心血管疾病基因治疗的基础研究Ⅵ．一种新的体内基因转移方法

将医用缝合线浸泡在质粒ＤＮＡ溶液中，然后缝合在大鼠股四头肌上，可以观察到外源基因在肌肉组织中高效、稳定地表达，其表达量高于磷酸钙共沉淀和ＤＮＡ沉淀块埋植法，较肌肉直接注射法表达量高１０倍左右。     

Expression pattern of Popdc2 during mouse embryogenesis and in the adult.

The Popdc2 gene is a member of the Popeye domain containing gene family encoding membrane proteins with prominent expression in striated and smooth muscle tissue. After introducing a LacZ reporter gene into the Popdc2 locus, expression was studied during embryonic development and postnatal life. At embryonic day (E) 7.5, expression was present in cardiac and extraembryonic mesoderm. At E10.5, expression was found in heart, somites, and mesothelial cells lining the coelom. At E12.5, expression was present in the coelomic mesothelium, pericardial and myocardial layer of the heart, skeletal muscle, bladder, gut, and umbilical vessels. Postnatal expression was found in cardiac and skeletal muscle and in the smooth muscle layer of colon, rectum, and bladder. In the stomach, Popdc2 was exclusively present in the pyloric epithelium. In conclusion, Popdc2 is expressed in various muscle and nonmuscle cell types during embryonic development and in postnatal life.     

False-Positive β-Galactosidase Staining in Osteoclasts by Endogenous Enzyme: Studies in Neonatal and Month-Old Wild-Type Mice

Escherichia coli β-galactosidase (β-gal), encoded by the lacZ gene, has become an essential tool in studies of gene expression and function in higher eukaryotes. lac-Z is widely used as a marker gene to detect expression of transgenes or Cre recombinase driven by tissue-specific promoters. The timing and location of promoter activity is easily visualized in whole embryos or specific tissues using the cleavable, chromogenic substrate, 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal). The tissue specificity of promoters in transgenic constructs is routinely tested by using a promoter of choice to drive lacZ. Alternatively, the targeted expression of Cre recombinase to perform in vivo recombination of loxP sites can be visualized by β-gal staining in mice carrying a Cre-activated lacZ transgene, such as the ROSA26 strain. In the course of our investigations, we examined β-gal activity in bone tissue from genetically normal mice using standard detection methodology and found very high endogenous activity in bone-resorbing osteoclasts. This was true in frozen, paraffin, and glycol methacrylate sections. X-gal staining colocalized with the osteoclast marker, tartrate-resistant acid phosphatase (TRAP). β-gal activity was present in osteoclasts in long bones, in the mandible, and in both neonatal and more mature animals. We present this brief article as a caution to those testing genetic models of skeletal gene expression using β-gal as a marker gene.     

腺病毒介导的β—半乳糖苷酶在小鼠气道上皮的转基因表达

目的：以β－半乳糖苷酶基因为标志基因研究重组复制缺陷腺病毒在 小鼠肺内气道上皮细胞中的转基因表达。方法：带有LacZ表达基因的重组复制缺陷腺病毒（AdCMVLacZ）,经鼻腔吸入法和气管内注射法两种途径分别感染小鼠肺组织,此后在不同时间取小鼠肺组织经多聚甲醛固定后,以X-gal染色。石蜡包埋切片后用核固红复染。结果：β－半乳糖苷酶经重组复制缺陷腺病毒AdCMVLacZ感染小鼠气道和肺泡上皮细胞,并高效表达目的基因,表达时间可长达31天,重复感染后仍可表达13天。结论：重组复制缺陷腺病毒可携带外源性基因在小鼠气道上皮细胞内进行高效、稳定和较为长期的转基因表达。     

Laminin-111 protein therapy prevents muscle disease in the mdx mouse model for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease caused by mutations in the gene encoding dystrophin. Loss of dystrophin results in reduced sarcolemmal integrity and increased susceptibility to muscle damage. The α₇β₁-integrin is a laminin-binding protein up-regulated in the skeletal muscle of DMD patients and in the mdx mouse model. Transgenic overexpression of the α₇-integrin alleviates muscle disease in dystrophic mice, making this gene a target for pharmacological intervention. Studies suggest laminin may regulate α₇-integrin expression. To test this hypothesis, mouse and human myoblasts were treated with laminin and assayed for α₇-integrin expression. We show that laminin-111 (α₁, β₁, γ₁), which is expressed during embryonic development but absent in normal or dystrophic skeletal muscle, increased α₇-integrin expression in mouse and DMD patient myoblasts. Injection of laminin-111 protein into the mdx mouse model of DMD increased expression of α₇-integrin, stabilized the sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscle from exercised-induced damage. These findings demonstrate that laminin-111 is a highly potent therapeutic agent for the mdx mouse model of DMD and represents a paradigm for the systemic delivery of extracellular matrix proteins as therapies for genetic diseases.     

Growth Characteristics in the Initial Stage of Micrometastasis Formation by Bacterial LacZ Gene-tagged Rat Prostatic Adenocarcinoma Cells

A micrometastasis model was established using a rat differentiated prostatic adenocarcinoma, designated PLS301Z, transfected with the lac Z gene encoding a bacterial β-galactosidase. The morphology, tumorigenicity and metastatic ability of PLS301Z were comparable to those of the parental cells. Micrometastatic foci could be specifically detected at the single cell level after X-Gal staining with a dissecting microscope. After intravenous injection, the number of X-Gal positive foci in the lung decreased progressively to a steady-state level (less than 1% of injected cells) by 4–7 days, while the size of persisting positive foci started to increase from 4 days after inoculation, as demonstrated by image analysis. X-Gal and BrdU double staining revealed that BrdU labeling indices of X-Gal-positive cells decreased transiently at the 2-day time point and increased again from 4 days after inoculation. Type IV collagen immunostaining showed the tumor cells to be surrounded by a basement membrane intravascularly at the time point when they started new growth. Electron microscopy confirmed that, 2 days post injection, most tumor cells were degenerative or dead, but on day 4, persisting tumor cells formed multicellular clumps in contact with the vascular basement membrane inside vessels. These results indicate that PLS301Z cells begin to grow intravascularly depending upon the presence of a basement membrane before extravasation at the initial stage of micrometastasis formation.     

Silk-elastinlike protein polymers for matrix-mediated cancer gene therapy

Silk-elastinlike protein polymers (SELPs) are recombinant polymers designed from silk fibroin and mammalian elastin amino acid repeats. These are versatile materials that have been examined as controlled release systems for intratumoral gene delivery. SELP hydrogels comprise monodisperse and tunable polymers that have the capability to control and localize the release and expression of plasmid DNA and viruses. This article reviews recent developments in the synthesis and characterization of SELP hydrogels and their use for matrix-mediated gene delivery.     

四环素调控复制缺陷性疱疹病毒载体介导LacZ基因在原代培养神经元中的表达

目的利用四环素调控复制缺陷性单纯疱疹病毒I型重组载体(QR9TO-LacZ)感染体外原代培养的大鼠皮层神经元,探讨其介导半乳糖苷酶(LacZ)基因的表达情况及基因调控效能;并检测该载体对神经元活力的影响。方法用RUL9-8细胞系扩增QR9TO-LacZ病毒株,收集后采用噬斑法测定病毒滴度;体外原代培养大鼠皮层神经元。将培养的原代神经元分为强力霉素(DOX)组和对照组。两组细胞均用QR9TO-LacZ转染,强力霉素组加入四环素衍生物强力霉素(0.5μg/ml),对照组加入等剂量空白对照液。同时根据感染复数(MOI),即MOI=3、5、10、30 PFU/cell,将两组培养细胞进一步分为亚组。病毒转染48h后行X-gal染色;光镜下观察神经元形态,随机选取10个高倍镜视野(×400),计算每高倍镜视野中平均蓝染细胞率,比较不同MOI病毒感染神经元的效率。采用四甲基偶氮唑盐微量酶反应比色法(MTT法)检测感染病毒48 h(MOI同上)后神经元的活性。结果在含有DOX的培养环境中,不同MOI亚组(3、5、10、30 PFU/cell)QR9TO-LacZ感染神经元出现蓝染细胞百分比分别为5.35%±0.84%、10.64%±1.92%、19.73%±2.87%、34.41%±2.58%;在无DOX的培养环境中,各亚组均未观察到蓝染细胞。上述MOI感染的神经元细胞存活率分别为:90.24%±8.16%、88.00%±5.69%、83.95%±3.82%、78.90%±5.49%。结论 QR9TO-LacZ载体能有效地将目的基因导入原代培养神经元中并表达;目的基因表达的开启受强力霉素调控;QR9TO-LacZ对体外原代培养神经元毒性较低。