溶藻弧菌热休克蛋白70基因真核表达载体的构建及鉴定

目的构建溶藻弧菌热休克蛋白70(HSP70)真核表达载体pcDNA3.1。方法提取溶藻弧菌基因组DNA,PCR扩增HSP70基因片段,克隆至TA载体,通过PCR、酶切及测序鉴定后,将HSP70基因片段用限制性内切酶切下,克隆至真核表达载体pcDNA3.1,构建pcDNA3.1重组质粒,通过PCR、酶切及序列分析对HSP70基因pcDNA3.1重组质粒进行鉴定。结果扩增出1 914bp的溶藻弧菌HSP70基因片段,并成功构建溶藻弧菌HSP70真核表达载体pcDNA3.1。结论成功构建了溶藻弧菌HSP70真核表达载体pcDNA3.1,为HSP70基因疫苗研制奠定了基础。     

SV40LT基因重组腺病毒载体的包装、鉴定及用于转染日本血吸虫童虫细胞的研究

目的构建携带SV40LT基因的重组腺病毒表达载体,制备具有感染力的重组腺病毒,观察其转染日本血吸虫（Schistosoma japonicum,Sj）童虫细胞后的表达情况。方法采用体外连接法构建好的携带SV40LT基因的重组腺病毒质粒（AdHu5-SV40LT）转化Stbl2感受态菌,获得重组腺病毒质粒后,经Pac I酶切线性化后转染293A细胞,获得出重组腺病毒（AdHu5-SV40LT）。将重组腺病毒感染Sj童虫细胞,采用RT-PCR和免疫组织化学检测SV40LT基因的表达情况。结果重组腺病毒载体质粒可转染293A细胞并可在293A细胞内进行有效的复制;提取病毒DNA进行PCR检测证实含有SV40LT目的基因。以重组腺病毒能感染Sj童虫细胞,经RT-PCR和免疫组化检测有SV40LT基因在细胞中的表达。结论成功包装了具有感染能力的含SV40LT基因的重组腺病毒,感染Sj童虫细胞后目的基因有表达,为进一步探索日本血吸虫细胞永生化提供了实验依据。     

Introduction of human β-defensin-3 into cultured human keratinocytes and fibroblasts by infection of a recombinant adenovirus vector

Cultured epidermal autografts and cultured skin substitute are vulnerable to infection. Human beta defensin (HBD)-3 is an antimicrobial peptide that exhibits a wide-spectrum antimicrobial activity against gram-positive/negative bacteria and fungi. This study determined whether normal human keratinocytes (NHKs) and human dermal fibroblasts (HDFs) transfected with the HBD-3 gene secrete HBD-3 peptide with an antimicrobial activity. An adenovirus vector with an HBD-3 cDNA inserted downstream of the CMV promoter (ADhBD3) was created. The HBD-3 gene was introduced into NHKs and HDFs via ADhBD3 infection. HBD-3 gene expression in each type of transfected cells was evaluated by RT-PCR. The presence of HBD-3 peptide in the culture supernatants of each type of transfected cells was evaluated by Western blotting. The antimicrobial activities of the culture supernatants of each type of transfected cells against several bacterial strains were also measured. Both NHKs and HDFs infected with ADhBD3 expressed the HBD-3 gene and secreted HBD-3 peptide into culture supernatants. These supernatants exhibited a strong bacteriocidal activity against a Staphylococcus aureus reference strain and methicillin-resistant S. aureus (MRSA). NHKs and HDFs transfected with the HBD-3 gene secrete HBD-3 peptide with an antimicrobial activity against S. aureus and MRSA.     

Breakthrough in three-dimensional scoliosis diagnosis: significance of horizontal plane view and vertebra vectors

Scoliosis is a multifactorial three-dimensional (3D) spinal deformity with integral and directly related vertebral deviations in the coronal, sagittal and horizontal planes. Current classification and diagnostic methods rely on two-dimensional (2D) frontal and lateral X-ray images; no routine methods are available for the visualization and quantitative evaluation of deviations in the horizontal plane. The EOS 2D/3D system presented here is a new, low-dose, orthopedic radiodiagnostic device based on Nobel prize-winning X-ray detection technology with special software for 3D surface reconstruction capabilities that finally led to a breakthrough in scoliosis diagnosis with high-quality, realistic 3D visualization and accurate quantitative parametric analysis. A new concept introducing vertebra vectors and vertebra vector parametric calculations is introduced that furnishes simplified visual and intelligible mathematical information facilitating interpretation of EOS 2D/3D data, especially with regard to the horizontal plane top view images. The concept is demonstrated by a reported scoliotic case that was readily characterized through information derived from vertebra vectors alone, supplemented with the current angulation measurement methods in the coronal and sagittal planes and axial vertebral rotation measurements in the horizontal plane, with a calibrated 3D coordinate system suitable for inter-individual comparisons. The new concept of vertebra vectors may serve as a basis for a truly 3D classification of scoliosis.     

Effect of Heat Shock Protein 72 Expression on Etoposide-induced Cell Death of Rat Retinal Ganglion Cells

Purpose

To assess whether the expression of heat shock protein 72 (Hsp72) protects rat retinal ganglion cells (RGC-5) from apoptotic cell death.

Methods

Hsp72 expression in RGC-5 cells transduced with replication-deficient recombinant adenovirus was analyzed by Western blot analysis and immunofluorescence. The effect of Hsp72 expression on etoposide-induced apoptotic cell death was examined by microscopic analysis and confirmed by cell proliferation assay.

Results

Western blot analysis and immunofluorescence clearly showed adenovirus-mediated Hsp72 expression in RGC-5 cells. Treatment with etoposide resulted in the death of a proportion of the cells by apoptosis. However, this apoptotic cell death was significantly reduced in cells expressing Hsp72, with the reduction in cell death correlating to the level of Hsp72 expression.

Conclusions

Over-expression of Hsp72 alone is sufficient to rescue neuronal cells from apoptotic cell death, suggesting that fine-tuning its expression may be an effective neuroprotective approach in retinal degenerative disease.     

Suppression of exposure to malaria vectors by an order of magnitude using microbial larvicides in rural Kenya

OBJECTIVE: To determine the contribution larviciding could make to reduce the burden of malaria, by conducting a trial of microbial larvicides in a 4.5 km2 area in and around a large village in rural western Kenya. METHOD: The abundance of immature and adult mosquitoes was monitored for 12 months under baseline conditions. Then microbial larval control was implemented for 28 months. After the intervention, the abundance of immature and adult mosquitoes was monitored for a further 12 months. RESULTS: Of the 419 mosquito larval habitats identified, 336 (80%) originated from human activities. Application of Bacillus thuringiensis var. israelensis and Bacillus sphaericus larvicides reduced the proportion of aquatic habitats containing Anopheles larvae from 51% during non-intervention periods to 7% during the intervention. The occurrence of late instar Anopheles in habitats was reduced from 39% and 33% in pre-intervention and post-intervention periods to 0.6% during intervention. Overall, larviciding reduced Anopheles larval density by 95% and human exposure to bites from adults by 92%. The estimated cost of providing this protection to the human population in the study area was less than US$ 0.90/person/year. CONCLUSION: Appropriately applied microbial larvicides can substantially and cost-effectively reduce human exposure to malaria in rural sub-Saharan Africa.     

IGF-1在股骨头再造关节软骨化生中的作用

目的观察分析大转子骨瓣表面骨膜及腱膜等纤维结缔组织向关节软骨转化的规律及胰岛素样生长因子-1(IGF-1)在软骨化生过程中的作用。方法制备液氮冷冻双侧股骨头缺血性坏死(Osteonecrosis of femoral head,ONFH)的动物模型。左侧股骨头造模后即缝合关节囊,右侧股骨头根据分组不同采用不同的处理方式:A组(骨瓣治疗组):带血管蒂大转子骨瓣进行股骨头再造;B组(骨瓣加Ad-IGF-1基因治疗治疗组):带血管蒂大转子骨瓣股骨头再造,关节内注入表达IGF-1的腺病毒载体(Ad-IGF-1);两组动物分别于3,6,12,18,24周每批4只处死,对骨瓣进行大体观察,组织病理学观察,免疫组化检测。结果所有动物左侧冷冻区组织坏死,纤维状物覆盖,碎片样组织修复。组织病理切片及免疫组化证实A组右侧骨瓣区自6周出现透明软骨细胞,B组右侧骨瓣区自3周出现透明软骨细胞,B组较A组修复效果好。结果经统计学处理有统计学意义。结论大转子表面的骨膜及腱膜能够向关节软骨化生,IGF-1对大转子表面的骨膜及腱膜向关节软骨化生有促进作用,为ONFH的外科治疗及生长因子的应用提供基础。     

人防御素-5基因的克隆和真核表达载体的构建

目的对人防御素5(HD-5)基因进行克隆,构建真核表达载体HD5-pPICZαA。方法从人cDNA文库中PCR扩增HD-5基因片段,用EcoRⅠ和XbaⅠ分别双酶切HD-5基因扩增产物和毕赤酵母表达载体pPICZαA,用T4DNA连接酶连接目的基因片段和pPICZαA,然后转化到E.coli Top10中,Zeocin筛选转化子并进行PCR、酶切和序列鉴定。结果提取阳性克隆的重组质粒,EcoR Ⅰ和Xba Ⅰ双酶切后跑电泳获得预期条带;同时重组质粒经序列测定,证实HD-5基因正确插入pPICZαA中,插入位置、方向均正确。结论成功构建人防御素5基因的真核表达载体HD5-pPICZαA,为HD-5蛋白的真核真核表达奠定基础。     

A review of near infrared spectroscopy and chemometrics in pharmaceutical technologies

Near-infrared spectroscopy (NIRS) is a fast and non-destructive analytical method. Associated with chemometrics, it becomes a powerful tool for the pharmaceutical industry. Indeed, NIRS is suitable for analysis of solid, liquid and biotechnological pharmaceutical forms. Moreover, NIRS can be implemented during pharmaceutical development, in production for process monitoring or in quality control laboratories.This review focuses on chemometric techniques and pharmaceutical NIRS applications. The following topics are covered: qualitative analyses, quantitative methods and on-line applications. Theoretical and practical aspects are described with pharmaceutical examples of NIRS applications.