p65基因序列shRNA慢病毒载体构建及其作用功能的初步研究

Objective To obtain shRNA sequences that can stably block the expression of Nuclear Factor kappa- B (p65) in the prostate cancer cell line LNCaP and construct the lentivirus vector.And validate the gene function of p65 in the cell line. Methods According to p65 genetic information, we design siRNA1, siRNA2, siRNA3 those three siRNA sequences targeting the ods area of p65 gene and then form the corresponding four pairs of complementary single strand DNA of shRNA, including the sense strand and the antisense strand. The synthetic shRNA sequence was inserted into the empty pSIH1-H1-copGFP shRNA Vector, and after transfecting the prostate cancer cells , the inhibitory effect of p65 mRNA by different sequences was detected through real-time PCR, and the inhibitory effect of p65 protein expression was detected by Western-blotting. Thus we can obtain highly effective shRNA sequences in the inhibition of p65 in prostate cancer cells. MTT, flow cytometry, transwell were chosen to test the cell growth, migration and invasive power in vitro to compare the difference of the experimental group, control group and negative group. Results The third shRNA sequence had the best inhibitory effect and the inhibitory effect of p65 mRNA in prostate cancer cell line was 59 % and the protein was 81%. It's position locates in p65 (NM_021975 ) 1096-1113 and it's stemloop sequence is 5'-GATCCGCCCTATCCCTTTACGTCATTCAAGAGATGACGTAAAGGGATAGGGCTTTTTG-3'. After transfecting, the prostate cancer cell line had the low expression of p65 stably. Through MTT, we got the growth curve, which showed that the growth ability of experimental group was significantly decreased compared with the control group and the Logarithmic growth didn't appear in the first 96 hours. Flow cytometry test displayed that the percentage of G0-G1-phase cells in experimental group was 61.49%, and the control group was 44.89%, idle group was 41.52%, which was increasing oberviously. The S-phase cells in the experimental group was 28.58%, compared with the 47.36% and 46. 10% diminished. The results of transwell showed that the experimental group had 16. 5000±6. 62076 cells and the other two groups had 45. 6333 13. 54159 and 36. 8333±5. 68412 cells, which showed the invasive power of experimental group was significantly declined(P＜0.05).Conclusions It's successful to obtain shRNA sequences that can stably block the expression of p65 in the prostate cancer cell line LNCaP and construct the lentivirus vector. p65 can positively regulates the biological behavior of prostate cancer LNCaP cell line in the cell growth, migration and invasive power.     

Lentivirus-mediated SMO RNA interference inhibits SMO expression and cell proliferation,and affects the cell cycle in LNCaP and PC3 cancer cell lines

Smoothened （SMO） is an important member of the Hedgehog signaling pathway. We constructed a specific recombinant lentiviral vector for RNA interference,targeting the SMO gene （NM_005631） to observe its effect on SMO expression,cell proliferation and the cell cycle in the human androgen-sensitive prostate cancer cell line,LNCaP,and in the androgen-independent prostate cancer cell line,PC3. Four siRNA sequences were designed and inserted into a lentiviral vector pGCSIL-GFP to construct four recombinant vectors. The vector with the highest interfering efficiency was co-transfected with packaging vectors （pHelper1.0 and pHelper2.0） in 293T cells to assemble lentivirus particles by liposome for infecting LNCaP and PC3 cell lines,respectively. The expression level of SMO mRNA,tumor cell proliferation and cell cycle were measured by quantitative realtime polymerase chain reaction （qRT-PCR）,3-（4,5）-dimethylthiahiazo （-z-yl）-3,5-di-phenytetrazoliumromide （MTT） assay and flow eytometry,respectively. Sequence results showed that recombinant lentiviral vectors were constructed successfully.pGCSIL-GFP-723 had the highest interfering efficiency,named Lv-SIL-SMO723 after co-transfection,with which LNCaP and PC3 cell lines were infected. Compared with the control groups,results showed significantly decreased （P〈0.05） SMO mRNA expressions of LNCaP and PC3,lower mean percentage of S-phase cells and higher mean percentage of G_2/M phase cells,as well as obviously slow proliferation （P〈0.01） of LNCaP in the infected group. Yet,the proliferation of PC3 was not altered （P〉0.05）. In conclusion,the recombinant lentivirus particles were able to suppress SMO expression,regulate the cell cycle in the LNCaP and PC3 cell lines and markedly inhibit proliferation of LNCaP cells but not PC3 cells.     

Androgen—independent growth in LNCaP cell lines and steroid uridine diphosphate—glucuronosyltransferase expression

Aim:To investigate the mechanism of adrogen-independent growth of prostate cancer after androgen ablation in LNCaP cells and the effect of glucuronidation activity.Methods:To establish androgen-independent growth in prostate cancer LNCaP-SF,continuous passage was performed in androgen-stripped medium and the cells were evaluated for glucuronidation activity.The expression vector of antisense uridine diphosphate glucuronosyltransferase(UGT)2B15 cDNA was also constructed and evaluated.Results:LNCaP-SF lead to a higher expression in UGT2B15 and their glucuronidation activity is 2.5 times higher than that of LNCaP cells.Significantly fewer LNCaP and LNCaP-SF than control were transfected with the antisense UGT2B15 cDNA,suggesting that UGT2B15 plays an important part in the glucuronidation activity of androgens in both cells.Conclusion:The alteration of UGT2B 15 expression in LNCaP-SF cells is proposed as a biological characteristic involved in the growth of hormonerefractory prostate cancer.     

Exogenous p27KIP1 expression induces anti-tumour effects and inhibits the EGFR/PI3K/Akt signalling pathway in PC3 cells

p27 is a cyclin-dependent kinase inhibitor that regulates the progression of cells from G1 to S phase of the cell cycle. Loss of p27 has been associated with disease progression and with an unfavourable outcome in prostate cancer. In this study, we investigated whether exogenous p27 expression in the human androgen-independent prostate cancer PC3 cell line had any effect on cell growth, and we studied the molecular mechanisms involved. p27 expression was restored in PC3 cells by plasmid delivery. Cell proliferation and apoptosis were assessed in PC3 cells transfected with p27. We also investigated the effects of p27 on the epidermal growth factor receptor （EGFR）/ phosphatidylinositol 3-kinase （PI3K）/Akt signalling pathway in PC3 cells. By restoring p27 expression in PC3 cells, we observed that p27 reduced proliferation and induced arrest in G0/G1 phase. Moreover, p27-transfected PC3 cells underwent apoptosis, as shown by flow cytometric analysis and western blotting analysis of Bcl-2, Bax, Bad, caspase-3 and poly（ADP-ribose）polymerase expression. Furthermore, the p27-induced anti-tumour action corre- lated with inhibition of the EGFR/PI3K/Akt signalling pathway, as confirmed by western blotting analysis and densitometry of EGFR, PI3K （p85）, Akt and p-Akts473 expression. Our results suggest that exogenous expression of p27 inhibits the proliferation of PC3 cells through induction of G1 arrest and apoptosis, and this process correlates with inhibition of the EGFR/PI3K/Akt signalling pathway.     

靶向Survivin的siRNA联合吉西他滨抑制胰腺癌细胞增殖

Objective To construct the siRNA eukaryotic expression vector targeting Survivin gene, and investigate the chemotherapy sensitivity of pancreatic cancer line Panc-1 treated by gemcitabine. Methods The siRNA eukaryotic expression vector targeting Survivin gene was constructed. Panc-1 cells were transfected with negative control vector or siRNA vector and selected by G418, and the cell growth curve was drawn. The expression of Survivin mRNA and protein was detected by RT-PCR and Western blot respectively. Panc-1 cells or transfected cells were treated with gemcitabine for 24 h,and then the growth inhibition rate was measured by MTT assay, and cell apeptosis rate was measured by flow cytometry. Results The result of endonuclease digestion and DNA sequencing revealed that the recombinant plasmid psiRNA-Survivin was constructed successfully. Survivin mRNA and protein levels were reduced by 79.2% and 83.6% respectively in stably transfected Panc-1 cells as compared with control group, and the cell growth curve was much smoother, and the growth inhibition rate [ ( 24.6±4.5 ) % / (38.7±5.2 ) % ] and apoptosis rate of these cells [ ( 16.7±2.5 ) %/( 26.8±3.4 ) % ] were significantly increased after treatment by gemcitabine (P < 0.05 ). Conclusion The constructed siRNA eukaryotie expression vector targeting Survivin could decrease the Survivin expression,inhibit the growth of Panc-1 cells significantly,and increase the chemotherapy sensitivity to gemcitabine.     

靶向Survivin的siRNA联合吉西他滨抑制胰腺癌细胞增殖

Objective To construct the siRNA eukaryotic expression vector targeting Survivin gene, and investigate the chemotherapy sensitivity of pancreatic cancer line Panc-1 treated by gemcitabine. Methods The siRNA eukaryotic expression vector targeting Survivin gene was constructed. Panc-1 cells were transfected with negative control vector or siRNA vector and selected by G418, and the cell growth curve was drawn. The expression of Survivin mRNA and protein was detected by RT-PCR and Western blot respectively. Panc-1 cells or transfected cells were treated with gemcitabine for 24 h,and then the growth inhibition rate was measured by MTT assay, and cell apeptosis rate was measured by flow cytometry. Results The result of endonuclease digestion and DNA sequencing revealed that the recombinant plasmid psiRNA-Survivin was constructed successfully. Survivin mRNA and protein levels were reduced by 79.2% and 83.6% respectively in stably transfected Panc-1 cells as compared with control group, and the cell growth curve was much smoother, and the growth inhibition rate [ ( 24.6±4.5 ) % / (38.7±5.2 ) % ] and apoptosis rate of these cells [ ( 16.7±2.5 ) %/( 26.8±3.4 ) % ] were significantly increased after treatment by gemcitabine (P < 0.05 ). Conclusion The constructed siRNA eukaryotie expression vector targeting Survivin could decrease the Survivin expression,inhibit the growth of Panc-1 cells significantly,and increase the chemotherapy sensitivity to gemcitabine.     

靶向Survivin的siRNA联合吉西他滨抑制胰腺癌细胞增殖

Objective To construct the siRNA eukaryotic expression vector targeting Survivin gene, and investigate the chemotherapy sensitivity of pancreatic cancer line Panc-1 treated by gemcitabine. Methods The siRNA eukaryotic expression vector targeting Survivin gene was constructed. Panc-1 cells were transfected with negative control vector or siRNA vector and selected by G418, and the cell growth curve was drawn. The expression of Survivin mRNA and protein was detected by RT-PCR and Western blot respectively. Panc-1 cells or transfected cells were treated with gemcitabine for 24 h,and then the growth inhibition rate was measured by MTT assay, and cell apeptosis rate was measured by flow cytometry. Results The result of endonuclease digestion and DNA sequencing revealed that the recombinant plasmid psiRNA-Survivin was constructed successfully. Survivin mRNA and protein levels were reduced by 79.2% and 83.6% respectively in stably transfected Panc-1 cells as compared with control group, and the cell growth curve was much smoother, and the growth inhibition rate [ ( 24.6±4.5 ) % / (38.7±5.2 ) % ] and apoptosis rate of these cells [ ( 16.7±2.5 ) %/( 26.8±3.4 ) % ] were significantly increased after treatment by gemcitabine (P < 0.05 ). Conclusion The constructed siRNA eukaryotie expression vector targeting Survivin could decrease the Survivin expression,inhibit the growth of Panc-1 cells significantly,and increase the chemotherapy sensitivity to gemcitabine.     

靶向Survivin的siRNA联合吉西他滨抑制胰腺癌细胞增殖

Objective To construct the siRNA eukaryotic expression vector targeting Survivin gene, and investigate the chemotherapy sensitivity of pancreatic cancer line Panc-1 treated by gemcitabine. Methods The siRNA eukaryotic expression vector targeting Survivin gene was constructed. Panc-1 cells were transfected with negative control vector or siRNA vector and selected by G418, and the cell growth curve was drawn. The expression of Survivin mRNA and protein was detected by RT-PCR and Western blot respectively. Panc-1 cells or transfected cells were treated with gemcitabine for 24 h,and then the growth inhibition rate was measured by MTT assay, and cell apeptosis rate was measured by flow cytometry. Results The result of endonuclease digestion and DNA sequencing revealed that the recombinant plasmid psiRNA-Survivin was constructed successfully. Survivin mRNA and protein levels were reduced by 79.2% and 83.6% respectively in stably transfected Panc-1 cells as compared with control group, and the cell growth curve was much smoother, and the growth inhibition rate [ ( 24.6±4.5 ) % / (38.7±5.2 ) % ] and apoptosis rate of these cells [ ( 16.7±2.5 ) %/( 26.8±3.4 ) % ] were significantly increased after treatment by gemcitabine (P < 0.05 ). Conclusion The constructed siRNA eukaryotie expression vector targeting Survivin could decrease the Survivin expression,inhibit the growth of Panc-1 cells significantly,and increase the chemotherapy sensitivity to gemcitabine.     

靶向Survivin的siRNA联合吉西他滨抑制胰腺癌细胞增殖

Objective To construct the siRNA eukaryotic expression vector targeting Survivin gene, and investigate the chemotherapy sensitivity of pancreatic cancer line Panc-1 treated by gemcitabine. Methods The siRNA eukaryotic expression vector targeting Survivin gene was constructed. Panc-1 cells were transfected with negative control vector or siRNA vector and selected by G418, and the cell growth curve was drawn. The expression of Survivin mRNA and protein was detected by RT-PCR and Western blot respectively. Panc-1 cells or transfected cells were treated with gemcitabine for 24 h,and then the growth inhibition rate was measured by MTT assay, and cell apeptosis rate was measured by flow cytometry. Results The result of endonuclease digestion and DNA sequencing revealed that the recombinant plasmid psiRNA-Survivin was constructed successfully. Survivin mRNA and protein levels were reduced by 79.2% and 83.6% respectively in stably transfected Panc-1 cells as compared with control group, and the cell growth curve was much smoother, and the growth inhibition rate [ ( 24.6±4.5 ) % / (38.7±5.2 ) % ] and apoptosis rate of these cells [ ( 16.7±2.5 ) %/( 26.8±3.4 ) % ] were significantly increased after treatment by gemcitabine (P < 0.05 ). Conclusion The constructed siRNA eukaryotie expression vector targeting Survivin could decrease the Survivin expression,inhibit the growth of Panc-1 cells significantly,and increase the chemotherapy sensitivity to gemcitabine.     

烧伤患者血清对单核细胞核因子κB核移位的影响

目的　观察烧伤患者血清 (以下称烧伤血清 )对单核细胞核因子κB(NF κB)异二聚体p5 0、p6 5核移位及核抑制因子κBα(IκBα)降解的影响 ,进一步探讨烧伤血清对单核细胞活化的作用。　方法　收集体外培养的人外周血单核细胞 (PBMC),分别用正常人血清、烧伤血清、烧伤血清 吡咯烷二硫代氨基甲酸盐 (PDTC)刺激PBMC(依次分为对照组、烧伤血清组、PDTC组 ) ,应用激光共聚焦显微镜观察血清刺激 30、6 0、12 0、4 80min后PBMC的p5 0、p6 5核移位 ;采用Western印迹法检测刺激 30、6 0、90、12 0min时PBMC的IκBα蛋白降解情况。　结果　与对照组比较 ,刺激 30min后烧伤血清组PBMC中p5 0、p6 5即发生核移位 ,30～ 6 0min达高峰 ,12 0min后核内聚集减少 ,回复至刺激前状态。刺激 30min后烧伤血清组PBMCIκBα发生降解 ,刺激 6 0min后含量几乎为零 ,与对照组比较差异有非常显著性意义 (P <0.0 1),12 0min后表达水平逐渐恢复。PDTC组PBMCIκBα降解 [刺激 6 0min后含量为 (11 5 7± 1.98)× 10 4积分灰度值 ]及p5 0、p6 5核移位程度比烧伤血清组轻 (P <0 0 1)。　 结论　烧伤血清可导致PBMCIκBα降解和 p5 0、p6 5核移位 ,进而活化NF κB,诱导PBMC分泌细胞因子。PDTC对此变化有抑制作用     

烧伤血清对内皮细胞核因子-κB核移位的影响

目的　了解烧伤血清对内皮细胞核因子 κB (NF κB)异二聚体 p5 0 /p6 5核移位 ,以及核抑制因子κB(IκB)α降解的影响 ,进一步探讨烧伤血清对内皮细胞的活化作用。方法　采用体外培养的人脐静脉内皮细胞株ECV 30 4 ,分别用正常人血清、烧伤患者血清、烧伤患者血清 吡咯烷二硫代氨基甲酸盐 (PDTC)刺激内皮细胞 ,并以正常培养的内皮细胞为对照。应用激光共聚焦显微镜观察刺激 30、6 0、12 0、4 80min后内皮细胞 p5 0 /p6 5核移位情况 ,采用Western印迹法检测刺激 30、6 0、90、12 0min后内皮细胞IκBα蛋白降解的情况。　结果　与对照组比较 ,烧伤血清刺激内皮细胞 30min后 ,p5 0、p6 5即发生核移位 ,30～ 6 0min达高峰 ,2h后恢复至刺激前状态 ;而烧伤血清刺激 30min后 ,IκBα发生降解 (P <0 .0 1) ,刺激 4 5～ 6 0min后最为明显 ,2h后表达逐渐恢复。PDTC能有效抑制烧伤血清作用 30、6 0min后 ,p5 0、p6 5的核移位和IκBα降解。 　结论　烧伤血清可导致内皮细胞NF κBp5 0、p6 5发生核移位 ,并使IκBα降解 ,进而活化NF κB ,诱导内皮细胞分泌细胞因子。PDTC对这一系列变化可能有抑制作用     

p65基因序列shRNA慢病毒载体构建及其作用功能的初步研究

目的 建立稳定阻断前列腺癌细胞株LNCaP中核因子NF-κB(p65)表达的shRNA序列,构建慢病毒载体,检测p65在前列腺癌细胞中的作用功能.方法 根据p65基因信息,设计siRNA1、siRNA2、siRNA3针对p65基因cds区的siRNA序列,组建shRNA对应的4对互补单链DNA将合成的序列插入空载体pSIH1-H1-copGFP shRNA Vector中转染前列腺癌细胞,采用RTPCR方法检测不同序列片断对p65 mRNA的抑制效率,免疫印迹方法检测其对p65蛋白表达的抑制效率.将获得的慢病毒载体感染LNCaP细胞,设对照组、空转组.采用MTT、流式细胞术、Transwell等方法检测p65的生物学作用.结果 设计的3条针对p65序列中,第3条序列对前列腺癌细胞株中p65 mRNA的干扰效率为59%,对蛋白表达的抑制率达81%.目的序列位于p65(NM_021975)的1096～1113,茎环序列为5'-GATCC GCCCTATCCCTTTACGTCATTCAAGAGAT-GACGTAAAGGGATAGGGCTTTTTG-3'.转染细胞后,细胞可以稳定低表达p65.MTT检测实验组细胞96 h内未出现对数增殖,生长能力低于对照组和空转组.流式细胞检测实验组G0～G1期细胞百分率为61.49%,高于对照组的44.89%和空转组的41.52%;而S期细胞百分率为28.58%,低于对照组的47.36%和空转组的46.10%,差异有统计学意义(P＜0.05).Transwell检测实验组透过细胞数为(16.5000±6.62076)个,低于对照组和空转组[(45.6333±13.54159)个,(36.8333±5.68412)个],差异有统计学意义(P＜0.05).结论 成功获得稳定阻断前列腺癌细胞株LNCaP中p65表达的shRNA序列,并构建慢病毒载体;初步验证了p65在前列腺癌细胞增殖、侵袭和转移中的促进作用.     

The role of p65 NF-kB/RelA in pancreatitis-induced kupffer cell apoptosis

Acute pancreatitis induces liver injury by upregulating Kupffer cell-derived Fas/FasL; on the other hand, acute pancreatitis induces apoptosis of Kupffer cells via NF-kB-dependent pathways. The balance between upregulation of Fas/FasL and Fas/FasL-induced apoptosis of its originator cell may determine the severity of pancreatitis-related liver injury. The aim of our study was to determine the role of p65 NF-kB/RelA in pancreatitis-induced Kupffer cell apoptosis. Acute pancreatitis was induced in NIH Swiss mice by a choline-deficient ethionine-supplement (CDE) diet. In vitro mouse Kupffer cell line was transfected with p65 siRNA and treated with pancreatic elastase to mimic pancreatitis. CDE pancreatitis upregulated nuclear translocation of p65 NF-kB/RelA, Fas/FasL, caspase-3, and DNA fragmentation in mice livers (all P<0.001). In vitro, pancreatic elastase mimicked CDE-pancreatitis by upregulating nuclear translocation of p65 NF-kB/RelA, Fas/FasL, caspase-3, DNA fragmentation, and apoptosis in Kupffer cells (all P<0.001). Transfection with p65 siRNA attenuated the elastase-induced nuclear translocation of p65 NF-kB/RelA, upregulation of Fas/FasL, caspase-3, DNA fragmentation, and apoptosis in Kupffer cells (all P<0.001). Acute pancreatitis activates p65 NF-kB/RelA and induces apoptosis of Kupffer cells. Inhibition of p65NF-kB/RelA attenuates elastase-induced upregulation of proapoptotic pathways and apoptosis in Kupffer cells. The ability of Kupffer cells to autoregulate their stress response by inducing self-apoptosis warrants further investigation. Presented at the Forty-Sixth Annual Meeting of The Society for Surgery of the Alimentary Tract, Chicago, Illinois, May 14–19, 2005 (poster presentation). Supported by VA Merit Award (M.M.) and Dr. Bob Haines Pancreatitis Research Fund (M.M.).     

硫喷妥钠对内毒素诱导小鼠肺组织NF-κB表达的影响

目的研究硫喷妥钠对内毒素（LPS）诱导小鼠肺组织NF-κB的表达的影响。方法雄性昆明小鼠24只，随机分为4组（n=6）：对照组（C组）、LPS组（L组）、硫喷妥钠处理组（L＋T组）、单纯硫喷妥钠处理组（T组）。C组腹腔注射生理盐水1ml／ks；L组腹腔注射LPS5mg／kg；L＋T组腹腔注射LPS5mg／kg 20min时再注射1％硫喷妥钠注射液60mg/kg；T组单纯腹腔注射1％硫喷妥钠注射液60mg／kg。在注射LPS后3h放血处死小鼠，立即开胸取肺。免疫蛋白印迹法测定肺组织NF-κB p65的表达；酶联免疫吸附法测定肺组织肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）含量。结果与C组相比，L、L＋T组肺组织NF-κB p65表达增加，L、L＋T、T组肺组织，TNF-α、IL-1β含量上升（P〈0．05）；与L组相比，L＋T、T组肺组织NF-κB p65表达降低，肺组织TNF-α，IL-1β含量降低（P〈0．05）。结论硫喷妥钠通过下调小鼠LPS诱导的肺组织NF．KB065表达，降低了TNF-α及IL-1β的释放。