表皮干细胞体外分离与培养

探索和建立表皮干细胞体外分离与培养的技术方法。通过酶消化法获得幼鼠表皮 ,并制成单表皮细胞悬液 ,以表皮干细胞培养基 ,即无钙EMEM培养基 (添加 9%FBS ,0 0 5mmol/LCaCl2 ,4ng/mlEGF ,0 5ng/ml硫酸庆大霉素及 5 0 %成纤维细胞条件培养基 )置细胞培养箱内培养。成纤维细胞条件培养基为无钙EMEM培养基(添加 9%FBS、0 0 5mmol/LCaCl2 、0 5 μg/ml硫酸庆大霉素 )培养幼鼠皮肤块 4 8h后所得培养液。将 1×10 6/ml上述培养的角质形成细胞经TGG细胞消化液 37℃下振荡消化后 ,接种至鼠Ⅳ型胶原包被的培养瓶内 ,37℃下静置 10～ 15min,留用快速贴壁细胞继续培养 ,所得细胞分别以能否呈克隆状生长和透射电镜观察其超微结构及β1整合素、K19免疫细胞化学染色进行鉴定。结果显示 ,鼠Ⅳ型胶原包被的培养瓶内快速贴壁细胞经继续培养 ,可见细胞呈克隆状生长。电镜观察其超微结构示非成熟细胞特征 ,β1整合素、K19免疫细胞化学染色阳性。研究表明 ,表皮干细胞可在体外分离与培养 ,本实验方法为较好方法之一。     

人工表皮构建及其结构的研究

目的 :以角朊细胞作为种子细胞 ,构建人工表皮 ,检测培养的人工表皮的结构 ,为进一步进行动物移植试验奠定基础。方法 :(1)采用分散酶 (dispase)分离表皮细胞 ,以无血清培养法培养表皮细胞。 (2 )人工表皮构建成功后 ,分别进行HE染色及透射电镜观察。 (3)角蛋白免疫组织化学染色。结果 :经过 2～ 3周的培养 ,表皮细胞连接成片 ,呈现典型的“铺路石”状 ;继续培养 5～ 7d后 ,将细胞膜消化下来 ,HE染色显示由 2～ 4层细胞组成 ,电镜可以观察到桥粒及张力微丝束的结构 ;免疫组织化学染色显示培养细胞和细胞膜角蛋白染色阳性。结论 :该人工构建表皮与人正常表皮结构类似 ,可作为移植物进行深入研究     

乙酰胆碱对人表皮细胞冷存的影响基础研究

目的：探讨乙酰胆碱（ Ach）对人表皮细胞冷存活力的影响。方法：应用人表皮细胞的分离、培养技术,并进行 MTT检测、病理切片以及电镜检测等手段进行观察。结果：不同浓度的 Ach对冷存的表皮细胞均有一定的保存活力作用,其中以 10－ 8～ 10－ 10mol/L的浓度为最佳。结论：认为 Ach对冷存中的人表皮细胞有一定的保护作用,它可提高表皮细胞的保存活力。     

乙酰胆碱对人表皮细胞冷存的影响

目的：探讨乙酰胆碱（Ach）对人表皮细胞冷存活力的影响。方法：应用人表皮细胞的分离、培养技术，并进行MTT检测、病理切片以及电镜检测等手段进行观察。结果：不同浓度的Ach对冷存的表皮细胞均有一定的保存活力作用，其中以10^-1-10^-10mol/L的浓度为最佳。结论：认为Ach对冷存中的人表皮细胞有一定的保存作用，它可提高表皮细胞的保存活力。     

移植培养表皮细胞片时创面局部抗生素的合理选用

目的：优选出适于移植培养表皮细胞片（ＣＥＡ）的局部抗菌用药方案。方法：将培养的人表皮细胞置于含不同浓度抗生素的培养液中培养３天后，用噻唑蓝（ＭＴＴ）法测定其细胞生长率，同时采用定量铂环法和Ｋ－Ｂ纸片扩散法分别测定其在创面上和在体外的抗菌效果；并以此分别准备烧伤或肉芽创面，观察了对ＣＥＡ早期成活的影响。结果：对培养的人表皮细胞毒性作用为：碘伏（ＰＶＰ－Ⅰ）＞磺胺嘧啶银（ＡｇＳＤ）氟哌酸银（ＡｇＮ）＞丁胺卡那霉素（ＡＫ）／ＡＫ与ＰＲ复合抗生素（ＤＡＢ）＞氧哌嗪青霉素（ＰＲ）；ＡｇＮ和ＤＡＢ在临床使用浓度时抗菌效果较好，以此分别准备烧伤或肉芽创面，显著地提高了ＣＥＡ早期成活率。结论：烧伤创面外用１％ＡｇＮ、肉芽创面外用０．１％ＡＫ和０．２％ＰＲ的ＤＡＢ液湿敷、术后局部预防性应用０．０１％ＡＫ和０．０２％ＰＲ的ＤＡＢ液，可有效地提高ＣＥＡ的早期成活率。     

苯丙氨酸二肽类化合物073对2．2．15细胞分泌HBsAg和HBeAg的影响

目的观察苯丙氨酸二肽类化合物073对2．2．15细胞分泌HBsAg、HBeAg的影响。方法以2．2．15细胞为研究对象,通过细胞毒性实验确定苯丙氨酸二肽类化合物073对细胞的最大无毒浓度（TC0）,在最大无毒浓度以下将药物用培养液对倍稀释成50、25、12．5μg·mL^-1加入2．2．15细胞进行培养,每4d换同浓度药物培养液;分别收集第4、8天以及停药后4天的培养液上清,酶联免疫法（ELISA）测定HBsAg、HBeAg含量。结果苯丙氨酸二肽类化合物073最大无毒浓度为50μg·mL^-1,在最大无毒浓度时对HBsAg有明显的抑制作用,第8天时的抑制率为59．3％,半数抑制浓度（IC50）为22．9μg·mL^-1,治疗指数（TI）为3．1;但对HBeAg无明显抑制作用,第8天时的抑制率仅为25．5％。结论苯丙氨酸二肽类化合物073对2．2．15细胞HBsAg有显著的抑制作用。     

丝素蛋白牟表皮细胞生长的影响

目的：了解丝素的生物学性能和丝素对表皮细胞生长的影响。方法：试验组采用培养液ＤＭＥＭ加５％丝素蛋白原液,对照组采用培养液ＤＭＥＭ加５％生理盐水的方法,对表皮细胞培养结果进行比较观察;观察指标为表皮细胞形态细胞生长曲线、＾３Ｈ－胸腺嘧啶核苷测定等。结果：培养后３天试验组大量细胞贴壁生长,而对照组在培养５天才大量贴壁生长;试验组细胞数在５天明显增多,对照组在７天才迅速增加;＾３Ｈ－胸腺嘧啶核苷测定试验     

水芹总酚酸对2．2．15细胞分泌HBsAg与HBeAg的抑制作用

目的观察水芹总酚酸对2．2．15细胞分泌HBsAg、HBeAg的抑制作用。方法以2．2．15细胞为研究对象,通过细胞毒性实验确定水芹总酚酸对细胞的最大无毒浓度（TC0）,在最大无毒浓度以下将药物用培养液对倍稀释成500、250、125μg·mL^-1加入2．2．15细胞进行培养,每4d换同浓度药物培养液;分别收集第4、8天以及停药后4d的培养液,酶联免疫法（ELISA）测定HBsAg、HBeAg含量。结果水芹总酚酸最大无毒浓度为500μg·mL^-1,在最大无毒浓度时对HBsAg、HBeAg具有明显的抑制作用,第8天时的抑制率分别为59．33％和90％,半数抑制浓度（IC50）分别为484．17、379．89μg·mL^-1,治疗指数（TI）分别为3．158、4．025,其中对HBeAg的抑制作用优于HBsAg。结论水芹总酚酸对2．2．15细胞分泌的HBsAg、HBeAg有显著的抑制作用。     

丝素蛋白对表皮细胞生长的影响

目的：了解丝素的生物学性能和丝素对表皮细胞生长的影响。方法：试验组采用培养液ＤＭＥＭ 加５％ 丝素蛋白原液,对照组采用培养液ＤＭＥＭ 加５％ 生理盐水的方法,对表皮细胞培养结果进行比较观察;观察指标为表皮细胞形态、细胞生长曲线、３Ｈ－ 胸腺嘧啶核苷测定等。结果：培养后３ 天试验组大量细胞贴壁生长,而对照组在培养后５ 天才大量贴壁生长;试验组细胞数在５ 天明显增多,对照组在７ 天才迅速增加;３Ｈ－ 胸腺嘧啶核苷测定试验组及对照组的增殖活力均在第７ 天达高峰,但同一时相点试验组增殖活力明显高于对照组。结论：丝素蛋白对表皮细胞生长具有促进作用。     

新型重组免疫抑制融合毒素蛋白B7-2-PE40KDEL的表达优化

目的：我们所研创的pRSETa-B7-2-PE40KDEL／BL21 pLysE工程菌通常情况下更多的是以包涵体形式为主，而少部分为可溶形式，本研究试图在不改变其结构而仅通过优化各种已知影响原核表达系统的相关因素而获得稳定、高效的几乎全部以可溶性形式表达的重组B7-2-PE40KDEL融合蛋白。方法：针对更换不同的培养基、降低或升高诱导温度、增加或减少IPTG的用量、延长或缩短诱导时间等诸多能够增加可溶性表达量的各种因素进行较为系统的探索。结果：该工程菌在无压力选择条件下传代50代后的表达稳定性约为60％；通过大量对比研究，我们最终确定了pRSETa-B7-2-PE40KDEL／BL21 pLysE菌种的最佳培养诱导表达方案：即过夜活化的菌种以2％浓度接种于2YT培养基，22℃培养至D值约为0．4时加入0．1mmol／L IPTG，诱导12h，由此可获得较高水平的、稳定的、几乎全部为可溶性表达的重组B7-2-PE40KDEL融合蛋白，目的蛋白量占菌体总蛋白的≥24％。结论：通过优化各种已知影响原核表达系统的相关因素，获得了稳定、高效的几乎全部以可溶性形式表达的重组B7-2-PE40KDEL融合蛋白的工程菌，为进一步有效分离纯化该蛋白奠定了基础。     

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.     

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.     

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).     

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.