Education, Nursing, Continuing ; General Surgery ; education ; Humans ; Physicians

Aim To screen and identify anti-CCR7 single chain fragments variable(scFv)from lager phage library and to detect the scFv efficiency.Methods The insert ratio of ScFv antibodies library was identified by PCR.The products digested by Sfi I/Not I double enzyme.Panning against breast cancer cell and CCR7 were performed four and three rounds respectively.Positive clones were transformed to E.coli HB2151, and their dissolvability was assayed.The soluble scFv was purified by affinity chromatography, and its relative molecular mass was determined by Western blot.The ELISA assay was used to identify the immunocompetence of the antibody.Immunocytochemical staining and radioimmunoimaging were employed to determine the affinities of scFv binding with CCR7 in cell line and in nude mice.Results The insert ratio of ScFv gene was 90%(18/20), enzyme digest reaction showed the aim products on 1% agarose gel.ScFvs were obviously enriched after 7 round panning.Western blot result showed soluble scFv's molecular mass was about 34 KD.ELISA analysis showed dissolved antibody had high immunocompetence to MDA-MB-435 s cells.Immunocytochemical staining and radioimmunoimaging indicated that ScFvs were bound efficiently to MDA-MB-435 s cells which expressed CCR7.Conclusions ScFvs against CCR7 are successfully acquired by screening the phage antibody library.The soluble ScFvs have high affinity and specifical binding to human breast cancer cells.

Objective To establish a stable HCV full-length genome replication cell model which is labeled with reporter gene and easyly to quantify intracellular HCV proteins and RNA level. Methodsneo gene was inserted into Luc-JC1 to make Luc-JC construct. Luc-JC RNA was obtained by in vitro transcription and then delivered into Huh7 cells by transfection. G418-resistant clones of Huh7 cells were obtained by selection. Clones of HCV full-length genome replication cell were confirmed by luciferase activity assay, Western blot and cleaveage of eYFP-MAVS by HCV NS3/4A protease. Then, HCV replication cell colonies were treated by different dose IFN-α in order to observe the change of luciferase activity, HCV protein and RNA level. Results At 3-4 weeks post-transfection, visible colonies were selected and stained by crystal violet. Luciferase activity and HCV NS3, NS5A protein were detected by luciferase activity assay and Western blot, respectively. Subcellular localization of eYFP-MAVS transferred from mitochondria to cytoplasms by cleavage of NS3/4A protease in cell colonies. Luciferase activity, HCV protein and RNA diminished obviously after IFN-α treatment. Conclusion A stable HCV full-length genome replication cell model labeled by reporter gene was successfully established and reporter activity can be used to indicate level of HCV proteins and RNA in cells. This cell model is a useful tool for the study on HCV pathogenesis and the screening of antiviral drugs.

Nanocrystal drugs have many advantages, such as no carrier materials, easy industrialization, diversified dosage forms, and can significantly improve the solubility and bioavailability of insoluble drugs, so many drugs have been on the market. The traditional nanocrystal preparation technology has the problems of low preparation efficiency and process limitation of the smallest achievable particle size. With the progress of pharmaceutical preparation technology, the preparation technology of nanocrystal drugs is constantly improving, and new preparation technologies are constantly emerging. The emergence of new technologies has greatly shortened the process time and makes it possible to prepare nanocrystal drugs with smaller particle diameters. In this paper, the preparation technologies of nanocrystal drugs, especially the new preparation technologies such as high gravity controlled precipitation, microfluidic reaction technology and various combination technologies, are reviewed from three aspects: "Top-down" technology, "Bottom-up" technology and combination technology. This article also prospects the development of new preparation technologies, hoping to provide reference for the related research of nano-preparations.

Background Platelet-derived growth facto(PDGF) affectthe proliferation of human lenepithelial cell(LECs),and human LECexpresPDGF-α recepto(PDGFR-α) throughoutheilifetime.The binding of activated PDGF-α receptowith PDGF promotethe synthesiof DNA.Othestudiedemonstrated thasilencing of PDGFR-α by antisense oligodeoxynucleotide(ASODN) inhibitthe growth of RPE cellin proliferative vitreoretinopathy (PVR),buwhethethitechnique ifeasible foLECiunclear.Objective Thistudy wato investigate the effecof the knockdown of the PDGFR-α on the proliferation of human LECin vitro,and to offean experimental basifothe gene therapy of posteriocapsule opacification.MethodHuman LECstrain SRA01/ 04 wacultured in α-MEM containing fetal bovine serum.The cellwere incubated in 6-well platea5 × 104 cells/ well and transfection of ASODN-containing liposome waperformed.The cellwere divided into the blank control group (with blank liposome),PDGFR-α missense oligodeoxynucleotide(MSODN) group (with PDGFR-α MSODN + liposome),0.5 μmol/L PDGFR-α ASODN group (with 0.5 μmol/L PDGFR-α ASODN+liposome) and 1.0 μmol/L PDGFR-α ASODN group (with 1.0 μ mol/L PDGFR-α ASODN+liposome).The morphology of LECwaexamined undean inverse microscope 24 houraftetransfection.The expression of PDGFR-α mRNin the cellwadetected by reverse transcription-PC(RT-PCR).The rate of proliferation (A490) of the cellwaassayed using Mtand the inhibitory rate of PDGFR-α ASODN on proliferation wameasured.The percentage of LECin G1 phase waanalyzed by flow cytometer.ResultThe LECgrew well and exhibited polygonal shape in the blank control group and PDGFR-α MSODN group 24 houraftetransfection.Buin the 0.5 μmol/L and 1.0 μmol/L PDGFR-α ASODN groups,the cellappeared round in shape and the numberof cellwere obviously decreased.The expression of PDGFR-α mRNdetected by RT-Pcdemonstrated highelevel in the blank control group and PDGFR-α MSODN group;however,the PDGFR-α mRNexpression waobviously lowein the 0.5 μmol/L and 1.0 μmol/L PDGFR-α ASODN groups.The A490 value wa0.661 ± 0.036,0.655 ± 0.016,0.529 ± 0.030 and 0.441 ± 0.039 in the blank control group,PDGFR-α MSODN group,0.5 μmol/L PDGFR-α ASODN group and 1.0 μmol/L PDGFR-α ASODN group,respectively,showing significandecline in the 0.5 μmol/L PDGFR-α ASODN group and 1.0 μ mol/L PDGFR-α ASODN group in comparison with the blank control group (F=34.08,P＜0.01).The percentageof LECin G1 phase were (47.73±1.18)％,(49.48±1.09)％,(53.31±1.30)％ and (59.98±0.95) ％ in the blank control group,PDGFR-α MSODN group,0.5 μmol/L PDGFR-α ASODN group and 1.0 μmol/L PDGFR-α ASODN group,showing significandifference among them (F =68.41,P＜0.01),and thain the 0.5 μmol/L PDGFR-α ASODN group o1.0 μmol/L PDGFR-α ASODN group showed significantly increase in comparison with the blank control group (P＜0.05).ConclusionPDGFR-α silencing could inhibithe proliferation of human LECin vitro.

BackgroundRetinal pigment epithelial(RPE) cells can secrete platelet-derived growth factor (PDGF) and PDGF receptor(PDGFR).Studies have shown that PDGF plays a key role in the formation of proliferative vitreous retinopathy(PVR). ObjectiveThis study was to investigate the proliferation and apoptosis changes of RPE after blockage of the PDGFR-α expression by antisense oligonucleotide ( ASODN ) in vitro. Methods Human RPE cells strain was cultured in low glucose DMEM with 10％ fetal bovine serum.Logarithmic phase cells were collected and incubated in 96-well plate at the density of 5 × 105 cells/hole.PDGFR-α ASODN was transfected into RPE cells at different concentrations for 48 hours.The cells of the blank control group were regularly cultured without any transfection.The changes of PDGFR-α expression were detected by reverse transcription-polymerase chain reaction(RT-PCR),and the proliferation of RPE was detected by MTT as the A490 value.Hoechst 33258 fluorescence staining was used to determine the apoptosis of RPE.Flow cytometry method (FCM) was applied to detect the change of cell cycle and apoptosis rate of RPE cells. ResultsThe A490 values of RPE cells were 1.45±0.12,1.07±0.06,0.65±0.05 in blank control group,1.0 μmol/L Lipo-ASODN group and 2.0 μmol/L Lipo-ASODN group with the significant difference(P=0.00 ),and that of 1.0 μmol/L Lipo-ASODN group and 2.0 μ mol/L Lipo-ASODN group were significantly lower than the blank control group ( P =0.00,0.00).Hoechst 33258 staining showed that the apoptosis cells were obviously more in Lipo-ASODN group compared with blank control group.PDGFR-α ASODN transfection induced an increase of percentage of RPE cells in G0/G1 phase( F =206.70,P =0.00),and the apoptosis rates in 1.0 μmol/L Lipo-ASODN group and 2.0 μmol/L Lipo-ASODN group were significantly enhanced in comparison with blank control group ( 37.8 ± 1.3 vs 10.5 ± 0.1,61.2 ± 1.9 vs 10.5 ± 0.1 ) ( F =1808.90,P =0.00 ).Expression intensity of PDGFR-α mRNA in RPE cells in Lipo-ASODN groups was lower. ConclusionsBlocking the PDGFR-α expression with ASODN technology can suppress proliferation and induce apoptosis of RPE cells.Intensity of PDGFR-α mRNA expression in RPE cells is ASODN dose-dependent.ASODN targeted to PDGFR-α offers an experimental basis of the gene therapy for PVR.

Objective To evaluate the CT findings of pancreatic carcinoid tumors.Methods The CT imaging data of five patients with pancreatic carcinoid tumors confirmed by pathology were retrospectively analyzed.Results The tumors ranged in maximum diameter from 2.0 to 11.0 cm with a mean of 6.4 cm. On unenhanced CT,the tumors were slightly hypodense relative to the pancreatic parenchyma,homogenous in 2 cases,and heterogenous in 3 cases.One tumor showed calcification.After contrast material injection, the solid component of the tumor showed marked heterogenous enhancement on the arterial phase scanning in 3 cases,and mild heterogenous enhancement in 2 cases.The degree of tumor enhancement was less intense than the surrounding pancreatic parenchyma due to necrosis of various degree,which led to the cystic appearance of the tumor in 1 ease.On the portal phase scanning,all tumors showed marked enhancement similar to that of the pancreatic parenchyma.On the delayed phase scanning,the degree of enhancement was more intense than the surrounding pancreatic parenchyma in 1 case.Liver metastases with retroperitoneal lymphadenopathy and peripancreatic vessels invasion were seen in 1 case.No dilatation of the biliary tract or pancreatic duct was present.Conclusion The CT features of pancreatic carcinoid tumors included infrequent dilatation of the biliary tract or pancreatic duct and unusual vascular involvement,calcification within the mass,marked enhancement similar to that of the surrounding pancreatic parenchyma during the portal phase scanning and more intense during the delayed phase scanning.

AIMTo analyze and compare the constituents of the volatile oil of Pogostemon cablin collected from different regions of cultivation and harvesting times in order to evaluate the quality of Shipai Huoxiang and to expound the chemical intension of Pogostemon cablin.

METHODSThe combination of GC and MS.

RESULTSThe volatile oil compositions of Herba Pogostemonis collected from various of cultivation regions and harvesting times are obviously different. Based on the chemical differences of the volatile oil compositions, Pogostemon cablin is divided into two chemotypes, Pogostone-type and Patchouliol-type. The former was cultivated in Guangzhou and Gaoyao regions, locally named as "Shipai Huoxiang"; the latter was locally named as "Hainan Huoxiang", cultivated in Wuchuan, Suixi and Leizhou regions of Guangdong Province and Wanning region of Hainan Province. The Pogostone-type contains rich oxygenated components, especially pogostone in the volatile oil compositions and poor non-oxygenated composition with patchouliol. The above chemical data may be used as evaluation standard for the authentic Shipai Huoxiang. The Patchouliol-type contains similar quantities of oxygenated and non-oxygenated composition, especially rich patchouliol with poor pogostone in oxygenated compositions, rich delta-guaiene and alpha-guaiene in non-oxygenation compositions. The contents of volatile oil and their constituents were varied due to different harvesting time. The yields of pogostone and volatile oil of Shipai Huoxiang was higher in July. The quality of the samples collected in this month was better.

CONCLUSIONAccording to the volatile oil compositions, there are two chemotypes (Pogostone-type and Patchouliol-type) in Pogostemon cablin plant. These two chemotypes correspond to the genotypes of plastid matK gene and nuclear 18s rRNA gene by cluster analysis.

Ecosystem ; Environment ; Lamiaceae ; chemistry ; classification ; Oils, Volatile ; analysis ; chemistry ; isolation & purification ; Plant Leaves ; chemistry ; Plant Stems ; chemistry ; Plants, Medicinal ; chemistry ; classification ; Quality Control ; Seasons

OBJECTIVETo study the effects of rhizoma Astilbes chinensis in abirritation, blood activation, cough relieving and sputum elimination.

METHODThe antalgic function of rhizoma A. chinensis was tested by hot-plate method and writhing reaction. The acute blood-stasis model rats were made by Injection of adrenaline hydrochloride along with stimulation by ice water. The effects of cough relieving and sputum elimination were observed by the ammonia water-induced tussive mice and excretion of phenol red in the airway of mice. The maximum tolerance dose of rhizoma A. chinensis was also determined during the acute toxicity test.

RESULTThe data were analyzed for statistical significance by t-test, which shows that the decoction of rhizoma A. chinensis is significantly effective in reducing the frequency of licking behavior of mice on hot-plate and writhing response induced by acetic acid, improving the hemarheology of acute blood-stasis model rats, prolonging the latent period, reducing the frequency of cough induced byammonia, and in increasing the quantity of phenol red output from the trachea in mice. The result acute toxicity test shows that maximum tolerance dose of gastrogavage in mice was 400 g x kg(-1). Which was 666. 7 times of that clinically used for human.

CONCLUSIONRhizoma A. chinensis has the effects of abirritation, blood activation, cough relieving and sputum elimination, and is safe in clinical application.

Animals ; Antitussive Agents ; isolation & purification ; pharmacology ; therapeutic use ; Blood Viscosity ; drug effects ; Cough ; drug therapy ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; therapeutic use ; Expectorants ; isolation & purification ; pharmacology ; therapeutic use ; Female ; Male ; Mice ; Mice, Inbred ICR ; Pain Threshold ; drug effects ; Plants, Medicinal ; chemistry ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Rhizome ; chemistry ; Saxifragaceae ; chemistry

Objective To investigate the correlation of plasma matrix metalloproteinase-3 (MMP-3)levels and MMP-3 Lys45Glu (rs679620) polyrnorphism with ischemic stroke and its TOAST subtypes.Methods The patients with large artery atherosclerotic stroke (LAA) and small artery occlusion stroke (SAO)according to TOAST etiological typing (ischemic stroke group) and healthy subjects (control group) were enrolled.The enzyme-linked immunosorbent assay was used to detect plasma MMP-3 level.The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to detect the genotypes of MMP-3 Lys45Glu.Results A total of 233 patients with ischemic stroke were enrolled,in which 162 were LAA and 71 were SAO; 200 healthy subjects were taken as controls.The plasma MMP-3 level in the ischernic stroke group was significantly higher than that in the control goup (253.99 ± 75.02 ng/ml vs.196.38 ± 78.17 ng/ml;t =7.813,P=0.000).The plasma MMP-3 level in the LAA group (262.81 ±69.23 ng/ml) was significantly higher than those in thegroups of SAO (233.85 ± 83.90 ng/ml,P =0.008) and control (P =0.000),and the plasma MMP-3 level in the SAO was also significantly higher than that in the control group (P =0.000).Multivariate logistic regression analysis showed that the increased serum MMP-3 level was an independent risk factor for ischemic stroke (odds ratio [OR] 1.012,95％ confidence interval [CI] 1.008-1.015; P =0.000).There was no significant difference in the frequencies of genotype (x2 =2.085,P =0.353) and allele (x2 =2.29,P =0.130) of MMP-3 Lys45Glu between the ischemic stroke group and the control group.However,there were significant difference in MMP-3 Lys45Glu genotype frequencies among.the groups of LAA,SAO and control (x2 =10.39,P=0.034).The AA + GA genotype frequency in the LAA group was significant higher than those in the groups of SAO (65.4％ vs.49.3％ ;x2 =5.375,P =0.020) and control (65.4％ vs.54.0％ ;x2 =4.84,P =0.028).There was no significant difference in the allele frequencies among the groups of LAA,SAO and control (x2 =3.887,P =0.143).Multivariate logistic regression analysis showed that MMP-3 Lys45Glu polymorphism was an independent risk factor for LAA (OR 1.783,95％ CI 1.183-2.688; P =0.006).The plasma MMP-3 level in patients with the genotypes AA (n =73),GA (n =176) and GG (n =184)were 235.70 ± 70.85 ng/ml,(244.20 ± 85.90 ng/ml and 207.98 ± 77.61 ng/ml.There were significant difference in the plasma MMP-3 levels among the patients with the genotypes AA,GA and GG (F=9.682,P =0.000).The plasma MMP-3 level in the patients with the genotype AA + GA was significantly higher than that in patients with genotype GG (241.71 ± 81.73 ng/ml vs.207.98 ± 77.61 ng/ml; t =4.336,P =0.000).Conclusions The plasma MMP-3 level increased in patients with LAA or SAO,especially in the patients with LAA.The MMP-3 Lys45Glu polymorphism might be associated with the plasma MMP-3 level and LAA.