血管生长抑制因子angiostatin在昆虫细胞中的表达及活性研究

背景与目的:抑制新生血管的形成,有助于抑制肿瘤的生长,减少和预防肿瘤转移的发生。血管抑素(angiostatin)是一种重要的内源性新生血管生成抑制剂,对血管内皮细胞增殖有较强的抑制作用。为获得具有高活性的Angiostatin表达,本研究通过杆状病毒表达系统表达重组的angiostatin,分析其在昆虫细胞中表达和生物活性。方法:用带有angiostatin的杆状病毒转移载体pBlueBacHis2B和病毒DNA共同转染Sf9细胞,构建重组病毒,蚀斑实验筛选,PCR分析确定后扩增产生大量高滴度的病毒贮存液;用SDS-PAGE电泳和Westernblot对感染不同时间后分泌的重组蛋白做时间表达分析;用ProBondTM纯化系统对表达的重组angiostatin进行纯化,分光光度计确定蛋白含量,SDS-PAGE电泳确定蛋白纯度;采用MTT法测定重组蛋白angiostatin对原代培养的人脐静脉内皮细胞(HUVEC)的抑制作用而后通过鸡胚尿囊膜实验进一步证实其抗血管形成作用。结果:成功构建了滴度为2×108pfu/ml的angiostatin重组杆状病毒,并在昆虫细胞Sf9中高效表达了分子量为53ku的angiostatin重组蛋白,纯度约为90%,重组angiostatin蛋白不仅在体外显著抑制内皮细胞的生长(IC50为2.3μg/ml),而且显著抑制鸡胚尿囊膜血管的生长。结论:此系统可制备高滴度angiostatin重组杆状病毒贮存液,并在Sf9昆虫细胞中高效表达该重组蛋白,经在体和离体细胞学实验验证其对内皮细胞的增殖具有抑制作用。     

人canstatin基因重组腺病毒载体的构建及其病毒制备

目的:利用HEK293细胞内同源重组法构建带有绿色荧光蛋白报告基因的人血管能抑素(canstatin)重组腺病毒载体,扩增并纯化重组腺病毒颗粒.方法:PCR法扩增cansta-tin全长基因,克隆至pGEM-T载体,经酶切和测序证实后,亚克隆到穿梭质粒pAdS-CMV中,获得穿梭质粒pAdS-CMV/canstatin.用PasI酶单独酶切穿梭质粒pad5-CMV/canstatin和腺病毒E3区带有绿色荧光蛋白表达元件的腺病毒骨架载体,采用标准的磷酸钙方法共转染HEK293细胞.7～10 d后收获细胞裂解物,在HEK293细胞中扩增,病毒颗粒经氯化铯密度梯度离心纯化后,分光光度计检测重组病毒滴度.结果:测序证实pEGM-T/canstatin基因片段与GenBank公布的can-statin基因序列一致.重组腺病毒质粒转染293细胞后24 h观察到绿色荧光,病毒纯化后制备出高滴度重组腺病毒(病毒滴度为2.0×1015pt/L).结论:成功构建了表达canstatin基因的重组腺病毒载体并制备了重组腺病毒颗粒,为研究该基因在肿瘤治疗中的作用奠定了基础.     

Effects of intra-articular injection of p38 mitogen-activated protein kinase inhibitor on matrix metalloproteinase in articular cartilage of a rat model of osteoarthritis

OBJECTIVE: To observe the effect of intra-articular injection of SB203580, a selective p38 mitogen-activated protein kinase inhibitor, on the expression of matrix metalloproteinase (MMP)-3, MMP-13 in a rat model of osteoarthritis (OA) and to explore the relationship between the MMP-3/MMP-13 expressions and the severity of OA. METHODS: Fourty SD rats underwent unilateral anterior cruciate ligament transection (ACLT) and then randomly divided into four groups, with 10 rats in each group. Group A received 0.1 ml intra-articular injection of SB203580 at a high concentration of 100 micromol/L (once a week) immediately after surgery, and group B were treated under the same condition using SB203580 with a low concentration of 10 micromol/ L Group C received 0.1 ml intra-articular normal saline, and group D were not injected as controls after ACLT. All rats were sacrificed seven weeks after the surgery. Macroscopic and immunohistochemical studies were performed on the cartilage. Protein expressions of MMP-3 and MMP-13 were determined by Western blot. RESULTS Cartilage degradation was significantly milder in group A and group B than in the control groups, as shown by morphological studies (P < 0. 05) and immunohistochemical studies (P < 0. 05). The protein expressions of MMP-3 and MMP-13 in cartilage were significantly lower in groups A and B than in groups C and D (P < 0.01). CONCLUSION: SB203580 can inhibit the expressions of MMP-3 and MMP-13 and thus protect the cartilage.     

吡洛芬的新合成法

报道了以苯乙腈为起始原料合成吡洛芬的新路线。具有反应条件温和,原料价廉易得等优点。总收率为4.8%。     

Effect of trisodium citrate on electrolytic deposition of hydroxyapatite coatings

Hydroxyapatite coatings were deposited on the titanium substrate by using various content Na?Cit as an organic modifier in electrolytes. The influence of the Na?Cit on the sizes of the HA crystals during electrolytic deposition was investigated under different molar ratio of Na?Cit to calcium ions. The experimental results showed that the size of HA crystal was well controlled by the addition of Na?Cit. When the molar ratio of Na?Cit to calcium ions was equal or greater than five, the obtained HA coating consisted of nanosized crystals. The HA nanoparticles were precipitated without a precursor phase when the molar ratio of Na?Cit to calcium ions was five; otherwise, the CaP coating experienced a phase conversion from octacalcium phosphate (OCP) to HA during the ELD. The effect of pH on saturation indexes with respect to hydroxyapatite, OCP, and dicalcium dihydrogen phosphate of these electrolytes was calculated by a computer program PHREEQC. The calculation showed the nucleation of OCP as the precursor during the deposition could be inhibited with increasing Na?Cit content in the electrolytes.     

临时中心静脉插管所致的血液透析并发症

目的 探讨临时中心静脉插管血液透析患者发生的导管相关并发症及其与插管时间、部位的关系.方法 将本院收入的临时插管血液透析患者753例为研究对象,分析血液透析导管相关并发症的危险因素,观察各种并发症与插管时间、部位的关系.结果 透析导管相关感染、导管功能不全、上腔静脉梗阻综合征与中心静脉插管部位、留置时间存在相关性.通过右颈内静脉置管可明显降低患者出现透析导管相关感染、上腔静脉梗阻综合征的机会,同时证明导管功能不全与置管位置无关而与置管时间有关.结论 导管留置部位、留置时间、年龄是透析导管相关感染、导管功能不全、上腔静脉梗阻综合征发生的重要危险因素.     

Extracorporeal shock wave treatment in nonunions of long bone fractures

We reviewed the clinical results of the past 7 years in order to investigate the effect of extracorporeal shock wave therapy (ESWT) in nonunions of long bone fracture. Sixty-nine patients with 69 nonunions (22 femora, 28 tibiae, 13 humeri, 5 radii, and 1 ulna) were treated with extracorporeal shock waves. The technical parameters were 6,000 to 10,000 impulses at 28 kV (0.62 mJ/mm² energy flux density) for the femur and tibia, 4,000 impulses at 24 kV for the humerus (0.56 mJ/mm² energy flux density), and 3,000 impulses at 24 kV (0.56 mJ/mm² energy flux density) for the radius and ulna. Sixty-six patients were followed up. The total successful rate of bony union was 75.4%. ESWT was successful in hypertrophic nonunions and seemed to have no evident effect in atrophic nonunions. We believe that extracorporeal shock wave therapy may be a good choice for nonunions of long bone fracture especially in hypertrophic nonunions.

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Résumé  L’objectif de cette étude a été d’évaluer cliniquement les résultats observés sur les 7 dernières années lde a technique extracorporelle “schock wave” (ESWT) sur le traitement des pseudarthroses des os longs. Matériel et méthode : 69 patients présentant 69 pseudarthroses (22 fémurs, 28 tibias, 13 humérus, 5 radius et 1 cubitus) ont été traités selon cette méthode. Les paramètres techniques étaient de 6000 et de 10000 impulsions à 28 kV (062 mJ/mm2) pour le fémur et le tibia, 4000 impulsions à 24 kV pour l’humérus (0.56 mJ/mm2), 3000 impulsions à 24 kV (0.56 mJ/mm2) pour le radius et le cubitus. Résultats : 66 patients ont été suivis pendant au moins 12 mois. Le taux de consolidation a été de 75,4%. La ESWT est une technique qui permet de traiter une pseudarthrose hypertrophique alors qu’elle a peu d’effet sur les pseudarthroses atrophique. Conclusion : nous pensons que cette technique est une technique de choix pour les pseudarthroses des os longs spécialement lorsque ces pseudarthroses sont hypertrophiques.

     

Association of estrogen receptor alpha gene polymorphisms with bone mineral density: a meta-analysis

Background  A number of studies have examined the association between estrogen receptor alpha (ESR-α) gene polymorphisms and bone mineral density (BMD), but previous studies of ESR-α gene XbaI (rs9340799) and PvuII (rs2234693) polymorphisms have been hampered by small sample size, regional restrictions and inconclusive results. Thus a meta-analysis is needed to assess their pooled effects.

Methods  This study reviewed all published articles indexed in Pubmed using the keywords in the title or abstract. All data were extracted independently by two reviewers using a standard form, the studies were meta-analyzed and minor discrepancies were resolved by authors’ discussion.

Results  Twenty seven eligible studies involving 8467 women and 2032 men were identified. The XbaI and PvuII polymorphisms were significantly associated with BMD of the lumbar spine. XX and PP homozygotes had a protective effect in comparison with carriers of the x and p alleles, the effects were more significant in premenopausal women or Western women. At the femoral neck, the results were different. XX served as a protective factor in postmenopausal women, Western women, Western postmenopausal women, and men, while PP was likely to serve as a risk factor in Eastern women, Eastern postmenopausal women, and men.

Conclusions  The XbaI polymorphism is correlated to BMD at diverse skeletal sites. PP had a protective role for the lumbar spine but might be a risk factor for the femoral neck.

     

膝关节内侧副韧带、前交叉韧带、后交叉韧带联合损伤的手术治疗

目的探讨关节镜下膝关节前交叉韧带(ACL)、后交叉韧带(PCL)、内侧副韧带联合损伤(Ⅲ度膝关节外翻伤)的手术治疗方法。方法关节镜下同时联合重建ACL、PCL并修复内侧副韧带和内侧关节囊，8例交叉韧带重建材料来源于自体双侧髌腱中1／3骨-腱-骨结构，8例采用同种异体髌腱重建，交叉韧带重建的同时进行膝关节内侧副韧带和关节囊的修补。术后佩戴可调式卡盘支具3个月，并给予严格的康复训练。结果术后随访6～18个月，所有患者主观感觉以及膝关节稳定程度恢复良好，均进行正常生活与工作，同种异体髌腱移植重建比自体髌腱移植重建前后交叉韧带手术时间短，效果更好，没有发现明显的排异反应和感染传染性疾病的现象。结论1期行关节镜手术治疗膝关节内侧副韧带、前交叉韧带／后交叉韧带联合损伤，使用安全，效果满意；同种异体髌腱是重建物是一个可以选择的来源，效果优于自体髌腱移植重建。