胰岛素样生长因子I放射免疫分析及其临床初步应用

建立血清胰岛素样生长因子Ｉ（ＩＧＦ－Ｉ）的放射免疫分析（ＲＩＡ）方法并初步将其应用于临床。采用血标本通过酸性乙醇提取冷冻再沉淀法,提取率达９１．５％±４．４％;用乳过氧化酶法以碘－１２５标记ＩＧＦ－Ｉ,ＲＩＡ反应液中含提取液（或标准液）５０μＬ,抗体２００μＬ（最终滴度１：１００００）,＾１２５Ｉ－ＩＧＦ－Ｉ２００μＬ（约１３０００ｃｐｍ）,４℃反应过放,双抗分离。ＲＩＡ回收率为９８．１％￣１０５     

缺氧—再给氧对大鼠胸主动脉环内皮依赖血管舒张反应的影响

生物测定法观察大鼠胸主动脉环经缺氧－再给氧、机械去内皮后，血管内皮分泌内皮细胞舒张因子（ＥＤＲＦ）的能力，ＥＤＲＦ前体Ｌ－精氨酸（Ｌ－Ａｒｇ）及拮抗剂ＮＧ－甲基－Ｌ－精氨酸（Ｌ－ＮＭＭＡ）对大鼠胸主动脉内皮ＥＤＲＦ合成和分泌的影响。发现缺氧－再给氧及去内皮的血管环对乙酰胆碱（Ａｃｈ）的舒张反应明显减弱或丧失，而对硝酸甘油的舒张反应仍保存。Ｌ－Ａｒｇ可加强正常内皮环和缺氧－再给氧环对Ａｃｈ的舒张反应     

试管固相RIA测定hCG

试管固相ＲＩＡ测定ｈＣＧ陈素娟，李振甲试管固相二抗法是李振甲等［１］最新研制成功的一种新技术。它是一种通用试管分离剂，可用于第一抗体为兔血清的分析方法，作者利用这项技术建立了试管固相ｈＣＧＲＩＡ，方法简便，只需在试管内加入标准（或样品），１２５Ｉ－ｈ...     

超抗原SEB和D—氨基半乳糖对小鼠肝细胞的作用观察

用超抗原葡萄球菌Ｂ型肠毒素（ＳＥＢ）、Ｄ－氨基半乳糖（Ｄ－ＧａｌＮ）及两者合用腹腔注射ＢＡＬＢ／ｃ小鼠，于２、６、１２、２４ｈ取肝脏和血标本，从形态学和生化指标来研究肝细胞的死亡模式；同时还检测了小鼠２４ｈ死亡率。结果发现单用ＳＥＢ可诱导肝细胞凋亡，其机制可能与ＴＮＦ、ＩＦＮ－γ等细胞因子的产生和作用有关。单用Ｄ－ＧａｌＮ可同时引起肝细胞凋亡和变性，ＳＥＢ和Ｄ－ＧａｌＮ合用２、６ｈ见肝细胞凋亡，１２ｈ后以肝细胞坏死为主，小鼠２４ｈ死亡率达５０％。因而，推测凋亡与坏死间存在一定的联系，在急性肝坏死的发病机制中大量肝细胞凋亡可能是坏死的前奏     

小鼠^60Co—γ射线照射后空肠组织中NO和cGMP含量变化

目的和方法：选用ＢＡＬＢ／ｃ小鼠，采用一氧化氮荧光分光光度法和放射免疫测定法，对不同剂量６０Ｃｏ－γ射线（８Ｇｙ、１２Ｇｙ、１４Ｇｙ）照射后的小鼠和８Ｇｙ照射后不同时间（分别为２４ｈ、４８ｈ、７２ｈ）小鼠空肠组织中ＮＯ－２和ｃＧＭＰ含量进行了测定。结果：８Ｇｙγ射线全身照射后，小鼠空肠组织中ＮＯ－２含量增加，照后４８ｈ含量最高，ｃＧＭＰ含量在照后２４ｈ最高，４８ｈ降至接近对照水平，在４８ｈ处，ｃＧＭＰ含量与照射剂量呈依赖性正相关。     

肿瘤坏死因子及磷脂酶A2在实验性急性肝衰竭中的作用

应用Ｄ－ＧａｌＮ＋ＥＴ复制急性肝衰竭（ＡＨＦ）动物模型，检测肝衰竭鼠血清中肿瘤坏死的子（ＴＮＦ）含量及肝组织匀浆中磷脂酶Ａ２（ＰＬＡ２）活性，探讨二者在ＡＨＦ的作用，结果发现，ＡＨＦ组鼠血清中ＴＮＦ含量及肝组织匀浆中ＰＬＡ２活性明显高于正常对照组（Ｐ〈０．０５，Ｐ〈０．０１），提示ＴＮＦ可能激活ＰＬＡ２后者与ＴＮＦ所致的肝损伤有关。     

小鼠α2—HS糖蛋白基因表达的研究

为了研究α２－ＨＳ糖蛋白（ＡＨＳＧ）基因的发育调节，我们用ＮｏｒｔｈｅｍＢｌｏｔ分析法，以小鼠ＡＨＳＧαｍＲＮＡ进行研究。结果发现（１）在发育的某些阶段，胚鼠的肢牙和脑有ＡＨＳＧｍＲＮＡ存在，（２）１２天胚鼠肝脏ＡＨＳＧｍＲＮＡ浓度最低，宵渐上升至出生后１－３个月达高峰，出生４个月维持在１个月时的５０％的水平。（３）胚鼠和成年鼠的多种组织都有ＡＨＳＧｍＲＮＡ表达，证实了肝外组织也是ＡＨＳＧ的合成场     

手持式r探测仪对荷人结肠癌裸鼠放射免疫定位研究

本文以手持式ｒ探测仪对荷人结肠癌细胞系Ｌｓ１７４Ｔ裸鼠进行体表放射免疫探测，为临床应用放射免疫引导外科手术（ＲＩＧＳ）提供了依据。以人结肠癌细胞系Ｌｓ１７４免疫ＢＡＬＢ／Ｃ小鼠制备ＭｃＡｂ ＣＬ－３，胃蛋白酶消化法制备ＣＬ－３Ｆ（ａｂ＇）２片段，Ｉｏｄｏｇｅｎ法以＾１２５Ｉ标记ＣＬ－３及其Ｆ（ａｂ＇）２。两组荷瘤裸鼠注射标记物后分别于２４、４８．７２、９６、１２０、１４４和１６８ｈ以手持式ｒ探测仪     

抗HBs-HBsAg复合物诱生体液免疫应答的研究

目的研究抗ＨＢｓ－ＨＢｓＡｇ复合物在ＢＡＬＢ／ｃ小鼠诱生体液免疫应答的特点。方法分别用鼠抗ＨＢｓ－ＨＢｓＡｇ免疫原性复合物（ＩＣ）或单独ＨＢｓＡｇ免疫ＢＡＬＢ／ｃ小鼠（加或不加氢氧化铝），分析其诱生的抗ＨＢｓＩｇＧ效价及ＩｇＧ亚类。结果用ＩＣ加氢氧化铝为佐剂免疫小鼠诱生的抗ＨＢｓ中以ＩｇＧ１亚类为主，而用ＨＢｓＡｇ免疫鼠则以ＩｇＧ２ｂ亚类较高。ＩＣ不加氢氧化铝佐剂诱生的抗ＨＢｓ中ＩｇＧ１及ＩｇＧ２ａ均高于单独ＨＢｓＡｇ免疫鼠。研究还显示，抗ＨＢｓＩｇＧＦｃ段是巨噬细胞（ＭΦ）等抗原提呈细胞摄取ＩＣ的主要途径。结论抗ＨＢｓ－ＨＢｓＡｇ复合物免疫诱生的体液免疫应答较单独ＨＢｓＡｇ免疫强。抗ＨＢｓ主要通过Ｆｃ段的介导作用，改变了机体抗原提呈细胞对ＨＢｓＡｇ的摄取、加工及抗原提呈，并可增强ＨＢｓＡｇ的免疫原性。     

分泌抗LDH-C4 pIgA和IgG抗体的杂交瘤细胞在生殖道的归巢

目的 探讨抗体产生细胞在生殖道的归巢。方法 用分泌抗ＬＤＨ－Ｃ４ＩｇＧ抗体的单克隆杂交瘤细胞ＳＧ２和分泌抗ＬＤＨ－Ｃ４ｐＩｇＡ抗体的单克隆杂交瘤细胞ＰＡ４标记后注入同系小鼠体内，３６ｈ后取材，以相对分布指数（ＲＤＩ）表示其归巢水平，检测啊细胞在部分淋巴结、肠、两性生殖器官的分布水平。结果 ＳＧ２和ＰＡ４细胞可以归巢在生殖道各部分，但三雌性生殖道各归巢水平比雄性生殖道分布水平高，雌性生殖道水平均ＲＤ     

人生长激素基因在小鼠体内的表达及其对血清中细胞因子浓度的影响

目的 :探讨体内高水平的人生长激素 (GH)对机体免疫功能的影响。方法 :构建人GH基因的真核表达质粒pcDNA3 hGH ,直接注射于小鼠的股四头肌中。注射后分别在第 5、15、2 5天分离血清 ,通过ELISA方法检测血清中GH的水平 ,同时检测血清中IL 2、IFN γ和TNF α水平的变化。结果 :所构建的质粒目的基因插入正确 ,转染细胞能分泌高水平的GH。接受质粒的小鼠血清中人GH的浓度与对照组相比较明显提高 ,但IL 2、IFN γ和TNF α的浓度没有显著变化。结论 :体内较高水平的GH对IL 2、IFN γ和TNF α的分泌没有显著影响     

Detection of circulating immune complexes with a Raji cell enzyme immunoassay

The primary aim of this work was to produce specific monoclonal antibodies to human growth hormone (hGH) for use in a diagnostic RIA of hGH levels in serum. Three different schedules were used for immunization of BALB/c mice and the splenocytes from each mouse were fused with myeloma cells Sp 2/0 Ag 14. Each fusion resulted in the production of hundreds of hybridomas secreting hGH-directed antibodies. Six antibodies have been fully characterized and have been grouped into pairs which recognize 3 different epitopes on the hGH molecule. One pair exhibits no cross reaction with the structurally related placental hormone, human placental lactogen (hPL), a second pair has low cross reaction with hPL (1.6-3%) and a third pair reacts equally well with hGH and hPL indicating binding to a common epitope in the 2 molecules. The highest affinity antibody, 74/6, which has an affinity constant of 4.4 X 10(10) l/mol and 3% cross-reactivity with hPL, has been used to establish a RIA for serum hGH measurements. Evidence is provided that hGH levels measured in this assay correlate well with those obtained in a conventional rabbit antiserum assay.     

In vivo delivery of recombinant human growth hormone from genetically engineered human fibroblasts implanted within Baxter immunoisolation devices

Continuous delivery of therapeutic peptide to the systemic circulation would be the optimal treatment for a variety of diseases. The Baxter TheraCyte^® system is a membrane encapsulation system developed for implantation of tissues, cells such as endocrine cells or cell lines genetically engineered for therapeutic peptide delivery in vivo. To demonstrate the utility of this system, cell lines were developed which expressed human growth hormone (hGH) at levels exceeding 1 microgram per million cells per day. These were loaded into devices which were then implanted into juvenile nude rats. Significant levels of hGH of up to 2.5 ng/ml were detected in plasma throughout the six month duration of the study. In contrast, animals implanted with free cells showed peak plasma levels of 0.5 to 1.2 ng four days after implantation with no detectable hGH beyond 10 days. Histological examination of explanted devices showed they were vascularized and contained cells that were viable and morphologically healthy. After removal of the implants, no hGH could be detected which confirmed that the source of hGH was from cells contained within the device. The long term expression of human growth hormone as a model peptide has implications for the peptide therapies for a variety of human diseases using membrane encapsulated cells.     

Monoclonal antibodies to human growth hormone can distinguish between pituitary and genetically engineered forms

Monoclonal antibodies (MABs) prepared against human pituitary growth hormone (hGH) have been compared for their binding to pituitary-derived and genetically engineered methionyl growth hormone (met-hGH). The antibodies bind to four non-overlapping epitopes of which two are completely shared with human choronic somatomammotropin (hCS). The determinant defined by MAB NA27 was expressed on met-hGH to a lesser degree than on hGH of pituitary origin. However, another antibody, QA68, which binds to a determinant closely related to NA27, failed to discriminate between hGH and met-hGH. A further two MABs (EB1 and NA71) were similarly ineffective in distinguishing between the two forms of the hormone. The determinant recognized by antibody EB2 was equally represented on hGH and met-hGH when assessed by a liquid-phase radioimmunoassay: however, measurement of the binding in a solid-phase assay resulted in a two-four-fold lower binding to met-hGH. Bioactivity assessed by both an in vitro cell proliferation assay and an in vivo cartilage sulphation bioassay failed to distinguish between the two hormones. It is therefore concluded that the NH2-terminal methionine on bacterially derived growth hormone results in altered antigenicity of the hormone without any measurable effect on bioactivity.     

Physiological effects of human growth hormone produced after hydrodynamic gene transfer of a plasmid vector containing the human ubiquitin promotor

Human growth hormone (hGH) deficiency causes retarded growth and abnormalities in body fat distribution and abundance, muscle growth, and bone mineralization. While injection of recombinant hGH may reverse or ameliorate symptoms of deficiency, therapy aiming at in vivo synthesis of hGH is still of considerable clinical interest. Hydrodynamic injection of a simple plasmid vector containing a human ubiquitin promotor and the hGH gene were found to result in high and prolonged expression. Synthesis of hGH was achieved both in NOD/SCID mice and in three different inbred strains of immune competent mice. Although hGH antibodies were produced in immunocompetent mice, physiological effects of the protein were documented by increase in body weight, increased serum levels of insulin-like growth factor I and relative increased weight of the internal organs. Nonviral gene therapy may be regarded as a potential future way of reconstituting hGH expression in deficient individuals.     

Age-independent effects of hyaluronan amide derivative and growth hormone on human osteoarthritic chondrocytes

Aim: Increased age is the most prominent risk factor for the initiation and progression of osteoarthritis (OA). The effects of human growth hormone (hGH) combined or not with hyaluronan amide derivative (HAD) were evaluated on human OA chondrocytes, to define their biological action and potentiality in OA treatment.

Material and methods: Cell viability, metabolic activity, gene expression and factors released were tested at different time points on chondrocytes treated with different concentrations of hGH (0.01–10?μg/ml) alone or in combination with HAD (1?mg/ml).

Results: We found that OA chondrocytes express GH receptor and that the different doses of hGH tested did not affect cell viability, metabolic activity or the expression of collagen type 2, 1, or 10 nor did it induce the release of IGF-1 or FGF-2. Conversely, hGH treatment increased the expression of hyaluronan receptor CD44. HAD combined with hGH reduced metabolic activity, IL6 release and gene expression, but not the suppressor of cytokine signaling 2 (SOCS2), which was significantly induced and translocated into the nucleus. The parameters analyzed, independently of the treatments used proportionally decreased with increasing age of the patients.

Conclusions: hGH only induced CD44 receptor on OA chondrocytes but did not affect other parameters, such as chondrocytic gene markers or IGF-1 or FGF-2 release. HAD reduced all the effects induced by hGH partially through a significant induction of SOCS2. These data show that GH or HAD treatment does not influence the response of the OA chondrocytes, thus the modulation of cellular response is age-independent.     

人生长激素基因修饰的鼠骨骼肌成肌细胞移植对心肌梗死大鼠血流动力学的影响

目的：探讨人生长激素（hGH）基因修饰的鼠骨骼肌成肌细胞移植对心肌梗死大鼠血流动力学和血管新生的影响。方法: 结扎大鼠左冠状动脉前降支制作大鼠心力衰竭模型。2周后将冠脉结扎后存活的30只大鼠随机分为3组,hGH基因修饰的成肌细胞移植组（hGH组）、绿色荧光蛋白(GFP)基因修饰的成肌细胞移植组（GFP组）、注射等体积培养液的对照组。治疗4周后,血流动力学检查各组心功能指标;心肌组织进行HE染色检测心肌梗死面积,Ⅷ因子免疫组织化学检测血管新生。RT-PCR检测bax和bcl〖STBX〗-2〖STBZ〗 mRNA的表达。Western blotting检测各组心肌hGH、血管内皮生长因子（VEGF）、caspase-3蛋白表达情况。结果: （1）与对照组和GFP组相比： hGH组血流动力学指标明显改善,心肌梗死面积缩小。（2）心肌组织Ⅷ因子免疫组织化学检查结果显示： hGH组血管密度显著高于GFP组和对照组。（3）RT-PCR检测结果显示hGH组bax mRNA水平显著低于GFP组和对照组, bcl-2 mRNA水平显著高于GFP组和对照组。（4）Western blotting检测结果显示hGH组大鼠心肌组织有hGH蛋白表达,其余两组心肌组织无hGH蛋白表达。与其它两组相比,hGH组caspase-3蛋白表达降低, VEGF蛋白表达增加。结论: hGH基因修饰的成肌细胞移植可以抑制细胞凋亡,与单独成肌细胞治疗相比可以诱导更大的血管化,更好地缩小心肌梗死面积,并改善心肌梗死大鼠血流动力学。成肌细胞治疗联合hGH基因治疗为心肌梗死后心力衰竭的治疗提供了一个新的途径。     

Changes in the Specificity of Antibodies in Mice Infected with Lactate Dehydrogenase-Elevating Virus

Infection with lactate dehydrogenase-elevating virus (LDV) modifies the isotypic distribution of antibodies (Ab) directed to several antigenic proteins with a preferential production of IgG2a. Because it was not known whether the virus could also affect the Ab specificity, the authors addressed this point using human growth hormone (hGH) as a model antigen. Anti-hGH monoclonal antibodies (MoAb) were used as probes to study the occurrence of Ab to three native hGH epitopes (3C11, F11 and 10D6) in sera from LDV-infected CBA/Ht and BALB/c mice immunized with hGH. Competition ELISA was used to determine the extent of Ab directed to cryptic hGH epitopes, i.e. antigenic determinants hidden in the native hormone. Results indicated that in LDV-infected CBA/Ht mice the titres of anti-hGH Ab were lower than in controls, although a consistent isotypic shift to IgG2a subclass was observed. Concurrently, the presence of Ab to epitopes 3C11, F11 and/or 10D6 were markedly reduced in infected animals and most anti-hGH Ab were directed to hGH cryptic epitopes. By contrast, LDV infection increased the amount of anti-KLH Ab elicited by CBA/Ht mice and did not affect Ab specificity, whilst control and LDV-infected BALB/c mice showed similar concentrations of anti-hGH Ab. Furthermore, the proportion of Ab to cryptic hGH epitopes did not change in infected animals even though an important shift to IgG2a was detected. Thus, data presented herein suggest that LDV infection modifies Ab specificity depending on the mice genetic background and on the antigenic characteristics of the immunogen.     

Comparative study of pituitary and bacteria-derived human growth hormone by monoclonal antibodies

We have established hybridoma lines which secrete mouse monoclonal antibodies (Mabs) to human pituitary growth hormone, hGH. Using indirect competitive ELISA and indirect passive hemagglutination inhibition twelve different Mabs were characterized with regard to cross-reactivity with the hGH-related hormones, human chorionic somatomammotropin, hCS, and human prolactin, hPRL. The reactivity of these Mabs with pituitary hGH was compared to that with either bacterially-produced methionyl-hGH or to that of reduced and S-carboxymethylated hGH, which has an altered conformation. None of the Mabs reacted with hPRL. Four did not react with hCS whereas the others showed varying degree of cross-reactivity with hCS. All Mabs reacted more weakly with reduced and S-carboxymethylated hGH than with the native form of the hormone, which was not seen with conventional rabbit antisera to hGH. Thus in the case of hGH the Mabs are superior to conventional antisera in revealing small conformational differences. However the pituitary and bacterially-derived methionyl-hGH were indistinguishable as determined by the 12 Mabs.