建立猪心脏移植至猕猴腹腔内的异位心脏移植模型

目的 评价改进后的猪心脏移植至猕猴腹腔内的异位心脏移植模型的效果。方法 建立21例猪心脏移植至猕猴腹腔内的异位心脏移植模型，其中采用传统手术方法11例，改进方法10例。改进方法为缩短供心缺血时间，调整了手术顺序，先游离受者供吻合的腹主动脉及下腔静脉段后，再行供心切取，并且改进了切取供心的方法。比较2种手术方法的热缺血时间、移植心功能恢复情况及术后并发症。结果 采用传统手术方法时，移植心准备时间为20～30min，热缺血时间为3～5min，总缺血时间为80～90min，再灌注后均未恢复正常的全心搏动，仅见右心收缩，移植后发生并发症较多（包括出血、吻合口血栓形成、肠道并发症及静脉回流不畅等）。改进手术方法后，移植心准备时间为5～10min，热缺血时间〈1min，总缺血时间为35～40min，再灌注后有6例恢复全心搏动，发生并发症较少。结论 改进后的猪心脏移植到猕猴腹腔内的异位心脏移植模型，其手术方法明显优于传统方法。     

异体软骨细胞复合透明质酸苄基酯凝胶修复关节软骨缺损

目的 :研究采用同种异体软骨细胞复合透明质酸苄基酯凝胶修复关节软骨缺损的效果。方法 :将体外培养的同种异体第二代软骨细胞与透明质酸苄基酯凝胶复合注入兔关节软骨缺损区作为实验组 ,以软骨细胞悬液组和不作任何处理组作为对照 ,于第 4、8、1 2周取材 ,进行大体、组织学、电镜检查 ,观察其修复效果。结果 :实验组术后 1 2周时 ,缺损区可见新生透明样软骨组织 ,表面光滑 ,与周围软骨界面难以辨认。软骨细胞悬液组和空白对照组组均未见明显修复。结论 :同种异体软骨细胞复合透明质酸苄基酯凝胶修复关节软骨缺损效果明显 ,将透明质酸苄基酯凝胶作为移植载体是可行的。     

重组合异种骨移植局部白细胞介素-8 mRNA和蛋白质的表达

目的：探讨在重组合异种骨(RBX)移植局部白细胞介素—8(IL-8)mRNA和蛋白质的表达规律．方法：将重组人骨形成蛋白(rhBMP2)复合于牛松质骨中制成RBX并植入小鼠股部肌肉内，于术后4，7，14和21d取材，应用原位杂交和免疫组织化学技术检测植骨局部IL-8 mRNA及蛋白的表达与细胞定位．标准对照组动物植入单纯牛松质骨，并另设空白对照．结果：术后4，7，14和21d，实验组和标难对照组动物植骨局部均可检测到IL—8mRNA及蛋白的表达．叫主要定位于单核细胞、巨噬细胞、中性粒细胞和成纤维细胞以及部分软骨细胞、成骨细胞和新生骨髓中部分骨髓细胞．结论：在RBX移植局部多种细胞表达IL—8，提示RBX可能通过新生骨中的多种细胞成分表达IL—8，参与异种骨移植的局部免疫反应。     

异种造血嵌合体模型的免疫耐受机制

目的 在已建立的大鼠→免异种造血嵌合体模型中，了解异种移植免疫耐受机制。方法 通过直接、间接免疫荧光染色技术及补体介导的细胞毒试验等方法，检测大鼠MHC－Ⅰ类分子阳性细胞以及体液免疫。结果 检测出大鼠MHC－Ⅰ类分子阳性细胞比例为31.0%-61.9%，且此细胞体液免疫功能正常；同时在此模型中检测出有较高浓度的兔抗大鼠单个核细胞抗体存在，且受体补体功能正常。结果 （1）用我们所建立的方法进行异种骨髓移植是成功的；（2）在受体补体活性正常的嵌合体模型中大鼠单个核细胞与兔抗大鼠单个核细胞抗体出现的共存现象（即适应accomm-oda-tion），是形成此嵌合体的原因。     

穿心莲转录因子ApNAC1的克隆、亚细胞定位及原核表达

从穿心莲叶片中克隆了1个NAC家族的转录因子,命名为ApNAC1,Gen Bank注册号为KY196416。生物信息学分析表明该基因的完整编码区包含972 bp,编码1个323个氨基酸的多肽,预测蛋白相对分子质量为35.9 k Da,等电点为6.14。原生质体瞬时表达研究证实了ApNAC1-GFP融合蛋白特异定位在穿心莲叶肉细胞的细胞核中,是一个核定位蛋白。实时定量PCR结果表明ApNAC1基因表达与穿心莲内酯的积累模式一致:主要在穿心莲的叶片里表达,而且茉莉酸甲酯处理能急剧上调其表达水平。ApNAC1基因在大肠杆菌中进行异源表达,并且被成功纯化。该研究为进一步探索ApNAC1蛋白参与穿心莲内酯生物合成的研究奠定了基础。     

异种骨基质明胶复合自体红骨髓治疗骨缺损的实验结果观察

目的观察异种骨基质明胶(bonematrixgelatin,BMG)复合自体红骨髓(redbonemarrow,RBM)治疗骨缺损的疗效,为临床应用提供证据。方法制备猪BMG,将之与自体红骨髓复合移植兔桡骨中段1cm骨缺损,通过影像学和组织形态学等检测,观察其成骨效果,并与单纯BMG移植及自体骨移植进行比较。结果BMG/RBM表现出较强的骨形成能力及骨传导作用,16周基本修复骨缺损,骨髓腔再通,其修复能力明显优于BMG组;与自体骨相近。骨修复过程为膜内成骨和软骨内成骨。结论使用BMG/RBM治疗骨缺损,可明显促进骨形成,加快骨修复,具有良好的诱导骨生成和骨传导作用。     

Experimental nodular ‘diabetic-like” glomerulosclerosis in guinea pigs following longacting,heterologous insulin immunization

Summary Renal lesions similar to Kimmelstiel-Wilson nephropathy have been found in guinea pigs immunized with long-acting heterologous insulin. — The animals were treated for periods ranging from 3 to 5 months, with monthly subcutaneous injections of highly purified bovine insulin in a mixture of lanolin and paraffin oil. — Kidney sections have been examined by means of light microscopy after standard and histochemical stainings, histoimmunological techniques and electron microscopy. — The following glomerular lesions have been detected : 1. PAS-positive hyaline nodules (55.5%); 2. Thickening of the basal membrane (100%); 3. aneurysmatic dilatation of the capillaries (88.8%); 4. diffuse glomerulosclerosis (61.1%); 5. increased number of mesangial cells (38.8%); 6. fibrinoid caps (44.4%); 7. capsular adhesions (33.3%). — Histo-chemical findings showed that there are differential characteristics between fibrinoid caps (exudative lesions) and hyaline nodular alterations: the former were rapidly digested by trypsin, whereas the nodules resisted the tryptic treatment for 6 h. — Histoimmunological staining of the lyophilized kidney sections with fluorescein-isothiocyanate-labelled anti-bovine insulin serum showed marked fluorescence of both the fibrinoid caps and hyaline nodules of the glomeruli, and also a less intense fluorescence of the basement membrane and of the intercapillary stroma. — Electron microscopy of the glomeruli demonstrated that the basal membrane was always irregularly thickened showing a patchy fibrillary structure, and that there were nodular areas and bands of basal-membrane-like material. Besides, dense osmiophilic deposits were noted in the mesangium. — It is concluded that insulin acting as an antigen brings about a nodular Kimmelstiel-Wilson-like nephropathy in experimental animals with normal or low blood sugar and lipids, through immunological mechanisms.

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Experimentell erzeugte pseudodiabetische noduläre Glomerulosklerose bei Meerschweinchen nach Immunisierung mit heterologem, protrahiert wirkendem Insulin

Zusammenfassung Nach Immunisierung durch heterologes Insulin mit protrahierter Wirkung ließen sich bei Meerschweinchen ähnliche Nierenveränderungen wie bei der Kimmelstiel-Wilson'schen Nephropathie nachweisen. — Die Tiere erhielten monatliche subcutane Injektionen von hochgereinigtem Rinderinsulin in einer Mischung von Lanolin und Paraffinöl über einen Zeitraum von 3–5 Monaten. — Die Untersuchung der Nierenschnitte erfolgte lichtmikroskopisch mit Hilfe von Standardund histochemischen Färbungen sowie durch histoimmunologische Verfahren und die Elektronenmikrosko pie. Dabei ließen sich an den Glomeruli folgende pathologische Befunde erheben: — 1. PAS-positive hyaline Knötchen (55.5%); 2. eine Verdickung der Basalmembran (100%); 3. aneurysmatische Kapillarerweiterungen (88.8%); 4. eine diffuse Glomerulosklerose (61.1%); 5. eine Vermehrung der mesangialen Zellen (38.8%); 6. Fibrinausfällungen (44.4%); 7. Kapseladhäsionen (33.3%). — Die histochemischen Untersuchungen zeigten Unterschiede zwischen den Fibrinausfällungen (exsudative Veränderungen) und den hyalinen Knötchen: erstere wurden durch Trypsin schnell zerstört, während die hyalinen Knötchen einer Trypsinbehandlung über 6 Std. standhielten. Histo-immunologische Anfärbung lyophlisierter Nierenschnitte durch Anti-Rinderinsulin-Serum, das mit Fluoreszin-Isothiozyanat markiert worden war, ließen eine ausgeprägte Fluoreszenz der Fibrinausfällungen, sowie der hyalinen Knötchen in den Glomeruli und eine schwächere Fluoreszenz der Basalmembran und des interkapillären Grundgewebes erkennen. Durch die Elektronenmikroskopie konnte nachgewiesen werden, daß die Basalmembran immer unregelmäßig verdickt war und stellenweise eine fibrilläre Struktur aufwies. Weiter fanden sich noduläre Bezirke und Streifen von membran-ähnlichem Material. Im Mesangium ließen sich ferner dichte osmophile Ablagerungen nachweisen. Es wird gefolgert, daß Insulin als Antigen über immunologische Mechanismen bei Versuchstieren mit normalen oder sogar erniedrigten Glucoseund Lipid-Serumspiegeln eine noduläre Eämmelstiel-Wilson ähnliche Nephropathie auslösen kann.

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Glomérulosclérose nodulaire expérimentale semblable à celle du diabète chez des cobayes après immunisation par de l'insuline hétérologue à action prolongée

Résumé Des lésions rénales semblables à la néphropathie de Kimmelstiel-Wilson ont été trouvées chez des cobayes immunisés avec de l'insuline hétérologue à action prolongée. — Les animaux ont été traités pendant des périodes de 3 à 5 mois par des injections mensuelles souscutan ées d'insuline bovine hautement purifiée, mélangée à de la lanoline et à de l'huile de paraffine. — Les coupes de rein ont été examinées à l'aide du microscope à lumière, après colorations standard et histochimiques, ainsi qu'à l'aide de techniques histoimmunologiques et au microscope électronique. — On a trouvé les lésions glomérulaires suivantes: 1. Nodules hyalins PAS-positifs (55.5%); 2. épaississement de la membrane basale (100%); 3. dilatation anévrismale des capillaires (88.8%); 4. glomérulosclérose diffuse (61.1%); 5. augmentation du nombre des cellules du mésangium (38.8%); 6. capsules fibrinoïdes (44.4%); 7. adhérences capsulaires (33.3%). — Les résultats histochimiques ont montré qu'il y a des caractéristiques diff érentielles entre les capsules fibrinoïdes (lésions exsudatives) et les altérations nodulaires hyalines: les premières sont digérées rapidement par la trypsine tandis que les nodules résistent au traitement de 6 h par la trypsine. — La coloration histoimmunologique des coupes de rein lyophilisé, avec du sérum anti-insuline de boeuf marqué avec del'isothiocyanate de fluorescéine montra une fluorescence prononcée des capsules fibrinoïdes et des nodules hyalins des glomérules, ainsi qu'une fluorescence moins intense de la membrane basale et du stroma intercapillaire. — La microscopie électronique des glomérules a montré que la membrane basale est toujours épaissie de façon irrégulière, présentant une structure en taches, et qu'il y a des aires nodulaires et des bandes de matière semblable à la membrane basale. En outre on a noté des dépôts osmophiles denses dans le mésangium. — On conclut que l'insuline agissant comme un antigène provoque, par l'intermédiaire de mécanismes immunologiques, une néphropathie nodulaire semblable à celle de Kimmelstiel-Wilson, chez les animaux d'expérience ayant un taux normal ou bas de sucre et de lipides.

     

Reconstitution of β-adrenergic regulation of CaV1.2: Rad-dependent and Rad-independent protein kinase A mechanisms

L-type voltage-gated Ca_V1.2 channels crucially regulate cardiac muscle contraction. Activation of β-adrenergic receptors (β-AR) augments contraction via protein kinase A (PKA)–induced increase of calcium influx through Ca_V1.2 channels. To date, the full β-AR cascade has never been heterologously reconstituted. A recent study identified Rad, a Ca_V1.2 inhibitory protein, as essential for PKA regulation of Ca_V1.2. We corroborated this finding and reconstituted the complete pathway with agonist activation of β1-AR or β2-AR in Xenopus oocytes. We found, and distinguished between, two distinct pathways of PKA modulation of Ca_V1.2: Rad dependent (∼80% of total) and Rad independent. The reconstituted system reproduces the known features of β-AR regulation in cardiomyocytes and reveals several aspects: the differential regulation of posttranslationally modified Ca_V1.2 variants and the distinct features of β1-AR versus β2-AR activity. This system allows for the addressing of central unresolved issues in the β-AR–Ca_V1.2 cascade and will facilitate the development of therapies for catecholamine-induced cardiac pathologies.

Cardiac excitation–contraction coupling crucially depends on the L-type voltage-dependent Ca²⁺ channel, Ca_V1.2. Influx of extracellular Ca²⁺ via Ca_V1.2 triggers Ca²⁺ release from the sarcoplasmic reticulum via the Ca²⁺ release channel (1). Activation of the sympathetic nervous system increases heart rate, relaxation rate and contraction force. The latter is largely due to increased Ca²⁺ influx via Ca_V1.2 (2, 3). Pathological prolonged sympathetic activation progressively impairs cardiac function, causing heart failure, partly due to misregulation of Ca_V1.2 (4, 5).Cardiac Ca_V1.2 is a heterotrimer comprising the pore-forming subunit α_1C (∼240 kDa), the intracellular Ca_Vβ₂ (∼68 kDa) and the extracellular α2δ (∼170 kDa) (Fig. 1A) (6, 7). The N and C termini (NT, CT respectively) of α_1C are cytosolic and vary among Ca_V1.2 isoforms. Further, most of the cardiac α_1C protein is posttranslationally cleaved at the CT, around amino acid (a.a.) 1800, to produce the truncated ∼210-kDa α_1C protein and the ∼35-kDa cleaved distal CT (dCT); however, the full-length protein is also present (8–11).Open in a separate windowFig. 1.cAMP regulation of Ca_V1.2 is enhanced by coexpression of Rad. (A) Ca_V1.2 and Rad. α_1C and α2δ subunits are shown schematically, with structures of β_2b (38) and Rad (74). The truncation in α_1CΔ1821 was at a.a. 1,821 (red cross mark) similar to naturally truncated cardiac α_1C, ∼a.a. 1800 (9). Ca_Vβ binds to the cytosolic loop I, L1, that connects repeat domains I and II. Rad exerts inhibitory action on the channel, in part through an interaction with Ca_Vβ. (B) Rad reduces the Ba²⁺ current of Ca_V1.2-α_1CΔ1821 (α_1CΔ1821, β_2b and α2δ; 1.5 ng RNA of each subunit) in a dose-dependent manner. Pearson correlation, r = −0.82, P = 0.023. Each point represents mean ± SEM from 7 to 10 oocytes recorded during 1 d. The linear regression line was drawn for nonzero doses of Rad. (C) Rad enhances the cAMP-induced increase in I_Ba. Diary plots of the time course of change in I_Ba (normalized to initial I_Ba) are shown before and after intracellular injection of cAMP in representative cells. No Rad: Upper; with Rad: Lower. (Insets) Currents at +20 mV before (black trace) and 10 min after cAMP injection (red trace). (D) “before–after” plots of cAMP-induced changes in I_Ba in individual cells injected Rad RNA while varying Rad:β_2b RNA ratio (by weight, wt/wt). Empty symbols–before cAMP; red-filled–after cAMP. n = 3 experiments; statistics: paired t test. (E) cAMP-induced increase in I_Ba at different Rad/β_2b RNA levels (summary of data from D). Each symbol represents fold increase in I_Ba induced by cAMP injection in one cell. Here and in the following figures, box plots show 25 to 75 percentiles, whiskers show the 5/95 percentiles, and black and red horizontal lines within the boxes are the median and mean, respectively. At all Rad:β_2b RNA ratios except 1:20, the cAMP-induced increase in I_Ba was significantly greater than without Rad (Kruskal–Wallis test; H = 36.1, 6 degrees of freedom, P < 0.001). (F) Summary of cAMP effects in 10 experiments without and with Rad at 1:2 and 1:1 Rad:β_2b RNA ratios (pooled). Number of cells: within the bars. Statistics: Mann–Whitney U test; U = 19.0, P < 0.001.The sympathetic nervous system activates cardiac β-adrenergic receptors (β-AR), primarily β1-AR (which is coupled to G_s, is globally distributed in cardiomyocytes, and mediates most of the β-AR-enhancement of contraction and Ca_V1.2 activity) and β2-AR, which can couple to both G_s and G_i (12). The cascade of adrenergic modulation of Ca_V1.2 comprises agonist binding to β-ARs, activation of G_s and adenylyl cyclase, elevated intracellular cAMP levels, and activation of protein kinase A (PKA) by cAMP-induced dissociation of its catalytic subunit (PKA-CS) from the regulatory subunit. However, the final step, how PKA-CS enhances Ca_V1.2 activity, remained enigmatic. A long-standing paradigm was a direct phosphorylation by PKA-CS of α_1C and/or Ca_Vβ subunits (3, 13–16). However, numerous studies critically challenged this theory. In particular, mutated Ca_V1.2 channels in genetically engineered mice lacking putative PKA phosphorylation sites on α_1C and/or β_2b, were still up-regulated by PKA (9, 17–21) (reviewed in refs. 6 and 22).One significant obstacle in deciphering the mechanism of PKA regulation of Ca_V1.2 was a recurrent lack of success in reconstituting the regulation in heterologous systems, which proved challenging and controversial (23). Studies in heterologous cellular models, including Xenopus oocytes, demonstrated that cAMP failed to up-regulate Ca_V1.2 containing the full-length α_1C, Ca_V1.2-α_1C (24–26). However, robust β-AR–induced up-regulation of Ca²⁺ currents was observed in oocytes injected with total heart RNA (27, 28), suggesting the necessity of an auxiliary protein, the “missing link” (24, 25). Interestingly, partial regulation was observed with dCT-truncated α_1C (16, 29). Intracellular injection of cAMP or PKA-CS in Xenopus oocytes caused a modest (30 to 40%) up-regulation of Ca_V1.2, containing a dCT-truncated α_1C, Ca_V1.2-α_1CΔ1821 (29). This regulation required the presence of the initial segment of the long-NT of α_1C but did not involve Ca_Vβ subunit. We proposed that this mechanism might account for part of the adrenergic regulation of Ca_V1.2 in the heart (29). Normally adrenergic stimulation in cardiomyocytes increases the Ca²⁺ current two- to threefold; thus, a major part of the regulation has remained unexplained.Recently, Liu et al. identified Rad as the “missing link” in PKA regulation of Ca_V1.2 (20). Rad is a member of the Ras-related GTP-binding protein subfamily (RGK) that inhibit high voltage-gated calcium channels Ca_V1 and Ca_V2 (30). Rad tonically inhibits Ca_V1.2, largely via an interaction with Ca_Vβ (31, 32). Ablation of Rad in murine heart was shown to increase basal Ca_V1.2 activity and rendered the channel insensitive to β-AR regulation, probably through a “ceiling” effect (33, 34). Liu et al. (20) reconstituted a major part of the Ca_V1.2 regulation cascade, initiated by forskolin-activated adenylyl cyclase in mammalian cells, ultimately attaining an approximately twofold increase in Ca²⁺ current. The regulation required phosphorylation of Rad, the presence of Ca_Vβ, and the interaction of Ca_Vβ with the cytosolic loop I of α_1C, suggesting that PKA phosphorylation of Rad reduces its interaction with Ca_Vβ and relieves the tonic inhibition of Ca_V1.2 (20, 35).Importantly, the complete adrenergic cascade, starting with β-AR activation, has not yet been heterologously reconstituted for Ca_V1.2. Also, the relation between the Rad-dependent regulation and the regulation reported in our previous study (29) is not clear. Here, we utilized the Xenopus oocyte heterologous expression system and successfully reconstituted the entire β-AR cascade. We demonstrate two distinct pathways of PKA modulation of Ca_V1.2 (Rad dependent and Rad independent) and characterize the roles of NT and CT of α_1C, β_2b, and Rad in the adrenergic modulation of cardiac Ca_V1.2 channels. Reproducing the complete β-AR cascade in a heterologous expression system will promote the identification and characterization of intracellular proteins that regulate the cascade, eventually assisting efforts to develop therapies to treat heart failure and other catecholamine-induced cardiac pathologies.     

补体在猪到猴异种心脏移植中的作用及机理

目的 探讨补体在异种大动物猪到猴心脏移植排斥反应中的作用及机理.方法 以梅山猪为供者,中国猕猴为受者,行异种腹腔异位心脏移植.随机将受者分为3组.A组(5只):为空白对照组,受者心脏移植后不作任何处理.B组(5只):为照射预处理组,受者于心脏移植前28 d、即1.5个月龄时接受60Coγ3 Gy全身剂量照射,其余同A组.C组(8只):为照射+胸腺注射预处理组,心脏移植前21 d,将供者的脾细胞(按照5×107个/只的数量)注入受者的两侧胸腺内,其余同B组.观察心脏移植术后各组移植心的存活时间;猪对猴单向混合淋巴细胞培养的刺激效应;采用双抗体夹心法检测补体C3和CD46的血清浓度;通过流式细胞术检测受者外周血细胞表面IgM、IgG阳性细胞百分比水平.结果 A、B、C三组移植心的存活时间分别为:(36.6±5.8)h、(65.6±6.5)h和(91.1±22.8)h,C组移植心的存活时间明显延长,与A组比较,P＜0.01,与B组比较,P＜0.05.C组在猪对猴单向混合淋巴细胞反应中的刺激效应较A、B组明显下降(P＜0.01).B、C组移植前补体水平(C3)无明显变化,但随着IgM、IgG水平的上升,发生排斥反应时C3和CD46水平显著降低.C组猕猴特异性抗猪抗体IgM及IgG的上升速度均较A、B组明显延缓.结论 对受者进行异种胸腺注射联合全身照射预处理在抑制T淋巴细胞免疫及体液免疫方面有重要作用,但无法抑制异种排斥反应中补体的激活,补体通过经典途径参与了延迟性异种排斥反应的发生.     

60Co辐射异种神经移植后皮肤感觉神经末梢的再形成

于梨状肌下缘１０ｍｍ处切除成年Ｗｉｓｔａｒ大白鼠右侧的坐骨神经５ｍｍ,移植入８ｍｍ长、经２×１０￣６ｒａｄ￣（６０）Ｃｏ辐射的成年日本大耳兔胫神经。异种神经移植术后８、１２、１６、２０、３２周,取出鼠手术侧的爪皮入１０％福尔马林固定、冰冻切片,按Ｇｒｏｓ