肢体负压对犬后肢动脉闭塞病影响的实验研究

目的为研究肢体负压疗法的疗效及探讨其作用机理,我们对犬后肢进行实验研究。方法。实验犬分三组,治疗组为左后肢缺血模型犬并行患者肢负压治疗;对照组同治疗组但不做治疗：自身对照组为双后肢缺血模型犬,左侧行负压治疗,右侧作为对照。     

质粒pcDNA3—HGF的大规模纯经制备研究

质粒ｐｃＤＮＡ３－ＨＧＦ具有潜在的临床治疗缺血性疾病的应用前景。大规模纯化制备是质粒ＤＮＡ应用于基因治疗的关键步骤。质粒大规模纯化制备流程包括：发酵、离心收集细胞、碱性裂解、Ｑ－Ｓｅｐｈａｒｏｓｅ ＸＬ捕获质粒ＤＮＡ、Ｓｏｕｒｃｅ １５Ｑ精制质粒ＤＮＡ,所得纯超螺旋质粒ｐｃＤＮＡ３－ＨＧＦ符合质量标准。该纯化制备方法避免使用动物源性的酶及有毒试剂。     

复方抗栓酶股动脉注射与静滴治疗下肢动脉闭塞性疾病101例分析

比较股动脉注射与静脉滴注两法给予复方抗栓确治疗下肢动脉闭性疾病的疗效。方法１０１例患者随机分成股动脉注射与静脉滴注复方抗栓酶两组。结果显效率股动脉组６２．３％,静脉组４１．７％。结论复方抗栓酶股动脉注射治疗下肢动脉闭塞性疾病的疗效明显优于静脉滴注（Ｐ＜０．０１）。     

蕲蛇酶治疗肢体闭塞性动脉硬化症46例

肢体闭塞性动脉硬化症（ASO）是周围血管病科常见病,由于肢体动脉血管发生狭窄或闭塞性病变而导致的肢体远端缺血,患者病程长、痛苦大、残肢率高,严重威胁着人类的健康。我科于2002-2004年对46例ASO病人用蕲蛇酶注射液配合中药治疗,取得一定疗效。现报告如下。     

重组质粒pUDKH的质量分析

目的:分析研究pUDKH的质量。pUDKH是由本实验室构建的携带人肝细胞生长因子(HGF)基因的真核表达质粒,具有治疗肢体动脉闭塞病的应用潜能。方法:用光密度法分析pUDKH的浓度及纯度;用0.8%琼脂糖凝胶电泳分析超螺旋pUDKH比例及RNA;以地高辛标记的核酸探针固相斑点杂交法测定残留宿主DNA含量;用酶联免疫法测定残留宿主菌蛋白;以4组限制性内切酶分析一致性;PCR扩增目的基因片段;抑菌圈测定板测定残余抗生素;用鲎试剂检测细菌内毒素;对CHO细胞进行基因转染;ELISA检测上清HGF表达量;用Transwells法分析表达的上清液诱导ECV304细胞迁移数。结果:03批次pUDKH的浓度为1.97mg/mL,D260nm/D280nm值为1.84,超螺旋pUDKH比例为95.5%;电泳图谱中未见RNA;残留宿主DNA含量低于质粒DNA的0.2%;菌体蛋白质残留含量低于质粒DNA的0.1%;限制性酶切图谱与自行制备的对照品一致;PCR扩增到HGF基因片段,长约2.2kb;未见残余抗生素(卡那霉素)抑菌圈;细菌内毒素≤0.03125EU/mL;转染的CHO细胞上清HGF表达量为39.32ng/mL;表达的上清诱导ECV304细胞迁移数高于对照组2倍。结论:03批次pUDKH的质量符合药学规格。     

脐血干细胞移植治疗肢体缺血性疾病的研究进展

脐血干细胞具有来源丰富、相对原始、免疫原性较弱、应用无伦理学问题等特点,脐血干细胞移植有助于缺血部位的血管新生,利用此治疗肢体缺血性疾病日益受到关注.免疫反应是脐血干细胞移植治疗肢体缺血性疾病存在的主要问题.与治疗其他疾病相比,脐血干细胞移植治疗肢体缺血性疾病的免疫反应具有特殊性.免疫反应在其中的作用及免疫反应的干预有待进一步深入研究.     

超选择性动脉溶栓治疗急性大脑中动脉脑梗死的疗效和安全性评价

目的:探讨超选择性动脉溶栓治疗急性大脑中动脉脑梗死的疗效和安全性。方法:收集我院就诊或住院治疗的120例急性大脑中动脉脑梗死患者,随机分为实验组和对照组,每组60例。两组患者入院后均给予相应的治疗措施,对照组患者给予静脉溶栓;实验组患者给予超选择动脉溶栓,观察并比较两组患者治疗前后神经功能缺损评分(NIHSS)以及患者治疗后临床疗效和并发症发生率。结果:与治疗前相比,两组患者治疗后的NIHSS水平均下显著降差异具有统计学意义(P0.05);与对照组相比,实验组患者的NIHSS水平、并发症的发生率和病死率较低而治疗有效率较高差异均具有统计学意义(P0.05)。结论:超选择性动脉溶栓治疗急性大脑中动脉脑梗死的临床疗效以及安全性较静脉溶栓更好。     

蕲蛇酶治疗血栓闭塞性脉管炎38例

血栓闭塞性脉管炎（TAO）多发于男性青壮年,以肢体动脉炎症性、节段性、周期性发作的慢性闭塞性疾病,致残率高。我科于2003～2005年10月对38例血栓闭塞性脉管炎患者用蕲蛇酶注射液配合中药治疗,取得一定疗效,现报告如下。     

腺病毒介导肝细胞生长因子基因转移治疗小型猪慢性心肌缺血的实验研究

血管生长因子基因转移诱导的血管新生已成为治疗心肌缺血的一种新的思路. 重组腺病毒介导的肝细胞生长因子基因转移能够有效诱导治疗性血管新生. 为评价重组腺病毒肝细胞生长因子基因(Ad-HGF)转移治疗冠心病效果, 本实验利用Ameroid 缩窄环建立了小型猪慢性心肌缺血的研究模型, 并评价了Ad-HGF对慢性心肌缺血的治疗作用. 18只小型猪随机分成3组: 手术对照组、模型组和治疗组. 模型组和治疗组分别在回旋支近端放置Ameroid缩窄环, 模型成功后分别直接注射安慰剂和Ad-HGF到缺血心肌部位进行治疗. 治疗4周后对心功能和血液供应的改善进行了评价. 结果表明, 与对照组和模型组相比, Ad-HGF组的心肌灌注有了明显改善. 在治疗后4周, Ad-HGF组新形成的血管密度和血管数量明显高于模型组. Ad-HGF组心肌缺血面积明显减少, 左心室射血分数显著改善. 这些结果为肝细胞生长因子基因治疗慢性心肌缺血提供了直接的证据.     

螺环酮联合帕罗西汀治疗焦虑症的疗效及安全性分析

目的:研究螺环酮联合帕罗西汀治疗焦虑症的疗效及其安全性。方法:选取2009年1月至2013年8月我院收治的符合诊断标准的焦虑症患者244例,按照知情同意原则随机分为治疗组(122例)和对照组(122例),治疗组给予丁螺环酮联合帕罗西汀治疗,对照组仅给予螺环酮治疗。治疗10周后,运用汉密尔顿焦虑量表(HAMA)及Montgomery-Asberg抑郁量表(MADS)评价疗效,运用治疗过程中不良反应症状量表(TESS)评价其安全性,比较两组患者的疗效及安全性。结果:治疗后两组的HAMA及MADS评分均低于治疗前,且治疗组低于对照组,差异均有统计学意义(P0.05);两组TEES评分在治疗第2、4、6、8、10周末均无统计学差异(P0.05)。结论:螺环酮联合帕罗西汀在治疗焦虑症时可提高疗效,且安全性高,可考虑在临床推广。     

人肝细胞生长因子基因表达质粒的构建及其活性研究

目的 :构建一种携带人肝细胞生长因子 (HGF)基因的表达质粒 (pUDKH) ,并对其体外活性进行研究 ,为其体内应用提供依据。方法 :从人胎盘cDNA文库用PCR方法克隆人HGF基因 ,并将其克隆至自行构建的真核表达载体 pUDK上 ,获得表达质粒 pUDKH。将pUDKH体外转染原代培养的骨骼肌细胞 ,分析其转染效率及表达上清中HGF、血管内皮生长因子 (VEGF)的表达水平 ,并采用MTT法分析不同剂量HGF表达产物对人脐静脉内皮细胞的作用。结果 :所克隆构建的携带人HGF基因的质粒 pUDKH可有效转染原代培养的骨骼肌细胞 (0 .0 5 7% ) ,并表达HGF(16～ 18ng/ 4× 10 5cells)和VEGF ,其表达产物对人脐静脉内皮细胞具有明显的增殖刺激活性 (P <0 .0 5 ) ,而且有剂量效应关系。结论 :初步证实本研究构建的质粒 pUDKH具有体内治疗缺血性疾病的应用潜力     

基因枪在大鼠闭塞性血管病基因治疗中的应用

探讨利用基因枪技术转移肝细胞生长因子基因治疗大鼠肢体闭塞性血管病的可行性,构建了携带人肝细胞生长因子（HGF）基因的重组真核表达载体（pUDKH),在制备好大鼠下肢闭塞性血管病模型后,通过基因枪或肌肉直接注射法,向局部缺血部位肌肉中转移pUDKH,每只5ug(基因枪）和12ug(肌肉注射）,应用常规组织病理切片（H．E．染色）及免疫组织化学方法观察血管形成及基因表达,转移pUDKH后第10天,用基因枪和直接注射法转移的局部肌肉组织的HGF的表达明显高于转移空白质粒（pUDK)的对照组,pUDKH组可见明显的小血管新生,而pUKD组至20天时仍未观察到或仅见到极少量的新生血管,基因枪与肌肉注射两组相比血管密度无明显差异,采用基因枪直接转移pUDKH裸露质粒子肢体缺血局部的方法是可行的,转移的基因可在局部有效表达,达到促进血管形成的预期目的。     

Protein-mediated isolation of plasmid DNA by a zinc finger-glutathione S-transferase affinity linker

The sequence-specific affinity chromatographic isolation of plasmid DNA from crude lysates of E. coli DH5alpha fermentations is addressed. A zinc finger-GST fusion protein that binds a synthetic oligonucleotide cassette containing the appropriate DNA recognition sequence is described. This cassette was inserted into the SmaI site of pUC19 to enable the affinity isolation of the plasmid. It is shown that zinc finger-GST fusion proteins can bind both their DNA recognition sequence and a glutathione-derivatized solid support simultaneously. Furthermore, a simple procedure for the isolation of such plasmids from clarified cell lysates is demonstrated. Cell lysates were clarified by cross-flow Dean vortex microfiltration, and the permeate was incubated with zinc finger-GST fusion protein. The resulting complex was adsorbed directly onto glutathione-Sepharose. Analysis of the glutathione-eluted complex showed that plasmid DNA had been recovered, largely free from contamination by genomic DNA or bacterial cell proteins.     

Gene therapy for pathological scar with hepatocyte growth factor mediated by recombinant adenovirus vector

A complementary DNA (cDNA) encoding human hepatocyte growth factor was introduced into a replication-defective type 5 adenovirus (lacking E1, E3 domains) vector by homologous recombination of intracellular plasmid DNA, thus a recombinant vector containing HGF (Ad-HGF) was obtained. Ad-HGF and Ad-GFP (adenovirus vector carrying green fluorescence protein gene) were expanded in 293 cells and purified by cesium chloride gradient centrifugation for large-scale preparation, then were infected to the primarily cultured scar fibroblast of rabbit ear to observe the transfer efficiency and expression level of HGF in vitro. To evaluate the effect of Ad-HGF on established scar Ad-HGF solution was injected into excessively formed scar, which bears some clinical and histologic similarities to human hypertrophic scars. The results showed that: (i) the transfer efficiency was 36.8% ±14.1% on day 3 in primarily cultured scar fibroblasts treated with Ad-GFP and lasted more than 20 d; (ii) high-level expression of HGF protein was detected by means of ELISA in supernatant of scar fibroblasts treated with Ad-HGF, the amount of expression was 76 ng/4.0 x 10⁵ cells on day 3; (iii) on day 32 after a single intradermal injection of Ad-HGF at different doses (8.6 x 10⁹ pfu, 8.6 x 10⁸ pfu, 8.6 x 10⁷ pfu, 8.6 x 10⁶ pfu) per scar, most of the scars in the former two dose groups were dramatically flattened, some were even similar to that of the normal skin. The value of Hl (hypertrophie index) showed that there was a therapeutic effect of Ad-HGF on scars at the dose of 10⁹ pfu and 10⁸ pfu. Whereas no therapeutic effects were seen at lower dose (10⁷ pfu and 10⁶ pfu of Ad-HGF) groups. In addition, clusters of hair were observed to different extent on healed wound treated with Ad-HGF. Histopathologic examination revealed that in most healed wounds of Ad-HGF treated group, the dermal layer was thinner, the amount of fibrous tissue was much fewer, and hair follicles growth and sebaceous glands were observed. In Sirius red-stained sections the amount of type I collagen in the Ad-HGF-treated scars was diminished markedly, compared to that in Ad-GFP group, in which a huge amount of type I collagen was still observed; (iv) immune response against HGF was absent. Antibody against HGF was not detectable by ELISA in serum from rabbit treated with Ad-HGF; (v) no local or systemic side-effects and toxicity associated with the gene transfer were found. These results demonstrated the potential use of treating pathologic scar by Ad-HGF, an alterative strategy of gene therapy for scar in clinical practice.     

Enhancement of bovine growth hormone gene expression by increasing the plasmid copy number

Effect of copy number on the expression of bovine growth hormone gene (bGH) was investigated using the copy number mutants such as pKBJ10, pBJ( tet)10, pUBJ10-1, and pUBJ10 plasmids. The cells harboring plasmids below 84 copies/cell did not produced detectable levels of bGH. When the ColE1 replicon was replaced with the mutated ColE1 replicon originated from pUC19 plasmid, the copy number was increased to about 300 copies/cell and bGH production was enhanced by 11.5% (pUBJ10-1) and 12.3% of total cell protein (pUBJ10). A large amount of mRNA caused by increment of copy number would be needed to overcome some inhibitory threshold and might be an important factor for regulating bGH expression.     

Gram-scale production of plasmid pUDK-HGF with current good manufacturing practices for gene therapy of critical limb ischemia

The demand of a plasmid encoding human hepatocyte growth factor gene (pUDK-HGF) in large quantities at high purity and concentration has increased for gene therapy of critical limb ischemia (CLI) in clinical trials. In this article, we produced pUDK-HGF in compliance with current good manufacturing practices at gram scale. The process included a 50-L batch fermentation, continuous alkaline lysis, and integrated three-step chromatography on Sepharose 6 Fast Flow, PlasmidSelect Xtra, and Source 15Q. The production process has been scaled up to yield 4.24?±?0.41?g of pharmaceutical pUDK-HGF from 1.0?kg bacterial cell paste and the overall yield reached range from 58.37 to 66.70%. The final pUDK-HGF product exhibited high purity with supercoiled percentage of >?95.8% and undetectable residual RNA, contaminated protein, and bacterial endotoxin. The phase I clinical study indicates that intramuscular injection of pUDK-HGF is safe, well tolerated, and may provide symptomatic relief to CLI patients. These results show that our manufacturing process of pUDK-HGF is efficient in producing pharmaceutical-grade plasmid DNA and is safe for clinical applications.     

肝细胞生长因子基因转染对人淋巴瘤细胞凋亡的影响

探讨肝细胞生长因子（HGF）基因转染人淋巴瘤细胞系Raji细胞后,拮抗足叶乙甙（VP－16）诱导细胞凋亡的研究。将三种细胞：未转染Raji细胞、空载体pVITR02－mcs转染细胞和HGF基因转染细胞,分成正常对照组和经vP－16处理的药物组。采用Westernblot法验证HGF蛋白的表达：CCK－8法检测诱导Raji细胞凋亡的药物浓度;通过透射电镜、流式细胞术、吖啶橙（A0）染色、苏木精咿红（HE）染色等方法观察Raji细胞的凋亡情况,并进行相关分析。结果显示：Westernblot法验证了HGF蛋白质的表达;CCK．8法显示100μg／mL足叶乙甙可明显抑制Raii细胞增殖;透射电镜下可发现典型的凋亡细胞;流式检测结果表明：给药组与正常组相比,三组细胞的凋亡率明显升高（P〈0．01）,提示VP－16具有诱导细胞凋亡的作用：但给药组间：HGF基因转染组凋亡率明显低于未转染组（P＜0．05）和空载体pVITR02．mcs转染组（P〈0．05）,提示嬲F基因转染可明显抑制VP-16诱导的Raji细胞的凋亡,AO染色和HE染色结果也同样提示HGF具有拮抗VP－16诱导的细胞凋亡效应。     

An evaluation of models for the effect of plasmid copy number on bacterial growth rate

The rates of growth of recombinant bacteria depend on their plasmid content. This is modelled by expressing the specific growth rate in terms of the number of copies of the plasmid per cell. Three models in common use have been tested with different Escherichia coli strains and one strain of Bacillus stearothermophilus containing different plasmids. While no particular model was decisively better than others for all data, that of Bentley & Quiroga (Biotechnol. Bioeng. 1993, 42: 222–234) was the best for specific growth rates which vary inversely with the plasmid copy number, and a modified form of the model of Satyagal & Agarwal (Biotechnol. Bioeng. 1989, 33: 1135–1144) was the best for growth rates which increase with the copy number. The differences appear to be linked to the plasmid replication mechanisms. Contrary to some claims, no model portrayed the experimentally observed inflection points.     

腺病毒介导人肝细胞生长因子对大鼠皮层神经元损伤的保护作用

目的:探讨腺病毒介导人肝细胞生长因子(Ad-HGF)对体外无血清培养所致大鼠皮层神经元损伤的保护作用.方法:用流式细胞仪测定不同感染强度(MOI)条件下携带绿色荧光蛋白的重组腺病毒(Ad-GFP)(25,50,100,200pfu/cell)对皮层神经元的转染效率,确定最佳MOI;ELISA测定Ad-HGF在最佳感染强度下皮层神经元中的表达规律;通过中性红染色和PI-Hoechst33342双染色法比较转染Ad-HGF组与对照组(转染Ad-GFP组和空白对照组)间无血清培养后6 h、12 h、24 h和48 h细胞的存活情况.结果:在感染强度为50 pfu/cell时,重组腺病毒对皮层神经元的转染率达99.3%,Ad-HGF能在皮层神经元中有效而持久地表达,接种后2 h转染Ad-HGF组的细胞死亡率和凋亡率在无血清培养后12 h明显低于两对照组(P＜0.05).结论:Ad-HGF对接种后2 h转染组无血清培养所致的损伤有显著的保护作用,对接种后第5 d转染的皮层神经元无血清所致的损伤虽亦具有一定的保护作用,但不明显.