Objective Nine VEGF siRNAs were designed and their efficiencys were evaluated for introducing new and effective design rules of siRNA.Methods Nine VEGF siRNA sequences with nineteen nucleic acids were designed.After construction in vitro,VEGF siRNAs were transferred into neuroglioma cells by lipofectamine.The level of VEGF protein in the media was determined by ELISA 48 hours after transferring.The relationship between the characteristics of VEGF siRNA and its' interfering effect were analyzed.Results A quantitative parameter called H-b index were used to characterize the secondary structure of VEGF mRNA.A low value of the H-b index would mean that the structure of mRNA is loose,in which the nucleotides of the targeting region are likely to be accessible by siRNA and the effects are better.The experiments showed a negative correlation between the suppression of VEGF protein expression and H-b index(r=-0.498).The H-b indexs of siRNAs targeting VEGF mRNA 386～406 and 401～421 position were less,however the effects of suppression were better.The siRNAs conferred a strong RNAi activity with A residue at position 6 and A/U residues at position 19 of sense strand of siRNAs.Thethermodynamic properties of siRNA play a critical role in the transferring effects.The lower average of internal stability values for positions 9～14 of the antisense strand is facilitating the interfering effect.The lower binding energy is the more stable incorporation between antisense strand of siRNA and mRNA present,the more favorable is for RISC cleaving mRNA.Conclusions The sequences of VEGF siRNA have been designed successfully and effectively inhibited the expression of VEGF protein.

AIM: To determine the inhibition of VEGF small interfering RNA (siRNA) on expression of VEGF protein and SMMC 7721 cell growth. METHODS: Nine VEGFsiRNA sequences with nineteen nucleotides were designed under the assistance of computer. The inhibitory effect of VEGFsiRNA was analyzed by CCK8. The protein level of VEGF in the media was determined by ELISA. The change of cell cycle was detected by flow cytometry. RESULTS: All the VEGFsiRNA were capable of inhibiting the proliferation of SMMC 7721 cells significantly compared with control and lipofectamine control, while the inhibitory effects of VEGFsiRNA2, VEGFsiRNA4, VEGFsiRNA6, VEGFsiRNA7, VEGFsiRNA8 and VEGFsiRNA9 were better than that of antisense oligodeoxynucleotide. All the VEGFsiRNA reduced the expression of VEGF protein. The effect of VEGFsiRNA7 and VEGFsiRNA2 were the best with the inhibitory rates of 52.65% and 50.43%, respectively. VEGFsiRNAs induced the S arrest in SMMC 7721 cells. The signs of apoptosis in SMMC 7721 cells induced by VEGFsiRNA were not observed. CONCLUSION: VEGFsiRNA sequences were designed and synthesized successfully. VEGFsiRNA effectively inhibited the proliferation of SMMC 7721 cells and reduced the level of VEGF protein.

Drugs, Chinese Herbal ; therapeutic use ; Glomerulosclerosis, Focal Segmental ; drug therapy ; Humans ; Sclerosis

AIM: To construct eukaryotic expression vector of human vascular endothelial growth factor (VEGF) gene, and to explore the effects of VEGF gene on proliferation and chemosensitivity of leukemia cells. METHODS: The recombinant eukaryotic express plasmid pEGFP-C1-hVEGF_(165) was constructed with conventional gene engineering methods. The pEGFP-C1-hVEGF_165 was transfected into leukemia cell K562. The proliferation of the recombinant eukaryotic expression plasmid was analyzed by CCK8 method. The level of VEGF mRNA was detected by reverse transcription and quantitative polymerase chain reaction (RT-PCR). The expression level of VEGF protein was determined by ELISA in cell culture medium and cells. The cell cycle was detected by flow cytometry. RESULTS: VEGF_(165) eukaryotic expression vector was successfully constructed, confirmed by enzyme digestion and sequencing. Compared with the K562 cells transfected pEGFP-C1, the K562 cells transfected with pEGFP-C1-hVEGF_(165) grew faster and expressions of VEGF mRNA and protein increased significantly. In addition, K562 cells transfected with pEGFP-C1-hVEGF_(165) had relatively higher cell survival rate to chemotherapy drugs homoharringtonine(HHT) and fluorouracil(FU). CONCLUSION: VEGF_(165) eukaryotic expression vector increases the level of VEGF mRNA and protein expression, accordingly promotes the proliferation of leukemia cells and decreases the sensitivity of leukemia cells to HHT and FU.

Npu DnaE intein was used to produce some large proteins,which were difficult to obtain through conventional expression systems.A T7 expression system was described,by which the gene of T7 RNA polymerase is split into two pieces,and each piece fuses with Npu DnaE N-and C-terminal sequences respectively.Functional T7 RNA polymerase is created by mixing the two kinds of fusion constructs in vitro.The approach of split intein-mediated production of large proteins,in theory,readily generalizable to the purification of other large,cytotoxic or membrane proteins.

Objective To explore the differentiation of rat bone marrow mesenchymal stem cells into dopaminergic neuron.Methods The mesenchymal stem cells were isolated from bone marrow. They were induced to differentiate into dopaminergic neurons by freated with bFGF, VitC and EGF at the third generation. Dopamine-associated protein and genes in the treated cells were examined by immumofluorescence and RT-PCR. Dopamine in the supernatant and endoplasm from culture system was determined by ELISA kit. Results The results showed that tyrosine hydroxylase, dopamine transporter and nerve neucleoprotein and Nestin,Nurr-1 genes were found. And the dopamine existed in the supernatant and cytoplasm from inducing culture system. Conclusion The rat bone marrow mesenchymal stem cells have the capacity of differenting into dopaminergic nurons.

Adolescent ; Adult ; Female ; Humans ; Male ; Occupational Exposure ; Organotin Compounds ; poisoning ; Poisoning ; diagnosis ; therapy ; Retrospective Studies ; Young Adult

From June 1997 to June 2001 intraoperative transesophageal echocardiography(TEE)was used in 493 patients undergoing different cardiovascular operations including correction of congenital heart defect, coronary artery bypass grafting(CABG),cardiac valve replacement and major vascular surgery.The clinical data of these cases were complete and detailed enough for retrospective review.Their cardiac functions were graded to be classⅡ-Ⅳaccording to New York Heart Association(NYHA)classification.The patients ranged in age between 1.6-74 yrs and weighed between 8.5-92 kg.The probe of TEE was inserted in esophagus after tracheal intubation.Preoperative diagnosis was found to be wrong by TEE in 20 cases(4.1%).Additional defect was found in 51 cases(10.3%).The type of operation planned before operation was altered in 52 cases(10.5%). During the operation real time TEE revealed that the operation performed failed to achieve the goal and revision surgery was needed in 24 cases(4.9%).Abnormal hemodynamics and cardiac function were found by TEE after the heart resumed spontaneous beat in 17 cases(3.5%)and appropriate treatment was instituted.Oral and pharyngeal mucous membrane bleeding occurred in 7 cases.There were no serious complications attributable to TEE.Intraoperative TEE is a very useful tool in formulating the surgery,monitoring hemodynamics and assessing immediate results of surgery and is safe and reliable in the hands of trained anesthesiologists.

Objective To evaluate the feasibility and effect of transthoracic echocardiography(TTE)on guiding the occlusion of the soft-rim atrial septal defect(ASD).Methods Sixty two patients with the soft-rim ASD were enrolled.The size of ASD was measured and rim of ASD was observed by TTE on various views by using color Doppler system with tissue harmonic function before occlusion,and filmy rim of ASD with flapping which could not sustain occluder was eliminated.The size of occluder was selected by integratively judging the size of ASD and"sustainable diameter of ASD"The waist size of occluder was measured after releasing occluder and compared with the longest diameter of ASD and"sustainable diameter of ASD"measured by TTE.Results The longest diameter of ASD measured by TTE before occlusion was 11-35 mm[average(21.6±5.2)mm],the "sustainable diameter of ASD"was 15-37 mm[average(25.6±5.(J)mm],the size of selected occluder was 18-44mm[average(30.7±5.5)mini and the waist size of released occluder was 13-35 mm[average(24.2±5.6)mm].Fine correlation was existed between the longest diameter of ASD measured by TTE and the waist size of released occluder(r=0.86,P＜0.000I).Morever,improved correlation was found between the"sustainable diameter of ASD"measured by TTE and the waist size of released occluder(r=0.89,P＜0.0001).Occluder was firmly fixed without falling in all patients.Conclusions TTE with tissue harmonic function can be used to measure the size of soft-rim ASD and the"sustainable diameter of ASD".It is a feasible,and effective method on guiding occlusion of soft-rim ASD.

Objective To observe the display and source of the artery located at anterolateral region next to pancreatic head on en-hanced CT,and to investigate its clinical value.Methods Imaging data of 200 consecutive patients who underwent abdominal CT scan were collected,and their CT images were analyzed.The reconstructed images were obtained at a post-processing workstation. After reconstruction,the display rate,source and branches of the artery located at anterolateral region next to pancreatic head were analyzed,and the diameters of these vessels were measured.Results The arterial vessel located at anterolateral region next to pan-creatic head was found on axial CT image in arterial phase with a display rate of 100%.By using post-reconstruction images,most vessels were gastroduodenal artery with mean diameter of 0.33 cm± 0.05 cm,and other ones were pancreaticoduodenal artery or right gastroomental artery.Conclusion The artery located at anterolateral region next to pancreatic head can be found on axial ab-dominal CT image in the arterial phase.Most vessels are gastroduodenal artery with a fixed position and traveling.