Objective:To construct K-ras-targeted siRNAs (K-ras siRNA) and to investigate the inhibitory effects of K-ras siRNAs on the growth and migration of lung cancer A549 cells (containing mutant K-ras gene) and NCI-H446 cells (containing wild-type K-ras gene). Methods: Four K-ras siRNAs (K-ras siRNA1～K-ras siRNA3 targeting wild-type K-ras and K-ras siRNA4 targeting mutant K-ras) were designed and artificially synthesized; they were used to transfect A549 cells and NCI-H446 cells. The expressions of Ras mRNA and protein were examined by RT-PCR and Western blot-ting. The inhibitory effects of K-ras siRNAs on the proliferations of A549 and NCI-H446 cells were determined by MTT assay. The effects of K-ras siRNAs on the cell migration and apoptosis were observed by Transwell assay and Hoechst 33258 staining, respectively. Results: Mutant K-ras-targeted siRNA (K-ras siRNA4) specifically inhibited the K-ras ex-pression but had no influence on H-ras and N-ras expression in A549 cells. K-ras siRNA4 inhibited the proliferation of A549 cells but did not inhibit that of NCI-H446 cells, which contained wild type K-ras gene. K-ras siRNA4 also induced apoptosis and inhibited migration of A549 cells. Conclusion: Mutant K-ras-targeted siRNA4 can inhibit the proliferation, migration and induce apoptosis of A549 cells. It may be a potential and personalized drug for the treatment against lung cancer containing mutant K-ras gene.

Objective To study the producing method for Euphorbia pekinensis promoted by endophytic fungi.Methods The tissue culture plantlets were innoculated with endophytic fungi,then it was transplanted to the pods and cultured for one year.The biomass and content of terpenoids(isoeuphpekinensin and euphol)were determined.Results The growth of the E.pekinensis was promoted by endophytic fungi E4(Fusarium sp.Ⅰ)and E5(Fusarium sp.Ⅱ).The fresh weight of the root increased 49.45% and 59.74% for E4 and E5 treated,respectively.The plants dry weight coefficient increased 2.99% and 6.48% for E4 and E5 treated,respectively.The contents of isoeuphpekinensin and euphol in the plants were increased for tissue cultured in both bottles and pods.For the plants cultured in pods for one year,the isoeuphpekinensin and euphol increased 92.79% and 40%,respectively treated by E4.The isoeuphpekinensin and euphol increassed 105.32% and 241.38%,respectively treated by E5.Conclusion The endophytic fungi E4 and E5 could promote the growth of E.pekinensis and accumulated terpenoids.

Objective To optimize the formulation of metoprolol succinate ( MS) controlled release pellets by central composite design-response surface methodology .Methods MS sustained-release pellets were prepared using sugar pellet cores as starter beads , ethyl cellulose as coating materials and MS itself as a pore former .The formulation of MS sustained-release pellets was optimized by a central composite design with two factors at five levels .These two factors ( two independ-ent variables) were the pore former level and coating level , and the evaluated indexes ( namely dependent variables ) included the in vitro cumulative release percentages of MS at 1, 4, 8, 12 and 16 h, respectively.Results and Conclusion The results of mathematical equation fitting suggested that the second-order quadratic model was the optimal fitting equa-tion.According to the response surfaces , the optimum values at the pore former level and coating level weve ranged from 16%to 18%and 20% to 25%, respectively .The in vitro cumulative release percentage of MS from the pellets at 1 h reached 9.15%,which consequently eliminated the lag phase in the initial release period and exhibited a good sustained-release effect.Central composite design-response surface methodology can be applied to optimizing the coating formulation for MS sustained release pellets .

High mobility group A2 protein (HMGA2), an architectural factor, is highly expressed in various cancer types including lung cancers. It is a candidate target for cancer therapy. RNAi is an effective gene silencing method with low cost and less time-consuming. It is possible to exploit this technology in therapy. Here, 5 siRNAs targeting Hmga2 gene (HMGA2 siRNA1-5) were designed and synthesized. MTT assay, colony formation assay, transwell assay and flow cytometry were used to evaluate the effects of these siRNAs on lung cancer cell lines (NCI-H446 and A549). Results from cell proliferation, clone formation, migration and apoptosis showed that HMGA2 siRNA1, 3, 5 could affect these aspects for both lung cancer cell lines. Among the five siRNAs, HMGA2 siRNA5 showed the greatest inhibition effects. The inhibition effects of HMGA2 siRNA5 are sequence specific and are not due to the induction of interferon response. Taken together, siRNAs targeting Hmga2 gene are potential candidates for lung cancer gene therapy.

The aim of this study was to reveal the protection role and the related mechanism of cytoglobin on the oxidation induced hepatic stellate cell damage. We applied siRNA to interfere the endogenous cytoglobin gene, used recombinant cytoglobin protein to treat the completely activated human hepatic stellate cell line LX-2 and the incompletely activated primary rat hepatic stellate cells, or over-expressed cytoglobin protein in LX-2 cells. We used two different oxidative-stress related models, the hydrogen peroxide model and the iron-overload model in our experiments and investigated the proliferation status and the intracellular superoxide level of the cells. The results showed that endogenous cytoglobin exerted significant protective effects on hydrogen peroxide or iron-overload induced LX-2 cell damage, confirming that upregulation of cytoglobin was the protective response of activated hepatic stellate cells to oxidative stress. Recombinant cytoglobin protein could protect LX-2 cells from oxidation induced damage, and prevent primary rat hepatic stellate cells from excessive proliferation and injury. The cytoplasmic reactive oxygen species (ROS) scavenging capacity of the recombinant cytoglobin protein was not as good as its capacity in scavenging ROS outside the cells, likely owing to the lack of active transporting mechanisms. Intracellular over-expression of cytoglobin protein could exert significant protective effect on LX-2 cells treated with hydrogen peroxide or iron-overload. Our results would accelerate the exploitation of new anti-fibrotic targets.
Animals ; Cell Line ; Globins ; genetics ; pharmacology ; Hepatic Stellate Cells ; cytology ; pathology ; Humans ; Hydrogen Peroxide ; toxicity ; Oxidative Stress ; drug effects ; Protective Agents ; pharmacology ; RNA, Small Interfering ; genetics ; Rats ; Reactive Oxygen Species ; metabolism

Recombinant adeno-associated viral vectors (rAAV) have been widely used as gene therapy vectors in clinical trials. Here, we reviewed the genomic structures and replication mechanisms of wt-AAV. Then, the assembly of capsid and the encapsidation of genomic DNA, two major events during AAV pakaging, was discussed in detail. Although the overall pattern of virus assembly and encapsidation is known, the molecular mechanisms and the structure-function relationship involved in these processes are not well understood. Further elucidatation of these processes may improve the production technology of rAAV and develop gene drug based on rAAV.
Capsid ; physiology ; Capsid Proteins ; genetics ; DNA, Viral ; genetics ; Dependovirus ; genetics ; physiology ; Genetic Vectors ; Genome, Viral ; Virus Assembly ; genetics ; physiology

Numerous studies and clinical trials have demonstrated the efficacy of recombinant adeno-associated virus gene delivery vectors. However, prior to expression, it is necessary to convert the single-stranded DNA genome into double-stranded DNA, which hinders the efficiency of these vectors. We can entirely circumvent this step through the use of self-complementary recombinant adeno-associated virus vector (scrAAV). ScrAAV packages an inverted repeat genome that can fold into double-stranded DNA without the requirement for DNA synthesis or base-pairing between multiple vector genomes. By using scrAAV, we could increase expression efficiency and reduce immune response caused by vectors themselves. Therefore, it is a promising vector for gene therapy. So far, it has been used in the treatment of hepatic diseases, central nervous system diseases, and eye diseases. It has also been used in the modifications of stem cells and as vectors for siRNA/miRNA and ribozymes. In this review, we focused on the preparation, expression and location of scrAAV both in vitro and in vivo. We mainly introduced the recent progress of scrAAV based therapy of Hemophilia B, in order to elucidate the potential and prospects of scrAAV in gene therapy.
Animals ; Base Sequence ; DNA ; genetics ; DNA, Complementary ; genetics ; DNA, Single-Stranded ; genetics ; Dependovirus ; genetics ; metabolism ; Gene Transfer Techniques ; Genetic Therapy ; methods ; trends ; Genetic Vectors ; genetics ; Hemophilia B ; therapy ; Humans ; Molecular Sequence Data

Objective To explore the relationship between anhedonia,alexithymia and self-efficacy in college students.Methods 260 college students were tested with Snaith-Hamilton Pleasure Scale (SHAPS),Temporal Experience of Pleasure Scale (TEPS),20-item Toronto Alexithymia Scale (TAS-20)and General Self-Efficacy Scale (GSES).Results (1) Males showed obviously higher level of SHAPS scores (24.91±5.14) and lower level of anticipatory pleasure of TEPS (TEPS-ANT) scores(31.81±5.79)than females (22.97±5.34 and 33.36±5.71,respectively).(2)Significant difference existed between the high-scoring,the middle-scoring and the low-scoring anhedonia group in TAS factor 1 (difficulty in identifying feelings) and GSES (F=3.11,5.30,P＜0.05 or 0.01),the scores of TAS factor 1 (difficulty in identifying feelings) in high-scoring anhedonia group were higher than that in low-scoring anhedonia group (20.24±4.94 vs 18.10±5.07,P＜0.01),and the scores of GSES in high-scoring anhedonia group were lower than that in low-scoring anhedonia group (24.97±6.72 vs 27.90±4.37,P＜0.01).(3)The SHAPS scores were positively related to TAS factor 2 (difficulty in describing feelings) and factor 3 (externally oriented thinking) (r =0.18,0.27,both P＜0.01),negatively related to GSES scores(r=-0.33,P＜0.01).Both TEPS-ANT and consummatory pleasure of TEPS (TEPS-CON) were negatively related to TAS factor1 (difficulty in identifying feelings)scores (r=-0.22,P＜ 0.01;r =-0.15,P＜ 0.05),positively related to GSES scores (r =0.28,0.26,both P＜0.01).(4)Layer multivariate linear regression analysis showed that the SHAPS scores were significant predictors of the general self-efficacy(β=-0.33,P＜0.01).Conclusions The anhedonia in college students has a closely relationship with alexithymia and self-efficacy,college students with high anhedonia have high level of alexithymia and low level of self-efficacy.

OBJECTIVETo study a reliable CT measuring method for quantitative diagnosis of unilateral zygomatic complex fracture and operation guiding.

METHODS20 zygomatic complex fracture patients needed operation were collected, three dimensional CT images of all patients were measured and analyzed by Mimics software before operation. Standard anatomy points of bilateral zygomatic complex and adjacent bone were selected, then the distance of two same selected points and the angles of three same selected points were measured by Mimics software. Compared to uninjured side, the different value of the distance and the angle of injured side zygomatic complex were acquired, zygomatic complex fractures were operated based on the different value. All patients were examined by three dimensional CT postoperatively, and the different value of the distance and the angle of both side were measured using same method to pre-operation. Therapy effects were analyzed based on the data of different value of the distance and the angle.

RESULTS1) The displacement of zygoma of most patients was backward and inward. The different value of the distance between uninjured and injured side was less than 2 mm, and the different value of the angle was less than 1 degrees. All patients reached the three-dimensional symmetric, surgical results were satisfactory in recovery of occlusion and mouth opening. 2) The zygomatic arch protrusion angle was about 138.50 degrees +/- 1.15 degrees, zygomatic prominent angle was about 132.72 degrees +/- 0.89 degrees. The two data were normal distribution.

CONCLUSIONThree dimensional CT measurements of zygomatic complex can be achieved for the quantitative measurement of spatial displacement, and have directive significance for the reduction of the zygomatic complex fractures.

Background: Inactivated vaccines are limited in preventing foot-and-mouth disease (FMD) due to safety problems. Recombinant virus-like particles (VLPs) are an excellent candidate for a novel vaccine for preventing FMD, given that VLPs have similar immunogenicity as natural viruses and are replication- and infection-incompetent. Objectives: The 3C protease and P1 polyprotein of type O FMD virus (FDMV) was expressed in yeast Hansenula polymorpha to generate self-resembling VLPs, and the potential of recombinant VLPs as an FMD vaccine was evaluated. Methods: BALB/c mice were immunized with recombinant purified VLPs using CpG oligodeoxynucleotide and aluminum hydroxide gel as an adjuvant. Cytokines and lymphocytes from serum and spleen were analyzed by enzyme-linked immunosorbent assay, enzyme-linked immunospot assay, and flow cytometry. Results: The VLPs of FMD were purified successfully from yeast protein with a diameter of approximately 25 nm. The immunization of mice showed that animals produced high levels of FMDV antibodies and a higher level of antibodies for a longer time. In addition, higher levels of interferon-γ and CD4 + T cells were observed in mice immunized with VLPs. Conclusions The expression of VLPs of FMD in H. polymorpha provides a novel strategy for the generation of the FMDV vaccine.