| Background:The modern operation of cataract is extracapsular extraction combined with intraocular lens implantation,and also the technology has become more and more mature, surgical techniques and equipment conditions gradually improved,p more and more patients with cataract and therefore benefit, but modern extracapsular cataract removed after resection of posterior capsule opacification (PCO), after cataract formation, although modern surgery through a variety of methods, such as changing capsule polishing, artificial crystal materials and shape design of, extent reduces the after cataract incidence, but after operation,the main complications of the surgery effect is still thethe PCO. The study showed that the incidence of the PCO cumulative incidence of 64 years of age after 3 years of artificial lens implantation in senile cataract was 38.5%,and in children and adolescents, the incidence can reach almost 100%[1]. At present, it is believed that after opacification is mainly due to the modern extracapsular cataract extirpation is required to open the anterior lens capsule, the process will start lens of the wound healing response, the residual lens epithelial cells (LECs) in the bag in the proliferation. migration. interstitial fibrosis, including the remaining part of the anterior capsule and all posterior region collapse, cloudy, lens epithelial cells also transitional. attached to the surface of intraocular lens (IOL), proliferation and, caused by cataract formation. lead to damaged visual acuity again.After the formation of the PCO. although the vast majority of patients with Nd:YAG (Neodymium-dopedYttriumAluminiumGarnet) laser incision and again to restore vision, but the YAG laser induced intraocular lens injury, intraocular pressure, choroidal damage, retinal damage, macular edema and other complications can not be ignored. Therefore, how to inhibit the proliferation of lens epithelial cells is the key to prevent the occurrence of the lens epithelial cells. Although there are already many kinds of technologies, methods and drugs used in the prevention of the clinical and experimental studies, but have not achieved very good results.Suicide gene/prodrug system was first applied in the field of tumor, the application of a large number such as HSV/GCV herpes herpes simplex virus thymidine kinase/ ganciclovir guanosine system, Escherichia coli cytosine deaminase gene/ 5-flucytosine (5-FC) system, because its have the specific killing effect to the abnormal proliferation cells, and the ophthalmologists have noticed these system soon.Many experiments have proved suicide gene/prodrug system can specifically kill the proliferation of LECs, its action principle for prodrug decency role to be suicide gene transfected target cells by effects on gene induce the apoptosis of the target cells. It is highly selective for the cells to kill the abnormal proliferation of cells, but it has some key points, such as the selection of gene carriers, and the second is the effect of the killing power, the third is the influence to the normal tissue, i At present, the vector of gene can be used as a virus or plasmid,if using the virus as the vector, its efficiency is higher, but the stability of earring the foreign gene is small, its stability is worse also, and the stability of the immune system will be caused by the immune system. Although the plasmid stability is good, it will not lead to immune response, but the main drawback is that the existence of short expression time and low efficiency of transfection. How to enhance the security, stability and destruction effectively, is the main problems that plagued gene transfection technology-specific cells. In this study, we used HSV/GCV, CD/5-FC two suicide gene/drug delivery system, and CD two kinds of suicide gene were transfected into cultured human lens epithelial cells. and combined with different concentrations of TK to study the cell killing, evaluate plasmid vector and double suicide gene combined with different concentrations or different concentrations, at the same time to provide a new way for prevention and treatment for PCO.Part 1 Transformation of plasmid vector and expression of target geneObjective:To explore whether the plasmid vector can successfully access and express the target gene.Methods:After using the LB medium melt cryopreservation of Escherichia coli DH5 bacteria liquid, CaCl2 Treatment legal under state bacteria. Then joining the plasmid pMD18-T-UL57 (PWZL plasmid)) vector, using centrifugal suspension cell culture. Selecting the best single colony was inoculated into the amp LB liquid medium, Omega kit step distribution of plasmid DNA was extracted BioPhotometer nucleic acid quantitative instrument to measure the concentration of CD and TK gene sequences of primers for PCR amplification and identification of CD and TK gene CD and TK band density and internal reference housekeeping gene density ratio represents the relative level, with simple liposomes import group as control, semi quantitative analysis for the determination of CD and TK at 24h,48h,72h.Results:By PCR reaction, the expression of CD, TK gene, gene was detected by 1% agarose gel electrophoresis. The expression of CD was detected in 48 hours,72 hours and 24 hours, and the expression level of TK gene was significantly higher than that of liposome group.Conclusion:In this study, the plasmid vector was used as the carrier, which can successfully access and express the target gene CD and TK, but the gene expression time is relatively short, and the expression of the gene in 72 hours after the transfection showed a clear downward trend, which is consistent with the short time.Part 2 In vitro culture of lens epithelial cells and plasmid transfectionObjective:To investigate the expression of the target gene in the transfected lens epithelial cells.Methods:Immortalized human lens epithelial cell (SRA01/04) of deep cryopreservation were released the frozen state and were recoveried.20% fetal bovine serum and 80%DMEM culture medium in the conventional culture. The cell density was cultured and the cell density was 70%～80%, which was mediated by liposome. The total RNA was extracted from lens epithelial cells transfected with plasmid, and the expression of CD was detected by two step RT-PCR, and the TK expression of mRNA gene was detected; Semi quantitative analysis was performed at 24 hours,48 hours and 72 hours, respectively, and the expression of CD and TK gene was quantified, and the gene expression of each time point was observed.Results:After plasmid transfection, LECs continued to develop, the light microscope showed that the growth of the cells was good, and the shape had no change. Semi quantitative analysis of the relative levels of gene expression in the density and the density of 24h after CD/TK with the density of the housekeeping gene, The results showed that the expression of CD/TK in 24h,48h and 72h after transfection, and the expression measure were different in each time point.Conclusion:To take the human lens epithelial cell line after freezing and thawing, it can meet the needs of the experiment. The double suicide gene CD and TK can be stably expressed in a number of time points. The expression of CD and TK gene is different, and the combination of double suicide gene can be used to make up the short time.Part 3 The killing effect of the drug on the lens epithelial cellsObjective:To investigate the specific killing effect of the drug on the transfection of lens epithelial cells in the transfected cells.Methods:Firstly, the concentrations of 5-FC and GCV were selected, and then the cells were divided into 6 groups,those were empty control group,liposome group, CD/TK group. GCV group,5-FC group,5-FC+GCV group, group and MTT group. The growth of LECs was observed in different time MTT colorimetric method was used to analyze the cell count, and the effect of bystander effect was analyzed by using different concentrations and combinations..Results:First days after transfection, the cell survival rate was significantly decreased in the group except for the GCV0.1 group.At the third days, the survival rate of cells were decreased, GCV100 g/ml+5-FC100 group g/ml cell survival rate is the lowest. On the 5th day, GCV0.1μ G group, the group GCV 1.0, GCV50μ G group, the cell survival rate is higher than the other groups, the survival rate of the lowest 5-FC80 μ g, GCV10μ g/ml+5-FC60 μ g/ml group, GCV 10 μ g/ml+5-FC100 μ g/ml and GCV100 μ g/ml+5-FC100 μ g/ml; On the 7th day, the cell survival rate of the lowest groups for GCV10 μ g/ml+5-FC60 μ g/ml, GCV10 μ g/ml+5-FC100 μ g/ml and GCV100 μ g/ml+5-FC100 μ g/ml. Longitudinal comparison:GCV1.0 μ G group, GCV0.1 μ G group, GCV10 μ G group, GCV50 μ G group, GCV100 μ group G the 3,5,7 days, the cell survival rate changes. There were no significant differences in; simply using 5-FC group except the 5-FC20μ g and 5-FC40 μ G Group on day 5 and day 7 cell survival rate had no significant difference, the other point of time statistics have differences; In other groups, there were some differences in the longitudinal comparison of time points, and the overall performance of the time was prolonged, and the survival rate of the cells decreased gradually. Bystander effect analysis showed that the cell survival rate was gradually increased with the increase of target gene transfection. The cell survival rate was not significantly different from that of the target gene at 60%.Conclusion:Alone or combined application of prodrug 5-FC and GCV were the transfection of lens epithelial cells produce specific killing effect. Combined with plasmid as vector of suicide gene and prodrug system to learn, in stable gene expression time to complete effective killer and a bystander, can either be reduced precursor drug dosage, and can make up for the shortcomings of low transfection efficiency; bystander effect of the suicide gene/prodrug system to further enhance, on target cell killing efficiency further improved. |
PDF Full Text Request