本文關(guān)鍵詞： Twist基因 結(jié)腸癌細胞株 細胞轉(zhuǎn)染 RT-PCR Western-blot　出處：《河北醫(yī)科大學》2011年碩士論文　論文類型：學位論文

【摘要】：目的:Twist基因是存在于人、果蠅、鼠等生物體內(nèi)的堿性螺旋-環(huán)-螺旋(Basichelix loop helix ,bHLH)蛋白,作為一種轉(zhuǎn)錄因子,主要表達于胚胎胎盤、中胚層及成人的某些中胚層來源的未分化組織。Twist表達的蛋白在人有202個氨基酸、小鼠有206個氨基酸、蟾蜍有166個氨基酸序列構(gòu)成,其中人類和小鼠的氨基酸序列具有96%的同源性,在不同的種屬中其DNA結(jié)合區(qū)域有著100%的序列保守,所有Twist基因都能結(jié)合E-box的DNA序列:CANNTG。Twist基因在轉(zhuǎn)錄水平上調(diào)控多種基因的表達[1],最初是在胚胎發(fā)育過程中發(fā)現(xiàn)它的生物學功能,它可以通過促進上皮-間葉變(epithelial mesenchymal transition , EMT)來調(diào)節(jié)胚胎的發(fā)育。近期研究結(jié)果證實[2,3]Twist具有癌基因的特性,能夠編碼凋亡抑制蛋白,通過多個信號系統(tǒng)參與腫瘤的發(fā)生:抑制分化,通過降低P53上游的調(diào)控因子ARF的表達阻斷P53信號通路,阻礙N-myc信號系統(tǒng)的傳遞,和抑制NF-κB通路等。它還參與了多種上皮來源的腫瘤細胞發(fā)生EMT并促進癌細胞侵襲轉(zhuǎn)移,同時Twsit基因在腫瘤進展中可以誘導一系列間充質(zhì)標志物的產(chǎn)生。研究表明, Twist基因高表達于多種實體瘤中(包括乳腺癌、前列腺癌、黑色素瘤以及骨肉瘤) ,并能夠促進人乳腺癌和黑色素瘤細胞系的凋亡。之前我們已經(jīng)有研究證實Twist基因在結(jié)腸癌細胞株HCT116中呈高表達,而在SW480細胞株中呈低表達,本實驗通過將高表達Twist基因的質(zhì)粒轉(zhuǎn)染入體外培養(yǎng)的人結(jié)腸癌SW480細胞株中,觀察Twist基因?qū)毎鲋臣扒忠u性的影響。 方法:本研究將高表達Twist基因的質(zhì)粒和陰性對照的空載質(zhì)粒瞬時轉(zhuǎn)染入SW480細胞株中,通過G418篩選后,分別命名為轉(zhuǎn)染組和對照組,經(jīng)過RT-PCR和Western-blot方法的鑒定,獲得穩(wěn)定轉(zhuǎn)染Twist基因的SW480細胞,再通過MTT法繪制轉(zhuǎn)染組細胞和對照組細胞的生長曲線,然后通過細胞劃痕實驗和Transwell侵襲實驗,觀察Twist基因?qū)毎w移能力和侵襲力的影響。 結(jié)果: 1 RT-PCR和Western-blot方法檢測結(jié)果顯示,穩(wěn)定轉(zhuǎn)染Twist基因的SW480細胞中TwistmRNA和Twist蛋白表達量均顯著高于未轉(zhuǎn)染組(P0.05)。 2通過MTT法繪制細胞生長曲線,結(jié)果顯示,從第4天開始,轉(zhuǎn)染Twist基因的SW480細胞生長速度明顯高于對照組(P0.05)。 3通過細胞劃痕實驗顯示,劃痕24小時后轉(zhuǎn)染組細胞遷移了(40.06±5.56),而對照組細胞遷移了(25.24±2.65),差異有統(tǒng)計學意義。48小時后,轉(zhuǎn)染組細胞遷移了(75.77±8.06),而對照組遷移了(35.37±6.79),差異有統(tǒng)計學意義(P0.05)。 4體外侵襲實驗結(jié)果顯示,轉(zhuǎn)染Twist基因的SW480細胞發(fā)生侵襲的個數(shù)為(154±12) ,未轉(zhuǎn)染組細胞侵襲個數(shù)為(73±14),差異有統(tǒng)計學意義(P0.05)。 結(jié)論: 1通過本實驗Twist基因成功轉(zhuǎn)入SW480細胞株并且呈穩(wěn)定表達。 2上調(diào)Twist基因表達后,SW480細胞體外增殖率明顯上升,說明Twist基因能夠增強SW480細胞的體外增殖能力。 3上調(diào)Twist基因表達后,SW480細胞體外遷移能力明顯增強,說明Twist基因能夠增強SW480細胞的遷移能力。 4上調(diào)Twist基因表達后,SW480細胞體外侵襲能力明顯增強,說明Twist基因能夠增強SW480細胞的體外侵襲能力。
[Abstract]:Objective: Twist gene is present in human, Drosophila, mouse basic helix loop helix and other organisms (Basichelix loop, helix, bHLH) protein, as a transcription factor that is expressed in embryonic placenta, some mesoderm mesoderm and adult undifferentiated tissue expression of.Twist protein has 202 amino acids in human mice, 206 amino acids, 166 amino acid sequence of a toad, the amino acid sequence of human and mouse has 96% homology, in different species in the DNA binding domain have 100% conserved sequences, all Twist genes can be combined with the DNA sequence of E-box: the expression of [1] CANNTG.Twist gene regulation at the transcriptional level a variety of genes, originally discovered its biological functions during embryonic development, it can promote epithelial mesenchymal transition (epithelial mesenchymal, transition, EMT) to regulate embryonic development. Nearly Results confirmed that [2,3]Twist has the characteristics of cancer gene, encoding apoptosis proteins through multiple signaling systems involved in tumor suppression and differentiation, blocking P53 pathway by lowering the expression of transcription factor ARF upstream of P53, blocking N-myc signal transmission system, and the inhibition of NF- B pathway. It is involved in the many kinds of epithelial tumor cells EMT and promoting cancer cell invasion and metastasis, while Twsit gene can induce a series of mesenchymal markers in tumor progression. The results show that Twist gene is highly expressed in a variety of solid tumors (including breast cancer, prostate cancer, melanoma and osteosarcoma). Apoptosis and can promote human breast cancer and melanoma cell lines. Before we have proved the high expression of Twist gene in colon cancer cell line HCT116 was a low expression in SW480 cell line, the In order to observe the effect of Twist gene on cell proliferation and invasion, we transfected the plasmid expressing high Twist gene into human colon cancer cell line SW480.
Methods: the study of Twist gene plasmid and negative control plasmid was transiently transfected into SW480 cell line in the high expression by G418 screening, named as transfection group and control group, identified by RT-PCR and Western-blot method, to obtain a stable transfection of Twist gene in SW480 cells, and then through the MTT method to draw the transfected cells the control group and the cell growth curve, followed by cell scratch assay and Transwell invasion assay, to observe the effect of Twist gene on the invasion and migration ability of cells.
Result:
1 RT-PCR and Western-blot detection results showed that the expression level of TwistmRNA and Twist protein in SW480 cells stably transfected with Twist gene was significantly higher than that in the untransfected group (P0.05).
2 the cell growth curve was plotted by MTT method. The results showed that the growth rate of SW480 cells transfected with Twist gene was significantly higher than that of the control group from fourth days (P0.05).
3 by cell scratch test showed that the scratch after 24 hours of transfection group cell migration was (40.06 + 5.56), while the control group, the cell migration (25.24 + 2.65), the difference was statistically significant.48 hours after transfection group cell migration (75.77 + 8.06), while the control group was (35.37 + 6.79) migration. The difference was statistically significant (P0.05).
4 in vitro invasion test showed that the number of SW480 cells transfected with Twist gene was 154 + 12, and the number of invasion in the untransfected group was (73 + 14), the difference was statistically significant (P0.05).
Conclusion:
1 through this experiment, the Twist gene was successfully transferred into the SW480 cell line and showed a stable expression.
2 after the expression of Twist gene was up-regulated, the proliferation rate of SW480 cells increased significantly in vitro, indicating that the Twist gene could enhance the proliferation ability of SW480 cells in vitro.
3 after the expression of Twist gene was up-regulated, the ability of SW480 cells to migrate in vitro was significantly enhanced, indicating that the Twist gene could enhance the migration ability of SW480 cells.
4 after the expression of Twist gene was up-regulated, the invasion ability of SW480 cells in vitro was significantly enhanced, indicating that the Twist gene could enhance the invasiveness of SW480 cells in vitro.

【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R346

【參考文獻】

相關(guān)期刊論文 前5條

1 關(guān)劍;陳孝平;王其;張必翔;張萬廣;張志偉;;Twist在人肝癌細胞系與正常肝細胞系中的表達差異[J];腹部外科;2007年02期

2 姚嬋;來茂德;;上皮間質(zhì)轉(zhuǎn)化(EMT)及其分子機制[J];國際遺傳學雜志;2006年04期

3 時昌文;李杰;孫京杰;曹莉莉;劉愛武;;Twist在胃癌中的表達及其與臨床病理學指標關(guān)系研究[J];中國現(xiàn)代普通外科進展;2007年03期

4 董銳增;大腸腺瘤癌變研究進展[J];實用腫瘤雜志;2004年01期

5 羅庚求;文繼舫;;Twist的研究進展[J];國際病理科學與臨床雜志;2006年05期

，

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