Progress and future challenges in gene vectors, gene therapy systems and gene expressions

Genetic engineering has made sizeable contributions to technical innovation, agriculture, and the development of pharmaceuticals. Various approaches were evolved to control the genetic cloth of cells using both viral and nonviral vector architectures. Gene therapy aims to reverse pathological traits with the aid of the use of viral and nonviral gene shipping mechanisms. Gene transfer motors have made massive strides in becoming more environmentally pleasant, much less risky, and nonimmunogenic, as well as making an allowance for lengthy-time period transgene expression. One of the most tough components of correctly enforcing gene healing treatments in the clinical putting is adjusting gene expression extremely tightly and constantly as and while it's required. This research work will cognizance on using viral vectors for gene concentrated on biological applications with various gene expressions. Due to improvements in viral vector engineering and superior gene regulatory systems to permit and adjust tightly therapeutic gene expression, the technology for using genes to offer a preferred treatment has confirmed to be an effective approach

Satellite cell activation and signaling pathway response in joint exercise athletes / Ativação de células satélites e resposta na via de sinalização nos exercícios articulares dos atletas / Activación de las células satélite y respuesta de la vía de señalización en los ejercicios articulares de los atletas

ABSTRACT Introduction: Skeletal muscle satellite cells are considered the unique source of stem cells for myogenic differentiation of adult skeletal muscle cells. Upon stimulation, the skeletal muscle satellite cell can be activated through specific signaling pathways, proliferate and differentiate into a muscle cell. An analysis of the effects of key signaling pathways could provide the basis for an in-depth study of skeletal muscle formation in athletes and muscle development. Objective: This paper analyzes the effects of key signaling pathways on skeletal muscle satellite cell proliferation and differentiation. Methods: We divided 32 athletes into four groups: control, stretching, experimental, and mixed groups. The control group received no training at all, the stretching group and the experimental group received stretching training on the right gastrocnemius. The mixed group also got weight climbing training in the stretching training, initial load 30% of the athlete's weight, increasing 25% each week until 100% of body weight, at the frequency of 3 times a week. After training, gene expression of live satellite cells was measured by intramuscular signaling. Results: The FGM level of the antagonistic group (3.56±0.21) was higher than in the control group (3.25±0.18). The gene expression of HGF mRNA was higher in the mixed group (2.16±0.24) followed by the antagonistic group (2.02±0.15), the stretching group (1.81±0.25), and the control group (1.03±0.06). Conclusion: Both stretching and antagonistic training can increase gene expression in signaling pathways. Antagonistic training significantly increased the expression of HGF, MGF, and mRNA. This activity can promote muscle bulking and skeletal muscle enlargements. Evidence Level II; Therapeutic Studies - Investigating the result.

RESUMO Introdução: As células satélites musculares esqueléticas são consideradas a única fonte de células-tronco para a diferenciação miogênica das células musculares esqueléticas adultas. Após a estimulação, a célula satélite muscular esquelética pode ser ativada através de vias de sinalização específicas, proliferar e diferenciar-se em célula muscular. Uma análise sobre os efeitos das principais vias de sinalização poderia estabelecer as bases para um estudo aprofundado da formação muscular esquelética nos atletas e do desenvolvimento muscular. Objetivo: Este artigo analisa os efeitos das principais vias de sinal na proliferação e diferenciação das células satélites musculares esqueléticas. Métodos: Dividimos 32 atletas em quatro grupos. Grupos controle, alongamento, experimental e grupo misto. O grupo controle não recebeu treinamento algum, o grupo de alongamento e o grupo experimental receberam treinamento de alongamento no gastrocnêmio direito. O grupo misto também obteve treinamento de escalada com peso no treino de alongamento, carga inicial de 30% do peso do atleta, aumentando 25% em cada semana até 100% do peso corporal. Na frequência de 3 vezes por semana. Após os treinos, a expressão genética das células satélites vivas foi medida por intermédio da sinalização proveniente de coleta intramuscular. Resultados: O nível de MGF do grupo antagônico (3.56±0.21) foi maior que no grupo controle (3.25±0.18). A expressão gênica do mRNA HGF foi maior no grupo misto (2.16±0.24) seguido pelo antagônico (2.02±0.15), o grupo de alongamento (1.81±0.25) e o grupo controle (1.03±0.06) Conclusão: Tanto o treinamento de alongamento quanto o treinamento antagônico podem aumentar a expressão genética nas vias de sinalização. O treinamento antagônico aumentou significativamente a expressão de HGF, MGF e mRNA. Essa atividade pode promover volume e hipertrofia muscular. Nível de evidência II; Estudos terapêuticos - investigação dos resultados do tratamento.

RESUMEN Introducción: Las células satélite del músculo esquelético se consideran la única fuente de células madre para la diferenciación miogénica de las células musculares esqueléticas adultas. Tras la estimulación, la célula satélite del músculo esquelético puede activarse a través de vías de señalización específicas, proliferar y diferenciarse en una célula muscular. Un análisis sobre los efectos de las vías de señalización clave podría sentar las bases para un estudio en profundidad de la formación del músculo esquelético en los atletas y del desarrollo muscular. Objetivo: Este trabajo examina los efectos de las vías de señalización clave en la proliferación y diferenciación de las células satélite del músculo esquelético. Métodos: Dividimos a 32 atletas en cuatro grupos. Grupos de control, de estiramiento, experimentales y mixtos. El grupo de control no recibió ningún entrenamiento, el grupo de estiramiento y el grupo experimental recibieron un entrenamiento de estiramiento en el gastrocnemio derecho. El grupo mixto también recibió entrenamiento de escalada con pesas en el entrenamiento de estiramiento, con una carga inicial del 30% del peso del atleta, aumentando un 25% cada semana hasta el 100% del peso corporal. Con una frecuencia de 3 veces por semana. Tras el entrenamiento, se midió la expresión génica de las células satélite vivas mediante la señalización de la recogida intramuscular. Resultados: El nivel de FGM del grupo antagonista (3,56±0,21) fue mayor que en el grupo de control (3,25±0,18). La expresión génica del ARNm del HGF fue mayor en el grupo mixto (2,16±0,24), seguido del grupo antagonista (2,02±0,15), el grupo de estiramiento (1,81±0,25) y el grupo de control (1,03±0,06) Conclusión: Tanto el entrenamiento de estiramiento como el antagonista pueden aumentar la expresión génica en las vías de señalización. El entrenamiento antagónico aumentó significativamente la expresión de HGF, MGF y mRNA. Esta actividad puede promover el aumento de volumen muscular y la hipertrofia. Nivel de evidencia II; Estudios terapéuticos - Investigación de resultados.

A Preliminary Investigation Of Htr1a Gene Expression Levels In Autism Spectrum Disorders

Objective: This study was conducted to explore the expression levels of HTR1A gene in a sample of Egyptian autistic children. Methods: Thirty autistic patients (18 boys, 12 girls) and 20 controls were enrolled in the study. From each child, we isolated RNA samples from whole blood. Quantitative Real-Time PCR (qRT-PCR) was used to measure the gene expressions of HTR1A and normalized to the house keeping gene, beta-actin. Results: The HTR1A gene expression of healthy controls and ASD subjects were varied significantly (p =0.0062). As compared to control healthy subjects, the HTR1A expressions were greatly reduced in samples of ASD. Conclusion: HTR1A gene expression level is a candidate gene for further studies to explore its potential roles in ASD related pathways.

Comparative analysis of biochemical parameters and gene expressions of novel markers in type II Diabetes mellitus

Background: Diabetic nephropathy (DN) is the most common cause of end stage renal disease (ESRD). Early detection of the disease and treatment of this chronic complication which would reduce the medical and economic burden. Early detection of kidney injury by evaluating gene expressions of Il-6, Il-10, LDLr, and CD36 in T2DM with pre-ESRD microalbuminuria minimizes the risk of DN. Methods: Present research work conducted at the Department of Biochemistry, School of Medicine, Navi Mumbai. This study includes 241 subjects (118 male, 123 women, and age ranges 30-70 years) were included after screening for T2DM by measurement of blood glucose in fasting, post-prandial, glycosylated haemoglobin. Microalbumin in urine and e-GFR is measured to eliminate patients of ESRD. Subjects were recruited after written consent and enrolled as per inclusion/exclusion criteria. Categorization of subjects in three study groups; group I (30-45 years), group II (46-70 years) were done on the basis of T2DM duration 3-6 years, glycosylated haemoglobin level (HbA1c) ≥ 7.0% with fasting blood glucose ≥126 mg/dl) and microalbuminuria (30-300 mg/dl) in study group, equal numbers of healthy volunteers enrolled in control group. Blood samples were processed for other renal parameters and RT-PCR to check expressions of novel genes Results: In study groups all renal, lipids parameters are within normal range except albumin/creatinine ratio (p <0.012), e-GFR (p <0.00) and cholesterol (p <.00). Descriptive analysis showed high significance (p <.00) of delta CT gene expressions, parameters in pre-ESRD microalbuminuria subjects. Conclusion: Screening biochemical renal parameters are not enough to prevent DN even in microalbuminuria. Early detection of gene expressions of novel biomarkers predicts risk of kidney injury. Early intervention may prevent morbidity and mortality of kidney due to diabetic nephropathy.

Establishment of a biodosimeter model based on multiple gene expressions in human peripheral blood cells after 60Co γ-rays irradiation / 中华放射医学与防护杂志

Objective To investigate a potential radiation biodosimetry based on multiple gene expressions.Methods Human peripheral blood were exposed to 60Co γ-rays at doses from 0 to 8 Gy.The mRNA expression levels of 10 selected genes were detected 6 and 12 h after irradiation by RT-PCR.Individual variation was also examined.An optimal mathematical model of the dose response of these gene expression levels at each time point was obtained by the stepwise regression method.A blind test was conducted to validate the statistical accuracy of dose estimation.Results The 10 selected genes expression levels at each time point were significantly increased along with dose from 0.5 to 8 Gy (R2 =0.61-0.97,P ＜ 0.05).Individual variations were evident in the gene expressions of TNFSF4,PHPT1 and FDXR.The gene expression levels of PCNA,CCNG1,TNFSF4,PHPT1,GADD45A and FDXR were incorporated into the model at 6 h after exposure (R2 =0.88,F =54.8,P ＜ 0.001);the gene expression levels of PCNA,CCNG1,TNFSF4,MDM2,GDF15 and TNFRSF10B were included in the model at 12 h after irradiation (R2 =0.82,F =42.767,P ＜ 0.001).These two statistical models can be utilized for the dose estimation accurately.Conclusions The multiple gene expressions have a potential as a radiation biodosimetry.

Effects of Triterpenoids from Luvunga scandens on Cytotoxic, Cell Cycle Arrest and Gene Expressions in MCF-7 Cells

Plant-derived triterpenoids commonly possesses biological properties such as anti-inflammatory, anti-microbial, anti-viral and anti-cancer. Luvunga scandens is one of the plant that produced triterpenoids. The aims of the study was to analyze cell cycle profile and to determine the expression of p53 unregulated modulator of apoptosis (PUMA), caspase-8 and caspase-9 genes at mRNA level in MCF-7 cell line treated with two triterpenoids, flindissol (1) and 3-oxotirucalla-7,24-dien-21-oic-acid (2) isolated from L. scandens. The compounds were tested for cell cycle analysis using flow cytometer and mRNA expression level using quantitative RT-PCR. The number of MCF-7 cells population which distributed in Sub G1 phase after treated with compound 1 and 2 were 7.7 and 9.3% respectively. The evaluation of the expression of genes showed that both compounds exhibited high level of expression of PUMA, caspase-8 and caspase-9 as normalized to β-actin via activation of those genes. In summary, the isolated compounds of L. scandens plant showed promising anticancer properties in MCF-7 cell lines.

Effects of long-term low-dose γ-rays exposure on radiosensitivity of human B lymphoblast cells / 中华放射医学与防护杂志

Objective To investigate the effects of long-term low-dose radiation (LDR) of γ-rays on the proliferation and radiosensitivity of human lymphoblast cells HMy2.CIR (HMy) and to elucidate the underlying mechanism.Methods HMy cells were divided into control group and long-term LDR group.For the long-term LDR treatment,HMy cells were fractionally exposed to a low dose of γ-rays,which could enhance cell proliferation,3 times per week for 4 weeks.After the long-term LDR exposure,part of the control and long-term LDR exposed cells were further irradiated with a challenging dose (2 Gy) of γ-rays.Then cell proliferation and radiosensitivity were assayed by CCK-8 kit,cell apoptosis,and γ-H2AX formation was measured by flow cytometry.Gene expressions of cyclinD1,PCNA,bcl-2 and bax were detected by RT-PCR.Results The long-term LDR significantly increased cell proliferation (t =9.607,P ＜ 0.01) accompanied with up-regulation of cell cycle regulation gene cyclinD1 (t =6.869,P ＜ 0.01),proliferation regulation gene PCNA (proliferating cell nuclear antigen) (t =9.229,P ＜ 0.01) and bcl-2 gene (t =2.662,P ＜ 0.05),but decreased the expression of pro-apoptotic gene bax (t =19.908,P ＜0.01) in HMy cells.Compared to untreated cells,the long-term LDR decreased cell radiosensitivity (t =8.896,P ＜ 0.01),including apoptosis induction (t =4.762,P ＜ 0.01) and γ-H2AX formation (t =10.264,P＜0.01).Conclusions The long-term LDR promoted cell proliferation by up-regulating cell cycle related genes,while it reduced the radiosensitivity of HMy cells with acquisition of apoptotic resistance.

Study of enhancer in gene expression regulation / 国际肿瘤学杂志

Enhancers are cis-acting elements of DNA which can enhance the transcription activity of promotors.Emerge of Bioinformatics brings great convenience for enhancers investigation.The utilization of tumor-specific promotors modified by enhancers to make the expression of theoretical gene or effector in specified organisms or cells,and to implement targeted therapy of tumor,is a significant and potential gene therapy method.

Signal transducer and activator of transcription 1 and matrix metalloproteinase 3's genetic expression and their clinical significance on urothelial tumor after renal transplantation / 中华泌尿外科杂志

Objective To investigate the signal transducer and activator of transcription 1(STAT1) and matrix metalloproteinase 3(MMP3)′s genetic expressions and their clinical significance on urothelial carcinoma after renal transplantation. Methods Fifty-one patients with urothelial carcinoma were recruited in this study. Sixteen of them who had renal transplant were in the experimental group and 35 of them without renal transplant were in the control group. All the cases had been proved postoperatively having transitional cell carcinoma by histopathological study. The human genome oligo arrays were used to analyze the gene expression spectrum of urothelial carcinoma after transplantation, aiming the STAT1 and MMP3's expression. Real time RT-PCR and immunohistochemical staining were used to compare the differences in the 2 groups. Results The experimental group showed that there were 35 genes up-regulated compared with the control group. Of them, 23had known gene function or partly known, and 12 had unknown gene function. There were 76 genes down-regulated. Of them, 46 had known gene function or partly known, and 30 had unknown gene function. After pathway analysis of the differentially expressed genes, there were 23 groups of pathways which had significant differences (P＜0.05), referring to the aspects of immunosuppressive and tumor growth. The levels of STAT1 and MMP3 expressions had significant differences between the 2groups(P＜0.05)as well. Conclusions The differential expression of urothelial tumor genes is obvious between patient who has had renal transplant and who has not. There are many aspects that are related to the tumor's growth like signaling pathways regulating proliferation, apoptosis of tumor cells, tumor angiogenesis and the tumor metastasis potential. STAT1 and MMP3 maybe become the targets of chemoprevention for post-transplantation urothelial carcinoma.

The expressions and clinical sig nificances of p27 and proliferating cell nuclear antigen and nuclear-associated antigen in synovial sarcoma / 中国地方病学杂志

Objective To investigate the association of the expressions of p27 and proliferating cell nuclear antigen(PCNA)and nuclear-associated antigen(ki-67)proteins in synovial sarcoma,and the relationship with its prognosis.Methods The expression of p27,PCNA and ki-67 in 36 synovial sarcoma and 10 normal synovial tissue were immunohistochemically examined.The relationship between the expressions of p27,PCNA and ki-67 proteins in synovial sarcoma with its prognosis by clinical stage,tissue grades and prognosis was investigated.Results (1)In normal synovial tissue,the expressions of PCNA(0) and ki-67(0) were positive,p27 were positive100%(10/10).but the expressions of PCNA[86.1%(31/36)]and ki-67[69.4%(25/36)]in most synovial sarcoma were positive,p27 were negative[11.1%(4/36)],significant differences were found among these expressions(χ2=25.16,χ2=9.18,P＜0.05).(2) p27 positive-expression correlated significantly wiht thehistological grade of synovial sarcoma(χ2=9.19,P＜0.05)and prognosis(χ2=5.98,P＜0.05).The positive expressions of PCNA and ki-67 were positively related with the clinical stage(χ2=7.40,χ2=8.12,P＜0.05)and tissue grades(χ2=7.17,χ2=9.18,P＜0.05)and prognosis(χ2=13.03,χ2=10.66,P＜0.05)of synovial sarcina(3) There were negative correlations between p27 and PCNA(r=-0.63,P＜0.05) and between p27 and ki-67(r=-0.53,P＜0.05).But positive correlation between PCNA and ki-67(r=0.63,P＞0.05)was found.Conclusions Expressions of PCNA and ki-67 in most synovial sarcomas are positive,and that of p27 is negative,each plays an important role in the proliferation of synovial sarcoma.Combination of measuring of the p27,PCNA and ki-67 is valuble in predicting the prognosis.

Inhibitory effects of recombinant human parvovirus H-1 vector expressing p21 (rhH1 Δ/p21) on the growth of human gastric cancer cell line HGC27 / 肿瘤

Objective: To investigate the effects of recombinant parvovirus H-1 vector expressing p21(rhH1 Δ p21) on human gastric cancer cell line HGC27, and to further reveal the biological function of p21wif1 to provide the basis for cancer gene therapy. Methods: The recombinant parvovirus H-1 vector expressing p21 (rhH1 Δ/p21) was constructed by reverse transcriptase-polymerase chain reaction (RT-PCR), and was transfected into HGC27 cell line. The morphological changes of HGC27 cell were observed. The transgene protein expressions in the gastric cancer cells were detected by Western blot. The inhibitory effects of rhH1 Δ/p21 on the growth of HGC27 cells were measured by MTT assay. The cell cycle distribution was determined by flowcytometry. Results: The rhH1 Δ/p21 was successfully constructed, with a titer of 3.5 × 107 PFU/mL. The transgene protein p21 was over-expressed in the HGC27 cells. The cell cycle distribution was changed. The proportion of cells in G1 phase significantly increased. The cell growth was significantly inhibited. Conclusion: rhH1 Δ/p21 induces G1 arrest and inhibits the proliferation of gastric cancer HGC27 cells. It indicates that rhH1 Δ/p21 gene therapy can effectively inhibit the growth of gastric cancer cells in vitro.

Expressions of heparanase and CD44v6 in osteosarcoma tissues and their correlations / 肿瘤

Objective: To study the expression of heparanase and CD44v6 in osteosarcoma and analyze the correlation between them. Methods: The expressions of heparanase and CD44v6 in human osteosarcoma were detected by immunohistochemical staining. Results: The positive rates of heparanase and CD44v6 protein in osteosarcoma tissues were significantly higher in osteosarcoma tissues than in osteochondroma (79.6% vs 20.0% and 55.1% vs 25.0%, P < O. 05). Expressions of heparanase and CD44v6 had positive correlation (r=0.663, P < 0.001). Both of them were closely associated with clinical stage and metastasis of osteosarcoma. Conclusion: Overexpressions of heparanase and CD44v6 in human osteosarcoma may stimulate the adhesion, invasion, and metastasis of osteosarcoma.

The Effect of Conjugated Linoleic Acid Isomers on the Cell Proliferation, Apotosis and Expressions of Uncoupling Protein (Ucp) Genes during Differentiation of 3T3-L1 Preadipocytes

It has been reported that CLA decreases fat deposition in vivo and in vitro experiments. Among CLA isomers, c9t11 and t10c12 have been shown to exert active biological activities. For example, t10c12 reduces body weight and increases lean body mass, whereas, c9t11 has little effect on body fattness. However, the underlying molecular mechanism for the anti-obesity action of CLA isomers are not well understood. The purpose of this study was to examine the effects of t10c12 and c9t11 on lipid accumulation, cell proliferation, cell death and the expression levels of Ucp genes which are proposed as targets for anti-obesity in 3T3-L1 preadipocytes. Isomers of CLA at 50 micrometer were added into preadipocyte differentiation medium for 3, 6 and 9days. Control cells received only the vehicle in the differentiation medium. Cytochemical analyses for lipid accumulation, cell proliferation and apotosis were carried out to compare lipidogenesis and cellular activity. RT-PCR analysis of GAPDH, Ucp 2, 3 and 4 were also performed to find any modulatory effects of CLA isomers on the metabolic genes. Lipid accumulation indicated by Oil Red-O staining was inhibited in CLA isomers as compared to the control. T10c12 isomer showed less lipidogenesis than c9t11 did. A decrease occurred in CLA isomers as shown by BrdU incorporation. Apotosis has occured at higher level in t10c12 when compared to that of t9c11. Ucp 2, 3 and 4 genes were also upregulated in CLA isomers. T10c12 showed higher level of Ucp gene expressions than the c9t11 did. The biological activities of CLA isomers were also found to be different during differentiation of 3T3-L1 preadipocytes, suggesting that different isomers may be active in certain stage of lipidogenesis. The results indicate that both c9t11 and t10c12 CLA isomers decrease lipidogenesis, inhibit cell proliferation, increase cell death and upregulate in Ucp gene expressions during 3T3-L1 preadipocyte differentiation. T10c12 isomer was more effective than c9t11 in overall anti-obesity activity.

Significance of the expressions of VEGF mRNA, bFGF mRNA,PDGF mRNA and their receptors in pancreatic carcinoma / 中国普通外科杂志

Objective To study the effect of intratumor angiogenesis and vascular growth factor(VEGF), basic fibroblase growth factor(bFGF), Platelet-derived growth factor (PDGF), and their receptors(flt-1 bFGFR.PDGFR) on the invasion, metaslasis of pancreatic carcimoma(PC) and the relationship between the expressions of three kinds of angiogenic factors, their receptors and microvessel count(MVC). Methods Tis- sue sections of 51 PC and 32 acute or chronic pancreatitis were examined by in situ hybridization for the expression of VEGF bFGF.PDGF, and by immunohistochemistry for the expression of the three kinds of angiogenic factor receptors and MVC. The correlation of the expressions and pathological characteristics of PC were also studied. Results The positive rate of VEGF mRNA,bFGF mRNA, PDGF mRNA and their receptors in PC were significantly higher than that in acute or chronic pancreatistis( P