Selection and identification of aptamers for mesenchymal stem cells from different species / 军事医学

Objective To screen an ssDNA aptamer for rabbit mesenchymal stem cells (MSCs) and to identify the ability of the aptamer to recognize MSCs of a variety of species origin.Methods MSCs were isolated from the thigh bone of immature rabbits and identified by induced osteogenic and adipogenic differentiation,respectively.Aptamers were screened by cell SELEX (systematic evolution of ligands by exponential enrichment) technique targeting to isolated MSCs.Enrichment of the 5th pool was evaluated through binding assay of FAM modified pool to MSCs by confocal microscopy.The enriched 5th pool was then cloned into pGE-T vector and the cloned sequences were determined randomly.The candidates were chosen based on primary sequence conservation and predicted secondary structure by RNA structure and MEME online analysis.Flow cytometry analysis was used to identify the aptamers binding to MSCs of rabbit, rat, and human origin.Results The isolated MSCs had the potential of osteogenic differentiation and adipogenic differentiation under certain conditions.Aptamer 5-1-12 from 5th enriched pool was characterized as MSCs recognizing aptamer binding to MSCs of rabbit, rat and human origin.Conclusion Aptamer 5-1-12 that recognizes MSCs of different species origin is obtained through live cell-SELEX.

Selection and identification of ssDNA aptamers specific to clinical isolates of Streptococcus mutans strains with different cariogenicity / 南方医科大学学报

<p><b>OBJECTIVE</b>To select and identify ssDNA aptamers specific to Streptococcus mutans strains with different cariogenicity isolated from clinical specimens.</p><p><b>METHODS</b>Subtractive SELEX technology targeting the whole intact cells was used to screen for ssDNA aptamers specific to the clinical isolates Streptococcus mutans strains with different cariogenicity. Radioactive isotope, flow cytometry, gene cloning and sequencing, MEME online software and RNA structure analysis software were employed to analyze the first and secondary structures of the aptamers and identify the screened aptamers.</p><p><b>RESULTS</b>Detection by radioactive isotope showed sufficient pool enrichment after 9 rounds of subtractive SELEX. Flow cytometry showed that the selected aptamers H1, H16, H4, L1, L10 and H19 were capable of binding specifically with highly cariogenic Streptococcus mutans strains but not with strains with a low cariogenicity. The aptamer H19 had the strongest binding capacity to highly cariogenic Streptococcus mutans strains, with a dissociation constant of 69.45∓38.53 nmol/L.</p><p><b>CONCLUSION</b>We have obtained the ssDNA aptamers specific to the clinical isolates of highly cariogenic Streptococcus mutans strains.</p>

A Novel Neutralizing epitope of Human cytomegalovirus glycoprotein M Screened by Phage Display / 生物化学与生物物理进展

Human cytomegalovirus glycoprotein complex Ⅱ (gC Ⅱ ) consists of two glycoproteins, gM and gN. Although gC Ⅱ specific IgG purified from HCMV positive patient sera can neutralize HCMV, there has been no report on the generation of virus-neutralizing antibodies by immunizing with one epitope of gM. The epitope, termed MAD, was screened from random phage peptide library by subtractive strategy. The peptide sequence of MAD was highly homologous with 32~38 amino acids of HCMV gM. Mice immunized with MAD coupled with keyhole limpet hemocyanin (KLH) could produce specific antibodies against MAD, and the antibodies obtained could bind not only native HCMV particles, but also the recombinant gM30～78 peptide. ELISA analysis results showed that MAD could specifically bind HCMV-positive human serum samples. Virus-neutralizing assay results demonstrated that the antibodies against MAD could inhibit HCMV strain AD169 entering the human embryonic lung cells. The results suggested that MAD could be used as a new potential protective antigen in the development of HCMV vaccine.

Conserved amino-acid residue mutations in epitope of human cytomegalovirus antigen M / 生物工程学报

We identified the critical amino-acid residues in antigen M derterminant (MAD) epitope of human cytomegalovirus protein M. On the basis of the peptide sequence of MAD, some conservative residues were mutated into the glycine residue. Then the gene fragment of mutants linked to amino terminal of Fc were cloned into the plasmid pET32-Fc and expressed by fusion with Fc. After purified by protein A affinity chromatography, the activity of mutants binding the goat polyclonal antibodies against human cytomegalovirus (HCMV) were detected by ELISA and Western blotting. Our results showed that when glutamine residue was mutated into glycine residue, the activity of MAD(Q --> G) binding the goat polyclonal antibodies against HCMV was reduced apparently. Other mutants did not have the same characteristics. The activity of MAD was closely related to the conformation of glutamine residue.

Advances in The SELEX Technique and Aptamers / 生物化学与生物物理进展

Aptamers are short single-stranded nucleic acid ligands that are capable of binding almost any targets with low nano- to picomolar affinities and exceptional specificities. They are selected out of a large combinatorial oligonucleotide library through an in vitro evolution process termed SELEX (systematic evolution of ligands by exponential enrichment). The unique advantages of high-throughput screening technique and aptamers in precise recognition as well as easiness to synthesis and modification bring aptamers a bright prospect in analytical chemistry, biology and medicine research. The recent advances in the SELEX technique and aptamers are reviewed.

Effects of free fatty acids on gene and protein expression and tyrosine phosphorylation of leptin-receptor in rat hepatic cells / 医学研究生学报

0.05). The protein abundance of leptin receptor in rat hepatic cells was significantly decreased compared with the control after incubated with palmitate and oleate for 36 hours( P 0.05),but significantly decreased after 24 or 36 hours( P

Therapeutic effect of phenol-croton oil on photoaging skin of hairless mice / 中华医学美学美容杂志

Objective To promote the use of chemical peeling in facial rejuvenation with the phenol and croton oil peeling agents to the UVA/B-irradiated skin of hairless mice, and to provide the experimental evidence for the clinical application of the treatment of irradiated skin.Methods Sixty BALB/C hairless mice were photo-aged by use of chronic ultraviolet A and ultraviolet B irradiation for 20 weeks. After irradiation the animals were randomly divided into two groups:untreated (10 mice) and treated (50 mice). The phenol and croton oil chemical peeling agents were applied to the dorsal skin of treated animal group while it was full anesthetized. Punch biopsies were taken at 7, 14, 30, 60, and 90 days after peel for histological analysis. At 60 days after irradiation, the skin wrinkling of animals were analyzed by macroscopy, cleavage line amplification, and computer imaging analysis system. Results The treated areas of irradiated skin recovered rejuvenation and exhibited a unique connective tissue layer composed of fine collagen fibers beneath the epidermis. Conclusion The mixture of phenol-croton oil may reverses the visible stigmata of photoaging skin. Our results will be of great help to promote the use of chemical peeling in facial rejuvenation.

Quantitative and quanlitative changes of wrinkles in ultraviolet irradiated skin of hairless mice / 中华医学美学美容杂志

Objective To study the changes of skin wrinkles in hairless mice while exposed to ultraviolet. Methods The hairless mice were irradiated under long-wave ultraviolet ray (UVA), medium-frequency wave ultraviolet ray (UVB) and the combination of the two for 20 weeks. Total dose of UVA was 222J/cm~2, and that of UVB was 5.9J/ cm~2. After irradiation, the skin wrinkling of animals were analysed by the naked eye, dermatoglyphics enlarges and applied color skin system of pathologic portrait quantitative analysis. Results Control group: The hairless mice skin were fine and delicate, the ditch and ridge of skin distributed even, and had no the obvious cornification. Long wave ultraviolet ray (UVA) set: The skin was slightly rough, skin ditch and ridge distributed still even, and had no obvious cornification; quantitative analysis had no the obvious difference from that of control group. Medium-frequency wave ultraviolet ray (UVB) set: The dermatoglyphics were disorderly, and the skin ditch deepened, widened, and the skin ridge increased the breadth and obvious cornification, and quantitative analysis had obvious difference from that of control group. Long wave and medium-frequency wave ultraviolet ray (UVA+ UVB) set: The dermatoglyphics was disorderly, and the skin ditch deepened, widened, the skin ridge increased the breadth, skin cornification was more obvious, quantitative analysis had obvious difference from that of control group. Conclusions The qualitative and quantitative changes of the wrinkles in the ultraviolet irradiated skin of hairless mice are related to ultraviolet B but not to UVA. UVB is a key factor of skin wrinkling in UV-irradiation.

Quantitative and quanlitative changes of collagen in ultraviolet irradiated skin of hairless mice / 中华医学美学美容杂志

Objective To study collagen changes in dermis of hairless mice that were exposed to ultraviolet. Methods The hairless mice was irradiated under UVA, UVB and the combination of the two for 20 weeks, total dose of UVA was 222J/cm~2, and that of UVB was 5.9J/cm~2. After irradiation, the dorsal skin's collagens of animals were analysed by computer imaging analysis system, histopathologic examination, specific stains and electorn microscopy. Results The hairless mice exposed to ultraviolet A were unchanged in dermis collagen. The hairless mice was irradiated under UVB and the both UVA and UVB, and the content of collagen was decreased with less affinity for collagen staining. These findings were supported by electron microscopy, which showed fraying, thickened, and proliferating collagen, coalesced into extensive denaturalization. The ratio of types Ⅲ/Ⅰ+Ⅲ collagen was significantly increased. Conclusion The qualitative and quantitative changes of the collagen in the ultraviolet irradiated skin of hairless mice are related to ultraviolet B but not to UVA. UVB is a key factor of skin collagen damage in UV-irradiation.