3.Construction of a rabbit model of steroid-induced osteonecrosis of the femoral head by combining different concentrations of glucocorticoids with horse serum
Liangquan CAO ; Bin DU ; Guangquan SUN ; Xin LIU ; Guoqing CHEN ; Lei GU ; Bing LIU
Chinese Journal of Tissue Engineering Research 2017;21(8):1229-1235
BACKGROUND: Glucocorticoid has been shown to be a major factor of osteonecrosis of the femoral head (ONFH), so constructing a reliable, effective and low mortality ONFH model will be helpful for searching for a better treatment strategy of ONFH.OBJECTIVE: To construct a rabbit model of early ONFH by intravenous injection of different concentrations of glucocorticoids and horse serum.METHODS: Thirty healthy male New Zealand rabbits were randomly allotted to six groups, followed by given the injection of 10 mg/kg horse serum combined with 5 mg/kg dexamethasone (group A), 10 mg/kg horse serum combined with 10 mg/kg dexamethasone (group B), 20 mg/kg horse serum combined with 5 mg/kg dexamethasone (group C), 20 mg/kg horse serum combined with 10 mg/kg dexamethasone (group D), 10 mg/kg dexamethasone (group E), and 2 mL/kg normal saline (control group) via ear veins, respectively.RESULTS AND CONCLUSION: Abnormal MRI signal of the femoral head appeared in the group D at postoperative 2 weeks, while abnormal signal was seen at postoperative 4 weeks in the other groups except the controls. Six weeks postoperatively, 80% rabbits in the group D showed abnormal signals, which were significantly more than those in the groups C (50%), B (40%), A (25%), and E (20%) (P < 0.05). The serum levels of triglyceride and total cholesterol in the groups A, B, C, D were significantly higher than those in the control group at 3, 7, 14 and 30 days after injection (P <0.05). Compared with the control group, the ratio of empty lacuna sigmificantly increased in the group D (P < 0.05).These results indicate that the injection of high concentration of horse serum combined with the high concentration of dexamethasone is successful and safe to make an animal model of early ONFH.
4.Studies on transdermal delivery of ferulic acid through rat skin treated by microneedle arrays.
Bing YANG ; Shou-ying DU ; Jie BAI ; Ke-xin SHANG ; Yang LU ; Peng-yue LI
China Journal of Chinese Materia Medica 2014;39(24):4773-4777
In order to investigate the characteristics of transdermal delivery of ferulic acid under the treated of microneedle arrays and the influence on permeability of rat skin capillaries, improved Franz-cells were used in the transdermal delivery experiment with the rat skin of abdominal wall and the length of microneedle arrays, different insertion forces, retention time were studied in the influence of characteristics of transdermal delivery of FA. The amount of FA was determined by HPLC system. Intravenous injection Evans blue and FA was added after microneedle arrays treated. Established inflammation model was built by daubing dimethylbenzene. The amount of Evans blue in the rat skin was read at 590 nm wavelength with a Multiskan Go microplate reader. Compared with passive diffusion group the skin pretreated with microneedle arrays had a remarkable enhancement of FA transport (P <0.01). The accumulation of FA increased with the enhancement of insertion force as to as the increase of retention time. Microneedle arrays with different length had a remarkable enhancement of FA transport, but was not related to the increase of the length. The research of FA on the reduce of permeability of rat skin capillaries indicated that the skin pretreated with microneedle arrays could reduce the content of Evans blue in the skins of rat significantly compared with the untreated group. The permeation rate of ferulic acid transdermal delivery had remarkable increase under the treated of microneedle arrays and the length of microneedle arrays ,the retention time so as to the insertion force were important to the transdermal delivery of ferulic acid.
Administration, Cutaneous
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Animals
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Coumaric Acids
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administration & dosage
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pharmacokinetics
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Male
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Needles
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Rats
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Rats, Sprague-Dawley
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Skin Absorption
5.Construction of eucaryotic expression plasmid carrying the BMP7 gene and expression in mesenchymal stem cells.
Shu-xun HOU ; Da-ming SUN ; Gui-xin DU ; Yi-gang TONG ; Xiao-bing FU
Chinese Journal of Surgery 2003;41(6):445-448
OBJECTIVESTo construct an eucaryotic expression plasmid carrying the BMP7 gene and express in MSCs.
METHODSThe BMP7 gene was cloned into the eucaryotic expression vector pcDNA3.1. At the same time, mesenchymal stem cells (MSCs) were isolated and cultured in vitro. The plasmid carrying the BMP7 gene was transfected into MSCs.
RESULTSPCR and digesting demonstrated that the eucaryotic expression plasmid -pcDNA-BMP7 was obtained. RT-PCR and immunohistochemical methods showed that the BMP7 gene was expressed in MSCs.
CONCLUSIONConstruction of an eucaryotic expression plasmid carrying BMP7 gene and expression in MSCs provide a sound basis for gene therapy using the BMP7 gene and the ideal seeds for tissue engineering.
Bone Morphogenetic Protein 7 ; Bone Morphogenetic Proteins ; genetics ; Genetic Therapy ; Humans ; Mesenchymal Stromal Cells ; metabolism ; Plasmids ; Polymerase Chain Reaction ; Tissue Engineering ; Transforming Growth Factor beta
6.Study of quality standards on Qianshan Huoxue plaster.
Xin-Tong FU ; Yan LI ; Xiao-Wei DU ; Xiao-Qian ZHANG ; Ren-Bing SHI
China Journal of Chinese Materia Medica 2006;31(17):1431-1433
OBJECTIVETo establish the methods of identification and assay in Qianshan Huoxue Gao.
METHODUsing TLC to identify Sanchi, Dragon's Blood and using HPLC to determine the content of ginsenoside Rg1.
RESULTThe linear range of ginsenoside Rg1 was from 0.153 9 to 1.026 microg. The average recovery was 97.4%, RSD was 2.1%.
CONCLUSIONThe methods are simple and have good reproducibility.
Administration, Topical ; Arecaceae ; chemistry ; Chromatography, High Pressure Liquid ; Chromatography, Thin Layer ; Drug Combinations ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; isolation & purification ; Ginsenosides ; analysis ; Panax notoginseng ; chemistry ; Plants, Medicinal ; chemistry ; Quality Control ; Reproducibility of Results
8.Diagnostic value of absent ductular reaction at hepatocellular-stromal boundaries in early stage hepatocellular carcinoma.
Qin ZHANG ; Zhe MA ; Qi XIN ; Gui-qiu LIU ; Bing-bing LIU ; Ying-tang GAO ; Chuan-shan ZHANG ; Zhi DU
Chinese Journal of Hepatology 2013;21(12):924-928
OBJECTIVETo investigate the role of absent ductular reaction (DR) at hepatocellular-stromal boundaries in early stage hepatocellular carcinoma (HCC) with cirrhosis in patients with chronic hepatitis B.
METHODSCytokeratin (CK)7 and CK19 expression was detected by the SP immunohistochemistry method in 112 hepatic nodules taken from 20 cases of early HCC, 26 cases of HCC with nodules more than 3 cm, 20 cases of high-grade dysplastic nodule (HGDN), 26 cases of low-grade dysplastic nodule (LGDN), and 20 cases of cirrhosis (CIR). DR/CK7 and DR/CK19 were assessed separately on a semi-quantitative scale and statistically analyzed.
RESULTSThe mean age of the patients in the study was 53.71 years-old, and the study population consisted of 73 males and 39 females. The follow-up time ranged from 3 to 90 months. Positive CK7 and CK19 staining was detected in the cytoplasm of DR-positive hepatobiliary cells, interlobular bile duct, and a portion of hepatic cells. All of the DR/CK7- and DR/CK19-positive cells were localized around the non-invasive nodules. Specimens with focal or diffuse DR/CK7- and DR/CK19-loss had more robust stromal invasion. Specimens from early HCC cases showed greater DR/CK19 loss than specimens from HGDN cases, LGDN cases and CIR cases (all P less than 0.01). DR/CK7 loss of early HCC was less than HCC with nodules more than 3 cm (P less than 0.05), and more than LGDN cases and CIR cases (both P less than 0.01).The area under the receiver operating characteristic curve of DR/CK7 was very similar to that of DR/CK19 (P more than 0.05). Pearson's correlation analysis indicated that DR/CK7 and DR/CK19 were positively correlated with tumor-free time (P less than 0.01) and negatively correlated with early recurrence time as well as death rate (both P less than 0.01). Furthermore, cases showing DR/CK7 or DR/CK19 loss had lower overall survival rate and tumor-free survival rate (P less than 0.01) and higher early recurrence rate (P less than 0.01).
CONCLUSIONDR/CK7 and DR/CK19 immunostaining may help to distinguish non-invasive HGDNs from both minimally-invasive and overtly-invasive HCCs by identifying small foci of invasion and predicting increased risk of invasiveness.
Adult ; Aged ; Aged, 80 and over ; Bile Ducts, Intrahepatic ; pathology ; Carcinoma, Hepatocellular ; diagnosis ; pathology ; virology ; Early Diagnosis ; Female ; Hepatitis B, Chronic ; pathology ; Humans ; Immunohistochemistry ; Keratin-19 ; metabolism ; Keratin-7 ; metabolism ; Liver Neoplasms ; diagnosis ; pathology ; virology ; Male ; Middle Aged
9.Association between CACNB2 gene polymorphisms and essential hypertension.
Qian SUN ; Xin WANG ; Ying HUANG ; Yunliang HU ; Jifei TANG ; Yan LIN ; Yuxin NIU ; Xiaoou WANG ; Bing DU
Chinese Journal of Medical Genetics 2013;30(3):340-344
OBJECTIVETo assess the association between single nucleotide polymorphisms (SNPs) of calcium channel β 2 subunit (CACNB2) gene and essential hypertension (EH) in ethnic Han Chinese in Wenzhou area, and to study the influence of rs7069292 alleles on gene expression with luciferase reporter technique.
METHODSSixty hundred and thirty seven Han Chinese with EH and 600 normal controls were enrolled. Genotypes of 6 SNP within CACNB2 gene including rs2228645, rs2357928, rs7069292, rs7099380, rs10764319 and rs11014166 were determined with matrix assisted laser desorption ionization/time of flight mass spectrometer (MALDI-TOF MS). A luciferase reporter gene plasmid containing the fragment flanking rs7069292 (-2831 bp to -2460 bp) in the 5' regulatory region of CACNB2 was constructed.
RESULTSCompared with the control, CT and TT genotypes for the rs7069292 locus were significantly more common in EH group (5.20% vs. 2.17%, 2.59% vs. 1.08%, P< 0.05). CC genotype was not found. Promoter activity for allele C of the rs7069292 locus was significantly increased compared with allele T (P< 0.05). No significant difference was detected for other 5 SNPs in terms of genotypes and allele frequency.
CONCLUSIONThe rs7069292 CT polymorphism of the CACNB2 gene is associated with EH in ethnic Han Chinese from Wenzhou area. A T>C mutation may affect the expression of CACNB2.
Aged ; Alleles ; Base Sequence ; Calcium Channels, L-Type ; genetics ; Case-Control Studies ; Cell Line ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Hypertension ; genetics ; Male ; Middle Aged ; Polymorphism, Single Nucleotide
10.Preparation of two antigens--Ag85b and HspX of Mycobacterium tuberculosis H37Rv and the effects of their co-administration with adjuvants in mice.
Lei CHEN ; Miao XU ; Wei-Xin DU ; Bao-Wen CHEN ; Zhi-Yu WANG ; Ya-Jun WANG ; Na DONG ; Cheng SU ; Xiao-Bing SHEN ; Guo-Zhi WANG
Acta Academiae Medicinae Sinicae 2009;31(4):403-409
OBJECTIVETo synthesize two antigens-Ag85b and HspX of Mycobacterium tuberculosis H37Rv with molecular biological methods and to observe their biologic activity after co-administration of adjuvants (aluminum and/or CpG) in mice.
METHODSRecombinant expression plasmids pET30a-Ag85b and pET30a-HspX were constructed. The objective DNA fragments was characterized with restriction enzyme. Then the recombinant plasmids were transformed into E. coli BL-21, and two proteins were expressed by induction of isopropyl beta-D-1-thiogalactopyranoside. After purification with anion exchange column Source30, QHP, and hydrophobic chromatography column, two proteins were identified by amino acid sequencing. After the successful preparation of these two antigens, they were co-administered in mice with adjuvants of aluminum and/or CpG (Ag85b, Ag85b + Al, Ag85b + CpG, Ag85b + Al + CpG; HspX, HspX + Al, HspX + CpG, HspX + Al + CpG); one group received normal saline and served as the control. Splenic lymphocytes were isolated for enzyme-linked immunosorbent spot assay to detect the secreted specific interferon-gamma (IFN-gamma); in addition, lymphocytes proliferation test was performed to observe lymphocytes proliferation after in vitro stimulated with two antigens.
RESULTSThe purity of two proteins reached 95% after purification. The N-terminal amino acid sequence (15 aa) of the purified proteins was same as the target sequence. For Ag85b, the secreted specific IFN-gamma from isolated splenic lymphocytes after having been stimulated in vitro with Ag85b (80 microg/ml) remarkably increased in Ag85b + CpG group, Ag85b + Al group, and Ag85b + CpG + Al group; the changes were significantly different between these three groups and control group (P < 0.05). For HspX, the changes were significantly different between HspX + Al + CpG group and normal sodium group, although remarked increase of IFN-gamma was also observed in HspX group, HspX + Al group, and HspX + CpG group.
CONCLUSIONSAg85b and HspX were successfully expressed and purified. A cell-mediated immunity may be induced when the antigens are co-administered with adjuvants of aluminum and/or CpG in mice, indicating that the recombinant proteins are bioactive.
Acyltransferases ; administration & dosage ; isolation & purification ; therapeutic use ; Adjuvants, Immunologic ; therapeutic use ; Animals ; Antigens, Bacterial ; administration & dosage ; isolation & purification ; therapeutic use ; Bacterial Proteins ; administration & dosage ; isolation & purification ; therapeutic use ; Escherichia coli ; Immunity, Cellular ; Interferon-gamma ; Mice ; Mycobacterium tuberculosis ; immunology ; metabolism ; Recombinant Proteins ; administration & dosage ; isolation & purification ; therapeutic use