异种（猪）脱细胞真皮基质敷料在门诊烧伤患者中的应用

目的探讨应用异种（猪）脱细胞真皮基质包扎治疗门诊小面积烧伤患者的可行性。方法2006年11月至2012年11月门诊收治的63例小面积烧伤患者分别应用异种（猪）脱细胞真皮基质治疗和传统聚维酮碘乳膏纱布换药治疗,根据烧伤的深浅和处理方法的不同分成四组：真皮基质治疗浅Ⅱ°组（13例）、聚维酮碘乳膏治疗浅Ⅱ°组（17例）、真皮基质治疗深Ⅱ°组（19例）、聚维酮碘乳膏治疗深Ⅱ°组（14例）。观察创面愈合时间、换药次数、创面疼痛情况、患者依从性等。结果真皮基质治疗小面积浅Ⅱ°创面,创面平均愈合时间为（6．23±2．21）天,真皮基质治疗小面积深Ⅱ°创面,创面平均愈合时间为（12．46±3．57）天;聚维酮碘乳膏治疗小面积浅Ⅱ°创面,创面平均愈合时间为（8．37±4．39）天,聚维酮碘乳膏治疗小面积深Ⅱ°创面,创面平均愈合时间为（15．45±6．32）天。真皮基质治疗小面积浅Ⅱ°创面,平均换药次数为（3．34±1．45）,真皮基质治疗小面积深Ⅱ°创面,平均换药次数为（3．69±2．48）;聚维酮碘乳膏治疗小面积浅Ⅱ°创面,平均换药次数为（6．67±3．72）,聚维酮碘乳膏治疗小面积深Ⅱo创面,平均换药次数为（8．54±6．21）。结论真皮基质能够有效促进创面愈合,减少换药次数,适合门诊应用。     

牛骨移植及牙种植体植入的实验研究

目的探讨牛骨块在牙种植领域应用的可能性。方法将２０块牛骨外置移植于兔胫骨干骺端，同时植入牙种植体固定骨块。术后按计划给动物注射三种荧光素，并通过组织学和组织测量学方法评价新骨形成。结果荧光素标记的新骨逐渐替代移植骨的松质骨和部分皮质骨，实验结束时新骨已达牙种植体肩台平面。实验８４天，４个上位螺纹平面的骨面积明显高于２１天的骨面积。平均矿物沉积率为１．９０μｍ／ｄ～２．７５μｍ／ｄ。结论牛骨外置移植结合螺钉型牙种植体植入可增加受区骨量。     

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。     

人羊膜上皮细胞移植用于兔损伤声带组织修复的初步研究

目的 研究人羊膜上皮细胞(human amniotic epithelial cells,hAEC)移植入兔声带损伤组织内的生长分布特点,探索hAEC促进声带损伤后修复再生的潜能.方法 分离和培养hAEC,慢病毒增强型绿色荧光蛋白(1entivirus enhanced green fluorescent protein,Lenti-EGFP)基因转染作标记.建立深及声韧带的兔声带损伤模型,设hAEC移植组(13侧声带)、损伤对照组(13侧声带)及正常对照组(4侧声带).荧光显微镜下连续观察hAEC在声带内的存活、分布情况,应用HE染色和免疫组化染色分析胶原、纤维连接蛋白等主要细胞外基质在损伤后3个月时的含量及分布.结果 hAEC原代培养6 d后呈铺路石样生长,植入声带损伤组织后可在固有层内持续存活2个月,细胞呈纵向排列,有趋向性.声带损伤2个月时免疫荧光显示hAEC移植组兔肌细胞标志结蛋白荧光阳性,提示hAEC向肌细胞分化;同时Ⅲ型胶原荧光阳性,提示hAEC植入后具有分泌Ⅲ型胶原功能.光镜观察见hAEC移植后3个月兔胶原纤维密度和排序较损伤对照组改善,但未及正常;免疫组化染色示hAEC移植组纤维连接蛋白含量和分布介于损伤对照组和正常对照组之间.结论 hAEC可在异种动物声带损伤组织内持续存活、生长,并有向声带组织分化和分泌部分细胞外基质的潜能,可能促进声带损伤后的修复再生.

Abstract：

Objective To investigate the survival, growth and distribution of human amniotic epithelial cells (hAEC) after injected into injured rabbit vocal folds, in addition, to assess the ability of hAEC to affect the components of lamina propria extracellular matrix (ECM) and prevent vocal fold scarring.Methods hAEC were isolated from human amnion and marked by Lenti-EGFP. Fifteen New Zealand rabbits were used for this experiment. EGFP-hAEC was injected into the left injured vocal folds in thirteen rabbits, and the contralateral thirteen vocal folds experienced an injured procedure only (" injured untreated control"), and four vocal folds were left as untreated controls. The survival, distribution, differentiation potential and secretion function of hAEC were examined by immunofluorescence method. HE staining and immunohistochemical staining were performed for the evaluation of collagen and fibronectin respectively.Results hAEC showed a cobblestone-like growth. After implanted into the injured vocal folds, hAEC could survive in vocal fold lamina propria for 2 months. The immunofluorescence analysis showed the evidence of hAEC differentiation into muscle cells as well as secretion the ECM protein. Three months postoperatively, the density of collagen was higher in the injured untreated control folds than that in the injured vocal folds injected with hAEC and the untreated controls. Besides, the content of fibronectin in the injured untreated control group was significantly increased. Conclusions hAEC survived in the vocal folds lamina propria,and had the potentiality to differentiate into vocal folds tissue and secret some ECM components. The histological improvement caused by the injected cells demonstrate that hAEC had the ability to promote the repairment and regeneration of injured vocal folds.     

Infantile Fibrosarcoma—a Misnomer?

Two cases of congenital or infantile fibrosarcoma are described that were incompletely excised at the time of primary excision and have not recurred or metastasized after 3 years. The tumors were composed of densely cellular spindle cells with a high mitotic index. Immunohistochemical stains were positive for vimentin but negative for desmin and S-100. The tumor cells were grown in vitro, and a karyotype was obtained. Both tumors had normal diploid modal karyotypes. In addition, fragments of the primary tumor from both cases were injected subcutaneously into nude mice; neither tumor could be heterotrans-planted. The clinical course and biologic features of these two tumors suggest that congenital or infantile sarcoma does not have the properties of a malignant neoplasm, and thus the designation of these tumors as a sarcoma may be a misnomer.     

非亲缘异基因骨髓移植治疗儿童白血病

目的　评价非亲缘异基因骨髓移植 (URD BMT)治疗儿童急性和慢性白血病的临床疗效。方法　 6例白血病患儿 ,其中慢性髓系白血病 2例 ,急性淋巴细胞白血病 3例 (第 1次完全缓解 ) ,急性早幼粒细胞白血病 1例 (第 2次完全缓解 ) ,由台湾慈济骨髓捐赠中心提供无关供者骨髓。预处理方案为马利兰 环磷酰胺 (Bu/Cy2 )方案 ,急性移植物抗宿主病 (aGVHD)预防为霉酚酸酯(MMF)、环孢菌素A(CsA)加氨甲喋呤 (MTX)联合方案 ;以前列素E1预防肝静脉闭塞病 (VOD) ,以巨细胞病毒 (CMV)抗原血症监测和更昔洛韦预防CMV病。供、受者间HLA基因位点型全相合 3例 ,1个基因位点型不合 2例 ,2个基因位点型不合 1例。结果　 6例患儿经DNA短串联重复序列多态性分析证明为供髓植入 ,中性粒细胞 >0 5× 10 9/L的中位天数为 14 5 (13～ 18)d ,血小板 >2 0×10 9/L的中位天数为 16 (11～ 2 3)d。发生Ⅱ～Ⅳ度aGVHD 2例 (33% ) ,局限性慢性移植物抗宿主病(cGVHD) 3例 ,未发生广泛性cGVHD。中位随访时间 4 12 (187～ 1338)d ,全部患儿均无病生存。结论非亲缘异基因骨髓移植是治疗儿童急性和慢性白血病的有效方法。     

非缝合式无支架异种主动脉生物瓣膜的研制

目的: 研制新型非缝合式无支架异种主动脉生物瓣膜,在体外对其功能进行初步研究。方法: 将猪主动脉瓣缝合到NiTi形状记忆合金支架上,在离体猪心脏内置入新型瓣膜,肉眼及彩超下观测瓣膜闭合、冠状动脉血流及瓣周漏情况。通过TH-1200型人工心脏瓣膜脉动流测试系统测定25#非缝合式无支架异种主动脉生物瓣膜平均跨瓣压差、返流百分比、瓣膜开口面积。结果: 新型瓣膜能较简单的植入离体猪心脏内。且不同压力（80～250mmHg）下,瓣膜闭合良好,无明显瓣周漏,冠状动脉血流良好;心脏彩超检测瓣周结构与主动脉壁之间可见无回声间隙,无明显的瓣周漏。体外脉动实验提示新型瓣膜与机械瓣膜相比跨瓣压差小,返流少,开口面积大。结论: 该型瓣膜制备相对简单,具有血流动力学良好,跨瓣压小,无需抗凝等优点,可简化手术操作,降低手术难度。     

乙型肝炎肝硬化与肝癌患者HBV DNA、AFP-L3、GP73的差异及HBV DNA与AFP-L3、GP73相关性分析

目的探讨乙型肝炎肝硬化与肝癌患者乙型肝炎病毒DNA(HBV DNA)、血清甲胎蛋白异质体L3(AFP-L3)、高尔基蛋白73(GP73)的差异及HBV DNA与AFP-L3、GP73相关性。方法回顾该院2015年10月至2017年10月收治的乙型肝炎肝硬化患者和肝癌患者的临床资料,其中80例乙型肝炎肝硬化患者设为对照组,68例肝癌患者设为观察组。所有患者均采用PCR法检测血清HBV DNA,采用酶联免疫吸附法检测血清AFP-L3、GP73水平。比较两组患者血清HBV DNA、AFP-L3、GP73表达水平及阳性率的差异,采用Pearson相关对HBV DNA、AFP-L3、GP73相关性进行分析,并采用ROC曲线分析HBV DNA与AFP-L3、GP73在肝癌诊断中的价值。结果观察组患者HBV DNA、GP73表达阳性率高于对照组,但差异无统计学意义(P>0.05);观察组患者AFP-L3表达阳性率明显高于对照组,差异有统计学意义(χ2=55.01,P<0.001)。观察组患者血清HBV DNA、AFP-L3、GP73水平明显高于对照组,差异均有统计学意义(P<0.05)。分别将患者HBV DNA水平与AFP-L3、GP73水平作Pearson相关性分析,结果显示,HBV DNA与AFP-L3无相关性(r=0.214,P=0.079);HBV DNA与GP73表达无相关性(r=0.155,P=0.208)。采用ROC曲线对两组患者3种指标的鉴别诊断价值进行分析,3种指标均有一定的临床诊断价值(AUC>0.5),且AFP-L3在鉴别诊断中的应用价值最高(AUC=0.971,95%CI:0.943~0.999)。结论乙型肝炎肝硬化与肝癌患者HBV DNA、AFP-L3、GP73的水平存在明显差异,且肝癌患者血清HBV DNA与AFP-L3、GP73水平无明显相关性,AFP-L3在乙型肝炎肝硬化与肝癌的鉴别诊断中价值最高,值得在临床推广应用。     

Elicitation of Neutralizing Antibody Responses to HIV-1 Immunization with Nanoparticle Vaccine Platforms

A leading strategy for developing a prophylactic HIV-1 vaccine is the elicitation of antibodies that can neutralize a large fraction of circulating HIV-1 variants. However, a major challenge that has limited the effectiveness of current vaccine candidates is the extensive global diversity of the HIV-1 envelope protein (Env), the sole target for HIV-neutralizing antibodies. To address this challenge, various strategies incorporating Env diversity into the vaccine formulation have been proposed. Here, we assessed the potential of two such strategies that utilize a nanoparticle-based vaccine platform to elicit broadly neutralizing antibody responses. The nanoparticle immunogens developed here consisted of different formulations of Envs from strains BG505 (clade A) and CZA97 (clade C), attached to the N-termini of bacterial ferritin. Single—antigen nanoparticle cocktails, as well as mosaic nanoparticles bearing both Env trimers, elicited high antibody titers in mice and guinea pigs. Furthermore, serum from guinea pigs immunized with nanoparticle immunogens achieved autologous, and in some cases heterologous, tier 2 neutralization, although significant differences between mosaic and single—antigen nanoparticles were not observed. These results provide insights into the ability of different vaccine strategies for incorporating Env sequence diversity to elicit neutralizing antibodies, with implications for the development of broadly protective HIV-1 vaccines.     

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.