低剂量12C6+辐射对小鼠免疫系统损伤和抗氧化剂氮乙酰半胱氨酸的保护作用研究

实验目的：随着科技的发展，人类活动范围已经逐渐向外太空扩展，对于人类太空探索的最大威胁是太空中的各种粒子辐射。这些辐射包括太阳辐射（质子和电子）和银河辐射（质子占85%，氦离子占14%，重离子占1%）。众所周知，重离子与常规X和γ射线相比有较高的传能线密度（linear energy transfer, LET）和相对生物学效应（relative biological effectiveness, RBE），对机体组织和器官有较强的影响。放射治疗是肿瘤治疗的重要手段之一，由于肿瘤细胞的异质性，其对放、化疗的反应相差悬殊。本研究的目的是： 1评估辐射对健康机体产生的生物学风险； 2研究抗氧化剂氮乙酰半胱氨酸（NAC）对机体辐射损伤的保护作用 3不同肿瘤细胞辐射敏感性的差异。实验方法： 1 X射线或12C6+离子对小鼠进行不同剂量的全身辐射。NAC处理组小鼠在照射前1小时腹腔注射200mg/kg的NAC，对照组注射等体积的生理盐水。照射后不同时间点取样，利用流式细胞仪检测小鼠免疫细胞周期和凋亡情况，单细胞电泳检测淋巴细胞DNA损伤，MTT法（3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide）检测脾脏NK（natural killer，NK）细胞活性，微核法检测淋巴细胞染色体损伤情况，小鼠体内干扰素－γ（Interferon-γ，IFN-γ）由ELISA方法得到，小鼠血清中超氧化物岐化酶(Surperoxide dismutase SOD）由分光光度法测定，并观察胸腺和脾脏指数变化。 2 不同剂量X射线和12C6+离子辐射人肺腺癌细胞H1299和A549，用细胞克隆法检测照射后细胞存活曲线，流式细胞仪检测细胞周期和凋亡，Western-blot 检测A549 细胞P53蛋白表达。 结果： 1小鼠外周血淋巴细胞、胸腺细胞和脾脏淋巴细胞周期随着X射线照射剂量的增大而被阻滞在了G0/G1期，相同剂量的12C6+离子辐射时外周血淋巴细胞周期被阻滞在S期，分次连续X射线照射时，外周血淋巴细胞周期随着累积剂量的增加被阻滞在G2/M期；细胞凋亡比例随着照射剂量的增加而增加。小鼠血清中IFN-γ水平和脾脏中NK细胞活性在重离子照射剂量为0.05Gy时有显著增加，脾脏NK细胞活性随着照射剂量的增加而减弱。 2重离子照射后，小鼠淋巴细胞DNA和染色体的损伤随辐射剂量和照射后时间的延长而加剧。脾脏NK细胞活性在照射后各个时间点减弱，血清中IFN-γ水平和SOD酶活性随着重离子照射剂量的增加而降低。预防性给予NAC，12C6+离子辐射对淋巴细胞DNA和染色体所致损伤，胸腺细胞周期和凋亡，脾脏NK细胞活性，血清中IFN-γ的水平和SOD酶的活性的损伤与盐水组比较均有显著改善。 3 X射线照射对肺腺癌H1299细胞周期和凋亡率未产生明显影响，重离子照射后随着照射剂量的增加细胞周期被阻滞在G2/M期，细胞凋亡率也呈剂量依赖性；X射线和12C6+离子照射A549细胞后，细胞周期均被阻滞在G2/M期，凋亡率剂量依赖性增加。A549细胞P53蛋白的表达水平随着重离子照射剂量的增加而增加。结论： 1重离子辐射造成细胞DNA和染色体损伤随着照射剂量的增加和照射后时间的延长而增加，比X射线辐射损伤复杂和难以修复，产生这种现象的机理为辐射导致活性氧分子簇的产生，细胞因子和与细胞氧化反应有关的酶活性的变化，同时这种损伤对胸腺细胞周期、凋亡和胸腺、脾脏指数以及机体免疫系统都有影响；低剂量重离子辐射（0.05Gy）对小鼠机体的免疫力有刺激作用，机体免疫能力随着照射剂量增加和照射后时间的推移而减弱，不同的免疫器官对辐射的敏感性也不同； 2 200mg/kg 的NAC对辐射所致小鼠免疫系统损伤有很好的保护作用； 3 肺腺癌细胞H1299比同系A549具有较强的辐射敏感性，A549细胞凋亡的增加与P53蛋白表达水平升高有关

抗HER2/neu单链抗体增强TNF-α对卵巢癌细胞的抗肿瘤活性

为了探索抗HER2/neu单链抗体与TNF-α联合应用对表面过度表达HER2/neu卵巢癌细胞的生物效应,同时构建了抗HER2/neu单链抗体scFvC6.5的原核表达载体和人TNF-α的原核表达载体,将上述两种重组子分别转化入感受态宿主菌BL21(DE3),得到稳定表达.表达产物主要以包含体形式存在;包含体经过溶解、变性、复性和纯化,得到了分纯的产物.SDS-PAGE和Western-blot检测结果证实,蛋白表达正确.ELISA法验证了scFvC6.5和人TNF-α具有与卵巢癌细胞SKOV-3的结合活性.MTT细胞毒活性试验进一步表明,相对于单独使用TNF-α,联合应用上述两个重组蛋白细胞毒效应明显提高,SKOV-3细胞对TNF-α的敏感性增强.这将为抗HER2/neu抗体的联合抗肿瘤疗法提供一种新的途径,具有潜在的临床应用价值.图5参23

抗HER2-hTNF-α新型免疫毒素的制备及功能研究

HER2/neu基因在肿瘤中的过度表达使其成为许多肿瘤的标志分子.为了增加过度表达HER2/neu的肿瘤细胞对肿瘤坏死因子(TNF)的敏感性和提高HER2/neu抗体的肿瘤杀伤效应,将抗HER2/neu单链抗体C6.5与人肿瘤坏死因子hTNF-α融合,构建了scFvC6.5-hTNF-α融合蛋白,完成了重组蛋白在大肠杆菌中的表达,产率为400μg/L菌液.经过亲和层析和柱复性,融合蛋白的纯度达95%以上.ELISA试验表明,scFvC6.5-hTNF-α能够特异结合HER2/neu阳性卵巢癌细胞SKOV-3和乳腺癌细胞MCF-7,而不结合HER2/neu阴性的黑色素瘤细胞A375.MTT试验表明,scFvC6.5-hTNF-α能够选择性地杀伤SKOV-3和MCF-7细胞,而不影响A375细胞的生长.这种肿瘤细胞特异性杀伤作用提示该免疫毒素具有肿瘤靶向治疗的潜在应用价值.

内源激素含量的变化与白桦成花关系的研究

采用酶联免疫吸附法(ELISA)测定有花史白桦大树与无花史白桦幼树的4种内源激素脱落酸(ABA)、细胞分裂素(iPA)、生长素(IAA)、赤霉素(GA3)含量的动态变化,研究其与白桦成花的关系。研究结果表明:白桦有花史大树与无花史幼树的4种内源激素含量动态变化明显不同。在雌、雄花芽的生理发端期,有花史大树的IAA含量保持相对稳定,而高含量的ABA和iPA含量的逐渐降低以及GA3含量的逐渐升高可能有利于白桦成花。

噬菌体呈现技术筛选抗黑色素瘤单链抗体

目的 :制备黑色素瘤单抗的单链可变区片段 ,用于肿瘤的诊断或靶向治疗。方法 :从杂交瘤细胞提取总RNA,分别扩增出轻重链可变区基因(variableregionofheavychain,VH和variableregionoflightchain,VLDNA) ,连接形成单链抗体 (singlechainfragmentvariableregion,ScFv)DNA,将ScFv与载体pCANTAB5E的连接产物转化大肠杆菌TG1 ,经M13K07超感染后 ,获得噬菌体抗体ScFvcDNA文库 ,用黑色素瘤细胞LiBr对重组的噬菌体抗体进行3轮吸附 -洗脱 -扩增亲和筛选后 ,随机挑选克隆经ELISA筛选鉴定。结果:VH、VL和ScFv分别约为360、330、750bp,从随机筛检的30个克隆中获得10个高亲和性噬菌体呈现型ScFv单克隆。结论:用噬菌体呈现技术制备的黑色素瘤ScFv ,可为肿瘤的诊断和靶向治疗奠定基础

梭形芽孢杆菌(Bacillusfusiforms)N141发酵产河豚毒素的研究

对分离筛选得到的产河豚毒素(TTX)的梭形芽孢杆菌(Bacillus fusiforms)N141菌株,在10L发酵罐中进行发酵产TTX的试验。发酵产物经提取和精制后,用高效薄层色谱、小鼠生物试验,以及河豚毒素单抗ELISA检测试剂盒,检测出发酵液中含有TTX。研究了菌体生长、pH、溶氧、以及产物TTX的变化规律。结果表明TTX是在菌体生长进入稳定期后才产生的,说明TTX是微生物产生的次级代谢产物。10L罐发酵产TTX的工艺技术,在500L发酵罐进行了验证,重现性良好。这为微生物源河豚毒素的发酵中试提供了科学依据。

鸭瘟病毒gB蛋白N端抗原域的高效表达及其免疫特性

在分析鸭瘟病毒(Duck plague virus,DPV)gB蛋白抗原性的基础上,设计1对引物克隆gB蛋白N端抗原性较好的抗原域编码基因,克隆到表达载体pET32a中,构建了原核表达质粒pET-gB1。将pET-gB1转化到感受态大肠杆菌(Escherichia coli)BL21(DE3)中,经IPTG诱导和SDS-PAGE分析,可见约42.4kD的目的蛋白以包涵体形式表达。Western blot分析发现,表达产物与抗鸭瘟的鼠阳性血清发生特异性反应。将包涵体溶解于8mol/L的尿素中,利用His·Bind试剂盒获得纯化的蛋白,将纯化的蛋白皮下注射免疫小鼠,间接ELISA法测得抗体的效价,MTT法检测免疫小鼠的T淋巴细胞增殖反应能力。结果说明,该融合蛋白能够诱导机体产生较强的体液免疫和细胞免疫。

植物生长调节剂对水曲柳组培苗生长及内源激素的影响

以水曲柳成熟种子为外植体材料,在不同增殖培养基中培养,应用间接酶联免疫法(ELISA)测定脱落酸(ABA)、吲哚乙酸(IAA)和玉米素核苷(ZR)3种内源激素,探讨不同细胞分裂素质量浓度和配比对水曲柳组培苗生长及内源激素的影响。研究结果表明:当年采收的水曲柳种子,消毒、浸泡处理后,播出种胚,可以解除水曲柳种子的休眠。增殖培养基中添加不同细胞分裂素都抑制了组培苗的高生长和生物量,但却可以解除组培苗顶端优势,诱导腋芽生长。与此同时,腋芽发生率与内源激素ABA、IAA和ZR质量分数不存在相关性,而与IAA/ZR比值存在负相关,但相关性并不显著。IAA/ZR比值的减少有可能有利于腋芽的诱导。

虎眼万年青多糖对小鼠免疫功能的调节作用

依据虎眼万年青组分中多糖对小鼠巨噬细胞吞噬功能的影响,筛选并优化出60%醇浓度中性多糖活性组分S3,进而研究其对体液免疫、细胞免疫及细胞因子变化的影响,并从细胞和分子水平探讨其作用的机理.方法:采用溶血素测定方法,测定S3对小鼠脾细胞溶血素抗体的诱导作用;采用3H-TdR渗入法,测定S3对ConA诱导小鼠脾淋巴细胞的增殖作用;采用3H-TdR后标记法,测定S3对NK细胞细胞毒活性的影响;以ELISA法测定S3对小鼠脾细胞IL-2产生的影响;利用流式细胞术,检测S3对T淋巴细胞亚群CD3、CD4、CD8、CD4/CD8阳性细胞百分率的影响;采用RT-PCR方法检测IL-2 mBNA的表达水平.结果:S3高、中剂量组能明显增强小鼠脾细胞溶血素抗体的形成(P<0.05);各剂量组均能明显增强ConA诱导的淋巴细胞增殖能力(P<0.001);各剂量组均能增强NK细胞的细胞毒活性并促进IL-2的产生(P<0.001,P<0.01);高、中剂量组CD8阳性细胞百分率明显降低(P<0.001),各剂量组均能显著地提高CD4/CD8阳性细胞百分率(P<0.001);高剂量组可明显促进脾细胞中细胞因子IL-2的mRNA表达,使表达量增加(P<0.05).结论:S3具有较强的增强机体多种免疫功能的作用,可利用开发为一种免疫增强药物.

时间分辨激光荧光光谱技术在免疫分析中的应用

本研究以时间分辨激发荧光光谱分析技术为基础,进行稀土离子标记的激光激发的时间分辨荧光免疫分析(TRFIA)研究.实验以自行合成的二乙三胺五醋酸酐(DTPAA)为双功能螯合剂.用Eu3+标记兔抗人(RAH)IgG抗体,依据解离增强原理(DELFIA),研究了Eu3+-β萘甲酰三氟丙酮(β-NTA)的荧光分辨体系,测定了荧光光谱和荧光寿命,建立了铕离子分析检出方法,其工作曲线范围为1×10-7～1×10-11 g·mL-1,检测限为1×10-13 g·mL-1,相对标准偏差为6.4%.结合TRFIA方法学研究,进行了人血清丙型肝炎病毒抗体(Anti-HCV)检测.并同酶联免疫法(ELISA)对比.取得TRFIA法阳性检测率明显高于ELISA法的结果.

Inhibitory effects and mechanisms of high molecular-weight phlorotannins from Sargassum thunbergii on ADP-induced platelet aggregation

We evaluated the effects of high molecular-weight phlorotannins from Sargassum thunbergii (STP) on ADP-induced platelet aggregation and arachidonic acid (AA) metabolism in New Zealand white rabbits and Wistar rats. The inhibition of STP on platelet aggregation was investigated using a turbidimetric method, and the levels of the terminal products of AA metabolism were measured using the corresponding kits for maleic dialdehyde (MDA), thromboxane B-2 (TXB2) and 6-keto-prostaglandin F-1 alpha (6-keto-PGF(1 alpha)) by colorimetry and radioimmunoassay, as appropriate. We found that STP could inhibit ADP-induced platelet aggregation, and the inhibitory ratio was 91.50% at the STP concentration of 4.0 mg/mL. Furthermore, STP markedly affected AA metabolism by decreasing the synthesis of MDA (P < 0.01) and increasing the synthesis of 6-keto-PGF(1 alpha), thus changing the plasma TXB2/6-keto-PGF(1 alpha) balance when the platelets were activated (P < 0.01). Therefore, STP altered AA metabolism and these findings

Effect of lanthanum on expression of recombinant allophycocyanin gene in Pichia pastotis

The enhancing effect of lanthanum on gene expression of recombinant allophycocyanin (rAPC), a potential antitumor medicine, in Pichia pastoris was studied. PCR and sequence analysis were used in order to prove whether the APC gene had integrated into the yeast genome. The expression level of the recombinant allophycocyanin (rAPC) in BMMY medium containing LaCl3 was detected by ELISA method. The recombinant allophycocyanin was determined by Western blot. The results show that the recombinant Pichia pastoris chromosome contained allophycocyanin gene. Expression efficiency of rAPC gene in Pichia pastoris was promoted by proper LaCl3 concentration like 2, 5, 10 mmol (.) L-1, among which 5 mmol (.) L-1 was the most effective. The highest expression yield of rAPC in the BMMY medium containing 5 mmol (.) L-1 LaCl3 was 4.4 mg (.) L-1 at 48 h, that was increased by 110% compared with 2.1 mg (.) L-1 of control, in the meantime, the optimum culture time is shortened from 72 to 48 h. The result of western blot analysis indicates that the rAPC consisted of two kinds of subunits with molecular weight of 19 and 21 kDa respectively.

Rapid detection of virulent protease secreted by Vibrio anguillarum by dot enzyme-linked immunosorbent assay

Dot enzyme-linked immunosorbent assay (dot-ELISA), indirect ELISA and Western blot were performed to detect the virulent protease secreted by Vibrio anguillarum which was isolated from the diseased left-eyed flounder, Paralichthys olivaceous. Sensitivity results showed that dot-ELISA is a more sensitive, rapid and simple technique for the protease detection. The minimal detectable amount of protease is about 7 pg in the dot-ELISA test, while 7.8 ng in the indirect ELISA and 6.25 ng in the Western blot respectively. Protease could be detected 2 h after incubation of V. anguillarum in the 2216E liquid medium but enzyme activity was very low at that period. From 6 to 12 h, the amount and enzyme activity of protease increased markedly and reached maximum at stationary phase. Analysis of serum samples periodically collected from the infected flounders showed that after 2 h of infection by V. anguillarum, the pathogenic bacteria could be detected in the blood of the infected flounders but no protease was found. It was 5 similar to 6 h after infection that the protease was detected in blood and then the amount increased as infection advanced. Quantitative detection of protease either incubation in the medium or from the blood of infected flounders could be accomplished in virtue of positive controls of quantificational protease standards ("marker") so that the alterations of protease secretion both in vitro and in vivo could be understood generally. In addition, the indirect ELISA and dot-ELISA were also performed to detect V. anguillarum cells. Results indicated that the sensitivity of indirect ELISA to bacteria cells is higher than that of the dot-ELISA, and that the minimal detectable amount is approximately 10(4) cell/mL in the indirect ELISA, while 10(5) cell/mL in the dot-ELISA.

Expression of hepatitis B surface antigen gene (HBsAg) in Laminaria japonica (Laminariales, Phaeophyta)

A transformation model for Laminaria japonica was established from 1993 to 1998, on the basis of which the transgenic kelp with heterologous gene encoding hepatitis B surface antigen (HBsAg) was obtained by using the micro-particle bombardment transformation method. Results of quantitative ELISA showed that HBsAg in transgenic kelp was 0.529 mug/mg soluble proteins on average and the highest value was 2.497 mug/mg, implying that recombinant HBsAg had natural epitope. Further support for the integration of HBsAg gene into kelp genome was obtained by PCR-Southern and total DNA hybridization. Prospect of kelp bio-reactor producing high value materials such as edible HBV vaccine was discussed as well.