Biomedical subjects

Lei Yu

Publications and source records attributed to Lei Yu.

77 records · Page 5Linked to original sources

PubMed2002

Expression and purification of recombinant bone morphogenetic protein-2 in E.coli.

OBJECTIVE: To explore the method for producing human bone morphogenetic protein-2 (hBMP-2) by gene engineering techniques. METHODS: E.coli BL21 was transformed with recombinant plasmid pYR (pBV220-hBMP-2) under different conditions, and SDS-PAGE analysis was conducted to observe the effects of the activation status and induction time of the bacterium on the target protein expression. The inclusion bodies obtained from E.coli were purified by anion exchange chromatography DEAE and molecular sieve S-300, and the recombinant protein was renatured by dialyse. RESULTS: SDS-PAGE analysis showed a conspicuous band after induction signifying a new foreign protein with relative molecular mass of approximately 13 000. After activation of the bacteria when D600 was about 0.45, most efficient expression of rhBMP-2 was achieved which reached the peak 4 h after induction with heat. Implantation of the purified recombinant hBMP-2 resulted in proliferation of mesenchymal cells and new cartilage and bone formation, as shown by histological analysis 4 weeks after implantation. CONCLUSION: hBMP-2 produced by gene engineering techniques possesses the biological capacity of ectopic bone formation.

Animals↗

PubMed2002

An angiogenic, endothelial-cell-targeted polymeric gene carrier.

Targeting is one of the primary considerations in designing a specific and efficient gene delivery system. Here, an angiogenic endothelial cell-targeted polymeric gene delivery carrier was developed by conjugating an alpha(v)beta3/alpha(v)beta5 integrin-binding RGD peptide, ACDCRGDCFC, into the cationic polymer polyethyleneimine (PEI) via a hydrophilic poly(ethylene glycol) (PEG) spacer. The incorporation of PEG into PEI improved the poor physicochemical properties of PEI-DNA complexes. At a neutral charge ratio, DNA complexes with PEI were polydisperse and substantially aggregated, whereas DNA complexes with PEI-g-1PEG-RGD were homogeneous with 100-200 nm effective diameter. Their surface charge was also significantly reduced due to the charge shielding effect of PEG. However, the extensive grafting of PEI with PEG was shown to inhibit the DNA condensation process, significantly decreasing transfection efficiency. In in vitro transfection experiments with angiogenic endothelial cells, PEI-g-1PEG-RGD showed an approximately fivefold increase in transfection efficiency over PEI, due to an integrin-mediated internalization pathway. PEI-g-1PEG-RGD also exhibited high specificity to angiogenic endothelial cells compared with normal endothelial cells, which was confirmed by in vitro transfection experiments with non-targeting PEI-g-1PEG-RAE in angiostatic endothelial cells.

Cells, Cultured↗

PubMed2002

[Expression of macrophage migration inhibitory factor mRNA in Schwann cells].

OBJECTIVES: To investigate the expression of macrophage migration inhibitory factor (MIF) mRNA in Schwann cells after peripheral nerve injury and roles of Schwann cells and MIF in macrophages activation and nerve regeneration. METHODS: Fifty SD rats were divided into 10 groups. One group served as normal control. The rest were anesthetized with 3% sodium pentobarbital (30 - 60 mg/kg, i.p) and sciatic nerves were transected distal to the obturator tendon respectively 1 h, 12 h, 1 d, 3 d, 7 d, 10 d, 14 d, 17 d and 21 d before being killed. Sciatic nerves were resected and connective tissues excised. Schwann cells were obtained by digesting the nerve tissues with trypsin and collagenase. RNA was isolated and reverse-transcription-polymerase chain reaction (RT-PCR) was carried out. cDNA was analyzed by automatic system and the parameters were assessed to define the status of MIF mRNA expression in different groups. RESULTS: The level of MIF mRNA started to increase 12 h after the nerve transection. The level remained high from day 7 up to 10 after the injury. During the period from days 10 to 21, MIF mRNA decreased slowly to the pre-transection level. CONCLUSION: After peripheral nerve injury, Schwann cells can secrete MIF which may play a pivotal role as an immunomodulatory cytokine in macrophage activation and inflammatory reaction.

Animals↗

PubMedDate not supplied

Rapid and direct quantitative RT-PCR method to measure promoter activity.

This Note describes a novel rapid and direct quantitative method for examining the activity of genetic response elements. This method will provide an alternative to the classically used "reporter gene" activity assays. We show that a transfected genetic cis-regulatory element that responds to the transcription factor p53 gives a quantitative read-out at the RNA level that parallels that of an endogenous p53 responsive gene, p21 waf1/cip1. The correlation between the endogenous p21 gene expression in response to p53 and the transfected cis element is remarkable. This method is more direct and potentially faster than traditional promoter-reporter assays.

Benzothiazoles↗

PubMedDate not supplied

Lattice-like array particles on Xenopus oocyte plasma membrane.

Plasma membrane from Xenopus laevis oocytes has been used as a model system to study membrane structure and particle components, including native and exogenously expressed proteins. Previous studies by electron microscopy (EM) and atomic force microscopy (AFM) compared intramembrane particles (IMPs) on uninjected oocyte membranes to oocytes expressing proteins of interest. These studies observed randomly distributed IMPs on the surface of the oocyte plasma membrane. In this paper, we introduce a novel technique to isolate oocyte membranes by bursting the oocyte and depositing its membrane on a flat mica substrate. The flat surface membrane preparation allows high-resolution AFM images to beobtained, revealing a novel structure of densely packed particles. These particles exhibit a regular, repeating pattern of a lattice-like array with orderly packing and are thus termed "lattice-like array particles" (LAPs). The LAPs are orderly yet imperfectly packed, are located in depressed pools, occur with a low frequency on the oocyte membrane surface, and have not previously been seen using other isolation and imaging methods. Histogram analysis of the center-to-center distance between LAPs suggest their size to be about 44 nm in diameter, considerably larger than other reported size estimates of IMPs. These results indicate that LAPs represent a novel membrane particle organization, which merits further study.

Animals↗