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Objective To investigate the expression and mechanism of miR-1470 in plasma of diabetic retinopathy (DR) patients.Methods Thirty patients with DR (DR group),30 patients with diabetes (DM group) and 30 normal healthy subjects (normal group) were enrolled in the study.Three groups of subjects were taken 5 ml of venous blood,and total plasma RNA was extracted and purified.The differentially expressed miRNAs in the plasma of DR patients were screened by gene chip,and the results of gene chip detection were verified by reverse transcription polymerase chain reaction (RT-PCR).Bioinformatics was used to predict potential target genes for miRNA regulation,and miR-1470 and its target gene epidermal growth factor receptor (EGFR) were screened.Human retinal microvascular endothelial cells (hREC) were divided into normal group (sugar concentration 5.5 mmol/L) and high glucose group (sugar concentration 25.0 mmol/L).hREC was transfected into miR-1470 mimics to establish a miR-1470 high expression cell model,which was divided into blank control group,high expression group and negative control group.The expression of miR-1470 was detected by RT-PCR.The expression of EGFR protein was detected by Western blot.The measurement data of the two groups were compared using the independent sample t test.The comparison of the measurement data between the two groups was analyzed by ANOVA.The comparison between the measurement data of the groups was compared by multiple comparisons.Results The results of RT-PCR were consistent with those of the gene chip.The expression of miR-1470 in the plasma of the DR group,the DM group and the normal group was statistically significant (F=63.486,P=0.049).Compared with the DM group and the normal group,the expression of miR-1470 in the DR group was significantly decreased,and the difference was statistically significant (q=l 11.2,73.9;P＜0.05).The expression of miR-1470 in hREC in the high glucose group was significantly lower than that in the normal group (t=42.082,P=0.015).The expression of EGFR protein in hREC of high glucose group was significantly higher than that of normal group (t=-39.939,P=0.016).The expression of miR-1470 (F=637.069,P=0.000) and EGFR (F=122.908,P=0.000) protein expression in hREC of blank control group,negative control group and high expression group were statistically significant.Compared with the blank control group and the negative control group,the expression of miR-1470 in hREC of high expression group was significantly increased (q=329.7,328.8;P＜0.05),and the expression of EGFR protein was significantly decreased (q=242.5,234.6;P＜0.05).There was no significant difference in the expression of miR-1470 and EGFR protein in hREC between the negative control group and the blank control group (q=1.5,7.9;P＞0.05).Conclusion The expression ofmiR-1470 in the plasma of patients with DR is significantly down-regulated,and the increase of EGFR expression may be related to it.
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Objective To prepare a lipoic acid modified polyarginine polypeptide nanocomplex for gene delivery system, and to observe its transfection efficiency and cytotoxicity on HEK293 cells. Methods We synthesized four disulfide cross-linked lipoic acid modified polyarginine peptide and histidine (LHRss) at different cross-linked degrees using different mol fraction of L-cysteine as cross-linking agent. The construction of LHRss was characterized by1H nuclear magnetic resonance (1H NMR) and gel permeation chromatography. The LHRss/plasmid DNA (pDNA) nanocomplexes were self-assembled with LHRss and pDNA at different nitrogen/phosphorus (N/P) ratios. The size and zeta potential of LHRss/pDNA nanocomplexes were characterized by particle size analyzer, and the pDNA enrichment capability was determined by electrophoretic mobility shift assay (EMAS). Then, the intracellular uptake and gene transfection efficiency of LHRss/pDNA nanocomplexes in HEK293 cells were investigated. CCK-8 method was used to determine the cytotoxicity of LHRss/pDNA nanocomplexes on HEK 293 cells. Results1H NMR results showed that LHRss was successfully synthesized. The nanocomplexes had a uniform distribution of particle size, and the zeta potential of LHRss3/pDNA and LHRss4/pDNA nanocomplexes were more than 30 mV when N/P≥40. EMAS results showed that pDNA could be completely wrapped by LHRss3 when N/P=5. When N/P=40, the intracellular uptake and transfection efficiency of LHRss3/pDNA nanocomplex by HEK293 cells was significantly higher than that of other three nanocomplexes and lipoic acid modified polyarginine peptide and histidine (LHR)/pDNA; the mean fluorescence intensity of LHRss3/pGL3 nanocomplexes was approximately 3. 98 times that of the LHR/pGL3 nanocomplex (P<0. 05). Cytotoxicity results showed that the cell survival rates were more than 80% at 24 h after transfected with LHR/pGL3 and LHRss/pGL3, and its toxicity was significantly lower than that of bPEI-25K, with the cell survival rates being about 25% at 24 h after transfected with 20 μg/mL bPEI-25K (P<0. 05). Conclusion The prepared lipoic acid modified polyarginine polypeptide nanocomplex is expected to become an efficient gene vector.
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Background Gene transfection is an effective therapeutic avenue to target many kinds of eye diseases.Non-viral vectors with high transfection efficiency,long-term expression,low toxicity and high expression levels are pivotal in gene therapy of corneal disease.Objective This study was to evaluate and compare the safety and efficiency between EntransterTM and liposome vectors for transfer of CD25 siRNA in rat cornea.Methods Eighty male SPF SD rats were randomly divided into EntransterTM-CD25 siRNA group,liposome-CD25 siRNA group,simple CD25 siRNA group and normal saline solution (NSS) group with the right eye as experimental eyes.Corneal epithelia of the rats were completely removed after ocular surficial anesthesia,and 50 μl EntransterTM-CD25 siRNA,liposome-CD25 siRNA,CD25 siRNA solution and NSS were topical administered in the eyes respectively.Ocular response and green fluorescence number on the corneas were examined under the slit lamp assisted microscope 12 hours,24 hours,3 days and 7 days after use of the drugs.The rats were sacrificed and the corneas were obtained,and corneal histopathological examination was performed by using hematoxylin eosin stain.The gene transferred efficiency in the corneas was evaluated by fluorescence technology,and the safety of EntransterTM and liposome carriers was assessed using TUNEL stain.The expression and location of CD11b in the corneas were detected by immunofluorescence technology.The use and care of the experimental animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Committee.Results The quantity and intensity of fluorescence staining in the corneas were significantly increased in the EntransterTMCD25 siRNA group in comparison with the liposome-CD25 siRNA group,and the corneal fluorescence appeared earlier in the simple CD25 siRNA group,but it disappeared in 24 hours after transfection.Corneal histopathological examination revealed that the corneal edema and inflammatory cell infiltration in corneal epithelium after gene transfection were more dominant in the liposome-CD25 siRNA group than those in the EntransterTM-CD25 siRNA group,simple CD25 siRNA group and NSS group,and no abnormality was seen in the stroma and endothelium.The number of inflammatory cells was more in the liposome-CD25 siRNA group than that in the EntransterTM-CD25 siRNA group,simple CD25 siRNA group and NSS group (all at P =0.00).The number of apoptosis cells was significantly more in the liposome-CD25 siRNA group than that in the EntransterTM-CD25 siRNA group,simple CD25 siRNA group and NSS group in 12 hours and 3 days after transfection (all at P =0.00).Immunofluorescence assay showed the expression of CD11b primarily located in the corneal epithelial and stromal layers.The expression of CD11b was gradually enhanced over time in the liposome-CD25 siRNA group and peaked in 24 hours after transfection.However,the expression was absent in the EntransterTM-CD25 siRNA group,simple CD25 siRNA group and NSS group.Conclusions EntransterTM nanometer material-mediated transfection of CD25 siRNA in corneas of normal SD rats appears to have high transfection efficiency,low toxicity and slight irritating response to corneas,and EntransterTM vector is currently available for the gene therapy of corneal disease.
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Background Research comfirmed that second mitochondrial activator of caspase (Smac) is a promoting tumor cell apoptosis protein.Our previous study showed that the expression level of Smac in LECs is obviously higher in cataract than that in normal eyes.We assumed that silencing Smac gene in LECs can inhibit the apoptosis of LECs.The way to transfect Smac siRNA into LECs is a key step.Objective This study was to construct siRNA lentiviral vector of Smac and identify its silencing efficiency in human lens epithelial B3 cell line (HLE-B3) and establish low-expressed Smac HLE-B3 line.Methods Based on the genebank and our previous study,siRNA sequence of Smac was designed and composed.The synthetic double-stranded DNA was linked to the lentiviral vector GVll8 by T4 DNA ligase and then transformed DH5α competent cells.The plasmids were transformed into the DH5α competent cells.Recombinant colonies were screened by PCR and sequenced.Recombinant plasmids and two other auxiliary plasmids were used to infect 293T cells.Cell culture supernatant was collected for the measurement of viral titer.Recombinant lentiviral vector was used to infect HLE-B3 cells to calculate the viral multiplicity of infection (MOI) under the fluorescence microscope.Transfection efficiency was examined by calculating the GFP-positive cells.HLE-B3 cells were divided into negative control group,siRNA plasmid tranfected group and GV118-Smac-siRNA1 tranfected group.The relative expression levels of Smac mRNA in the cells were detected and compared among the three groups by real-time fluorescent quantitative PCR.Results GV118-Smac-siRNA was successfully constructed with the positive colonies 340 bp and blank vector colonies 299 bp,and viral titer was 3.0× 108 TU/ml.At a MOI of 100,the infecting efficiency of the vector on HLE-B3 cells was about 82％ and the cytotoxicity was low.The relative expression levels of Smac mRNA were (101.290±8.349)％,(92.330±6.320)％ and (32.540±4.221)％ in the negative control group,siRNA plasmid tranfected group and GV118-Smac-siRNA1 tranfected group,respectively,showing a significant difference among the three groups(F =32.871,P＜0.01),and the relative expression level of Smac mRNA was significantly lower in the GV118-Smac-siRNA1 tranfected group than that in the negative control group (P =0.000).However,no significant difference was found in the Smac mRNA expression between the blank plasmid group and the negative control group (P=0.535).Conclusions GV118-Smac-siRNA lentiviral vector is successfully constructed.Smac-siRNA can effectively inhibit the expression of Smac mRNA in human LECs.
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BACKGROUND:Studies have shown that stem cels transfected with human telomerase reverse transcriptase (hTERT) gene can stably express high-level telomerase activity and strengthen cel proliferation, which lays the foundation to establish geneticaly engineered immortalized stem cel lines. OBJECTIVE:To explore the effects of hTERT transfection on proliferation and cel cycle of rat fetal liver stem cels in vitro. METHODS:Rat fetal liver stem cels culturedin vitro were transfected by recombinant adeno-associated virus carrying hTERT genes. RT-PCR and western blot assay were used to detect the expression of hTERT mRNA and protein, respectively. Cel Counting Kit-8 method and cel growth curve were used to detect cel growth and proliferation. Changes in cel cycle distribution were determined by flow cytometry. RESULTS AND CONCLUSION:Compared with the control group and empty virus group, in the hTERT infection group, hTERT expressed at gene and protein levels, the growth rate of the cels increased, and the number of cels at G0/G1 phase and S phase was decreased and increased, respectively. The results show that recombinant adeno-associated virus as a vector of hTERT gene used for gene transfection can promote the in vitro proliferation of rat fetal liver stem cels and play an optimal role in cel culture.
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Advantages of DNA vaccination against infectious diseases over more classical immunization methods include the possibilities for rapid manufacture, fast adaptation to newly emerging pathogens and high stability at ambient temperatures. In addition, upon DNA immunization the antigen is produced by the cells of the vaccinated individual, which leads to activation of both cellular and humoral immune responses due to antigen presentation via MHC I and MHC II molecules. However, so far DNA vaccines have shown most efficient immunogenicity mainly in small rodent models, whereas in larger animals including humans there is still the need to improve effectiveness. This is mostly due to inefficient delivery of the DNA plasmid into cells and nuclei. Here, we discuss technologies used to overcome this problem, including physical means such as in vivo electroporation and co-administration of adjuvants. Several of these methods have already entered clinical testing in humans.
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Animals , Humans , Adjuvants, Immunologic , Antigen Presentation , Communicable Diseases , DNA , Electroporation , Gene Transfer Techniques , Immunity, Humoral , Immunization , Plasmids , Rodentia , Vaccination , Vaccines, DNAABSTRACT
Methods and techniques employed in gene therapy are reviewed in parallel with pertinent ethical conflicts. Clinical interventions based on gene therapy techniques preferentially use vectors for the transportation of therapeutic genes, however little is known about the potential risks and damages to the patient. Thus, attending carefully to the clinical complications arising as well as to security is essential. Despite the scientific and technological advances, there are still many uncertainties about the side effects of gene therapy. Moreover, there is a need, above all, to understand the principles of bioethics as both science and ethics, in accordance with its socioecological responsibility, in order to prioritize the health and welfare of man and nature, using properly natural resources and technology. Therefore, it is hard to determine objective results and to which extent the insertion of genes can affect the organism, as well as the ethical implication.
Métodos e técnicas empregadas na terapia gênica são revisados em paralelo a conflitos éticos pertinentes. Intervenções clínicas com base em técnicas de terapia gênica são usadas preferencialmente em vetores para o transporte de genes terapêuticos; porém, pouco se sabe sobre os possíveis riscos e danos para o paciente, sendo necessário atender cuidadosamente às complicações clínicas resultantes, bem como à segurança. Apesar dos avanços científicos e tecnológicos relacionados à terapia gênica, ainda há muitas incertezas sobre os efeitos colaterais do uso dessa terapia. Além disso, é necessário, acima de tudo, compreender os princípios da bioética como uma ética da ciência para com a responsabilidade socioecológica, a fim de priorizar a saúde e o bem-estar do homem e da natureza, utilizando adequadamente recursos naturais e tecnologia. Portanto, é difícil afirmar qual é o rendimento real, bem como os resultados do aumento da genética inserida no organismo e as implicações éticas.
Subject(s)
Humans , Bioethical Issues , Gene Transfer Techniques , Genetic Therapy , Attitude of Health Personnel , Patient Safety , Risk Assessment/standards , Social ResponsibilityABSTRACT
Objective: To explore the protein component change in normal thyroid follicular epithelial cell line Nthy-ori 3-1 transfected with oncogene PAX8/PPARgamma fusion (PPFP) gene using proteomics technologies in order to find the proteins regulated by PPFP gene, providing clues to looking for follicular thyroid carcinoma (FTC) marker proteins or proteins as drug targets. Methods: The Nthy-ori 3-1PPFP cells, Nthy-ori 3-1vector cells and Nthy-ori 3-1 cells were collected to exract total proteins. The two-dimensional gel electrophoresis (2-DE) maps of total proteins in cells in three groups were created. The 2-DE maps of the three groups were analyzed and compared using PDQuest software to find out differentially expressed protein spots. These differentially expressed candidate proteins were identified by matrix-assisted laser desorption-ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS). Five protein spots [prohibitin, galectin-1, cytokeratin 8, cytokeratin 19 and heat shock protein 27 (HSP27)] which were considered to be more closely related to the occurrence and development of FTC and have a higher MALDI-TOF-MS score and a greater coverage were selected and detected by Western blotting. Results: The 2-DE maps of total proteins in Nthy-ori 3-1PPFP, Nthy-ori 3-1vector and Nthy-ori 3-1 cells were successfully established. Thirty-eight differentially expressed protein spots with a difference in expression more than twice between Nthy-ori 3-1PPFP cells and the Nthy-ori 3-1 and Nthy-ori 3-1vector cells were identified by using PDQuest 2-D Analysis Software. Combination of MALDI-TOF- MS and database search identified a total of 28 differentially expressed protein spots. Of all the identified protein spots, 19 proteins were upregulated and 9 proteins were down-regulated in Nthy-ori 3-1PPFP cells as compared with the Nthy-ori 3-1vector and Nthy-ori 3-1 cells. This result was fully consistent with the results of proteomics. Conclusion: Twenty-eight differentially expressed proteins in Nthy-ori 3-1PPFP cells transfected with PPFP gene are identified. These proteins may be dominated and regulated by PPFP gene. This finding provides a basis for clarifying the molecular mechanism of the occurrence and development of FTC.
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Objective To observe the effects of transplantation of bone marrow stem cells(BMSCs)transfected with bone morphogenetic protein-2(BMP-2)on fracture healing in rats with diabetes so as to provide a new therapy for diabetic fractures.Methods Fifty male adult Wistar rats aged six weeks randomized to the control group and experimental group were all employed to establish models with diabetic fractures.Under high glucose condition,BMSCs were transfected with BMP-2 by adenovirus vector in vitro.BMSCs transfected by BMP-2 were transplanted into the fracture area of rats in the experimental group,while non-transfected BMSCs into the corresponding area of rats in the control group.X-ray examination was performed at 1,2,3,4 and 6 weeks after transplantation.Bony calluses were collected for HE staining and gray scales of BMP-2 in calluses were determined by immunohistochemical method.Meanwhile,serum levels of BMP-2 were measured by ELtSA.Results The gray scales of BMP-2 in the calluses were 83±3 in the experimental group and 118±4 in the control group at four weeks(P＜0.01).The serum concentrations of BMP-2 were(203.80±8.96)ng/L in the experimental group and(139.15±4.19)ng/L in the control group at four weeks(P＜0.01).Conclusion Transplantation of BMSCs transfected by BMP-2 promotes fracture healing in diabetic rats.
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Objective To investigate the efficiency of transduction of recombinant adenovirus-mediated human endothelial nitric oxide synthase (eNOS) into lung tissue by repeated intratracheal transfection in rats.Methods Sixty 3-4 month old male Wistar rats weighing 220-280 g were randomly divided into 2 groups:control group (group C,n =10) and eNOS gene transduction group (group T,n =50).The animals were anesthetized with intraperitoneal 10％ chloral hydrate 35 mg/kg,tracheally intubated and mechanically ventilated (VT 2.5 ml,RR 60 bpm,FiO2 1.0).Recombinant adenovirus carrying human eNOS gene was given as gift by Professor Gerard from Texas University,Southwest Medical Center.In group T 50 μl of the recombinant adenovirus in concentration of 5 × 109 PFU/ml was instilled into trachea every 5 minutes for 12 times,while in group C equal volume of vector conservation solution was instilled instead.Pulmonary arterial blood samples were obtained at 2,5,7,14 and 21 d after intratracheal transfection (n =10 at each time point) for determination of serum NO concentration.The animals were immediately sacrificed after blood sample collection for determination of expression of eNOS protein in the lung tissue and RNA.The eNOS expression in the trachea,bronchus,lung,liver,spleen and kidney was detected by immuno-histochemistry.Results The serum NO concentrations were significantly higher at all time points in group T than in group C.The eNOS expression was detected in the epithelial cells of trachea and bronchi,and endothelial cells of alveoli and pulmonary blood vessels in group T but not in group C.eNOS expression was not detected in liver,spleen and kidney at 7 d after intratracheal transfection in group T.Conclusion Human eNOS gene mediated by recombinant adenovirus was transducted into rat lung tissue with normal enzyme activity by repeated intratracheal administration without being detected in distant organs.
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Objective To explore the effects of 14-3-3 γ gene on dopaminergic neurons of PD rat model.Methods 6 hydroxydopamine (6-OHDA) was injected into the corpus striatum of 60 SD rats to establish PD model.A week later,Ad/14-3-3 γ was injected into the corpus striatum of 16 rats,while PBS and Ad-LacZ were injected into the corpus striatum of 16 and 28 rats,respectively,as control.X-gal dyeing was utilized to examine the LacZ reporter gene expression in the corpus striatum at 3 day,2 week and 6 week.Real-time PCR was utilized to test the expression level of 14-3-3 γmRNA at 2 weeks after Ad/14-3-3 γ injection.Immunohistochemistry technique was used to detect TH positive neurons and fibers in the corpus striatum and substantial nigra.Western blotting was performed to check the expression of 14-3-3 γ in the corpus striatum at 2 weeks and caspase-3 at 6 veeks after Ad/14-3-3 γ injection.High performance liquid chromatography (HPLC) method was used to examine the contents of DA and DOPAC in the corpus striatum.The rats underwent rotational ethological examination at 1,2 and 6 weeks after the second injection.Results The expression of β-gal,which showed the successful LacZ gene transfection,was found in the corpus striatum of LacZ groups.The 14-3-3 γ mRNA and protein expression in the corpus striatum were significantly higher in the Ad/14-3-3 γ group than in the other two groups.The TH-positive cell ratio of substania nigra to contralateral area in the lesion side was 0.44±0.17 and the optical density (OD) of TH-positive fibers was 0.62±0.14 in the Ad/14-3-3 γ group,both higher than those in PBS group (0.16±0.13 and 0.36±0.15) and LacZ group (0.15±0.09 and 0.30±0.11) (all P＜0.01).The contents of DA and DOPAC in the lesion sides of corpus striatum were increased in the Ad/14-3-3 γ group [(248± 116)ng/g and (752±177) ng/g] than in PBS group [(106±35) ng/g and (724±159) ng/g] and LacZ group [(136±49) ng/g and (765±163) ng/g] (P＜0.01).The DA and DOPAC ratios of the lesion side of corpus striatum to the contralateral side were 0.37±0.14 and 0.38±0.17 in the Ad/14-3-3 γgroup,higher than those in PBS group (0.15±0.08 and 0.13±0.10,respectively) and LacZ group (0.19±0.11 and 0.16±0.09 (all P＜0.01).The expression level of caspase-3 was decreased in Ad/14-3-3 γ group than in PBS and LacZ groups at 6 weeks after the second injection.The turns/min induced by apomorphine in Ad/14-3-3 γ group were 9.4 ± 2.5 at 1 week after the Ad/14-3-3 γinjection,and reduced to 4.6±2.2 at 6 weeks later.While in PBS and LacZ group,the turns were 14.5±4.9 and 13.8±3.5 at 1 week after the second injection,6 weeks later rised to 18.7±5.2 and 20.6± 6.7 respectively,significantly higher than those in Ad/14-3-3 γ group (all P＜0.01).Conclusions 14-3-3γ gene transfer has a protective effect on the dopaminergic neurons and it may be a promising candidate gene for curing PD.
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Hepatocyte growth factor (HGF), also known as scatter factor, was identified during the experimental attempts to explore a phantom factor acting as a trigger for liver regeneration after partial hepatecotmy. HGF is synthesized and secreted as a single-chain inert precursor by cells of mesodermal origin, and extracellularly processed to the two-chain functional heterodimer by proteolytic cleavage at a specific site. The binding of HGF to the c-MET, the HGF receptor, induces activation of tyrosine kinase and autophosphorylation of tyrosine residues. c-MET activation propagates an intricate system of signaling pathways that controls a range of cellular processes as diverse as cell proliferation, differentiation, transformation and apoptosis. In the aspect of kidney, the HGF/c-MET signaling pathway plays important roles in renal development and in the maintenance of normal adult kidney structure and function. In various injury and disease models, HGF has been reported to promote cell survival, tissue regeneration, and fibrosis suppression. Neutralization of HGF by the antibody may accelerate renal failure or fibrosis while HGF administration may lead to remarkable amelioration. Thus, HGF is not only the endogenous safeguard protecting normal tissues against the fibrotic process after injury, but also a therapeutic option to prevent organ failure. If insufficient production of HGF is causative for renal fibrosis, administration of recombinant human HGF protein or HGF gene therapy may improve renal fibrosis and dysfunction. HGF gene therapy requires appropriate delivery systems, and biodegradable polyester amine based on glycerol dimethacrylate and polyethylenimine is being tested for the HGF gene carrier in experimental models.
Subject(s)
Adult , Humans , Apoptosis , Cell Proliferation , Cell Survival , Fibrosis , Gene Transfer Techniques , Genetic Therapy , Glycerol , Hepatocyte Growth Factor , Hepatocytes , Kidney , Kidney Diseases , Liver Regeneration , Mesoderm , Models, Theoretical , Polyesters , Polyethyleneimine , Protein-Tyrosine Kinases , Proto-Oncogene Proteins c-met , Regeneration , Renal Insufficiency , TyrosineABSTRACT
Objective To investigate the role and the mechanism of Apr-1 gene on cholangiocarcinoma QBC939 cell lines proliferation and cell cycle regulation. Methods Apr-1 gene was transfected into QBC939 cells by using liposomes to establish a QBC939 cell model ( QBC939-Apr-1 ) stably expressing Apr-1 gene. Apr-1 mRNA expression and the changes in cell cycle and cell growth of QBC939 cells were analyzed by RT-PCR, flow cytometry ( FCM ) and growth curve before and after transfection. The regulatory effect of Apr-1 gene on the expression of cell cycle-related genes was investigated in QBC939 cells before and after Apr-1 transfection using cell cycle gene microarrays. Results Significant suppression of cell growth was observed with the cell model stably expressing Apr-1 gene. Apr-1 over-expression caused cell arrest from 9% to 13% (P ＜0. 01 ) increase in G2 population. Cell cycle gene microarrays demonstrated that the expression of Skp2 、UBE1 was up-regulated, while the expression of MRE11A 、CKS2 、CDK8 、CDC45 was down-regulated by more than 3 folds. Conclusions Apr-1 gene suppresses QBC939 cell proliferation in vitro, QBC939 cells presented with differences in the expression of cell cycle-related genes after Apr-1 gene transfection.
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Objective To construct small hairpin RNA (shRNA) expression plasmid targeting rat opticin gene. Methods Four pairs of opticin oligonucleotides were synthesized and inserted into the plasmid vector, resulting into four plasmids: shRNA-1, shRNA-2, shRNA-3 and shRNA-4. Then the four constructed shRNA expression vectors and empty vector were transfected into rat ciliary non-pigment epithelium (NPE) cells by lipofectmaine 2000. Non-transfected NPE cells were set as control group. The expression of opticin mRNA and protein were measured by Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot respectively. Results The opticin mRNA expression of the shRNA-1,shRNA-2, shRNA-3, shRNA-4 group were decreased compared with the control group (F = 10. 239, P = 0. 000);the inhibitory rate were 85.7% ,62. 87% ,54.87% and 48.77% respectively. The opticin protein expression of the shRNA-1,shRNA-2, shRNA-3, shRNA-4 group were also decreased compared with the control group (F=17.870, P= 0.000);the inhibitory rate were 78.7%, 34.6%, 31.1% and 16.8% respectively.Conclusions The shRNA-1 expression plasmid has most potent inhibitory effect on opticin expression in rat ciliary NPE cells.
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Objective:To investigate the effect of exogenous extopic fragile hisdidine triad (FHIT) gene on the apoptosis of gastric cancer cells induced by octreotide and elucidate the underlying mechanism. Methods: Human gastric cancer cell line MGC-803, which was loss of FHIT gene, was transfected with plasmid pRcCMV-FHIT and pRcCMV via lipofectamine mediation. After transfection, Western blotting was used to analyse the expression of FHIT protein in positive cells screened out by G418. The cells were treated with octreotide 10~(-10), 10~(-9), 10~(-8), 10-7, and 10~(-6) mol/L for 24, 48 and 72 h. Cell proliferation was evaluated by MTT assay and apoptosis by flow cytometry. The expression of bcl-2 and bax protein was detected with Western blotting.Results:FHIT protein expression was detected in MGC-803 gastric cancer cells after FHIT gene transfection. After treatment with octreotide 10~(-8) mol/L for 48 h, the apoptotic rate of MGC-803 cells transfected with FHIT gene was (26.777±1.702)%, significantly higher than that in empty vector group and untransfected group [(13.800±0.511)% vs (12.634±0.479)%, F=245.789, P<0.05]. The expression of bcl-2 protein was decreased while the expression of bax protein was increased in FHIT gene transfection group after octreotide treatment. Conclusion:The exogenous FHIT gene and octreotide had strong synergistic effects on apoptosis of MGC-803 cell lines, and their action mechanism may be related to the alteration of the expression of apoptosis-related proteins of bcl-2 family.
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As distrofias hereditárias de retina abrangem um amplo número de doenças caracterizadas por lenta e progressiva degeneração da retina. São o resultado de mutações em genes expressos em fotorreceptores e no epitélio pigmentado da retina. A herança pode ser autossômica dominante, autossômica recessiva, ligada ao X recessiva, digênica ou herança mitocondrial. Atualmente não há tratamento para essas doenças e os pacientes convivem com a perda progressiva da visão. O aconselhamento genético e o suporte para reabilitação têm indicação nestes casos. Pesquisas envolvendo a base molecular e genética dessas doenças está continuamente em expansão e ampliam as perspectivas para novas formas de tratamento. Dessa forma, a terapia gênica, que consiste na inserção de material genético exógeno em células de um indivíduo com finalidade terapêutica, tem sido a principal forma de tratamento para as distrofias hereditárias de retina. O olho é um órgão peculiar para a terapia gênica, pois é anatomicamente dividido em compartimentos, imunologicamente privilegiado e com meios transparentes. A maioria das doenças oculares tem defeitos em genes conhecidos. Além disso, há modelo animal bem caracterizado para algumas condições. Propostas para pesquisa clínica em terapia gênica nas degenerações retinianas hereditárias com defeito no gene RPE65, recentemente tiveram aprovação ética e os resultados preliminares obtidos trouxeram grandes expectativas na melhora da qualidade de vida dos pacientes.
The inherited retinal dystrophies comprise a large number of disorders characterized by a slow and progressive retinal degeneration. They are the result of mutations in genes that express in either the photoreceptor cells or the retinal pigment epithelium. The mode of inheritance can be autosomal dominant, autosomal recessive, X linked recessive, digenic or mitochondrial DNA inherited. At the moment, there is no treatment for these conditions and the patients can expect a progressive loss of vision. Accurate genetic counseling and support for rehabilitation are indicated. Research into the molecular and genetic basis of disease is continually expanding and improving the prospects for rational treatments. In this way, gene therapy, defined as the introduction of exogenous genetic material into human cells for therapeutic purposes, may ultimately offer the greatest treatment for the inherited retinal dystrophies. The eye is an attractive target for gene therapy because of its accessibility, immune privilege and translucent media. A number of retinal diseases affecting the eye have known gene defects. Besides, there is a well characterized animal model for many of these conditions. Proposals for clinical trials of gene therapy for inherited retinal degenerations owing to defects in the gene RPE65, have recently received ethical approval and the obtained preliminary results brought large prospects in the improvement on patient's quality of life.
Subject(s)
Animals , Humans , Genetic Therapy/methods , Retinal Degeneration/therapyABSTRACT
Objective To explore the repair mechanism of olfactory ensheathing cells (OECs)-neurotrophin-3 (NT-3) gene engineering cell on neuron myeline and axon of experimental allergic encephalomyelitis (EAE).Methods OECs-NT-3 gene engineering cell, constructed by ueurotrophin-3 transinfecting OECs inducted by retrovirus, was transplanted into lateral ventricle.The migration and distribution were observed and compared with control group and OECs transplantation group.Then myeline repair and axon regeneration were evaluated in the aspects of function score, morphological structure, SYN grey level Results (1) OECs-NT-3 could survive, diffuse, migrate with axons, spread in the focus diffusely on the 28th day after transplantation.(2) OECs-NT-3 survived and migrated to the transcription level of NT-3 mRNA in transgene group, being (212.3±16.1)×10-2, significantly higher than OECs group ((1.98±0.19)×10-2) and the contrast group ((1.23±0.13)×10-2, t = - 31.161, -31.928, P < 0.01).(3) The myeline of transgene group was kept complete and the number of inflamatory focus was lower than those of other groups (t = 11.388-22.728, P <0.01).(4) The SYN grey level of transgene group was obviously higher (P < 0.01).Conclusion OECs-NT-3 cell expresses NT-3 in EAE stably and effectively, which contributes to the repair of myeline and the regeneration of axon.
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Objective To study the enhancing effect of ultrasound plus microbubble on small interference RNA(siRNA)transfection in vitro and in vivo.Methods Human vascular epithelial growth factor(VEGF)siRNA with 2'deoxy modification(VEGF 2'-eoxy siRNA,VdsR)was used.The human CNE cells(fromnasopharyngeal carcinoma)line was used for in vitro cell-based experiments and in vivo mouse xenograft model.Two different microbubble agents.BR14 and Levovist.were used together with the RNA transfection reagent RNA-mate.ELISA and RT-PCR assays were used to assess VEGF gene expression.Immunohistochemical staining (IHC)was performed to assess CD31 expression in xenograft tumors.Results VdsR transfection in CNE cells abolished VEGF expression as determined by ELISA experiments.In the first mouse xenograft experiment,ultrasound exposure dramatically enhanced VdsR-mediated tumor inhibition.In the second mouse xenograft experiment,when VdsR was mixed with the microbubble reagents and then injected into xenografts,ultrasoundexposure significantly reduced tumor growth in BR14-mixed VdsR group but not in the Levovist-mixed VdsR group compared to the control.RT-PCR experiments demonstrated that VEGF expression in ultrasound-exposed tumors was significantly lower than that in the control.Meanwhile,VEGF expression in the tumor tissue treated by BRl4-mixed VdsR declined as compared with the controls.Tumor vascular density as measured by CD31 immunostaining was significantly decreased in ultrasound-exposed tumors compared to the control.Conclusions Ultrasound exposure and/or microbubble can significantly enhance delivery and the efficiency of VdsR-mediated anti-tumor effects,and should be a location-specific enhancement approach for siRNA-based anti-cancer therapy.
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Objective To study the optimized condition of transfection efficiency for MCF-7 cells enhanced by ultrasound(US) irradiation and contrast agent combined with polyethyleneimine(PEI) and observe whether the combination can have a synergistic effect to increase DNA transfection. Methods MCF-7 cells were transfected with the compounds prepared by the vector of plasmid DNA encoding luciferase (pCMV-luciferase-GL3) and PEI.SonoVue microbubble was added to the cell suspension to serve as nucleation sites for aeoustic cavitation before US irradiation. The DNA expression of luciferase plasmid and viability of cells were evaluated. The strategy of US irradiation was optimized. Furthermore, the influencing factor, such as the concentration of plasmid, incubation time, serum, the type of solvent and the volume of culture media, were examined. Results The viability of cells and US-induced enhancement of luciferase activity were influenced by the US intensity,exposure time and duty cycle.US irradiation under an appropriate condition enables ceils to accelerate the permeation of the PEI/DNA complex through the cell membrane, resulted in enhanced transfection efficiency of plasmid DNA. Optimal US condition for the enhancement was determined to be 1 W/cm2,10% DC for 3 min. In contrast to the PEI/DNA complex alone without US irradiation or US irradiation alone, the combination of US irradiation with contrast agent and PEI had a significantly enhanced luciferase activity (P<0.01). The 2 h pre-irradiation incubation with PEI/DNA complex for MCF-7 ceils exhibited a significantly enhanced lueiferase activity (P<0.01). Besides,serum,type of solvent and the volume of culture media did affect the transfection efficiency. Conclusions The optimized parameters of US and transfection provide efficient gene delivery in MCF-7 cancer cells. The combination of US irradiation with contrast agent and PEI has a synergistic effect to increase DNA transfection. This is a simple and promising method to enhance the gene expression of plasmid DNA.
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Objective To investigate different ultrasound parameters and transfection conditions that would affect transfection rate of red fluorescent protein(RFP)and cell viability of cancer cells.Methods In this study,Hela cells were cultured using two different protocols:(A)24 h culture for complete adherence;(S)suspension.Subsequently,cells were transfected following different ultrasound exposure protocols[1.0W/cm2;duty cycle(DC):10%,20%and 50%;exposure 1min or 3 min].Gene transfection and cell viability were evaluated.Treatment parameters optimized in Hela cells were applied for delivery RFP in 4 other cell lines(HepG2,Ishikawa,MCF-7 and B16-F10).Results Cell injury were found to increase progressively with DC and exposure time in group A.Cell detachment was significantly accompanied by ultrasound exposure in adherent HeLa cells.Cells in group S were found more prone to be transfected than group A with the same ultrasound parameters,while the survival rate was not decreased apparently.The ideal ultrasound conditions were noted to be at 1.0 W/cm2 irradiated 3 min with 20%DC using suspended protocol,producing maximum efficiency[transfection=(28.04±2.27)%]in gene delivery with minimum cell toxicity[cell viability=(81.20±1.73)%].These experiments also revealed different response to ultrasound treatment,but for all tested cell lines,dead and transfected cells in the treated groups were significantly different from the non-irradiated groups.Conclusions Ultrasound parameters and transfection conditions have a great impact on the gene delivery and cell viability.Gene delivery of ultrasound-mediated microbubble enhance should be optimized to improve the efficiency.