OBJECTIVETo study the effects of zuogui pill (ZP) contained serum on the gene expressions of type-II collagen and proteoglycan during the differentiation of mesenchymal stem cells (MSCs) towards chondrocytes.

METHODSMSCs isolated from rat bone marrow were in vitro induced differentiation towards chondrocytes and stimulated with high- (57 g/kg), middle- (28.5 g/kg), and low-dose (9.5 g/kg) ZP contained serums and serum of blank rats. The proliferation of MSCs was analyzed by CCK-8 method. The 3rd-passage MSCs were divided into the blank control group (by adding serum of the blank group rats), the induction control group (by adding the induction fluid and serum of the blank group rats), and the ZP contained serum group (by adding the induction fluid and middle-dose ZP contained serum). The expressions of type-II collagen and proteoglycan were determined using reverse transcriptase PCR, Real-time PCR, and immunohistochemistry.

RESULTSCompared with the blank control group, the proliferation of MSCs could be promoted by ZP contained serum at different doses (P < 0.05), with the most obvious effect shown in the middle-dose ZP contained serum group (P < 0.05). The mRNA and protein expressions of type-II collagen could be identified in the induction control group and the ZP contained serum group on the 21st day of the induction. Of them, the mRNA expression of type-II collagen in ZP contained serum groups was obviously higher than that of the induction control group. Results of Real-time PCR showed that on the 21st day of the induction, the mRNA expression quantitation of proteoglycan in ZP contained serum groups was about 16-fold and 3-fold of the levels on the 7th day and the 14th day (P < 0.05), obviously higher than those of the induction control group (P < 0.05).

CONCLUSIONZP contained serum could induce MSCs proliferation, the gene expressions of type- II collagen and proteoglycan, which might be one of its molecular bases for protecting the cartilage.

Animals ; Cell Differentiation ; drug effects ; Cells, Cultured ; Chondrocytes ; cytology ; drug effects ; metabolism ; Collagen Type II ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Female ; Male ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Proteoglycans ; metabolism ; Rats

For epidemiological investigation of the rabies virus carrier rates of domestic dogs, cats and wild animals like rodent animals and bats,three kinds of regions where rabies had higher incidence (Hunan and Guizhou Provinces), lower incidence (Jiangsu Province, Wuhan City) and provisionally rabies-free (Shenyang City) were selected. Then the antigenic types, the genovariation of the isolaled viruses and the currently vaccine matching of the virus strains were analyzed. The results showed that in China the principal host of rabies is dog,the total virus carrier rate of the captured dogs was 2.56%, and the highest positive isolation rate was 20.0% in some monitoring site. However,there was no evidence about the rabies virus carrier rate in rodent animals,bats or other wild animals. The rabies vaccines which prepared from aG and CTN strains have already been produced successfully in China. The research showed that the nucleotide sequences of the newly isolated viruses were more similar with the glycoprotein gene of CTN strain. In order to evaluate the safety and the efficacy of the vaccines currently used, two groups (50 people each) were injected with vaccine of aG strain and CTN strain respectively in five surveillance points. The neutralizing antibody tested were 0.49 IU/mL-0.52 IU/mL and 6.7 IU/mL-7.53 IU/mL after the 7 and the 14 days of vaccine injection respectively. In addition, the rates of antibody positive seroconversion were 45.1%-47.9% and 100% respectively, and there was no moderate or severe adverse reactions observed. These data showed the vaccines have satisfactory effect on safety and protection.
Animals ; Antibodies, Viral ; blood ; Carrier State ; epidemiology ; veterinary ; Cats ; virology ; Cercopithecus aethiops ; Dogs ; virology ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique ; Rabies Vaccines ; immunology ; Rabies virus ; classification ; genetics ; isolation & purification ; Vero Cells

OBJECTIVETo analyze a Duchenne muscular dystrophy(DMD) patient's muscular regeneration, dystrophin expression and locomotive variation before and after he underwent umbilical cord blood stem cell transplantation in order to assess the therapeutic effect.

METHODSA 12-year-old DMD boy who could not walk for 3 years was confirmed by gene analysis and dystrophin protein immune test on his muscle. He had no other chronic disease. By HLA matching, a piece of umbilical cord blood stem cell with 6 HLA sites matching to the boy was found in Guangdong Umbilical Cord Blood Bank. The number of the nucleated cells of the umbilical cord blood stem cell was 24.08x 10(8). After pretreatment for the DMD boy with busulfan, cyclophosphamide and rabbit anti-human thymocyte globulin, the allergenic cord blood stem cells were transplanted into him by intravenous injection. Cyclosporin A, methylprednisolone, MMF, prostaglandin E1 and ganciclovir were given after the transplantation. At the same time, Gran, the granulocytic cell stimulating factor, and gamma globulin were administered. The biochemistry profile including serum creatine kinase (CK), the reconstruction of blood making, the deletion exon of DMD gene, the regenerating muscles, the dystrophin protein expression, and the locomotive function of the DMD boy were tested regularly.

RESULTS(1) The white blood cells (WBC) of peripheral blood decreased gradually to zero after pretreatment. In a period of 15 days after transplantation, the neutrophil increased to 0.5x 10(9)/L; at 25 days, WBC increased to normal level. Blood platelet was more than 20x 10(9)/L at 22 days. The hemoglobin rose to 85-100 g/L. At 140 days, sternal puncture revealed the rapid growth of neutrophil, blood platelet and hemoglobin. (2)At 140 days, the blood type of the DMD boy transformed from type O to type AB (the donor's blood type being AB). There was no grafe versus host reaction. (3) At 18, 30, 43, 55, 74 and 233 days after transplantation, the PCR-short tandem repeat test of the boy's peripheral blood DNA showed that his genotype was completely the same as the donor's. The results of PCR-short tandem repeat tests of the bone marrow cells DNA by sternal puncture at 140, 183 and 235 days were the same as those of the blood DNA. (4) At 60 days, DMD gene analysis by PCR showed that the defected DMD gene (exon 19 deletion) had been corrected by the umbilical cord stem cells transplantation. (5) At 75 days, the biopsy of calf muscle showed there were myoblast cells and muscular tubes growing. The dystrophin expressions were weak, but a few of them were strong. DNA analysis showed that the donor's gene DNA accounted for 1%-13%. At 126 days, obviously increased dystrophin positive muscular fibers of the boy were found. The donor's fibers rose to 2.5%-25%. (6) The serum CK of the boy declined from 5735 U/L to 274 U/L. (7) At 100 days, physical examination revealed improvement in his arms and legs.

CONCLUSIONThe therapy of Duchenne muscular dystrophy with allogeneic umbilical cord blood hematopoietic stem cell transplantation may reset up the blood-making function, decrease the serum CK level, restore the dystrophin in muscles, and improve the locomotive function of the DMD boy. These data suggest that the allogeneic umbilical cord blood hematopoietic stem cell transplantation may benefit the DMD boys.

Alprostadil ; therapeutic use ; Busulfan ; therapeutic use ; Child ; Combined Modality Therapy ; Cord Blood Stem Cell Transplantation ; methods ; Cyclosporine ; therapeutic use ; Dystrophin ; genetics ; Ganciclovir ; therapeutic use ; Humans ; Male ; Methylprednisolone ; therapeutic use ; Muscular Dystrophy, Duchenne ; genetics ; therapy ; Polymerase Chain Reaction ; Treatment Outcome

AIMTo synthesize and identify artificial antigen of podophyllotoxin for the production of podophyllotoxin polyclonal antibody.

METHODSThe hapten was synthesized by two different chemical approaches and characterized by TLC, IR, NMR, and MS. Mixed anhydride reaction (MAR) and active ester method (AEM) were used to couple the podophyllotoxin to carrier proteins (BSA and OVA). Characterization of artificial antigens was done by using spectroscopy and electrophoresis. The anti-podophyllotoxin polyclonal antibodies were obtained through immunizing rabbits.

RESULTSThe results from IR, NMR and MS showed that 4-O-succinoyl podophyllotoxin (hapten) was successfully synthesized. The coupling molar ratios of the hapten and carrier proteins were 88.6 for Hapten-BSA1, 40.3 for Hapten-BSA2, 17.8 for Hapten-OVA1, and 54.2 for Hapten-OVA2. Hapten conjugates coupled with BSA yielded two sets of the specific and affinitive polyclonal antibodies. One set of antibodies showed an IC50 value of 2.21 microg.mL(-1) with a detection limit of 0.12 microg.mL(-1).

CONCLUSIONAntigenic conjugates were artificially synthesized, and based on these artificial antigens, polyclonal antibodies against podophyllotoxin were raised from rabbits immunized with two different immunogens and characterized with an indirect ELISA format.

Animals ; Antibodies ; analysis ; Antibody Affinity ; Antibody Formation ; Antineoplastic Agents, Phytogenic ; immunology ; Enzyme-Linked Immunosorbent Assay ; Haptens ; chemistry ; immunology ; Immune Sera ; chemistry ; Male ; Ovalbumin ; immunology ; Podophyllotoxin ; immunology ; Proteins ; immunology ; Rabbits ; Serum Albumin, Bovine ; immunology

OBJECTIVETo study influenza epidemic and analyze antigenic and genetic characterization of the predominant strains in Wuhan area in 2003.

METHODSEpidemiological data and specimens from influenza patients were collected from surveillance sites weekly. Viruses were isolated from the specimens. Three H3 isolates were chosen to do antigenic analysis by hemagglutination inhibition (HI) test and their HA1 region was sequenced.

RESULTSTotally 58 influenza viruses were isolated from 418 specimens, 57 of them were identified as H3 subtype and 1 of them was B subtype; both monthly positive rate and numbers of influenza like illness had two peaks of winter and summer, the highest peak appeared in July. The 3 new H3 isolates were antigenically different from vaccine strain A/Panama/2007/99, 14 amino acid changes have been found in HA1 domain of these 3 strains compared with A/Panama/2007/99, phylogenetic analysis also confirmed the difference in HA1 domain.

CONCLUSIONSInfluenza epidemic had two peaks in Wuhan area in 2003. The activity of H3 virus was strengthened remarkably. And they are antigenically and genetically different from the vaccine strain.

Amino Acid Sequence ; Antigens, Viral ; immunology ; China ; epidemiology ; Genes, Viral ; Glycosylation ; Hemagglutination Inhibition Tests ; Humans ; Influenza A Virus, H3N2 Subtype ; genetics ; immunology ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Molecular Sequence Data ; Phylogeny ; Sequence Analysis, Protein