Construction and identification of ST8Sial gene over-expression vector and its effect on proliferation of human melanoma cells / 中国药理学通报

Aim To construct different over-expression vectors of ST8Sial gene and establish a cell line with stable ST8Sial over-expression , and to check the proliferation of cells with ST8Sial over-expression. Methods Polymerase chain reaction ( PCR) was used to amplify the code region of ST8Sial. The amplified ST8Sial fragment and pEGFP-Cl vector, pHBLV-CMV-MCS-3flag-EFl-ZsGreen-T2A-Puro vector were digested with restriction enzymes. The target gene fragment and the different vectors were ligated to obtain the ST8Sial-pEGFP-Cl recombinant vector and the recombinant lentiviral vector, respectively. PCR and DNA sequencing were used to identify recombinant vectors. Melanoma cells WM451 were transfected with different vectors. Cell line with stable ST8Sial over-expressing was established. ST8Sial mRNA level was checked by real-time PCR. STSSial protein expression level was checked by Western blot. Proliferation of the stable cells was assayed by CCK-8 method. The clony formation of stable cells was also performed. Results Both ST8Sial-pEGFP-Cl recombinant plasmid and STSSial over-expression lentiviral vectors were successfully constructed. The transfection efficiency of ST8Sial over-expression lentiviral vector was much higher than that of ST8Sial-pEGFP-Cl recombinant plasmid. A WM451 cell line with stable ST8Sial over-expression lentiviral vectors was established. Results showed that the over-expression of ST8Sial promoted the proliferation and colony formation of cells. Conclusions ST8Sial over-expression vectors are successfully constructed. The over-expression of ST8Sial promotes the proliferation and colony formation of WM451 cells.

Over-expressing root-specific β-amyrin synthase gene increases glycyrrhizic acid content in hairy roots of glycyrrhiza uralensis / 中草药·英文版

Objsective: Glycyrrhizia uralensis, one of the most widely-used traditional Chinese medicines, is mainly cropped in China. However, many cultivars are less in glycyrrhizic acid than Chinese Pharmacopoeia requires. In this paper, we improved glycyrrhizic acid by regulating β-amyrin synthase gene (GuBAS). Methods: Tobacco root-specific promoter TobRB7 and GuBAS cDNA were obtained and combined with linearized pCAMBIA1305.1 to construct root-specific plant expression vector which was later transformed into Agrobacterium rhizogenes ACCC10060 by electrotransformation. The cotyledons and hypocotyls of G. uralensis were infected by the recombinant A. rhizogenes ACCC10060 to induce hairy roots. The GA content was quantified by HPLC. Results: The PCR and sequencing results both showed that three transgenic hairy root lines were obtained. The copy number of GuBAS in these transgenic hairy roots was intended by qRT-PCR to be 3, 7, and 4. GA was detected by HPLC, and the results showed that GA was present in the three transgenic hairy roots, while absent in wild hairy roots. Conclusion: Over-expressing GuBAS root-specifically in hairy roots of G. uralensis enhanced GA accumulation.

Construction and screening of fibroblast-like synoviocytes in human rheumatoid arthritis stably overexpressing lncRNA HOTAIR / 医学研究生学报

Objective　The HOTAIR gene is closely related to pannus formation in rheumatoid arthritis (RA). This study aimed to construct and screen fibroblast-like synoviocytes in human RA (HFLS-RA) stably overexpressing lncRNA HOTAIR, and to pave the way for further study of the role of lncRNA HOTAIR in the pathogenesis of RA.　Methods　LncRNA HOTAIR was cloned and linked to the PMT406 vector digested by BamHI-HF-HF and XhoI. The constructed plasmids were sequenced, identified and then transfected into 293T cells to pack lentivirus. The HFLS-RA cells were infected with the recombinant and empty vector lentiviruses, and purinomycin was employed to screen the lncRNA HOTAIR-overexpressed and control cell lines. The total RNA was extracted from the blank, negatively transfected and overexpressed cells by Trizol, and the cDNA obtained by reverse transcription was amplified by qPCR, followed by determination of the expression of lncRNA HOTAIR.　Results　The relative expression of lncRNA HOTAIR was significantly higher in the overexpression group than in the blank control and negative transfection groups (30.329 ± 3.860 vs 1.001 ± 0.048 and 0.892 ± 0.247, P 0.05).　Conclusion　The HFLS-RA cell line stably overexpressing lncRNA HOTAIR was successfully constructed, which has provided some experimental evidence for further investigation of the role of lncRNA HOTAIR in the pathogenesis of RA.

Effects of 3β-HSD gene silence or overexpression on DEHP induced oxidative damage in MCF-7 cells / 中华劳动卫生职业病杂志

Objective@#To study the oxidative damage of di- (2-ethylhexyl) phthalate (DEHP) on MCF-7 cells, and to investigate the effects of 3β-hydroxysteroid dehydrogenase (3β-HSD) gene silence or overexpression on DEHP-induced oxidative damage.@*Methods@#MCF-7 cells, 3β-HSD gene silencing cells and 3β-HSD gene overexpression cells were treated with different doses of DEHP (0,0.05,0.1,0.2,0.4,0.8 mmol/L) for 24h, then intracellular oxidative damage index such as MDA, SOD, GSH, GSH-PX were detected, DNA repair gene hOGG1, hMTH1 mRNA expression were tested by Q-PCR, hOGG1, hMTH1 protein expression were detected by western blot.@*Results@#After MCF-7 cells were treated by DEHP, MDA levels increased; SOD activity, GSH content, GSH-PX activity decreased, hOGG1 and hMTH1 mRNA expression levels increased, hOGG1 and hMTH1 protein expression levels increased, the differences were statistically significant when compared with control (P<0.05 or P<0.01) . In 3β-HSD gene silencing cells which were treated by DEHP, when compared with the same dose group of MCF-7 cells, MDA content increased, SOD activity, GSH content, GSH-PX activity decreased, hOGG1 and hMTH1 mRNA expression levels decreased, hOGG1 and hMTH1 protein expression levels decreased, the difference were statistically significant (P<0.05 or P<0.01) . In 3β-HSD gene overexpression cells which were treated by DEHP, when compared with the same dose group of MCF-7 cells, MDA content decreased; SOD activity, GSH content, GSH-PX activity increased, of hOGG1 and hMTH1 mRNA expression levels increased, hOGG1 and hMTH1 protein expression levels increased, the difference were statistically significant (P<0.05 or P<0.01) .@*Conclusion@#DEHP could cause oxidative damage in MCF-7 cells, induce the changes of related genes and proteins, 3β-HSD plays an antioxidant role in the process of DEHP ox-idative damage.

Over-expression of PGC-1α reverses mitochondrial function reduction and apoptosis in OGD/R-induced neurons / 中国病理生理杂志

AIM:To investigate the effect of over-expression of peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α) on mitochondrial morphology and cell apoptosis in the cortical neurons with oxygen glucose depriva-tion/reoxygenation(OGD/R). METHODS:The whole gene sequence of PGC-1α was obtained from the cerebral cortex of C57BL/6 mice by RT-PCR and cloned into the eukaryotic expression vector pEGFP-N1. The pEGFP-N1-PGC-1α was iden-tified by PCR,and transfected into cortical neurons. The level of PGC-1α expression was identified by Western blot. The cortical neurons transfected with pEGFP-N1 and pEGFP-N1-PGC-1α vectors were treated with OGD/R. The mitochondrial mass,reactive oxygen species (ROS) and ATP production,cell apoptosis and changes of cleaved caspase-3 were detected by MitoTracker Red staining,flow cytometry,ATP metabolic assay kit and TUNEL. RESULTS:Over-expression of PGC-1α inhibited the decrease in mitochondrial biogenesis capacity and the ROS formation of OGD/R neurons(P<0.05),en-hanced the ability of ATP synthesis (P<0.01),inhibited neuronal apoptosis (P<0.01) and decreased the activation of caspase-3 (P<0.01). CONCLUSION:PGC-1α over-expression inhibits neuronal apoptosis with OGD/R treatment by promoting mitochondrial biogenesis,inhibiting the production of ROS and maintaining mitochondrial function. PGC-1α may be used as a target for the development of cerebral ischemia/reperfusion injury drugs.

Over-expression of ROBO4 reduces permeability of human renal glomerular endothelial cells in high glucose medium / 解放军医学杂志

Objective To investigate the effect of over-expression of ROBO4 on permeability of human renal glomerular endothelial cells (HRGECs) in high glucose medium.Methods HRGECs infected with recombinant lentiviral vector ROBO4 were cultured in high glucose or low glucose medium in vitro.The protein levels of ROBO4 and ARF6 in each group were detected by Western blotting.The endothelial permeability was measured by the effiux of fluorescein isothiocyanate-dextran (FITC-Dextran)permeated through the monolayer endothelial cells using Transwell cell model system.The cell viability after lentivirus transfection was measured by CCK8 assay.Results The transfection rate of lentiviruses in HRGECs reached 80％ 72h after,and obvious overexpression of ROBO4 protein was in transformed cells compared with the empty vector group (P＜0.05).The lentivirus-mediated ROBO4 transfection did not affect cell viability of HRGECs.Compared with the low glucose group,the expression of ROBO4 increased obviously after 12h,but declined after 24h (P＜0.05),and reached to minimun after 72h (P＜0.05).On the contrary,the expression of ARF6 increased after 12h,and the increase reached to the maximum after 72h (P＜0.05).Furthermore,the vascular permeability increased gradually after 24h,and reached to the maximum after 72h (P＜0.05) in high glucose group.Compared with the empty vector group,the over-expression of ROBO4 inhibited the expression of ARF6 significantly,and the FITC-Dextran permeability reduced obviously.Conclusion Over-expression of ROBO4 may significantly enhance the barrier functions of HRGEC in high glucose medium,and ROBO4 activation may be a potential therapeutic approach in diabetic nephropathy.

Over-expression and suppression of HMGBI affect proliferation and invasion of endometrial carcinoma and underlying mechanisms / 中南大学学报(医学版)

Objective:To investigate effects of over-expression and suppression of HMGB1 on proliferation and invasion of endometrial carcinoma HEC-1A cell and underlying mechanisms.Methods:Over-expression or silence of HMGB 1 in HEC-1A cell lines were established by lentiviral vector containing HMGB 1 recombinant plasmid or by HMGB 1 shRNA,respectively.Cell counting kit-8,Transwell,and wound healing assay were used to analyze proliferation,invasion,and migration of HEC-1A cells,respectively.Western blot and reverse transcription-PCR (RT-PCR) were used to detect the expression of NF-κB,VEGF,and matrix metalloproteinase 2 (MMP2) in the cells.Results:Over-expression of HMGB1 promoted the proliferation,invasion,and migration of HEC1A cell,and up-regulated NF-κB,VEGF,and MMP2 expressions,while suppression of HMGB1 inhibited the proliferation,invasion,and migration of HEC-1A cells and down-regulated NF-κB,VEGF,and MMP2 expressions.Conclusion:HMGB1 plays an important role in proliferation,invasion,and migration of HEC1A cell via NF-κB/VEGF/MMP2 pathway.HMGB 1 might be a potential target for endometrial carcinoma therapy.

The effect of CRELD1 gene on the related genes in endocardial cushion development / 中华实用儿科临床杂志

Objective To investigate the effect of alteration of CRELD1 gene expression on the related genes in the endocardial cushion development.Methods Over-expression and silence of CRELD1 gene were realized by the construction of lentiviral vector.Afterwards,the lentiviral vectors were used to infect the human fetal lung fibroblasts (HFL)-I.All of the cells were divided into the following 5 groups:the blank control group,the negative control group of interference,the interference group,the negative control group of over-expression,and the over-expression group.Western blot and real-time fluorescent quantitative polymerase chain reaction were applied to examine the mRNA and protein expression of CRELD1,Sox9,Aggrecan,Scleraxis and Tenascin-C.Results The DNA sequences of 2 recombinant plasmids pLV3-shRNA-CRELD1 and pLV4-CRELD1 matched very well with those which were designed according to the DNA sequence analysis.HFL-I was successfully infected with lentiviral vectors and displayed fluorescent green light under inverted fluorescence microscope.The results of real-time PCR detection and Western blot test were consistent:expressions of Sox9 and Aggrecan in the interference group were significantly higher than those in the negative control group of interference,while the expressions of the 2 genes in the over-expression group were significantly lower than those in the negative control group of over-expression.Expressions of Scleraxis increased in both the interference group and the over-expression group when compared with the negative control groups respectively.Compared to the corresponding negative control groups,Tenascin-C expression decreased markedly in the interference group,whereas it increased significantly in over-expression group.Conclusions CRELD1 gene has negative effect on the expression of the related genes Sox9 and Aggrecan in the endocardial cushion development,whereas it has positive effect on the Tenascin-C expression.It serves as a theoretical framework to illustrate the effect of CRELD1 gene on the atrioventricular septal defect.

Construction of a eukaryotic expression vector of fibroblast activation protein and establishment of its stable over-expression in the oral squamous cell carcinoma / 华西口腔医学杂志

<p><b>OBJECTIVE</b>This study aimed at constructing fibroblast activation protein (FAP) over-expression lentivinus vectors to investigate transfection in SCC9 cell lines and establish a stable FAP over-expression oral squamous cell line.</p><p><b>METHODS</b>The cDNA of FAP gene from an oral squamous cell carcinoma (OSCC) tissue was amplified by polymerase chain reaction (PCR) and subcloned into eukaryotic expression vector pCDH-CMV-MCS-EF1-copGFP. The recombinant plasmid was sequenced and then transfected into an SCC9 cell line. Subsequently, the SCC9 cell line that over-expressed FAP stably was established by fluorescence activated cell sorting (FACS). The expression of green fluorescent protein (GFP) was detected with fluorescence microscopy, and the over-expression of FAP was identified by real-time PCR and Western blot.</p><p><b>RESULTS</b>The FAP gene was amplified by PCR and then cloned into the vector, whose sequence was identical to that in the GenBank. GFP was expressed in the transfected cells. Furthermore, FAP over-expression in the transfected cells was detected by real-time PCR and Western blot.</p><p><b>CONCLUSIONS</b>The recombinant eukaryotic expression vector pCDH-FAP was constructed successfully. This result provides a foundation for further studies on the function of FAP in vitro.</p>

The expression of CREB in the Glioma / 中国神经精神疾病杂志

Objective To investigate the clinical significance and abnormal expression of the CREB in different grade gliomas. Methods The expression of CREB was examined by using immunohistochemistry in brain tissues from the brain injury (5 cases) and different grade gliomas (55 cases).The mRNA and protein levels of CREB were further as?sessed using Western blot and RT-PCR in brain tissues from the patients with brain injury (10 cases) and those with dif?ferent grade gliomas (30 cases). Results The positive rates of CREB immunohistochemistry were 2/5 in control, 10/15 inⅠ-,Ⅱ11/12 in Ⅲ, 28/28 in Ⅳ. The positive rates of CREB were significantly different among different groups (H=28.183,P<0.05).The mRNA levels of CREB were 1.00 ± 0.000 in control, 1.35 ± 0.068 inⅠ-Ⅱ, 2.88 ± 0.111 in Ⅲand 3.75 ± 0.196 in Ⅳ. The expression of CREB was higher in the glioma than in control group, and the mRNA levels of CREB were significantly different among different groups(F=1.208,P<0.05). The protein levels of CREB were 0.311 ± 0.014 in control, 0.469±0.026 inⅠ-Ⅱ, 0.641±0.028 inⅢand 0.896±0.024 inⅣ. The protein levels of CREB were sig?nificantly different among different groups(F=1.123,P<0.05). Conclusion The expression of CREB is elevated in glio?mas with different differentiation degrees. The expression of CREB was positively correlated with the degree of differentia?tion, indicating that CREB may have an important regulatory role in the progress of gliomas.

Establishment and identification of HUVEC cell strains with over-expression and low expression of RACK 1 / 基础医学与临床

Objective To establish several human umbilical vein endothelial cell ( HUVEC ) strains with over-ex-pression or low expression of receptor for activated C kinase 1 ( RACK1 ) , which will provide an effective tool for future studying the function of RACK1 in arrhythmia.Methods The full-length cDNA sequence of RACK1 gene was amplified and inserted into pIRES2-EGFP.At the same time, designed and synthesised complementary DNA sequences of 3 pairs of short hairpin structure and a pair of negative control sequence , then subcloned into the plas-mid pGenesil-1 .The HUVEC cells were transfected with these plasmids and screened by using G 418 .And the expression of RACK1 mRNA and protein in the cells were assayed by qRT-PCR and Western blot , respectively . Results RACK1 eukaryotic expression vector and siRNA expression vectors of RACK 1 were constructed success-fully.After a 48 h transfection of HUVEC cells with the recombinant vectors and G 418 selection, the positive cell clones were obtained .qRT-PCR and Western blot showed that over-expression vector and interference vectors could effectively enhanced and knocked-down RACK1 expression in HUVEC strains .Conclusions HUVEC cell strains with over-expression and low expression of RACK 1 have been successfully established .

Effects of CDT1 gene over-expression on cell apoptosis and cell cycle in irradiation-induced genomic instable liver cells / 中华放射医学与防护杂志

Objective To investigate the effect of CDT1 gene over-expression on the apoptosis and cell cycle distribution in liver cells with a characteristic of genomic instability induced by radiation(GIR).Methods Lentivirus particles were transferred into liver cells of GIR to up-regulate the expression of CDT1 gene.The apoptosis and the cell cycle were detected by flow cytometry (FCM).The expression changes of p53,ATM,ATR,Bcl-2,and Caspase-3 genes were analyzed by real-time fluorescence quantitative PCR.Results CDT1 gene was efficiently increased by the gene transfection(t =15.56,P ＜ 0.05).In the CDT1 over-expressed cells,while the apoptosis ratio was increased (t =4.19,P ＜ 0.05),the expressions of p53 and Bcl-2 gene were decreased (t =-4.21,-2.06,P ＜ 0.05),but the expression of ATM,ATR and Caspase-3 changed with no significant difference compared with control.Conclusions Over-expression of CDT1 could regulate genomic instability through apoptosis pathway and checkpoint independent of p53.

Enhancing ergosterol production in Pichia pastoris GS115 by overexpressing squalene synthase gene from Glycyrrhiza uralensis / 中国天然药物

The present study was designed to determine the effects of copy number variations (CNVs) of squalene synthase 1(SQS1) gene on the mevalonate (MVA) pathway. SQS1 gene from G. uralensis (GuSQS1) was cloned and over-expressed in Pichia pastoris GS115. Six recombinant P. pastoris strains containing different copy number of GuSQS1 were constructed. HPLC was used to assay the level of ergosterol in all transgenic P. pastoris strains containing GuSQS1. HPLC analysis showed that the contents of ergosterol in all of the transgenic P. pastoris containing GuSQS1 were higher than that in the negative control. And with the increase of copy number of GuSQS1, the content of ergosterol showed an increasing-decreasing-increasing pattern. The contents of ergosterol in 10-copy-GuSQS1 P. pastoris and 47-copy-GuSQS1 P. pastoris were significantly higher than that in the rest recombinant P. pastoris strains. In conclusion, the CNVs of GuSQS1 influence the content of secondary metabolites in the MVA pathway. The present study provides a basis for over-expressing GuSQS1 and increasing the content of glycyrrhizin in G. uralensis cultivars.

Construction of C3a secrectory expression vector and tubular epithelial cell line stably expressing C3a / 医学研究生学报

[Abstract ] Objective Evidence from previous studies indicated that over-activation of C3a/C3aR axis existed in the renal tubular epithelial cells of patients with renal diseases including diabetic nephropathy .However , the pathological significance of over-ac-tivating C3a/C3aR axis still remains to be elucidated .In this study, we constructed a renal tubular epithelial cell line over-expressing C3a in a secretory manner in order to provide a cell model to investigate the pathological significance of over -activating C3a/C3aR axis under various pathological scenes . Methods We designed a synthesized C3a secretory expression unit and cloned it into the multi-clonal site of lentivirus expression vector pLenti 6.3-MCS-IRES2-EGFP.After identification by sequencing , recombinant lentivirus was packaged by using pLenti 6.3-C3a-IRES2-EGFP and packaging plas-mid in 293T cells.Then, the recombinant lentivirus was used to in-fect HK2, a cell line of human renal tubular epithelial cells .After screening in medium with blasticidin , blasticidin resistant cell clones were obtained .Real-time PCR and ELISA method were applied to analyze the expression and secretion of stable transfected cells cloned C3a and identify renal tubular epithelial cell lines with stable over-activating C3a. Results ①C3a secretory expression unit was suc-cessfully synthesized and correctly cloned into the multi-clonal site of pLenti6.3-IRES2-EGFP; ②C3a secrectory expression recombi-nant lentivirus LV-C3a was successfully packaged with a high titer of 5 ×108/mL;③HK2 Cell clones resistant for blasticidin were ob-tained;according to the analysis of Real-time PCR and ELISA, the C3a mRNA level in HK2-C3a cell lines was significantly higher than that of HK2 cells(1.0 ±0.5 vs 1321.0 ±18.0, P<0.01) and the secreted C3a level increased significantly ([0.3 ±0.2]ng/mL vs [249.0 ±37.0] ng/mL, P<0.01). Conclusion The present study successfully constructed C 3a secretory expression vector pLenti6.3-C3a-IRES2-EGFP and C3a over-expression renal tubular epithelial cell line HK 2-C3a, which is very useful in further study of the function and significance of C 3a/C3aR axis not only in renal tubular epithelial cells but also in other cell types .

Hormone Receptor-negative Metastatic Breast Cancer Presented as Cancer from an Unknown Primary Site / 대한내과학회지

A 37-year-old woman was admitted to our hospital because of cervical and axillary lymphadenopathy that developed after delivery. An axillary lymph node biopsy revealed metastatic adenocarcinoma. Immunohistochemical staining indicated that the tumor cells expressed c-ErbB-2, but were negative for the estrogen and progesterone receptors. No definite evidence of breast cancer was detected. The patient underwent chemotherapy for suspected metastatic breast cancer. She complained of swelling in the left breast 22 months later, and a biopsy showed invasive ductal carcinoma. Here, we report a case of hormone receptor-negative occult breast cancer in a patient with cervical and axillary lymphadenopathy presenting as a cancer with an unknown primary site.

The research progress of Sox2 gene in malignant tumor / 实用肿瘤学杂志

Sox2 is one of the important members of the Soxfamily ,It plays an import role in the regulation of early embryo and normal tissue development ,maintain the versatility of stem cells and progenitor cells self -re-newal ability and decide cell fate ,etc.Sox2 genetic mutation or missing may lead to dysplasia or congenital disea-ses.Previous studies showed that Sox 2 expressed in a variety of malignant tumors ,and associated with the inci-dence of malignant tumor ,lymph node metastasis and pathological grading and clinical staging .There fone Sox2 is considered as a potential carcinogenic factor ,its over expression may form one of development of mechanisms for the occurrence and different kinds of malignant tumor .

Construction of retrovirol vector containing novel gene mgt-16 and its expression in mouse mesenchymal stem cells / 第二军医大学学报

Objective To construct the retroviral vector carrying novel gene mgt-16 and to investigate its expression in mouse mesenchymal stem cells C3H/10T1/2(10T1/2). Methods DNA sequences containing mouse novil gene mgt-16 was used as a template for PCR amplification of full length mgt-16 cDNA. Then the DNA fragment was cloned into pEGFP-N1 vector to produce pEGFP-N1-16 vector after T-A cloning with pMD18T plasmid and sequencing. The pEGFP-N1-16 vector was confirmed by PCR, restriction enzyme digestion and sequencing analysis. The retroviral vector, pLEGFP-N1-16, was constructed using retroviral vector, pLEGFP-N1, and pEGFP-N1-16 vector including mgt-16 gene. The pLEGFP-N1-16 vector was verified by restriction enzyme digestion, sequenced, and then transfected into packaging cell line Phoenix to prepare EGFP fused mgt-16 retrovirus particles, whichwere collected and used to infect 10T1/2 cells. G418 (400 (μg/mL) continuous selection was conducted to obtain 10T1/2 cell clones stably overexpressing EGFP fused mg-16. Fluorescence microscope was employed to determine the expression and subcellular localization of MGT-16 in Phoenix and 10T1/2 cells. Results A band of about 300 bp size was observed by agarose gel electrophoresis after PCR amplification for mgt-16 gene, and the result of sequencing showed that the sequence of insert fragment in T-A clones was identical to mg-16 gene reported in Genbank. PCR, restriction enzyme digestion and sequencing revealed that the pEGFP-Nl-16 plasmid was successfully constructed. Restriction enzyme digestion and sequencing revealed that the pLEGFP-Nl-16 plasmid was also successfully constructed. Phoenix and 10T1/2 cells overexpressing MGT-16 showed green fluorescence distribution in thecytoplasmic, especially around the perinucleararea. Conclusion We have successfully constructed a recombinant retroviral vector carrying novll gene, mg-16, and expressed it in mouse mesenchymal stem cells, which provides a basis for studying the role of novel gene mg-16 in mesenchymal stem cells.

Effect of galectin3 on proliferation and migration of esophageal cancer Eca109 cells / 国际肿瘤学杂志

Objective To investigate galectin3 on proliferation and migration of esophageal cancer Eca109 cells.Methods A lentiviral vector for over-expression of RNA targeting galectin3 was designed to transfect Eca109 cancer cells following plasmid-mediated transfection manual (Eca109/Gal3 cells).Inverted fluorescence microscope was used to observe the expression of EGFP.The proliferation of Eca109 cells was measured by cell counting Kit-8 assay.Eca109 cells apoptosis was determined by Annexin-V/7-AAD doublestaining.The migration capacity of Eca109 cells was determined in transwell assays.Western blot analysis was used to measure the expression of galectin3 protein.Results Galectin-3 expression was detected in Eca109 cells,with the Galectin3 expression in Eca109/Gal3 cells much more than non-transfected cells (t =14.33,P ＜ 0.05 ; t =10.28,P =0.037).Compared with non-transfected Eca109 cells,proliferation increased significantly in Eca109/Gal3 cells (t =-17.277,P ＜ 0.05 ; t =-13.4,P ＜ 0.05).Galectin3 evidently decreased in Eca109 cell apoptosis (t =3.053,P ＜ 0.05 ; t =5.446,P ＜ 0.05).Transwell migration assay showed that a greater number of Eca109/Gal3 cells crossed the artificial basement membrane compared with non-transfected Eca109 cells and negative control Eca109 cells (t =3.465,P ＜ 0.05; t =3.252,P ＜ 0.05).Conclusion Galectin3 expression is detected in transfected esophageal cancer Eca109 cells,whose overexpression can result in enhanced proliferation,migration,invasion as well as reduced apoptosis.These data indicate that in-depth research of galectin-3 may prove to be a potential molecular target for the treatment of esophageal cancer.

Construction of lentiviral vector containing mouse aqua porin-1 gene and its expression in Schwann cells / 第二军医大学学报

Objective To construct a lentiviral vector carrying mouse aquaporin-1 (AQP1) gene and use it for infecting Schwann cells of C57BL/6 mouse, so as to provide AQP1 + Schwann cells for further studying the relationship of AQP1 with peripheral nerve system injury edema. Methods Mouse AQP1 gene was inserted into lentiviral vector pCDH-CMV-MCS-EFl-copGFP, and the products were confirmed by PCR and sequencing analysis. pCDH-CMV-MCS-EFl-copGFP-AQPl and the virus packaging plasmids psPAX2 and pMD were cotransducted into 293T cells to prepare lentivirus-AQPl, and the latter was used to infect C57BL/6 mouse Schwann cells in vitro. The expression of AQP1 mRN A and protein was detected by quantitative real-time PCR and Western blotting analysis in infected mouse Schwann cells. Results PCR and sequencing revealed that the pCDH-CMV-MCS-EFl-copGFP-AQPl plasmids were successfully constructed. The expression of AQP1 mRNA and protein in Schwann cells was significantly increased than that in cells infected with control Antiviruses (P<0. 05). Conclusion We have successfully constructed a recombinant lentivirus carrying AQP1 gene, which can be used to infect mouse Schwann cells, leading to increase of AQP1 mRNA and protein expression.

Biochemical study of CymJ, the putative regulator of cyclodextrin metabolism in Klebsiella oxytoca M5a1.

The products of ten genes clustered in two divergently oriented operons are required for the metabolism of cyclic oligoglucosides, the cyclodextrins, by Klebsiella oxytoca. The function of CymJ, the product of the promoter distal gene in one of the operons was studied. Over expression of cymJ in K. oxytoca led to strong reduction of the expression of the cym operons. This repression could be alleviated by addition of high concentration of α-cyclodextrin into the medium. There is a possible relationship between CymJ and CymD since the absence of CymD in a CymD deletion mutant prevented the repression effect of CymJ. An intriguing finding was that the presence of CymJ in large amount in the cell caused severe cell division inhibition leading to retardation of growth. This morphological change was paralleled by a significant increase in the susceptibility of K. oxytoca to ampicillin.