本文關鍵詞： 喉癌 RNA干擾 STAT3 穩(wěn)定轉染 細胞增殖 細胞侵襲　出處：《河北醫(yī)科大學》2010年碩士論文　論文類型：學位論文

【摘要】： 目的:應用RNAi(RNA干擾)技術沉默STAT3(信號轉導子和轉錄激活子3),篩選出能穩(wěn)定表達siRNA-STAT3基因的細胞系,觀察對Hep-2細胞增殖力及侵襲力的影響,在蛋白水平上進一步探討STAT3作用于Hep-2細胞增殖和侵襲可能的機制。以尋找抑制喉癌增殖和侵襲的新方法,為臨床通過基因沉默技術治療喉癌提供實驗支持和理論依據(jù)。 方法:構建重組質粒真核表達載體PGPU6/GFP/Neo-siRNA-STAT3,將重組表達質粒用脂質體LipofactaminTM2000轉染人喉癌Hep-2細胞,通過G418持續(xù)單克隆篩選,Western-blot印跡法鑒定,進而篩選出穩(wěn)定表達siRNA-STAT3的細胞系。實驗分為三組:siRNA-STAT3組、siRNA-shNC組及空白對照組。應用流式細胞術檢測Cyclin Dl、P21、ICAM-1、VEGF蛋白水平的表達情況及細胞周期的變化;采用MTT法觀察接種培養(yǎng)板1d、2d、3d、4d、5d、6d后喉癌Hep-2細胞增殖狀態(tài);MTT法觀察其粘附性的變化;細胞劃痕法觀察其遷移性;Transwell法檢測其侵襲能力的變化。 結果: (1)構建了重組pGPU6/GFP/Neo-siRNA-STAT3質粒載體通過模板退火和載體的線性化等步驟,構建了重組質粒表達載體。酶切結果表明,所有質粒均為陽性重組載體。 (2)細胞轉染與G418篩選熒光顯微鏡下觀察瞬時轉染6小時后較多Hep-2細胞內有綠色熒光蛋白(GFP)表達,24小時后GFP表達明顯增強。G418篩選結果顯示空白對照組的細胞全部死亡,而siRNA-STAT3轉染組和siRNA-shNC轉染組的細胞出現(xiàn)明顯而且數(shù)量較多的細胞克隆集落形成。 (3)Western-blot鑒定p-STAT3的表達結果顯示siRNA-STAT3組的p-STAT3的表達明顯減少,而siRNA-shNC組和空白對照組細胞p-STAT3的表達未見有下降。 (4)MTT檢測細胞增殖狀態(tài)Hep-2細胞在穩(wěn)定轉染siRNA-STAT3后,同空白對照組和轉染siRNA-shNC無關質粒組比,2天后就開始表現(xiàn)出較明顯的抑制效應(P0.05),即使6天后在對照組細胞生長變緩時,siRNA-STAT3組Hep-2細胞生長抑制仍較明顯(P0.05),故其抑制效應隨時間延長愈明顯,并表現(xiàn)出一定的時間抑制效應關系。 (5)平板克隆形成試驗2周后,觀察三組均有一定的單克隆集落形成。結果顯示siRNA- STAT3組Hep-2細胞克隆形成率為(15.17±2.32)%明顯低于siRNA-shNC組(24.33±1.97)%及空白對照組(26.33±2.16)%(P0.05)。 (6)流式細胞術(FCM)檢測細胞周期應用FCM檢測細胞周期分布,轉染重組siRNA-STAT3質粒的Hep-2細胞G1期比率由(60.7±0.9)%上升至(66.0±2.0)%(P0.05),S期所占比率由(34.8±1.1)%降至(24.6±1.9)%(P0.05)。 (7)FCM檢測Cyclin Dl、P21、ICAM-1、VEGF蛋白的表達觀察siRNA-STAT3組Cyclin Dl、ICAM-1、VEGF蛋白表達熒光指數(shù)分別為0.71±0.08(P0.05)、0.83±0.06(P0.05)、0.74±0.02(P0.05),均低于siRNA-shNC組,siRNA-STAT3組P21蛋白表達熒光指數(shù)(1.39±0.10)則高于siRNA-shNC組(P0.05)。 (8)細胞黏附試驗應用MTT法檢測細胞在Matrigel基質上的粘附性,接種96孔培養(yǎng)板20分鐘后觀察各組細胞的黏附率相差不大(P0.05),但有一定的趨勢性,40分鐘后siRNA-STAT3組的黏附率小于siRNA-shNC組和空白對照組(P0.05),這種抑制效應在60分鐘后更加明顯(P0.05)。 (9)細胞遷移試驗在培養(yǎng)板生長48小時后,熒光顯微鏡下見空白對照組和siRNA-shNC組的Hep-2細胞向劃痕區(qū)生長速度明顯快于siRNA-STAT3組,而空白對照組和siRNA-shNC組的Hep-2細胞向劃痕區(qū)生長速度差異不明顯。 (10)體外侵襲試驗各組細胞接種Transwell小室上室內24小時后,400×光學顯微鏡下隨機5個視野中觀察siRNA-STAT3組穿過Matrigel基質和濾膜的細胞(85.20±4.92)明顯少于空白對照組(122.8±7.50)和siRNA-shNC組(126.4±8.73)(P0.05)。 結論: 1、成功構建了重組pGPU6/GFP/Neo-siRNA-STAT3質粒載體。 2、應用G418篩選出穩(wěn)定表達siRNA-STAT3基因的喉癌Hep-2細胞系。 3、抑制STAT3的活性后Hep-2細胞周期形成G1期阻滯,其增殖能力和單克隆形成能力均明顯下降。說明喉癌細胞具有無限增殖能力與STAT3的高表達有一定的關聯(lián)。 4、本實驗應用RNA干擾技術沉默STAT3活性發(fā)現(xiàn)能抑制喉癌Hep-2細胞的粘附、遷移及侵襲能力。推測表達STAT3基因的喉癌細胞因提高了其黏附、遷移和降解基質的能力而增加了侵襲到達周圍的組織及遠處轉移并定植生長的機會。說明STAT3在喉癌的侵襲及轉移中起著重要的作用。 5、阻斷STAT3信號通路后,STAT3下游調控蛋白CyclinD1、VEGF、ICAM-1的表達降低,而P21蛋白的表達升高。提示CyclinD1、P21、VEGF、ICAM-1蛋白可能在STAT3的誘導下參與了STAT3信號通路對喉鱗癌細胞增殖、侵襲及浸潤轉移的作用。
[Abstract]:Objective: to apply RNAi (RNA interference) silencing STAT3 (signal transducer and activator of transcription 3), screened stable expression of siRNA-STAT3 gene in cell lines, to observe its effect on Hep-2 cell proliferation and invasiveness, at the protein level to further explore the mechanism of STAT3 on proliferation and invasion of Hep-2 cells. New method for inhibiting the proliferation and invasiveness of laryngeal carcinoma, and provide experimental and theoretical basis for the gene silencing technique for clinical treatment of laryngeal cancer.
Methods: to construct the recombinant plasmid eukaryotic expression vector PGPU6/GFP/Neo-siRNA-STAT3, the recombinant expression plasmid by liposome transfection of LipofactaminTM2000 human laryngeal carcinoma Hep-2 cells, sustained by G418 monoclonal screening, Western-blot blotting, and then screened siRNA-STAT3 stable expression cell lines. The experiment was divided into three groups: siRNA-STAT3 group, siRNA-shNC group and control group by flow. Detection of Cyclin Dl, P21 ICAM-1, cytometry, change the expression level of VEGF protein and cell cycle; MTT assay was used to observe the inoculated plates 1D, 2D, 3D, 4D, 5D, Hep-2 cell proliferation in laryngeal carcinoma after 6D; observe the changes of adhesion of MTT; cell scratch test was used to observe the movement change; the invasion ability was detected by Transwell.
Result:
(1) a recombinant plasmid vector was constructed by template annealing and vector linearization. The recombinant plasmid expression vector was constructed. The results of pGPU6/GFP/Neo-siRNA-STAT3 digestion showed that all plasmids were positive recombinant vectors.
(2) cells transfected with G418 were observed under fluorescence microscope 6 hours after transient transfection of Hep-2 cells with more green fluorescent protein (GFP) expression, GFP expression was significantly enhanced.G418 test showed the control group cells all died 24 hours after siRNA-STAT3 transfection group and siRNA-shNC transfection group the cell clone and obvious a large number of colony formation.
(3) the expression of p-STAT3 was identified by Western-blot. Results showed that the expression of p-STAT3 in siRNA-STAT3 group was significantly reduced, while the expression of p-STAT3 in siRNA-shNC group and blank control group did not decrease.
(4) MTT detection of cell proliferation in Hep-2 cells stably transfected with siRNA-STAT3, blank control group and transfection of siRNA-shNC plasmid to group, 2 days later began to exhibit obvious inhibitory effect (P0.05), even after 6 days in the control group the cell growth slowed, siRNA-STAT3 group was still significantly inhibited the growth of Hep-2 cells (P0.05), the inhibition effect increased with time is more obvious, and show some time inhibition effect relationship.
(5) after 2 weeks, the colony formation of three groups was observed. The results showed that the colony formation rate of Hep-2 cells in siRNA- STAT3 group was (15.17 + 2.32)%, which was significantly lower than that in siRNA-shNC group (24.33 + 1.97)% and blank control group (26.33 + 2.16)% (P0.05).
(6) flow cytometry (FCM) was used to detect cell cycle. The cell cycle distribution was detected by FCM. The G1 phase ratio of Hep-2 cells transfected with recombinant siRNA-STAT3 plasmid increased from (60.7 + 0.9)% to (66 + 2)% (P0.05), and the proportion of S phase decreased from (34.8 + 1.1)% to (24.6 + 1.9)% (P0.05).
(7) FCM Cyclin Dl P21, ICAM-1 assay, and the expression of VEGF protein was observed in the siRNA-STAT3 group Cyclin Dl, ICAM-1, VEGF protein expression and fluorescence index were 0.71 + 0.08 (P0.05), 0.83 + 0.06, 0.74 + 0.02 (P0.05) (P0.05), siRNA-shNC group was higher than that in siRNA-STAT3 group the expression of P21 protein fluorescence index (1.39 + 0.10) was higher than that of siRNA-shNC group (P0.05).
(8) adhesion to detect cell adhesion test using MTT method on Matrigel matrix, inoculated in 96 well culture plates for 20 minutes were observed after the cell adhesion rate difference (P0.05), but there is a certain trend, 40 minutes after the adhesion rate of siRNA-STAT3 group was lower than siRNA-shNC group and blank control group (P0.05) this inhibitory effect is more obvious, in 60 minutes (P0.05).
(9) cell migration test in culture plates after 48 h of growth under fluorescence microscope showed that the control group and the siRNA-shNC group of Hep-2 cells to wound area faster than the growth rate of siRNA-STAT3 group and blank control group and siRNA-shNC group of Hep-2 cells to wound area growth rate was not significantly different.
(10) in vitro invasion assay cells were inoculated with Transwell cells on the indoor 24 hours later, 400 x optical microscope 5 random field observation group siRNA-STAT3 through Matrigel matrix and cell membrane (85.20 + 4.92) were significantly less than the control group (122.8 + 7.50) and siRNA-shNC group (126.4 + 8.73) (P0.05).
Conclusion:
1, the recombinant plasmid vector of pGPU6/GFP/Neo-siRNA-STAT3 was successfully constructed.
2, using G418 to screen the Hep-2 cell line of the larynx cancer that stably expressed the siRNA-STAT3 gene.
3, after inhibiting STAT3 activity, the Hep-2 cell cycle formed G1 phase arrest, and its proliferative capacity and monoclonal ability were significantly decreased. This indicates that the proliferation ability of laryngeal carcinoma cells is correlated with the high expression of STAT3.
4, we used RNA interference technology to silence STAT3 activity that can inhibit the adhesion of laryngeal carcinoma Hep-2 cells, migration and invasion ability. It is suggested that the expression of STAT3 gene in laryngeal carcinoma cells by improving the adhesion, migration and degradation ability of matrix and increased the invasion to the surrounding tissue and distant metastasis and opportunity for colonization and growth. It shows that STAT3 plays an important role in the invasion and metastasis of laryngeal carcinoma.
5, after blocking the STAT3 pathway downstream of STAT3 regulatory protein CyclinD1, VEGF, decreased the expression of ICAM-1, while the expression of P21 protein increased. P21, VEGF, CyclinD1, ICAM-1 in the induction of STAT3 protein may be involved in STAT3 signaling pathway on the proliferation of laryngeal squamous cell carcinoma, invasion and metastasis effect.

【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】：2010
【分類號】：R739.6

【參考文獻】

相關期刊論文 前10條

1 劉家云,李慶霞,黃紅艷,馬龍洋,鮑煒,賈林濤,薛采芳,劉軍,王成濟,楊安鋼;瞬時轉染和穩(wěn)定轉染對RNAi抑制乙型肝炎病毒S基因表達的影響[J];第四軍醫(yī)大學學報;2005年11期

2 王俊閣;李曉明;路秀英;;JAK激酶抑制劑AG490聯(lián)合順鉑對喉癌細胞STAT3信號轉導通路的抑制作用[J];第四軍醫(yī)大學學報;2007年21期

3 軒小燕;李珊珊;鄭獻召;李娜;王豐;高遠;張紅燕;閆愛華;;RNA干擾Stat3基因對EC-1細胞侵襲轉移和增殖能力的影響[J];第四軍醫(yī)大學學報;2009年07期

4 王俊閣;李曉明;路秀英;皮麗宏;;Stat3信號傳導通路在喉癌中的表達及意義[J];中國耳鼻咽喉頭頸外科;2009年03期

5 洪珍珍;唐瞻貴;李金茂;俞志維;;口腔鱗癌Stat3基因的表達研究和SH2功能區(qū)編碼序列測定[J];臨床口腔醫(yī)學雜志;2007年06期

6 步明強;徐偉;呂正華;李會政;;下咽鱗癌Ets-1、磷酸化STAT3的表達及其與侵襲轉移的關系[J];山東大學耳鼻喉眼學報;2008年06期

7 郭慧;杜波;李曉明;路秀英;;攜EGFP的STAT3載體構建、表達及其對HEP-2細胞的增殖抑制作用[J];山東醫(yī)藥;2008年03期

8 李素梅;鐘雪云;林琛蒞;劉坤平;彭輝;方茅;;Stat3、CyclinD1及Bcl-2在食管鱗癌組織芯片中的表達及意義[J];現(xiàn)代腫瘤醫(yī)學;2009年04期

9 楊俊波;毛志福;蔡享道;;STAT3在食管鱗狀細胞癌中的表達和意義[J];消化外科;2006年03期

10 岳利華;程金妹;張鵬飛;林功標;張榕;易自翔;;STAT3,STAT5在EBV相關鼻NK/T細胞淋巴瘤中的表達[J];中國耳鼻咽喉顱底外科雜志;2006年02期

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