The optimal antisense accessible sites （AAS） of uroplakin Ⅱ （UP Ⅱ ） mRNA, a specific gene expressed in bladder urothelium, were selected in order to provide a novel method for targeted therapy of transitional ce...The optimal antisense accessible sites （AAS） of uroplakin Ⅱ （UP Ⅱ ） mRNA, a specific gene expressed in bladder urothelium, were selected in order to provide a novel method for targeted therapy of transitional cell carcinoma （TCC） of bladder. The 20 mer random oligonucleotide library was synthesized, hybridized with in vitro transcripted total UPⅡ cRNA, then digested by RNase H. After primer extension and autoradiography, the AAS of UPⅡ were selected. The RNADraw soft- ware was used to analyze and choose the AAS with obvious stem-loop structures, according to which the complementary antisense oligonucleotides （AS-ODN） were synthesized. With the AS-ODN0 designed only by RNADraw as a control, the AS-ODNs were transferred into UP Ⅱ highly-expressing cell line RT4. The cellular expression of UPⅡ mRNA was detected by RT-PCR and Western blot. Twelve AAS of UPⅡ mRNA were selected in vitro. Four AAS with stem-loop structures were chosen, locating at 558-577, 552-571, 217-236 and 97-116 bp of UP Ⅱ mRNA respectively. After transfection with the corresponding AS-ODN （AS-ODN1, AS-ODN2, AS-ODN3 and AS-ODN4） for 18 h, the UPⅡ mRNA levels in RT4cells were reduced by 29,3%, 82.7%, 71.3% and 70.9%, while UP Ⅱ protein levels were decreased by 20.2%, 78.5%, 65.2% and 64.4% respectively, which were significantly higher than those of AS-ODN0 （14.3%, 12.1% respectively） （P〈0.01）. The AAS of UPⅡ mRNA was effectively selected in vitro by random oligonucletide library/RNase H cleavage method in combination with computer software analysis, which had important reference values for further studying biological functions of UP Ⅱ gene and targeted therapeutic strategy for TCC of bladder.展开更多
目的探讨Salubrinal对大鼠脑缺血再灌注模型LC3-ⅡmRNA及LC3-Ⅱ/LC3-ⅠmRNA表达的影响。方法用线栓法建立大鼠大脑中动脉脑缺血(MCAO)缺血再灌注模型。大鼠随机分为假手术组、模型组、Salubrinal组(Sal组),各组又分为再灌6、12、24、72 ...目的探讨Salubrinal对大鼠脑缺血再灌注模型LC3-ⅡmRNA及LC3-Ⅱ/LC3-ⅠmRNA表达的影响。方法用线栓法建立大鼠大脑中动脉脑缺血(MCAO)缺血再灌注模型。大鼠随机分为假手术组、模型组、Salubrinal组(Sal组),各组又分为再灌6、12、24、72 h 4个亚组。各组于再灌相应时间点行神经功能缺损评分,并断头取脑行HE染色及Real time-PCR检测。结果神经功能缺损:与假手术组比较,模型组、Sal组各时间点均有神经功能缺损(P<0.01);与模型组比较,再灌24、72 h Sal组神经功能缺损评分降低(P<0.05)。HE染色:模型组神经元减少、缺失,细胞肿胀,部分破裂,细胞核固缩、偏移、深染,胶质细胞增生,经Salubrinal干预后,再灌各时间点神经元增多,胞膜较完整,核固缩现象较轻,水肿轻微。Real time-PCR检测:与假手术组比较,模型组、Sal组各指标于再灌各时间点均增高(P<0.01)。与模型组比,Sal组LC3-ⅡmRNA表达于再灌12、24、72 h下降明显(P<0.05);Sal组LC3-Ⅱ/LC3-ⅠmRNA于再灌后各时间点下降均明显(P<0.01)。结论 Salubrinal通过下调LC3-ⅡmRNA及LC3-Ⅱ/LC3-ⅠmRNA比值,对大鼠脑缺血再灌注损伤起保护作用。展开更多
目的研究强心安神汤对Wistar慢性心衰模型大鼠AngⅡ含量及AT1 m RNA表达的影响,探讨其可能的作用机制。方法除空白组外,将Wistar大鼠造模后分为模型组、西药组、强心安神汤高剂量组及强心安神汤低剂量组,干预4周后,抽静脉血以免疫组化...目的研究强心安神汤对Wistar慢性心衰模型大鼠AngⅡ含量及AT1 m RNA表达的影响,探讨其可能的作用机制。方法除空白组外,将Wistar大鼠造模后分为模型组、西药组、强心安神汤高剂量组及强心安神汤低剂量组,干预4周后,抽静脉血以免疫组化法测定大鼠血清中AngⅡ的含量;处死大鼠,提取心肌组织,以RT-PCR法检测心肌组织AT1 m RNA的表达。结果强心安神汤组大鼠血清AngⅡ含量及AT1 m RNA表达与模型组及西药组比较,差异有统计学意义(P<0.05或P<0.01)。结论强心安神汤治疗心衰的机制可能与其抑制AngⅡ引起的AT1 m RNA表达上调有关。展开更多
基金supported by grants from the National Natural Sciences Foundation of China (No. 30200284, No. 30600278, No. 30772359)Program for New Century Excellent Talents in University (NCET-06-0641)Scientific Research Foundation for the Returned Overseas Chinese Scholars (2008889)
文摘The optimal antisense accessible sites （AAS） of uroplakin Ⅱ （UP Ⅱ ） mRNA, a specific gene expressed in bladder urothelium, were selected in order to provide a novel method for targeted therapy of transitional cell carcinoma （TCC） of bladder. The 20 mer random oligonucleotide library was synthesized, hybridized with in vitro transcripted total UPⅡ cRNA, then digested by RNase H. After primer extension and autoradiography, the AAS of UPⅡ were selected. The RNADraw soft- ware was used to analyze and choose the AAS with obvious stem-loop structures, according to which the complementary antisense oligonucleotides （AS-ODN） were synthesized. With the AS-ODN0 designed only by RNADraw as a control, the AS-ODNs were transferred into UP Ⅱ highly-expressing cell line RT4. The cellular expression of UPⅡ mRNA was detected by RT-PCR and Western blot. Twelve AAS of UPⅡ mRNA were selected in vitro. Four AAS with stem-loop structures were chosen, locating at 558-577, 552-571, 217-236 and 97-116 bp of UP Ⅱ mRNA respectively. After transfection with the corresponding AS-ODN （AS-ODN1, AS-ODN2, AS-ODN3 and AS-ODN4） for 18 h, the UPⅡ mRNA levels in RT4cells were reduced by 29,3%, 82.7%, 71.3% and 70.9%, while UP Ⅱ protein levels were decreased by 20.2%, 78.5%, 65.2% and 64.4% respectively, which were significantly higher than those of AS-ODN0 （14.3%, 12.1% respectively） （P〈0.01）. The AAS of UPⅡ mRNA was effectively selected in vitro by random oligonucletide library/RNase H cleavage method in combination with computer software analysis, which had important reference values for further studying biological functions of UP Ⅱ gene and targeted therapeutic strategy for TCC of bladder.
文摘目的探讨Salubrinal对大鼠脑缺血再灌注模型LC3-ⅡmRNA及LC3-Ⅱ/LC3-ⅠmRNA表达的影响。方法用线栓法建立大鼠大脑中动脉脑缺血(MCAO)缺血再灌注模型。大鼠随机分为假手术组、模型组、Salubrinal组(Sal组),各组又分为再灌6、12、24、72 h 4个亚组。各组于再灌相应时间点行神经功能缺损评分,并断头取脑行HE染色及Real time-PCR检测。结果神经功能缺损:与假手术组比较,模型组、Sal组各时间点均有神经功能缺损(P<0.01);与模型组比较,再灌24、72 h Sal组神经功能缺损评分降低(P<0.05)。HE染色:模型组神经元减少、缺失,细胞肿胀,部分破裂,细胞核固缩、偏移、深染,胶质细胞增生,经Salubrinal干预后,再灌各时间点神经元增多,胞膜较完整,核固缩现象较轻,水肿轻微。Real time-PCR检测:与假手术组比较,模型组、Sal组各指标于再灌各时间点均增高(P<0.01)。与模型组比,Sal组LC3-ⅡmRNA表达于再灌12、24、72 h下降明显(P<0.05);Sal组LC3-Ⅱ/LC3-ⅠmRNA于再灌后各时间点下降均明显(P<0.01)。结论 Salubrinal通过下调LC3-ⅡmRNA及LC3-Ⅱ/LC3-ⅠmRNA比值,对大鼠脑缺血再灌注损伤起保护作用。
文摘目的研究强心安神汤对Wistar慢性心衰模型大鼠AngⅡ含量及AT1 m RNA表达的影响,探讨其可能的作用机制。方法除空白组外,将Wistar大鼠造模后分为模型组、西药组、强心安神汤高剂量组及强心安神汤低剂量组,干预4周后,抽静脉血以免疫组化法测定大鼠血清中AngⅡ的含量;处死大鼠,提取心肌组织,以RT-PCR法检测心肌组织AT1 m RNA的表达。结果强心安神汤组大鼠血清AngⅡ含量及AT1 m RNA表达与模型组及西药组比较,差异有统计学意义(P<0.05或P<0.01)。结论强心安神汤治疗心衰的机制可能与其抑制AngⅡ引起的AT1 m RNA表达上调有关。
