大鼠肺泡Ⅱ型上皮细胞的分离、纯化、原代培养及鉴定

目的建立大鼠肺泡Ⅱ型上皮细胞（alveolar typeⅡepithelial cell,AT-Ⅱ）分离、纯化、原代培养及鉴定方法。方法取Wistar大鼠肺脏,采用胰蛋白酶消化法分离AT-Ⅱ,通过差速离心法、红细胞裂解法、贴壁选择法、免疫黏附选择法纯化AT-Ⅱ,倒置相差显微镜下观察细胞形态,鞣酸染色法进行细胞鉴定。结果与结论AT-Ⅱ呈单个或岛状生长,细胞呈圆形或椭圆形,细胞浆内有大量的反差明显的细胞小颗粒,细胞核明显。鞣酸染色法鉴定阳性。通过改进的酶消化法获得的AT-Ⅱ生长状态良好,纯度高,符合体外实验要求。     

人类小肠上皮细胞原代培养方法探讨

 介绍一种人类小肠上皮细胞原代培养方法.采用16～24周引产胎儿小肠,比较3种小肠上皮细胞分离方法,探讨最佳培养条件.结果发现应用胶原酶/中性蛋白酶消化可获满意的上皮细胞分离效果,细胞贴壁良好.培养10～12d细胞达到融合,并保持对碱性磷酸酶的表达.提示小肠上皮细胞繁殖十分依赖于培养基和底物质量.     

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.     

子宫内膜异位症异位内膜细胞原代培养方法的改良

目的：建立一种简易的人异位子宫内膜细胞体外原代培养方法。方法：通过胰蛋白酶消化、贴壁纯化等技术,分离及培养10例子宫内膜异位症患者的异位子宫内膜细胞。在光学显微镜下观察细胞形态,以免疫细胞化学法鉴定细胞类型。结果：8份标本获得成功,培养的异位内膜细胞均有腺上皮细胞和间质细胞两种形态细胞。膜上皮细胞和基质细胞分别经角蛋白单抗和波形蛋白单抗免疫组化染色为阳性,腺上皮细胞平均生长时间为5周,间质细胞平均生长时间为14周。结论：成功建立了子宫内膜异位症异位内膜细胞体外原代培养方法,该改良培养法经济、简单、高效。     

改良酶消化法在人脑胶质瘤细胞快速原代培养中的应用

目的 建立一种人脑胶质瘤细胞原代培养的改良酶消化法.方法 基于文献并结合实际工作中经验,改良既往文献报道的酶消化法,对32例不同级别的胶质瘤进行消化后行原代细胞培养,通过倒置相差显微镜观察肿瘤细胞的形态学特点,传代时采用差速贴壁法对原代细胞进行纯化.采用细胞免疫荧光法对原代细胞进行鉴定,通过细胞增殖实验(CCK-8法)绘制生长曲线,研究体外培养细胞的增殖情况.结果 采用改良的酶消化法成功培养28例,失败4例,成功率为87.5％.培养成功的细胞贴壁生长,细胞状态稳定,形态各异,生长状态良好,并可传代.细胞免疫荧光检测显示胶质纤维酸性蛋白(GFAP)表达阳性,证明培养的细胞为胶质瘤细胞;细胞增殖实验显示细胞在体外增殖活跃,肿瘤病理级别越高,细胞增殖能力越强.结论 改良的酶消化法用于人脑胶质瘤细胞原代培养具有简单、高效、成功率高等优点.     

改良法体外培养大鼠成肌细胞的实验研究

目的:探索一种新型体外分离培养新生大鼠骨骼肌成肌细胞的途径.方法:取新生Wistar大鼠的四肢骨骼肌适量,采用混合酶一步消化分离获取成肌细胞,将差速贴壁法和胰酶消化法相结合进行纯化,观察分离纯化成肌细胞的形态学特点、生长状况,绘制细胞生长曲线,利用扫描电镜观察成肌细胞表面结构并进行结蛋白免疫细胞化学特异性鉴定.结果:采用改良方法分离成肌细胞产量高、耗时短;在培养过程中双重纯化获取的细胞生长状态良好,接种后3～5 d增殖达高峰;在扫描电镜下可观察到成肌细胞膜表面有微绒毛突起;体外培养的成肌细胞中94%的细胞胞质呈结蛋白抗体染色强阳性.结论:改良方法分离纯化大鼠骨骼肌成肌细胞效果理想,是成肌细胞体外培养的有效途径之一.     

肌卫星细胞体外培养及其对胰岛素样生长因子Ⅰ刺激的反应

目的原代分离、纯化、培养大鼠肌卫星细胞，观察体外培养肌卫星细胞的增殖与分化特性及对类胰岛素样生长因子Ⅰ（IGF-Ⅰ）刺激的反应。方法选用Wistar大鼠，切取双下肢及背部肌肉，两步酶消化法分离卫星细胞，不连续密度梯度离心法纯化后体外培养，观察其生长与分化特性，绘制生长曲线及融合率曲线，MTT法测定卫星细胞对不同浓度IGF-Ⅰ刺激的反应，并对其生长分数及融合率给予观察。结果此方法可以获得足量、纯度较高的肌卫星细胞。结论适宜浓度（100ng／ml）的IGF-Ⅰ对肌卫星细胞有较强的促增殖及促分化作用。     

大鼠肺微血管内皮细胞原代培养方法的改进

目的：改进大鼠肺微血管内皮细胞（pulmonary microvascular endothelial cells,PMVECs）原代培养方法,以获取纯化的大鼠肺微血管内皮细胞。方法：应用Wistar大鼠,采用改进的组织块贴壁法分离、培养肺微血管内皮细胞;光镜观察细胞形态;Ⅷ因子相关抗原免疫细胞化学法及植物凝集素BSI结合实验鉴定培养的PMVECs。结果：体外培养的原代PMVECs在光镜下呈短梭形或多角形,形成单层后呈铺路石样排列,但随着传代或培养条件的改变,细胞形态发生变化;Ⅷ因子相关抗原染色阴性,异植物凝集素BSI结合实验阳性。结论：通过改进的PMVECs分离、培养方法获得的细胞生长状态良好,纯度高,且能够稳定地传代培养。细胞形态观察结合免疫细胞化学法是目前较为理想的PMVECs鉴定方法。     

利用动物耳皮肤组织培养成纤维细胞的研究

对采集的动物耳部皮肤样品采用将组织块剪碎后直接贴附于细胞培养瓶底部培养的方法与酶消化法对动物体细胞进行原代培养 ,前者使得对不同品种绵羊、山羊、牛、北山羊的原代体细胞出现率及可传代率均达到 1 0 0 %。两种培养方法在成纤维细胞出现及可进行传代时间上有所差别。对于在体细胞传代培养中出现的上皮样细胞与成纤维样细胞混合生长的问题根据两者贴壁紧实程度的不同 ,用 0 .0 5%的胰酶—EDTA对其进行不同时间的消化 ,将两种不同类型的细胞进行分离和纯化。纯化的体细胞经数次传代培养后进行冷冻—解冻后仍具有正常的传代能力。对不同代次绵羊和牛的体细胞进行的核型分析表明 ,在体外培养至 2 0～ 30代 (绵羊胎儿细胞培养至 45代 )成纤维细胞的染色体倍性及数目未发现异常     

兔原代胸主动脉平滑肌细胞培养方法及不同浓度血清培养基对其增殖的研究

目的探讨体外分离培养兔原代胸主动脉平滑肌细胞（vascular smooth muscle cells, VSMCs）技术及在不同浓度血清培养基下生长情况。方法采用组织贴块法进行原代培养,分别用不同浓度血清培养基（10％、15％、20％）对原代VSMCs进行培养．胰酶消化法传代,用倒置显微镜对原代培养兔平滑肌细胞进行形态学观察并鉴定,MTT法测定增殖能力。结果体积分数20％血清的DMEM培养液培养的原代细胞生长出现明显对数期。培养5代的兔平滑肌细胞纯度达97％以上：镜下可见平滑肌细胞生长,免疫组化提示平滑肌细胞肌动蛋白阳性表达,细胞生长第4～5d内光密度值变化较明显。结论本法能有效提高兔动脉平滑肌细胞原代培养的成功率,为研究血管平滑肌细胞生物学行为提供了有效模型。     

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.