慢病毒转染TLR_3基因的小分子RNA对人肺腺癌A549细胞增殖及免疫逃逸影响的研究
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摘要
第一部分TLR3在A549细胞中的表达
目的：Toll样受体家族(Toll-like receptors，TLRs)是非常重要的天然免疫分子，并被视为联系天然免疫和获得性免疫的桥梁，近年来随着天然免疫研究的不断发展，人们逐渐关注TLRs和机体肿瘤的关系。本研究旨在探讨TLR_3在人肺腺癌细胞(A549细胞)和人支气管上皮细胞(HBE细胞)中的表达情况及意义。
方法：体外培养A549细胞和HBE细胞，分别采用细胞免疫组化技术、实时定量PCR(Real-time Quantitative Reverse Transcriptase-Polymerase Chain Reaction，Real-Time Quantitative PCR)及western blot检测TLR_3 mRNA和蛋白在2种细胞中表达的差异。
结果：细胞免疫组化染色，TLR_3主要表达于2种细胞的细胞膜和胞浆中，成棕黄色颗粒状，A549细胞中TLR_3 mRNA的表达高于HBE细胞，TLR_3蛋白在A549细胞中的表达高于HBE细胞。(p＜0.01)
结论：TLR_3在A549细胞中表达高于HBE细胞，提示TLR_3可能参与促进人肺腺癌细胞的生长进展过程，具体机制有待进一步深入研究。
第二部分TLR_3基因RNAi慢病毒载体的构建
目的：观察慢病毒表达载体介导的RNA干扰(RNAi)对人肺腺癌细胞株A549细胞Toll样受体3(Toll-like receptor 3，TLR_3)表达的影响，为后续的以TLR_3基因为靶点的肺癌研究和治疗奠定基础。
方法：应用基因工程技术，筛选出4条针针对TLR_3基因的RNAi靶序列，分别与pCCL-CFP载体连接，构建4个重组慢病毒表达载体TLR3-RNAi-LV 1#，TLR3-RNAi-LV 2#，TLR3-RNAi-LV 3#，TLR3-RNAi-LV 4#；将连接产物转化到DHSα感受态细胞，经PCR筛选阳性克隆、测序鉴定。将TLR3-RNAi-LV、pHelper 1.0、pHelper 2.0共转染293T细胞，包装产生慢病毒颗粒并测定病毒滴度。将包装产生的4种重组慢病毒分别感染A549细胞，实时定量PCR和Western印迹检测A549细胞TLR3 mRNA和蛋白的表达，并与未转染及空转染细胞进行比较。
结果：4个慢病毒载体PCR和测序结果与预期结果一致，经包装产生的病毒滴度分别为2E+8、2E+8、2E+8、1E+8 TU／ml。感染A549细胞后，TLR3基因mRNA和蛋白的表达量与未感染慢病毒的细胞组及空载体感染组相比均明显下降(P＜0.05)，其中TLR3-RNAi-LV 4#作用较明显，使mRNA表达下降80％，蛋白表达下降68％(P＜0.05)。
结论：成功构建针对TLR3基因的4个慢病毒载体TLR3-RNAi-LV，体外感染A549细胞后可有效抑制TLR3基因和蛋白的表达。
第三部分转染TLR3-RNAi慢病毒载体对A549细胞增殖及免疫逃逸的影响
目的：探讨通过RNA干扰技术，使人肺腺癌细胞中TLR3的表达发生变化，对人肺腺癌细胞的增殖、抗凋亡及免疫逃逸方面的影响。
方法：利用第二部分实验构建并筛选出的TLR3-RNAi慢病毒载体，转染A549细胞，建立TLR3基因沉默的人肺腺癌稳转细胞株，同时给予慢病毒转染组、未转染组和阴性对照组TLR3的配体Poly(I：C)刺激后，分别通过流式细胞术检测B7-H1的表达，实时定量PCR方法检测PGE-2的表达，western blot方法检测磷酸化P38的含量，以及用Annexin V／PI双染色法检测A549细胞的凋亡。
结果：在Poly(I：C)刺激时间及浓度相同的情况下，转染TLR3-RNAi慢病毒载体的A549细胞组中B7-H1阳性细胞百分比为23.35±0.071％，未转染慢病毒载体组为55.25±0.015％，阴性对照组为48.75±0.020％，慢病毒转染组明显小于未转染组及阴性对照组(p＜0.01)；运用2-~(△△)Ct数据分析法，转染TLR3-RNAi慢病毒载体的A549细胞组中PGE-2表达明显少于未转染慢病毒载体组及阴性对照组(p＜0.01)；转染TLR3-RNAi慢病毒载体的A549细胞组中磷酸化P38的百分比相对值为6.37±0.28％，未转染慢病毒载体组中磷酸化P38的百分比相对值为33.63±6.752％，阴性对照组中磷酸化P38的百分比相对值为17.36±2.24％，病毒载体转染组磷酸化P38的含量明显低于未转染慢病毒载体组及阴性对照组(p＜0.01)；凋亡实验显示，转染TLR3-RNAi慢病毒载体的A549细胞组细胞凋亡率为44.4±1.41％，未转染慢病毒载体组细胞凋亡率为34.7±0.89％，阴性对照组细胞凋亡率为36.5±1.12％，慢病毒转染组明显高于未转染慢病毒载体组和阴性对照组。(P＜0.01)，
结论：TLR3基因沉默抑制了A549细胞中P38、PGE-2、B7-H1的表达，并降低了细胞的抗凋亡能力。
PartⅠ: The expression and significance of TLR_3 in humanpulmonary adenocarcinoma cells line A549
Objective : Being considered as a bridge between the innate immunity and acquiredimmunity, Toll-like receptors are very important innate immunity moleculars. Recentresearches on the innate immunity have focused on the relationship between Toll-likereceptors and human tumor, This paper investgates the expression and significance ofTLR_3 in human pulmonary adenocarcinoma cell (A549 cell) and human bronchialepithelial cell (HBE cell).
Methods: After culturing A549 cell and HBE cell in vitro, the expression of TLR_3mRNA and protein in both cells were detected by immunocytochemistry, real-timequantitative reverse transcriptase-polymerase chain reaction (Real-Time Quantitative PCR)and western blot, respectively.
Results: By immunocytochemistry staining, TLR_3 is mainly expressed in both cells'cell membrane and endochylema as brown-yellow material. It shows that the expressions of TLR_3 mRNA and protein in A549 cell are stronger than those in HBE cell (p＜0.01).
Conclusion: The resusts suggest TLR_3 might cause the progression ofhuman pulmonary adenocarcinoma, and the mechanism needs to be furtherinvestgated.
PartⅡ: Construct lentiviral RNAi vectors targeted againstToll-like receptor 3 gene
Objective : To observe the influence of lentiviral vector-mediated RNA interferenceon expression of human TLR3(Toll-like receptor 3) gene in human pulmonaryadenocarcinoma cell line A549, so as to pave a way for TLR3 gene-targeted gene therapyof pulmonary adenocarcinoma.
Methods: Gene engineering technique was used to screen four RNA interferencesequences targeting TLR3 gene; the sequences were separately cloned into the pCCL-GFPvector to construct TLR3-RNAi-LV 1# , TLR3-RNAi-LV 2# , TLR3-RNAi-LV 3#,TLR3-RNAi-LV 4#, which were subsequently confirmed by PCR and DNA sequencinganalysis. The titer of lentivirus was determined after 293T cells were contransfected withTLR3-RNAi-LV、pHelper 1.0、pHelper 2.0. The four kinds of recombinant lentiviruseswere injected into A549 cells and the TLR3 mRNA and protein expression were examinedby real-time RT-PCR and Western blotting, respectively, and the result was compared withthose of the non-transfected and blank vector transfected A549 cells.
Results: PCR analysis and DNA sequencing confirmed that the four TLR3 vshRNAsequences were successfully inserted into the lentiviral vectors. The titer of concentratedvirus were 2E+8、2E+8、2E+8、1E+8 TU/ml ,respectively. TLR3 expression in A549 cellswas significantly inhibited at both mRNA and protein levels compared with thenon-tansfected and empty vector transfected A549 cells.(P＜0.05) After transfection withTLR3-RNAi-LV 4#, TLR3 mRNA expression decreased by 80％, TLR3 protein expression decreased by 68％. (P＜0.05)
Conclusion: Four lentiviral RNAi vectors of TLR3 gene have been successfullyconstructed, and they can effectively inhibit the expression of TLR3 gene in A549 cellsin vitro.
PartⅢ: Effect of lentivirus-induced siRNA targeted againstToll-like receptor 3 on the proliferation and immune escape ofhuman pulmonary adenocarcinoma cells line A549
Objective: To evalute the effect of Toll-like receptor 3 on the cell proliferate、anti-apoptotic and immune escape in human pulmonary adenocarcinoma cells line A549with different expressions of Toll-like receptor 3.
Methods: To transfecte A549 cell line with TLR3-RNAi lentiviral vector in thesecond experiment, and transfected、non-transfected and blank vector transfected A549cells were all incubated with Poly(I:C),the ligand of TLR3. The expressions of B7-H1、PGE-2 and phosphorylase p38 were respectively evaluated by flow cytometry、real-timequantitative PCR and western blot experiments. Apoptosis of those cells was detected byflow cytometry with AnnexinV FITC/PI double staining.
Results: Under the condition of the same time and concentration of stimulation withPoly(I:C), the percentage of B7-H1-positive cells of TLR3-RNAi lentiviral vectortransfected A549 cells is 23.35±0.071％, those of the non-transfected and blank vectortransfected A549 cells were 55.25±0.015％、48.75±0.020％, respectively。Theexpression of B7-H1 of TLR3-RNAi lentiviral vector transfected A549 cells wassignificantly lower than those of the non-transfected and blank vector transfected A549cells (p＜0.01); To analyze with the 2-ΔΔCt method, The expression of PGF-2 ofTLR3-RNAi lentiviral vector transfected A549 cells was significantly lower than thoseof the non-transfected and blank vector transfected A549 cells (p＜0.01); the relativelypercentage of phosphorylase p38 of TLR3-RNAi lentiviral vector transfected A549 cellsis 6.37±0.28％those of the non-transfected and blank vector transfected A549 cells were 33.63±6.752％、17.36±2.24％, respectively。The expression of phosphorylase p38of TLR3-RNAi lentiviral vector transfected A549 cells was significantly lower thanthose of the non-transfected and blank vector transfected A549 cells (p＜0.01); Thepercentage of the apoptosis cells of TLR3-RNAi lentiviral vector transfected A549 cellsis 44.4±1.41％, those of the non-transfected and blank vector transfected A549 cellswere 34.7±0.89％、36.5±1.12％, respectively。The percentage of the apoptosis cells ofTLR3-RNAi lentiviral vector transfected A549 cells was significantly higher than thoseof the non-transfected and blank vector transfected A549 cells (p＜0.01 )
Conclusions: Down-regulation of TLR3 led to inhibit the expression ofB7-H1、PGE-2 and phosphorylase p38 in human pulmonary adenocarcinoma cells lineA549, weaken the anti-apoptosis ability of human pulmonary adenocarcinoma cells lineA549.
目的：Toll样受体家族(Toll-like receptors，TLRs)是非常重要的天然免疫分子，并被视为联系天然免疫和获得性免疫的桥梁，近年来随着天然免疫研究的不断发展，人们逐渐关注TLRs和机体肿瘤的关系。本研究旨在探讨TLR_3在人肺腺癌细胞(A549细胞)和人支气管上皮细胞(HBE细胞)中的表达情况及意义。
方法：体外培养A549细胞和HBE细胞，分别采用细胞免疫组化技术、实时定量PCR(Real-time Quantitative Reverse Transcriptase-Polymerase Chain Reaction，Real-Time Quantitative PCR)及western blot检测TLR_3 mRNA和蛋白在2种细胞中表达的差异。
结果：细胞免疫组化染色，TLR_3主要表达于2种细胞的细胞膜和胞浆中，成棕黄色颗粒状，A549细胞中TLR_3 mRNA的表达高于HBE细胞，TLR_3蛋白在A549细胞中的表达高于HBE细胞。(p＜0.01)
结论：TLR_3在A549细胞中表达高于HBE细胞，提示TLR_3可能参与促进人肺腺癌细胞的生长进展过程，具体机制有待进一步深入研究。
第二部分TLR_3基因RNAi慢病毒载体的构建
目的：观察慢病毒表达载体介导的RNA干扰(RNAi)对人肺腺癌细胞株A549细胞Toll样受体3(Toll-like receptor 3，TLR_3)表达的影响，为后续的以TLR_3基因为靶点的肺癌研究和治疗奠定基础。
方法：应用基因工程技术，筛选出4条针针对TLR_3基因的RNAi靶序列，分别与pCCL-CFP载体连接，构建4个重组慢病毒表达载体TLR3-RNAi-LV 1#，TLR3-RNAi-LV 2#，TLR3-RNAi-LV 3#，TLR3-RNAi-LV 4#；将连接产物转化到DHSα感受态细胞，经PCR筛选阳性克隆、测序鉴定。将TLR3-RNAi-LV、pHelper 1.0、pHelper 2.0共转染293T细胞，包装产生慢病毒颗粒并测定病毒滴度。将包装产生的4种重组慢病毒分别感染A549细胞，实时定量PCR和Western印迹检测A549细胞TLR3 mRNA和蛋白的表达，并与未转染及空转染细胞进行比较。
结果：4个慢病毒载体PCR和测序结果与预期结果一致，经包装产生的病毒滴度分别为2E+8、2E+8、2E+8、1E+8 TU／ml。感染A549细胞后，TLR3基因mRNA和蛋白的表达量与未感染慢病毒的细胞组及空载体感染组相比均明显下降(P＜0.05)，其中TLR3-RNAi-LV 4#作用较明显，使mRNA表达下降80％，蛋白表达下降68％(P＜0.05)。
结论：成功构建针对TLR3基因的4个慢病毒载体TLR3-RNAi-LV，体外感染A549细胞后可有效抑制TLR3基因和蛋白的表达。
第三部分转染TLR3-RNAi慢病毒载体对A549细胞增殖及免疫逃逸的影响
目的：探讨通过RNA干扰技术，使人肺腺癌细胞中TLR3的表达发生变化，对人肺腺癌细胞的增殖、抗凋亡及免疫逃逸方面的影响。
方法：利用第二部分实验构建并筛选出的TLR3-RNAi慢病毒载体，转染A549细胞，建立TLR3基因沉默的人肺腺癌稳转细胞株，同时给予慢病毒转染组、未转染组和阴性对照组TLR3的配体Poly(I：C)刺激后，分别通过流式细胞术检测B7-H1的表达，实时定量PCR方法检测PGE-2的表达，western blot方法检测磷酸化P38的含量，以及用Annexin V／PI双染色法检测A549细胞的凋亡。
结果：在Poly(I：C)刺激时间及浓度相同的情况下，转染TLR3-RNAi慢病毒载体的A549细胞组中B7-H1阳性细胞百分比为23.35±0.071％，未转染慢病毒载体组为55.25±0.015％，阴性对照组为48.75±0.020％，慢病毒转染组明显小于未转染组及阴性对照组(p＜0.01)；运用2-~(△△)Ct数据分析法，转染TLR3-RNAi慢病毒载体的A549细胞组中PGE-2表达明显少于未转染慢病毒载体组及阴性对照组(p＜0.01)；转染TLR3-RNAi慢病毒载体的A549细胞组中磷酸化P38的百分比相对值为6.37±0.28％，未转染慢病毒载体组中磷酸化P38的百分比相对值为33.63±6.752％，阴性对照组中磷酸化P38的百分比相对值为17.36±2.24％，病毒载体转染组磷酸化P38的含量明显低于未转染慢病毒载体组及阴性对照组(p＜0.01)；凋亡实验显示，转染TLR3-RNAi慢病毒载体的A549细胞组细胞凋亡率为44.4±1.41％，未转染慢病毒载体组细胞凋亡率为34.7±0.89％，阴性对照组细胞凋亡率为36.5±1.12％，慢病毒转染组明显高于未转染慢病毒载体组和阴性对照组。(P＜0.01)，
结论：TLR3基因沉默抑制了A549细胞中P38、PGE-2、B7-H1的表达，并降低了细胞的抗凋亡能力。
PartⅠ: The expression and significance of TLR_3 in humanpulmonary adenocarcinoma cells line A549
Objective : Being considered as a bridge between the innate immunity and acquiredimmunity, Toll-like receptors are very important innate immunity moleculars. Recentresearches on the innate immunity have focused on the relationship between Toll-likereceptors and human tumor, This paper investgates the expression and significance ofTLR_3 in human pulmonary adenocarcinoma cell (A549 cell) and human bronchialepithelial cell (HBE cell).
Methods: After culturing A549 cell and HBE cell in vitro, the expression of TLR_3mRNA and protein in both cells were detected by immunocytochemistry, real-timequantitative reverse transcriptase-polymerase chain reaction (Real-Time Quantitative PCR)and western blot, respectively.
Results: By immunocytochemistry staining, TLR_3 is mainly expressed in both cells'cell membrane and endochylema as brown-yellow material. It shows that the expressions of TLR_3 mRNA and protein in A549 cell are stronger than those in HBE cell (p＜0.01).
Conclusion: The resusts suggest TLR_3 might cause the progression ofhuman pulmonary adenocarcinoma, and the mechanism needs to be furtherinvestgated.
PartⅡ: Construct lentiviral RNAi vectors targeted againstToll-like receptor 3 gene
Objective : To observe the influence of lentiviral vector-mediated RNA interferenceon expression of human TLR3(Toll-like receptor 3) gene in human pulmonaryadenocarcinoma cell line A549, so as to pave a way for TLR3 gene-targeted gene therapyof pulmonary adenocarcinoma.
Methods: Gene engineering technique was used to screen four RNA interferencesequences targeting TLR3 gene; the sequences were separately cloned into the pCCL-GFPvector to construct TLR3-RNAi-LV 1# , TLR3-RNAi-LV 2# , TLR3-RNAi-LV 3#,TLR3-RNAi-LV 4#, which were subsequently confirmed by PCR and DNA sequencinganalysis. The titer of lentivirus was determined after 293T cells were contransfected withTLR3-RNAi-LV、pHelper 1.0、pHelper 2.0. The four kinds of recombinant lentiviruseswere injected into A549 cells and the TLR3 mRNA and protein expression were examinedby real-time RT-PCR and Western blotting, respectively, and the result was compared withthose of the non-transfected and blank vector transfected A549 cells.
Results: PCR analysis and DNA sequencing confirmed that the four TLR3 vshRNAsequences were successfully inserted into the lentiviral vectors. The titer of concentratedvirus were 2E+8、2E+8、2E+8、1E+8 TU/ml ,respectively. TLR3 expression in A549 cellswas significantly inhibited at both mRNA and protein levels compared with thenon-tansfected and empty vector transfected A549 cells.(P＜0.05) After transfection withTLR3-RNAi-LV 4#, TLR3 mRNA expression decreased by 80％, TLR3 protein expression decreased by 68％. (P＜0.05)
Conclusion: Four lentiviral RNAi vectors of TLR3 gene have been successfullyconstructed, and they can effectively inhibit the expression of TLR3 gene in A549 cellsin vitro.
PartⅢ: Effect of lentivirus-induced siRNA targeted againstToll-like receptor 3 on the proliferation and immune escape ofhuman pulmonary adenocarcinoma cells line A549
Objective: To evalute the effect of Toll-like receptor 3 on the cell proliferate、anti-apoptotic and immune escape in human pulmonary adenocarcinoma cells line A549with different expressions of Toll-like receptor 3.
Methods: To transfecte A549 cell line with TLR3-RNAi lentiviral vector in thesecond experiment, and transfected、non-transfected and blank vector transfected A549cells were all incubated with Poly(I:C),the ligand of TLR3. The expressions of B7-H1、PGE-2 and phosphorylase p38 were respectively evaluated by flow cytometry、real-timequantitative PCR and western blot experiments. Apoptosis of those cells was detected byflow cytometry with AnnexinV FITC/PI double staining.
Results: Under the condition of the same time and concentration of stimulation withPoly(I:C), the percentage of B7-H1-positive cells of TLR3-RNAi lentiviral vectortransfected A549 cells is 23.35±0.071％, those of the non-transfected and blank vectortransfected A549 cells were 55.25±0.015％、48.75±0.020％, respectively。Theexpression of B7-H1 of TLR3-RNAi lentiviral vector transfected A549 cells wassignificantly lower than those of the non-transfected and blank vector transfected A549cells (p＜0.01); To analyze with the 2-ΔΔCt method, The expression of PGF-2 ofTLR3-RNAi lentiviral vector transfected A549 cells was significantly lower than thoseof the non-transfected and blank vector transfected A549 cells (p＜0.01); the relativelypercentage of phosphorylase p38 of TLR3-RNAi lentiviral vector transfected A549 cellsis 6.37±0.28％those of the non-transfected and blank vector transfected A549 cells were 33.63±6.752％、17.36±2.24％, respectively。The expression of phosphorylase p38of TLR3-RNAi lentiviral vector transfected A549 cells was significantly lower thanthose of the non-transfected and blank vector transfected A549 cells (p＜0.01); Thepercentage of the apoptosis cells of TLR3-RNAi lentiviral vector transfected A549 cellsis 44.4±1.41％, those of the non-transfected and blank vector transfected A549 cellswere 34.7±0.89％、36.5±1.12％, respectively。The percentage of the apoptosis cells ofTLR3-RNAi lentiviral vector transfected A549 cells was significantly higher than thoseof the non-transfected and blank vector transfected A549 cells (p＜0.01 )
Conclusions: Down-regulation of TLR3 led to inhibit the expression ofB7-H1、PGE-2 and phosphorylase p38 in human pulmonary adenocarcinoma cells lineA549, weaken the anti-apoptosis ability of human pulmonary adenocarcinoma cells lineA549.
引文
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