The Expression of the Plasmid DNA Encoding TGF—β1 in Endothelium after Injection into the Anterior Chamber

The method of gene transfer into corneal endothelium was investigated to provide a foundation for the study of TGF-β1 gene transfer to inhibit corneal graft rejection.Two days after direct injection of pMAM TGF-β1 mediated by liposome into the anterior chamber of rabbits,one half of corneas were made into paraffin slides and the endothelial layer was carefully torn from the other half to make a single layer slide of endothelia.By means of immunohistochemical technique,the plasmid pMAM TGF-β1 expression product TGF-β1 in the endothelia was detected.Specific TGF-β1 expression was positive in the endothelia on both the paraffin slide and the single layer slide.The results showed that by direct injection into the anterior chamber,foreign plasmid DNA could be transferred into the endothelia and its expression was obtained.This may provide a foundation for further study on TGF-β1 participating in local induction of corneal immune tolerance.     

Immune response to plasmid DNA encoding HPV16-L1 protein

Objective To test the immunogenicity of recombinant plasmid DNA containing human papillomavirus type 16-L1 (HPV16-L1) coding sequence of mice.Methods The HPV16-L1 encoding sequence was generated by polymerase chain reaction (PCR), and inserted into TA cloning vector PCR Ⅱ, then cloned in the eukaryotic expression vector pcDNA3.1 with CMV promoter. The recombinant plasmid DNA pcDNA-L1 was transferred into Cos-7 cells and used to immunize BALB/c mice via muscular injection. The expression of HPV16-L1 in transferred cells was identified by immunospot and immunocytochemistry, which tested specific anti-HPV16-L1 antibody in the serum of immunized mice. Results Using the immunospot technique, we found L1 protein expression in pcDNA-L1 transferred cells. The immunocytochemistry studies demonstrated that the L1 protein was located in nuclei. In immunized mice, specific anti-HPV16-L1 antibodies could be detected by immunospot and immunocytochemistry 28 days after the first immunization and last at least 41 days. Conclusions We constructed HPV16-L1 eukaryotic expressing plasmid whose DNA could induce immuno-humoral response in mice. This observation will be helpful in designing HPV16 prophylactic vaccine.     

CONSTRUCTING AAV-TGFβ1 AND COMPARISING ITS BIOLOGICAL EFFECTS ON PROTEOGLYCAN SYNTHESIS OF NUCLEUS PULPOUS CELLS WITH AV-TGFβ1

Objective To construct adeno-associated virus express system for TGFβ1 （AAV-TGFβ1） and compare its biological effects on proteoglycan synthesis of the rabbit lumbar disc nucleus pulpous （NP） cells with adenovirus （Ad） express system for TGFβ1 （AV-TGFβ1）. Methods TGFβ1 gene was obtained by polymerase chain reactions （PCR）. The upstream of TGFβ1 contained restriction enzyme site of EcoR Ⅰ, and the restriction enzyme site of Sal Ⅰ was at the downstream of TGFβ1. Using the multiple cloning sites （MCS） in plasmid AAV and the corresponding contained restriction enzyme site in PCR product of TGFβ1, TGFβ1 gene was subcloned into AAV. The recombinant plasmid AAV-TGFβ1 was detected by restriction enzyme digestion and DNA sequencing. Then, AAV-TGFβ1 virus was packaged and TGFβ1 expression mediated by AAV was detected by immunofluence analysis in H293 cells. AAV transfection rate to NP cells was evaluated with AAV-PEGF. After NP cells were respectively transfected by AAV-TGFβ1 virus and AV-TGFβ1 virus, proteoglycan synthesis was detected and compared by using Antonopulos methods. Results DNA sequencing revealed that the PCR-amplified TGFβ1 gene was consistent with NCBI Gene Bank. The recombinant plasmid was proved to be constructed successfully by restriction enzyme digestion. AAV could be transfected into NP cells and mediate an efficient expression of TGFβ1 protein. AV-TGFβ1 virus could quickly enhance the proteoglycan synthesis of the NP cells, but its biological effect was transient. AAV-TGFβ1 virus could enhance stably proteoglycan synthesis. Conclusion AAV-TGFβ1 virus was successful constructed and enhanced stably proteoglycan synthesis of NP cells.     

Nerve Growth Factor Modulate Proliferation of Cultured Rabbit Corneal Endothelial Cells and Epithelial Cells

Summary： In order to investigate the effect of nerve growth factor （NGF） on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF.MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570 nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner. 50 U/mI. and 500 U/mI. NGF got more increase of proliferation than that of 5 U/mL NGF did. Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.     

The Chondrogenic Potential of Rabbit Bone—mar—orw—derived Mesenchymal Stem Cells（MSCs）As—sayed under the Influence of Growth Factors

Objective To establish a method of inducing rabbit bone marrow mesenchymal stem cells(MSCs)to express chondrocytic phenotype.Methods MSCs were seperated from bone marrow extracted from the iliac of New Zealand rabbit,TGF-β1,IGF-Ⅰand Vitamin C were applied into culture medium to induce proliferation and transformation of MSCs,Colony forming efficiency(CFE)was measured to reflect cell proliferation rate;procollagenαl（Ⅱ）mRNA in cells was detected by RT-PCR;ultrastructure of cells was observed under scanning electronic microscope(SEM);alkline phosphatase(ALP)in culture medium was also detected.Results Under the influence of TGF-β1,IGF-Ⅰand VitamineC,CFEof MSCs rose from control level of 3/106to21/106,expression of articular cartilage specific procollagenαl（Ⅱ）mRNA by cells was promoted;At the same time,floccu-lent matrixes synthesized were observed between connecting polygonal MSCs,ALP in culture medium ed-clined to control level with culture time,Conclusion Expression of chondrocytic phenotype by MSCs could be induced by the symergistic action of TGF-β0281and Vitamine C.Growth factor-induced MSCs could be a good cell source for tissue-engineered cartilage.     

Role of Connective Tissue Growth Factor in Extracellular Matrix Degradation in Renal Tubular Epithelial Cells

In order to investigate the effects of connective tissue growth factor (CTGF) antisense oligodeoxynucleotide (ODN) on plasminogen activator inhibitor-1 (PAI-1) expression in renal tubular cells induced by transforming growth factor β1 (TGF-β1) and to explore the role of CTGF in the degradation of renal extracellular matrix (ECM), a human proximal tubular epithelial cell line (HKC) was cultured in vitro. Cationic lipid-mediated CTGF antisense ODN was transfected into HKC. After HKC were stimulated with TGF-β1 (5 μg/L), the mRNA level of PAI-1 was detected by RT-PCR. In-tracellular PAI-1 protein synthesis was assessed by flow cytometry. The secreted PAI-1 in the media was determined by Western blot. The results showed that TGF-β1 could induce tubular CTGF and PAI-1 mRNA expression. The PAI-1 mRNA expression induced by TGF-β1 was significantly inhib-ited by CTGF antisense ODN. CTGF antisense ODN also inhibited intracellular PAI-1 protein syn-thesis and lowered the levels of PAI-1 protein secreted into the media. It was concluded that CTGF might play a crucial role in the degradation of excessive ECM during tubulointerstitial fibrosis, and blocking the biological effect of CTGF may be a novel way in preventing renal fibrosis.     

Construction of eukaryotic expression plasmid of hTGF-β3 and its inducing effect on differentiation of precartilaginous stem cells into chondroblasts

This study examined the construction of eukaryotic expression plasmid of human transforming growth factor-β3(hTGF-β3) and its inducing effect on the differentiation of precartilaginous stem cells(PSCs) into chondroblasts.hTGF-β3 gene was amplified by using polymerase chain reaction(PCR) and then inserted into the eukaryotic expression plasmid pcDNA3.1 to construct the eukaryotic expression plasmid pcDNA3.1(+)-hTGF-β3.Rat PSCs were isolated and purified by employing an immunomagnetic cell sorting system.pcDNA3.1(+)-hTGF-β3 was transfected into purified PSCs with the use of linear polyamines.The expression of TGF-β3 and cartilage-specific extracellular matrix(ECM) components was detected after transfection by real-time quantitative PCR,ELISA,immunochemistry and Western blotting,respectively.The results showed that the eukaryotic expression plasmid pcDNA3.1(+)-hTGF-β3 was successfully established as identified by enzyme digestion and DNA sequencing.Real-time quantitative PCR and ELISA revealed that hTGF-β3 was strongly expressed in pcDNA3.1(+)-hTGF-β3-transfected PSCs.Real-time quantitative PCR,immunochemistry and Western blotting showed that the cartilage-specific ECM markers,i.e.,cartilage oligomeric matrix protein(COMP),Aggrecan,collagen type Ⅹ and Ⅱ were intensely expressed in the pcDNA3.1(+)-hTGF-β3transfected cells.It was concluded that hTGF-β3 could be stably expressed in pcDNA3.1(+)-hTGFβ3-transfected PSCs and induce the differentiation of PSCs into chondroblasts.     

Construction of Eukaryotic Expression Plasmid pcDNA3-FHIT and Its Transient Expression in COS-1 Cells

Objective: To construct eukaryotic expression plasmid pcDNA3-fragile histidine triad(FHIT) and obtain its transient expression in COS-1 cells. Methods: FHIT gene was cloned from normal human thyroid tissue by RT-PCR and then inserted into eukaryotic expression vector pcDNA3. After the sequence was confirmed, the recombinant plasmid pcDNA3-FHIT was transfected into COS-1 cells by cation liposome. The transient expression in the cells was measured by immunocytochemistry. Results: The sequence of FHIT in pcDNA3 was correct and high expression was obtained in COS-1 cells. Conclusion: The eukaryotic expression plasmid pcDNA3-FHIT was constructed successfully and could highly express FHIT protein in COS-1 cells. This will be potentially useful for the research on gene therapy.     

Construction of eukaryotic expression vector encoding ATP synthase lipid-binding protein-like protein gene of Sj and its expression in HeLa cells

Objective： To clone and construct the recombinant plasmid containing ATP synthase lipid-binding protein-like protein gene of Schistosomajaponicum,（SjAslp） and transfer it into mammalian cells to express the objective protein. Methods： By polymerase chain reaction （PCR） technique, SjAslp was amplified from the constructed recombinant plasmid pBCSK＋/SjAslp, and inserted into cloning vector pUCm-T. Then, SjAslp was subcloned into an eukaryotic expression vector pcDNA3.1（＋）. After identifying it by PCR, restrictive enzymes digestion and DNA sequencing, the recombinant plasmid was transfected into HeLa cells using electroporation, and the expression of the recombinant protein was analyzed by immunocytochemical assay. Results： The specific gene fragment of 558 bp was successfully amplified. The DNA vaccine of SjAslp was successfully constructed. Immunocytochemical assay showed that SjAslp was expressed in the cytoplasm of HeLa cells. Conclusion： SjAslp gene can be expressed in eukaryotic system, which lays the foundation for development of the SjAslp DNA vaccine against schitosomiasis.     

Construction of Recombinant Plasmid Harboring APP717 Mutation and Preliminary Study of APP Proteolysis

In order to investigate the pathogenesis of Alzheimer disease （AD） and study the enzymatic progress of amyloid precursor protein （APP）, the fluorescent eukaryotic expression plasmid of C99 was constructed containing APP717 mutation. The fragment encoding the last 99-aa of APP （which was named C99 containing APP717 mutation）, together with the fragment encoding yellow fluorescence protein （which was named YFP） were amplified by PCR. The two fragments （YFP and C99） were inserted into the vector pcDNA3.0. The recombinant plasmid pcDNA3.0-YFP-C99 was accomplished and its authenticity was confirmed by enzyme digestion and sequencing. Then SH-SY5Y cells were transiently transfected with the recombinant plasmid pcDNA3.0-YFP-C99. The expression of the fusion gene was detected by laser confocalmicroscopy. Amyloid-β （Aβ） was detected by both microscopy and immunochemistry. The authenticity of the construct was confirmed by the endonuclease digestion and DNA sequencing. The YFP fluorescence could be seen and proved the expression of fusion gene. Aβ labeled by YFP was detected by confocalmicroscopy and confirmed by immunocytochemistry. It was found that Aβ accumulated and deposited in the intracytoplasm, membrane and outside of the cell. Furthermore, Aβ accumulated mainly within the cell ahead of the deposition in the cell space and the cell shape was rough. It was suggested that Aβ could be generated within the cells. Aβ accumulated in the cell at the early stage before the deposition outside of the cells Intracellular Aβ accumulation induced the secondary damage to the cells and caused the cell shape rough. Taken together, the recombinant plasmid, pcDNA3.0-YFP-C99 could be a useful tool to further study the cleavage mechanism of APP and to explore the pathogenesis of AD.     

Role of protein kinase C in advanced glycation end products-induced epithelial-mesenchymal transition in renal proximal tubular epithelial cells

The role of protein kinase C （PKC） activation in advanced glycation end products （AGEs）-induced epithelial-mesenchymal transition in renal proximal tubular epithelial cells was investigated. HKC cells were divided into three groups： normal group, AGE-BSA group （100 mg/L AGE-BSA） and AGE-BSA＋PKC inhibitor （10 μmol/L chelerythrine chloride） group. PKC activity was measured by PKC assay kit. The expression of Vimentin, and phosphorylated β-catenin was detected by using Western blotting, and the content of TGF-β1 was examined by ELISA method. The intracellular disposition of Vimentin was observed by fluorescence microscopy. As compared with normal group, PKC activity was increased significantly in AGE-BSA group. The expression of Vimentin, phosphorylated β-catenin, and TGF-β1 was enhanced significantly in AGE-BSA group. The expression of Vimentin, phosphorylated β-catenin, and TGF-β1 was significantly blocked by chelerythrine chloride. High expression of Vimentin, phosphorylated β-catenin, and TGF-β1 induced by AGE-BSA may be mediated via the activation of PKC signal transduction pathway.     

Construction of ER-β siRNA Expression Vector and Its Expression in Human Periodontal Ligament Fibroblast Cells

Objective To construct vectors expressing siRNA against human ER-β gene and study the inhibition effect of ER-β-siRNA on expression of ER-β in human periodontal ligament （HPDL） cells. Methods ER-β-specific siRNA gene was synthesized and cloned into pSilencer3.1-Hl-neo vector. The plasmid was identified by DNA sequencing. The constructed ER-β-siRNA was transfected into HPDL cells. The expression of ER-β in the levels of mRNA was detected by RT-PCR. Results It was confirmed by sequencing that the plasmid had been constructed successfully. The result of RT-PCR showed that Sequence-specific siRNAs targeting ER-β significantly down regulated the expression of ER-β gene in HPDL cells. Conclusion The ER-β siRNA plasmid was constructed successfully. ER-β gene expression of HPDL cells was inhibited by RNAi.     

Promotion of chondrogenesis of marrow stromal stem cells by TGF-β3 fusion protein in vitro

The purpose of this study was to investigate the repair of the osteoarthritis（OA）-induced car- tilage injury by transfecting the new TGF-β3 fusion protein （LAP-MMP-mTGF-β3） with targeted ther- apy function into the bone marrow-derived mesenchymal stem cells （MSCs） in rats. The recombinant of plRES-EGFP-MMP was constructed by combination of DNA encoding MMP enzyme cutting site and eukaryotic expression vector plRES-EGFP. LAP and mTGF-β3 fragments were obtained from rat em- bryos by RT-PCR and inserted into the upstream and downstream of MMP from plRES-EGFP-MMP respectively, so as to construct the recombinant plasmid ofplRES-EGFP-LAP-MMP-mTGF-β3, plRES- EGFP-LAP-MMP-mTGF-β3 was transfected into rat MSCs. The genetically modified MSCs were cul- tured in medium with MMP-1 or not. The transfected MSCs were transplanted in the rat OA models. The OA animal models were surgically induced by anterior cruciate ligament transaction （ACLT）. The pathological changes were observed under a microscope by HE staining, Alcian blue, Safranin-fast Gre- en and graded by Mankin＇s scale, plRES-EGFP-LAP-MMP-mTGF-β3 was successfully constructed by means of enzyme cutting and sequencing, and the mTGF-β3 fusion protein （39 kD） was certified by Western blotting. Those genetically modified MSCs could differentiate into chondrocytes induced by MMP and secrete the relevant-matrix. The transfected MSCs could promote chondrogenesis and matrix production in rat OA models in vivo. It was concluded that a new fusion protein LAP-MMP-mTGF-β3 was constructed successfully by gene engineering, and could be used to repair the OA-induced cartilage injury.     

Construction of Porcine CCK pDNA and Its Expression in COS-7 Cells

CCK correlates with the generation and progression of pancreatic cancer. The research aims to construct eukaryotic expression plasmid pIRES2-EGFP/CCK (CCK pDNA) and transiently express it in COS-7 cells. Total RNA was extracted from porcine intestinal mucosa. RT-PCR was used to amplify the aimed segments CCKcDNA which was then digested with EcoR1 and BamH1 and inserted into a eukaryotic expression plasmid pIRES2-EGFP to construct CCK pDNA. The con- structed plasmid was transfected into COS-7 cells by lepofectamineTM2000-mediated transfer method. The expression of CCK in transfected COS-7 cells was detected 24, 48 and 72 h post-transfection with fluorescence microscopy and the expression level of CCK mRNA in transfected COS-7 cells was assayed by using RT-PCR. The results showed CCK pDNA was successfully constructed and expressed transiently in COS-7 cells. Green fluorescent protein could be detected in the COS-7 cells transfected with porcine CCK pDNA 24 h post-transfection. At 48th h post-transfection, the number of positive cells was increased significantly and much brighter green fluorescence could be detected. And 72 h post-transfection, the green fluorescence of positive cells became even stronger, while no green fluorescence was detected in the control group. The expression of CCK mRNA in the cells was detectable by using RT-PCR. In COS-7 cells transfected with CCK pDNA a high level of porcine CCK mRNA was detected while no expression of porcine CCKmRNA was found in the cells trans- fected with null plasmid. It was concluded CCK pDNA was expressed successfully in COS-7 cells, which lays a foundation for further research on the relationship between CCK and tumor.     

Adenoviral vector mediated-expression of caspase-3 siRNA on apoptosis induced by lipopolysaccharide

Objective： To construct the recombinant adenovirus expressing small RNA of rats caspase-3 and observe the down-regulation effect of caspase-3 in neurons induced by lipopolysaccharide（LPS） in vitro. Methods： pShuttleHl-siCas3 containing Oligo DNA of the targeting sequences and pEGFPC1-Cas3 containing caspase-3 and EGFP sequences were constructed respectively, pShuttleH 1-siCas3 and pEGFPC 1-Cas3 were co-transfected to the 293 cells by liposomes to determine interfering efficacy by flow cytometry, pShuttleHl-siCas3 was linearized and transformed into E. coli B J5183 cells containing backbone plasmid pAdEasy-1. The recombinant plasmid was transfected into 293 cells to package the adenovirus Ad-siCas3. The titers of adenovirus were determined by the specific 50% tissue culture infection dosage method. After virus infected the cultured hippocampus neurons, LPS-induced apoptosis and caspase-3 mRNA expression were observed. Results： It was identified that the sequence of target gene was correctly inserted into the genome of virus. The expression of green fluorescence protein was reduced by pShuttleHl-siCas3 in 293 cells. The titer of recombinant adenovirus was 1.06×10^10pfu/ml. After virus infection, caspase-3 mRNA was greatly reduced and neurons apoptosis was suppressed. Conclusion： The recombinant adenovirus expressing rats caspase-3 siRNA were successfully constructed, which may probably be further used in pain therapy by its anti-apoptosis effect.     

Expression of NF-κB in hRPE Cells Stimulated by PDTC and IL-1β

The constitutive expression of nuclear-factor-κB (NF-κB) in human pigment epithelial (hRPE) cells cultivated in vitro and the possible changes when incubated with PDTC and IL-I were investigated. The synchronized hRPE cells in vitro were divided into two groups. In nonPDTC group, hRPE cells were exposed respectively to IL-1β and NS (for detecting the constitutive expressions of NF-κB in hRPE cells) ; In PDTC group, PDTC-pretreated hRPE cells were exposed respectively to IL-1β?Aand NS. (for detecting the constitutive expression of NF-κB in PDTC-pretreated hRPE cells). The expression of NF-κB in hRPE cells in two groups was detected by immunofluorescence stain and flow cytometry. The results showed that the constitutive expression of NF-κB in hRPE cells in vitro was 8.05 %, and increased to 30.26 % by IL-1β. After PDTC pretreatment, the constitutive expression of NF-κB in hRPE cells was decreased to 3.74%, and 3.66 % by IL-l,respectively. It was concluded that the expressions of NF-κB in hRPE cells could be increased significantly by IL-1βand depressed effectively by PDTC. Also, PDTC could significantly inhibit the activation of NF-κB induced by IL-1β.     

Transforming growth factor-β1 involved in urotensin II-induced phenotypic differentiation of adventitial fibroblasts from rat aorta

Background Urotensin Ⅱ （UⅡ） is a new vasoconstrictive peptide that may activate the adventitial fibroblasts.Transforming growth factor-β1 （TGF-β1） is an important factor that could induce the phenotypical transdifferentiation of adventitial fibroblasts. This study aimed to explore whether TGF-β1 is involved in UⅡ-induced phenotypic differentiation of adventitial fibroblasts from rat aorta.Methods Adventitial fibroblasts were prepared by the explant culture method. TGF-β1 protein secretion from the cells was determined by enzyme-linked immunosorbent assay （ELISA）. The mRNA and protein expression of α-smooth nuscle actin （α-SM-actin）, the marker of phenotypic differentiation from fibroblasts to myofibroblasts, were determined using real-time quantitative RT-PCR （real-time RT-PCR） and Western blotting, respectively.Results UⅡ stimulated the secretion of TGF-β1 in cultured adventitial fibroblasts in a time-dependent manner. The secretion reached a peak at 24 hours, was higher by 69.8% （P ＜0.01）, than the control group. This effect was also concentration dependent. Maximal stimulation was reached at 10-8 mol/L of UⅡ （P ＜0.01）, which was increased by 59.9%,compared with in the control group （P ＜0.01）. The secretion of TGF-β1 induced by UⅡ was significantly blocked by SB-710411 （10-7 mol/L）, a specific antagonist of UⅡ receptor. In addition, both UⅡ （10-8 mol/L） and TGF-β1 significantly stimulated α-SM-actin mRNA and protein expression. Moreover, the α-SM-actin induced by UⅡ was inhibited by the specific neutralizing antibody （20 μg/ml） of TGF-β1, while the α-SM-actin expression stimulated by TGF-β1 （20 ng/ml）was inhibited by SB-710411 （10-7 mol/L）, the UⅡ receptor antagonist.Conclusion This study suggests that UⅡ could induce TGF-β1 secretion in adventitial fibroblasts via UT activation, and TGF-β1 might be involved in phenotypic differentiation from adventitial fibroblasts into myofibroblasts induced by UⅡ, and TGF-β1 signaling might be one of the important pathways by which UⅡ is involved in vascular fibrosis.