经导管动脉注入脂质体介导的p53基因治疗肝癌的实验研究

目的 以兔VX2肝癌模型为对象,探讨经导管动脉注入脂质体介导的p53基因治疗肝癌的可行性及转染和表达情况.方法 将pCMV-myc-p53质粒、阳离子脂质体LipofectAMINE以及pCMV-myc-p53和LipofectAMINE的复合体分别注入兔VX2肝癌模型的肿瘤供血动脉,并提取肿瘤组织蛋白,采用蛋白印迹法及免疫组化检测基因转染及其表达.以不同量的pCMV-myc-p53与LipofectAMINE形成的复合体分别注入兔VX2肝癌模型的肿瘤供血动脉内,同法检测基因的转染及其表达.结果 脂质体介导的p53基因经动脉途径成功转染了兔VX2肝癌模型的肿瘤组织并进行表达,其转染效率明显高于单纯基因导入,基因的量与转染效率之间存在量效关系.结论 经动脉途径导入脂质体介导的p53基因治疗肝癌是可行、有效的,具有广阔的应用前景.     

经动脉导人脂质体联合转铁蛋白介导的p53基因治疗肝癌的实验研究

目的 探讨经导管动脉内联合应用转铁蛋白对脂质体介导的p53基因治疗肝癌的转染效率及治疗效果的影响.方法 25只荷瘤兔(VX2肝癌模型兔)采用随机数字表法分为5组,每组5只.取0、100、200、300、400 μg的转铁蛋白(Tf)分别与阳离子脂质体(LipofectAMINE)和真核细胞表达质粒(pCMV-myc-p53)的复合体混合后,分别注入5组兔VX2肝癌模型的肿瘤供血动脉内,48 h后提取肿瘤组织蛋白,采用免疫斑点法(Western blot)和免疫组化法检测基因的转染及其表达水平.对5个不同Tf剂最组p53表达水平的比较,采用x2检验.另取10只荷瘤兔分为2组,1组经肿瘤供血动脉注入Tf200 μg及LipefectAMINE 45 μl和pCMV-myc-p53质粒15 μg形成的复合体,另1组仅注入后两者的复合体,观察2组兔的肝功能、肿瘤体积和生存期.对2组实验兔同一时间点的肝功能输测结果采用成组t检验;肿瘤体积检查结果采用配对t检验;生存期采用单一样本均数t检验.结果 Western blot半定量分析显示,含Tf组的实验兔p53基因的转染及表达水平均较不含Tf组高.免疫组化染色显示,含0、100、200、300、400 μg Tf的5组实验兔,p53基因高度表达的阳性率分别为58.33%、69.44%、80.00%、83.33%、81.67%,含Tf组与不含Tf组之间差异有统计学意义(总体x2=42.37,P<0.01);随着Tf的量从0增加到200 μg,053基因高度表达的比例逐渐增加,但Tf自200 μg增加到400 μg时,p53基因高度表达比例差异无统计学意义(x2分割:前3组x2=4.8161,P<0.05,后3组x2=0.67,P>0.05).含Tf及不含Tf组实验兔术前及术后各时间点肝功能结果比较的差异无统计学意义(P值均>0.05),术后第3、7、10、14、20、25、30天2组实验兔平均肿瘤体积分别为(4.33±0.72)、(5.65±0.85)、(7.12±0.99)、(8.44±1.23)、(9.21±1.51)、(10.32±1.78)、(11.55±1.90)cm3和(4.36±0.66)、(4.86±0.68)、(5.12±0.73)、(5.72±0.93)、(6.13±1.26)、(6.68±1.38)、(7.02±1.55)cm3,在同一时间点差异有统计学意义(t=4.59,P<0.05),2组实验兔的平均生存时间分别为(28.6±2.2)及(35.8±3.1)d,差异有统计学意义(t=1.52,P<0.05).结论 经动脉途径联合应用Tf是安全的,可明显提高脂质体介导的p53基因的转染效率和治疗效果.     

微小核糖核酸-7调控表皮细胞生长因子受体对肝癌细胞增殖和转移的影响

目的 探讨微小核糖核酸(miR)-7是否通过靶向调控表皮细胞生长因子受体(EGFR)对人肝癌细胞侵袭和增殖产生影响.方法 传代培养HepG2人肝癌细胞,脂质体转染miR-7后使其表达上调或下调.荧光定量聚合酶链反应检测转染效率.蛋白印迹法检测miR-7上调或下调后肝癌细胞EGFR表达变化.溴化噻唑蓝四氮唑(MTT)法检...     

iASPP干扰RNA转染HepG-2细胞后细胞凋亡的变化

目的:观察iASPP基因干扰RNA转染肝癌细胞HepG-2后的干扰效果及细胞凋亡的变化.方法:设计特异性siRNA序列,将序列克隆至PGCsilencerTM H1/Neo/GFP质粒中,用脂质体将重组子转染至HepG-2细胞中,用RT-PCR方法检测iASPP表达的变化,Western blot检测蛋白表达的变化,流式细胞仪检测细胞凋亡的变化.结果:iASPP干扰质粒转染HepG-2细胞后,iASPP表达下降,凋亡细胞增加.结论:抑制内源性的iASPP,能有效地恢复肝癌细胞HepG-2中p53的抑癌功能.     

iASPP干扰RNA转染HepG-2细胞后细胞凋亡的变化

目的：观察iASPP基因干扰RNA转染肝癌细胞HepG-2后的干扰效果及细胞凋亡的变化。方法：设计特异性siRNA序列,将序列克隆至PGCsilencer^TM H1／Neo／GFP质粒中,用脂质体将重组子转染至HepG-2细胞中,用RT—PCR方法检测iASPP表达的变化,Westernblot检测蛋白表达的变化,流式细胞仪检测细胞凋亡的变化。结果：iASPP干扰质粒转染HepG-2细胞后,iASPP表达下降,凋亡细胞增加。结论：抑制内源性的iASPP,能有效地恢复肝癌细胞HepG-2中p53的抑癌功能。     

p53与HBV相互作用对7721细胞凋亡及p21启动子的影响

为了观察HBV与p53是否存在相互作用的关系，以进一步揭示与原发性肝细胞肝癌发生发展的相关机制，选用7721肝癌细胞系（表达野生型p53，HBV阴性）为靶细胞，应用磷酸钙转染法，将pCMVp53单独或者与野生型HBV质粒（pCMVHBVa）、突变型HBV质粒（pCMVHBVb）共转染细胞，用annexin-V-FITC标记及流式细胞仪检测细胞凋亡的变化。各组另与含有p21基因启动子的荧光素酶报告基因质粒p21-luc共转染细胞，通过检测荧光毒酶的表达，了解p21启动子的活化情况。结果表明，单独的pCMVp53质粒转染7721细胞系能够促进p21报告基因的表达，细胞凋亡率升高；pCMVp53与pCMVHBV。共同转染时，对p21基因启动子的激活作用增强、细胞凋亡率明显增加，而与pCMVHBVb共转染时则否。提示HBV在该细胞内的复制能够诱导增强p53的作用并对其下游基因p21的转录有促进作用，导致细胞凋亡的增强。     

非编码基因MEG3稳定表达细胞株建立及其对p53转录活性的影响

目的构建人类长链非编码RNA母系表达基因3（MEG3）的真核表达载体,筛选MEG3稳定高表达的肝癌细胞株,分析MEG3过表达对p53转录活性的影响。方法根据GenBank中MEG3的基因序列设计合成特异引物,从肝癌细胞中扩增MEG3的cDNA序列,将其构建入pcDNA3.0真核表达载体中,得到pcDNA3.0-MEG3表达载体,转染肝癌细胞SK-Hep-1,用G418筛选稳定株,采用RT-PCR技术检测转染细胞中MEG3基因表达,采用荧光素酶报告基因技术分析MEG3过表达细胞中对p53介导的基因转录活性的影响。结果成功构建了MEG3真核表达质粒pcDNA3.0-MEG3,筛选获得了稳定高表达MEG3基因的SK-Hep-1肝癌细胞株,双荧光报告实验结果显示MEG3过表达能增强p53介导的转录激活作用。结论本研究获得了稳定高表达MEG3基因的SK-Hep-1肝癌细胞株,为深入分析MEG3的功能及其作用机制奠定了基础。     

外源性PTEN基因体外转染C6胶质母细胞瘤株及稳定表达蛋白产物的鉴定

目的 探讨外源性PTEN抑癌基因转染胶质母细胞瘤株并表达活性蛋白产物的可行性.方法 对获赠的包含有人PTENcDNA全长序列的PRK-PTEN质粒进行扩增和酶切鉴定后,用脂质体介导PRK-PTEN质粒转染大鼠C6胶质母细胞瘤株,荧光染色检测转染细胞中PRK-PTEN质粒携带的绿色蛋白荧光报告基因,免疫组化染色鉴定稳定转染后C6细胞中PTEN功能蛋白.结果 转染后的C6胶质母细胞瘤株稳定表达绿色荧光蛋白和PTEN功能蛋白.结论 PRK-PTEN质粒载体在脂质体的介导下可以将PTEN基因整合到C6胶质母细胞瘤的基因序列中去,并且通过宿主基因序列的表达稳定地产生PTEN功能蛋白,为以PTEN基因为靶点的胶质瘤基因治疗提供了实验理论依据.     

E4F1真核表达载体的构建及与p53的相互作用

目的：构建E4 F1野生型及缺失32～81位氨基酸的真核表达载体,检测其与p53的相互作用及对下游基因p53的调节。方法分别用普通PCR和重组PCR方法从乳腺文库中扩增E4F1野生型编码序列及缺失32～81位氨基酸的突变型序列,分别将其以正确相位构建到pXJ40-MYC载体中,得到重组质粒MYC-E4F1和MYC-E4F1（Δ32-81）,分别转化大肠杆菌DH5α,重组质粒经酶切鉴定并转染293 T细胞, Western印迹检测蛋白的表达。将MYC-E4F1和MYC-E4F1（Δ32-81）质粒与FLAG-p53质粒共转染293T 细胞,免疫共沉淀实验检测其相互作用,野生型及缺失突变型表达载体共转染骨肉瘤细胞U2OS,检测其对下游基因p53的调节。结果 E4F1重组质粒及其突变型构建成功,在293 T细胞中鉴定表达正确,野生型及突变型E4 F1均与p53存在相互作用,突变型的结合能力增强,野生型E4F1升高p21蛋白表达水平,而突变型不改变p21蛋白水平。结论成功构建E4F1野生型及缺失32～81位氨基酸的突变型真核表达载体,二者都能与p53相互作用且突变型结合能力更强,野生型E4F1升高基因p53的靶基因p21的蛋白表达水平,而突变型不改变靶基因p21的蛋白水平。该研究为进一步研究E4 F1对p53的调节及其机制奠定了基础。     

利用CRISPR／Cas9系统构建H22细胞GP73基因敲除稳定株及功能鉴定

目的：利用CRISPR／Cas9基因编辑系统敲除小鼠源肝癌细胞H22中的GP73基因,构建H22细胞GP73基因敲除的稳定细胞株。方法根据CRISPR／Cas9靶点设计原则,设计2条特异性识别GP73基因启动子的上下游sgRNA,利用载体pX459质粒,构建2对重组真核表达质粒。经酶切和测序鉴定后,将重组质粒转染至H22细胞内,使用嘌罗霉素加压筛选稳定敲除GP73的H22细胞株,利用免疫印迹检测重组质粒对内源GP73的敲除效果。MTT实验检测GP73被敲除后对细胞增殖能力的影响,再利用划痕实验检测细胞的迁移能力。结果免疫印迹结果说明敲除GP73基因的小鼠源H22细胞株内无GP73蛋白的表达;并且GP73被敲除后,H22细胞的增殖能力和迁移能力减慢。结论通过CRISPR／Cas9系统获得了靶向GP73基因的重组质粒,并且筛选出了稳定干扰GP73表达的细胞株,从而为探讨GP73在肝癌发生中的作用奠定基础。     

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.