ABSTRACT
OBJECTIVE@#To explore the effects of Ena/VASP gene family on the expression of glycoprotein (GP) Ib-IX complex in human megakaryoblastic leukemia Dami cells.@*METHODS@#SiRNAs targeting Ena/VASP gene family were designed and synthesized to interfere Enah, EVL and VASP gene expression. When the siRNAs were transfected into Dami cells by using LipofectamineTM 2000 for 48 h, the expression of GPIb-IX complex was detected by quantitative real-time PCR, Western blot and flow cytometry.@*RESULTS@#We successfully established siVASP , siEVL and si Enah Dami cell lines. And it was found that the expression of GPIb-IX complex had no evident reduction in siEVL or siVASP Dami cells at both mRNA and protein level, while the total protein and membrane protein of GPIb-IX complex were obviously reduced when Enah was knocked down.@*CONCLUSION@#Enah could affect the expression of GPIb-IX complex in human megakaryoblastic leukemia Dami cells, but the underlying mechanism still needs to be further explored.
Subject(s)
Humans , Cell Line , Platelet Glycoprotein GPIb-IX Complex/metabolism , Leukemia/metabolism , Blood Platelets/metabolismABSTRACT
@#Abstract: Transfection of Plasmodium falciparum is helpful to study the function of its genes, such as drug resistance. However, transgenic manipulation has been very challenging, mainly due to the high A/T base sequence structure (A+T content of about 82%) and low transfection efficiency of the Plasmodium genome. Electroporation-based transfection of Plasmodium falciparum has been successfully applied in the study of certain genes, and electroporation by preloading is currently the preferred method for introducing foreign DNA into Plasmodium falciparum. The site-directed editing of Plasmodium genes mostly adopts the method of two-plasmid transfection. It is generally believed that successful transfection of Plasmodium requires a large amount of high-purity plasmid DNA and an accurate transfection system. In addition to the evaluation of the current commonly used electrotransfection methods, this paper also introduces a new transfection method, namely lyse-reseal erythrocytes for transfection (LyRET). This paper also review the role of factors such as plasmid DNA concentration, the use of transfection reagents, the setting of transfection parameters, the addition of fresh red blood cells, and the markers of successful transfection in improving the success rate and efficiency of Plasmodium transfection, in the hope of providing a reference for study in this field.
ABSTRACT
Objective:To explore the expression of long non-coding RNA (lncRNA) CDK5RAP3 in gastric cancer tissue and its regulatory effect on gastric cancer cell proliferation and invasion.Methods:The expression differences of CDK5RAP3 in gastric cancer tissues and adjacent tissues were analyzed by TCGA database. By transfecting the pcDNA3.1-CDK5RAP3 plasmid into Hs-746T cells, a gastric cancer cell line overexpressing CDK5RAP3 (CDK5RAP3 group) was constructed, and the pcDNA3.1 plasmid was transfected into Hs-746T cells as a control group. The changes of CDK5RAP3 expression in the two groups of cells were detected by real-time quantitative PCR (qRT-PCR). The effects of overexpression of CDK5RAP3 on the proliferation and invasion of Hs-746T cells were detected by CCK-8 assay and Transwell assay, respectively. The binding sites of CDK5RAP3 and miR-223-3p were predicted by the starBase v2.0 database. The direct binding of CDK5RAP3 and miR-223-3p was verified by dual-luciferase reporter gene experiment. The expression levels of miR-223-3p in Hs-746T cells in each group were detected by qRT-PCR. Western blot was used to detect the expression levels of proliferation proteins and invasion proteins in Hs-746T cells in each group. The experimental data were analyzed by SPSS 17.0 software, and the measurement data conforming to the normal distribution were expressed as Mean±SD. The t-test was used to compare between two groups, and the one-way analysis of variance was used to compare the means of multiple groups. Results:Compared with adjacent tissues, the expression level of CDK5RAP3 in gastric cancer tissues was significantly lower ( P<0.01). The expressions of CDK5RAP3 in Hs-746T cells in the control group and CDK5RAP3 group were (1.08±0.77) and (10.63±2.14), respectively, and the difference was statistically significant ( P<0.01). Up-regulation of CDK5RAP3 significantly decreased the proliferation activity of Hs-746T cells ( P<0.05). The number of invasive cells in the control group and CDK5RAP3 group were (137.80±28.72) and (57.76±24.95), respectively, and the difference was statistically significant ( P<0.01). CDK5RAP3 could directly bind miR-223-3p ( P<0.01). The expression of miR-223-3p in Hs-746T cells in control group and CDK5RAP3 group were (6.22±1.20) and (1.01±0.98), respectively, and the difference was statistically significant ( P<0.01). Compared with the control group, up-regulation of CDK5RAP3 significantly reduced the expression levels of proliferation and invasive proteins. Conclusion:The expression of CDK5RAP3 is low in gastric cancer tissue, and CDK5RAP3 inhibits the proliferation and invasion of gastric cancer Hs-746T cells by targeting miR-223-3p.
ABSTRACT
Objective:To explore the effect of down-regulation of long non-coding RNA (lncRNA) CTB-191K22.5 on the proliferation and invasion of colorectal cancer SW480 cells and the molecular mechanism.Methods:The TCGA database was used to analyze the expression differences of CTB-191K22.5 in colorectal cancer tissues and normal tissues. The CTB-191K22.5 inhibitor (Anti-CTB-191K22.5) and negative inhibitor (Control) were transfected into colorectal cancer SW480 cells, denoted as Observation group and Control group, real-time quantitative polymerase chain reaction (qRT) -PCR) was used to evaluate the inhibitory effect. MTT method and Transwell chamber method were used to evaluate the proliferation and invasion of SW480 cells. Western blot was used to evaluate the protein levels of PI 3K/AKT/mTOR signaling pathway in SW480 cells. The bioinformatics software starBase v2.0 was used to predict the target genes of CTB-191K22.5. qRT-PCR was used to evaluate the expression of CTB-191K22.5 target gene in SW480 cells. Measurement data were expressed as Mean±SD, and t-test was used for comparison between two groups. Results:Compared with normal tissues, the expression of CTB-191K22.5 in colorectal cancer tissues was significantly increased ( P<0.01). The expression of CTB-191K22.5 in SW480 cells of the Control group and Observation group were 6.60±0.85 and 1.08±0.21, respectively. The expression level of CTB-191K22.5 decreased after transfection with Anti-CTB-191K22.5 ( P<0.01). Compared with the Control group, the SW480 cell proliferation ability of the Observation group decreased ( P<0.01). The invasion numbers of SW480 cells in the Control group and Observation group were (135.4 ± 16.29) and (42.24±14.59), respectively. The invasion ability of SW480 cells decreased after transfection with Anti-CTB-191K22.5 ( P<0.01). Compared with the Control group, the expression levels of PI 3K/AKT/mTOR signaling pathway protein in SW480 cells in the Observation group decreased. miR-326 may be the target gene of CTB-191K22.5. Compared with the Control group, transfection with Anti-CTB-191K22.5 significantly increased the expression level of miR-326 in SW480 cells ( P<0.01). Conclusion:CTB-191K22.5 is highly expressed in colorectal cancer tissues, and down-regulation of CTB-191K22.5 may inhibit the proliferation and invasion of colorectal cancer SW480 cells by targeting miR-326.
ABSTRACT
Objective:To observe the inhibitory effect of lentivirus (LV)-mediated miR-191 on the proliferation and angiogenesis of human retinal vascular endothelial cells (hREC) cultured in vitro.Methods:The hREC cell lines were cultured in vitro and divided into control group, hypoxia group, LV-empty vector (LV-vector) group, and LV-miR-191 (LV-191) group. The LV-vector group and LV-191 group were transferred to the corresponding lentiviral vector respectively. Flow cytometry was used to detect cell transfection efficiency. Cell Counting Kit-8 (CCK-8) test was used to detect cell proliferation ability. Scarification test and invasion chamber (Transwell) test were used to detect cell migration ability. Matrigel test was used to detect cell lumen formation ability. Real-time quantitative polymerase chain reaction (qPCR) was used to detect the relative expression of miR-191 and relative mRNA expression of its downstream target genes p21, vascular endothelial growth factor (VEGF), cell division protein kinase (CDK) 6, cyclin-D1 (Cyclin D1). Independent sample t test was used for pairwise comparison. Results:The results of flow cytometry showed that the transfection efficiency of cells in the control group and the LV-191 group were 0.615% and 99.400%, respectively. The results of CCK-8, scarification, Transwell and Matrigel test showed that, compared with the control group, the number of cell proliferation ( t=6.130, 4.606), the cell mobility ( t=4.910, 6.702), the number of stained cells on the microporous membrane ( t=7.244, 6.724) and the lumen formation ability cells ( t=8.345, 9.859) were significantly increased in the hypoxia group and the LV-vector group ( P<0.01), while the LV-191 group showed completely opposite performance ( t=14.710, 6.245, 5.333, 5.892; P≤0.01). The qPCR test results showed that, compared with the control group and the LV-vector group, the relative expression of miR-191 mRNA in the cells of the LV-191 group was significantly up-regulated ( t=44.110, 42.680), the relative expression of Cyclin D1 mRNA ( t=29.940, 14.010) and CDK6 mRNA ( t=15.200, 7.645) decreased significantly, and the difference were statistically significant ( P<0.01); the relative expression of p21 mRNA increased, however, the difference was not statistically significant ( t=2.013, 2.755; P>0.05). There was no significant difference in the relative expression of VEGF mRNA in the 4 groups of cells ( F=0.966, P>0.05). Conclusions:LV-191 can inhibit the proliferation, migration and tubing of hREC by up-regulating p21 and down-regulating CDK6 and Cyclin D1.
ABSTRACT
Objective:To investigate the expression of zinc finger protein 580 (ZNF580) in oxygen-glucose deprivation (OGD) model of SH-SY5Y cell line and its overexpression on the apoptosis of hypoxic-ischemic neurons and the possible mechanism.Methods:The study was divided into two parts: (1) Human neuroblastoma SH-SY5Y cell line was cultured and divided into the model group and control group. The model group was incubated at 37 ℃ for 6 h in a three-gas incubator of 95% N 2, 5% CO 2, and 0.1% O 2 to establish OGD model, and proteins were extracted at 6, 12, and 24 h after OGD. The expression of ZNF580 was quantified by Western blot. (2) Effects of ZNF580 overexpressed with lentivirus transfection on the apoptosis and cleaved caspase-3 expression: Cells were collected from the control group and model group 24 h after OGD. Overexpressed ZNF580 cells were constructed by lentivirus transfection as the overexpression group and then treated with OGD. Flow cytometry was used to detect the apoptosis rate in the three groups and Western blot was used to detect the expression of cleaved caspase-3. Two independent sample t-test, one-way variance analysis, and LSD- t for pairwise comparison were used for statistical analysis. Results:(1) ZNF580 expression was significantly increased at 6, 12, and 24 h after OGD compared with the control group (1.36±0.05, 2.12±0.07, 1.69±0.05 vs 1.00, LSD- t=9.20, 28.26, and 19.21, all P<0.001). (2) Apoptosis rates of the control, model, and overexpression groups were (1.07±0.56)%, (21.51±1.65)%, and (3.42±0.93)%, respectively, and relative expression levels of cleaved caspase-3 were 1.00, 2.47±0.59, and 1.70±0.25, respectively. Compared with the control group, apoptosis rate and cleaved caspase-3 relative expression level were significantly increased in the model group (LSD- t=21.98 and 8.17, both P=0.001), while the two figures were significantly decreased in the overexpression group when compared with the model group (LSD- t=19.45, P=0.001; LSD- t=4.28, P=0.005). Conclusion:Hypoxia and ischemia could lead to the overexpression of ZNF580, which may reduce the apoptosis of hypoxic-ischemic neurons by inhibiting the expression of cleaved caspase-3 and affecting its enzymatic activation.
ABSTRACT
In the past 20 years, with the development of molecular biology, the treatment of non-small cell lung cancer (NSCLC) has been developing. Targeted therapy has improved the survival period of patients with positive mutation of tumor driver gene. More and more targets have been found gradually. Drugs targeting different driving genes have brought the treatment of NSCLC into a promising target era. Among the many driving genes of NSCLC, the fusion of transfection proto oncogene (RET) is the addition of the epidermal growth factor receptor (EGFR), analytic lymphama kinase (ALK) and c-ros oncogene 1-receptor tyrosine kinase (ROS1) are emerging targets. Targeted drugs for RET gene fusion have been constantly updated. Recently, new high selective RET inhibitors blu-667 and loxo-292 have made important breakthroughs. This paper will review the review of the fusion mutation of RET gene in NSCLC, the detection methods, clinicopathological characteristics, targeted treatment and the research progress after drug resistance. .
ABSTRACT
OBJECTIVE: To construct the RNA interference(RNAi) lentiviral vector of suppressor of variegation 3-9 homolog 1(Suv39 h1) and verify its interfering efficiency by transfecting it to the bone marrow-derived mesenchymal stem cells(BMSCs). METHODS: The oligonucleotides of RNA plasmid were designed and synthesized according to the gene sequence of Suv39 h1 and short hairpin RNA design principles. Three kinds of LV-Suv39 h1-RNAi recombinant plasmids with different lentivirus knockdown targets(KD1, KD2 and KD3) were constructed. After identification by restriction analysis and sequencing, the packaged lentivirus vectors with the three kinds of Suv39 h1 gene were transfected into rat BMSCs at logarithmic growth stage, and were named KD1, KD2 and KD3 transfection groups. The control group was transfected with the negative control virus. After 72 hours transfection, the transfection efficiency was evaluated, and the relative mRNA levels of Suv39 h1 were determined by quantitative real-time polymerase chain reaction(qPCR). RESULTS: Sequencing analysis demonstrated that three kinds of LV-Suv39 h1-RNAi recombinant plasmids were constructed correctly. The results of transfection efficiency evaluation showed that more than 80.00% green fluorescence was expressed in the BMSCs transfected with the three lentiviral vectors with a multiplicity of infection of 20. These results indicated that lentivirus was successfully constructed and transfection efficiency was high. The results of qPCR showed that the relative expression of Suv39 h1 mRNA in BMSCs of KD1, KD2 and KD3 transfection groups was lower than that in the control group(all P<0.05), and the relative expression of Suv39 h1 mRNA in KD1 and KD3 transfection groups was lower than that in KD2 transfection group(both P<0.05). However, there was no significant difference in the relative expression of Suv39 h1 mRNA between KD1 and KD3 transfection groups(P>0.05). CONCLUSION: The constructed lentiviral vector with low expression of Suv39 h1 was constructed successfully. This vector can be expressed in rat BMSCs, which lays a foundation to study the effect of Suv39 h1 gene in acute myeloid leukemia.
ABSTRACT
BACKGROUND: Studies have shown that the loss of sex combing protein 1 (Asxl1) can lead to the occurrence of bone dysplasia and bone defects, but the relationship between this factor and bone destruction in the microenvironment of apical periodontitis has not been reported. OBJECTIVE: To study the effect of Asxl1 on proliferation and differentiation of osteoblasts in an inflammatory microenvironment. METHODS: MC3T3-E1 cells were excited by lipopolysaccharide to establish an in vitro inflammatory microenvironment. The best concentration and optimal action time of lipopolysaccharide were screened by cell counting kit-8 test. MC3T3-E1 cells were then stimulated with 20 mg/L lipopolysaccharide for 24 hours. The expression levels of Asxl1 protein and mRNA were detected by immunofluorescence and real-time PCR, respectively. After lipopolysaccharide stimulated the formation of inflammatory microenvironment, Asxl1-Si RNA was transfected for 24 hours, cell counting kit-8 was applied to detect the activity of cell proliferation, and real-time PCR was used to detect the expression levels of Asxl1 and osteogenic related genes ALP and RUNX2 mRNA. RESULTS AND CONCLUSION: After lipopolysaccharides stimulation of MC3T3-E1 cells, the expression levels of Asxl1 protein and mRNA were decreased. Under the inflammatory microenvironment, the proliferation activity of MC3T3-E1 cells transfected with Asxl1-Si RNA for 24 hours was significantly decreased, and the expression levels of Asxl1, ALP and RUNX2 mRNA were markedly decreased. These findings indicate that Asxl1 may influence the proliferation and differentiation of osteoblasts by involvement in the process of inflammatory reaction, thereby participating in bone destruction.
ABSTRACT
BACKGROUND: In the research of human embryonic stem cells, introducing exogenous molecules such as DNA into cells is a common research method, but the transfection efficiency is relatively low. It is crucial to answer the question of how to optimize the existing conditions to improve the transfection efficiency. OBJECTIVE: To compare the effects of two different passaging methods on H9 transfection efficiency, in order to optimize the conditions required for embryonic stem cell transfection. METHODS: Human embryonic stem cell lines H9 were cultured for 48 hours after small clone passaging or single-cell passaging. Lipofectamine 3000 was used to transfect pAdTrack-AKT1 fluorescent plasmid into human embryonic stem cells. After 2 days of transfection, the expression of fluorescent plasmids was observed by fluorescence microscope and the transfection efficiency was detected by flow cytometry. RT-qPCR and western blot were used to detect the mRNA and protein expression levels of AKT1 respectively. RESULTS AND CONCLUSION: Under the fluorescence microscopy, the number of cells expressing fluorescent plasmids in the single-cell passaging group was more than that in the small clone passaging group, and the flow cytometry analysis showed that the transfection efficiency of cells in the single-cell passaging group was (47.18±2.00)%, which was significantly higher than (19.52±0.86)% in the small clone passaging group (P < 0.01). RT-qPCR and western blot analysis showed that the expression levels of AKT1 mRNA and protein in the single-cell passaging group were significantly higher than those in the small clone passaging group (P < 0.01). These findings indicate that single-cell passaging can increase the contact area between cells and transfection reagent liposomes, and improve the transfection efficiency of human embryonic stem cells.
ABSTRACT
BACKGROUND: Human amniotic mesenchymal stem cells have a wide variety of sources, low immunogenicity, and multilineage differentiation potential. Studies have confirmed that Scleraxis gene can induce human amniotic mesenchymal stem cells to differentiate into ligaments and accelerate tendon-bone healing. OBJECTIVE: To explore whether Scleraxis induces human amniotic mesenchymal stem cells to promote tendon-bone healing in vivo in rabbits, providing new options for clinical treatment of tendon-bone healing. METHODS: The study protocol was approved by the Ethic Committee of the Affiliated Hospital of Zunyi Medical University, and written informed consent was obtained from each puerpera. The healthy full-term maternal placenta was taken and cultured, and human amniotic mesenchymal stem cells were isolated and cultured by trypsin digestion twice. Then the morphology of the cells was observed under an inverted microscope, and the cells were further cultured until the third generation for subsequent experiments. The lentivirus carrying the Scleraxis gene was transfected into human amniotic mesenchymal stem cells in vitro. Expression levels of ligament-related genes were detected by real-time fluorescent quantitative PCR, and the expression levels of related proteins were detected by immunofluorescence. Human amniotic mesenchymal stem cells transfected with Scleraxis gene were injected into the extraarticular tendon-bone model of rats. After 3 months, specimens were taken to observe the tendon-bone healing. RESULTS AND CONCLUSION: (1) Human amniotic mesenchymal stem cells from passage to third generation showed long fusiform and vortex-like adherent growth under the inverted phase contrast microscope. (2) The third-generation human amniotic mesenchymal stem cells expressed green fluorescence after 24 hours of infection with the Scleraxis gene lentivirus, and the fluorescence expression was strong and stable. (3) Cell counting kit-8 findings indicated that lentivirus transfection of Scleraxis gene showed no influence on the cell growth rate. (4) Real-time fluorescent quantitative PCR findings showed that the mRNA expression of Scleraxis and ligament-related genes type I collagen, type III collagen, Fibronectin and Tenascin-C was significantly increased after lentivirus transfection of Scleraxis gene. (5) The results of immunofluorescence showed that the expression levels of ligament-related proteins type I collagen, type III collagen, Fibronectin and Tenascin-C were increased after lentivirus transfection of Scleraxis gene. To conclude, in vivo animal experiments have confirmed that the lentivirus transfection of Scleraxis gene can accelerate the tendon-bone healing of the rabbit extraarticular tendon-bone model.
ABSTRACT
BACKGROUND: P75 Neurotrophin receptor (P75NTR) is one of the receptors for nerve growth factor (NGF). P75NTR plays a dual role in promoting proliferation or apoptosis in various cell tissues, and is highly expressed at fracture nonunion sites. However, excessive NGF can shut down P75NTR receptor, thereby saving damaged cells. Therefore, the study regarding co-transfection of silenced P75NTR and NGF overexpression is of great significance for the proliferation of bone marrow mesenchymal stem cells and provides new ideas for clinical treatment of fracture nonunion. OBJECTIVE: To observe the effect of lentivirus-mediated silencing of P75NTR combined with NGF overexpression on the proliferation of bone marrow mesenchymal stem cells in Sprague-Dawely rats. METHODS: Bone marrow mesenchymal stem cells from Sprague-Dawley rats were cultured to the third generation in vitro and were divided into blank control group, negative control group, silent p75NTR group, NGF overexpression group, and silent p75NTR combined with NGF overexpression group. Lentivirus-mediated silencing of P75NTR and overexpression of NGF were transfected into rat bone marrow mesenchymal stem cells to induce P75NTR silencing and NGF overexpression. Inverted fluorescence microscopy was used to observe changes in cell morphology on day 3 after transfection. Flow cytometry was used to detect transfection efficiency and western blot method was applied to detect the expression of P75NTR and NGF. Finally, the cell proliferation activity was detected by MTT method and cell counting kit-8 method. RESULTS AND CONCLUSION: Cell growth and distribution were good after co-transfection of lentivirus. The transfection efficiency of the double-gene lentiviral vector exceeded 70%. Compared with the blank control and negative control groups, the expression of P75NTR protein was significantly down-regulated, and the expression of NGF was profoundly up-regulated in the silent p75NTR combined with NGF overexpression group. Compared with the blank control and negative control groups, cell proliferation was significantly increased in the other three groups (P < 0.05), and the fastest proliferation was observed in the silent p75NTR combined with NGF overexpression group. To conclude, silencing P75NTR combined with NGF overexpression co-transfection can promote the proliferation of bone marrow mesenchymal stem cells from Sprague-Dawley rats.
ABSTRACT
Objective@#To explore the mechanism of llinc00052 regulating Wnt pathway affecting invasion and metastasis of gastric cancer.@*Methods@#SGC-7901 cells were selected from gastric cancer cell lines, and siRNAs related to INC0005 and had invasion and metastasis effects in gastric cancer cells were screened by binding of IncRNA expression profiles to qRT-PCR.Ilinc00052 and miRNA expression changes were studied by in vitro cell transfection experiments.Through molecular experiments, the expression of llinc00052 and the effect on Wnt/β-catenin expression were investigated to explore whether llinc00052 could affect the invasion and metastasis of gastric cancer cells by regulating miRNAs affecting Wnt/β-catenin signaling pathway.@*Results@#Transwell chamber test showed that the number of transmembrane cells in the untransfected plasmid group was (134.10±4.29), and the number of transmembrane cells in the overexpressed llinc000522 plasmid group was (169.24±6.99)(t=8.956, P=0.001). The scratch test showed that the migration distance in the llinc000522 overexpression transfection plasmid group was significantly higher than that in the no-load plasmid transfection group(r=0.907, P<0.01). The clone formation rate of llinc000522 overexpressed transfected plasmid group was significantly higher than that of the empty plasmid group[(92.75±6.32)% vs.(73.34±9.14)%](t=5.998, P<0.05). Compared with the transfection of blank plasmid, the expressions of Wnt1, Wnt3a, Wnt2 and β-catenin mRNA in the llinc000522 overexpression transfection group were significantly up-regulated(P<0.05), while the miRNA transfection group had no significant effect on the expression.The expressions of Wnt1, Wnt3a, Wnt2, and β-catenin mRNA were significantly increased [(1.82±0.11), (1.52±0.15), (1.42±0.21), (1.71±0.19)](P<0.05), but their expressions were still lower than those of the genes transfected with llinc000522 alone.Compared with the blank plasmid transfection, the expressions of Wnt1, Wnt3a, Wnt2 and β-catenin protein in the llinc000522 overexpression transfection group were significantly up-regulated(all P<0.05), while the miRNA transfection group had no significant effect on its expression.The protein expressions of Wnt1, Wnt3a, Wnt2 and β-catenin were also significantly increased[(1.53±0.09), (1.4±0.21), (1.33±0.07), (1.47±0.19)](P<0.05), but their expressions were still lower than those of the gene transfected with llinc000522 alone.@*Conclusion@#In gastric cancer cells, llinc00052 can affect the invasion and metastasis of gastric cancer by regulating the level of miRNA and affecting the Wnt/β-catenin pathway.
ABSTRACT
Dengue virus(DENV) has been identified by World Health Organization as a major public health problem in tropical and subtropical regions. In recent years, dengue outbreaks have become more and more frequent in the world. In 2019, dengue outbreaks of varying degrees have occurred in the Philippines, Thailand, Bangladesh, Myanmar and Chongqing City in China. The laboratory diagnostic method of DENV is of great significance to the prevention and control of dengue epidemic. Therefore, the methods and strategies of DENV laboratory diagnosis are reviewed in this paper. By reviewing the traditional diagnostic methods and looking forward to the emerging diagnostic strategies, this paper aims to provide a reference to select the appropriate laboratory diagnostic scheme for the outbreak of dengue.
ABSTRACT
Cordycepin was the first adenosine analogue used as an anticancer and antiviral agent, which is extracted from Cordyceps militaris and hasn't been biosynthesized until now. This study was first conducted to verify the role of ribonucleotide reductases (RNRs, the two RNR subunits, RNRL and RNRM) in the biosynthesis of cordycepin by over expressing RNRs genes in transformed C. militaris. Quantitative real-time PCR (qRT-PCR) and western blotting results showed that the mRNA and protein levels of RNR subunit genes were significantly upregulated in transformant C. militaris strains compared to the control strain. The results of the HPLC assay indicated that the cordycepin was significantly higher in the C. militaris transformants carrying RNRM than in the wild-type strain, whereas the RNRML was preferentially downregulated. For the C. militaris transformant carrying RNRL, the content of cordycepin wasn't remarkably changed. Furthermore, we revealed that inhibiting RNRs with Triapine (3-AP) almost abrogated the upregulation of cordycepin. Therefore, our results suggested that RNRM can probably directly participate in cordycepin biosynthesis by hydrolyzing adenosine, which is useful for improving cordycepin synthesis and helps to satisfy the commercial demand of cordycepin in the field of medicine.
ABSTRACT
Objective: To observe and compare the cytological and biological differences between human normal and degenerated nucleus pulposus (NP), and to investigate the repair effect of insulin-like growth factor 1 (IFG-1) and platelet derived growth factor (PDGF) on human degenerated NP. Methods: Human degenerative and normal NP tissues were obtained from operative patients, a portion of which were processed into tissue sections and HE staining was performed to observe the morphological changes of nucleus pulposus cells (NPCs) before and after degeneration of NP. Immunohistochemistry staining was used to determine the expression levels of collagen type Ⅰ, collagen type Ⅱ, B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X (Bax) proteins. Another portion of tissues were isolated and cultured and NPCs morphology was observed under inverted microscope. Western blot analysis was used to detect collagen type Ⅱ protein expression. Then, the gene transfection experiments were launched, including 4 groups, with group A designed as degenerated NPCs only, and groups B, C, and D of degenerated NPCs transfected with IGF-1 gene lentiviral particles, PDGF gene lentiviral particles, and lentiviral particles carrying IGF-1 and PDGF double genes, respectively. At 21 days after transfection, the cell morphology of each group was observed under inverted microscope, the positive rates of IGF-1 and PDGF of each group were measured by flow cytometry, and the expression of collagen type Ⅱ protein was detected by using immunohistochemistry staining and Western blot. Results: HE staining showed that there were a large number of notochordal cells and a small number of chondrocytes in the central NP tissue of normal group, while the NPCs in degeneration group were significantly reduced, and a large proportion of fibrocartilage tissues were found in NP tissue. Immunohistochemistry staining showed that the percentages of collagen type Ⅰ and Bax protein-positive cells in degeneration group were significantly higher than those of normal group, while the percentages of collagen type Ⅱ and Bcl-2 protein-positive cells were significantly lower than those of normal group ( P<0.05). Western blot showed that the relative expression level of collagen type Ⅱ protein in degeneration group was significantly lower than that in normal group ( t=65.493, P=0.000). At 21 days after gene transfection, compared with group A, the cell viability of groups B, C, and D increased and the morphology became more regular. Flow cytometry showed that the percentages of IGF-1-positive cells in groups B and D were significantly higher than that in group A, and the percentages of PDGF-positive cells in groups C and D were significantly higher than that in group A ( P<0.05). Immunohistochemistry staining showed that the positive stainings of collagen type Ⅱ in groups A, B, C, and D was (±), (+), (+), and (++), respectively. Western blot showed that the relative expression of collagen type Ⅱ protein in groups A, B, C, and D increased by degrees, and the differences between groups were significant ( P<0.05). Conclusion: Both IGF-1 and PDGF can reverse the degeneration of intervertebral discs NPCs and they have synergistic effects, providing experimental basis for its application in clinical treatment approaches for degenerative disc disease.
ABSTRACT
Objective To explore the mechanism of llinc00052 regulating Wnt pathway affecting invasion and metastasis of gastric cancer.Methods SGC-7901 cells were selected from gastric cancer cell lines ,and siRNAs related to INC0005 and had invasion and metastasis effects in gastric cancer cells were screened by binding of IncRNA expression profiles to qRT -PCR.Ilinc00052 and miRNA expression changes were studied by in vitro cell transfection experiments.Through molecular experiments ,the expression of llinc00052 and the effect on Wnt／β-catenin expression were investigated to explore whether llinc 00052 could affect the invasion and metastasis of gastric cancer cells by regulating miRNAs affecting Wnt／β-catenin signaling pathway.Results Transwell chamber test showed that the number of transmembrane cells in the untransfected plasmid group was (134.10 ±4.29),and the number of transmembrane cells in the overexpressed llinc 000522 plasmid group was (169.24 ±6.99)(t＝8.956,P＝0.001). The scratch test showed that the migration distance in the llinc 000522 overexpression transfection plasmid group was significantly higher than that in the no -load plasmid transfection group (r＝0.907,P＜0.01).The clone formation rate of llinc000522 overexpressed transfected plasmid group was significantly higher than that of the empty plasmid group[(92.75 ±6.32)% vs.(73.34 ±9.14)%] (t＝5.998,P＜0.05).Compared with the transfection of blank plasmid,the expressions of Wnt1,Wnt3a,Wnt2 and β-catenin mRNA in the llinc000522 overexpression transfection group were significantly up -regulated(P＜0.05),while the miRNA transfection group had no significant effect on the expression.The expressions of Wnt1,Wnt3a,Wnt2,and β-catenin mRNA were significantly increased [(1.82 ± 0.11),(1.52 ±0.15),(1.42 ±0.21),(1.71 ±0.19)] ( P＜0.05),but their expressions were still lower than those of the genes transfected with llinc000522 alone.Compared with the blank plasmid transfection ,the expressions of Wnt1,Wnt3a,Wnt2 and β-catenin protein in the llinc000522 overexpression transfection group were significantly up-regulated(all P＜0.05),while the miRNA transfection group had no significant effect on its expression.The protein expressions of Wnt1,Wnt3a,Wnt2 and β-catenin were also significantly increased [(1.53 ±0.09),(1.4 ±0.21), (1.33 ±0.07),(1.47 ±0.19)](P＜0.05),but their expressions were still lower than those of the gene transfected with llinc000522 alone.Conclusion In gastric cancer cells, llinc00052 can affect the invasion and metastasis of gastric cancer by regulating the level of miRNA and affecting the Wnt ／β-catenin pathway.
ABSTRACT
Objective To investigate the therapeutic effect of SPK1 gene transfected adipose derived mesenchymal stem cells(ADMSC)on experimental autoimmune encephalomyelitis mice and the effect on T helper cell 17(Th17)/regulatory T(Treg) cells balance. Methods EAE was induced by myelin oligodendrocyte glycoprotein 35-55 in mice.Totally 44 mice were randomly divided into four groups:normal control group(NC group),model group(EAE group),ADMSC group,and ADMSC-SPK1 group.Forty days after injection,the pathological changes of brain and spinal cord,Th17/Treg-related inflammatory markers in brain tissue,expressions of interleukin-17A(IL-17A)and forkhead box protein p3(Foxp3)in brain and spinal cord tissue,and flow cytometric results of spleen immune cells were detected. Results Forty days after the injection,serious inflammatory cell infiltration and demyelination occurred in the brain and spinal cord of EAE group,whereas demyelination and axonal injury were improved in ADMSC group and ADMSC-SPK1 group.Compared with EAE group,the ADMSC group and ADMSC-SPK1 group had significantly improved levels of IL-17A(
Subject(s)
Animals , Mice , Adipose Tissue/cytology , Cytokines , Encephalomyelitis, Autoimmune, Experimental/therapy , Interleukin-17 , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Mice, Inbred C57BL , Phosphotransferases (Alcohol Group Acceptor)/genetics , T-Lymphocytes, Regulatory/cytology , Th17 Cells/cytology , TransfectionABSTRACT
Aim: This study was conducted to investigate the quelling of tri4 and tri5 genes associated with deoxynivalenol, most common class B trichothecene, via siRNA application. Methodology: In this study, one Fusarium graminearum (4F) and two F. culmorum (9F and 20F) isolates, expressing high levels of tri4 and tri5 genes, were targeted for siRNA-based silencing. Full length tri4 and tri5 genes were amplified from all isolates, sequenced and then subjected to CLUSTALW analysis for designing siRNA molecules. Totally designed six siRNAs were separately co-transfected together with helper plasmids (pEGFP75 and pAN7-1) as 36 combinations into protoplast cultures and quelling was analysed via real time polymerase chain reaction, thin layer chromatography and spectrofluorimetric analysis. Results: Alignment of both tri4 and tri5 genes’ sequencing revealed high levels of similarity between isolates (94-99% and 95-100%, respectively). Transfection efficiency ranged from 50±0.00/5x104 to 99.33±10.06/5x104 and relative fluorescence unit values were calculated between 1.37±0.07 and 2.89±0.06. The 0.7 kb length fragments of hph and GFP were amplified from transfectants. The real time PCR revealed that tri4 was completely suppressed in 30 experimental sets whereas down-regulations of tri5, ranging from 74.5 to 99%, was detected in remaining 6 experiment groups. Thin layer chromatography assay also showed that siRNA-transfected Fusarium samples showed no deoxynivalenol spots. Interpretation: This study revealed that tri4 and tri5 genes can be targeted in the development of less deoxynivalenol (DON)-producing strains through RNAi. Moreover, current study has great importance as it demonstrates that quelling can be used as a potential strategy in controlling diseases caused by phytopathogenic species.
ABSTRACT
Aim: To establish a HEK-293T cell line model stably expressing TRPA1 channel, and to verify the successful establishment of the model. Methods: The eukaryotic expression plasmid of TRPA1 was constructed and transfected into HEK-293T cells by liposome transfection method, the stable expression strain was screened by G418, and the transcription and protein expression of TRPA1 gene in HEK-293T cells were detected by RT-PCR and immunohistochemical techniques. Results: After restriction enzyme digestion and sequencing, it proved recombinant cloning plasmid of TRPA1 gene was successfully constructed; the results of PCR and immunohistochemistry showed that this recombinant plasmid could be transferred into HEK-293T cells with TRPA1 gene stable expression. Conclusions: A HEK-293T cell line with stable expression of TRPA1 channel is successfully constructed, which lays the foundation for studying the physiological and pathological functions of TRPA1 in vitro and screening of relevant TR-PA1 channel regulators.