belongs to IV. The average survival time of patients with grade glioma is 1-2 years after the diagnosis of GBM. At present, the treatment of.GBM is mainly a comprehensive treatment based on surgical treatment, which has high mortality and easy recurrence. The cancer cells have a certain chemotherapeutic resistance and radiation resistance, and the prognosis is not good. The basic and clinical research of glioma is of great significance in finding a more effective treatment for glioma.
U251 cells and U87 cells are two common tool cells in vitro experiment of glioma. The two cell lines are isolated from GBM. The strong representative.U251 cells are derived from an elderly male GBM patient, and U87 originated from a female GBM patient. All of them were established by Professor Ponten.J and his colleagues. Since the establishment of the two cell line, it has been established. A large number of studies on two kinds of cells have made the biological learning of GBM proliferation, invasion, migration and apoptosis more and more clear. From the present study, although both of them are derived from GBM patients, the U251 and U87 cell lines are different in cell biological characteristics, showing the U251 observed under the microscope under normal conditions. The cell morphology of the cells is not consistent with the cell morphology of the U87 cells. The cell body of the U251 cell is a skirt shape, the protuberance of the dendritic axon is relatively short, the cell body of the U87 cell is spindle shaped, and there are many longer axons. Compared with the U251, U87 cells have stronger proliferation, migration and invasion ability, and the sensitivity to TMZ is stronger than that of U251, and the results observed in the experiment. In the same way, it can be seen that although U251 and U87 cells are from GBM, the biological characters are not exactly the same, especially the invasive ability, and the difference of the ability of tumor formation is significant. Since U251 and U87 are invasive and the difference of the ability of tumor formation is significant, then the two groups are used as a tool to explore the molecular mechanism of GBM invasion, and it will be used to treat GBM. Treatment provides potential molecular targets. However, a literature search shows that the difference in protein expression between U251 cells and U87 cells is not clear.
Therefore, in this study, U251 cells and U87 cells were used to compare the expression of two cell protein expression profiles by means of proteomic method protein two-dimensional electrophoresis and isotopic relative labeling and absolute quantitative technique (isobaric tags forrelative and absolute quantitation, iTRAQ). The expression of two cell differential proteins was detected. By bioinformatics analysis of GO and pathway enrichment analysis of differential proteins, the reasons of cell biological differences, such as cell morphology, proliferation, migration and invasion ability and drug sensitivity of two cells, were analyzed, and some proteins were selected from the differential proteins selected to verify the protein expression. The expression level of U251 cells and U87 cells was disturbed, and the biological characteristics of U251 cells and U87 cells after interference were observed.
research method
First, U251 cells and U87 cells were cultured. MTT was used to detect the proliferation ability of two cell lines and their sensitivity to different concentrations of TMZ. The migration ability was detected by scratch test.
Two, two-dimensional gel electrophoresis2-DE was used to detect the protein expression profiles of U251 and U87 cell lines, and to verify whether their histological sources were consistent.
Three, iTRAQ compared the differential protein expression profiles of U251 cells and U87 cell lines. The results were found by using line matrix assisted laser analytical ionization time of flight mass spectrometry (MALDI-TOF-MS) to find differential protein points, and the analysis of two cell differential proteins by G0 enrichment analysis and Pathway enrichment analysis.
Four, qPCR technology was used to verify the difference between U251 cells and U87 cell line 10 in gene TPM4, FLNC, C10orf58, PDLIM1, MYH10, PSIP1, YA61, SYNM, and PSIP1.
Five, the expression of TPM4, PDLIM1, PSIP13 gene was expressed by siRNA interference, and the interference efficiency was detected by qPCR technique. The scratch test was used to detect the cell migration ability of U251 and U87 cells after siRNA interference.
Six, statistical processing: SPSS19.0 software was used for statistical processing. The measurement data were expressed in X + s, and single factor analysis of variance was used in groups. P0.05 was defined as statistical significance.
Research results
1. Under the same culture conditions, U87 has faster proliferation and stronger invasiveness than U251 cells, and the former is more sensitive to TMZ than the latter.
Two, Image Master detected the difference between the protein expression profiles of U251 and U87 cell lines, and found that the protein expression profiles were basically the same, suggesting that the histologic sources of U251 and U87 cells were the same, as described by the ATCC database, and there were a few differential proteins, suggesting that there were differences in their biological properties.
Three, isotopic relative labeling and absolute quantification analysis of the two cell line protein group, the total protein number is 3660. According to the protein abundance level, when the difference multiple reaches more than 1.5 times, and the p-value value is less than 0.05 by statistical test, the difference in the amount of protein is apparent in the amount of the 507 proteins, and the relative U87 of the U251 cells is relative to U87. The number of upregulated proteins was 244 and the number of down regulated proteins was 263.
Four, qPCR results showed that in the 10 genes, the expression of C10orf58, FLNC, PDLIM1, YA61, TPM4 in U251 cells was higher than that in U87 cells, MYH10, PSIP1, SYNM, SLC9A3R2and, and the expression in the cells was lower than that in the U251 cells, and the difference was more than 4 times. The above 10 differences were compared. The level of protein expression was consistent with that in two cells.
Five, siRNA interference TPM4, PDLIM1, PSIP1 expression after the scratch test suggested that U251 interference TPM4 expression group cells (hereinafter referred to as T group) migration is the fastest, interfering with the PSIP1 expression group cells (hereinafter referred to PS group), interfering with PDLIM1 expression group cells (hereinafter referred to PD group) and negative group (NC group) cell migration is the slowest, the latter two groups of cell migration speed did not There are obvious differences.
research conclusion
The U251 and U87 cell lines are derived from glioblastoma, and their histological origin is consistent, and the protein expression profiles are different. The differential expression proteins have reached 507 species, of which the expression of PDLIM1, TPM4 and relative protein expression in U251 cells is higher than that in U87 cells, PSIP1 gene and relative protein table. The expression in U251 cells is lower than that expressed in U87 cells. PDLIM1 and TPM4 have negative regulation on U87 migration, which can inhibit the migration of U87 cells. The expression of PSIP1 gene is low in two cells, and there is no obvious effect on the mobility of U87 cells.
The significance of this study
First, provide data for protein level differences based on U251 cell and U87 cell lines.
Two, because the biological characteristics of the two GBM are not completely consistent, further elucidation of the role of differential proteins is helpful to the analysis of GBM specific functions.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R739.41

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相關(guān)期刊論文 前1條

1 王惠麗;苑召虎;陳志江;姚芳;胡子有;吳炳義;;槲皮素誘導(dǎo)U87細胞凋亡及對MDM2-p53負反饋調(diào)節(jié)的影響[J];南方醫(yī)科大學(xué)學(xué)報;2014年05期

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本文編號：1834608