Objective To prepare a peptide monoclonal antibody (McAb)against human papillomavirus 18E6 separately,and identify its specificity and pathogenicity.Metheds The advantage epitope peptide was designed and synthesized by ABCpred and Bcepred,and then used to immunize BALB/c mice after coupling with bovine serum albumin (BSA).And the McAb was prepared by hybridoma technique.HPV18E6 gene was amplified from cervical swab specimen containing HPV18 and insert-ed into expression vector pET-28a.The constructed recombinant plasmid was transformed to E.coli BL21(DE3)for expres-sion under induction of isopropyl thio-β-D-galactoside.The expressed protein was used to identified the McAb had been pre-pared.Results The hybridoma cell lines could constantly produce MAbs against HPV18E6 peptides.Sequencing proved that recombinant plasmid pET-28a-HPV18E6 was constructed correctly.Western blotting showed that the anti-HPV18E6 pep-tides antibody could specifically recognize HPV18E6.Conclusion A monoclonal antibody against the advantage epitope pep-tide of human papillomavirus 18E6 prepared could specifically recognize HPV18E6 specifically.

Objective To explore the expression of HCK gene during the cardiomyocyte differentiation of mouse embryonic stem cells and analyze the role of HCK gene in maintenance of pluripotency of embryonic stem cells.Methods Mouse embryonic stem cells were cultured,then induced to differentiate into cardiomyocytes.Total RNAs were isolated from mouse embryonic stem cells in the differentiation days:0 day(D0),the second day(D2),the fourth day(D4),the sixth day(D6),the eighth day(D8),respectively.The levels of HCK mRNAs were assessed by the method of semi-quantitive reverse transcriptase-polymerase chain reaction(RT-PCR).In the meanwhile,Total proteins were also isolated from mouse embryonic stem cells in the differentiation D0,D2,D4,D6,D8,and the levels of HCK proteins were evaluated by Western-blot.Results HCK mRNAs could be detected in the mouse embryonic stem cells in D0 and D2,however,they were undetectable from D4 to D8.The expression of HCK mRNAs was rapidly down-regulated during cardiomyocyte differentiation of mouse embryonic stem cells.Expression of HCK proteins,which coincided with HCK mRNAs,down-regulated during differentiation and couldn't be detected in D4.Conclusions With the cardiomyocyte differentiation of mouse embryonic stem cells,the expression of HCK in the levels of mRNA and proteins are sharply down-regulated;HCK may play an important role in maintaining the pluripotency of embryonic stem cell.

BACKGROUND:Hypertrophic differentiation of chondrocytes is the sign of starting endochondral ossification, and it is also an essential step in endochondral ossification, which is a cascade reaction and difficult to be blocked once started. The end result is the formation of bone structure. RNA interference is a post-transcriptional gene silencing. Relevant studies have shown that the use of RNA interference to block the expression of core binding factorα1 (Cbfα1) can effectively inhibit the formation of heterotopic ossification. OBJECTIVE:To use RNA intereference technology to suppress Cbfα1 expression so as to achieve the purpose of blocking the hypertrophic diferentiation of chondrocytes. METHODs: We constructed an adenovirus containing siRNA against Cbfα1 (Ad-Cbfα1-siRNA). Retinoic acid and interleukin-1α were used to induce hypertrophic differetiation of chondrocytes, and then Ad-Cbfα1-siRNA was utilized to inhibit the hypertrophic differentiation of chondrocytes. Immunohistochemistry method was used to analyze the expression of Cbfα1. RESULTS AND CONCLUSION:After induction with retinoic acid and interleukin-1α, the chondrocytes in the negative control virus group appeared to have hypertrophy and the expression of Cbfα1 was positive. In the Ad-Cbα1-siRNA group, the expression of Cbfα1 was negative. These findings suggest that the inhibition of Cbfα1 by RNA interference can be a powerful way to prevent the hypertrophic differentiation of chondrocytes .

To explore the treatment of refractory anemia (RA), 7 cases of myelodysplastic syndrome (subtype of refractory anemia) were treated in combination of cyclosporin A (CsA) with stanozolol. Duration of treatment with CsA was 5 months-3 years (mean 13 months). The results showed that among 7 cases 6 were effective, 1 case no responded to treatment. 3 cases out of 6 effective cases achieved complete remission without transfusion dependence, 1 cases achieved partial remission, 2 cases were improved. During the investigation signs of leukemia ot other malignant tumors not were found in all cases. In conclusion, CsA treatment is effective for part cases of RA, side effects of drugs are tolerable for patients.
Adolescent ; Adult ; Aged ; Anemia, Refractory ; drug therapy ; Cyclosporine ; administration & dosage ; Drug Therapy, Combination ; Female ; Humans ; Male ; Middle Aged ; Stanozolol ; administration & dosage

OBJECTIVETo explore the risk factors for nosocomial infection caused by extended-spectrum beta-lactamases (ESBLs)-producing bacteria in hospitals of Zhejiang province.

METHODSOne hundred and eighty-five cases with nosocomial infection (108 men and 77 women, with an average age of 55 +/- 17 years) caused by positive-ESBLs bacteria, including 59 cases of respiratory infection, 71 with urinary infection, ten with blood infection, 30 with wound infection and 59 with other infection, and 77 controls with nosocomial infection (54 men and 23 women, with an average age of 54 +/- 20 years) caused by negative-ESBLs bacteria, including 38 cases of respiratory infection, 20 with urinary infection, six with blood infection, eight with wound infection and five with other infection, from six hospitals in Zhejiang Province were studied during May 1999 to May 2000. Data were analyzed with unconditional logistic regression and principal component analysis (PCA).

RESULTSMultivariate unconditional logistic regression analysis showed that the independent risk factors for nosocomial infection were use of the third generation cephalosporins for more than three days (odds ratio, OR 4.52, 95% confidence interval of OR 2.30 - 8.89), combined use of antibiotics (OR 2.86, 95% CI 1.51 - 5.43), use of quinolones for more than three days (OR 2.44, 95% CI 1.18 - 5.04), use of adrenal cortical hormone (OR 2.16, 95% CI 1.08 - 4.31) and oxygen inhalation (OR 2.56, 95% CI 1.14 - 5.72). Five principal components were extracted from the 14 risk factors for nosocomial infection with ESBLs-producing bacteria by principal component analysis, with a contribution of cumulative variance of 60.2%, and arranged in an order as follows, use of ventilator, tracheal intubation or tracheotomy, oxygen inhalation, retaining needle in vein, indwelling urethral catheter, use of the third generation cephalosporins over three days, hospitalization over ten days, use of quinolones over three days, combined use of antibiotics, use of aminoglycosides antibiotic over a week, use of adrenal cortical hormone, catheterized examination and prophylactic use of antibiotics.

CONCLUSIONSNosocomial infection with ESBLs-producing bacteria could attribute to multiple factors, mainly to invasive manipulation and use of antibiotics.

Case-Control Studies ; Cephalosporins ; pharmacology ; China ; epidemiology ; Cross Infection ; epidemiology ; microbiology ; Drug Resistance, Bacterial ; physiology ; Drug Therapy, Combination ; pharmacology ; Drug Utilization ; Female ; Humans ; Length of Stay ; Male ; Microbial Sensitivity Tests ; Middle Aged ; Multivariate Analysis ; Risk Factors ; beta-Lactamases ; metabolism

Objective:To improve the genetic stability of HBV gene in transgenic mice.Methods:HBV transgenic mice were bred by backcross and double cross.The HBV gene expression and replication were studied with real-time PCR,ELISA and chemiluminescence.Results:The HBV transgenic mice have stably bred to 23rd generation.The serum HBsAg level is 4122.31?2044.74IU/ml;The rate of HBV transgenic mice whose serum HBV DNA reach 104~106copies/ml was 93.93%.The HBV replication and expression were improved markedly.There is no difference between male and female mice about serum HBsAg level.Conclusion:After breeding the HBV gene was expressed stably with high-level in transgenic mice.

OBJECTIVETo observe effects of blood circulation promoting compounds combined with medicinal guides on content of bone glaprotein (BGP), bone morphogenetic protein-2 (BPM-2) and expression of BMP-2 mRNA in rabbits with femoral head necrosis, and explore its mechanism.

METHODSNinety-eight healthy Spragur-Dawley male rabbits were collected and weighted 2.2 to 2.8 kg. Eighty-four rabbits were built femoral head necrosis model by freezing left femoral head in liquid nitrogen, then randomly divided into 6 groups, 14 in each group. The 6 groups included model group,promoting blood circulation to remove meridian obstruction group,promoting blood circulation to remove meridian obstruction combined with achyranthes bidentata group,radix angelicae pubescentis, asarum group, and platycodon grandiflorum group,other 14 rabbits were sham operation group. While drug groups were administrated corresponding Chinese herb after molding,model group and shamp operation group were given saline. Recombinant human granulocyte-colony stimulating factor ( 30 microg x kg(-1) x d(-1))were injected into all rabbits for 7 days. Samples were taken on the second and fourth week,the content of BGP and BMP-2 were detected by enzyme-linked immunosorbent assay (ELSA) and radioimmunoassay (RIA), histopathological changes of left femoral head were observed by Hematoxylin and Eeosin staining (HE), and expression of BMP-2 mRNA were tested by fluorescence in situ hybridization.

RESULTSCompared with sham operation group, the rate of empty lacunae femoral head were obviously increased in model group, and the content of BGP were increased on the second week, and BMP-2 and BMP-2 mRNA were decreased on the fourth week. Compared with model group, the content of BGP, BMP-2 and BMP-2 mRNA were higher both of the second and fourth week in promoting blood circulation to remove meridian obstruction group. The rate of empty la- cunae femoral head were lower in achyranthes bidentata group, BGP, BMP-2 and BMP-2 mRNA were higher on the fourth week. The rate of empty lacunae femoral head were lower in platycodon grandiflorum group, and BGP were decreased on the second and fourth week, BMP-2 were lower on the second week ,while BMP-2 mRNA were decreased on the fourth week; the content of BMP-2 and BMP-2 mRNA were increased in radix angelicae pubescentis group on the second week; while there was no change in asarum group.

CONCLUSIONRadix angelicae pubescentis can increase the content of BGP, BMP-2 and expression of BMP-2 mRNA ,which is an effective mechanism of preventing femoral head necrosis.

Animals ; Blood Circulation ; drug effects ; Bone Morphogenetic Protein 2 ; analysis ; genetics ; Femur Head Necrosis ; drug therapy ; pathology ; physiopathology ; Male ; Meridians ; Osteocalcin ; blood ; Osteogenesis ; drug effects ; Rabbits

This paper is aimed to investigate the feasibility of applying the small intestine submucosa (SIS) as the scaffold in constructing tissue engineering cartilage in vitro. We obtained SIS from the small intestine of specific pathogen-free pigs. Then we isolated tunica submucosa layer from the mucosal, muscular, and serosal layers by gentle mechanic abrasion. The SIS was made acellular by combination of detergent and enzyme digestion. The chondrocytes were seeded onto the SIS and were cultured for 3 weeks. The cell growth, attachment and distribution were detected by histochemical stain, immunohistochemical stain and scan electron microscope. The chondrocytes could adhere and grow well on the matrix surface, and synthesize a large of the GAG and type U collagen. However, the chondrocytes grew only on the surface andsuperficial layer of the scaffold, they did not move into the inner part of the scaffold. It could be concluded that SIS has good cellular compatibility without cytotoxicity and provides temporary substrate to which these anchorage-dependent cells can adhere, and stimulate the chondrocytes anchored on the scaffold to proliferate and keep differentiated phenotype. Further study will be needed to promote the ability of chondrocyte chemotaxis in order to distribute the chondrocytes into the whole scaffold uniformly.
Animals ; Cell Adhesion ; Cell Culture Techniques ; Cell Proliferation ; Chondrocytes ; cytology ; Chondrogenesis ; physiology ; Intestinal Mucosa ; cytology ; Intestine, Small ; cytology ; Swine ; Tissue Engineering ; methods ; Tissue Scaffolds

OBJECTIVETo evaluate and compare the histopathology effects and mechanisms of the methods of "invigorating spleen to remove phlem & promoting blood circulation to remove meridian obstruction" and "invigorate the kidney & promoting blood circulation to remove meridian obstruction" preventing hormone induced femoral head necrosis in hens.

METHODSSixty-four healthy hens were randomly divided into 4 groups: blank control group, model group, Jianpi group (with therapeutics of invigorating spleen to remove phlem), Bushen group (with the effect of warming kidney for duresis). All hens were injected intramuscularly with Medrat once a week for 16 weeks but normal saline in blank control group. Bilateral femoral heads were dissected on 8 weeks or 16 weeks. Paraffin tissue sections were prepared to detect histopathologic change with hematoxylin and eosin, or mason staining.

RESULTSHistological analysis showed that Huogu recipe I and Huogu recipe II can promote osteogenesis and repair osteonecrosis, increase blood circulation of bone marrow, and inhibit pimelosis of bone marrow. Compared with blank control group, the areas of adipose cells increased significantly (t = -12.9, P < 0.01), the area of immature collagen increased significantly (t = -2.0, P < 0.05) and the ratio of empty lacuna in medullary cavity (t = -3.7, P < 0.05). Compared with model group, both the area of adipose cells and the ratio of empty lacuna decreased in Jianpi group and Bushen group (F = 26.8, 13.5, P < 0.01), so it was with the Bushen group immature collagen (F = 4.6, P < 0.01).

CONCLUSIONBoth the methods of "invigorating spleen to remove phlem & promoting blood circulation to remove meridian obstruction" and "invigorate the kidney & promoting blood circulation to remove meridian obstruction" can prevent hormone induced femoral head necrosis. The time taking effect in the method of "invigorating spleen to remove phlem" was shorter.

Animals ; Chickens ; Drugs, Chinese Herbal ; Female ; Femur Head ; pathology ; Femur Head Necrosis ; chemically induced ; drug therapy ; pathology ; Glucocorticoids ; adverse effects ; Medicine, Chinese Traditional

OBJECTIVETo study the mechanism of Huogu I formula (I) in treating osteonecrosis of femoral head.

METHODSForty-eight healthy female Leghorn chickens were randomly divided into control group, model group and Huogu I group, and each group consisted of 16 chickens. At the meantime of model establishment, chickens of the Huogu I group were administrated with decoction, while the model and control group with distilled water by gavage. At the 8th and 16th week after medication, blood samples were obtained for blood lipid detection while both sides of femoral head were harvested for the rest of examinations. Specifically, expressions of bone morphogenetic protein-2 (BMP2), transforming growth factor beta1 (TGFβ(1)), Smad4 and Smad7 were evaluated by immunohistochemistry, while expression of osteoprotegerin/receptor activator of nuclear factor kappaB ligand (OPG/RANKL) mRNA was detected by in situ hybridization.

RESULTSCompared with the control group, serum levels of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) in the model group rose significantly. Positive cell counting of BMP2, TGFβ(1), Smad4 and OPG in femoral head of the model group dropped prominently. Positive cell counting of Smad7 and RANKL increased dramatically. In contrast with the model group, levels of TC, TG and LDL-C in Huogu I group reduced significantly. Positive cell counting of BMP2, TGFβ(1), Smad4 and OPG in femoral head of the Huogu I group increased prominently. Indices of Smad7 and RANKL both decreased significantly. Especially at the 8th week, these variations were more significant.

CONCLUSIONHuogu I formula is effective in promoting repair of necrotic femoral head by regulating the expressions of BMP2, TGFβ(1), Smads and OPG/RANKL of osteoclast in femoral head.

Animals ; Bone Morphogenetic Protein 2 ; metabolism ; Bone Regeneration ; drug effects ; physiology ; Chickens ; Chondrocytes ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; Female ; Femur Head Necrosis ; chemically induced ; drug therapy ; metabolism ; Lipid Metabolism ; physiology ; Osteocytes ; metabolism ; Osteoprotegerin ; genetics ; metabolism ; Receptor Activator of Nuclear Factor-kappa B ; genetics ; metabolism ; Smad4 Protein ; metabolism ; Smad7 Protein ; metabolism ; Steroids ; pharmacology ; Transforming Growth Factor beta1 ; metabolism