真皮成纤维细胞成骨分化的实验研究

目的比较不同诱导剂对真皮成纤维细胞成骨分化的不同影响,探讨成纤维细胞成骨分化机制。方法取新生大鼠皮肤进行组织块培养,真皮成纤维细胞分离培养及鉴定,并分别由地塞米松、1,25(OH)2D3以及地塞米松和1,25(OH)2D3进行成骨分化诱导。分别于诱导后14d行ALP含量测定,21d行茜素红染色,并进行TAZ表达检测。结果真皮成纤维细胞表达波形蛋白,不表达角蛋白;成纤维细胞诱导14d后,1,25(OH)2D3诱导组及地塞米松+1,25(OH)2D3诱导组ALP含量与对照组有显著差异;成骨诱导21d,地塞米松诱导组仅见少量散在红色钙结节形成,1,25(OH)2D3诱导组钙结节数量增高,地塞米松+1,25(OH)2D3诱导组钙结节数量明显增高,直径变大;1,25(OH)2D3诱导组及地塞米松+1,25(OH)2D3诱导组,经TAZ免疫荧光染色,可见部分细胞核表达TAZ。结论地塞米松可促进1,25(OH)2D3诱导真皮成纤维细胞成骨分化。     

1,25(OH)_2D 是否参与内分泌激素释放的调节

近年来,有关维生素D 在内分泌学领域中的重要进展之一,就是发现1,25(OH)_2D 受体不仅存在于经典的靶器官小肠、骨骼、肾脏,而且也存在于某些内分泌腺体,如垂体、胰腺、甲状旁腺、性腺以及胎盘组织。因此,有人认为,1,25(OH)_2D 可能参与了上述腺体组织中激素的合成和释放的调节。为了阐明1,25(OH)_2D 是否对人的内分泌调节具有直接的作用,Hochberg 等对1,25(OH)_2D 受体有缺陷或缺乏该受体、具有典型的抗1,25(OH)_2D 综合征的患儿进行了观察。     

蒙药乌力吉-18对大鼠下丘脑-垂体-卵巢轴相关激素及受体表达的影响

目的:探讨蒙药乌力吉-18对大鼠下丘脑-垂体-卵巢轴相关激素及受体的影响。方法:选取40只健康雌性未孕SD大鼠,随机分为空白组、对照组、乌力吉-18高、低2个剂量组,每组10只。空白组灌胃等体积蒸馏水,对照组灌胃逍遥丸,高、低剂量组分别灌胃2.0 g·kg^-1·d^-1、1.0 g·kg^-1·d^-1乌力吉-18,连续给药31学艺术d。采用酶联免疫吸附法测定血清促性腺激素释放激素(GnRH)、促卵泡生成素(FSH)、黄体生成素(LH)、雌二醇(E₂)及孕酮(PROG)的含量;免疫组化法检测下丘脑组织促性腺激素释放激素(GnRH)、垂体组织促性腺激素释放激素受体(GnRHR)的表达;以蛋白免疫印迹技术检测卵巢组织促卵泡生成素受体(FSHR)、黄体生成素受体(LHR)蛋白表达量。以实时荧光定量PCR检测卵巢组织中FSHR、LHR基因表达量。结果:与空白组比较,乌力吉-18低剂量组可明显升高血清LH含量(P<0.05),上调下丘脑组织GnRH、垂体组织GnRHR表达及卵巢组织FSHR、LHR蛋白表达(P<0.05);乌力吉-18高剂量组可显著升高血清FSH、LH、E₂含量(P<0.05),上调下丘脑组织GnRH表达及卵巢组织FSHR表达量(P<0.05),并可显著升高卵巢组织中FSHR、LHR基因表达量(P<0.05);对照组可明显升高血清E₂含量(P<0.05)。结论:蒙药乌力吉-18可明显升高血清FSH、LH及E₂的含量,促进下丘脑组织GnRH、垂体组织GnRHR及卵巢组织中FSHR、LHR的表达,表明乌力吉-18能够对下丘脑-垂体-卵巢轴相关激素及受体表达产生影响。     

胰岛素作用原理的研究——Ⅶ.不同种属红细胞胰岛素受体与胰岛素的相互作用

参照Gambhir等人的报告建立了分离豚鼠、兔和鸡红细胞的方法。比较了豚鼠、兔和鸡红细胞胰岛素受体与猪胰岛素的相互作用,初步观察到不同种属动物的红细胞含胰岛素受体的数目似乎与其自身的胰岛素活力成反比。     

胰岛素作用原理的研究 Ⅶ.不同种属红细胞胰岛素受体与胰岛素的相互作用

参照Gambhir等人的报告建立了分离豚鼠、兔和鸡红细胞的方法。比较了豚鼠、兔和鸡红细胞胰岛素受体与猪胰岛素的相互作用,初步观察到不同种属动物的红细胞含胰岛素受体的数目似乎与其自身的胰岛素活力成反比。     

不明原因复发性流产再次妊娠患者血清1,25(OH)2D3、sTim-3与Th17/Treg免疫失衡和妊娠结局的关系

摘要 目的：探讨不明原因复发性流产（URSA）再次妊娠患者血清1,25-二羟维生素D3[1,25（OH)）₂D₃]、可溶性T细胞免疫球蛋白黏蛋白分子3（sTim-3）与辅助性T细胞17（Th17）/调节性T细胞（Treg）免疫失衡和妊娠结局的关系。方法：选择于湖南省妇幼保健院2020年1月~2022年1月就诊的62例URSA再次妊娠患者作为研究组,另选择同期进行孕检的正常早孕妇女30例作为对照组。比较两组孕早期血清1,25(OH) ₂D₃、sTim-3及外周血Th17细胞、Treg细胞水平、Th17/Treg比值。Pearson法分析URSA再次妊娠患者血清1,25(OH) ₂D₃、sTim-3与外周血Th17细胞、Treg细胞水平、Th17/Treg比值平的相关性。根据URSA再次妊娠患者妊娠结局的不同分为妊娠成功分娩组和妊娠再次流产组,比较两组孕早期血清1,25(OH) ₂D₃、sTim-3与外周血Th17细胞、Treg细胞水平、Th17/Treg比值。受试者工作特征（ROC）曲线分析血清1,25(OH) ₂D₃、sTim-3与外周血Th17细胞、Treg细胞水平、Th17/Treg比值对妊娠结局的预测价值。结果：研究组血清sTim-3、外周血Th17细胞水平、Th17/Treg比值高于对照组,血清1,25(OH) ₂D₃、外周血Treg细胞水平低于对照组（P<0.05）。Pearson相关分析显示,URSA再次妊娠患者血清1,25(OH) ₂D₃与血清sTim-3、外周血Th17细胞水平、Th17/Treg比值呈负相关,与Treg细胞水平呈正相关（P<0.05）;血清sTim-3与外周血Treg细胞水平呈负相关,与Th17细胞水平、Th17/Treg比值呈正相关（P<0.05）。妊娠再次流产组血清sTim-3、外周血Th17细胞水平、Th17/Treg比值高于妊娠成功分娩组,血清1,25(OH) ₂D₃、外周血Treg细胞水平低于妊娠成功分娩组（P<0.05）。ROC曲线分析显示,血清1,25(OH) ₂D₃、sTim-3及外周血Th17细胞、Treg细胞水平及Th17/Treg比值均可预测URSA再次妊娠患者妊娠再次流产的发生风险,且上述指标联合检测的预测效能更高。结论：血清1,25(OH) ₂D₃水平异常降低、sTim-3水平异常升高可导致Th17/Treg免疫失衡,导致URSA再次妊娠患者再次发生流产。上述指标联合检测对URSA再次妊娠患者妊娠再次流产的预测效能更高。     

鸡Toll样受体21单克隆抗体的研制及其初步应用

【目的】研制鸡Toll样受体21(chTLR21)单克隆抗体,考察禽致病性大肠杆菌(APEC)及高致病性禽流感病毒(HPAIV)感染鸡组织中的chTLR21蛋白的表达情况。【方法】以人工合成的多肽免疫原chTLR21(203–225aa)与KLH偶联蛋白免疫6周龄BALB/c小鼠,以多肽免疫原chTLR21 (203–225 aa)与BSA偶联蛋白作为检测抗原,通过间接ELISA技术筛选阳性杂交瘤细胞株,同时,采用间接免疫荧光技术(IFA)检测单克隆抗体的荧光反应性;上述单克隆抗体用于chTLR21在鸡巨噬细胞(HD11)中的定位;在APEC O1血清型E516菌株、HPAIV H5N6亚型毒株感染35日龄SPF鸡模型中,应用获得的单克隆抗体以免疫印迹(Western blotting)和免疫组化(IHC)方法检测感染鸡组织中chTLR21的表达情况。【结果】建立了间接ELISA检测方法,确定最适抗原包被浓度为2.5μg/mL,最适血清稀释度为1:6400。经ELISA、IFA筛选,获得4株阳性杂交瘤细胞株,分别命名为1G3、2C10、3B6和4F11。上述单克隆抗体亚类分别为IgG2...     

银灵通胶囊调节实验性高脂饮食大鼠血脂的研究

目的:探究银灵通胶囊对脂质代谢的影响及其机制。方法:建立大鼠高脂模型,用药后检测其血脂、丙二醛(MDA)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)的活性,检测肝脏组织的高密度脂蛋白受体SR-BI、低密度脂蛋白受体(LDLR)、氧化低密度脂蛋白(ox-LDH)受体CD36蛋白表达的mRNA表达水平,检测血管组织学变化。结果:高脂饮食明显升高大鼠血清中总胆固醇(CH)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)和动脉硬化指数(AI值),银灵通胶囊组可降低上述指标,且呈一定的浓度依赖;高脂饮食可增加肝脏中SR-BI及CD36表达,降低LDLR表达,银灵通胶囊引起SR-BI的过度表达,使LDLR表达增加,CD36表达下降。高脂饮食使血清中MDA的含量增加,给予银灵通胶囊后,明显降低血清MDA的含量。结论:银灵通胶囊具有调节脂质代谢,抗动脉粥样硬化(AS)及抗脂质过氧化作用。其机制与银灵通胶囊能引起肝脏中SR-BI的过度表达及LDLR表达增加,降低肝脏中CD36表达和血清MDA含量有关。     

胆汁酸的合成调控及其在生理与病理中的功能机制

胆汁酸是一类胆固醇的代谢物,在机体胆固醇与能量代谢平衡和小肠营养物质吸收等方面起着重要作用。肝脏是合成胆汁酸的主要场所。饥饿条件下,胆汁酸从肝脏分泌进入胆管并被储存到胆囊;进食后胆囊收缩,贮存的胆汁酸被排出进入小肠。在小肠中,95%的胆汁酸会被小肠重新吸收,通过肝门静脉返回肝脏,这一过程被称为胆汁酸的肝肠循环。胆汁酸一方面作为乳化剂促进小肠中脂类等物质的吸收及转运,同时也作为重要的信号分子与多种受体结合,包括核受体法呢醇X受体(farnesoidXreceptor,FXR)、维生素D受体(vitaminD receptor,VDR)、孕烷X受体(pregnaneXreceptor,PXR)以及细胞膜表面受体G蛋白偶联受体(cellmembrane surface receptor-G protein coupled receptor, TGR5)等,在调节体内胆汁酸的代谢平衡、糖脂代谢与能量代谢平衡等方面发挥重要作用。肝细胞生长因子(hepatocyte growth factor, HGF)、白介素1-(interleukin-1, IL-1)及肿瘤坏死因子(tumor necrosis factor, TNF-)等协同作用构成了胆汁酸合成的精密调控网络。本文主要综述了胆汁酸的合成调控及其功能方面的最新研究进展,旨在为胆汁酸代谢相关研究提供参考。     

仙台病毒黑龙江省地方株的分离与鉴定

目的自本省普通级实验动物中分离并鉴定出仙台病毒地方毒株,为建立仙台病毒血清抗体检测方法奠定基础。方法通过鸡胚尿囊腔传代自普通级小鼠肺脏分离病毒,经血凝实验、血凝阻断实验和结构基因序列测定对分离得到的病毒进行鉴定;大量繁殖病毒并通过蔗糖密度梯度离心纯化,免疫动物制备阳性血清,用标准试剂盒检测阳性血清效价。结果自150份小鼠肺脏分离到2株有血凝性的病毒,经形态学、血清学和结构基因序列测定鉴定为仙台病毒,命名为SV-HLJ。SV-HLJ与标准毒株Fushimi核蛋白基因(N)的核苷酸、氨基酸同源性分别为99·6%和99·0%。结论分离并鉴定出了仙台病毒黑龙江省地方毒株,为检测试剂盒的研制奠定了基础。     

Vitamin D receptors in isolated rat parotid gland acinar cells

Rat parotid gland was examined for the presence of 1α,25-dihydroxycholecalciferol receptors using sucrose density gradient ultracentrifugation techniques. [³H]DHCC bound specifically and with high affinity to a 3.2 S protein present in nuclear and cytosolic fractions of isolated parotid acinar cells. Values for the equilibrium dissociation constant and for the receptor concentration were determined to be approx. 0.1 nM, and 12 fmol/mg protein, respectively. In competitive inhibition experiments, the 3.2 S protein displayed 100-fold lower affinity for 25-hydroxycholecalciferol than for DHCC, and did not bind estradiol or methylprednisolone. These results suggest that rat parotid gland acinar cells contain classical DHCC receptors. A similar approach failed to provide evidence of DHCC receptors in isolated pancreas acinar cells, lacrimal gland or submandibular gland. It has been previously reported that vitamin D is essential for normal exocrine secretion from the rat parotid gland (Tenenhouse, A. and Afari, G. (1978) Biochim. Biophys. Acta 538, 631–634). The present findings suggest that this effect is the result of a direct action of DHCC on the parotid gland acinar cell. The absence of DHCC receptors in other exocrine cells suggests that tissue sensitivity to DHCC is not a general property of exocrine systems.     

Long-term cultures of chicken bone marrow cells

We report an adaptation to cultures of chicken bone marrow cells of the Dexter culture technique for obtaining long-term hemopoiesis in vitro. Cells were seeded in DMEM supplemented with fetal calf serum (20%) and hydrocortisone (10(-6) M) with or without chicken serum (1%). Cultures were incubated at 37 degrees C and fed every 2 weeks. An adherent cell layer composed of macrophages, fibroblasts, and adipocytes became established, over which hemopoietic cells formed foci and were released into the supernatant. Granulocytes and monocytes-macrophages differentiated in a constant proportion until Week 6, whereafter differentiation became progressively restricted to the monocytic lineage. As demonstrated by the generation of colony-forming cells, hemopoiesis was maintained for either 12 or 28 weeks.     

Purification of cone visual pigments from chicken retina

A novel method for purification of chicken cone visual pigments was established by use of a 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate-phosphatidylcholine (CHAPS-PC) mixture. Outer segment membranes isolated from chicken retinas were extracted with 0.75% CHAPS supplemented with 1.0 mg/mL phosphatidylcholine (CHAPS-PC system). After the extract was diluted to 0.6% CHAPS, it was loaded on a concanavalin A-Sepharose column. Elution from the column with different concentrations of methyl alpha-mannoside yielded three fractions: the first was composed of chicken violet, blue, and red in roughly equal amounts, the second predominantly contained chicken red, and the third was rhodopsin with a small amount of chicken green, which was separated from rhodopsin by DEAE-Sepharose column chromatography. Since CHAPS has little absorbance at both ultraviolet and visible regions, we could demonstrate the absolute absorption spectra of chicken red (92%) and rhodopsin (greater than 96%) in these regions. The maximum of the difference spectrum between either chicken red or rhodopsin and its photoproduct (all-trans-retinal oxime plus opsin) was determined to be 571 or 503 nm, respectively. Although chicken green was contaminated with a small amount of rhodopsin having a similar spectral shape, the maximum of its difference spectrum was located at 508 nm by taking advantage of the difference in susceptibility against hydroxylamine between these pigments. Although chicken blue and chicken violet were minor pigments present in the first fraction from the concanavalin A column, their maxima in the difference spectra were determined to be at 455 and 425 nm, respectively, by a partial bleaching method.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo function of VDR in gene expression-VDR knock-out mice

Vitamin D exerts many biological actions through nuclear vitamin D receptor (VDR)-mediated gene expression. The transactivation function of VDR is activated by binding 1,25-dihydroxyvitamin D₃[1,25(OH)₂D₃], an active form of vitamin D. Conversion from 25(OH)D₃ is finely regulated in kidney by 25(OH)D₃ 1-hydroxylase[25(OH)D 1-hydroxylase], keeping serum levels of 1,25(OH)₂D₃ constant. Deficiency of vitamin D and mutations in the genes like VDR (type II genetic rickets) are known to cause rickets like lowered serum calcium, alopecia and impaired bone formation. However, the molecular basis of vitamin D–VDR system in the vitamin D action in intact animals remained to be established. In addition, the 1-hydroxylase gene from any species had not yet been cloned, irrespective of its biological significance and putative link to the type I genetic rickets. We generated VDR-deficient mice (VDR KO mice). VDR KO mice grew up normally until weaning, but after weaning they developed abnormality like the type II rickets patients. These results demonstrated indispensability of vitamin D–VDR system in mineral and bone metabolism only in post-weaning life. Using a newly developed cloning system, we cloned the cDNA encoding a novel P450 enzyme, mouse and human 1-hydroxylase. The study in VDR KO mice demonstrated the function of liganded VDR in the negative feed-back regulation of 1,25(OH)₂D₃ production. Finally, from the analysis of type I rickets patients, we found missense genetic mutations in 1-hydroxylase, leading to the conclusion that this gene is responsible for the type I rickets.     

Organotypic slice culture based on in ovo electroporation for chicken embryonic central nervous system

Organotypic slice culture is a living cell research technique which blends features of both in vivo and in vitro techniques. While organotypic brain slice culture techniques have been well established in rodents, there are few reports on the study of organotypic slice culture, especially of the central nervous system (CNS), in chicken embryos. We established a combined in ovo electroporation and organotypic slice culture method to study exogenous genes functions in the CNS during chicken embryo development. We performed in ovo electroporation in the spinal cord or optic tectum prior to slice culture. When embryonic development reached a specific stage, green fluorescent protein (GFP)‐positive embryos were selected and fluorescent expression sites were cut under stereo fluorescence microscopy. Selected tissues were embedded in 4% agar. Tissues were sectioned on a vibratory microtome and 300 μm thick sections were mounted on a membrane of millicell cell culture insert. The insert was placed in a 30‐mm culture dish and 1 ml of slice culture media was added. We show that during serum‐free medium culture, the slice loses its original structure and propensity to be strictly regulated, which are the characteristics of the CNS. However, after adding serum, the histological structure of cultured‐tissue slices was able to be well maintained and neuronal axons were significantly longer than that those of serum‐free medium cultured‐tissue slices. As the structure of a complete single neuron can be observed from a slice culture, this is a suitable way of studying single neuronal dynamics. As such, we present an effective method to study axon formation and migration of single neurons in vitro.     

Comparative studies of type X collagen expression in normal and rachitic chicken epiphyseal cartilage

The levels of type X collagen in mineralizing normal chicken epiphyses and nonmineralizing rachitic chicken tibial epiphyses were measured and compared. Qualitative immunoperoxidase studies with anti-chick type X collagen monoclonal antibodies on sections from normal and rachitic cartilage demonstrated that the type X collagen levels in rachitic growth plates are reduced. Northern hybridization of mRNA and biosynthetic studies have confirmed that type X collagen synthesis in rickets is also decreased. In hypocalcemic rickets, the level of type X collagen mRNA is reduced by 80% whereas the level of type X collagen mRNA is only reduced by 50% in normocalcemic rickets. These observations provide additional evidence that type X collagen is involved in the process of cartilage mineralization and also suggest that the partial recovery of type X collagen synthesis in normocalcemic rickets may be related to the elevated plasma concentration of calcium. Calcium concentration may therefore play an important role in the control of type X collagen synthesis.     

Apparent Efficacy of Food-Based Calcium Supplementation in Preventing Rickets in Bangladesh

To determine whether increased Ca intakes can prevent rickets in a susceptible group of children living in a rickets-endemic area of Bangladesh, we conducted a 13-month long, double-blind, clinical trial with 1-to 5-year-old children who did not present with rickets but ranked in the upper decile of plasma alkaline phosphatase (AP) activity of a screening cohort of 1,749 children. A total of 158 children were randomized to a milk-powder-based dietary supplement given daily, 6 days/week, and providing either 50, 250, or 500 mg Ca, or 500 mg Ca plus multivitamins, iron, and zinc. Upon initial screening, 194 healthy children presented with no rachitic leg signs and had serum AP in the upper decile (>260 u/dl) of the cohort. When 183 of those subjects were re-screened after a 7-month pre-trial period, 23 (12.6%) had developed rachitic leg signs, suggesting an annual risk of 21.5% in this cohort. Of those still not presenting with leg signs and completing 13 months of dietary intervention, none showed rachitic leg signs, none showed significant radiological evidence of active rickets, and all showed carpal ossification normal for age after that intervention. These results are consistent with even the lowest amount of supplemental Ca (50 mg/day) being useful in supporting normal bone development in this high-risk population.     

Bone mineralization by OST-6 (OsteoCare), a herbomineral preparation, in experimentally induced rickets in rats.

In the present study the efficacy of OST-6 (OsteoCare), a herbomineral preparation, on bone mineralization in experimental rickets has been evaluated. This was accomplished by feeding pregnant rats and subsequently their pups with vitamin D and calcium deficient (VDCD) with low phosphorus diet. The parameters such as serum and bone mineral contents (calcium and inorganic phosphorus), serum alkaline phosphatase, sex hormones and histology of bone were considered. VDCD resulted in a significant reduction in bone and serum calcium and inorganic phosphorus, increased serum alkaline phosphatase and decreased sex hormones (testosterone in males, progesterone and oestrogen in females). Histologically the bone showed osteodystrophic changes and disproportionate cartilaginous proliferations in the epiphyseal region. Incorporation of OST-6 into feed at 5% concentration resulted in a complete reversal of rickets, which was substantiated by biochemical and histological observations. It has been concluded that OST-6 is useful in the management of rickets in a natural way through herbal resources.     

A preliminary study on viral decontamination of chicken serum using gamma-irradiation

A preliminary experiment was carried out to determine whether a decontamination procedure using gamma irradiation, similar to that adopted in the European guideline for bovine serum contaminated by pestivirus, could be applied to chicken serum. Chicken sera spiked with known amounts of enveloped and non-enveloped chicken viruses were gamma irradiated. The remaining live viruses were then measured by titration and the virus reduction capacity of the irradiation process was established for both enveloped and non-enveloped virus models. In parallel with the irradiation procedure, a classical in vivo extraneous agent test was also evaluated in order to see if it has the capacity to detect low enough levels of live viruses to be used for testing irradiated serum. The results suggest that the principles of the bovine serum decontamination procedure may be applied to chicken serum. Further studies are required to determine if this process would provide an acceptable solution for the viral ‘decontamination’ of chicken serum.     

Circadian rhythms of melatonin release from chicken pineal in vitro: modified melatonin radioimmunoassay

An improved and simplified radioimmunoassay for measuring pineal, serum, and in vitro cultured medium melatonin is described. Using 2-[125I]iodomelatonin as radiolabeled ligand and a polyclonal rabbit antimelatonin antiserum, melatonin concentrations were determined in all three types of samples by a 2-day direct equilibrium double-antibody assay method without prior extraction. Serial dilutions of pineal homogenates, serum, and cultured medium all gave parallel displacement curves. Cross-reactivity of the antisera with other indoles was negligible. Intraassay coefficients of variation (n = 3) were 5.09, 3.32, and 5.05% at 7.81, 62.5, and 500 pg/tube, respectively, and the interassay coefficients of variation (n = 20) were 12.18% at 62.5 pg/tube. A characteristic diurnal rhythm of melatonin was observed using this direct assay for measuring daytime and nighttime chicken pineal and serum samples. An in vitro incubation of chicken pineal glands with a lighting cycle of 12-hr light:12-hr dark showed that the diurnal rhythm of melatonin secretion into the cultured medium was maintained. The direct assay method described in this report for measuring chicken melatonin using 2-[125I]iodomelatonin as radiolabeled ligand coupled with the in vitro cultured chicken pineal gland clearly offers great potential for studying the chicken pineal circadian oscillator and its underlying mechanism.