大鼠胰岛分离与纯化的实验研究

为探讨胰岛移植物的制备方法，采用胰管注射胶原酶、胰腺静止消化方法，分离成年大鼠胰岛，葡聚糖离心纯化。纯化后胰岛收获量为６１０～８２０个／胰腺，纯度达９２％；胰岛形态结构完整，内分泌细胞超微结构保持良好；对葡萄糖刺激反应胰岛素释放量是基础分泌水平的８倍；异种移植可逆转实验性糖尿病小鼠的高血糖达１周。结果表明：胰管注射胶原酶胰腺静止消化可获得高产量、高纯度、功能良好的胰岛。为临床胰岛移植开辟了新的途径。     

大鼠胰岛细胞原代培养方法比较研究

目的：比较减碎消化法和原位灌注消化法在大鼠胰岛细胞原代培养中的优缺点,探讨一种简单易行的大鼠胰岛细胞培养方法。方法：雄性SD大鼠30只随机分成减碎组（CC组,n=15）,胰腺剪为小组织块后,Ⅴ型胶原酶逐级消化分离;原位灌注组（YY组,n=15）,采用胆总管逆行灌注Ⅴ型胶原酶分离胰岛。双硫腙（DTZ）染色检测胰岛纯度,葡萄糖刺激胰岛素释放试验检测胰岛功能。结果：2组获得的胰岛对于葡萄糖刺激均具有良好的分泌活性（P〈0.01）;YY组比CC组获得的胰岛数量多（P〈0.01）,形态较好,等量胰岛的胰岛素释放量亦多（P〈0.05）。结论：胆总管原位灌注Ⅴ型胶原酶消化可以获取数量多,活性和纯度较好的胰岛。     

CD4+T细胞抗体标记超顺磁性氧化铁MR成像诊断异种胰岛移植免疫排斥反应的实验研究

目的 探讨利用抗人分化抗原簇(CD)4+T细胞抗体标记的纳米免疫超顺磁性氧化铁(SPIO)作为MRI生物探针,诊断胰岛细胞移植后免疫排斥反应的可行性.方法 27只BALB/C裸鼠,每只左肾被膜下移植分离纯化后的猪胰岛细胞2000个,3周后MRI可观察到肾包膜下移植物大小,然后采用数字表法随机将实验动物分为实验组(21只)和对照组(6只).将人T淋巴细胞注入实验组鼠腹腔内重建人的T淋巴细胞免疫系统,其中20只重建成功.实验组动物免疫系统重建前及重建后3、7和14 d,经尾静脉注射纳米免疫SPIO,30 min后行MRI T2WI,观察移植物的MRI信号改变.MRI结束后立即安乐死方法牺牲动物,取下左肾行移植物病理组织检查;对照组动物胰岛细胞移植后,不进行免疫处理,与实验组动物相同时间点给予相同检查.并以病理检查为金标准,用临床诊断一致性检验计算MRI对异种胰岛细胞移植后免疫排斥反应诊断的灵敏度、特异性、Youden指数和符合率.结果 2组动物在移植胰岛细胞3周后MRI可显示移植物.实验组BALB/C裸鼠,重建免疫系统前及重建成功后3 d注入纳米免疫SPIO,MRI移植物局部未见异常信号;免疫系统重建成功后7 d,MRI显示移植物对应区域信号减低,苏木精.伊红及CD4+T免疫组织化学染色显示移植物局部大量淋巴细胞浸润,提示免疫排斥反应发生.对照组动物移植物所有MRI均未见异常;病理组织学检查移植物局部未见炎性细胞浸润.MRI对免疫反应检测的灵敏度、特异度、Youden指数和符合率分别为:(72.96±0.24)%、100%、0.73±0.24、(88.46±0.13)%(Kappa值为0.76).结论 通过抗人CD4+T细胞抗体标记的纳米免疫SPIO牛物探针MRI,可以检查异种胰岛细胞移植后免疫排斥反应,并能早期、实时、无创性的对移植受体免疫排斥反应进行诊断.     

大鼠肝脏原代Kupffer细胞的分离培养

目的 建立稳定而有效的大鼠肝脏Kupffer细胞分离、纯化的实验方法.方法 用无Ca2+灌注液和浓度为0.05%的胶原酶灌注液进行肝脏的灌注及消化,用Percoll密度梯度离心法进行Kupffer细胞的分离、纯化,并鉴定大鼠肝脏原代Kupffer细胞.结果 分离培养后得到纯化的原代Kupffer细胞,生长状态良好,DAB染色实验证实经大鼠肝脏灌流得到稳定生长的原代Kupffer细胞.结论 通过大鼠肝脏灌注建立了有效的大鼠肝脏Kupffer细胞分离、纯化的实验方法.     

模拟微重力条件下大鼠胰岛培养及体内移植的实验研究

目的研究经模拟微重力条件培养的胰岛是否能够降低胰岛移植过程中引发的免疫排斥反应，进而延长胰岛的体内存活时间。方法分离新鲜胰岛，应用培养皿对胰岛进行静态培养，同时应用旋转式生物反应器对胰岛进行三维立体培养。应用吖啶橙一碘化丙啶（AO-PI）染色和葡萄糖刺激实验对3种条件下培养得到的胰岛进行生物学活性研究。2000当量胰岛在旋转式生物反应器中培养5d后，将其移植入经链脲佐菌素（STZ）处理的糖尿病模型SD大鼠。肾被膜下作为实验组，以新鲜分离的胰岛和静态培养5d的胰岛移植入经STZ处理的糖尿病模型SD大鼠肾被膜下作为对照组。在不同时间点上，检测各组SD大鼠血糖变化情况；对各移植组中大鼠进行糖耐受实验。切除。肾被膜下的移植物组织作为标本，进行苏木精一伊红（HE）染色及胰岛素组织化学染色检测。结果体外检测结果表明，与新鲜分离的胰岛和静态培养的胰岛相比，微重力条件下培养的胰岛仍保持良好的胰岛素合成及分泌功能。体内实验表明，微重力条件下培养的胰岛体内移植组的血糖保持正常的时间明显高于接受静态培养的胰岛和新鲜分离的胰岛移植组。将微重力培养条件下胰岛移植大鼠后，取。肾被膜下移植区组织标本进行HE染色，未见明显淋巴细胞浸润，胰岛素免疫组织化学均可见阳性细胞。而新分离的胰岛以及静态培养的胰岛移植后，大鼠。肾被膜下移植物组织标本可见淋巴细胞浸润，移植物的厚度明显变小。结论胰岛在微重力条件下培养可降低移植引起的免疫排斥反应，进而延长移植物体内存活时间。     

不同活性的胶原酶对大鼠心肌细胞分离的影响

目的 研究不同活性的胶原酶对大鼠心肌细胞存活率的影响.方法 用4种不同活性的胶原酶以常规酶解法分离成年大鼠心肌细胞,观察分离即刻(D时点)、复钙60min后(E时点)和电刺激10min后(F时点)心肌细胞的存活率,采用IonOptix单细胞动缘检测系统同步检测心肌细胞收缩幅度(ph)、ph占单个心肌细胞长度(bl)的百分比(以ph/bl表示)、心肌细胞收缩速率( dL/dt)和心肌细胞舒张速率(-dL/dt)等指标.结果 观察D、E、F时点心肌细胞存活率,结果表明299U/mg胶原酶的分离效果286U/mg、203U/mg和403U/mg的胶原酶(P＜0.05).D时点4组心肌细胞的存活率没有差别,E、F时点时存活率降低.在心肌细胞收缩/舒张功能的检测中,299U/mg的胶原酶分离效果最佳,203、286、299U/mg和403U/mg组的ph分别是0.13±0.05,0.14±0.06、0.14±0.06μm和0.11±0.05μm;ph/bl分别是8.8%±2.4%、8.9%±2.3%、9.2%±3.5%和7.5%±2.2%; dL/dt分别是1.8±0.6、1.9±0.6,2.0±0.7μm/s和1.7±0.5μm/s,-dL/dt分别是1.9±0.6、2.0±0.7、2.1±0.6μm/s和1.7±0.6μm/s.结论 采用一定范围(203～299U/mg)的胶原酶对心肌细胞进行分离,酶活性越大,消化时间越短,心肌细胞存活率高、收缩/舒张功能好,其中以299U/mg活性的胶原酶效果最好.活性偏高或偏低的胶原酶分离心肌细胞的存活率和收缩/舒张功能均明显降低.     

Bcl-2基因转染胰岛细胞移植的实验研究

目的探讨Bcl-2基因转染胰岛细胞同种移植治疗糖尿病大鼠的效果。方法腺病毒介导胰岛细胞的Bcl-2基因转染,以表达Bcl-2的胰岛细胞经门静脉肝内移植治疗链脲菌素诱导的糖尿病大鼠。通过观察受体大鼠的血糖变化和组织学改变来了解排斥反应和移植物的功能。结果胰岛细胞植入后48h内,基因转染组和未转染组糖尿病大鼠血糖均有明显下降。与未转染组比较,基因转染组大鼠移植物存活有效时间明显延长(16·4±4·3天vs6·8±2·2天,P<0·05),移植部位可见到结构和功能完好的胰岛细胞,局部炎性反应也明显减轻。结论胰岛细胞的Bcl-2基因转染对移植物的存活和功能有保护作用。     

帕金森病大鼠模型细胞移植的PET在体显像研究

目的 探讨PET在体显示移植于帕金森病(PD)大鼠模型脑内的视网膜色素上皮(RPE)细胞分化结果及治疗效果.方法 将RPE细胞移植于治疗组PD模型大鼠(12只,其中1只用于组织化学染色)纹状体内,对照组(11只)移植相同体积的生理盐水.移植前后分别进行11C-雷氯必利(raclopride)、"C-N-甲基-213-甲基酯-3B-(4-F-苯基)托烷(a-CFT)PET显像,并用感兴趣区(ROI)法分析2组移植前后显像变化.移植后观察大鼠行为学改善情况及分析细胞免疫组织化学染色结果.结果 移植前PET显像示大鼠模型损伤侧纹状体11C-raclopride摄取增高,而11C-B-CFF摄取下降.移植RPE细胞后,治疗组大鼠损伤侧纹状体11C-raclopride摄取降低,11C-B-CFT摄取上升.ROI分析纹状体/小脑放射性比值11C-raclopride由1.870±0.465(移植前)降至1.601±0.257(移植后);11C-B.CFT由1.827±0.347(移植前)升至2.336±0.326(移植后).大鼠旋转行为有所改善.免疫组织化学染色显示移植的RPE细胞可分化为多巴胺神经元.结论 PET显像可为监测移植细胞存活、分化及转归提供一种在体、无创、可视化评价工具.     

负载猪源性CTLA4Ig的猪胰岛细胞阻断直接识别途径在异种移植中的作用

目的：探讨负载供体源性CTLA4Ig的胰岛细胞通过阻断直接识别途径,诱导异种胰岛细胞抑制免疫耐受的可行性及有效性。方法：使用本实验小组前期构建的携带供体源性（猪）CTLA4-Ig的腺病毒载体（Adv-pCT-LA4-Ig）转染猪胰岛细胞,植入糖尿病大鼠肾包膜下,建立猪-大鼠异种胰岛移植模型,监测移植术后大鼠血糖变化及IL-4、γ-IFN的水平变化,检测移植物中pCTLA4-Ig、猪源性胰岛素（p-Insulin）表达情况。结果：1.大鼠血糖在移植术后即开始明显下降,于第3 d左右降至正常,三组大鼠血糖分别在移植术后6、7、35 d左右开始升高;2.三组大鼠术后胰岛平均存活时间为7.43±1.72 d、7.22±1.72 d、34.50±4.14 d,实验组胰岛细胞存活时间较对照组明显延长（P〈0.01）;3.细胞因子变化：（1）γ-IFN：对照组在移植术后第1 d开始轻度升高,于第3 d后开始明显升高,第7 d达到高峰,此后缓慢升高;实验组在移植术后第1～28 d波动较小,同术前相比无明显变化,在移植后第28 d开始上升,第35 d开始急剧上升;（2）IL-4：实验组在移植术后第1 d开始下降,第7 d达到谷值;对照组在移植术后第3～5 d轻微上升,在移植后第28 d开始下降至移植术前水平,第35 d开始急剧下降;4.病理学检查：实验组移植物无明显破坏,炎细胞浸润不明显;免疫组化可见大量pCTLA4-Ig、p-Insulin表达。结论：转染供体源性CTLA4-Ig的胰岛细胞通过阻断直接识别途径,可诱导异种胰岛细胞产生较长时间的免疫耐受。     

少突胶质前体细胞移植治疗对大鼠脊髓损伤轴突髓鞘化的影响

目的 探讨少突胶质前体细胞(oligodendrocyte precursor cells,OPCs)移植治疗对大鼠脊髓损伤轴突髓鞘化的影响.方法 采用Allen法制作大鼠T10脊髓损伤模型,亚急性期OPCs移植治疗.通过HE染色、免疫组化染色、髓鞘染色及透射电镜观察等方法,研究OPCs移植治疗对大鼠脊髓损伤轴突髓鞘化的影响.结果 移植术后8周时大鼠损伤脊髓内检测到移植细胞分布,HE染色显示OPCs移植组大鼠脊髓组织结构较对照组改善,勒克司坚牢蓝(LFB)髓鞘染色表明OPCs移植组大鼠脊髓内髓鞘含量(7 802.42±1 085.58)明显高于对照组(5 055.98±916.74)(P＜0.01),OPCs移植组大鼠脊髓组织内髓鞘碱性蛋白(MBP)表达水平(8 544.44±812.78)较对照组(5 243.83±808.27)显著增高(P＜0.01),透射电镜示OPCs移植组大鼠脊髓组织髓鞘化超微结构改善也较对照组明显.结论 OPCs移植治疗有助于改善大鼠脊髓损伤轴突髓鞘化情况.     

Knee injury and Osteoarthritis Outcome Score (KOOS) - validation of a Swedish version

The Knee injury and Osteoarthritis Outcome Score (KOOS) is a self-administered instrument measuring outcome after knee injury at impairment, disability, and handicap level in five subscales. Reliability, validity, and responsiveness of a Swedish version was assessed in 142 patients who underwent arthroscopy because of injury to the menisci, anterior cruciate ligament, or cartilage of the knee. The clinimetric properties were found to be good and comparable to the American version of the KOOS. Comparison to the Short Form-36 and the Lysholm knee scoring scale revealed expected correlations and construct validity. Item by item, symptoms and functional limitations were compared between diagnostic groups. High responsiveness was found three months after arthroscopic partial meniscectomy for all subscales but Activities of Daily Living.     

The acutely limping child

Acute limping may be the result of multiple pathologies in children. The differential diagnosis varies based on the age of the child. Irrespective of age, the initial imaging work-up includes AP and frog leg radiographs of the pelvis and ultrasound; MRI may sometimes be helpful. In children less than 3 years, infections and trauma are most frequent. MRI is the imaging modality of choice when osteomyelitis is clinically suspected. Between the ages of 3 and 10 years, transient synovitis of the hip and Legg-Calvé-Perthes disease are main considerations but infection, inflammation and focal bony lesions are also considered. In children over 10 years, slipped capital femoral epiphysis also is considered.     

Do Balance Board Training Programs Reduce the Risk of Ankle Sprains in Athletes?

Introduction Ankle sprains are the most common musculo-skeletal injury that occurs in athletes,particularly in sports that require jumping and landing on one foot such as soccer,and basketball(1-4).These injuries often result in significant time loss from participation,long-term disability,and have a major impact on health care costs and resources(5-8).     

High Intensity Interval Training:New Insights

KEY POINTS ·High-intensity interval training(HIT)is characterized by repeated sessions of relatively brief，intermittent exercise.often performed with an“a11 out”effort or at an intensity close to that which elicits peak oxygen uptake(i.e.，≥90%of VO2 peak).     

Endovascular management of carotid-cavernous fistulas

Objective To investigate endovascular treatment of traumatic direct carotid-cavernous fistulas （CCF） and their complications such as pseudoaneurysms. Methods： Over a five-year period, 22 patients with traumatic direct CCFs were treated endovascularly in our institution. Thirteen patients were treated once with the result of CCF occluded, 8 twice and 1 three times. Treatment modalities included balloon occlusion of the CCF, sacrifice of the ipsilateral internal carotid artery with detachable balloon, coll embolization of the cavernous sinus and secondary pseudoaneurysms, and covered-stem management of the pseudoaneurysms. Results All the direct CCFs were successfully managed endovascularly. Four patients developed a pseudoaneurysm after the occlusion of the CCF with an incidence of pseudoaneurysm formation of 18.2% （4/22）. A total number of 8 patients experienced permanent occlusion of the ICA with a rate of ICA occlusion reaching 36.4% （8/22）. Followed up through telephone consultation from 6 months to 5 years, all did well with no recurrence of CCF symptoms and signs. Conclusion Traumatic direct CCFs can be successfully managed with endovascular means. The pseudoaneurysms secondary to the occlusion of the CCFs can be occluded with stent-assisted coiling and implantation of covered stents.     

Developmental validation of the PowerPlex(®) ESI 16 and PowerPlex(®) ESI 17 Systems: STR multiplexes for the new European standard

In response to the ENFSI and EDNAP groups’ call for new STR multiplexes for Europe, Promega^® developed a suite of four new DNA profiling kits. This paper describes the developmental validation study performed on the PowerPlex^® ESI 16 (European Standard Investigator 16) and the PowerPlex^® ESI 17 Systems. The PowerPlex^® ESI 16 System combines the 11 loci compatible with the UK National DNA Database^®, contained within the AmpFlSTR^® SGM Plus^® PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to reduce the amplicon size of the loci found in the AmpFlSTR^® SGM Plus^® kit. This design facilitates increased robustness and amplification success for the loci used in the national DNA databases created in many countries, when analyzing degraded DNA samples. The PowerPlex^® ESI 17 System amplifies the same loci as the PowerPlex^® ESI 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpFlSTR^® SGM Plus^® kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex^® ESI 16 and ESI 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5 pg of a fully heterozygous single source DNA template. This high level of sensitivity was found to impact on mixture analyses, where 54–86% of unique minor contributor alleles were routinely observed in a 1:19 mixture ratio. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of data obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors.     

Bronchial stump fistula :treatment with covered retrievable hinged metallic stents-preliminary clinical experience

Objective To evaluate the preliminaily clinical efficacy and retrievability of a retrievable hinged covered metallic stent in the treatment of the bronchial stump fistula （BSF）. Methods Between April 2003 and March 2005, 8 patients with bronchial stump fistula after pneumonectomy or lobectomy were treated with two types （A and B） of retrievable hinged covered metallic stents. Type A stent was placed in 6 patients and type B in 2 under fluoroscopic guidance. The stent was removed with a retrieval set when BSF was healed or complications occurred. Results Stent placement in the bronchial tree was technically successful in all patients, without procedure-related complications. Immediate closure of the BSF was achieved in all patients after the procedure. Stents were removed from all patients but one. Removal of the stents was difficult in two patients due to tissue hyperplasia. Patients were followed up for 6 - 21 months. Placement of the stents remained stable in all patients except one due to severe cough. Permanent closure of BSF was achieved in 7 （87.5%） of 8 patients. Conclusion Use of a retrievable hinged covered expandable metallic stent is a simple, safe, and effective procedure for closure of the BSF. Retrieval of the stent seems to be feasible. （J Intervent Radiol, 2007, 16： 253-257）     

Prevalence and severity of hip and groin pain in sub‐elite male football: a cross‐sectional cohort study of 695 players

The purpose of this study was twofold: (a) to investigate the prevalence of hip and groin pain in sub‐elite male adult football in Denmark and (b) to explore the association between prevalence and duration of hip and groin pain in the previous season with the Copenhagen Hip and Groin Outcome Score (HAGOS) in the beginning of the new season. In total 695 respondents from 40 teams (Division 1–4) were included. Players completed in the beginning of the new season (July–Sept 2011) a self‐reported paper questionnaire on hip and/or groin pain during the previous season and HAGOS. In total 49% (95% CI: 45–52%) reported hip and/or groin pain during the previous season. Of these, 31% (95% CI: 26–36%) reported pain for >6 weeks. Players with the longest duration of pain during the previous season had the lowest HAGOS scores, when assessed at the beginning of the new season, P < 0.001. This study documents that half of sub‐elite male adult football players report pain in the hip and/or groin during a football season. The football players with the longest duration of pain in previous season displayed the lowest HAGOS scores in the beginning of the new season.