Osteogenic differentiation effects on rat bone marrow-derived mesenchymal stromal cells by lentivirus-mediated co-transfection of human BMP2 gene and VEGF165 gene

We proposed a novel combined gene therapy of human vascular endothelial growth factor 165 gene (hVEGF165) and human bone morphogenetic protein 2 gene (hBMP2) for bone regeneration by lentivirus-mediated co-transfection of both genes into rat bone marrow-derived mesenchymal stromal cells (MSCs). Both genes were successfully co-expressed in MSCs confirmed by real-time PCR and ELISA. And the alkaline phosphatase activity of MSCs was significantly augmented by the co-transfection with both genes than any single gene transfection (P < 0.01). These results demonstrated the feasibility of the combined gene therapy by using MSCs lentivirally co-transfected with hVEGF165 and hBMP2 for bone regeneration.     

hBMP2转基因载体的构建及其在兔骨髓基质细胞中的表达

目的构建hBMP2真核表达载体pcDNA3-hBMP2,将其转染兔骨髓基质细胞(Marrow Stromal Cells,MSCs)并检测其表达效率。方法将hBMP2的cDNA构建于真核表达载体pcDNA3,形成重组真核表达载体pcDNA3-hBMP2,酶切鉴定后体外转染培养状态下的兔骨髓基质细胞,用原位杂交和免疫组织化学方法鉴定表达情况并用计算机图像分析系统测定其表达效率。结果pcDNA3-hBMP2载体酶切鉴定与预期片段相符,表明成功构建了pcDNA3-hBMP2转基因载体。用该载体转染兔骨髓基质细胞后获得了瞬时表达和稳定表达。结论载体pcDNA3-hBMP2可以在骨髓基质细胞中表达,为下一步将其用于转基因骨组织工程研究奠定基础。     

一种双顺反子表达载体的构建及应用的研究

将表达载体pEC34中的一段寡核苷酸序列,其中包括翻译增强子序列、SD序列、终止码、起始码及两端的限制性内切酶位点,插入GST基因后,构建成双顺反子的表达载体．利用此载体表达了非融合的人骨形成蛋白2A(hBMP2A)和人骨形成蛋白3(hBMP3)C端肽段,将第一顺反子基因（GST基因）切小到原来的1／3时,则位于下游的第二顺反子基因编码的蛋白质在大肠杆菌中的表达量增加一倍。     

Identification of a vitamin D responsive element in the promoter of the rat cytochrome P450(24) gene.

Mitochondrial cytochrome P450(24) expression in the vitamin D-degradation pathway is induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. The molecular basis of this enzyme regulation was investigated by isolating the rat P450(24) gene and examining the 5'-flanking region for possible cis-acting regulatory elements involved in the induction process. Constructs containing different lengths of 5'-flanking region of the gene were linked to a luciferase reporter gene and transiently co-transfected with a human vitamin D receptor (hVDR) expression vector (pRSV-hVDR) into COS-1 cells. These experiments showed that the flanking region from -298 to -122 directed a 24-fold increase in luciferase activity in response to 1,25-(OH)2D3 provided that the cells were co-transfected with pRSV-hVDR. Within this region, the sequence from position -171 to -123 conferred 1,25-(OH)2D3 responsiveness to both the native P450(24) promoter and the heterologous thymidine kinase promoter. Mutagenesis revealed that the sequence from position -150 to -136 is required for induction by 1,25-(OH)2D3 and that this sequence shares similarity to other vitamin D responsive elements (VDREs) reported for other genes. Gel shift mobility assays showed this region specifically bound a nuclear protein complex from 1,25-(OH)2D3 treated COS-1 cells that had been co-transfected with pRSV-hVDR. The retarded band was specifically competed with the well characterized VDRE from the mouse osteopontin gene. A VDRE at position -150 to -136 in the promoter of the rat P450(24) gene is identified in this study and found to be important in mediating the enhanced expression of the gene by 1,25-(OH)2D3.     

Effects of codon modification on human BMP2 gene expression in tobacco plants

Bone morphogenetic protein 2 (BMP2) has great potential in therapeutic applications. We are working on generating transgenic plants as a bioreactor to produce BMP2. We have studied the effects of codon optimization on the expression of human BMP2 (hBMP2) in tobacco plants. Three modified hBMP2 genes were transformed into tobacco under the control of either cauliflower mosaic virus 35S (CaMV35S) promoter or double-CaMV35S promoter plus alfalfa mosaic virus (AMV) enhancer. The fused β-glucuronidase (GUS) reporter gene was used to facilitate the assay of protein expression. The results indicated that codon optimization could increase the protein expression level obviously under CaMV35S promoter. However, under relatively stronger initiation condition (double-CaMV35S promoter plus AMV enhancer), only the gene with the lowest degree of codon optimization could increase the protein expression level. Our findings suggest that the action of codon optimization may be influenced by the factors of promoter strength and A+T content in tobacco plants.     

Regulated genes in mesenchymal stem cells and gastric cancer

AIM: To investigate the genes regulated in mesenchymal stem cells(MSCs) and diffuse-type gastric cancer(GC),gene expression was analyzed. METHODS: Gene expression of MSCs and diffuse-type GC cells were analyzed by microarray. Genes related to stem cells, cancer and the epithelial-mesenchymal transition(EMT) were extracted from human gene lists using Gene Ontology and reference information. Gene panels were generated, and messenger RNA gene expression in MSCs and diffuse-type GC cells was analyzed. Cluster analysis was performed using the NCSS software.RESULTS: The gene expression of regulator of G-protein signaling 1(RGS1) was up-regulated in diffuse-type GC cells compared with MSCs. A panel of stem-cell related genes and genes involved in cancer or the EMT were examined. Stem-cell related genes, such as growth arrest-specific 6, musashi RNA-binding protein 2 and hairy and enhancer of split 1(Drosophila), NOTCH family genes and Notch ligands, such as delta-like 1(Drosophila) and Jagged 2, were regulated.CONCLUSION: Expression of RGS1 is up-regulated, and genes related to stem cells and NOTCH signaling are altered in diffuse-type GC compared with MSCs.     

CXCR4慢病毒载体的构建及其在人脐带血间充质干细胞的表达

目的：构建趋化因子CXC亚族CXCR4的慢病毒表达载体并观察其转染人脐带间充质干细胞后的表达。方法：用逆转录PCR方法获取CXCR4基因编码区片段,将构建的慢病毒载体质粒pLVTHM-EGFP-CXCR4与包装质粒psPAX2和包膜质粒pMD2．G共转染293T细胞,包装生产慢病毒。用相同滴度的慢病毒转导等量间充质干细胞（Mesenchymal Stem Cell,MSCs）,后采用Real time PCR检测CXCR4 mRNA、Western Blot方法检测蛋白质的表达。结果：PCR、酶切和测序结果表明成功的构建了CXCR4基因重组慢病毒载体。同时用该慢病毒载体转染MSCs后可有效地增加MSCs中CXCR4的表达。结论：成功构建了CXCR4的慢病毒表达载体并能在MSCs中表达,为进一步研究其在干细胞移植中的应用奠定基础。’     

Specific adeno-associated virus serotypes facilitate efficient gene transfer into human and non-human primate mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) show great promise for ex vivo gene and cell-mediated therapies. The immunophenotype and in vitro differentiation capacity of primary baboon MSCs was demonstrated to be near-identical to that observed in human MSCs. To optimize gene transfer efficiency, we compared the efficiency of serotypes 1, 2, 3, 4, 5, 6, and 8 of adeno-associated virus (AAV) vectors for their ability to mediate transduction of human and baboon MSCs. AAV serotype 2 vectors were the most efficient in transducing MSCs from humans and baboons. As a reference, human Ad293 cells were transduced with these seven AAV serotypes, and were found to have the highest transduction levels followed by baboon MSCs, and then human MSCs. The order of increasing transduction efficiency for the serotypes tested was similar for human and baboon MSCs, but was different for human Ad293 cells. The transduction efficiency of MSCs isolated from different individuals was comparable within the same species. We also demonstrated that baboon MSCs transduced with AAV serotype 2 vectors retain their potential to differentiate into adipocytes in vitro, and can incorporate into injured muscle tissue of NODSCID mice in vivo. We detected beta-galactosidase reporter gene expression in host muscle tissue for up to 9 weeks in this study, indicating engraftment of transduced baboon MSCs and sustained transgene expression in vivo.     

Comparison of cis and trans tat gene expression in HIV LTR-based amplifier vectors

The long terminal repeat (LTR) of the human immunodeficiency virus (HIV) drives highly efficient gene expression in the presence of the transactivator, Tat. Thus, tat-containing vectors may be very useful tools in gene therapy. However information about the optimal way of delivering the tat gene is limited. In this study, we compared the effects of cis and trans expressions of the tat gene and its effects on HIV LTR-driven gene expression in different cell lines using non-viral vectors. The human interleukin-2 (IL-2) gene was used as a reporter gene under the control of the HIV2 LTR (pHIV2-IL-2). The tat gene, driven by a cytomegalovirus (CMV) promoter, was either co-transfected separately (pCMV-Tat) or inserted downstream of the IL-2 gene (pHIV2-IL-2-neo-C-Tat). Our results showed that HIV2 LTR-Tat-based vectors were much more potent than CMV promoter-based vectors in transient expression. The co-transfection of both plasmids was comparable to a single transfection of pHIV-IL-2-neo-C-Tat in both high and low transfection efficiency cells. In conclusion, the co-placement of HIV2 LTR and tat genes on a single plasmid allows for gene expression as efficiently as a two-plasmid system, suggesting that HIV2 LTR-Tat-based vectors may be attractive tools for gene therapy.     

人骨形态发生蛋白7（hBMP7）在毕赤酵母中的分泌表达

依据酵母密码子使用偏好性,利用重叠延伸PCR(OE-PCR)介导的定点突变方法,对人骨形态发生蛋白-7(human Bone Morphogenetic Protein-7,hBMP7)成熟肽编码序列进行改造,将毕赤酵母低频使用的精氨酸密码子CGG或CGA突变为高频使用的同义密码子AGA,明显提高了hBMP7成熟肽在毕赤酵母中的表达量摇瓶培养表达量为25.45mg/L,是改造前序列的4.6倍;TricineSDS-PAGE及Western-blotting结果表明,rhBMP7成熟肽分子量为18kD,以单体形式存在,具有良好的免疫原性;利用梯度浓度G418筛选到一株高拷贝整合的转化子,该转化子摇瓶表达量为45.45mg/L,约为单拷贝转化子的2倍。表达上清经阳离子交换介质SPSepharoseR○FastFlow纯化后,目的蛋白纯度达到90%。纯化后的样品与I型胶原混合冻干后埋植于小鼠股部肌袋内,能异位诱导间充质细胞分化形成软骨细胞。     

Phenotypic analysis of cell surface markers and gene expression of human mesenchymal stem cells and chondrocytes during monolayer expansion

Both chondrocytes and mensenchymal stem cells (MSCs) are the most used cell sources for cartilage tissue engineering. However, monolayer expansion to obtain sufficient cells leads to a rapid chondrocyte dedifferentiation and a subsequent ancillary reduced ability of MSCs to differentiate into chondrocytes, thus limiting their application in cartilage repair. The aim of this study was to investigate the influence of the monolayer expansion on the immunophenotype and the gene expression profile of both cell types, and to find the appropriate compromise between monolayer expansion and the remaining chondrogenic characteristics. To this end, human chondrocytes, isolated enzymatically from femoral head slice, and human MSCs, derived from bone marrow, were maintained in monolayer culture up to passage 5. The respective expressions of cell surface markers (CD34, CD45, CD73, CD90, CD105, CD166) and several chondrogenic-related genes for each passage (P0-P5) of those cells were then analyzed using flow cytometry and quantitative real-time PCR, respectively. Flow cytometry analyses showed that, during the monolayer expansion, some qualitative and quantitative regulations occur for the expression of cell surface markers. A rapid increase in mRNA expression of type 1 collagen occurs whereas a significant decrease of type 2 collagen and Sox 9 was observed in chondrocytes through the successive passages. On the other hand, the expansion did not induced obvious change in MSCs gene expression. In conclusion, our results suggest that passage 1 might be the up-limit for chondrocytes in order to achieve their subsequent redifferentiation in 3D scaffold. Nevertheless, MSCs could be expanded in monolayer until passage 5 without loosing their undifferentiated phenotypes.     

Stable gene expression by self-complementary adeno-associated viruses in human MSCs

Genetically modified mesenchymal stem cells (MSCs) are potentially valuable tools for the novel treatment of human illnesses. Here, we investigated whether gene transfers by self-complementary adeno-associated viruses (scAAV) lead to promising genetic modification in human bone marrow and umbilical cord blood MSCs. Of the various scAAVs, scAAV2, and scAAV5 effectively and safely expressed transgenes in both hMSCs. Transduction efficiency with scAAV2 at 1000 multiplicity of infection was 66.3+/-9.4% and 67.6+/-6.7% in bone marrow and umbilical cord blood MSCs, respectively. A co-infection study showed that the distinct scAAV2 and scAAV5 can effectively express different transgenes in the same hMSC. hMSCs transduced by scAAVs showed long-term gene expression for three months in rat brains. Genetic modification by scAAVs did not affect osteogenic differentiation of hMSCs. Therefore, the present study strongly supports the promising potential of scAAVs as a technical platform for safe, long-term transgene expression in hMSCs.     

Stable gene expression from a mammalian artificial chromosome

We have investigated the potential of PAC-based vectors as a route to the incorporation of a gene in a mammalian artificial chromosome (MAC). Previously we demonstrated that a PAC (PAC7c5) containing α-satellite DNA generated mitotically stable MACs in human cells. To determine whether a functional HPRT gene could be assembled in a MAC, PAC7c5 was co-transfected with a second PAC containing a 140 kb human HPRT gene into HPRT-deficient HT1080 cells. Lines were isolated containing a MAC hybridizing with both α-satellite and HPRT probes. The MACs segregated efficiently, associated with kinetochore proteins and stably expressed HPRT message after 60 days without selection. Complementation of the parental HPRT deficiency was confirmed phenotypically by growth on HAT selection. These results suggest that MACs could be further developed for delivering a range of genomic copies of genes into cells and that stable transgene expression can be achieved.     

Identification of novel TGF-beta /Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach

Despite major advances in the understanding of the intimate mechanisms of transforming growth factor-beta (TGF-beta) signaling through the Smad pathway, little progress has been made in the identification of direct target genes. In this report, using cDNA microarrays, we have focussed our attention on the characterization of extracellular matrix-related genes rapidly induced by TGF-beta in human dermal fibroblasts and attempted to identify the ones whose up-regulation by TGF-beta is Smad-mediated. For a gene to qualify as a direct Smad target, we postulated that it had to meet the following criteria: (1) rapid (30 min) and significant (at least 2-fold) elevation of steady-state mRNA levels upon TGF-beta stimulation, (2) activation of the promoter by both exogenous TGF-beta and co-transfected Smad3 expression vector, (3) up-regulation of promoter activity by TGF-beta blocked by both dominant-negative Smad3 and inhibitory Smad7 expression vectors, and (4) promoter transactivation by TGF-beta not possible in Smad3(-/-) mouse embryo fibroblasts. Using this stringent approach, we have identified COL1A2, COL3A1, COL6A1, COL6A3, and tissue inhibitor of metalloproteases-1 as definite TGF-beta/Smad3 targets. Extrapolation of this approach to other extracellular matrix-related gene promoters also identified COL1A1 and COL5A2, but not COL6A2, as novel Smad targets. Together, these results represent a significant step toward the identification of novel, early-induced Smad-dependent TGF-beta target genes in fibroblasts.