發(fā)布時(shí)間：2018-06-04 10:01

  本文選題：ILK + RNA干擾�。� 參考：《現(xiàn)代口腔醫(yī)學(xué)雜志》2015年01期

【摘要】：目的研究整合素連接激酶(ILK)在細(xì)胞中的功能,構(gòu)建ILK基因sh RNA慢病毒載體,并對(duì)其在舌鱗癌細(xì)胞株Tca-8113中沉默效果進(jìn)行鑒定。方法針對(duì)ILK基因有效靶序列的3個(gè)位點(diǎn),設(shè)計(jì)合成3對(duì)oligo DNA,退火形成雙鏈DNA,與線性化p ENTR/U6載體連接產(chǎn)生sh ILK-LV慢病毒載體,篩選出陽(yáng)性克隆測(cè)序鑒定,用sh ILK-LV載體、包裝質(zhì)粒Packaging Mix共轉(zhuǎn)染293T包裝細(xì)胞,包裝產(chǎn)生慢病毒,以293T細(xì)胞中綠色熒光蛋白(GFP)的表達(dá)數(shù)目測(cè)定病毒滴度并確定恰當(dāng)?shù)腗OI值。獲得重組慢病毒后感染人舌鱗癌細(xì)胞株Tca-8113,用q PCR及Western blot檢測(cè)ILK在Tca-8113細(xì)胞中的表達(dá)。結(jié)果測(cè)序證實(shí)成功構(gòu)建了3個(gè)ILK-sh RNA慢病毒載體,分別轉(zhuǎn)染Tca-8113細(xì)胞后用sybr法檢測(cè)出sh RNA-ILK-370組ILK的表達(dá)明顯下降,ΔΔCt值為0.268。用sh RNA-ILK-370慢病毒載體包裝病毒,測(cè)定病毒滴度,并得出當(dāng)MOI=30-60時(shí),細(xì)胞陽(yáng)性比率最高。用病毒感染Tca-8113細(xì)胞后再用抗生素篩選穩(wěn)轉(zhuǎn)株,后用q PCR檢測(cè)干擾效率達(dá)90.5%,Western blot檢測(cè)ILK表達(dá)明顯被抑制。結(jié)論成功構(gòu)建sh ILK-LV慢病毒載體并建立了穩(wěn)定的Tca-8113-sh ILK細(xì)胞模型,為研究ILK在舌鱗癌信號(hào)轉(zhuǎn)導(dǎo)通路中的作用提供了堅(jiān)實(shí)基礎(chǔ)。
[Abstract]:Objective to study the function of integrin linked kinase (ILK) in cells, construct ILK gene sh RNA lentivirus vector, and identify its silencing effect in tongue squamous cell carcinoma cell line Tca-8113. Methods three pairs of ILK DNA pairs were designed and synthesized for three sites of the effective target sequence of ILK gene. After annealing, shILK-LV lentivirus vector was generated by ligating with linearized p ENTR/U6 vector to produce sh ILK-LV lentivirus vector. The positive clones were sequenced and identified by sh ILK-LV vector. The packaging plasmid Packaging Mix was co-transfected into 293T packaging cells to produce lentivirus. The virus titer was determined by the expression number of green fluorescent protein (GFP) in 293T cells and the appropriate MOI value was determined. Human tongue squamous cell carcinoma cell line Tca-8113 was infected with recombinant lentivirus. The expression of ILK in Tca-8113 cells was detected by Q PCR and Western blot. Results three ILK-sh RNA lentivirus vectors were successfully constructed by sequencing. After transfection into Tca-8113 cells, the expression of ILK in sh RNA-ILK-370 group was detected by sybr method, and 螖 Ct value was 0.268. Sh RNA-ILK-370 lentivirus vector was used to package the virus, the titer of the virus was determined, and when MOI=30-60 was used, the positive rate of the cells was the highest. The stable transgenic strain was screened with antibiotics after infection with virus, and the interference efficiency of Q PCR was 90.5%. The expression of ILK was obviously inhibited by Western blot. Conclusion the sh ILK-LV lentivirus vector was successfully constructed and a stable Tca-8113-sh ILK cell model was established, which provides a solid foundation for the study of the role of ILK in the signal transduction pathway of tongue squamous cell carcinoma.
【作者單位】： 蘭州大學(xué)第一臨床醫(yī)學(xué)院;蘭州大學(xué)第一醫(yī)院腫瘤外科;甘肅省腫瘤醫(yī)院;蘭州大學(xué)第一醫(yī)院口腔科;
【基金】：甘肅省衛(wèi)生廳衛(wèi)生行業(yè)科研計(jì)劃資助項(xiàng)目(GSWST2010-04)
【分類號(hào)】：R739.8

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