彗星试验检测DNA交联及其修复效应的研究

ＤＮＡ交联一般分为ＤＮＡ 蛋白质交联 (ＤＮＡ ｐｒｏｔｅｉｎｃｒｏｓｓｌｉｎｋｓ,ＤＰＣ)和ＤＮＡ ＤＮＡ交联 (ＤＮＡ ＤＮＡｃｒｏｓｓｌｉｎｋｓ,ＤＤＣ)两类 ,它们都是环境污染物和化学致癌物引起的生物大分子物质发生遗传损害的结果。与其他类型ＤＮＡ损伤相比 (如ＤＮＡ链断裂 ) ,ＤＮＡ交联较难修复或易于发生易错修复 ,在细胞周期中维持时间较长。当ＤＮＡ复制时 ,一些重要的基因容易丢失 ,且经常发生染色体断裂 ,甚至致死性突变[1 ,2 ] 。故筛检环境化学物的ＤＮＡ交联性损伤和观察其修复效应具有极其重要的意义。然而 ,由于检…     

一种更新的检测DNA交联的尝试

ＤＮＡ交联是外来化学物作用于ＤＮＡ所致的重要遗传损伤 ,与其他类型ＤＮＡ损伤相比 ,ＤＮＡ交联较难修复或易于发生易错修复 ,在细胞周期中维持时间较长。当ＤＮＡ复制时 ,一些重要的基因容易丢失 ,且经常发生染色体断裂 ,甚至致死性突变[1 ,2 ] 。故筛检环境化学物的ＤＮＡ交联性损伤具有极其重要的意义。然而 ,由于检测方法的局限 ,极大地制约了其应用。近年来 ,国内外偶有将彗星试验 (Ｃｏｍｅｔａｓｓａｙ)用于检测ＤＮＡ交联的报道[3,4 ] ,但很不系统和全面。本室在深入挖掘彗星试验的应用前景时 ,首次探讨了通过增加电压或延长电泳…     

芥子气对人皮肤细胞 DNA 交联形成与细胞毒性的关系

芥子气对人皮肤细胞ＤＮＡ交联形成与细胞毒性的关系浦跃朴林嫔嫔１ＩｓａｄｏｒｅＡＢＥＲＮＳＴＥＩＮ１金锡鹏２（南京铁道医学院预防医学系，南京２１０００９；１美国密执安大学公共卫生学院；２上海医科大学公共卫生学院，上海２０００３２）本研究结果表明，芥子气...     

溴乙锭荧光法测定环境污染物的 DNA 交联作用

溴乙锭荧光法测定环境污染物的ＤＮＡ交联作用尹立红浦跃朴杨薇（南京铁道医学院预防医学系，南京２１０００９）ＤＮＡ交联溴乙锭荧光分析法（ＥＦＡ）是一种灵敏，快速，简便检测ＤＮＡ交联作用的方法，已被广泛用于化学物质ＤＮＡ损伤机制的研究．该方法是基于双链间交...     

大麻性别连锁的特异DNA标记的初步研究

目的··:寻找大麻性别连锁的特异DNA标记 ,为大麻早期性别鉴别研究提供进一步研究基础。方法··:分别提取5个♀和5个♂大麻干标本的基因组DNA ,运用RAPD标记技术 ,分别用17个随机引物对取自我国不同地区的大麻进行了检测。结果··:在引物OPX -09中发现1条长约820bp与♀性别连锁的特异带。结论··:该特异带可被用来作为性别鉴别的特异性分子遗传标记。     

重组人组织型纤溶酶原激活剂中细胞残余DNA的检测

采用地高辛标记探针,以核酸分子班点杂交技术检测重组人组织型纤溶酶原激活剂（ｒｔ－ＰＡ）中细胞残余ＤＮＡ的方法。从样品中去掉蛋白提取ＤＮＡ的方法能有效的避免蛋白对检测结果的干扰,其灵敏度和结果都能较好地达到ＷＨＯ的要求。     

^125I—人胸腺素的标记及其在小鼠体内的分布与代谢研究

介绍了一种应用预载的ＩＯＤＯ－ＢＥＡＤ进行人胸腺素（ＨＴＮ）蛋白质酷氨酸位＾１２５Ｉ标记的Ｉｏｄｏｇｅｎ改良方法，并就用本法椟记的ＨＴＮ的生物活性与用经的氯胺ＴＩ地作了对比，对这两种方法的优缺点作了初步讨论。结果表明，前者是一种较理想的标记方法，可以伯者对被标记物进行示踪分布与代谢观察研究。     

抗胃癌单克隆抗体与血卟啉衍生物的交联物实验研究

血卟啉衍生物(HPD)是当前用于光动力学疗法的光敏剂。HPD与胃癌单抗(3H_(11))的交联物的体内外抗肿瘤作用,表明单克隆抗体(McAb)与血卟啉衍生物的交联物的光动力学效应明显优于游离HPD,充分显示了McAb的导向治疗作用。同时我们还观察了3H_(11)——HPD与游离HPD在血液、肿瘤及其它正常组织中分布特点,发现24小时后血中交联物浓度比游离HPD高,滞留时间长,符合药物动力学原则(大分子药物透过血管壁速度慢)。在肿瘤组织中交联物含量在24—96小时内,均明显高于游离HPD,为McAb的导向作用又提供了更直接的证据,即光敏导向疗法是提高光动力疗法的主要途径。     

重组人纤溶酶原Kringle 5的125Ⅰ标记及其体内代谢

目的:探讨放射性碘标记纤溶酶原Kringle 5(K5)的生物学活性及其体内药代动力学.方法:采用基因工程方法制备重组人纤溶酶原Kringle 5(rhK5),以小剂量Iodogen多次重复标记法标记rhK5,细胞增殖抑制试验和亲和力试验检测125Ⅰ-rhK5活性,并研究其在大鼠体内的药代动力学.结果:125Ⅰ-rhK5的标记率为86%,放射化学纯度为96%.细胞增殖抑制试验表明,125Ⅰ-rhK5的生物活性与未标记rhK5相当.大鼠单次股静脉注射2vg125Ⅰ-rhK5后,血药浓度-时间曲线符合两室模型,T1/2α为(0.31±0.03)h,T1/2β为(14.48±0.73)h,AUC为(436.58±34.6)ng·h·ml-1.结论:小荆量10dogen多次重复125Ⅰ标记不影响rhK5的生物学活性.125Ⅰ-rhK5大鼠体内单次静脉注射的药代动力学符合两室模型,半衰期约为1 5h.125Ⅰ-rhK5为肿瘤的显像和靶向治疗奠定了基础.     

氯化镍在体内诱导大鼠DNA-蛋白质交联

目的　了解NiCl2 在体内对大鼠细胞的DNA产生何种损伤以及与聚腺苷二磷酸核糖聚合酶(PARP)活性的关系。方法　以 10 ,2 0 ,30mg·kg- 1NiCl2 ip给大鼠进行染毒 ,2 4h后处死动物 ,分离外周血淋巴细胞和肺细胞 ,应用单细胞凝胶电泳 (彗星试验 ) ,检测DNA单链、双链断裂和DNA 蛋白质交联 ,同时用 [3H]NAD渗透法检测PARP活性的变化。结果　外周血淋巴细胞和肺细胞均没有出现明显的DNA单链和双链断裂。所有剂量组的外周血淋巴细胞都出现了不同程度的DNA 蛋白质交联 ,交联率为 14 %～ 36 %,但没有剂量反应关系。 2 0mg·kg- 1NiCl2 也能诱导肺细胞DNA 蛋白质交联 ,交联率达 30 %。所有剂量组的NiCl2 均能明显抑制大鼠外周血淋巴细胞PARP酶的活性 ,酶活性下降至对照组的 38%～ 5 7%,但没有表现出剂量反应关系。大鼠肺细胞中该酶活性不受NiCl2 染毒的影响。结论　NiCl2 在体内环境下能诱导大鼠外周血淋巴细胞和肺细胞的DNA损伤 ,主要是引起DNA 蛋白质交联 ,而不直接造成DNA链的断裂 ;NiCl2 还能明显抑制外周血淋巴细胞的PARP酶活性 ,进而可能影响DNA修复。     

DNA-protein crosslinks induced by nickel compounds in isolated rat lymphocytes: role of reactive oxygen species and specific amino acids

Isolated rat lymphocytes in salts-glucose medium (pH 7.2) were incubated with nickel chloride, nickel acetate, nickel sulfate, and a soluble form of nickel subsulfide (0-2 mM) at 37 degrees C for 2 h. The soluble form of nickel subsulfide induced a significant increase in DNA-protein crosslinks (DPXLs) (111%) beginning at 0.5 mM and a maximum increase of 700% from that of the control value was reached at a 2 mM concentration, whereas nickel sulfate produced only a 65% increase of such crosslinks at the 2 mM concentration only. No significant reduction in viability of rat lymphocytes (as measured by trypan blue exclusion) due to these nickel compounds was observed at any concentration used. Time-course studies of DPXLs and cellular viability due to 2 mM nickel subsulfide indicate that DPXL formation may not be due in part to cellular necrosis. Coincubation of nickel subsulfide (2 mM) with l-histidine (16 mM), l-cysteine (4 or 8 mM), or l-aspartic acid (24 mM) significantly reduced the DPXLs induced by 2 mM nickel subsulfide. But Mg(2+) even at 24 mM failed to antagonize nickel subsulfide-induced increase in DPXLs. High concentrations of these amino acids significantly decreased the accumulation of Ni(2+) from nickel subsulfide in lymphocytes, suggesting that such reduction of cellular uptake of Ni(2+) by these amino acids is partly responsible for the potent protective effects of these amino acids against such genotoxicity of nickel subsulfide. In vitro exposure of lymphocytes to nickel subsulfide (0-2 mM) increased the formation of reactive oxygen species (ROS) in a concentration-dependent manner. Furthermore, coincubation of 2 mM nickel subsulfide with catalase, dimethylthiourea, mannitol, or vitamin C at 37 degrees C for 2 h resulted in a significant decrease of nickel subsulfide-induced formation of DPXLs, suggesting that nickel subsulfide-induced DPXLs formation in isolated rat lymphocytes is caused by the formation of ROS. The amino acid treatment also abrogated Ni(3)S(2)-induced generation of ROS. Deferoxamine (a highly specific iron chelator) treatment prevented nickel subsulfide-induced DNA-protein crosslink formation, suggesting that Ni(2+)-induced DPXL formation in rat lymphocytes is caused by the induction of Fenton/Haber-Weiss reaction, generating hydroxyl radicals. The potent protective effects of these specific amino acids against nickel subsulfide-induced DPXL formation in isolated rat lymphocytes may be due in part to impaired cellular uptake of Ni(2+), inhibition of the binding of Ni(2+) to deproteinized DNA, and a reduction in reactive oxygen species.     

术中~(125)Ⅰ粒子瘤体内植入治疗晚期胰腺癌

目的探讨术中~(125)I放射性粒子永久性组织间植入近距离照射治疗晚期无法切除的胰腺癌的治疗方法和临床效果。方法回顾性分析我院2004～2006年间11例手术无法切除的晚期胰腺癌患者行术中B超引导下~(125)I放射性粒子永久性组织间植入近距离照射的临床资料。结果11例接受手术中~(125)I粒子植入的患者术后1、3个月CT复查,肿瘤平均比术前缩小70%。其中有4例患者肿瘤在术后2个月后完全消失;3例明显缩小;2例肿瘤体积无明显变化,但未进一步增长;2例患者因粒子分布不均匀而肿瘤增大,但在~(125)I粒子分布区肿瘤缩小,无~(125)I粒子分布的区域肿瘤生长明显。生存时间:最短者5个月,最长者13个月,平均9．6个月。结论对手术不能切除的晚期胰腺癌患者行术中~(125)I放射性粒子永久性肿瘤组织间植入近距离照射治疗,可使肿瘤明显缩小或消失,延长患者的生存期,减轻晚期痛苦,提高其生活质量。     

[~(125)I]Spiro-I脑靶向脂质体在小鼠体内分布(英文)

研究[125 I]Spiro-I经长循环脂质体(SSL)和脑靶向脂质体(SSTL)包载后的组织分布, 尤其考察其脑摄入。采用薄膜超声分散法制备[125 I]Spiro-I脂质体, RMP-7通过共价键连在DSPE-PEG上进一步形成靶向脂质体。[125 I]Spiro-I-SSL及 SSTL的包封率分别为97.47%±4.01%, 93.02%±2.98%, 粒径分别为(66.47±0.76) nm, (71.40±0.45) nm。给药后, [125 I]Spiro-I迅速从血液中清除, 长循环组延长了药物在血液中的保留时间, RMP-7提高了[125 I]Spiro-I的脑摄取量。[125 I]Spiro-I-SSTL组的AUC较游离药物组提高了1.52倍。SSTL有望拓展显像剂在中枢神经系统的应用。     

A competitive enzyme-linked immunosorbent assay (ELISA) to detect retronecine and monocrotaline in vitro

Antibodies to the nonesterified pyrrolizidine nucleus, retronecine (155 mol.wt), were produced in rabbits and detected using an avidin-biotin antibody ELISA. A competitive ELISA for the detection of retronecine and the cyclic diester monocrotaline was also developed using the antiserum produced against the hapten conjugate, retronecine-bovine serum albumin. Retronecine was obtained by hydrolysis of monocrotaline, succinylated and directly coupled to bovine serum albumin or ovalbumin. Antibodies to the pyrrolizidine nucleus, retronecine, can be detected within 5 min after the addition of substrate using the avidin-biotin ELISA. Competitive inhibition of antibodies to retronecine is obtained by the addition of known amounts (0-11.42 micrograms/microliters) of either the homologous antigen, retronecine, or the heterologous antigen, monocrotaline, however, retronecine acts as the better competitor.     

重组人纤溶酶原Kringle 5的~(125)I标记及其体内代谢

目的:探讨放射性碘标记纤溶酶原Kringle 5(K 5)的生物学活性及其体内药代动力学。方法:采用基因工程方法制备重组人纤溶酶原Kringle 5(rhK 5),以小剂量Iodogen多次重复标记法标记rhK 5,细胞增殖抑制试验和亲和力试验检测125I-rhK 5活性,并研究其在大鼠体内的药代动力学。结果:125I-rhK 5的标记率为86%,放射化学纯度为96%。细胞增殖抑制试验表明,125I-rhK 5的生物活性与未标记rhK 5相当。大鼠单次股静脉注射2μg125I-rhK 5后,血药浓度-时间曲线符合两室模型,T 1/2α为(0.31±0.03)h,T 1/2β为(14.48±0.73)h,AUC为(436.58±34.6)ng.h.m-l 1。结论:小剂量Iodogen多次重复125I标记不影响rhK 5的生物学活性1。25I-rhK 5大鼠体内单次静脉注射的药代动力学符合两室模型,半衰期约为15h。125I-rhK 5为肿瘤的显像和靶向治疗奠定了基础。     

AUTORADIOGRAPHIC ANALYSIS OF (-)-[125I]-CYP BINDING IN MOUSE KIDNEY

The distribution and binding characteristics of the radioligand (-)-[125I]-cyanopindolol (CYP) have been examined in slide mounted mouse kidney sections, using the technique of in vitro labelling and autoradiography. (-)-[125I]-CYP binding to sections was of high affinity (KD = 55.8 pmol/l, s.e.m. = 8.1, n = 4) to a single population of non-interacting sites (nH = 0.95, s.e.m. = 0.01, Bmax = 0.74 fmol/section, s.e.m. = 0.12, n = 4) and stereoselective with respect to the (-)- and (+)-isomers of both propranolol and pindolol. Autoradiographic studies showed that (-)-[125I]-CYP binding was localized to areas in the renal cortex and medulla. Both cortical and medullary binding were abolished by the inclusion of (-)-propranolol (1 mumol/l) in the incubation medium, whereas (-)-isoprenaline (200 mumol/l) selectively abolished cortical binding. Medullary binding could be prevented by the inclusion of the lipophilic compounds cinanserin (10 mumol/l), haloperidol (10 mumol/l) or phentolamine (10 mumol/l), either alone or together or by washing at 37 degrees C. These results suggest that medullary binding sites are lipid rather than receptor-related. In conclusion, in mouse kidney sections, (-)-[125I]-CYP binds to discrete areas in the cortex and medulla. Cortical binding sites have the molecular characteristics of beta-adrenoceptors while medullary binding sites are lipid-related. Caution should therefore be exercised when defining non-specific binding of lipophilic radioligands. The autoradiographic technique is useful for discriminating between receptor and non-receptor binding sites.     

Macromolecular DNA-damage in murine and human leukemic and lymphoid cells after in vitro exposure to ASTA Z 7557 (INN mafosfamide)

Summary Cyclophosphamide (CPA) is widely used against leukemic and lymphoproliferative diseases, but in vitro studies on response to this agent so far have been limited to instable derivatives with poor galenic properties. ASTA Z 7557 is a newly synthesized activated cyclophosphamide that circumvents the need for hepatic activation and has good stability. The critical cytotoxic lesions after exposure to bifunctional alkylating agents presumably are DNA interstrand crosslinks (ISC). We have, therefore, examined the formation and apparent removal of ISC after in vitro treatment with ASTA Z 7557 by use of the highly sensitive alkaline elution technique. Survival of murine L1210 cells was determined after 1 hour in vitro exposure with a D 37 value of 5.7 g/ml (from the initial shoulder part of the survival curve) and a Do value of 1.5 g/ml (from the exponential part of the curve). Previous labelling of L1210 cells by ¹²⁵IUdR simplified the alkaline elution procedure but there was some cytotoxicity of the radiochemical itself with a reduction of cloning efficiency from 77% to 61 %. The maximum of ISC was observed at 6 h after initiation of treatment with much of the damage apparently removed at 24 h. The simultaneous presence of DNA single strand breaks (SSB), however, confounds the analysis of DNA damage at 24 h and early cytolysis and unaided death of human lymphocytes often preclude the analysis of macromolecular damage at this time. Human peripheral blood cells isolated from patients with leukemic or lymphoproliferative diseases showed a remarkable heterogeneity with regard to the formation of ISC at 3 h. Thus, analysis of macromolecular damage may become an additional prognostic factor for response to CPA beyond the morphologic classification of these diseases.     

乙型肝炎导向干扰素在荷瘤鼠体内的分布实验

将2，2，15细胞接种裸鼠，制得抗乙型肝炎病毒（HBV）动物模型。用125I标记乙型肝炎导向干扰素（T－IFN），观察放射标记物（125I－T－IFN）在荷瘤鼠体内的分布情况，以评价T－IFN在体内的抗HBV导向治疗作用。结果显示，125I－T－IFN在各组织浓度顺序依次为：瘤＞心＞肝＞脾＞肾，且瘤组织与其它各组织比较，有明显差异（P＜0．01）。结果表明，在HBV动物模型体内，T－IFN相对浓集于可分泌HBsAg的瘤区。实验结果显示了T-IFN导向治疗HBV感染的可行性。     

Ser~(125)白介素-2的免疫及抑瘤活性研究

研究 Ｓｅｒ１２５白介素－２（Ｓｅｒ１２５ＩＬ－２）的免疫活性及抗瘤作用。方法：小鼠给药后,观察药物对巨噬细胞和淋巴细胞转化的影响;小鼠体内接种Ｓ１８０肉瘤和Ｂ１６黑色素瘤后,观察其抑瘤作用。结果：Ｓｅｒ１２５ＩＬ－２可明显提高小鼠腹腔巨噬细胞的吞噬率和淋巴细胞的转化率（Ｐ＜０． ０１）,显著抑制 Ｓ１８０肉瘤和 Ｂ１６黑色素瘤的体内生年（Ｐ＜０． ０１）。结论：Ｓｅｒ１２５ＩＬ－２ 有增强细胞免疫和抑制肿瘤生氏的作用。