Stargardt disease (STGD) is an inherited disorder of retinal pigment epithelium.Three genes have been found to be implicated in STGD including Abca4 (adenosine triphosphate-binding cassette,sub-family A,member 4),Elovl4 (elongation of very long chain fatty acids protein 4) and Prom1 (prominin-1).Target genes can be delivered to the retina by various methods such as lentivirus (LV) vectors,adeno-associated virus (AAV) vectors and non-viral nano-particles.The Abca4-/-,Elovl4-/-and Prom1-/ mice model are used to study the pathogenesis mechanism and treatment of STGD.Retinal function improved significantly upon gene therapy in these models.Based on these works using animal model,phase Ⅰ /Ⅱ a clinical trial of Abca4-associated STGD gene therapy are underway.As a LV vector,equine infectious anemia virus (EIAV) is used to carry the Abca4 gene.These studies will evaluate three dose levels of the EIAV vector for safety,tolerability and biological activity.Moreover,some preclinical attempts to deliver Abca4 via AAV have been made using a modified AAV vectors because of the large size of the ABCA4 cDNA.The good responses in animal models render STGD a very attractive object for human gene therapy after the successful of the phase Ⅰ /Ⅱ clinical trials of Leber's congenital amaurosis.

Acute Disease ; Adult ; Aged ; Female ; Humans ; Hydrogen Sulfide ; poisoning ; Male ; Middle Aged ; Occupational Diseases ; Young Adult

Adolescent ; Adult ; Control Groups ; Female ; Humans ; Male ; Middle Aged ; Noise, Occupational ; statistics & numerical data ; Power Plants ; Young Adult

Background Trans-corneally subretinal injection in rodent model is a useful method for genetic therapy,stem cell transplantation and the study on the ophthalmic research.Standarized operation process is critical for the successful treatment.However,there is no literature to report the detailed procedure and the influence of this technique on morphology and function of retina.Objective This sudy was to introduce a method of trans-corneally subretinal injection and evaluate its influence on the morphology and function of retina.Methods Trans-corneallly subretinal injection was performed on the left eyes of 2-month-old SPF C57BL/6J mice after dilation of pupils.A 301/2G disposable needle was used to puncture the cornea within the pupil area near limbus and avoid touching the lens and irises under eye surgery microscope.Then,a 33G blunt needle was used to insert into the vitreous and toward subretinal space via corneal puncture.Normal saline with 0.1％ fluorescein sodium of 1 μl was slowly injected into the space,and 2.5％ hydroxypropyl methylcellulose was dropped on ocular surface for the observation of the fundus clearly.According to the percentage of the retina filled with subretinally injected solution,the experimental eyes were divided into 80％-100％ area group,50％-70％ area group after injection,and the mice in the pseudo-injected group,in which injection procedure stopped just before the solution was pushed in to the subretinal space did not inject any solution after punctured.The right uninjected eyes of the mice served as normal control group.Four eyes were selected for each group.The structural changes were evaluated by optical coherance tomograpby (OCT) 1 day,2 days,3 days and 5 weeks after injection,and retinal function was assessed by the recored of electroretinography (ERG) 5 weeks after injection.The retinal sepcimens were prepared to examin the morphological changes by hematoxylin and esosin staning.The use of care followd the Regulations for the Administration of Affair Concerning Experimental Animals of Zhejiang Province.Results About 70％ of the injected eyes showed that retinal blebs filled with injected green fluorescein solution occupied 50％ or more retinal area with minimal damages.The focal detachment between neurosensory retinal layer and retinal pigment epithelium (RPE) was exhibited 1 day postinjection,and almost all the retinas retached 2 days after injection.In the fifth week after injection,the amplitudes of ERG b wave were (386.25±37.88),(357.50±41.03),(324.25±53.45) and (410.50±14.88) μV in the sham operation group,50％-70％ area group,80％-100％ area group and normal control group,respectively,showing a significant difference among the 4 groups (F=3.574,P=0.047),and the amplitudes of b wave in the normal control group were higher than those in the 80％-100％ area group (all at P ＜ 0.05).The detachment between retinal neuroepithelium layer and RPE layer,cell proliferation and transposition in the outer nuclear layer were dispalyed under the light microscope in the sham operation group,50％-70％ area group and 80％-100％ area group,and the disordered outer segment of photoreceptors at the injecting area was seen in the 50％-70％ area and 80％-100％ area groups at five weeks after injection.However,retinal sructure and morphology were normal at the non-injection area.Conclusions Trans-corneally subretinal injection is an effective and safe way for subretinal injection.

Objective Chiral recognition for the glimepiride and glimepiride-cis-isomer by applying three amino acid (D-Lysine,L-Glutamine and L-Tyrosine) as chiral probes based on electrospray ionization mass spectrometry (ESI-MS) was achieved.Methods glimepiride/glimepiride-cis-isomer solutions were mixed with three amino acid solutions.The complex was extracted by ESI-MS and then the fragmentation abundance was investigated applying collision induced dissociation (CID) by MS/MS,which is the basis of chiral recognition for glimepiride and glimepiride-cis-isomer.Results Chiral recognition effect was achieved with the recognition rate (R) 1.61,2.92 and 2.17 for D-Lysine,L-Glutamine and L-Tyrosine respectively.Conclusion 3 kinds ofchiral amino acids were used as probes to distinguish between stereoisomers,and rapid identification of glimepiride and glimepiride cis isomer by mass spectrometry come true for the first time.

Objective To study the expression characteristics of Let-7 genes in breast cancer.Methods Twenty-eight patients with breast cancer were randomly selected,and their cancer tissue and adjacent normal tissue were collected.TRIzol was used to extract the total RNA and real-time quantitative PCR was used to detect the relative gene expression levels.Results The expression of precursor Let-7 in cancer tissue was (9.65 ± 2.31),which was lower than that in normal tissue (10.05 ± 2.81),P =0.048.Precursor Let-7 had dependence relationship with the long menstruation (b =0.816,P =0.029).The menarche age showed a positive correlation with precursor Let-7 in normal tissue (r =0.502,P =0.048) and a negative correlation in cancer tissue (r =-0.484,P =0.049) Conclusions The expression of precursor Let-7 in cancer tissue is lower than that in normal tissue.The period of menstruation is a protective factor to breast cancer.

OBJECTIVE:To establish a RP-HPLC analytical method for the determination of pazufloxacin mesilate in human plasma and urine.METHODS:The plasma proteins were precipitated with methanol and the supernatant liquid ob-tained from the serum centrifugate was subjected to chromatographic analysis.The supernatant liquid obtained from the diluted urine centrifugate was subjected to sample introduction.The analytical column was Diamonsil C 18 ,the mobile phase consisted of acetonitrile-0.05mol/L potassium dihydrogen phosphate(containing1%tetrabutylammonium bromide)(8∶92,V/V)with a flow rate at1.4ml/min,excitation wavelength at320nm and emission wavelength at400nm.RESULTS:The linear range of pazufloxacin mesilate in both plasma and urine was31.25～10000ng/ml(r=0.9999).The relative recoveries of pazu-floxacin mesilate in human plasma and urine were97.77%～99.87%and98.31%～100.82%,respectively with RSD less than1.0%～3.0%.CONCLUSION:This method is accurate,reliable and simple and it is suitable for the pharmacokinetic study and routine monitoring of blood concentration of pazufloxacin mesilate.

0.05),but the numbers of CD41+ cells and platelets were increased significantly(P

OBJECTIVETo study the metastasis-associated molecules differentially expressed in highly and poorly metastatic sublines and the mechanism of metastasis in lung giant cell carcinoma.

METHODSHighly and poorly metastatic sublines (PLA801D and PLA801C)were used as metastasis model. Cell motility and invasion assay in vitro were first compared between the two sublines. Then, gelatin zymography analysis was used to determine the MMP-2 and MMP-9 activity. The protein expression level of secreted MMP-2, MMP-9, TIMP-1, TIMP-2 and intracellular expression level of p53, p16, PCNA, CD44(V6) isomeride, E-cadherin, CK18, nm23-H1 as well as the mRNA expression level of MMP-2, MMP-9, TIMP-1, TIMP-2, VEGF were compared through Western blot. Semi-quantitative RT-PCR analysis was used to determine the intracellular mRNA expression of MMP-2, MMP-9, TIMP-1, TIMP-2 and VEGF.

RESULTSThe in vitro cell invasion potential of highly metastatic subline PLA801D was significantly higher than that of poorly metastatic subline PLA801C by about 4 folds, while the cell motility potential was similar. The secreted MMP-2 activity was notably higher in PLA801D, which was initiated by the higher expression of MMP-2 at protein and mRNA level. In addition, the expression level of p53, PCNA, CK18 protein and VEGF mRNA were significantly higher, while the expression level of p16, E-cadherin and nm23-H1 protein were significantly lower in PLA801D. Some molecules such as MMP-9, TIMP-1, TIMP-2, CD44(V6) isomeride, which had been reported to be associated with tumor metastasis, were not observed to change significantly between the two sublines.

CONCLUSIONThere are significant differences in metastatic potential and phenotypes between highly and poorly metastatic sublines of lung giant cell carcinoma. Some differentially expressed molecules might be playing roles in promoting or inhibiting metastasis of lung giant cell carcinoma, which may be useful to elucidate the mechanism of metastasis.

Carcinoma, Giant Cell ; metabolism ; pathology ; Cell Line, Tumor ; Humans ; Interleukin-8 ; genetics ; Lung Neoplasms ; metabolism ; pathology ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Neoplasm Invasiveness ; Neoplasm Metastasis ; RNA, Messenger ; analysis ; Tissue Inhibitor of Metalloproteinase-1 ; analysis ; Vascular Endothelial Growth Factor A ; genetics

OBJECTIVE To study the mechanism of drug-resistance of multi-resistant Pseudomonas aeruginosa,and provide the guideline for treatment and control of P.aeruginosa infection in hospital.METHODS Fifty strains of multi-resistant P.aeruginosa were selected with K-B susceptibility method.The three-dimensional method was taken to differentiate the various beta-lactamases.The relative drug-resistance gene was detected by polymerase chain reaction(PCR).RESULTS Among 50 strains of multi-resistant P.aeruginosa,there were 2 strains(4%)producing ESBLs,20 strains(40%)producing AmpC beta-lactamases,and 11 strains(22%) producing ESBLs and AmpC beta-lactamases at the same time.There were 8 positive genes in the detected drug-resistance gene,the most common sources of gene were CTX(56%),OprD(60%) and aac(6′)-Ⅱ(60%),respectively.CONCLUSIONS The main beta-lactamases are AmpC beta-lactamases and the main genotype is CTX in the multi-resistant P.aeruginosa cultured in our area.The main course of imipenem-resistance was deletion of outer membrane proteins,and the aminoglycoside modifying enzyme gene and disinfectant-resistance gene in multi-resistant P.aeruginosa are acquired.In order to reduce the drug-resistance strains and control the infection of P.aeruginosa,antibiotics should be used reasonably according to drug susceptibility testing clinically.