Vectors used to carry foreign genes play an important role in gene therapy, among which, the adeno-associated virus (AAV) has many advantages, such as nonpathogenicity, low immunogenicity, stable and long-term expression and multiple-tissue-type infection, etc. These advantages have made AAV one of the most potential vectors in gene therapy, and widely used in many clinical researches, for example, Parkinson's disease. This paper introduces the biological characteristics of AAV and the latest research progress of AAV carrying neurotrophic factor, dopamine synthesis related enzymes and glutamic acid decarboxylase gene in the gene therapy of Parkinson's disease.

In this paper, the preliminary study on antioxidant, enhancement of antioxidant enzymes activity, reducing the content of oxygen free radicals, delaying skin aging of the recombination cytoglobin (rCygb) purified by our lab were investigated through human keratinocyte cell line (HaCAT) H2O2 oxidative stress model, mouse skin aging model caused by continuous subcutaneous injection D-gal, rat acute liver injury model induced by CCl4 and rat skin wound healing model. The results showed that rCygb improved the activities of total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), reduced the activities of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) as well as decreased the content of malondialdehyde (MDA). Skin biopsy showed that rCygb promoted angiogenesis, increased expression of collagen and improved the anti-inflammatory ability. All results displayed that rCygb improved the oxygen free radical scavenging ability, delayed skin aging and promoted wound healing.

he aim of this study was to obtain a cell-penetrating cytoglobin (Cygb), which combines the transmembrane function of cell-penetrating peptides TAT with the anti-aging and anti-fibrotic role of cytoglobin. The Cygb gene was complexed with TAT gene by overlapping PCR, inserted into the vector pET22b to construct the recombinant expression plasmid (pET22b-TAT-Cygb) and then transformed into Escherichia coli BL21 (DE3). The fusion protein TAT-Cygb, whose expression was induced by lactose, was purified by CM Sepharose Fast Flow Protocol and verified by Western blotting. The final TAT-Cygb had a molecular weight of 23 kDa with 95% purity, as shown by SDS-PAGE. As demonstrated by bioactivity experiments, TAT-Cygb exhibited a high specific peroxidase activity up to (422.30 ± 0.36) U/mg. Both TAT-Cygb and Cygb pretreatment group could protect Hacat cells against oxidation of H2O2, but only TAT-Cygb treatment group could remedy cells injuried by H2O2 (RGR = 98%), which was significantly different from Cygb treatment group (RGR = 79%). We successfully obtained the bioactive and cell-penetrating fusion protein TAT-Cygb that has the potential application in anti-aging, anti-fibrotic and anti-cancer.
Blotting, Western ; Cell Line ; Cell-Penetrating Peptides ; biosynthesis ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; metabolism ; Gene Products, tat ; Genetic Vectors ; Globins ; biosynthesis ; Humans ; Hydrogen Peroxide ; Recombinant Fusion Proteins ; biosynthesis

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 improving effect of curcumin on cardiac function and its influence on myocardial collagen concentration in rats with myocardial infarction (MI).Methods:A total of 60 healthy adult male SD rats were randomly divided into sham operation group (n=10,survived rats n=9)and operation group [n=50,left an-terior descending artery was ligated,they were randomly divided into following four groups,on 28 d the treatment and survived rats in every group were:myocardial infarction (MI)group (n=9),control group (received intraper-itoneal injection of mixed solution,n= 10)and curcumin group (received intraperitoneal injection of curcumin, 100mg·kg-1 · d-1 n= 12)].After four weeks,M-mode echocardiography was used to measure left ventricular shortening fraction (LVFS),left ventricular ejection fraction (LVEF)and heart rate (HR).Masson staining was used to detect the myocardial fibrosis alteration after MI.Immunohistochemistry was used to measure expressions of myocardial collagen type Ⅰ and Ⅲ,and ratio of collagen Ⅰ/Ⅲ.Results:Compared with MI group and control group,after four weeks,there were significant rise in LVFS [(17.23±1.97)%,(19.34±0.83)% vs.(26.70± 1.15)%]and LVEF [(42.08±5.50)%,(41.63± 1.81)% vs.(56.76±2.49)%],significant reductions in HR [(433.16±20.05)beats/min,(433.04±24.17)beats/min vs.(403.96±7.08)beats/min],concentrations of myo-cardial collagen Ⅰ and Ⅲ,and ratio of collagen Ⅰ/Ⅲ [(13.5±0.9),(13.9±1.0)vs.(10.3±1.6)]in curcumin group,P <0.01 all.Masson staining indicated that acute myocardial infarction can cause significant left ventricular interstitial fibrosis.Conclusion:Curcumin could significantly improve cardiac function and inhibit myocardial fibro-sis level in rats with acute myocardial infarction.

Objective: To explore independent risk factors of coronary artery calcification (CAC) and analyze correlation among risk factors of CAC and serum osteopontin (OPN) level. Methods: According to results of 64-slice spiral computed tomography (MSCT) coronary angiography, a total of 65 patients were continuously enrolled and divided into CAC group (n=37) and non-CAC control group (n=28). Enzyme linked immunosorbent assay (ELISA) was used to measure serum level of OPN. Single factor and multiple factor Logistic regression analysis’s were used to analyze risk factors of CAC. Spearman’s straight line analysis was used to analyze correlation between risk factors of CAC and serum OPN. Results: 1、 The age, hypertension, diabetes, poor eating habits，lack of exercise, overweight, etc., which were independent risk factors of CAC （OR=3.47～12.96, P=0.018～0.003）by single factor Logistic regression analysis, were inducted to multiple factor Logistic regression analysis, its result showed that age, overweight, poor sleep quality, poor eating habits were independent risk factors of CAC, OR=35.31～5.17, P＜0.01～0.05; 2、Serum level of OPN in CAC group was significant higher than that of non-CAC control group ［(39.919±11.879) μg /L vs. (24.000±6.000) μg /L，P<0.01］; 3、The Spearman straight line correlation analysis indicated that serum level of OPN was correlated with risk factors of CAC : positively correlated with LDL-C, overweight, age, TC（r=0.487～0.286,P＜0.001～＜0.05）, and positively correlated with poor sleep quality, diabetes, poor eating habits, lack of exercise（r=4.10～2.24， P<0.01～0.05）; negatively correlated with HDL-C（r=-0.250,P＜0.05）. Conclusion: Correlation analysis indicates that age, overweight, poor sleep quality, poor eating habits etc. are independent risk factors of CAC；Serum OPN level is correlated with LDL-C, overweight，age, diabetes, lack of exercise etc., so these indicate that must decrease OPN level and risk factors of CAC to relieve CAC and slow down its development.

Objective To investigate the relationship between hepatitis B virus (HBV) genotype,the mutation in basic core promoter(BCP)region/pre-core(Pre-C)region and the incidence of hepatocellular carcinoma(HCC) in Fusui county of Guangxi Zhuang Autonomous Region (Guangxi),a area with high incidence of HCC. Methods In this case-control study,53 HCC patients and 70 asymptomatic HBV carriers were enrolled. Blood samples were collected from them for serum separation and HBV DNA extraction. The DNA sequences of the S region and BCP/Pre-C region of HBV was determined by direct sequencing following nested-PCR amplification. The relationship between the genotype,gene mutation of HBV and the incidence of HCC was analyzed. Results The mutation rates of the A1762T/G1764A in the BCP region and the T1858C in the Pre-C region of HBV were significantly higher in HCC group than in control group(94.3%vs. 75.7%,P=0.006;50.9%vs. 31.4%,P=0.029). The mutation rate of A1775G was significantly higher in control group (28.6%) than in HCC group (13.2%)(P=0.041). Multiple logistic regression analysis indicated that A1762T/G1764A and T1858C mutations are the risk factors for the development of HCC (OR=5.459,95%CI:1.397- 21.332,P=0.015;OR=3.881,95%CI:1.462-10.305,P=0.006). A1775G is the protective factor in the development of HCC(OR=0.192,95%CI:0.059-0.622,P=0.006). Conclusion The present investigation showed that BCP A1762T/G1764A,A1775G and Pre-C T1858C mutations are correlated with the incidence of HCC in Fusui county of Guangxi.

Purpose To investigate human soluble TRAIL(sTRAIL)protein expression and purification and its potential anti-tumor activity on hepatocellular carcinoma(HepG2).Methods Soluble TRAIL gene ligated with expression vector pPIC9 was transfected into GS115(his4)and the recombination strain expressing sTRAIL was screened by MD plate.The effects of different media,methanol inducement period,methanol concentration,and pH were investigated and optimized using shaking flask.The anti-tumor activity of sTRAIL with HepG2 cells Was analyzed after purification.Results The highest expression of sTRAIL was obtained at pH 6.0,1% methanol in BMMY medium,with the concentration of(58.7±2.4)mg/L at 48 h.Recombinant sTRAIL protein could induce HepG2 cells apoptosis and inhibit HepG2 cells proliferation effectively.Conclusion The optimized condition of human sTRAIL expression and purification Was developed and the obtained recombinant sTRAIL protein may be a promising therapeutic agent for hepatocellular carcinoma.

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