人甲状旁腺激素相关蛋白（1-34）对去卵巢大鼠骨密度和骨生物力学性能的影响

目的：观察人甲状旁腺激素相关蛋白氨基端肽(PTHrP 1-34)对去卵巢的骨质疏松大鼠骨密度(BMD)和骨生物力学性能的影响。 方法： 80只4月龄Wistar健康雌性大鼠，其中60只行双侧卵巢摘除术，20只做假手术，6周后各处死10只证实骨质疏松造模成功。剩余50只骨质疏松模型鼠随机分为5个治疗组，每组10只，其它10只假手术组作对照。PTHrP治疗组（PTHrP 20组， PTHrP 40组， PTHrP 80组）分别用20、40、80 μg/kg剂量，每日皮下注射1次PTHrP 1-34；雌二醇治疗组（E2组）用苯甲酸雌二醇40 μg/kg, 每3 d注射1次；安慰剂组及假手术对照组分别用生理盐水，每3 d注射1次。治疗3个月后，测定并比较股骨、腰椎BMD、骨生物力学参数及血清钙、磷、碱性磷酸酶（ALP）水平。结果： 双侧卵巢摘除6周后，大鼠腰椎BMD及腰椎压缩最大载荷明显低于假手术组(P<0.05)。3个月治疗后，PTHrP 40组和PTHrP 80组大鼠股骨、腰椎BMD及骨生物力学性能明显高于安慰剂组，与雌二醇治疗组无显著差异，腰椎BMD明显高于PTHrP 20组（P<0.05）； PTHrP 40组与PTHrP 80组无明显差异。结论： 每日每公斤体重皮下注射40和80 μg PTHrP 1-34对去卵巢骨质疏松大鼠有明显治疗作用。     

甲状旁腺激素1-34、阿仑膦酸钠、辛伐他汀治疗大鼠骨质疏松效果的比较

BACKGROUND:Bone resorption drugs in the treatment of osteoporotic fracture are still controversial. The development of new drugs, especially drugs in the promotion of bone formation, become more important and urgent.  OBJECTIVE: To compare the effects of parathyroid hormone (1-34), alendronate sodium and simvastatin on bone loss in ovariectomized rats. METHODS: Thirty female Sprague-Dawley rats aged 3 months were randomized into five groups of six animals in each group. All rats but those in sham group received dual ovariectomy (OVX) and treated by vehicle (OVX+V), parathyroid hormone (1-34) (5 days per week at a dose of 20 μg/kg, OVX+P), alendronate sodium (70 μg/kg/wk, OVX+A), simvastatin (5 mg/kg/d, OVX+S). Three months later, all rats were sacrificed. Dual energy X-ray method was used to measure bone density of L4 vertebra. Bone histomorphometry was utilized to analyze cancellous bone mass and bone microstructure. Compression test was performed on the L5 vertebra for the maximum loading and elastic modulus.   RESULTS AND CONCLUSION:(1) Bone density: The bone density from high to low levels was OVX+P group, sham group, OVX+A group, OVX+S group, and OVX+V group. No significant difference in bone density was detectable between the OVX+P and sham groups, but significant differences in bone density were determined between any other two groups (P < 0.05). (2) Bone histomorphometry: Relative volume of trabecular bone was significantly lower in the OVX+V group than in other groups. Relative volume of trabecular bone was remarkably higher in the OVX+P group than in the OVX+A group and OVX+S group. (3) Biomechanical properties: Biomechanical properties were noticeably lower in the OVX+V group than in other groups. Biomechanical properties were remarkably higher in the sham group and OVX+P group than in the OVX+S group and OVX+A group. (4) These findings indicated that under the dosage and intervention period used in the present study, parathyroid hormone (1-34), alendronate sodium and simvastatin showed anti-osteoporosis effect on OVX rats in a descending manner. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

人重组骨形态发生蛋白-2在脊柱融合中的作用

脊柱融合是治疗脊柱结核、感染、畸形、椎间盘退行性病变脊柱疾患的有效手段,促进脊柱融合成骨率的方法之一就是使用自体骨移植,但其存在来源有限、取骨处疼痛等弊端,目前人重组骨形态发生蛋白-2(The role of recombinant human bone morphogenetic 2,rh BMP-2)负载于载体是一种理想的自体骨移植材料的替代物,广泛应用于脊柱融合术中,但近来发现了与其相关的一些副反应。本文就其在脊柱融合中的应用的安全性、有效性及相关的副反应进行综述。     

人重组骨形态蛋白-2在脊柱融合中并发症的研究

自体骨移植物具有天然的骨诱导和骨传导特性,是骨移植材料中的金标准,但其存在局限性。目前重组人骨形态发生蛋白-2(rhBMP-2)被广泛应用于临床中以提高脊柱融合率。但其应用范围远超美国食品药品监督管理局(FDA)标准,同时也出现了很多并发症。因此研制出新型骨修复材料成为了目前亟待解决的问题。     

重组人骨形态发生蛋白-2结合纳米晶胶原基骨材料植骨融合在腰椎不稳症中的临床应用

目的观察重组人骨形态发生蛋白-2﹙rhBMP-2﹚结合纳米晶胶原基骨材料﹙nHAC﹚行腰椎间融合治疗腰椎不稳症的疗效。方法 2008年1月~2010年12月23例﹙27个节段﹚腰椎不稳患者,所有患者均行腰椎椎弓根螺钉固定系统,辅助以rhBMP-2结合nHAC椎间植入融合椎体。结果全部病例获随访,手术后平均随访13.5月,按照JOA评价标准,优14例,良6例,可3例。优良率86.9%。结论 rhBMP-2结合nHAC行腰椎间融合治疗腰椎不稳症是一种比较理想的治疗方法。     

ACTH(1-24)对脂多糖致ECV304细胞损伤的保护作用

目的研究ACTH(1-24)对脂多糖(LPS)损伤的脐静脉内皮细胞(ECV304)的保护作用。方法采用体外培养的ECV304为模型,分别观察对照组、LPS损伤组和ACTH(1-24)保护组的细胞活性及NO、TNF-α、细胞间黏附分子ICAM-1、核因子NF-κB家族成员p50、p52及趋化因子SCYA3、SCYB10的表达情况。结果LPS(50μg/ml)可使ECV304的细胞活性降低,细胞的NO、TNF-α、NF-κB、SCYA3、SCYB10及ICAM-1的表达增强。ACTH(1-24)可以提高损伤细胞的活性,抑制上述细胞因子的高表达。结论ACTH(1-24)可以阻断LPS对细胞的损伤,减少炎性介质和细胞因子的过度释放,对细胞具有明显的保护作用。     

不同剂量培哚普利对缺血性心功能障碍家兔心功能及ACE2/Ang-(1-9)/Ang-(1-7)轴的影响

 目的: 探讨不同剂量培哚普利对缺血性心功能障碍家兔心功能的影响。方法: 采用结扎冠状动脉前降支的方法制作缺血性心功能障碍家兔模型。利用随机数字表法将30只家兔随机分为培哚普利大、小剂量组和心功能障碍组。大、小剂量组分别给予培哚普利生理盐水溶液(浓度分别为1 g/L、0.33 g/L)2 mL·kg^-1·d^-1灌胃;心功能障碍组给予等量生理盐水灌胃。4周后心脏超声测定心功能;real-time PCR检测血管紧张素转换酶2(angiotensin-converting enzyme 2,ACE2)和血管紧张素2型受体(angiotensin type 2 receptor,AT2R) mRNA表达;ELISA检测家兔血清血管紧张素(angiotensin,Ang)-(1-9)和Ang-(1-7)水平。结果: 与心功能障碍组相比,不同剂量培哚普利均可改善心功能(P<0.01),大剂量培哚普利比小剂量培哚普利改善心功能的效果显著(P<0.05);心功能障碍家兔应用培哚普利后,血清Ang-(1-9)和Ang-(1-7)水平均增高(P<0.01),ACE2和AT2R mRNA表达均增加(P<0.01);与小剂量组相比,大剂量组心肌ACE2和AT2R mRNA表达均增高(P<0.01),血清Ang-(1-9)水平增高(P<0.05),血清Ang-(1-7)水平无明显增加。相关性分析发现,左室射血分数与血清Ang-(1-9)、ACE2及AT2R水平呈正相关关系(P<0.01),与血清Ang-(1-7)水平无相关关系。结论: 大剂量培哚普利较小剂量培哚普利可以更有效改善缺血性心功能障碍家兔心功能,其心功能的改善可能与Ang-(1-9)水平增多,引起AT2R活化相关。     

肌醇多磷酸5-磷酸酶(PIPP)在人下咽癌组织的表达及临床意义

目的研究肌醇多磷酸5-磷酸酶(PIPP)在人下咽癌中的表达,探讨其临床意义。方法对15例人下咽癌组织学标本和15例正常的癌旁组织进行免疫荧光和免疫印迹检测。结果 PIPP的表达量及荧光强度在下咽癌组织中明显低于正常组织。结论 PIPP在下咽癌组织中低表达可能为临床提供新的诊断指标。     

人禽流感病毒H5N1多价重组痘苗病毒(天坛株)疫苗在小鼠中的免疫原性研究

目的 研发高效广谱的人高致病性禽流感病毒H5N1实验疫苗.方法 首先构建了含H5N1(安徽株)结构基因[血凝素(HA)、神经氨酸酶(NA)、基质蛋白M1与M2]的两个双顺反子(HAop/M2,NAop/M1)重组痘苗病毒(rTTV天坛株)疫苗,采用不同剂量(104 PFU或107PFU)或组合(疫苗单独或联合)方式于0、4周二次免疫BALB/c小鼠,初步比较分析抗原特异的体液(HA血凝抑制抗体、NA特异性抗体、中和抗体)与细胞免疫应答(IFN-γ ELISPOT)特点.结果 重组痘苗病毒疫苗可有效表达H5N1靶抗原;高剂量组的重组痘苗病毒疫苗可快速激发较强的针对各个抗原的抗体与针对血凝素与神经氨酸酶蛋白的细胞免疫应答,含血凝素蛋白的重组痘苗病毒疫苗亦可诱导明显的中和抗体;但各组重组痘苗病毒疫苗所激发的针对基质蛋白(M1,M2)的细胞免疫应答均较弱;两个双顺反子(HAop/M2,NAop/M1)重组痘苗病毒疫苗联合应用所激发的针对基质蛋白2(M2)的体液免疫应答明显强于单双顺反子(HAop/M2)疫苗单独应用.结论 本研究中制备的各组重组痘苗病毒疫苗可诱导多个抗原特异的体液与细胞免疫应答,该研究为新型H5N1疫苗的研发及免疫方案的优化奠定了基础.     

应用酵母双杂交系统研究与血管紧张素-(1-7)相互作用的蛋白

目的用酵母双杂交系统筛选与血管紧张素-(1-7)相互作用的蛋白,为该7肽在体内如何发挥功能提供线索。方法构建Ang-(1-7)的真核表达载体pBD—Ang-(1—7),转化酵母菌YRG-2,进行毒性和自身非特异激活性检验。与大鼠心肌细胞文库质粒共同转化酵母细胞,在营养缺陷培养基上进行双杂交筛选,选择既能在3重营养缺陷培养基上生长,也能使X—gal变蓝的克隆为阳性,提取靶质粒后进行复交,将真阳性质粒测序,进行生物信息学分析。结果诱饵载体构建成功并转化酵母,对酵母无毒性,无自身激活现象。筛选出的蛋白主要参与细胞代谢和蛋白合成。结论Ang-(1-7)可能影响细胞内某些蛋白合成和细胞代谢,发挥对血管紧张素Ⅱ的结抗作用。酵母双杂交结果为Ang-(1—7)的作用途径的进一步研究提供了分子生物学线索。     

Parathyroid hormone(1-34)-induced apoptosis in neuronal rat PC12 cells: implications for neurotoxicity

Based on accumulated evidence, we speculate that a high concentration of parathyroid hormone (PTH) may cause neurotoxicity in patients with uremia through apoptosis-induced neuropathy. In this study, we demonstrated that in vitro stimulation with PTH(1-34) induced a significant decrease in PC12 cell numbers in both dosage- and time-dependent fashions when these cells were treated with PTH(1-34) at concentrations of 0.01, 0.1 or 1.0 μM for 24, 48, 72, and 96 h, respectively, as assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Decreased numbers of PC12 cells were caused by PTH(1-34)-induced apoptotic and cytotoxic processes, as determined by DNA fragmentation, flow cytometry, and lactate dehydrogenase (LDH)-leakage assays. Upregulation of the extracellular signal-regulated kinase (ERK) and p38 signaling pathway was the underlying mechanism responsible for 1.0 μM PTH(1-34)-induced apoptosis in PC12 cells, as elucidated by Western blotting analysis and confirmed with ERK and p38 inhibitors. Furthermore, 1.0 μM PTH(1-34)-induced apoptosis was accompanied by a release of cytochrome c and subsequent caspase-3 activation. These data suggest that a high concentration of PTH(1-34)-induced cytotoxicity and apoptosis in PC12 cells was associated with upregulation of ERK and p38 through a mitochondria-mediated apoptotic pathway.     

Effect of parathyroid hormone PTH (1-34) on hemopoietic and stromal stem cells

Long-term administration of parathyroid hormone causing activation and proliferation of osteoblasts to mice increases the concentration of primitive hemopoietic precursor cells (cobblestone area-forming cells) in long-living bone marrow culture after 28–35 days. The concentrations of later precursors forming colonies in the spleen and the concentration of cobblestone-area forming cells in long-living bone marrow culture after 7 days decrease, while the concentration of more differentiated cells forming colonies in the culture does not change. Transplantation of the bone marrow from mice treated with parathyroid hormone under the renal capsule of syngeneic recipients results in the formation of a focus of ectopic hemopoiesis not differing by size from the control. Injection of parathyroid hormone to mice during the growth of the ectopic focus did not modulate its size. These foci tolerate retransplantation procedure similarly as controls. Hence, parathyroid hormone has no effect on mesenchymal stem cells responsible for transfer of the stromal microenvironment. Therefore, the number of stem hemopoietic cells in the body is regulated by not stromal stem cells, but their better differentiated descendants.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 12, pp. 645–648, December, 2004This revised version was published online in April 2005 with a corrected cover date.     

Effect of 1-34 human parathyroid hormone upon first trimester placental human chorionic gonadotrophin secretion in vitro: potentiation by epidermal growth factor

Human chorionic gonadotrophin (HCG) secretion by the early placentais under multifactorial control. Epidermal growth factor (EGF)has been reported to be involved in regulating the formationand secretion of HCG by first trimester placental explants inculture. The effect of the amino-terminal fragment of parathyroidhormone (1–34 PTH), a calciotrophic factor upon HCG secretion,and its possible interaction with EGF were examined in thisstudy, both in static cultures and in superfusion, where ithas previously been demonstrated that HCG secretion is spontaneouslypulsatile. Gestational age-dependent effects of 1–34 PTHwere noted in both models. In static cultures, 1–34 PTHstimulated HCG secretion in 7–9 week placenta, in a biphasicfashion, the maximal effect being noted at 10–25 ng/mlconcentrations (250–270%), while at 1 and 100 ng/ml, theeffect was mild. In superfusion, the effect of 1–34 PTHadded overnight was also stimulatory, as shown by the significantlyincreased pulse amplitude and area under the curve. Effectsof 1–34 PTH at 11–14 weeks were inhibitory. In staticcultures at 7–9 weeks, the stimulatory effect of 25 ng1–34 PTH was increased by 70% when EGF (100 ng/ml) wasadded. However at 11–14 weeks, this combined effect wasinhibitory. We conclude that 1–34 PTH has an endocrineeffect on secretion of HCG by the first trimester placentaltissue, and this effect is potentiated by the addition of EGF.     

Monoclonal antibodies to human parathyroid hormone (1-34) and their use in the immunocytochemical detection of parathyroid tumours

Monoclonal antibodies against the biologically active N-terminal fragment of human parathyroid hormone, hPTH (1-34), were produced. The procedure included the use of novel secondary immunization in vitro of mouse spleen cell cultures. Dissociated spleen cells from primary immunized Balb/c mice, were cultured for five days in the presence of thymocyte conditioned media (TCM) and synthetic hPTH (1-34). Contrary to previous findings by other workers, in our hands Balb/c mice responded well. Following immunization the spleen cells were fused with NSl myeloma cells and cultured for eleven days before screening for antibody. Using an enzyme linked immunosorbent assay (ELISA) a number of positive clones were detected. Positive cells were cloned by limiting dilution and fifteen specific monoclonal hybridomas were produced. The immunoglobulin class of the different monoclonal antibodies was found to be IgGl. The immunocytochemical reaction was tested with chief cell carcinoma tissue and found to be clearly positive.     

Effect of angiotensin-(1-7) and angiotensin II on the proliferation and activation of human endometrial stromal cells in vitro

Recent studies have shown that angiotensin II (Ang II) or angiotensin-(1-7) [Ang-(1-7)] has effect on the proliferation and activation of a variety of cells, however, the exact mechanisms that the role of Ang II or Ang-(1-7) in human endometrial stromal cell (ESCs) remains elusive. Here we demonstrated that Ang II could promote proliferation and activation of ESCs, up-regulated the expression of a-SMA, TGF-β1 and IGF-I, increased the secretion of extracellular matrix [Type I collagen (Col I) and fibronectin (FN)] of ESCs; Ang-(1-7) could inhibit Ang II induced the proliferation and activation of ESCs, down-regulated the expression of a-SMA, TGF-β1 and IGF-I, decreased the secretion of extracellular matrix (Col I and FN) of ESCs. These findings suggest that Ang-(1-7) can inhibits Ang II induced the proliferation of ESCs, Ang-(1-7) can inhibits the Ang II induced activation of ESCs and decreases secretion of Col I and FN by suppressing TGF-β1 and IGF-I expression.     

PTH对破骨细胞骨吸收功能的影响及成骨细胞介导作用

采用分离、培养兔破骨细胞和成骨细胞的方法，体外研究甲状旁腺激素（ＰＴＨ）对破骨细胞骨吸收功能的影响，以及成骨细胞和破骨细胞之间的相互作用。结果表明，ＰＴＨ对破骨细胞的骨吸收功能无直接影响，但在成骨细胞参与下，ＰＴＨ对破骨细胞性骨吸收有明显的促进作用。说明成骨细胞在ＰＴＨ调节破骨细胞功能活动中有着重要的介导作用。     

Expression and function of B7-1 (CD80) and B7-2 (CD86) on human epidermal Langerhans cells

In addition to T cell receptor triggering, activation of T cells requires co-stimulatory signals that have been shown to be mainly initiated through CD28. We analyzed the expression and function of the two ligands for CD28, B7-1 (CD80) and B7-2 (CD86), on human Langerhans cells (LC), the antigen-presenting cells from epidermis. Human LC freshly isolated from epidermis (fLC) expressed significant level of B7-2, which was increased upon a short culture in vitro. In contrast, B7-1 was undetectable on fLC but appeared at the cell surface after a 3-day culture in vitro. Pre-incubation of 18-h cultured LC with anti-B7-2 monoclonal antibodies (mAb) was sufficient to abrogate the binding of CTLA4-Ig fusion protein, while a combination of both mAb against B7-1 and B7-2 was necessary to obtain a complete inhibition of CTLA4-Ig binding on 3-day cultured LC, showing the absence of a third CTLA4 ligand. The function of B7-1 and B7-2 on human LC has been analyzed by adding mAb at the beginning of mixed epidermal cell lymphocyte reactions. Anti-B7-2 mAb and CTLA4-Ig, but not anti-B7-1 mAb, strongly inhibited allogeneic, as well as recall antigen-induced T cell proliferation supported by fLC or 3-day cultured LC. Collectively, these results demonstrate that B7-2 is the major ligand for CD28/CTLA4 at the LC surface and that it plays a crucial role in human LC co-stimulatory function with little, if any, dependence on B7-1 expression.     

Stimulation of growth hormone secretion with human growth hormone releasing factors (GRF1–44, GRF1–40, GRF1–29) in normal subjects

Summary Peptides of different chain lengths (GRF^1–44, GRF^1–40, GRF^1–29) were given as a 50 µg i.v. bolus to five normal volunteers. Blood was collected before and until 120 min after GRF injection. No serious side effects were recorded. All GRF peptides led to a clearcut and significant increase of GH levels compared to placebo controls with the maximum occurring 15–30 min after GRF injection. There was no significant difference in the maximal GH increase after the different GRF peptides. When GRF^1–44 was administered to five normal subjects over 2 days in 6- and 18-h intervals, respectively, significant increase of GH levels were recorded after each injection compared to placebo controls. Again, there was no significant difference between the maximal GH rises during the different tests. These findings show that the short GRF^1–29 peptide may be used for diagnosis and therapy. In addition, in contrast to continuous infusion and administration of GRF in short intervals, GRF application in 6-h intervals leads to adequate GH responses.Abbreviations (h) GH (human) growth hormone - hpGRF human pancreatic growth hormone releasing factor Supported by the Deutsche Forschungsgemeinschaft (We 439/4-1) and the Fraunhofergesellschaft     

In vitro and in vivo effect of parathyroid hormone analogue (1-14) containing -amino-iso-butyric acid residue (Aib)1,3

Firstly, parathyroid hormone (1-14) [PTH (1-14)] analogue containing various -amino-iso-butyric acid residue (Aib) was synthesized by exchanging the 1st and 3rd Ala residues of alpha carbon of PTH (1-14). This analogue revealed to have the quite tight and stable -helical structure using the nuclear magnetic resonance (NMR) analysis. The biological activities of these analogues were examined using a cAMP- generating assay in LLC-PK1 cell lines stably transfected with the wild- type human PTH1 receptor. Only the PTH analogue substituted with methyl moiety without acetylation showed significant cAMP generating action with 15.0 +/- 3.414 of EC50. Then, we used an ovariectomized rat model system to compare the in vivo effects of parathyroid hormone analogue with that of PTH (1-84). Daily subcutaneous administration of the unacetylated Aib1,3PTH (1-14) for 5 weeks in 30 nM/kg subcutaneously with positive control group receiving PTH (1-84) with 8 nM/ kg were performed. However, there was no significant change in spinal or femoral bone mineral density assessed by dual x-ray absorptiometry (DXA) in the Aib1,3PTH (1-14) group where definite increase of these parameters shown in the PTH (1-84) group (p < 0.001). Assessment of bone strength was evaluated with no significant differences among all groups. It was quite disappointing to see the actual discrepancies between the result of significant pharmacokinetic potency and the in vivo clinical effect of the Aib1,3PTH (1-14). However, there are several limitations to mention, such as the short duration of treatment, matter of dosage, and insufficient effect of tight -helical structures with absence of C-terminus. In conclusion, our findings suggest that unacetylated Aib1,3PTH (1-14) did not exhibit any anabolic effects at the bones of ovariectomized rats.