胞嘧啶脱氨酶的原核表达和多克隆抗体的制备

目的：在原核系统中表达并纯化大肠杆菌胞嘧啶脱氨酶（cytosine deaminase,CD)，制备鼠抗大肠杆菌CD多克隆抗体，方法：亚克隆CD基因到原核表达载体pMAL-c2和pBV222中，并转化入大肠杆菌DH5α内，诱导表达并纯化MBP-CD和6his-CD融合蛋白，用纯化的MBP-CD融合蛋白免疫小鼠制备多克隆抗体。结果：通过重组质粒的酶切筛选出重组阳性克隆，成功地表达和纯化出MBP-CD和6his-CD融合蛋白，用纯化的MBP-CD成功制备了鼠抗CD多克隆抗体，并用6his-CD和GST-CD重组蛋白进行Western印迹分析，证实了抗体的正确性，结论：应用多克隆抗体可以检测体内外CD基因的表达，为临床前和临床上深入开展CD基因的生物治疗研究提供重要的实验材料。     

组织特异性胞嘧啶脱氨酶和5-氟胞嘧啶热化疗对裸鼠结肠癌肝脏转移的影响

Objective To investigate the effects of tissue specific cytosine deaminase/5-fluorocytosine (CD/5-FC) thermotherapy on hepatic metastasis of colonic carcinoma in nude mice. Methods Forty-five nude mice were randomly divided into control group, 5-FC group and 5-FC thermotherapy group according to the random number table (15 mice in each group). Mice models of hepatic metastasis of colonic carcinoma were established by portal vein injection of LoVo/CEACD cells. The hepatic metastasis rate and number of metastatic nodules of the 3 groups were compared by ehi-square test and one-way ANOVA. The pathological changes in tumor tissues and apoptotic index of tumor cells were observed. The expression of the CD gene in tumor tissues was detected by fluorescent quantitative RT-PCR and Western blot. Results The number of metastatic nodules and liver metas-tasis rate were 4.6±1.3 and 100.0% in control group, 2.2±1.0 and 60.0% in 5-FC group, 0.5±0.8 and 13.3% in 5-FC thermotherapy group, with statistical difference among the 3 groups (F=25.898, χ2=5.208, 19.548, 5.168, P<0.05). The mean apoptotic indexes of tumor cells of the 3 groups were 4.6%, 9.9% and 17.4%, respectively. Vacuolar degeneration, cell necrosis, cytolysis and apoptotic bodies were mostly observed in the 5-FC thermotherapy group. The expression of CD gene in tumor tissue was detected in all the groups. Conclusion Tissue specific CD/5-FC thermotherapy has inhibitory effects on the hepatic metastasis of LoVo cells transfected with CD gene.     

Therapeutic effect of 5-FC on YCD modified murine P388 leukemia in vivo

Cytosine deaminases (CDs) in bacteria andfungi are found to deaminate prodrug 5 - FC intocytotoxic agent 5 - FU .Yeast CD (YCD) is clonedfrom Saccharomyces cerevisiae.It has been shownpreviously that YCD was more efficient inconversing5 - FC than bacterial CD(b CD) [1-3 ] .As anovel suicide gene therapy system,YCD/ 5 - Fcsystem may be promising in cancer therapy andprevention of graft versus host disease.In thepresent study,we established a P388/ DBA murineleukemia model by infusin…     

TK/GCV、CD/5-Fc双自杀基因系统治疗结肠癌的实验研究

目的观察胸苷激酶/丙氧鸟苷(TK/GCV)、胞嘧啶脱氨酶/5-氟胞嘧啶(CD/5-Fc)双自杀基因系统对结肠癌的治疗效果,并结合研究结果进行作用机理的分析。方法采用培养细胞移植法,将人结肠癌细胞系SW480接种于裸鼠背部皮下,建立裸鼠人结肠癌移植瘤模型。将32只裸鼠随机分为4组:空白对照组,TK/GCV治疗组,CD/5-Fc治疗组,TK/GCV CD/5-Fc联合治疗组,每组8只。TK/GCV CD/5-Fc采用逆转录病毒介导,采用瘤体内直接注射法,同时腹腔注射GCV、5-Fc治疗,观察各组小鼠的生存状况及肿瘤体积、瘤重、肿瘤生长抑制率、常规病理、生存期等指标,比较观察各治疗组的治疗效果及对荷瘤裸鼠存活的影响。结果各治疗组裸鼠人结肠癌移植瘤的生长均受到显著抑制,治疗后各组体积数增加(P<0.05),治疗组裸鼠存活期显著延长,TK/GCV CD/5-Fc联合治疗组效果最好,疗效q值=1.675>1.15,即TK/GCV、CD/5-Fc有交互协同作用。结论TK/GCV与CD/5-Fc对人结肠癌SW480细胞有明显抑制作用,TK/GCV、CD/5-Fc双自杀基因系统效果更强。     

红细胞同工酶ADA－EAP一AK_1同步电泳分型方法的建立

综合、改良了分别分型的方法，建立了红细胞同工酶ＡＤＡ一ＥＡＰ一ＡＫ１同步电泳分型方法，为ＡＤＡ，ＥＡＰ和ＡＫ１３种红细胞同工酶在法医学鉴定中更广泛的应用，提供了一种可靠、经济、实用的方法。     

黄芪、女贞、旱莲合剂对小鼠免疫功能的影响

本文报道以黄芪、女贞子、墨旱莲三合剂(AFE)大小剂量(9g/kg、20g/kg)分别给予小鼠灌胃7天,观察其对机体细胞免疫及体液免疫功能的影响。结果表明,两种剂量的AFE均能非常明显地增加小鼠细胞免疫功能(P<0.01),促进其免疫器官正常发育(P<0.05),提高血清IgG水平(>40％)。增加小鼠免疫生化酶一腺苷脱氨酶(ADA)活性,其中大剂量组对ADA的影响具有非常显著性意义(P<0.01)。该合剂还具有恢复环磷酰胺对小鼠免疫器官所致的萎缩作用。提示.该中药合剂可保护、提高小鼠免疫功能。     

胞嘧啶脱氨酶基因修饰神经干细胞及其表达的离体实验研究

目的 探讨胞嘧啶脱氨酶(cytimidine deaminase，CD)基因修饰神经干细胞及其基因表达。方法 通过构建真核表达质粒pCMVCD，限制性内切酶消化鉴定后，采用Lipofectamine 2000脂质体介导法转染新生大鼠室管膜下区神经干细胞(Neural stem cells，NSCs)，G418筛选阳性克隆，加入不同浓度的5-氟胞嘧啶(5-Flourocytosine，5-FC)，MTT比色法测定NSCs的生存率。结果 本实验成功地培养并鉴定了神经干细胞，并将CD基因成功地转染了神经干细胞，G418阳性NSCs对低浓度5-FC高度敏感。结论 CD基因修饰神经干细胞的离体实验研究为干细胞治疗研究提供依据。     

Characterization of adenosine deaminase (ADA)-negative B-lymphoblastoid cells cocultured with ADA-positive fibroblasts

Abstract: A cell line, BAD05, derived from B lymphocytes of an adenosine deaminase (ADA; EC 3,5,4,4)-deficient patient could not proliferate in a serum-free medium containing 100 μmol/l deoxyadenosine. When BAD05 was cultured with ADA-positive fibroblasts, the proliferation of BAD05 was improved. BAD05 cell density increased when the initially mixed ratio of fibroblasts/BAD05 was 1/10 or higher, but decreased when the ratio was 1/20 or lower. Deoxyadenosine concentrations in the medium and ATP and deoxyATP (dATP) levels in the BAD05 were measured after 4 hours of coculture at initial BAD05 cell densities of 1 × 10⁵and 1 × 10⁶cells/ml. Deoxyadenosine concentrations in the medium decreased as the density of fibroblasts increased. The dATP level decreased as the mixed ratio rose. The ratio of fibroblasts/BAD05 rather than the cell density of fibroblasts had a larger effect on the dATP levels in BAD05. Under our experimental conditions, ADA-negative cells proliferated well when the ratio of ADA-positive cells/ADA-negative cells was over 1/10.     

The regional distribution of adenosine-regulating enzymes in the left and right ventricle walls of control and hypertrophic heart

Summary The transmural distribution of the adenosine-generating enzyme 5-nucleotidase (5N) and of the adenosine-degrading enzymes adenosine deaminase (ADA), AMP deaminase (AMP-D) and adenosine kinase (Ado-K) were determined across the walls of left and right ventricles of control and hypertrophic rat hearts.The enzyme distribution across the left ventricle wall (but not across the right wall) of normal hearts was not uniform: 5N activity shows its highest levels in the subepicardial and in the subendocardial regions, whereas all the other enzyme activities show their lowest levels. A similar pattern of transmural distribution was also detected in other mammalian species (ox and pig).In the experimental cardiac hypertrophy, caused by two different types of chronic cardiac overload, the levels and the profiles of transmural distribution of 5N and ADA enzyme activities may significantly change across the rat left ventricle wall.     

Adenosine and adenine nucleotides are independently released from both the nerve terminals and the muscle fibres upon electrical stimulation of the innervated skeletal muscle of the frog

The independent release of adenosine and adenine nucleotides upon electrical stimulation was studied in the innervated sartorius muscle of the frog after blockade of the extracellular catabolism of adenosine monophosphate (AMP) through exo-AMP deaminase and ecto-5-nucleotidase. Nerve stimulation (30 min, 0.2Hz) induced the release of both adenosine (19±3 pmol) and adenine nucleotides (101±7 pmol). Experiments performed in the presence of tubocurarine (5 M) to prevent purine release due to nerve-evoked muscle twitching, or under direct stimulation of the muscle in low calcium solutions to prevent pre-synaptic release of purines, showed that there was an evoked release of adenosine and adenine nucleotides both from the nerve endings and from the twitching muscle fibres. Removal of ecto-5-nucleotidase inhibition shows that the catabolism of adenine nucleotides released during stimulation contributes in about 50% to the amount of endogenous extracellular adenosine. When only one of the enzymes catabolizing AMP (ecto-5-nucleotidase or exo-AMP deaminase) was inhibited, the evoked release of adenine nucleotides was undetectable, suggesting that each enzyme is able to catabolize all the AMP formed from adenine nucleotides released upon stimulation. It is concluded that the concentration of endogenous extracellular adenosine is under the control of the relative activities of exo-AMP deaminase and ecto-5-nucleotidase.Brief accounts of some of the results in this study have been published previously (refs. [6, 7]).