Objective To compare the short-term and long-term efficacy of chemotherapy with biotherapy and same chemotherapy followed by biotherapy on malignant melanoma in stage Ⅲ and Ⅳ.Methods 82 cases with malignant melanoma were treated with chemotherapy(dacarbazing,cis-platin and carmustine)or biotherapy(interleukin-2,interferon ? and dendritic cell vaccine)or biochemotherapy(chemotherapy plus biotherapy).Among them,32 cases were received biochemotherapy,26 for biotherapy and 24 for chemotherapy.Therapentic response was assessed every 3 cycles.The median time of follow up was 2 years(1-4 years).Results Response rate was 71.9% for biochemotherapy,46.2% for biotherapy and 54.2% for chemotherapy(P

Objective To study the difference of oxygen free radical metabolism between healthy native Tibetans and mi- grated Hans at different altitude.Methods The activity of Total-antioxidation capability(T-AOC),superoxide dis- mutase(SOD),glutathione peroxidase(GSH-PX)and the content of,reactive oxygen species(ROS),malondialdehyde (MDA)and nitric oxide(NO)in serum in healthy native Tibetans and migrated Hans at different altitude were meas- ured.Results The activity of T-AOC,SOD,GSH-PX and the content of NO were increased in serum in native Tibet- ans group than that in migrated Hans group(P

Objective:To investigate the inhibitory effect of RNA interference (RNAi) on the expression of multidrug resistance (MDR1) gene and analyze the altered sensitivities of human renal carcinoma cell line to cisplatin.Methods:Three small interfering RNA (siRNA) sequences targeted MDR1 gene were synthesized and transfected into renal carcinoma A498 cells. The expression level of MDRl mRNA was measured by RT-PCR to identify the most effective siRNA sequence. The recombinant plasmid was packed by lentivirus and transfected into A498 cells. RT-PCR was used to screen the A498 cells with the optimal silencing efficacy. The MDR1 protein expression level in the cloned cells was verified by Western blotting. The inhibitory effect of cisplatin on the proliferation of A498 cells was assessed by MTT assay and the IC_(50) value was calculated. Results:The 3 siRNA sequences suppressed MDR1 gene expression at different degrees. The siRNA 1 sequence silenced MDR1 gene more effectively with a significant reduction of 67%. The MDR1 protein expression greatly decreased in screened A498 cells compared with non-transfected cells (P<0.01), and the IC_(50) value of cisplatin on screened A498 cells was significantly decreased by 83.37% (P<0.01). Conclusion: The RNAi could effectively inhibit the expression of MDR1 gene and increase the sensibility to cisplatin in human renal carcinoma A498 cell line, which make it possible to reverse the resistance of renal carcinoma to chemotherapy.

Objective To investigate whether thalidomide inhibits the over expression of type I collagen in pulmonary fibrosis rats via inhibiting the JNK signaling pathway,thereby reducing bleomycin induced pulmonary interstitial fibrosis in rats.Methods 90 healthy male SD rats were randomly divided into normal control group (group N),model group (group M),thalidomide group (group T),SP600125 group (group SP) and thalidomide+SP600125 group (group T+SP).The pulmonary fibrosis models were prepared via intratracheal injection of 5mg/kg bleomycin,and rats in groups were given corresponding drugs from the first day after preparing model.Rats were randomly sacrificed at 7,14 and 28 days after treatment.The degree of pulmonary alveolitis and fibrosis was evaluated by H&E and trichrome masson stainings.The level of hydroxyproline in the lung tissue was detected by applying alkaline hydrolysis technique,and expression levels of p-JNK and type I collagen were tested by Western bloting for protein expression and real-time polymerase chain reaction (RT-PCR) for mRNA expression.Results In group M,alveolitis was the most serious on day 7; a marked pulmonary fibrosis formed on day 28; the level of hydroxyproline also peaked on day 28,and the contents of p-JNK and type I collagen were higher than in group N(F=277.87,472.51,both P＜ 0.01).Group T,SP and T+SP showed mild alveolitis and fibrosis at all time points,and their levels of hydroxyproline,p-JNK and type I collagen were remarkably decreased as compared with group M (F=14.77,61.59,101.73,all P＜0.01;F=10.33、79.12、57.48,all P＜0.01).No significant difference in p JNK was found between group SP and group T+SP.Conclusions Thalidomide may inhibit the over expression of type I collagen in pulmonary fibrosis rats via inhibiting the JNK signaling pathway,thereby reducing bleomycin induced pulmonary interstitial fibrosis in rats.

AIM: To explore the effects of adipose-derived stem cell (ADSC) transplantation on the expression of IL-10 amd TNF-α after cerebral ischaemia in Middle cerebral artery occlusion (MCAO) rats. METHODS: 72 male adult Sprague-Dawley rats were randomly divided into 4 groups: sham group, MCAO group, Vehicle group and ADSC group (n=18). Rat's cerebral ischemia model was established by MCAO with Longa' s method. ADSC were labeled by DAPI before transplantation. One day after MCAO, 30 μL of cell suspension containing 1×10~6 ADSCs were injected into the lateral ventricle of ADSC group and the same dose of PBS was given to the Vehicle group. At day 4, day 7 and day 14 after MCAO, the rats were decapitated to detect the expression of IL-10 and TNF-α in ischaemic rat' s brain by ELISA, immunohistochemistry and RT-PCR. RESULTS: Compared with sham group, the expression of IL-10 and TNF-α significantly up-regulated at 4 d, 7 d of MCAO group(P<0.05). There was no statistical difference of IL-10 and TNF-α expression between MCAO and vehicle group ant any time point(P>0.05). Compared with Vehicle group, the expression of IL-10 significantly up-regulated while TNF-α expression significantly decreased of ADSC-treated group at any timepoint post MCAO(P<0.05). CONCLUSION: The transplantation of ADSC could up-regulate the expression of IL-10 and down-regulate the expression of TNF-α in MCAO rat' s brain, which might contribute to its protective role upon cerebral ischaemia.

OBJECTIVETo determine whether the sonic hedgehog (Shh) signaling could regulate the expression of histone demethylases in the head and neck squamous cell carcinoma(SCC).

METHODSHuman recombinant SHH-N protein or over-expression of the mutant 2 smoothened (M2-SMO) was applied to activate the Shh signaling in tongue squamous cell carcinoma cell line-SCC-6 in this study. Cyclopamine was used to block the Shh signaling in SCC-6. The real-time reverse transcription (RT)-PCR was used to detect the expression of histone demethylases at the mRNA level.

RESULTSThe data showed that activation of the Shh signaling up-regulated the expression of histone demethylase, lysine-specific demethylase 8 (KDM-8) at the mRNA level by human recombinant SHH-N protein (1.841 ∼ 3.591 fold compare with untreated group; P < 0.01), over-expression of the M2-SMO also increased the expression of KDM-8 (1.358 ∼ 3.013 fold compared with empty vector group; P < 0.05), and after the Shh signaling was blocked by Cyclopamine, the expression of KDM-8 was down regulated (decreased 25.6% ∼ 66.6% compared with control cells, P < 0.05).

CONCLUSIONSHistone demethylase KDM-8 was downstream target gene of Shh signaling in head and neck squamous cell carcinoma cell line SCC-6, and its expression was positively regulated by the Shh signaling.

Carcinoma, Squamous Cell ; genetics ; metabolism ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Head and Neck Neoplasms ; genetics ; metabolism ; Hedgehog Proteins ; metabolism ; Histone Demethylases ; genetics ; metabolism ; Humans ; Mutant Proteins ; metabolism ; RNA, Messenger ; genetics ; Receptors, G-Protein-Coupled ; metabolism ; Recombinant Proteins ; metabolism ; Signal Transduction ; Smoothened Receptor ; Veratrum Alkaloids ; pharmacology

The aim of the manuscript was to optimize formulations and preparation technologies of cataplasm of white mustard seed varnish, and to evaluate its anti-asthma effect on rats. The single factor experiments included spreading thickness, types of crosslinking agents, dihydroxyaluminum aminoacetate amount, sodium polyacrylate amount, types of adhesive agents with human sense as the evaluation index. Blank cataplasm matrix was optimized by the orthogonal experiment with the amount of glycerine, citric acid, and sodium carboxymethylcellulose as the major influential factors. Initial adhesive force, peeling strength and human sense were as the evaluation index. The optimized formulation of blank cataplasm were as followings: glycerine-water-ethanol-PEG400-dihydroxyaluminum aminoacetate-citric acid-sodium carboxymethylcellulose-sodium carboxymethylcellulose 2 : 8 : 0.8 : 0.4 : 0.07: 0.15 : 0.1 : 0.5. The active ingredients of white mustard seed, corydalis, and gansui root were extracted by alcohol extraction method. Asiasarum volatile oil was extracted by oil extractor. The optimized drug loading amount was 11% with initial adhesive force, peeling strength and human sense as the evaluation index. Asthma rats model were established by sensitized with ovalbumin and nose-scratching time as the evaluation index. High dose (17%) group of drug-loaded cataplasm had the obvious inhibition effect on nose-scratching time of rats (P = 0.037 < 0.05). In comparison, middle dose (11%), low dose (4%) and positive-control groups had no obvious inhibitive effect on rats. White mustard seed cataplasm supplied a novel choice for anti-asthma therapy. And the overall pharmacodynamics assessment will be carried out on molecular level in near future.
Animals ; Anti-Asthmatic Agents ; administration & dosage ; chemistry ; Asthma ; drug therapy ; Chemistry, Pharmaceutical ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; Female ; Humans ; Male ; Mustard Plant ; chemistry ; Rats ; Rats, Sprague-Dawley ; Seeds ; chemistry

Background and purpose:Ionizing radiation(IR) activates the early growth response- I(Egr1) promoter through specific cis-acting sequences termed CArG elements by production of radical oxygen intermediates(ROls).Egr-EG,an expression vector pCIneo containing CArG elements cloned upstream of the cDNA for human recombinant GM-CSF,was used to treat hematopoietic damage due to chemotherapy.Commonly used chemotherapeutic agents can cause tumor cell death by producing DNA damage and generating ROIs.We therefore hypothesized that clinically employed chemotherapeutic agents that increase ROIs could also be employed to activate Egr-EG in a chemoinducible gene therapy strategy.This study was done to explore the chemo-protective effect of the expression of hematopoietic growth factors regulated by Egr-1 promoter on chemotherapy induced damage. Methods:The human GM-CSF cDNA and EGFP cDNA were linked together with internal ribosome entry site(IRES) and then inserted into the eukaryotic expression vector pCI-neo with the Egr-1 promoter(Egr-EG),and was further transduced into human bone marrow stromai cell lines HFCL(HFCL/EG).The HFCL/EG cells were transplanted i.v.into BI6 melanoma in C.B-17 combined immunodeficient(SCID) mice.5-FU was given i.p.on day 3 and 4.The white blood cell amount in peripheral blood,the expression of EGFP and GM-CSF in human stromal cell engrafted in recipient mice were detected by flow cytometry,RT-PCR,Western blot and colony-forming units for granulocytes and macrophages(CFU-GM),respectively.Results:In contrast to the two control groups,HFCL/EG(the Egr-regulatory element-derived expression of GM-CSF gene therapy) resulted in a proportional increase in the number of the white blood cell after chemotherapy,no significant diifferences were found for CFU-GM in bone marrow cells and the inhibition ratio on tumor in recipient mice.Chemotherapy could markedly increase the expression of EGFP and GM- CSF mRNA/protein as compared with that of non-chemotherapy control groups and HFCL group.Conclusion: Chemoinducible GM-CSF gene therapy regulated by Egr-1 promoter can ameliorate the toxic effect on 5-FU chemotherapy-inducible hematopoietic damage.

BackgroundObstruction of aqueous humor out flow pathway or abnormality of the extracellular matrix( ECM ) of trabecular meshwork cells causes high intraocular pressure. The balance of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases(TIMPs) is critical for the metabolism of ECM. Interleukin1α(IL-1α) can influence outflow of aqueous humor by regulating MMPs level. Objective This study was to investigate the effect of interleukin-1α on the expression of MMP-2,MMP-3 and TIMP-I in cultured swine trabecular meshwork cells.Methods Swine sclera with trabecular meshwork tissue was isolated from 20 swine eyes and cultured with explant cultured method. Cultured cells were passaged and third generation cells were identified by fibronectin ( FN ) and laminin ( LN ) staining. After 24 hours of serum starvation, trabecular meshwork cells treated with IL-1α at the concentration of 10 mg/L were regarded as the IL group,and serum-free culture medium used to treat trabecular meshwork cells was regarded as the control group. The expression of MMP-2, MMP-3 and TIMP-1 proteins in trabecular meshwork cells were detected by immunohistochemistry,and the expression of MMP-2 mRNA, MMP-3 mRNA and TIMP-1 mRNA were detected by RT-PCR. The examination results were compared between the two groups. ResultsThe third generation of cells were positive for FN and LM. Compared with the control group, the expression levels of MMP-3 and TIMP-1 proteins(A value) in trabecular meshwork cells were significantly higher in the IL group than the control group(t=-7. 694,t =-5. 199,P＜0. 05) ,but no obvious difference was found in the expression of MMP-2 between the two groups( t=-2. 365, P＞0.05 ). The higher expression levels in MMP-3 mRNA and TIMP-1 mRNA (A value) in trabecular meshwork cells were seen in comparison with the control group (t =-3. 025,t=-1. 921 ,P＜0. 05). However,similar results were found in the expression of MMP-2 mRNA between the two groups(t =- 1. 173, P＞0.05 ). ConclusionsThe overexpression of MMP-3 and TIMP-1 proteins and their mRNA leads to the imbalance of MMP-3/TIMP-1 and promotes the decomposition of ECM in the trabecular meshwork, and therefore increases aqueous outflow.

Background The rapid diagnosis can win more treating opportunities for patients with fungal keratitis.Even though the fungal culture is the gold standard for the diagnosis of fungal keratitis,it is difficult in early diagnosis due to the long duration of cultivation and false-negative rate.Objective This trial was to explore the clinical value in the rapid diagnosis of fungal keratitis by the combination of corneal scraping with laser scanning confocal microscopy.Methods Corneal scraping and laser scanning confocal microscopy were separately performed in 167 eyes of 167 patients with fungal keratitis.All the eyes were examined by the slit lamp,followed by laser scanning confocal microscope,and then the 10％ KOH corneal smear was examined under the optical microscope.Results The positive rate of diagnosis was 75％ (125/167) by corneal scraping,and that by laser scanning confocal microscopy was 91％ (152/167).The positive rate of examining outcome was significantly higher in laser scanning confocal microscopy than that of corneal scraping (x2 =14.88,P =0.00).The positive results were 114 cases and negative results were 4 cases by two methods,with the concordance rate 70.7％ (118/167).The hyphae or spore were seen in 32 cases by laser scanning confocal microscopy in 42 negative cases by corneal scraping,and in 15 negative cases by confocal laser scanning microscopy,11 positive outcomes were offered by corneal scraping.Conclusions The combined application of corneal scraping with confocal laser scanning microscopy can improve and speed up the diagnosis positive rate of fungal keratitis.