穿膜肽标记异硫氰酸荧光素及钆喷替酸葡甲胺行MR分子显像的探讨

目的选择穿膜肽的基本序列之一，构建目的多肽序列标记异硫氰酸荧光素（FITC）及钆喷替酸葡甲胺（Gd-DTPA），探讨穿膜肽携带FITC及Gd-DTPA跨膜转运性能及MR分子显像的价值。方法在穿膜肽9聚精氨酸[arginine]基础上，构建新目的多肽序列CPP13：LAGRRRRRRRRRK，固相合成多肽后标记FITC（记为CPP13-FITC）和Gd-DTPA（记为CPP13-DTPA-Gd）；分别取CPP13-FITC和FITC溶于无血清Dulbecco最低必须培养液（DMEM）培养基中，待HEPG2细胞及鼠骨髓干细胞爬片上生长至80％-90％汇合时，取2只30mm^2皿，弃去培养皿中培养液，一皿中加入CPP13-FITC／DMEM溶液，另一皿中加入FITC／DMEM溶液，37℃CO2孵箱中抚育10、30、60min时依次取出1片，磷酸平衡盐溶液（PBS）冲洗爬片后置于倒置荧光显微镜上观察细胞内出现荧光素分布的时间和部位。CPP13-DTPA-Gd作用肝癌细胞株HEPG2后，MRI研究CPP13-DTPA-Gd在细胞内转运性能及MRI的特点，将CPP13-DTPA-Gd溶于无血清DMEM培养基中，浓度为3mg／ml。待HEPG，细胞在100mm。培养皿中长至80％-90％汇合时，取3只皿分别加入CPP13-DTPA-Gd的DMEM溶液、DTPA-Gd／DMEM溶液和DMEM溶液，孵育30min后弃去培养液，以0．1MPBS反复冲洗，胰酶消化，加入2m 1l％琼脂糖／PBS溶液混匀，装入2ml离心管中，将3管固定于装有150ml 1％琼脂糖／PBS溶液的微量加样枪头盒中，待凝固后测定MR信号。结果固相合成多肽成功，测定分子量为1792．78，与理论值接近。纯度达到95％以上；标记荧光素后，倒置荧光显微镜观察细胞摄取显示10min时肝癌细胞株HEPG2和鼠骨髓干细胞胞质与细胞核内均出现荧光分布；CPP13标记的Gd-DTPA，作用细胞30min后MRI显示CPP13-DTPA-Gd作用HEPG2细胞组呈短TI、短T2信号。3层面内3组细胞感兴趣区T1信号强度（Ii）与琼脂糖T1信号强度（Io）的比值及统计学分析如下：（1）号管3层面内Iil／Io（为CPP—DTPA-Gd作用cell组管内信号值与管外烧杯内琼脂糖背景信号值的比值）分别为：2．84、2．60、2．48；（2）号管3层面内Ii2／Io（为Gd-DTPA作用cell组管内信号值与管外烧杯内琼脂糖背景信号值的比值）分别为1．15、1．11、1．12；（3）号管3层面内Ii3／Io（为空白cell对照组管内信号值与管外烧杯内琼脂糖背景信号值的比值）分别为1．13、1．11、1．11。两两组间F检验：（1）与（2）：F（I．2）=201．88（P〈0．001）；（1）与（3）：F（1．3）=206．37（P〈0．001）；（2）与（3）：F（2．3）=0．529（P=0．507）。说明T1WI信号强度与Gd-DTPA作用组及与单纯细胞组信号强度比较差异有统计学意义；HEPG2细胞不摄取Gd-DTPA，30min时信号强度与单纯细胞组信号强度间差异无统计学意义。结论在穿膜肽基本序列基础上成功地重新构建的多肽能够携带荧光素和MR对比剂，并有效地跨膜转运进入细胞，为MR分子显像提供了1种新的重要手段。     

聚乙二醇修饰多肽的电泳分析方法

目的 建立聚乙二醇修饰多肽的电泳分析方法。方法 采用不同浓度和不同交联度的凝胶进行SDS-聚丙烯酰胺凝胶电泳,然后依次进行碘化钡染色和考马司亮蓝染色,并采用图像分析仪对所得结果进行处理。结果 采用5％浓缩胶,15％分离胶(C=3％),可以将不同分子量聚乙二醇与聚乙二醇修饰的小分子多肽分离;采用3.125％浓缩胶(C=20％),15％分离胶(C=5％),可以将聚乙二醇与小分子多肽分离。结论 建立了有效分离聚乙二醇修饰的小分子多肽的SDS-PAGE分析方法。     

老年人血浆内皮素降钙素基因相关肽水平的研究

探讨血浆内皮素（ＥＴ）、降钙素基因相关肽（ＣＧＲＰ）和神经肽（ＮＰＹ）与老年人动脉硬化之间的关系，提高动脉硬化的预防和治疗效果。方法用放射免疫法对３０例５５岁以上老人进行血浆ＥＴ、ＣＧＲＰ和ＮＰＹ的测定，并与５０例健康青年人做对照。结果老年组ＥＴ升高，ＣＧＲＰ、ＮＰＹ降低（Ｐ＜０．０５～０．０１）。结论ＥＴ升高，ＣＧＲＰ、ＮＰＹ降低表明老年人心血管分泌及调节功能失调，提示该改变可能与老年人心脑血管疾病的发生有关。     

Immunocytochemical study of endocrine cells in pelvic ileal reservoirs

The distribution and morphology of intestinal endocrine cells was investigated in the mucosa of pelvic ileal reservoirs using immunocytochemical methods. Endoscopic biopsies were obtained from 15 patients after the construction of a modified J-pouch. The mucosa of the reservoir showed a variable degree of colonic metaplasia in all cases. No relevant quantitative variations of gut endocrine cells were detected, as revealed by immunostaining for the general marker, chromogranin, compared with normal ileal mucosa. Immunostaining for different peptide-containing cells resulted in normal number and morphology of serotonin, enteroglucagon, peptide tyrosine-tyrosine, and somatostatin-containing cells. Neurotensin cells were less numerous than in normal mucosa. The role played by gastrointestinal hormones in the adaptive response of the intestine to pouch construction is, presently, unclear. Further studies involving measurements of fasting and meal-stimulated levels of gut hormones in pouch patients might clarify this aspect.     

乙酰胆碱受体α亚单位125～147段多肽致实验性自身免疫性重症肌无力

目的　用多肽免疫动物建立实验性自身免疫性重症肌无力动物模型。方法　合成电鳗乙酰胆碱受体α亚单位Tα1 2 5～ 1 4 7片段 ,并用其免疫接种长耳白兔 ,观察接种动物的临床症状、抗体滴度及神经电传导功能变化。结果　实验兔强化接种后第 2～ 5天渐出现肌无力症状 ,新斯的明试验阳性。接种免疫原组抗多肽Tα1 2 5～ 1 4 7抗体吸光度 (0 745 8± 0 1 681 )远高于对照组 (0 1 2 79± 0 0 1 73 ,P <0 0 5) ,3、5、1 0Hz下重复神经电刺激实验 (RNS)动作电位衰减率和单纤维肌电图检查颤抖值 (MCD)均较对照组明显延长 (P <0 0 5)。结论　Tα1 2 5～ 1 4 7能作为免疫原诱导动物产生重症肌无力模型     

Anorexic action of a new potential neuropeptide Y antagonist [ -Tyr, -Thr]-NPY (27–36) infused into the hypothalamus of the rat

Neuropeptide Y (NPY) produces a vigorous feeding response in several species when it is injected into hypothalamic structures involved in eating behavior. The purpose of this study was to determine whether a unique carboxy terminal fragment of NPY would alter the pattern of eating induced in the rat either by NPY injected into the hypothalamus or by a 24-h period of food deprivation. In this case, two -tyrosine residues and one t.-threonine residue of the NPY_27–36 fragment were transformed to their D-conformation to produce [ -Tyr^27,36, -Thr³²]-NPY (27–36), i.e., D-NPY_27–36. Guide cannulae for microinjection were implanted stereotaxically just dorsal to the paraventricular nucleus (PVN) or ventromedial hypothalamus (VMH) of 24 adult male Sprague-Dawley rats. Following postoperative recovery, a microinjection of artificial CSF or 1.1 jig or 3.3 μg of a peptide was made directly into the PVN or VMH as follows; native NPY; D-NPY_27–36; or [L-Tyr^27,36 L-Thr³²]-NPY (27–36), i.e., L-NPY_27–36. Food intakes were measured at intervals of 0.25, 0.5, 1.1, 2.0, 4.0, and 24 h. When D-NPY_27–36 was microinjected at NPY reactive sites in the PVN or VMH of the rat 15 min before a similar microinjection of NPY, the intense eating response induced by the peptide was reduced significantly. Not only was the effect dose dependent, but D-NPY_27–36 also augmented the latency to feed. A mixture of the two doses of NPY and DNPY_27–36 injected at the same hypothalamic loci did not attenuate the intake of food but tended to enhance the feeding response in the rats. After the rats were deprived of food for 24 h, D-NPY_27–36 microinjected in the same hypothalamic sites similarly caused a dose-dependent suppression of normal feeding behavior. However, the CSF control vehicle and L-NPY_27–36 microinjected in the PVN or VMH were without effect on the pattern of eating. Further, D-NPY_27–38 injected in the same hypothalamic sites affected neither body temperature nor water intakes of the rats significantly. These results demonstrate that the D substitution of this C-fragment of the NPY molecule, i.e., D-NPY_27–36, serves to inhibit feeding evoked in the rat by hypothalamic NPY as well as the natural eating response to food deprivation. Thus, the D-NPY_27–36 molecule may act as an antagonist at one or more subtypes of the NPY receptor in the brain of the rat.     

肺癌组织心钠素、血清降钙素含量测定及T淋巴细胞亚群浸润程度结果分析

为了解４种不同组织学类型肺癌组织心钠素（ＡＮＰ），血清降钙素（ＣＴ）含量与Ｔ淋巴细胞亚群浸润相关程度，用放免分析法及免疫组化ＡＢＣ法测定５８例肺癌组织ＡＮＰ血清ＣＴ含量Ｔ３＋，Ｔ４＋，Ｔ８＋淋巴细胞浸润程度。结果：５８例肺癌组织ＡＮＰ，ＣＴ含量均明显高于对照组（Ｐ＜０．０１）。肺癌不同组织学类型ＡＮＰ，ＣＴ含量均高于对照组（Ｐ＜０．０１）且有不同程度相关。肺癌相同组织学类型Ｔ３＋，Ｔ４＋，Ｔ８＋／浸润程度不同，以Ｔ３＋淋巴细胞最为明显。肺癌组织ＡＮＰ，血清ＣＴ含量与Ｔ３＋，Ｔ４＋，Ｔ８＋淋巴细胞浸润程度之间有不同程度相关。表明肺癌组织ＡＮＰ，ＣＴ及Ｔ淋巴细胞浸润之间有一定内在联系。     

Reversed-phase liquid chromatography of the opioid peptides. 3. Development of a microanalytical system for opioid peptides involving microbore liquid chromatography, post-column derivatization and laser-induced fluorescence detection

A microanalytical system has been developed for the determination of peptides in small samples. Naphthalene-2,3-dicarboxaldehyde-beta-mercaptoethanol (NDA-BME) was used as the labelling reagent system as an alternative to NDA-cyanide (NDA-CN) because of the faster labelling when CN was replaced by a thiol. The fluorescence characteristics of the NDA-thiol adducts, N-substituted 1-alkylthiobenz[f]isoindoles (TBIs), were found to be different from the previously described cyanobenz[f]isoindole (CBIs) adducts formed by the reaction of primary amines with NDA-CN. The excitation maximum of the TBI adducts was at 460 nm, which was closer to the 457.9 nm argon-ion laser line, than the 440-nm maximum of the CBI adduct. The limit of detection for leucine enkephalin was 36 fmol (S/N = 3) and linearity was proven for greater than 2 orders of magnitude, from 45 fmol to 9 pmol for an injection volume of 60 nl. The detectability was limited by the high background noise produced by the post-column derivatization system. The utility of the system was demonstrated for the analysis of methionine enkephalin and its potential oxidation products, using leucine enkephalin as a suitable internal standard.     

131I-层粘连九肽的合成及在荷乳腺癌裸鼠中的显像和分布

目的用１３１Ｉ层粘连九肽进行荷乳腺癌裸鼠的肿瘤受体显像及其生物分布研究。方法以固相法合成层粘连九肽，经ＨＰＬＣ纯化，用氯胺Ｔ法进行１３１Ｉ标记。标记物以ＳｅｐｈａｄｅｘＧ１０纯化。尾静脉注入荷乳腺癌裸鼠体内，分别在１、２、３、４、５、６小时进行肿瘤显像，划出感兴趣区，测其Ｔ／Ｂ值。静脉给药后分别在３、６小时观察生物分布，计算其每克组织摄取注射剂量百分数（％ＩＤ／ｇ）和肿瘤与非肿瘤的放射性比值（Ｔ／ＮＴ值）。结果化学合成的层粘连九肽经ＨＰＬＣ、ＦＡＢＭＳ纯化鉴定，Ｍ＋１＝９６６３。尾静脉注射后４～５小时肿瘤部位有明显浓聚，Ｔ／Ｂ值达４６２。肿瘤／肌肉的Ｔ／ＮＴ值在６小时为３４５。结论１３１Ｉ层粘连九肽有希望用于诊断人体乳腺癌