Objective To construct small interfering RNA（siRNA） eukaryotic expression vector specific for human hnRNP K gene，and to observe its silencing effects on hnRNP K gene in A549 cells.Methods The expression vectors of pSUPER/hnRNP K siRNAa，pSUPER/hnRNP K siRNAc and pSUPER/siRNAn were constructed by gene recombination and then transfected into the A549 lung carcinoma cell line by using Lipofectamine2000（a and c respectively represented A and C fragments in hnRNP K coding sequence contained 19 nts，n represented nonsense fragment as control）.The mRNA and protein were harvested after 24 h and analyzed for the expression of hnRNP K by RT-PCR and Western blotting respectively.Results The siRNA vector targeted to hnRNP K successfully decreased hnRNP K mRNA and protein levels 24 h after transfection in A549 cells.Relative expressed doses of hnRNP K mRNA in lung cancer cells transfected by hnRNP K siRNAa and hnRNP K siRNAc respectively were 0.24±0.53 and 0.28±0.57 after 24 h，which were significantly lower than that in the control group（both Plt;0.01）.The gray scale values of hnRNP K protein were 0.23±0.11 and 0.28±0.09 respectively，which were also significantly lower than those in the control group（both Plt;0.05）.And pSUPER/hnRNP K siRNAa was the most effective one.Conclusion Eukaryotic expression vector of siRNA specific for hnRNP K is successfully constructed，which lays the basis for the function study of hnRNP K gene and its application in the treatment of lung carcinoma.
OBJECTIVE: To construct eukaryotic expression vector of rat myogenin gene for further study on its functions in skeletal muscle denervated atrophy and repair. METHODS: The cloning vectors (containing full length of myogenin cDNA and two restriction sites: Hind III and Xho I) were first cut by two restriction endonuclease: Hind III and Xho I, and the same as the eukaryotic expression vector; then, the myogenin cDNA and the digested vector were ligated by T4 DNA ligase, and recombinant eukaryotic expression vector was formed. Its length was certificated by agarose gel electrophoresis analysis, digestion with Hind III and Xho I, PCR; and the rightness of the myogenin cDNA sequence was confirmed by sequencing. RESULTS: The results of agarose gel electrophoresis analysis, digestion, and PCR confirmed the right length of inserted DNA, which was the same as the myogenin cDNA, and the sequencing result of pcDNA3-myogenin was identical with the reported. CONCLUSION: pcDNA3-myogenin a eukaryotic expression vector, is successfully constructed.
Objective To construct the eukaryotic expressive vector of human tissue factor （TF），and to abserve the effect of TF on invasion and metastasis of gastric cancer cells line. Methods The human TF cDNA was obtained from human placenta by nest PCR, and the constructed eukaryotic expressive vector TF-pcDNA3 was transfected into SGC7901 cells by lipofectamine. Stable-transfected cells were screened by G418. The expressions of TF mRNA and protein on the cells were detected by RT-PCR and Western blot. Cell motility was assessed by using Transwell experiments and wound-healing assays. Results The eukaryotic expressive vector TF-pcDNA3 was successfully constructed and transfected into SGC7901. Compared with blank control group and negative control group, the expressions of TF mRNA and TF protein in transfection group were increased, the cell motility in vitro was enhanced. Conclusion TF can enhance the ability of invasion and metastasis of gastric cancer cells in vitro.
Objective To research the transfer of adenovirus human bone morphogenetic protein 4 (Ad-hBMP-4) to human degenerative lumbar intervertebral disc cells in vitro and analyze its effect on the proteoglycan, collagen type II, and Sox9 of intervertebral disc cells. Methods Identified Ad-hBMP-4 was amplified and detected. Degenerative lumbar intervertebral disc cells were aspirated from the degenerative lumbar intervertebral disc of patients with Modic III level disc protrusion (aged, 27-50 years). The expressing position of collagen type II was identified in the intervertebral disc cells through the laser confocal microscope. The intervertebral disc cells at passage 1 were transfected with Ad-hBMP-4 as experimental group. After 3 and 6 days of transfection, RT-PCR was used to detect the mRNA expressions of proteoglycan, collagen type II, and Sox9, and Western blot to detect the expressions of proteoglycan and collagen type II proteins. Non-transfected cells at passage 1 served as control group. Results The virus titer of Ad-hBMP-4 was 5 × 106 PFU/mL. No morphological changes in the cells after transfection by Ad-hBMP-4. Collagen type II mainly expressed in the cell cytoplasm. The mRNA expressions of the proteoglycan, collagen type II, and Sox9 in experimental group at 3 and 6 days after transfection were significantly higher than those in control group by RT-PCR (P lt; 0.05), and the expressions of proteoglycan and collagen type II proteins were significantly higher than those in contorl group by Western blot (P lt; 0.05). There were significant differences between 3 days and 6 days in experimental group (P lt; 0.05). Conclusion Ad-hBMP-4 could transfect human degenerative lumbar intervertebral cells with high efficiency and promote collagen type II, proteoglycan, and Sox9 expressions. hBMP-4 may play an important role in the repair process during early disc degeneration.
Abstract: Objective To generate a eukaryotic expression plasmid-pcDNA3.1/human tissue inhibitor of metalloproteinase-1(hTIMP-1)enhanced green fluorescent protein (EGFP), carrying hTIMP-1 and labeled with EGFP, and to examine the expression of hTIMP-1 in vascular smooth muscle cells (SMCs) transferred with hTIMP. Methods The recombinant plasmids of pcDNA3.1/hTIMP-1-EGFP were obtained bypolymerase chain reaction (PCR) amplification, splicing, and insertion of complementary deoxyribonucleic acid (cDNA) fragments of hTIMP-1 and EGFP. The target gene was transferred to the primarily cultured SMCs (pcDNA3.1/hTIMP-1-EGFP transferred group) by using cationic liposome mediated gene transfection technique. EGFP expression was detected by fluorescence microscopy, and the transfection rate was determined by flow cytometry. Reverse transcriptase polymerase chain reaction (RTPCR), Western blotting, and other techniques were used to detect the expression of hTIMP-1 gene. The biological activity of matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9(MMP-9) were studied by zymographic analysis of gelatinases. Blank plasmidpcDNA3.1 transferred SMCs (blank plasmid pcDNA3.1 transferred group) and untransferred SMCs (untransferred group) were used as control. Results In cDNA3.1/hTIMP-1-EGFP transferred group,the growth ability of SMCs was profoundly inhibited, bright green fluorescence was observed by fluorescence microscopy 24 hours after transfection in SMCs,the rate of transfection analyzed with flow cytometry was 15%,RT-PCR results showed that the genome of hTIMP-1 transferred SMCs contained a 646 bp specific fragment of hTIMP-1 gene, Western blotting results proved hTIMP-1 protein expression in SMCs transferred by hTIMP-1, zymographic analysis of elatinases showed decreased activity of MMP-2 and MMP-9, compared to those in blank plasmidpcDNA3.1 transferred group and untransferred group, significant differences were observed (Plt;0.05). Conclusion The generation of a eukaryotic expression plasmid carrying TIMP-1 gene and its expression in SMCs provide a sound basis for hTIMP-1 gene therapy.
Objective To study the effect of knockdown of signal transducer and activator of transcription 3 (STAT3) expression by short hairpin RNA (shRNA) on proliferation, apoptosis and invasion of human gastric cancer cell line MKN-45 in vitro . Methods Specific shRNA plasmids to STAT3 were constructed, and then transfected into MKN-45 cells by lipofectamine methods. Cells were divided into three groups: control group, psiRNA-H1 transfected group as negative group and psiRNA-H1/STAT3 transfected group. Semi-quantitative RT-PCR and Western blotting were used to detect the expression of STAT3 mRNA and protein, respectively. Proliferation and apoptosis of the transfected cells were observed by methyl thiazolyl tetrazolium (MTT) method and flow cytometry (FCM), respectively. The invasion of the transfected MKN-45 cells was measured by Boyden chamber. Results Compared with the negative control cells, semi-quantitative RT-PCR and Western blotting showed that the expressions of STAT3 mRNA and protein were down-regulated in the psiRNA-H1/STAT3 transfected group ( P ＜ 0.05) . The subcloned recombinant plasmid expressing shRNA effectively inhibited MKN-45 cell growth and proliferation while empty plasmid had no such specific effect. Cell apoptosis rate increased significantly in psiRNA-H1/STAT3 transfected group （ P ＜ 0.01）, and the invasion of MKN-45 cells was efficiently inhabited in psiRNA-H1/STAT3 transfected group as compared with control group and psiRNA-H1 transfected group( P ＜ 0.01).Conclusion Recombinant plasmid psiRNA-H1/STAT3 shRNA significantly inhibits the proliferation and invasion of MKN-45 cells and promotes their apoptosis.
ObjectiveTo establish a cell inflammation model induced by tumor necrosis factor-α (TNF-α) in human bronchus epithelial cells, and investigate the effects of glutathione S-transferase mu 5 (GSTM5) on the inflammation and oxidative stress. Methods16HBE cells were treated with TNF-α (10 ng/mL, 24 h) in the absence or presence of the constructed GSTM5 eukaryotic expression vector (1 μg/mL). The concentration of malondialdehyde (MDA) and total antioxidation capacity (T-AOC) were detected by colorimetric method. The survival rate of cells was assessed by the methyl thiazolyl tetrazolium (MTT) assay. The transcription level of NADPH oxidase-1 (NOX1), NOX2, NOX3, NOX4, NOX5, dual oxidase-1 (DUOX1) and DUOX2 were evaluated by RT-PCR. Western blot was performed to investigate the protein levels of NOX1 and NOX2. ResultsTNF-α simulation significantly increased the level of MDA in cells, and decreased the level of T-AOC and survival rate of 16HBE. When transfected with the GSTM5 eukaryotic expression vector, the concentration of MDA significantly decreased (P < 0.05), and the activation of T-AOC increased dramatically (P < 0.05). Consequently, the survival rate of 16HBE in the GSTM5 group improved (P < 0.05). The 16HBE cells transfected with the constructed GSTM5 eukaryotic expression vector had a lower transcription and protein levels of NOX1 and NOX2 (all P < 0.01). There were no significant changes in the mRNA expressions of NOX3, NOX4, NOX5, DUOX1 or DUOX2. ConclusionGSTM5 may down-regulate the transcription level of NOX1 and NOX2 to reduce the inflammation and oxidative stress induced by TNF-α.
Objective To clone human bone morphogenetic protein 2 ( BMP-2) gene and construct the gene’s eukaryotic expression vector. Methods The total RNA was extracted from human osteosarcoma cells, the human BMP-2 cDNA was amplified by RT-PCR and inserted into pGEM-T vector. The positive clones were screened out, and the n the recombinant plasmid was confirmed by restriction enzyme digestion, PCR and the analysis of nucleotide sequence. The BMP-2 cDNA in the pGEM-T cloning vec tor was inserted into the pcDNA3.1(+) eukaryotic expression vector. Results The agarose electrophoresis showed that the fragments of BMP-2, pGEMT and pcDNA3.1(+) were 1.2 kbp, 4.0 kbp and 5.0 kbp, respectively. The result of nucleotide sequence confirmed that the cDNA sequence, which was inserted into pGEM-T and pcDNA3.1(+) plasmid was human BMP-2. Conclusion The pcDNA3.1(+)-hBMP-2 eukaryotic vector can be successfully constructed.
Objective To construct the lentiviral vector to co-express enhanced green fluorescent protein (EGFP) gene and human insul in (insulin) gene, and to explore the condition to transfect human umbil ical cord mesenchymal stem cells (hUCMSCs) so as to lay a foundation for tissue engineered adipose reconstruction and transplantation in vivo infuture. Methods The insulin gene was cloned to lentiviral expression vector with EGFP [pLenti6.3-internal ribosome entrysite (IRES)-EGFP] by recombinant DNA technology, the positive clones were screened, and lentiviral packaged systems and target gene plasmid were co-transfected to package virus in 293T cells by lipofectin. The reporter gene expression was observed by fluorescent inverted phase contrast microscope, virus supernatant was collected, purificated and concentrated, and the titer of recombinant viruses was determinated. hUCMSCs from umbilical cord tissue of mature neonates were isolated and cultured by different multiple of infection (MOI, 0, 1, 3, 5, 7, 10, 15, and 20). By recombinant lentiviral infected hUCMSCs with reporter gene green fluorescent protein expression, the best MOI was screened; recombinant lentiviral infected hUCMSCs at the best MOI, then real-time PCR and Western blot methods were appl ied to detect insulin gene and insul in protein expression levels in cells. Results The recombinant lentiviral vector of co-expressing insulin gene and EGFP gene (pLenti6.3-insulin-IRESEGFP) was successfully constructed. Virus could be packaged, purificated and concentrated successfully. The virus titer was 1.3 × 108 TU/mL. The best MOI was 10 and the transfer efficiency was up to 90% in the same time. Real-time PCR results showed that insulin gene expression of transfected group was positive and non-transfected group was negative; Western blot detection confirmed that insul in protein expression of transfected group was positive in cells and supernatant, but that of non-transfected group was both negative. Conclusion Lentiviral vector pLenti6.3-insulin-IRES-EGFP carrying recombinant insulin gene could effectively transfect hUCMSCs and express insul in protein.
目的 构建小鼠甲状腺转录因子-2（TTF-2）转基因动物表达载体（pBROAD3-TTF-2），观察其在小鼠骨髓间充质干细胞（BMSC）中的表达。 方法 从C57BL/6J小鼠肝脏组织中提取基因组DNA，利用聚合酶链式反应方法扩增出TTF-2基因1 113 bp开放阅读框，通过DNA重组技术将TTF-2基因片段插入克隆载体pMD18-T中，经测序正确后，再重组于pBROAD3-mcs中，构建转基因动物表达载体pBROAD3-TTF-2，用酶切电泳分析对其进行鉴定。运用脂质体转染试剂将其转染BMSC后，蛋白质印迹法检测TTF-2基因的表达。 结果 ① DNA测序证实目的基因序列正确无突变，酶切电泳分析得到相应的目的片段，大小与理论计算值一致，成功构建转基因动物表达载体pBROAD3-TTF-2。② 蛋白质印迹法显示转染的BMSC高表达TTF-2蛋白。 结论 成功构建了pBROAD3- TTF-2转基因动物表达载体，显示其转染BMSC后TTF-2基因的表达，为下一步建立TTF-2转基因小鼠模型奠定了基础。