Diagnosis, Differential ; Esophageal Neoplasms ; pathology ; surgery ; Granuloma, Pyogenic ; pathology ; surgery ; Hemangioma, Capillary ; pathology ; Hemangiosarcoma ; pathology ; Humans ; Male ; Middle Aged ; Sarcoma, Kaposi ; pathology

OBJECTIVETo establish and evaluate a rabbit model of fecal incontinence.

METHODSTwelve normal adult male New Zealand rabbits were randomly divided into experimental group and control group. The nerve innervating the external anal sphincter, namely the fourth sacral nerve, was functionally located and selectively damaged with local injection of 50 g/L ropivacaine in the experimental group, and normal saline injection was administered in the control group. The changes in the resting anal pressure was examined before and after the surgery, and the electromyogram (EMG) of the external anal sphincter was recorded for comparison with the pathological changes of the fourth sacral nerve.

RESULTSCompared with the control group, the experimental group exhibited significantly decreased resting anal pressure after the surgery. The EMG of the experimental group showed abnormal nerve conduction velocity of the fourth sacral nerve, suggesting successful nerve block. Transmission electron microscope revealed irreversible pathological changes in the ultrastructure of the axons of the fourth sacral nerve.

CONCLUSIONThis method allows successful establishment of fecal incontinence in rabbits, which facilitates further in vivo study of artificial sphincters for treatment of anal incontinence.

Amides ; administration & dosage ; Anal Canal ; physiopathology ; Animals ; Disease Models, Animal ; Electromyography ; Fecal Incontinence ; Lumbosacral Plexus ; Male ; Nerve Block ; Rabbits ; Random Allocation

In order to efficiently control the quality of the Tibetan medicine Gentianae Szechenyii Flos, the quality standard was established in this study. The tests of water content, total ash and ethanol-soluble extractives of the crude drugs were carried out based on the methods recorded in appendix of Chinese Pharmacopeia (2010 edition, volume 1). The TLC method was established by using reference drug and gentiournoside A as reference substance, and a mixture of ethyl acetate-methanol-water-formic acid (7: 1.5: 1: 0.2) as the developing solvent system on silica gel G TLC plate. The content of gentiournoside A was assayed by HPLC on a Ultimate XB-C18 (4.6 mm x 250 mm, 5 μm) column, using methanol-water (0.02% phosphoric acid) (52:48) as the mobile phase at a flow rate of 1.0 mL x min(-1). The column temperature is 25 degrees C and the detection wavelength is at 240 nm. As a result, gentiournoside A and the other constituents were separated and presented the same fluorescence light comparing with the reference substance on TLC detected under the UV light(366 nm). The methodology validation for the assay of gentiournoside A showed that it was in a good linear correlation in the range of 10.01-400.32 mg x L(-1) with the regression equation of Y = 1 539.5X - 33.339 (r = 0.999 7), and the average recovery was 99.68% (RSD 1.92%). The mass fractions of gentiournoside A, water content, ethanol-soluble extractives of 19 batches samples were varied in the ranges of 14.48-31.51 mg x g(-1), 11.25% -12.74% and 24.21% - 31.60%, respectively, and total ash was 4.64% - 6.12% detected from 10 batches samples. The recommended standards of quantitative indexes are that the mass fractions of gentiournoside A and extractives are not less than 15.0 mg x g(-1) (1.5%) and 21.0%, respectively; the water and total ash are not more than 13.0% and 6.0%, respectively.
Chromatography, High Pressure Liquid ; Chromatography, Thin Layer ; Drugs, Chinese Herbal ; chemistry ; standards ; Flowers ; chemistry ; Gentiana ; chemistry ; Medicine, Tibetan Traditional ; Quality Control

Gentianae Urnulae Herba, dried whole herb of Gentiana urnula,is a commonly used Tibetan medicine. However, only the character identification is used as quality control standard officially at present. As a part of project for the Chinese Pharmacopoeia (2015 edition), the quality standard of this species was established in this study. The tests of water content, total ash, acid-insoluble ash and ethanol-soluble extractives of the crude drugs were carried out following the methods recorded in appendix of Chinese Pharmacopeia (2010 edition, volume 1). The TLC identification method was established by using gentiournoside A as reference substance, and a mixture of ethyl acetate-methanol-water-formic acid(7:1. 5:1: 0. 2) as the developing solvent system on silica gel G TLC plate. The content of gentiournoside A was assayed by HPLC on an Agilent Zorbax SB-C18 (4.6 mm x 250 mm,5 μm) column, using acetonitrile-water (0.1% phosphoric acid) (26:74) as the mobile phase at a flow rate of 1.0 mL x min(-1). The column temperature is at 30 degrees C and the detection wavelength is at 240 nm. As a result, gentiournoside A and the other constituents were separated and presented the same fluorescence light comparing with the reference substance on TLC detected under the UV light(366 nm). The methodology validation for the assay of gentiournoside A showed that it was in a good linear correlation in the range of 0.009 95-0.398 g x L(-1) with the regression equation of Y = 1 467.1X +41.407(r = 0.999 9), and the average recovery was 98. 3% (RSD 2.2%). The mass fractions of gentiournoside A, water content, ethanol-soluble extractives of 15 batches samples were varied in the ranges of 0.175% -1.83%, 8.60% - 9.93% and 29.2% - 35.2%, respectively. Total ash and acid-insoluble ash were 10.2% - 17.2% and 5.26% - 10.8% detected from 10 batches samples. The recommended standards of quantitative indexes are that the mass fractions of gentiournoside A and extractives are not less than 0.80% and 26.0%, respectively; the water, total ash and acid-insoluble ash are not more than 12.0%, 15.0% and 8.0%, respectively.
China ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; standards ; Humans ; Medicine, Tibetan Traditional ; standards ; Plants, Medicinal ; chemistry ; Quality Control

OBJECTIVETo study the selective cytotoxic effect of lentivirus-mediated double suicide gene (CD/TK) against human gastric carcinoma cells SGC-7901 in vitro.

METHODSSGC-7901 cells were infected with FGW-KDRP-CD/TK vector and the infection efficiency was observed under a fluorescence microscope. The morphological changes of the infected cells were observed by Giemsa staining. Flow cytometry (FCM) was employed for cell cycle analysis, and the expression of CD/TK was detected by RT-PCR. The infected cells were then treated with the prodrugs ganciclovir (GCV) and/or 5-fluorocytosine (5-FC) at different concentrations, and the cytotoxic effects were evaluated using MTT method.

RESULTSThe infection efficiency of the lentiviral vector in SGC-7901 cells increased with the titer of the virus, which produced no significant effect on the cancer cell morphology in vitro or on the percentages of G0-G1, G2-M and S phase cells (P>0.05). RT-PCR demonstrated the expression of CD/TK gene in SGC-7901 cells infected by FGW-KDRP-CD/TK. The infected cells were highly sensitive to the prodrugs with a dose-dependent cytotoxic effect within a specific concentration range of the drugs, whereas the non-infected cells were not sensitive to the prodrugs. Combined use of the two prodrugs produced an obviously stronger inhibitory effect than either of the them (P<0.05). When combined, GCV and 5-FC at the concentration of 0.1+40, 1+80, 10+160, and 100+320 mg/L demonstrated a synergetic effect with a CDI<1.

CONCLUSIONLentivirus-mediated CD/TK fusion gene system can selectively kill gastric cancer cells, and the two prodrugs show a synergistic cytotoxic effect.

Adenocarcinoma ; genetics ; pathology ; Cell Line, Tumor ; Cytosine Deaminase ; biosynthesis ; genetics ; Cytotoxins ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; Lentivirus ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Stomach Neoplasms ; genetics ; pathology ; Thymidine Kinase ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; metabolism

Aged ; External Fixators ; Female ; Follow-Up Studies ; Fractures, Bone ; complications ; physiopathology ; surgery ; therapy ; Humans ; Male ; Middle Aged ; Osteoporosis ; complications ; Tomography, X-Ray Computed ; Treatment Outcome

OBJECTIVETo evaluate the effect of adenovirus-mediated double suicide gene (CD/TK) for selective killing of breast cancer cells.

METHODSVascular endothelial growth factor (VEGF)-expressing MCF-7 cells and normal human mammary epithelial cells that did not express VEGF were infected with the adenovirus containing VEGFP-CD/TK-GFP genes. CD/TK gene expression in the infected cells was detected by RT-PCR. After treatment of the infected cells with GCV and/or 5-FC, the cell growth status was evaluated using MTT assay, and the cell cycle changes were detected with flow cytometry. In nude mice bearing human breast cancer, the recombinant adenovirus vector was injected directly into the tumor followed by intraperitoneal injection of the prodrugs GCV and/or 5-FC, and the subsequent tumor growth was observed.

RESULTSThe recombinant adenovirus achieved similar infection rates in MCF-7 and human mammary epithelial cells, and the rates increased gradually with the multiplicity of infection (MOI) of the virus. RT-PCR demonstrated the presence of CD/TK gene product in infected MCF-7 cells, but not in the infected mammary epithelial cells. The infected MCF-7 cells, but not the mammary epithelial cells, were highly sensitive to the pro-drugs. The CD/TK fusion gene system showed significantly greater efficiency than either of the single suicide gene in killing the target cells (P<0.01). At the MOI of 100, treatment of the infected cells with the pro-drugs resulted in increased cell percentage in G(0)-G(1) phase and decreased percentage in S phase. In nude mice bearing MCF-7 cell-derived subcutaneous tumor, treatment with the double suicide gene system significantly inhibited the tumor growth, showing much stronger effect than either of the single suicide gene (P<0.01).

CONCLUSIONThe adenovirus-mediated CD/TK double suicide gene driven by VEGF promoter combined with GCV and 5-FC treatment can be an effective therapy against experimental breast cancer, and produces much greater efficacy than the single suicide gene CD/TK combined with GCV or 5-FC.

Adenoviridae ; genetics ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cytosine Deaminase ; genetics ; metabolism ; Female ; Flow Cytometry ; Flucytosine ; pharmacology ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Recombinant Fusion Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Thymidine Kinase ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

To improve the targeting of adenovirus vector for gene therapy, a fusion gene sCAR-EGF, in which epidermal growth factor gene was fused to the 3' end of extracellular Coxsackie virus-adenovirus receptor gene, was constructed and cloned into shuttle plasmid pDC315 to obtain a recombinant plasmid pDC315-sCAR-EGF. With the AdMax system, AD-293 cells were co-transfected with pDC315-sCAR-EGF and adenovirus genomic plasmid pBHGloxdeltaE13cre. Through high efficiency site specific recombination, a replication-defective adenovirus Ad5-CMV-sCAR-EGF was constructed. The recombinant adenovirus was analyzed by PCR and Western blotting, the results indicated that Ad5-CMV-sCAR-EGF contained the fusion gene sCAR-EGF, and the adenovirus infected cells was induced to produce and secrete the fusion protein into the supernatant. We have demonstrated that the fusion protein sCAR-EGF is helpful for elevating the infection efficiency of Ad5-CMV-luc with the reporter gene in vitro, which providing a new approach to the gene therapy for tumors overexpressing EGFR.
Adenoviridae ; genetics ; Cell Line ; Coxsackie and Adenovirus Receptor-Like Membrane Protein ; Enzyme-Linked Immunosorbent Assay ; Epidermal Growth Factor ; analysis ; genetics ; Genetic Therapy ; Humans ; Neoplasms ; therapy ; Polymerase Chain Reaction ; Receptors, Virus ; analysis ; genetics ; Recombinant Fusion Proteins ; biosynthesis

ObjectiveTo investigate the influence of recombinant thioredoxin (TRX)on apoptosis of myocardium cell in viral myocarditis of mice.MethodsTwenty-four Balb/c mice,weighting 12 - 14 g,were randomly divided into 3 groups:the control group,the virus group and the protective group,8 mice in each group.The virus group and the protective group were injected with 0.1 ml 100TCID50 Coxackie virus B3 (CVB3)intraperitoneally,and the control group was injected equal volume of saline.Therewithal the protective group was injected with TRX(2 mg/kg) by tail vein,and the virus group was injected saline the same way.After 14 days all mice were killed and hearts were taken.Changes of myocardial histopathology was observed with optical microscope,cell apoptosis was checked by TUNEL technique,and the expression of apoptosis-related proteins (Bcl-2,caspase-3)in infiltrated cell of myocardium was determined by immunohistochemistry.Results(①)Lymphocyte infiltration and necrosis were observed in survivals of the virus group,sporadic coagulation necrosis and ballooning degeneration of cells were observed in the protective group,however no myocardial lesion was found in the control group.(②)TUNEL technique showed that the positive ratio of apoptosis in the virus group and the protective group[(90.23 ± 3.63)％,(20.02 ± 2.41)％] was significantly higher than that of the control group(0.00 ± 0.00,all P ＜ 0.05),the positive ratio of apoptosis in the protective group was significantly lower than that of the virus group (P ＜ 0.05 ).(③)Immunohistochemistry showed that the expression of protein Bcl-2(+,++,+++) in the virus group and the protective group was significantly higher than that of the control group (all P ＜ 0.05).The expression of protein Bcl-2 in the protective group was significantly higher than that of the virus group(P ＜ 0.05).The expression of caspase-3 (+,++) was significantly higher in the virus group and the protective group than the control group (all P ＜ 0.05).Compared with the virus group,the expression of caspase-3 in the protective group was significantly lower(P ＜ 0.05).ConclusionTRX could inhibit cardiomyocyte apoptosis in viral myocarditis mice and the inhibition is related to regulation of apoptosis-related protein expression.

OBJECTIVETo study the inhibitory effect of adenovirus-mediated fusion gene system driven by KDR promoter on the proliferation of human gastric adneocarcinoma SCG7901 cells and observe the bystander effect in vitro.

METHODSSCG7901, ECV304 and HepG2 cells were infected with Ad-KDR-CDglyTK and Ad-CMV-CDglyTK at a multiplicity of infection (MOI) of 100, and the infection efficiency and the mRNA expressions of the transferred fusion gene were investigated. GCV and/or 5-FC at different concentrations were added into the culture medium of the infected cells to observe the targeted antitumor effect and bystander effect of CDglyTK suicide gene driven by KDR promoter.

RESULTSWith the MOI of the adenovirus of 100, the fluorescence emitted by green fluorescent protein (GFP) was observed in 95% of the infected SCG7901, ECV304 and HepG2 cells. All the cells infected by Ad-CMV-CDglyTK and SCG7901 and ECV304 cells infected by Ad-KDR-CDglyTK were highly sensitive to the prodrugs. In comparison, HepG2 cells infected with Ad-KDR-CDglyTK did not show much sensitivity to the two prodrugs. Following treatment with the prodrugs at the same concentration, the infected SCG7901 and ECV304 cells exhibited gradually lowered survival rates as the culture time was prolonged, whereas the transgenic HepG2 cells showed no such time-dependent changes. When the non-infected cells were cocultured with the transgenic cells, the bystander effect of CDglyTK gene was observed, which increased with the ratio of the transgenic cells. In these mixed cell culture systems, GCV and 5-FC showed obvious synergetic effect in suppressing the cell survival.

CONCLUSIONThe CDglyTK fusion gene system driven by KDR promoter can inhibit the proliferation of SCG7901 and ECV304 cells with obvious bystander effect in vitro. The combination of the prodrugs produces obvious synergetic effect against the cell survival.

Adenocarcinoma ; genetics ; pathology ; therapy ; Adenoviridae ; genetics ; metabolism ; Cell Line, Tumor ; Cytosine Deaminase ; biosynthesis ; genetics ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; Promoter Regions, Genetic ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Stomach Neoplasms ; genetics ; pathology ; therapy ; Thymidine Kinase ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; metabolism