Chitosan-DTPA-Gd舌间质磁共振淋巴造影显示兔颈淋巴的研究

目的：探讨磁共振间质淋巴造影剂Chitosan-DTPA-Gd经舌黏膜下注射后,显示颈淋巴的应用价值。方法：选取健康成年纯种新西兰大白兔12只,麻醉并仰卧位固定磁共振平扫,再于每只兔双侧舌缘中后交界处粘膜下各注射0.1 mL造影剂,注射部位按摩30秒,按摩后5、10、15、20、25、30、35、40 min行三维增强磁共振淋巴造影成像,测量增强前后不同时间颈部淋巴结的信号强度（SI）,计算相对应的信号强化率（E%）,采用SPSS软件进行数据的统计学分析。结果：Chitosan-DTPA-Gd于兔双侧舌缘粘膜下注射后很快吸收进入颈部淋巴引流区,淋巴结及淋巴管明显、均匀的强化,血管无显影。颈部淋巴结于注射后15 min信号强度达到峰值,并可保持一段时间,25 min后信号强化率开始明显降低,40分钟后淋巴系统显影与周围组织无差别。结论：间质磁共振淋巴造影剂Chitosan-DTPA-Gd用量小,强化效应明显,能够有效的显示颈部淋巴结形态及淋巴管走形。     

兔VX2肝癌模型微波消融后磁共振灌注成像的可行性与应用价值

目的通过兔VX2模型探讨肿瘤消融治疗后动态变化过程中,磁共振灌注成像动态量化研究的可行性及其价值。方法16只新西兰大白兔分为实验组12只,对照组4只。实验组在兔肝脏种植VX2肿瘤后,观察肿瘤直径超过2.0 cm时行微波消融治疗。对比术后当天7、d、14 d及28 d实验组与对照组磁共振灌注成像量化指标—最大增强斜率（MSI）的动态变化差异,并与病理结果对照分析。结果对照组兔及实验组兔术后当天肝实质灌注MSI差异无显著性;实验组兔术前肿瘤与术后当天残留肿瘤的平均MSI差异无显著性;实验组兔残留肿瘤与良性强化组织的MSI差异有显著性。残留肿瘤的时间-信号强度曲线表现为快速上升型;良性强化组织的时间-信号强度曲线表现为缓慢上升型。结论磁共振灌注成像的动态量化研究是可行的,量化指标MSI与消融治疗后各种组织的病理结果相吻合,可更为准确地量化表达病变组织的病理状态的改变。     

山羊IL-2基因SYBR GREEN Ⅰ实时荧光定量RT-PCR检测方法的建立及应用

为建立山羊IL-2基因SYBR GREENⅠ实时荧光定量RT-PCR检测方法,根据Gen Bank中山羊IL-2基因(登录号:KT934548),设计1对特异性引物,用于扩增目的基因,并将目的基因克隆于p MD-19-T载体,转化至大肠杆菌DH5α,经质粒PCR及序列测定鉴定后获得阳性重组质粒,作为标准品模板建立SYBR GREENⅠRTFQ-PCR标准曲线和溶解曲线,进行特异性、重复性和敏感性试验。并应用所建立的方法,检测Con A刺激健康山羊PBMC后0 h、2 h、4 h、6 h、8 h、10 h、12 h、24 h和48 h不同时间点IL-2基因转录的动态变化。结果表明,当质粒标准品稀释度在7.2×10~9~7.2×10~5 copies/μL扩增曲线的Ct值与浓度间具有良好的线性关系,相关系数为-0.996;熔解曲线为特异性单峰,组内变异系数为0.306%~1.458%,组间变异系数为0.514%~1.191%,最低检测限为7.2×10~2 copies/μL。山羊IL-2基因的mRNA转录量在0~2 h呈现上升趋势,在2 h达到峰值,2~6 h呈现下降趋势。6~12 h未能检测到IL-2基因的mRNA转录;12~48 h检测到IL-2基因的mRNA转录量呈逐渐上升趋势。研究结果将为山羊IL-2基因的定量分析提供技术平台。     

大鼠脑缺血/再灌注过程中血流量和脑组织含水量的变化趋势*

目的:研究大鼠脑缺血/再灌注过程中血流量及与脑组织水含量变化的趋势。方法:选取5只成年SD雄性大鼠(n=5),参照改良Zea-Longa线栓法制备大鼠大脑中动脉缺血/再灌注模型,2 h后拔出线栓。利用PeriCam PSI血流灌注成像系统实时监测大鼠在缺血前及缺血5 min、30 min、1 h、2 h、再灌注5 min、30 min、1 h、2 h、4 h、6 h及24 h的血流灌注量,记录在ROI(感兴趣区)测量的数值。再选取15只成年SD雄性大鼠,分为Control组、缺血2 h、再灌注30 min、4 h及24 h组(n=3)。正常组不做任何处理,实验组按上述线栓法制备MCAO模型。取新鲜脑组织用干湿重法测定其左、右半球的水含量。结果:栓塞时缺血侧血流量逐渐下降,缺血2 h下降最低(P＜0.05);再灌注早期血流量恢复较大(P＜0.05),30 min时显著下降(P＜0.05),4 h明显上升(P＜0.05),24 h再次上升(P＜0.05)但低于缺血前血流量(P＞0.05)。脑组织水含量测量,缺血2 h组和再灌注30 min组与正常组无明显差异(P＞0.05);再灌4 h组和再灌24 h组明显增高(P＜0.05),且再灌24 h组明显高于再灌4 h组(P＜0.05)。结论:大鼠脑缺血/再灌注过程中血流量和脑组织中水含量的变化存在一定的规律,且脑组织中水含量与再灌注过程中血流量的变化有一定关系。     

大鼠脑缺血再灌注后Cathepsin D和caspase-9表达的动态变化

目的:探讨大鼠缺血再灌注后溶酶体组织蛋白酶D(Cathepsin D CD)、caspase-9在不同时段蛋白质及mRNA表达变化。方法:将60只S D大鼠随机分为三组:正常组(10只),假手术组(10只),脑缺血再灌注组(40只),线栓法制备大脑中动脉梗死模型(MCAO),免疫组化及RT-PCR法分别检测Cathepsin D、caspase-9的蛋白和mRNA表达。结果:与正常组和假手术组比较,模型组大鼠脑缺血再灌注损伤后6h Cathepsin D的蛋白和mRNA表达明显增强(P<0.05),24h达高峰,48h仍保存高水平。caspase-9蛋白和mRNA6h开始明显升高,12h达高峰,此后缓慢下降,但48h组仍显著高于对照组(P<0.05),结论:Cathepsin D、caspase-9在大鼠脑缺血再灌注后表达增强,溶酶体可能参与了脑缺血再灌注损伤后神经细胞凋亡的过程。     

颅脑创伤后大鼠脑组织脑红蛋白表达变化及其与神经元凋亡的关系研究

目的：研究大鼠弥漫性颅脑创伤后脑组织中脑红蛋白的表达变化情况,探究创伤后脑红蛋白表达变化及其与神经元凋亡的关系。方法：采用雄性SD大鼠50只,随机分为10组（n=5）空白对照组、伤后30min、1h、2h、6h、12h、24h、48h、72h和5d组。以Marmarou’s自由落体打击装置复制颅脑创伤模型,采用免疫组化技术检测伤后不同时间脑组织中脑红蛋白的表达情况及神经元凋亡相关基因Bax、Bcl-2表达情况,并对所得数据进行统计学分析。结果：致伤区皮层神经元脑红蛋白表达分别于伤后2h、72h呈现出两次高峰表达;伤后30min～1h、48～72h期间大脑皮层区脑红蛋白表达的上调均伴随着Bax/Bcl-2比值上升趋势减缓甚至呈现下降趋势。结论：弥漫性颅脑创伤后脑组织中脑红蛋白的高表达在一定程度上可以拮抗创伤应激及伤后继发缺血、缺氧性损伤所导致的神经元凋亡,在颅脑创伤的超早期（〈3h）、急性期（〈72h）可能具有一定的神经保护作用。     

大鼠脑缺血再灌注后Cathepsin D和caspase．9表达的动态变化

目的：探讨大鼠缺血再灌注后溶酶体组织蛋白酶D（Cathepsin DCD）、caspase-9在不同时段蛋白质及mRNA表达变化。方法：将60只SD大鼠随机分为三组：正常组（10只）,假手术组（10只）,脑缺血再灌注组（40只）,线栓法制备大脑中动脉梗死模型（MCAO）,免疫组化及RT—PCR法分别检测CathepsinD、caspase-9的蛋白和mRNA表达。结果：与正常组和假手术组比较,模型组大鼠脑缺血再灌注损伤后6h Cathepsin D的蛋白和mRNA表达明显增强（P〈0．05）,24h达高峰,48h仍保存高水平。caspase．9蛋白和mRNA6h开始明显升高,12h达高峰,此后缓慢下降,但48h组仍显著高于对照组（P〈0．05）,结论：Cathepsin D、caspase．9在大鼠脑缺血再灌注后表达增强,溶酶体可能参与了脑缺血再灌注损伤后神经细胞凋亡的过程。     

肢体缺血预处理对大鼠海马CA3区和齿状回区p38 MAPK和HSP 70表达的影响

目的:观察肢体缺血预处理(LIP)后大鼠海马CA3区和齿状回区(DG)p38MAPK和HSP70的表达,以进一步探讨CA3区和DG区的缺血耐受机制。方法:96只Wistar大鼠随机分为sham组和LIP组,LIP组进一步分为LIP6 h、LIP 12h、LIP1 d、LIP2d、LIP3 d、LIP4d和LIP5 d亚组。方法:应用免疫组织化学和Western blot方法观察大鼠海马CA3区及DG区p38MAPK和HSP70的表达。结果:免疫组化及Western blot结果显示,与sham组相比,LIP后大鼠海马CA3区及DG区p-p38MAPK及HSP70的表达呈现出动态的变化,其中p-p38MAPK的表达于LIP后1 d明显增加、3 d达到高峰,LIP后4 d逐渐下降;HSP 70的表达则于LIP后2 d明显增加、3 d达到高峰,LIP后4 d逐渐下降。结论:LIP上调了大鼠海马CA3区和DG区p38MAPK和HSP70的表达。     

荚膜唾液酸对猪链球菌激活巨噬细胞TLR2-AKT-NF-κB信号通路影响的研究

[目的]探索猪链球菌2型感染后单核/巨噬细胞启动信号转导通路机制,探讨荚膜唾液酸对细菌激活巨噬细胞TLR2-AKT-NF-κB信号通路的影响.[方法]以小鼠巨噬细胞系RAW264.7细胞为研究对象,采用RT-PCR、Western blotting、免疫荧光和ELISA法分别检测猪链球菌2型野毒株、唾液酸缺失突变株、唾液酸回复突变株感染后不同时间点巨噬细胞TLR2 mRNA转录水平、AKT磷酸化水平、NF-κB激活程度以及前炎症因子TNF-α分泌水平;再分别用TLR2阻断剂和PI-3K抑制剂预处理巨噬细胞,检测上述分子的表达水平.[结果]唾液酸缺失株可选择性的活化信号转导通路途径.RT-PCR结果表明,缺失株TLR2 mRNA表达水平自1h开始升高,1.5 h达高峰后有所下降;Westernblotting显示,缺失株TLR2蛋白表达水平7h达高峰,9h下降;p-AKT水平1.5-5 h持续稳定在高峰水平,7h后开始下降;免疫荧光可见15 min NF-κB激活-核转运程度较高;ELISA结果显示,10h之后TNF-α的水平显著高于野生株和回复株.使用TLR2阻断剂和PI-3K抑制剂,三菌株通路活化程度均明显受抑制.[结论]荚膜唾液酸可抑制宿主免疫细胞TLR2-AKT-NF-κB信号通路的激活,藉此参与细菌逃避宿主的免疫防御作用.     

雄激素受体在大鼠睾丸中转录模式的分析

目的：研究Ar（雄激素受体）基因在大鼠睾丸组织中的转录模式。方法：取刚出生的雄性Wistar大鼠,于出生2~65日的不同时间点,脊椎脱臼处死3只不同窝别的幼鼠,提取睾丸组织总mRNA;将mRNA反转录成cDNA,随后采用Real-time PCR方法检测大鼠睾丸组织中Ar mRNA的转录情况。结果：Ar mRNA表达在出生后第2天开始缓慢上升到第16天达到最高值,第16天到第30天迅速下降,第31天出现一个小高峰后到第65天持续缓慢下降。结论：Ar基因在大鼠早期发育睾丸组织中表达量逐渐升高,可能与精原干细胞的增殖及初级精母细胞的发生相关。     

哈萨克族成人腰稚间盘高度的放射学测量及其临床意义

目的：为人工椎间盘的设计提供形态学依据。方法：对56例哈萨克族成人腰椎（L）间盘高度进行放射学测量。结果：56例哈萨克族L1-2椎间盘高度男女性之间差异无统计学意义（P〉0．05）,L3-5椎间盘高度男、女性之间差异有统计学意L（P〈0．005-0．001）;哈萨克族与汉族腰椎间盘高度之间差异均有统计学意义（P〈0．005）。结论：哈萨克族腰椎间盘高度均大于汉族,临床上可通过对腰椎间盘间高度的测量,为人工椎间盘假体设计提供参数．     

Immunolocalization of type X collagen in human lumbar intervertebral discs during ageing and degeneration

 Type X collagen has so far not been reported to occur in human intervertebral discs. The objective of this study was therefore to investigate the occurrence of type X collagen in human lumbar intervertebral discs during ageing and degeneration. Ninety intervertebral discs with adjacent endplates were excised in toto from individuals (0–86 years) without known spinal disease and were processed for routine decalcified histology. Appropriate slices of each disc were processed for immunohistochemistry using a type-spec ific, monoclonal antibody raised against human type X collagen. Each intervertebral disc was examined for macroscopic and histomorphological features of disc degeneration. Immunohistochemically, a positive specific type X staining was observed in the hypertrophic zone of the growth plate and only in the interstitial matrix of juvenile (<2 years) nucleus pulposus. In adult discs, type X collagen could be localized in conjunction with advanced disc degeneration and first occurred in the disc matrix (i.e., pericellular region) of a 47-year-old specimen. Positive type X staining of the disc matrix was more frequently found in senile (>70 years) discs with end stages of disc degeneration. This study provides the first evidence for the occurrence of type X collagen in human lumbar intervertebral discs and it appears that type X collagen is re-expressed in late stages of disc degeneration. Accepted: 24 April 1997     

Artificial Cervical Vertebra and Intervertebral Complex Replacement through the Anterior Approach in Animal Model: A Biomechanical and In Vivo Evaluation of a Successful Goat Model

This was an in vitro and in vivo study to develop a novel artificial cervical vertebra and intervertebral complex (ACVC) joint in a goat model to provide a new method for treating degenerative disc disease in the cervical spine. The objectives of this study were to test the safety, validity, and effectiveness of ACVC by goat model and to provide preclinical data for a clinical trial in humans in future. We designed the ACVC based on the radiological and anatomical data on goat and human cervical spines, established an animal model by implanting the ACVC into goat cervical spines in vitro prior to in vivo implantation through the anterior approach, and evaluated clinical, radiological, biomechanical parameters after implantation. The X-ray radiological data revealed similarities between goat and human intervertebral angles at the levels of C2-3, C3-4, and C4-5, and between goat and human lordosis angles at the levels of C3-4 and C4-5. In the in vivo implantation, the goats successfully endured the entire experimental procedure and recovered well after the surgery. The radiological results showed that there was no dislocation of the ACVC and that the ACVC successfully restored the intervertebral disc height after the surgery. The biomechanical data showed that there was no significant difference in range of motion (ROM) or neural zone (NZ) between the control group and the ACVC group in flexion-extension and lateral bending before or after the fatigue test. The ROM and NZ of the ACVC group were greater than those of the control group for rotation. In conclusion, the goat provides an excellent animal model for the biomechanical study of the cervical spine. The ACVC is able to provide instant stability after surgery and to preserve normal motion in the cervical spine.     

Changes in intervertebral disc morphology persist 5 mo after 21-day bed rest

As part of the nutrition-countermeasures (NUC) study in Cologne, Germany in 2010, seven healthy male subjects underwent 21 days of head-down tilt bed rest and returned 153 days later to undergo a second bout of 21-day bed rest. As part of this model, we aimed to examine the recovery of the lumbar intervertebral discs and muscle cross-sectional area (CSA) after bed rest using magnetic resonance imaging and conduct a pilot study on the effects of bed rest in lumbar muscle activation, as measured by signal intensity changes in T(2)-weighted images after a standardized isometric spinal extension loading task. The changes in intervertebral disc volume, anterior and posterior disc height, and intervertebral length seen after bed rest did not return to prebed-rest values 153 days later. While recovery of muscle CSA occurred after bed rest, increases (P ≤ 0.016) in multifidus, psoas, and quadratus lumborum muscle CSA were seen 153 days after bed rest. A trend was seen for greater activation of the erector spinae and multifidus muscles in the standardized loading task after bed rest. Greater reductions of multifidus and psoas CSA muscle and greater increases in multifidus signal intensity with loading were associated with incidence of low back pain in the first 28 days after bed rest (P ≤ 0.044). The current study contributes to our understanding of the recovery of the lumbar spine after 21-day bed rest, and the main finding was that a decrease in spinal extensor muscle CSA recovers within 5 mo after bed rest but that changes in the intervertebral discs persist.     

Morphometric analysis of vertebrae and intervertebral discs as a basis of disc replacement

In this study measurements were obtained from 359 vertebrae and 215 intervertebral discs in an attempt to classify discs by their size. At the cervical and thoracic levels, this attempt was unproductive because of extensive variations. In the lumbar spine, discs were allocated to six size-matched groups and to two height-matched groups. The breadth of marginal rims were also measured with a view to provide surgeons operating on the spine with precise data on disc morphology to facilitate disc replacement.     

Growth related increase in rat intervertebral disc size: a quantitative radiographic and histologic comparison

Methods were developed for measuring the width of lumbar intervertebral discs in Sprague-Dawley rats. Rates of growth of the L4-5 and L5-6 discs in a rostral-caudal direction in an approximately midsagittal plane were reported from analysis of radiographic and histologic data. The radiographic data correlated with histologic data (p less than 0.001). It was concluded that the L4-5 and L5-6 intervertebral discs continue to increase in size throughout the first 18 months of life of the rat, well past the age of rapid gain in body weight.     

Effect of in vitro and in vivo culture on embryo development from prepubertal goat IVM-IVF oocytes

The aim of this study was to analyze different culture systems on embryo development of prepubertal goat oocytes. We compare (i) the effect of the age of donor (goat) of oocytes on in vitro maturation, fertilization and subsequent embryo development, (ii) the effect of the origin of oviduct cells from coculture of prepubertal goat embryo development, and (iii) the effect of in vivo culture in rabbit oviducts for 1, 2 and 3 days on the development of prepubertal goat embryos produced in vitro. In Experiment 1, at 24 h post-insemination (hpi), oocytes from adult goats were allocated in TCM199 with oviduct cells from adult goats, and oocytes from prepubertal goats were randomly placed in drops with oviduct epithelial cells from adult (aOEC) or prepubertal (pOEC) goats. Cleavage rate and embryo development were evaluated at 48 hpi and after 7 days coculture, respectively. In Experiment 2, at 24 hpi, prepubertal oocytes were allocated in TCM 199 with pOEC. At 40-42 hpi, a group of embryos remained in the coculture (control group), and the rest were transferred to rabbit oviducts (three rabbits for replicate) for culturing in vivo for 24, 48 and 72 h. After these in vivo cultures, embryos were recovered, evaluated and placed in TCM199 with pOEC until Day 8 post-insemination. The maturation, fertilization and blastocyst rates did not differ significantly between oocytes obtained from adult and prepubertal goats. The percentage of blastocysts obtained from prepubertal goat embryos cocultured with aOEC or pOEC was also similar (12.1% versus 12.2%). The transfer of prepubertal goat embryos to rabbit oviducts for 1, 2 and 3 days did not improve the blastocyst rate compared to the control group (9.7, 10.9, 4.1 and 11.5%, respectively). In conclusion, in our conditions, there were no significant differences in embryo development between oocytes obtained from prepubertal and adult goats, and the embryo development from prepubertal goat oocytes were similar in the different culture systems compared.     

Restoration of Insular Ecosystems: Control of Feral Goats on Aldabra Atoll, Republic of Seychelles

The control of feral goats (Capra hircus) was studied on Aldabra Atoll, Republic of Seychelles, from October 1993 to May 1994 and November 1994 to May 1995. A total of 832 goats was killed on Aldabra using the Judas goat technique and traditional hunting methods. Twenty-eight goats equipped with radio transmitters (Judas goats) were used to locate and kill other feral goats. The remnant goat populations on Ile Picard (n=13) and Ile Malabar (n=19) were eradicated during the first season. On Grande Terre, a total of 798 (374 M : 424 F) goats were killed. Mean group size was 3.2 with a range of 1–20. Judas goat hunting became increasingly important over time with 18.0% (n=85) of goats killed in the presence of Judas goats in the first season compared to 42.3% (n=126) of goats killed during the second season. The overall kill rate for the project was almost 2 times greater for Judas goat hunting (0.61 goats killed/h) than traditional hunting (0.32 goats killed/h). The home range size of each Judas goat and the number of goats killed in association with it was significantly related. Using the Leslie–Davis removal method of population estimation, 84 goats were estimated to remain on all of Grande Terre at the end of thebreak project. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effects of buck teasing on synchronization of estrus in goats after intravulvo-submucosal administration of cloprostenol

A study was conducted on 35 East African shorthorned female goats to determine if a combination of buck teasing and low doses of a prostaglandin (PGF(2) alpha) analogue, cloprostenol, given intravulvo-submucosally (i.v.s.m.) would be suitable for synchronization of estrus. Goats were allotted, with the onset of estrus, to seven groups (n = 5 goats per group). Five of the seven groups received varying doses of cloprostenol: Group 1 (125 mug cloprostenol i.m. per goat); Group 2 (62.5 mug cloprostenol i.v.s.m. per goat); Group 3 (62.5 mug cloprostenol i.v.s.m. per goat plus buck teasing); Group 4 (31.25 mug cloprostenol i.v.s.m. per goat); Group 5 (31.25 mug cloprostenol i.v.s.m. per goat plus buck teasing); Group 6 (buck teasing); Group 7, (2 ml physiological saline i.v.s.m. per goat, control group). Plasma progesterone concentration was measured on day of treatment and for 6 d thereafter. All goats in groups 1, 2, 3 and 5 exhibited estrus within 68 h. Thus, the number of goats receiving low doses of PG-cloprostenol intravulvo-submucosally observed in estrus increased (P < 0.05) with exposure to bucks. Exhibition of behavioral signs of estrus was maximal between 2 and 20 h after onset of signs of estrus. The exposure of females to males prior to intrauterine penetration was an advantage because copious mucus eased penetration.