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1.
APE1基因RNAi慢病毒载体的构建与鉴定 总被引:1,自引:0,他引:1
目的构建APE1基因慢病毒载体,为其后续的体内外实验研究提供基础。方法应用基因工程技术,筛选出两条针对APE1基因的RNAi靶序列KD1、KD2,分别与pGCIL-GFP载体连接,经转化筛选鉴定后,包装产生慢病毒颗粒并测定病毒滴度,分别命名为LV-APE1-shRNA1、LV-APE1-shRNA2,两种病毒颗粒分别感染MHCC97-H细胞,设为感染LV-APE1-shRNA1、LV-APE1-shRNA2细胞组,同时设未感染病毒组和感染空载体细胞组。Real-time PCR和Western blot检测MHCC97-H细胞中APE1基因的mRNA和蛋白的表达。结果 PCR及测序结果与预期结果一致,LV-APE1-shRNA1、LV-APE1-shRNA2的病毒滴度为4×10^8TU/mL和7×10^8TU/mL。与未感染病毒组和感染空载体细胞组相比,2组慢病毒组APE1基因mRNA和蛋白的表达均明显下降,LV-APE1-shRNA1、LV-APE1-shRNA2对APE1基因的mRNA表达的抑制率分别为75%,90%,对APE1蛋白表达的抑制率达90%,95%。结论成功构建高效阻断APE1基因表达的RNAi慢病毒表达载体,为应用RNAi进一步研究APE1基因在肝癌中的作用机制和基因治疗奠定基础。 相似文献
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目的观察新型甲磺酸二氢麦角碱缓释剂培磊能对伴有认知功能障碍的脑血管功能不全患者的疗效及其安全性。方法采用自身对照设计方法,对43例脑血管功能不全所致认知功能障碍的患者给予口服培磊能,1粒/次,2次/d,于治疗后45d和90d观察该药物对认知功能和主观症状的改善程度及药物的副作用。结果(1)对认知功能的影响:与治疗前相比,治疗后45d和90d患者的MMSE总评分提高(P<0.001);但治疗后90d与45d相比,差异无显著性意义(P>0.05)。治疗后45d以记忆力、计算力及言语功能障碍改善最为明显(P<0.05或P<0.01);治疗后90d,除注意力外,其余各单项评分均明显提高(P<0.05)。治疗后90d记忆力和定向力的改善程度优于45d(P<0.05)。(2)对主观症状的影响:治疗后90d,患者头痛、头晕、睡眠障碍、易疲劳和情绪不佳等主观症状明显改善;在治疗后45d和90d,对头痛、头晕两项症状改善的总有效率分别为66.7%、61.5%和83.3%、84.6%;对睡眠障碍和易疲劳改善的总有效率分别为22.2%、12.5%和44.4%、50.0%;但对情绪不佳的疗效欠佳,治疗后90d的总有效率仅为16.6%。(3)药物安全性:治疗期间发生的相关不良反应为恶心、鼻塞、口干等,症状较轻。结论培磊能是治疗血管性认知功能障碍安全、有效的药物。 相似文献
3.
J. Schrader S. Nees E. Gerlach 《Pflügers Archiv : European journal of physiology》1978,378(2):167-171
A sensitive and specific radioimmunoassay for adenosine has been developed. Antibodies directed against adenosine (titer 1400–1700) were obtained by immunizing rabbits with adenosine, conjugated via its vicinal hydroxyl groups to bovine serum albumin (periodate oxidation). Interfering adenosine deaminase activity was removed from the antisera by treatment with DEAW-cellulose. Free and antibody bound3H-adenosine was separated by either the second antibody precipitation method or by a simple filtration step. The sensitivity and assay range for adenosine was 1–100 pmoles per assay tube. Structurally related purine compounds (adenine nucleotides, adenine) crossreacted with adenosine binding and were removed by a single chromatographic step. Analysis of the adenosine content in normoxic guinea pig hearts yielded 2.53 nmoles/g, a value which was confirmed by spectrophotometric analysis. 相似文献
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通过观察正常兔、四氧嘧啶糖尿病兔及胰岛素(INS)治疗兔的血糖含量、体重和生长素受体(GHR)含量的变化,研究INS、GHR及胰岛素样生长因子-Ⅰ(IGF-Ⅰ)三者的关系,以探讨INS缺乏所致生长素(GH)抵抗现象的机理。结果表明,当INS完全缺乏,血糖极度升高时,体重严重丢失,降至原体重的75%,而GHR仅降至正常值的42%。提示,INS对GH促生长效应具有双调节作用,既能直接刺激GHR生成,又可作用于受体后机制,直接促进IGF-Ⅰ生成。 相似文献
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Phenolic compounds are strong environmental toxicants, which are found in food, drinking water as well as in the indoor and outdoor air environment. In this work we investigated the effect of low concentrations of 0.2, 1 and 5 μg/ml of 2,4,5-trichlorophenol (2,4,5-TCP), pentachlorophenol (PCP), 4,6-dichloroguaiacol (4,6-DCG), tetrachloroguaiacol (TeCG), 4,5-dichlorocatechol (4,5-DCC) and tetrachlorocatechol (TeCC) on DNA bases oxidation in human peripheral blood lymphocytes. The analysis was performed using alkaline single cell gel electrophoresis (the comet assay). To detect oxidized pyrimidynes and purines we used the repair enzymes such as endonuclease III and formamidopyrimidine-DNA glycosylase. DNA oxidation was expressed as a percentage of comet tail, which was formed after the xenobiotics treatment. The obtained results showed that all the compounds examined were able to oxidize DNA bases in human lymphocytes. It was also observed that pyrimidine bases were more strongly oxidized in comparison to purine ones. Finally, it was found that chlorinated catechols and TeCC in particular, revealed a higher oxidative potential in comparison to chlorophenols and chloroguaiacols, and a rise in the number of chlorine atoms in the compound from each group examined led to an increase in DNA bases damage. 相似文献
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New targets for pyrimidine antimetabolites in the treatment of solid tumours
1: Thymidylate synthase
1: Thymidylate synthase
Thymidylate synthase forms the target for anticancer therapy with fluoropyrimidines. Anticancer activity can be increased by the use of different modulators of fluoropyrimidine metabolism, which lead to an enhanced inhibition of thymidylate synthase.In vitro andin vivo studies with fluoropyrimidines and two of these modulators, folinic acid (leucovorin) and interferon, are summarized. The promise of these preclinical results is reflected by the response data of several clinical trials. The biochemical effects of these modulators are described and illustrated by the fluoropyrimidine-mediated inhibition of thymidylate synthase in tumour samples, which is clearly enhanced by folinic acid. The regulation of thymidylate synthase synthesis may also be crucial for total blockade of thymidylate synthase activity. This regulation may be influenced by interferon-. Although the addition of modulators increases the activity of fluoropyrimidines at the level of thymidylate synthase, most solid tumours, especially colorectal carcinomas, are resistant to these combinations. For this reason, new, more potent inhibitors of thymidylate synthase have been developed, the antifolates. Preclinical data show that some of these compounds have good antitumour activity, but they still have to prove their value in the clinic. These two approaches, the use of modulators and new compounds, have shown activity preclinically and the extension of these findings to clinical studies stresses the importance of thymidylate synthase as a target in fluoropyrimidine therapy of solid tumours. 相似文献
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目的 建立人类慢性粒细胞白血病(CML)细胞系K562细胞在伊马替尼(Imatinib mesylate)处理前、后的DNA损伤模型,探讨伊马替尼对K562细胞的DNA损伤修复功能的影响.方法 用噻唑蓝(MTT)法确定伊马替尼预处理K562细胞的浓度;用免疫印迹法(Western blot)检测伊马替尼处理后K562细胞BCR/ABL的磷酸化状态,以反映BCR-ABL酪氨酸激酶活性受抑制情况;用彗星实验检测不同浓度过氧化氢(H2O2)诱导的K562细胞、伊马替尼预处理K562细胞的DNA损伤模型;用彗星实验对各组细胞在DNA损伤后的修复进行动态观测.结果 MTT实验结果显示,伊马替尼预处理K562细胞的最佳终浓度为1 μmol/L,作用时间24 h;Western blot实验结果显示,该浓度的伊马替尼可有效抑制BCR/ABL融合蛋白第177位酪氨酸激酶磷酸化,密度比为0.100±0.018,与对照组(0.425±0.039)相比,降低了(77.11±5.59)%,差异有统计学意义(t=4.57,P<0.05);彗星实验确定了各组细胞DNA损伤模型的建立条件,采用10 μmol/L终浓度的H2O2对K562细胞和伊马替尼预处理后K562细胞进行染毒,H2O2作用温度和时间为4℃、10 min.修复结果显示,经伊马替尼预处理的K562细胞修复时间为120 min,与未处理组修复时间60 min相比,前者DNA损伤修复时间明显延长(F=97.79,P<0.05).结论 本研究建立了伊马替尼处理前、后的白血病K562细胞的DNA损伤模型,BCR/ABL融合蛋白的酪氨酸激酶抑制剂伊马替尼能减弱K562细胞的DNA损伤后修复能力. 相似文献
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Lassiter LK Tummala MK Hussain MH Stadler WM Petrylak DP Carducci MA 《Clinical genitourinary cancer》2008,6(1):31-35
BACKGROUND: Piritrexim is reported to have a response rate of 38% in patients with chemotherapy-naive disease and 23% for second-line therapy after chemotherapy failure. We report the results of a multiinstitutional, open-label, 2-stage, phase II study that further evaluates oral piritrexim in patients with urothelial carcinoma and who proved nonresponsive to standard chemotherapy. PATIENTS AND METHODS: Eligible patients included those with bi-dimensionally measurable disease and an Eastern Cooperative Oncology Group performance status of 0-2, transitional cell carcinoma or adenocarcinoma of the urothelium, and nonresponse to > or = 1 previous standard chemotherapy regimen. Patients received piritrexim orally at 25 mg 3 times daily (every 8 hours regularly) for 5 consecutive days each week for 3 weeks, followed by a 1-week rest period. Treatment was continued until disease progression, unacceptable toxicity, or patient refusal. RESULTS: Of the 23 patients enrolled, 19 patients and 22 patients were assessable for toxicity and response, respectively. Two patients required dose reduction because of toxicity, 2 patients discontinued study because of toxicity, and 6 patients had > or = 1 serious adverse event. Except for grade 1/2 pain and fatigue, gastrointestinal toxicities were the most commonly reported events, followed by fever, delirium, and myelosuppression. No objective responses were observed, with 2 patients demonstrating stable disease after 2-4 cycles. By the statistical design of the trial, further enrollment was halted because of lack of activity. CONCLUSION: Regardless of modest side effects, oral piritrexim in heavily pretreated patients is inactive at this dose and schedule, confirming the results of a recent cooperative group trial. 相似文献
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Albert Leyva Jan H. Schornagel Ineke Kraal Sybe K. Wadman Herbert M. Pinedo 《Journal of cancer research and clinical oncology》1984,107(3):211-216
Summary In a clinical study of high-dose thymidine (TdR) treatment, toxic effects, TdR metabolism, and the influence of TdR on pyrimidine and purine metabolism were examined. Ten patients with solid tumors were treated with continuous infusion of TdR at 34–75 g/m2/day for 3 to 5 days. Hematologic toxicity occurred with 5-day TdR infusion at 75 g/m2/day but not when plasma TdR concentration failed to reach millimolar levels. In three patients who received similar TdR doses, plasma TdR levels were related to elimination rates of TdR and its metabolites from plasma. In one patient in whom urinary excretion was studied, 100% of the TdR dose given was recovered in the form of TdR, thymine (Thy), -aminoisobutyrate, and 5-hydroxymethyluracil (5-HMUra). The latter metabolite, which had not been previously described in high-dose TdR treatment, was also found in plasma at levels from 5% to 10% of those of TdR. No effects of high-dose TdR infusion on purine levels in plasma were observed, while a substantial increase in uracil levels was noted both in plasma and urine. These data provide further information on high-dose TdR treatment with regard to clinical, pharmacokinetic, and biochemical effects. 相似文献