An electrochemical sensor based on molecularly imprinted poly(o-phenylenediamine) for the detection of thymol.

A molecularly imprinted electrochemical sensor was facilely fabricated for the detection of thymol (THY). o-Phenylenediamine (oPD) was used as the functional monomer and electropolymerized on the surface of the glassy carbon electrode (GCE) by using THY as the templates. After the THY templates were removed with 50 % (v/v) ethanol, imprinted cavities complementary to the templates were formed within the poly(o-phenylenediamine) (PoPD) films. The resultant molecularly imprinted PoPD/GCE (MI-PoPD/GCE) was used for the detection of THY, and a wide linear range from 0.5 to 100 µM with a low limit of detection (LOD) of 0.084 µM were obtained under the optimal conditions. The developed MI-PoPD/GCE also displays high selectivity, reproducibility and stability for THY detection. Finally, the content of THY in the real samples was accurately determined by the as-fabricated MI-PoPD/GCE, demonstrating its high practicability and reliability.

RTP4, a Biomarker Associated with Diagnosing Pulmonary Tuberculosis and Pan-Cancer Analysis.

Background: Pulmonary tuberculosis (PTB) is a global epidemic of infectious disease; the purpose of our study was to explore new potential biomarkers for the diagnosis of pulmonary tuberculosis and to use the biomarkers for further pan-cancer analysis. Methods: Four microarray gene expression sets were downloaded from the GEO public databases and conducted for further analysis. Healthy control (HC) samples and samples of pulmonary tuberculosis (PTB) were calculated with enrichment scores in folate biosynthesis pathways. The scores acted as a new phenotype combined with clinical information (control or PTB) for subsequent analysis. Weight gene coexpression network analysis (WGCNA) was used to seek the modules mostly related to PTB and folate biosynthesis in training sets. Twenty-nine coexistence genes were screened by intersecting the genes in the green-yellow module of GSE28623 and the brown module of GSE83456. We used the protein-protein interaction network analysis to narrow the gene range to search for hub genes. Then, we downloaded the unified and standardized pan-cancer data set from the UCSC database for correlations between biomarkers and prognosis and tumor stage differences. Results: Eventually, RTP4 was selected as a biomarker. To verify the reliability of this biomarker, an area under the ROC (AUC) was calculated in gene sets (GSE28623, GSE83456, and GSE34608). Lastly, to explore the difference in RTP4 expression before and after antituberculosis treatment, the GSE31348 gene set was enrolled to compare the expressions in weeks 0 and 26. The results showed significant differences between these two time points (p < 0.001). RTP4 was significantly upregulated in the pulmonary tuberculosis group compared to the healthy control group in three gene sets and downregulated after antituberculosis therapy in one gene set. These results suggest that RTP4 can be used as a potential biomarker in diagnosing tuberculosis. The results of pan-cancer analysis showed that high expression of RTP4 in 4 tumor types was positively correlated with poor prognosis and high expression of RTP4 in 6 tumor types was negatively correlated with poor prognosis. We found significant differences in the expression of the RTP4 gene at different stages in 5 types of tumors. Conclusion: RTP4 might be a new potential biomarker for diagnosing pulmonary tuberculosis.

Meta-analysis of corneal endothelial changes after phacoemulsification in diabetic and non-diabetic patients.

BACKGROUND: Currently, there is still controversy about the differential changes in corneal endothelium function and morphology after phacoemulsification between Diabetes Mellitus (DM) and non-Diabetes Mellitus (non-DM) patients. In this study, we aimed to evaluate the influence of phacoemulsification on the corneal endothelium in DM and non-DM patients. METHODS: Databases of PubMed, Embase, Web of Science, and the Cochrane Library were searched for studies published between January 1, 2011 and December 25, 2021. The weighted mean difference and 95% confidence interval were used to estimate the outcomes of statistical analyses performed. RESULTS: Thirteen studies involving 1744 eyes were included in this meta-analysis. No significant difference was observed in the central corneal thickness (CCT), endothelial cell density (ECD), coefficients of variation (CV), or hexagonal cell percentage (HCP) between the DM and non-DM groups (CCT: P = 0.91; ECD: P = 0.07; CV: P = 0.06; HCP: P = 0.09) preoperatively. The CCT was significantly thicker in the DM group at 1 month (P = 0.003) and 3 months (P = 0.0009) postoperatively, and there was no significant difference at 6 months postoperatively (P = 0.26) than non-DM group. The CV was significantly higher and HCP was significantly lower in the DM group at 1 month (CV:P < 0.0001, HCP: P = 0.002), with no significant difference at 3 months (CV: P = 0.09, HCP: P = 0.36) and 6 months (CV: P = 0.32, HCP: P = 0.36) postoperatively than non-DM group. DM patients had lower ECD than non-DM patients at all postoperative time points (1 month, 3 months: P < 0.00001, 6 months: P < 0.0001). CONCLUSIONS: The influence of phacoemulsification on corneal endothelial damage is greater in diabetic patients. Moreover, the recovery of corneal endothelial function and morphology is delayed in these patients. Clinicians should be more attentive to the corneal health of DM patients when considering phacoemulsification.

Inhibitory effect of anti-Scg3 on corneal neovascularization: a preliminary study.

BACKGROUND: Corneal neovascularization (CNV) is an important disease that causes blindness. Secretogranin III (Scg3) has emerged as a new influencing factor of neovascularization. This study analyzed the Scg3 antibody's inhibitory effect on CNV and and explored its preliminary mechanism. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with Scg3 and anti-Scg3. Cell proliferation, wound healing migration and tube formation assays were performed. Healthy adult New Zealand rabbits were randomly selected to be alkali burned and establish the corneal neovascularization (CNV) model. The rabbits were randomly divided into 3 groups (the high concentration group, low concentration group and control group). Different doses of anti-Scg3 and PBS were administered to the rabbits. Clinical examinations, immunostaining, quantitative real-time polymerase chain reaction (qPCR) and western blotting analyses were performed postoperatively. RESULTS: In the in vitro study, the Scg3 antibody mixture inhibited Scg3-induced endothelial cell proliferation and angiogenesis. In the in vivo study, significant CNV was observed in the control group. Confocal microscopy also revealed considerable active neovascularization in the control group. There was no obvious CNV growth in the high concentration group. Additionally, CD31, LYVE1 and CD45 expression was significantly inhibited after treatment with a high concentration of Scg3 antibody. The qPCR and western blotting analyses revealed that the levels of ERK in the low concentration group and high concentration group were higher than those in the control group at 7 days and 14 days. The levels of VEGF in the control group were significantly increased compared with those in the high concentration group. In all three groups, the levels of Akt were not significantly different at any time point. CONCLUSION: The expression of Scg3 could affect the growth of HUVECs in vitro. Treatment with a high concentration (0.5 µg/mL) of Scg3 antibody reduced the inflammatory response and inhibited the growth of corneal neovascularization after corneal alkali burn injury in rabbits. The MEK/ERK pathway might play an important role in the inhibitory effect of anti-Scg3.

Association of four gene polymorphisms in Chinese Guangxi population with diabetic retinopathy in type 2 diabetic patients.

BACKGROUND: Diabetic retinopathy (DR) is one of the most common chronic microvascular complications of diabetes. Many studies have suggested that genetic factors are important in the context of DR. This study evaluated the associations of GWAS (Genome-wide association study) -identified DR-associated SNPs in a Chinese population in Guangxi Province with type 2 diabetes mellitus (T2DM). METHODS: A total of 386 hospitalized T2DM patients without proliferative diabetic retinopathy (PDR) and 316 hospitalized T2DM patients with PDR were included in this case-control study. Four tag SNPs, including rs1800896 in the IL-10 gene, rs2010963 in the VEGFA gene, rs2070600 in the RAGE gene and rs2910164 in the miR-146a gene, were examined using KASP (kompetitive allele specific PCR) genotyping assays. RESULTS: There were no significant differences in the genotype or allele frequencies of the miR-146a polymorphism (rs2910164) between subjects with PDR and those without DR. The TC genotype of rs1800896 was determined to be associated with an increased risk of PDR (the odds ratio (OR) was 2.366, with a 95% confidence interval (CI) ranging from 1.144 to 4.894). The CG genotypes of rs2010963 was associated with an decreased risk of PDR (the OR was 0.588, with a 95% CI ranging from 0.366 to 0.946). Regarding rs2070600, 2 genotypes (TT and CT) were associated with a decreased risk of PDR (the OR of the TT genotype was 0.180, with a 95% CI ranging from 0.037 to 0.872, and the OR of the CT genotype was 0.448, with a 95% CI ranging from 0.266 to 0.753). CONCLUSIONS: The rs1800896 polymorphisms in the IL-10 gene, rs2010963 in the VEGFA gene and rs2070600 in the RAGE gene are associated with the risk of PDR in the Han Chinese population of Guangxi Province. Our findings provide suggestive evidence that these polymorphisms may be involved in the pathogenesis of PDR and should be investigated further.

DPEP1 promotes the proliferation of colon cancer cells via the DPEP1/MYC feedback loop regulation.

DPEP1 is highly expressed in the colorectal carcinoma tissues and colon cancer cells. However, the function and underlying mechanism of DPEP1 in the colon cancer cells are still poorly understood. Here, we found that transcription factor MYC could occupy on the DPEP1 promoter and activate its activities, and DPEP1 was up-regulated by MYC proteins in mRNA and protein levels in a dose-dependent manner in colon cancer cells. The expression levels of DPEP1 were positively correlated with that of MYC in colorectal tumor tissues. Moreover, Laser confocal images and Co-immunoprecipitation (Co-IP) revealed that DPEP1 and MYC proteins could bind to each other in the colon cancer cells. In turn, DPEP1 could enhance the stability of MYC proteins by extending the half-life of MYC proteins in colon cancer cells. Thus, DPEP1 and MYC proteins might form a positive feedback loop to maintain their high expression levels in colon cancer cells. In function, the MTT, EdU, Clone Formation assays and xenograft tumors assays demonstrated that DPEP1 could boost the proliferation of colon cancer cells through the DPEP1/MYC positive feedback loop in vitro and in vivo. Theoretically, DPEP1 may serve as a colon cancer biomarker and a novel target of colorectal carcinogenesis therapy.

Correlation between leptin and IFN-γ involved in granulosa cell apoptosis in PCOS.

OBJECTIVES: Our study aimed to explore the relationship between leptin and IFN-γ in PCOS patients, and confirmed the effect of leptin-induced IFN-γ on granulosa cells furtherly. METHODS: 29 patients with PCOS and 36 healthy controls were enrolled. Leptin level and the proportion of Th1 cells were detected and association between them were analyzed. Meanwhile, peripheral blood mononuclear cells (PBMCs) isolated from PCOS patients were treated with leptin and then the proportion of Th1 was analyzed. Besides that, the apoptotic level of KGN cells was monitored after IFN-γ treatment. RESULTS: In the circulation of PCOS patients, leptin level dramatically increased compared with controls. And, this was associated with upregulated Th1 cells proportion and IFN-γ level. In vitro, Th1 cells proportion increased after leptin treated PBMCs from PCOS patients. Furthermore, for KGN cells, the percentage of live cells decreased and later apoptosis cells increased after IFN-γ treatment. CONCLUSIONS: Our results indicated that leptin takes part in process of PCOS via inducing expression of IFN-γ. Our findings highlight the importance of the connection between leptin and inflammation in PCOS and provide new insights therapeutic strategy for this disease.

Expression regulation of multiple key genes to improve L-threonine in Escherichia coli.

BACKGROUND: Escherichia coli is an important strain for L-threonine production. Genetic switch is a ubiquitous regulatory tool for gene expression in prokaryotic cells. To sense and regulate intracellular or extracellular chemicals, bacteria evolve a variety of transcription factors. The key enzymes required for L-threonine biosynthesis in E. coli are encoded by the thr operon. The thr operon could coordinate expression of these genes when L-threonine is in short supply in the cell. RESULTS: The thrL leader regulatory elements were applied to regulate the expression of genes iclR, arcA, cpxR, gadE, fadR and pykF, while the threonine-activating promoters PcysH, PcysJ and PcysD were applied to regulate the expression of gene aspC, resulting in the increase of L-threonine production in an L-threonine producing E. coli strain TWF001. Firstly, different parts of the regulator thrL were inserted in the iclR regulator region in TWF001, and the best resulting strain TWF063 produced 16.34 g L-threonine from 40 g glucose after 30 h cultivation. Secondly, the gene aspC following different threonine-activating promoters was inserted into the chromosome of TWF063, and the best resulting strain TWF066 produced 17.56 g L-threonine from 40 g glucose after 30 h cultivation. Thirdly, the effect of expression regulation of arcA, cpxR, gadE, pykF and fadR was individually investigated on L-threonine production in TWF001. Finally, using TWF066 as the starting strain, the expression of genes arcA, cpxR, gadE, pykF and fadR was regulated individually or in combination to obtain the best strain for L-threonine production. The resulting strain TWF083, in which the expression of seven genes (iclR, aspC, arcA, cpxR, gadE, pykF, fadR and aspC) was regulated, produced 18.76 g L-threonine from 30 g glucose, 26.50 g L-threonine from 40 g glucose, or 26.93 g L-threonine from 50 g glucose after 30 h cultivation. In 48 h fed-batch fermentation, TWF083 could produce 116.62 g/L L-threonine with a yield of 0.486 g/g glucose and productivity of 2.43 g/L/h. CONCLUSION: The genetic engineering through the expression regulation of key genes is a better strategy than simple deletion of these genes to improve L-threonine production in E. coli. This strategy has little effect on the intracellular metabolism in the early stage of the growth but could increase L-threonine biosynthesis in the late stage.

Developing an l-threonine-producing strain from wild-type Escherichia coli by modifying the glucose uptake, glyoxylate shunt, and l-threonine biosynthetic pathway.

Wild-type Escherichia coli MG1655 usually does not accumulate l-threonine. In this study, the effects of 13 genes related to the glucose uptake, glycolysis, TCA cycle, l-threonine biosynthesis, or their regulation on l-threonine accumulation in E. coli MG1655 were investigated. Sixteen E. coli mutant strains were constructed by chromosomal deletion or overexpression of one or more genes of rsd, ptsG, ptsH, ptsI, crr, galP, glk, iclR, and gltA; the plasmid pFW01-thrA*BC-rhtC harboring the key genes for l-threonine biosynthesis and secretion was introduced into these mutants. The analyses on cell growth, glucose consumption, and l-threonine production of these recombinant strains showed that most of these strains could accumulate l-threonine, and the highest yield was obtained in WMZ016/pFW01-thrA*BC-rhtC. WMZ016 was derived from MG1655 by deleting crr and iclR and enhancing the expression of gltA. WMZ016/pFW01-thrA*BC-rhtC could produce 17.98 g/L l-threonine with a yield of 0.346 g/g glucose, whereas the control strain MG1655/pFW01-thrA*BC-rhtC could only produce 0.68 g/L l-threonine. In addition, WMZ016/pFW01-thrA*BC-rhtC could tolerate the high concentration of glucose and produced no detectable by-products; therefore, it should be an ideal platform strain for further development. The results indicate that manipulating the glucose uptake and TCA cycle could efficiently increase l-threonine production in E. coli.

Deletion of arcA, iclR, and tdcC in Escherichia coli to improve l-threonine production.

l-Threonine is an important amino acid supplemented in food, medicine, or feed. Starting from glucose, l-threonine production in Escherichia coli involves the glycolysis, TCA cycle, and the l-threonine biosynthetic pathway. In this study, how the l-threonine production in an l-threonine producing E. coli TWF001 is controlled by the three regulators ArcA, Cra, and IclR, which control the expression of genes involved in the glycolysis and TCA cycle, has been investigated. Ten mutant strains were constructed from TWF001 by different combinations of deletion or overexpression of arcA, cra, iclR, and tdcC. l-Threonine production was increased in the mutants TWF015 (ΔarcAΔcra), TWF016 (ΔarcAPcra::Ptrc), TWF017 (ΔarcAΔiclR), TWF018 (ΔarcAΔiclRΔtdcC), and TWF019 (ΔarcAΔcraΔiclRΔtdcC). Among these mutant strains, the highest l-threonine production (26.0 g/L) was obtained in TWF018, which was a 109.7% increase compared with the control TWF001. In addition, TWF018 could consume glucose more efficiently than TWF001 and produce less acetate. The results suggest that deletion of arcA, iclR, and tdcC could efficiently increase l-threonine production in E. coli.

Long noncoding RNA expression profile in HLE B-3 cells during TGF-ß2-induced epithelial-mesenchymal transition.

BACKGROUND: Recent evidence has shown that long noncoding RNAs (lncRNAs) are involved in the process of epithelial-mesenchymal transition (EMT). However, little research has focused on the expression profile of lncRNAs during EMT in human lens epithelial cells (LECs) and their functions have not yet been described. METHODS: Dysregulated lncRNAs and mRNAs in normal human lens epithelial B-3(HLE B-3) cells and during transforming growth factor ß2(TGF-ß2)-induced EMT were analyzed via lncRNA microarray. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analyses of differentially expressed mRNAs were performed to identify their functions and pathologic pathways. Six candidate lncRNAs were validated via quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR) to confirm the microarray data. RESULTS: A total of 775 lncRNAs (325 up-regulated and 450 down-regulated) and 935 mRNAs (329 up-regulated and 606 down-regulated) were differentially expressed in HLE B-3 cells during TGF-ß2-induced EMT compared to normal HLE B-3 cells. GO and KEGG Pathway analyses indicated the functions of differentially expressed mRNAs in the TGF-ß2-induced EMT in HLE B-3 cells. qRT-PCR confirmed the trends indicated in microarray analysis for all 6 candidate lncRNAs. CONCLUSION: Our study lays the foundation for future research in lncRNAs related to EMT in HLE B-3 cells and could provide new avenues for the prevention and treatment of posterior capsule opacification (PCO).

Cytoprotective effects of high dose of α-galactosylceramide against activation-induced CD4+ T and CD8+ T cell death as an adjuvant.

OBJECTIVE: To investigate the cytoprotective effects of high dose of α-galactosylceramide (α-GC) on the activation-induced CD4+ T and CD8+ T cell death. METHODS: Experimental autoimmune encephalomyelitis (EAE) was induced using adoptive transfer of MOGCD4+ cells treated using α-GC into recipient C57BL/6 mice while the MOGCD4+ cells treated using 0.5% polysorbate were set as vehicle group, based on which to investigate the effects of α-GC on activation induced CD4+ T cell death. Additionally, an EG7 tumor-bearing mice model is established using adoptive transfer of CD8+ T cells, based on which to investigate the effect of α-GC on the apoptosis of CD8+ T cells. RESULTS: A higher induction rate was noticed after adoptive transfer of MOGCD4+ cells treated using α-GC together with the severity of EAE compared with the conventional methods. Longer survival duration was noted in the green fluorescent protein (GFP) labeled MOGT in the α-GC group compared with the vehicle group (P < 0.05). Severe inflammatory cell infiltration and myelinoclasis was noted in the white matter of nervous system in the α-GC group. In the EG7 tumor model, more adoptive CD8+ T cells were survived in α-GC group compared with that of vehicle group. The growth of tumor mass was significantly inhibited in α-GC group. CONCLUSIONS: high dose of α-GC could be used as an adjuvant for inhibiting activation-induced CD4+ T and CD8+ T cell death. Our study could provide helpful information for the development of adoptive cell therapy with reduced programmed cell death.

Association of thrombomodulin Ala455Val dimorphism and inflammatory cytokines with carotid atherosclerosis in the Chinese Han population.

BACKGROUND AND METHODS: It has been reported that C/T dimorphism at position 1418 of the thrombomodulin gene causes a cytosine (C) transition to thymidine (T), resulting in an alanine (A) to valine (V) substitution at amino acid position 455 (TM455). TM455 had been found not only in African American and American whites, but also in whites in The Netherlands and Sweden. Among these populations, the C/C genotype is predominant, although the distribution of this dimorphism is different. Thrombomodulin is an important anticoagulant protein that is downregulated in endothelial cells overlying atherosclerotic plaques and is also an anti-inflammatory molecule. TM455 is located in the last epidermal growth factor-like repeat of thrombomodulin, which is functionally important for protein C activation and thrombin binding. The distribution of thrombomodulin polymorphism and association between TM455, inflammatory cytokines, and carotid atherosclerosis in the Chinese Han population is unclear. METHODS: This thrombomodulin dimorphism was analyzed by allele-specific amplification in 144 patients with carotid atherosclerosis and in 384 healthy controls. TM455 was found in the Chinese Han population, but the genotype frequency and distribution of each genotype in this population differed substantially from that in other ethnic subgroups. The C/T and T/T genotypes were predominant in the Chinese Han population, and the frequency of the T allele in this population (63.8%) was much higher than that in whites in The Netherlands (18%), Sweden (26.1%), and the US (18.4%), and in blacks in the US (7.6%). The frequencies of these single nucleotide polymorphisms complied well with the Hardy-Weinberg equilibrium in healthy individuals. The C allele was significantly more common among patients with carotid atherosclerosis than in controls (P < 0.05). The frequency of the C allele was 45.5% in patients and 36.2% in controls. The thrombomodulin Ala455 genotypes C/C and C/T were significantly more common than the T/T genotype in patients with carotid atherosclerosis in the Chinese Han population. In addition, higher baseline levels of tumor necrosis factor alpha (55.45 ± 11.58 pg/mL versus 52.70 ± 10.74 pg/mL; P < 0.05), interleukin-6 (31.53 ± 10.51 pg/mL versus 27.73 ± 8.37 pg/mL; P < 0.01), and C-reactive protein (6.65 ± 2.01 mg/L versus 4.06 ± 1.03 mg/L; P < 0.01) were observed in patients with carotid atherosclerosis than in controls. Interestingly, compared with baseline inflammatory cytokine levels in those with the Val/Val genotype, higher baseline tumor necrosis factor alpha, interleukin-6, and C-reactive protein levels were observed for the Ala/Ala genotype in both patients with carotid atherosclerosis and healthy controls. CONCLUSION: Our results support a significant association between thrombomodulin Ala455Val dimorphism, inflammatory cytokines, and carotid atherosclerosis in the Chinese Han population.

[Mechanism of DADS in the bystander effect of HSV-tk/GCV suicide gene therapy system in lens epithelial cells].

OBJECTIVE: To investigate the mechanism and effect of diallyl disulfide (DADS) on the bystander effect of herpes simplex virus kinase/ganciclovir (HSV-tk/GCV) suicide gene therapy system which was mediated by recombinant adeno-associated virus (rAAV) in lens epithelial cells. METHODS: Immunohistochemistry method was used to determine the distribution of connexin 43 (Cx43) protein in cultured rabbit lens epithelial cells and rabbit lens epithelial cells transfected by HSV-tk suicide gene. Cx43 protein was measured and analyzed in cultured rabbit lens epithelial cells and rabbit lens epithelial cells transfected by HSV-tk suicide gene that was induced by various DADS. Cell survival was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. RESULTS: DADS increased the Cx43 protein expression in cultured rabbit lens epithelial cells and rabbit lens epithelial cells transfected by HSV-tk suicide gene, and especially in 50 µmol/L DADS. After combining with DADS, the bystander effect was significantly improved (P<0.05). CONCLUSION: DADS can elevate the expression of Cx43 protein and enhance the bystander effect of HSV-tk/GCV suicide gene therapy system.

[Inhibitory effects of HSV-tk/GCV system mediated by recombinant adeno-associated virus 2 on rabbit lens epithelial cells].

OBJECTIVE: To study the inhibitory effects of recombinant adeno-associated virus 2 (rAAV2)-mediated herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system on the rabbit lens epithelial cells (N/N1003A) in vitro and to investigate the mechanism of cell death. METHODS: After N/N1003A cells had been transfected with rAAV2-EGFP, expression of enhanced green fluorescent protein (EGFP) were observed by inverted fluorescent microscope and the transfection efficiency was detected by flow cytometry. N/N1003A cells were infected by recombinant virus rAAV2/HSV-tk as the treated group, and the uninfected N/N1003A cells were used as the controls. The dose- and time-dependent efficiency and bystander effect of HSV-tk/GCV system on the cells were studied by MTT assay. Apoptosis and necrosis were observed by phase contrast microscope, electron microscope and Hoechst33258 stain. Apoptotic cell rate and cell cycle were detected by flow cytometry. RESULTS: rAAV2 vector encoding EGFP gene could be transfected into N/N1003A cells stably and efficiently. The effects of GCV on these two groups were dose-dependent (F = 13.076. 239, P < 0.001). The difference of percentages of survival cells between the study group and the control group at various doses of GCV was statistically significant (F = 53,47.119, P < 0.001). The 50% of the inhibitory concentration (IC50) of GCV in the study group was 2 mg/L and was 524 mg/L in the control group. The killing efficiency of GCV increased with the prolongation of time and showed significant bystander effect. Cell apoptosis and necrosis were observed in N/N1003A-tk cells transfected by GCV, and the percentage of apoptotic cells was significantly higher than that of the control group (t = 3.83, P < 0.01). The percentages of N/N1003A-tk cells in the S phase of the cell cycle was significantly higher than that of the control group (t = 3.55, P < 0.01). Whereas the percentages of the G0/ G1 phase in GCV treated cells was significantly lower than that of the control group ( t = 4.29, P < 0.01). CONCLUSIONS: GCV can kill efficiently the N/N1003A cells infected by recombinant virus rAAV2/HSV-tk, and there is strong bystander effect. Recombinant adeno-associated virus-mediated HSV-tk/GCV suicide gene system may provide an effective approach for the treatment of lens posterior capsular opacification.

[Construction and expression of recombinant adeno-associated virus vector containing HSV1-TK gene].

OBJECTIVE: To construct the recombinant adeno-associated virus(rAAV) vector plasmid pSNAV2.0-TK containing HSV1-TK gene, to produce recombinant adeno-associated virus rAAV2/HSV1-TK, and to detect the integration and expression of HSV1-TK gene in lens epithelial cells transfected by rAAV2/HSV1-TK, and to provide foundation for gene therapy of posterior capsular opacification. METHODS: The recombinant vector plasmid constructed by gene recombinant technology was analyzed by PCR and restriction enzyme digestion. The cell strain BHK-21/TK was screened by G418 after the plasmid was transfected into BHK-21 cells,with the helper virus HSV1-rc/UL2 to produce the recombinant virus rAAV2/HSV1-TK. The purity of rAAV2/HSV1-TK was detected by SDS-PAGE and HPLC, and the titre of rAAV2/HSV1-TK was observed by dot blot hybridization. The HSV1-TK gene in lens epithelial cells transfected by rAAV2/HSV-TK was investigated by PCR and RT-PCR. RESULTS: The recombinant plasmid proved successful by PCR and restriction enzyme digestion. The recombinant virus rAAV2/HSV1-TK was produced successfully and its titre was 1 x 10(12) v.g./mL by dot blot hybridization. The HSV1-TK gene was integrated and expressed in lens epithelial cells. CONCLUSION: The recombinant adeno-associated virus vector plasmid containing HSV1-TK gene is successfully constructed, and high titre recombinant adeno-associated virus (rAAV2/HSV1-TK) is obtained. The HSV1-TK gene in lens epithelial cells is expressed after being transfected by rAAV2/HSV1-TK.