免疫测定

Nighty rats were randomly divided into the groups of control, myocardial infarction and doxycycline, which were given orally 48 hours before and 48 hours after MI with 30mg/Kg per day. Protein and mRNA extraction was done on left ventricular samples containing scar and myocardium together. Samples were assayed from 1 day to 4 weeks post-Mi. The activity of MMPs was measured by zymography and collagen amount by the method of chloramine T, the ratio of II III collagen was assessed by immunohistochemical stain. Protein and mRNA of MMP-2, 9, TIMP-1 were determined by Western-Blot and RT-PCR.

分为对照组、心梗组和治疗组(D组，术前两天至术后两天，口服Doxycycline，30mg／kg／天)，分别于术后第一天、术后一周、术后两周、术后四周取心肌组织，采用免疫组化测定胶原含量和Ⅰ／Ⅲ胶原比例，酶谱法测定心梗后MMP-2，9活性蛋白的表达规律，Western Blotting进一步确定酶谱法中所消化条带蛋白的属性，逆转录-聚合链反应法测定心梗后MMP-2，9和TIMP-1的mRNA的变化规律，运用Acuson Sequoia 512超声心动图机分别测定心梗组、治疗组在两周和四周时左室前壁、后壁厚度以及左心室舒张末内径和左室射血分数。

We detected fasting plasma glucose by glucose oxidase,fasting insulin by chemiluminescent immunoassay, triglyceride and high density lipoprotein-cholesterol by zymology,tumor necrosis factor-αby enzyme linked immunosorbent assay, the reverse of the product of FPG and FIns is used as the insulin sensitivity index,that is,ISI=1/FPG×FIns.

血糖测定采用葡萄糖氧化酶法；胰岛素测定采用化学发光法；血脂测定采用酶法；肿瘤坏死因子-α测定采用双抗体夹心酶联免疫法；胰岛素敏感指数采用空腹血糖与空腹胰岛素乘积的倒数，即ISI=1/FPG ×FIns。

We detected fasting plasma glucose by glucose oxidase, fasting insulin by chemiluminescent immunoassay, triglyceride and high density lipoprotein-cholesterol by zymology, tumor necrosis factor-αby enzyme linked immunosorbent assay, the reverse of the product of FPG and FIns is used as the insulin sensitivity index, that is, ISI=1/FPG×Fins.

血糖测定采用葡萄糖氧化酶法；胰岛素测定采用化学发光法；血脂（甘油三酯TG、高密度脂蛋白HDL-C）测定采用酶法；肿瘤坏死因子-α测定采用双抗体夹心酶联免疫法；胰岛素敏感指数采用空腹血糖与空腹胰岛素乘积的倒数，即ISI=1/FPG ×FIns。

Get high purity DCs by Cultured plastic-adherent monocytes isolated from healthy human peripheral blood with GM-CSF and IL-4 for 7 days. To observe the morphology of DCs by inverted phase contrast microscope ,electron microscope and laser confocal microscope. Analyse phenotype of DCs with flow cytometry. Investigate the endocytosis ability of DCs as a group by Horseradish peroxidase endocytosis assay. To appraise allogeneic mixed lymphocytes reaction of DCs by MTT reduction assay. Analyse the levels of IL-12 and TNF in liquids of cultured medium by ELISA and MTT reduction assay respectively. Soluble antigens of HCCs was obtained by 3 freeze-and-thaw cycles. Biological characteristics of HC soluble antigens pulsed DCs were monitored by flow cytometry. According to MTT reduction assay estimated the cell proliferation of self lymphocytes activated by HC antigens pulsed DCs. Get high purity BCG HSP 70 protein by SDS-PAGE electrophoresis and determined its biological activity with ELISA. Analyse phenotype of antigen pulsed DCs primed by BCG HSP70 with flow cytometry. By MTT reduction assay estimated the cell proliferation of self lymphocytes and the MLR of DC based vaccine. Analyse expression of HLA-DR molecule on surface of HCC lines. The IFN-γ mRNA in lymphocytes after actived by DC vaccine and the Fas-L expression on DC and DC vaccine primed lymphocytes were detected by in situ hybridization and flow cytometry respectively. Specific cytotoxity lysis of T lymphocytes and nonspecific inhibition of liquids in culture medium against HCC lines were also tested. Detect expression of hAFP on four HCC lines with Cell-ELISA. Induce apoptosis of HCCs with actinomycin-D. Interaction of DCs and apoptotic cells was observed under transmission electron microscope. Growth inhibition test of DC against HCC lines was also performed. Establish the nude mouse model bearing human HC xenografts and indentify the characteristic of tumour by histochemistry and immunohistochemistry techniques. Prevent and treat transplanted human HC on nude mouse with Freezing and anabiotic HC specific lymphocytes.

用GM-CSF和IL-4从健康人外周血诱导DC；分别用倒置相差显微镜、电子显微镜及激光共聚焦显微镜观察DC形态；流式细胞术检测DC表型；HRP吞噬实验测定DC的群体内吞能力；MTT法检测同种异体混合淋巴细胞反应；ELISA法和MTT法分别测定DC培养上清液中IL-12和TNF水平；冻融法制备肝癌细胞可溶性抗原；流式细胞术检测负载肝癌可溶性抗原后DC的生物学特性；MTT法检测DC负载肝癌抗原后对自身淋巴细胞增殖的影响；SDS-PAGE制备电泳纯化BCG HSP70并鉴定纯度，ELISA测定活性；流式细胞术检测负载抗原DC经BCGHSP 70活化后的表型；MTT法检测肝癌DC疫苗对自身淋巴细胞增殖的影响和混合淋巴细胞反应；流式细胞术检测肝癌细胞表面HLA-DR表达；MTT法检测肝癌DC疫苗对自身淋巴细胞的活化；原位杂交法检测肝癌DC疫苗活化后的淋巴细胞IFN-γmRNA表达；流式细胞术检测DC和肝癌DC疫苗活化后淋巴细胞表面Fas-L；MTT法分别检测肝癌DC疫苗活化的淋巴细胞和其培养上清对肝癌细胞的特异性杀伤和非特异性抑制作用；Cell-ELISA检测人肝癌细胞hAFP表达；MTT法检测负载AFP表位肽和凋亡肝癌细胞DC对自身淋巴细胞增殖的影响；ELISA法和MTT法分别测定活化后淋巴细胞培养上清中TNF和IL-12水平；肝癌细胞凋亡的诱导和检测；DC吞噬凋亡肝癌细胞后的电子显微镜观察；DC对肝癌细胞的生长抑制试验；人肝癌裸鼠皮下移植瘤动物模型的建立及其组织学和免疫组织化学鉴定；DC及肝癌特异性淋巴细胞预防和治疗人肝癌裸鼠皮下移植瘤；冻存和复苏后的肝癌特异性淋巴细胞预防和治疗人肝癌裸鼠皮下移植瘤。

A novel method for the measurement of insulin-link growth factor I with capillary zone electrophoresis was developed. The molecular interaction between IGF-I and its polyclonal antibody was primarily studied with immunocapillary electrophoresis and the binding constant was determined. The work shows the possibility of applying CE to immuno-analysis, which could be a new clinical analytical method for diagnosis, treatment and monitoring for the endocritic and metabolic diseases.

以分子间相互作用理论为依托，以胰岛素样生长因子-Ⅰ及其抗体为研究体系，建立了用毛细管区带电泳测定IGF-I的方法，并首次用免疫毛细管电泳方法研究了IGF-I与它的多克隆抗体的相互作用，测定了它们的结合常数，进一步证实了在毛细管电泳中实施免疫分析的可能性，为内分泌及代谢性疾病等威胁人类健康的常见病的临床诊断及治疗观察提供了新的检测方法。

The PSCA_3 fragment was selected for its superior expression level in eukaryotic cells.Then the sig-PSCA_3-Fc-GPI genetic fragment was cloned into pVAX1-neo-IRES-GM/B7 vector to construct the final immunological inhanced DNA vaccine pVAX1-PSCA_3-FcGB. Immunofluorescence and flow cytometry were used to confirm the expression of PSCA_3 fragment by transfected into Cos7 cell.Finally,the anti-tumor effect of pVAX1-PSCA_3-FcGB was tested in murine prostate cancer model generated by RM-1 cell line.The animal was immunized with pVAX1-PSCA_3-FcGB DNA vaccine by intramuscular injection plus electroporation,pVAX1 and pVAX1-PSCA_1-FcGB plasmid were used as control.The inhibitory effect of tumor was investigated by observion of forming time,volume and inhibition ratio of tumor.Results:DNA sequencing conformed that the heterological PSCA fusion antigen fragment which was synchronized by overlapping-extending-PCR,was consistent to design.Enzyme digestion analysis showed that the 1 to 4 copies heterological PSCA fusion antigen fragments were constructed successfully.

方法(1)检索GenBank，选择包含人主要T细胞抗原表位序列的人PSCA基因片段，应用异种化抗原设计技术，保留人T细胞抗原表位，设计异种化PSCA融合抗原片段；(2)根据核酸序列按中心模板法设计引物，应用重叠延伸PCR技术拼接合成异种化PSCA融合抗原片段基因，以PCR、限制性酶切和DNA序列测定法进行鉴定：(3)利用DNA限制性内切酶BssHⅡ和MluⅠ酶切后粘端互补的特点，采用同尾酶法构建1—4拷贝异种化PSCA融合抗原片段(PNCA_1-PSCA_4)，并将上述片段分别插入真核表达载体pCI-neo-Fc-GPI中，转染293T细胞，借助免疫荧光+流式细胞术考察插入片段表达效率，最终选定PSCA_3片段进行下一步研究；(4)将sig-PSCA_3-Fc-GPI基因片段自pCI-PSCA_3-Fc-GPI质粒上切下，插入pVAX1-neo-IRES—GM/B7载体中，构建免疫增效DNA疫苗pVAX1-PSCA_3-FcGB，并应用转染Cos7细胞+免疫荧光/流式细胞术方法鉴定其在真核细胞中的表达情况；(5)给8周龄雄性C57BL/6小鼠皮下种植RM-1细胞，制备小鼠前列腺癌模型，并采用股四头肌肌肉注射+电脉冲法(Electroporation，EP)接种DNA疫苗质粒pVAX1-PSCA_3-FcGB，同时接种pVAX1空载体质粒和pVAX1-PSCA_1-FcGB质粒作为对照，通过观察计算免疫动物的成瘤时间、肿瘤体积和抑瘤率，来评价该DNA疫苗在小鼠体内的抑瘤效果。

It is demonstrated CAV 2 adenovirus and named CAV 2 XN 3,after analysing the morphological,physical,chemical and biological features.The authors replicate the virus into hypotoxic and immunize 8 local young canines of two months old,without harmful reactivity being found.

实验将该病毒复制成弱毒疫苗，免疫 8只 2月龄当地幼犬，经临床观察证明，试验犬未出现不良反应；免疫后 14天采血测定血清中和抗体效价达到 1∶12 8，免疫后 2 5天血清中和抗体效价达到 1∶2 5 6 ，免疫后 10个月降至 1∶32 ，但仍能耐受强毒攻击，而对照组 4只犬则发病死亡。

Methods 45 patients with nonobstructive azoospermia, 40 patients with obstructive azoospcrmia and 40 healthy subjects were involved in this study. Serum FSH, LH and T levels were measured by chemiluminescence immunoassay, and serum INHB was measured by sandwich enzyme-linked immunosorbent assay.

采用化学发光免疫分析法测定45例非梗阻性无精子症、40例梗阻性无精子症及40名正常生育男性血清FSH、LH及T的浓度，以双抗体夹心酶联免疫吸附试验测定血清INHB水平。

There were no significant differences for the firing rates in the site of contralateral TNC neurons among during pre-CSD,CSD,and post-CSD (P＞0.05).For flunarizine group,the firing rates in the site of ipsilateral TNC neurons during pre-CSD were higher as compared with during CSD(P＜0.05).2.1 There were statistical differences on palasma levels of CGRP and SP among the three groups(P＜0.05).The levels of CGRP and SP in CSD group were higher than control group(P＜0.05).No significant differences on the levels of CGRP and SP in ipsilateral trigeminal ganglia were found among the three groups(P＞0.05).2 The number of neurons with positive CGRP and SP immunoreactivity was statistically different in right-sided trigeminal ganglia among the three groups (P＜0.05).The number in fight-sided trigeminal ganglia in CSD group was higher as compared with control group(P＜0.05).The number in right-sided trigeminal ganglia was statistically higher than that in left-sided trigeminal ganglion in CSD group(P＜0.05).3.1 Altered ReHo in ipsilateral pons and other brain regions response to pain such as basal nuclei,thalamus,cingulated gyms and prefrontal cortex was detected during the acute spontaneous attack as compared with during headache remission(P＜0.05,corrected by Monte Carlo simulation). 2 Positive functional connectivity was detected between ipsilateral pons and other brain regions related to pain within pain state and within non-pain state (P＜0.05,corrected by false discovery rate,FDR).Increased functional correlation between ipsilateral pons and other pain-related brain regions such as ipsilateral prefrontal cortex and contralateral subcallosal gyrus was detected during the acute spontaneous attack as compared with during headache remission(P＜0.05,corrected by Monte Carlo simulation).

结果1。对照组未发现CSD；同侧TNC放电频率，CSD中＞CSD后＞CSD前P＜0.05对侧TNC放电频率，CSD前、中、后无统计学差异(P＞0.05氟桂利嗪组同侧TNC放电频率，CSD前＞CSD中(P＜0.05)，CSD前与CSD后及CSD中与CSD后之间无统计学差异(P＞0.05)。2.1关于放免测定，各组血浆CGRP、SP水平有统计学差异(P＜0.05)，CSD组高于对照组(P＜0.05)，CSD组与氟桂利嗪组、对照组与氟桂利嗪组之间均无统计学差异P＞0.05各组之间同侧三叉神经节中CGRP、SP水平未见变化(P＞0.05.2关于免疫组化研究，右侧三叉神经节CGRP、SP免疫阳性细胞数三组之间有统计学差异(P＜0.05)，多重两两比较结果CSD组大于对照组(P＜0.05)，CSD组与氟桂利嗪组之间、对照组与氟桂利嗪组之间无统计学差异P＞0.05左侧三叉神经节CGRP、SP免疫阳性细胞数三组之间无统计学差异(P＞0.05CSD组中右侧三叉神经节CGRP、SP免疫反应阳性细胞数大于左侧(P＜0.05)。3.1局部一致性分析发现两组患者头痛疼痛状态较非疼痛状态脑活动发生变化的脑区有同侧脑桥以及其他疼痛相关脑区如基底节区、丘脑、扣带回、前额叶皮层等(P＜0.05，蒙特卡罗模拟校正)。2功能连接分析发现疼痛状态与非疼痛状态下主要疼痛相关脑区均与同侧脑桥有功能联系P＜0.05，false discovery rate，FDR校正疼痛状态与非疼痛状态比较，同侧前额叶皮层、对侧胼胝下回等疼痛相关脑区与同侧脑桥之间功能联系增强(P＜0.05，蒙特卡罗模拟校正。

Methods KC and FE of tympanic membrane were cultured on the surface of CCM prepared by torrefaction, then the cells biological characteristics were detected by immunofluorescence stain of keratin and vimentin, and the cells proliferative activity were identified by immunofluorescence stain of PCNA.

方法热干法制备CCM，传代的大鼠鼓膜上皮KC和FB分别接种于培养板及CCM表面，通过角蛋白、细胞膜波形蛋白免疫荧光染色鉴定细胞特异性，通过细胞核增殖抗原(proliferating Cell nuclear antigen， PCNA)免疫荧光染色测定膜上细胞的增殖能力，通过细胞培养液上清的经脯氨酸测定评价对FB胶原分泌功能的影响。

However, as the name（read－only memory）implies, CD disks cannot be written onorchanged in any way.

然而，正如其名字所指出的那样，CD盘不能写，也不能用任何方式改变其内容。

Galvanizes steel pallet is mainly export which suits standard packing of European Union, the North America. galvanizes steel pallet is suitable to heavy rack. Pallet surface can design plate type, corrugated and the gap form, satisfies the different requirements.

镀锌钢托盘多用于出口，替代木托盘，免薰蒸，符合欧盟、北美各国对出口货物包装材料的法令要求；喷涂钢托盘适用于重载上货架之用，托盘表面根据需要制作成平板状、波纹状及间隔形式，满足不同的使用要求。