Two novel alternatively spliced 9-bp exons in the COL4A5 gene

The existence of an alternatively used 18-bp sequence has been described in type IV collagen α5 chain mRNA from human kidney (Guo et al., Kidney Int 44, 1993). In this study we have shown that this sequence is encoded by two exons, termed 41A and 41B, that are located in a 9-kb intron 41 that was sequenced in its entirety. The sequences of exon 41A and 41B were shown to be present in mRNAs from a variety of tissues. Received: 13 April 2000 / Revised: 20 June 2000 / Accepted: 20 June 2000     

Effect of expansion medium on ex vivo gene transfer and chondrogenesis in type II collagen-glycosaminoglycan scaffolds in vitro

OBJECTIVE: Growth factors can profoundly affect the behaviour of chondrocytes during expansion and subsequent growth in three-dimensional (3-D) scaffolds. Prolonging such effects has stimulated investigation of the transfer of growth factor genes to chondrocytes. This study evaluated the effects of the monolayer expansion medium on the proliferation and cartilage matrix molecule synthesis of chondrocytes in 3-D pellet culture and in type II collagen-glycosaminoglycan (CG) scaffolds, and on ex vivo insulin-like growth factor-1 (IGF-1) gene transfer to articular chondrocytes in monolayer. The possibility of transfecting cells in 3-D culture using CG scaffolds was also investigated and the resulting effect of IGF-1 overexpression on glycosaminoglycan (GAG) biosynthesis in 3-D culture was assessed. METHODS: Two expansion media were compared-one that has been widely used for growing chondrocytes (Medium 1) and one that has been found to increase chondrocyte proliferation rates and preserve the redifferentiation potential of monolayer-expanded chondrocytes when subsequently placed in pellet cultures (Medium 2). Chondrocytes were expanded in monolayer culture and then 1) redifferentiated in 3-D culture, or 2) infected with the IGF-1 gene in monolayer or in type II CG scaffolds. RESULTS: The cell count for first passage chondrocytes was more than 3-fold higher when using Medium 2. In 3-D culture, cells expanded with Medium 2 and seeded in CG scaffolds produced more total GAG/DNA and displayed more intense immunohistochemical staining for collagen type II. Gene transfer and IGF-1 release kinetics from infected cells in monolayer were significantly affected by the composition of the expansion medium, the gene transfer method and time. IGF-1 gene transfer in CG scaffolds resulted in a 35-fold elevation in accumulated IGF-1 released from transfected Medium 2-expanded chondrocytes over controls, and resulted in a 40% increase in accumulated GAG/DNA. CONCLUSION: The composition of the expansion medium significantly affects monolayer proliferation of adult canine chondrocytes, GAG synthesis when the cells are subsequently grown in CG scaffolds, and ex vivo IGF-1 gene transfer.     

腺病毒介导的NT-3基因在骨髓间质干细胞中的表达

目的:研究腺病毒介导的NT-3基因在培养的大鼠骨髓间质干细胞(mesenchymal stem cells,MSCs)中的表达.方法:在293细胞中培养扩增NT-3重组腺病毒(adenovirus vector for NT-3,Ad-NT-3),测定病毒滴度,然后用Ad-NT-3感染传代培养的MSCs,RT-PCR技术检测NT-3基因的表达.结果:Ad-NT-3扩增后获得了较高滴度的病毒,MSCs经Ad-NT-3感染后有NT-3 mRNA的转录.结论:腺病毒介导的NT-3基因可转入培养的MSCs并高效表达,为NT-3基因治疗的研究奠定了基础.     

Successful renal transplantation in a family with a novel mutation in COL4A3 gene and autosomal recessive Alport syndrome

Alport syndrome (AS) is an inherited disorder of basement membranes caused by mutations affecting specific proteins of the type IV collagen family, presenting with nephropathy and extrarenal manifestations such as sensorineural deafness and ocular anomalies. Ten percentage to 15% of the patients with AS have autosomal recessive (ARAS) due to mutation in either COL4A3 or COL4A4 gene. We report a novel mutation in the COL4A3 gene in an Indian family with ARAS. The above‐mentioned genetic anomaly was a missense variation in exon 26 of the COL4A3 gene (chr2:228137797G>A; c.1891G>A) that resulted in the amino acid substitution of Arginine for Glycine at codon 631 (p.Gly631Arg) that was present in the heterozygous state in the asymptomatic parents and homozygous state in the male offspring who presented with early‐onset end‐stage renal disease, lenticonus and hearing loss. The patient (male offspring) underwent successful renal transplantation with his mother as a donor.     

Ex vivo and in vivo gene transfer to the peritoneal membrane in a rat model.

The efficacy of peritoneal dialysis (PD) depends on preserving the structural integrity and dialysing capacity of the peritoneal membrane. Membrane structure and function can change on PD, resulting in decreased membrane performance and possible discontinuation of PD as a treatment modality. We hypothesized that a gene therapy strategy might be an innovative and promising approach to maintaining membrane integrity and dialysing capacity in the PD population. We have characterized two methods of genetic modification of the peritoneal membrane in a rat model to test the feasibility of this approach. In ex vivo gene transfer, mesothelial cells are isolated from the peritoneal membrane, genetically modified in culture and subsequently re-implanted back onto the peritoneal membrane of syngeneic recipients. In in vivo gene transfer, genetic modification of the membrane is accomplished in situ through adenovirus-mediated delivery of the genetic material into the peritoneal cavity. The peritoneal membrane can be genetically modified to produce factors that may be of therapeutic value in maintaining the fibrinolytic balance in the peritoneal cavity, moderating peritoneal inflammation, and studying the development of peritoneal fibrosis. These models provide a basis for studying the contribution of specific molecules to peritoneal membrane physiology. Optimally, they will be paired with other PD-relevant model systems to understand peritoneal physiology, identify ways to prevent membrane damage, and maintain dialysing performance. While gene transfer can be used as a tool to understand the individual roles of factors or pathways in peritoneal membrane physiology, it can also be developed as a therapy platform for improving membrane characteristics and enhancing the therapy of peritoneal dialysis.     

Safe and efficient gene transfer into porcine hepatocytes using Sendai virus-cationic liposomes for bioartificial liver support

Establishment of a bioartificial liver support system using genetically modified hepatocytes is a potential approach to improve the treatment of severe liver failure. We describe the development of an efficient ex vivo method of gene transfer into a large number of porcine hepatocytes using hemagglutinating virus of Japan (HVJ)-liposome. The transfection efficiency of HVJ-liposome into isolated porcine hepatocytes attached to microcarrier beads was evaluated by beta-galactosidase (beta-gal) staining, fluorescence activated cell sorting analysis for beta-gal and luciferase assay, respectively. To examine the function and cellular damage of transduced hepatocytes, we used enzyme-linked immunosorbent assay for porcine albumin synthesis, lidocaine clearance test (P-450 activity), aspartate aminotransferase, and lactic dehydrogenase release assays. The optimal conditions for gene transfer into the beads-attached hepatocytes using HVJ-liposome included 4 microg of deoxyribonucleic acid with 200 microg of lipid/2 x 105 cells and exposure duration of 90 min. Under these conditions, beta-gal and luciferase genes were transduced to 2.5 x 108 isolated porcine hepatocytes following attachment to the beads. Positive beta-gal staining was observed in more than 30% of the beads-attached hepatocytes. The gene transfer activity of HVJ-liposome method determined by luciferase activities was about 100-fold of that of the lipofection method. Transfected porcine hepatocytes remained functional without any significant cell damage. Our results demonstrated that HVJ-liposome mediated gene transfer into microcarrier-attached porcine hepatocytes is an efficient and nontoxic method suitable for a bioartificial liver support sytem.     

改良的大鼠近端肾小管上皮细胞分离纯化方法

目的　建立纯度高、操作简便的大鼠近端肾小管上皮细胞分离纯化方法。 方法 Wistar大鼠无菌取肾前先心脏抽血替代原位肾脏灌注,分离肾皮质,经Ⅰ型胶原酶消化后用45% Percoll分离液制备细胞悬液直接连续密度梯度离心。所得细胞用含10%胎牛血清的DMEM-F12培养基原代培养并传代,根据细胞形态、细胞角蛋白18（CK18）和水通道蛋白1（AQP1）免疫细胞化学染色及碱性磷酸酶化学染色进行鉴定。 结果 此简化方法获得的肾小管细胞中肾小球掺杂明显减少,培养4～5 d后细胞融合长满,呈典型上皮样细胞的鹅卵石状,其CK18、AQP1免疫细胞化学染色及碱性磷酸酶化学染色几乎均呈阳性,证实所培养细胞为近端肾小管上皮细胞。 结论 改良后的分离纯化方法可获得大量高纯度近端肾小管上皮细胞,操作简便。     

Nine novel <Emphasis Type="Italic">COL4A3</Emphasis> and <Emphasis Type="Italic">COL4A4</Emphasis> mutations and polymorphisms identified in inherited membrane diseases

Both thin basement membrane nephropathy (TBMN) and autosomal recessive Alport syndrome result from mutations in the COL4A3 and COL4A4 genes, and this study documents further mutations and polymorphisms in these genes. Thirteen unrelated children with TBMN and five individuals with autosomal recessive Alport syndrome were examined for mutations in the 52 exons of COL4A3 and the 47 coding exons of COL4A4 using single-stranded conformation polymorphism (SSCP) analysis. Amplicons producing different electrophoretic patterns were sequenced, and mutations were defined as variants that changed an amino acid but were not present in 50 non-hematuric normals. Three further novel mutations were identified. These were IVS 22-5 T>A in the COL4A3 gene in a consanguineous family with autosomal recessive Alport syndrome, and R1677C and R1682Q in the COL4A4 gene. In addition, six novel polymorphisms (G455G, I462I, G736G and IVS 38-8 G>A in COL4A3, and L658L and A1577A in COL4A4) were demonstrated. Many different COL4A3 and COL4A4 mutations cause TBMN and autosomal recessive Alport syndrome. The identification of polymorphisms in these genes is particularly important to enable diagnostic laboratories to distinguish mutations from uncommon normal variants.     

血管内皮生长因子重组腺病毒转染鼠骨髓基质细胞及其产物促内皮细胞增殖的研究

目的探讨腺病毒介导血管内皮生长因子165(VEGF165)基因转染骨髓基质细胞(BMSCs)后对VEGF基因的转录、表达及其产物生物活性的影响。方法体外培养4只大鼠骨髓基质细胞,用携带VEGF165基因的重组腺病毒转染培养细胞;通过逆转录聚合酶链反应、免疫印记法和酶联免疫吸附法检测VEGF在转染细胞内的转录、表达和细胞外分泌情况;通过血管内皮细胞增殖实验测定转染VEGF165基因后的BMSCs培养上清中VEGF蛋白的生物活性。结果腺病毒介导VEGF165基因转染BMSCs后可以获得有效的转录及表达,其分泌于培养上清中的表达产物可明显促进大鼠主动脉血管内皮细胞的增殖(P<001),具有很强的生物学活性。结论腺病毒可安全、有效地转染BMSCs,VEGF165基因转染的鼠BMSCs可有效表达具有生物活性的VEGF蛋白。     

Adenovirus vector-mediated ex vivo gene transfer of brain-derived neurotrophic factor to bone marrow stromal cells promotes axonal regeneration after transplantation in completely transected adult rat spinal cord

The aim of this study was to evaluate the efficacy in adult rat completely transected spinal cord of adenovirus vector-mediated brain-derived neurotrophic factor (BDNF) ex vivo gene transfer to bone marrow stromal cells (BMSC). BMSC were infected with adenovirus vectors carrying β-galactosidase (AxCALacZ) or BDNF (AxCABDNF) genes. The T8 segment of spinal cord was removed and replaced by graft containing Matrigel alone (MG group) or Matrigel and BMSC infected by AxCALacZ (BMSC-LacZ group) or AxCABDNF (BMSC-BDNF group). Axons in the graft were evaluated by immunohistochemistry and functional recovery was assessed with BBB locomotor scale. In the BMSC-BDNF group, the number of fibers positive for growth associated protein-43, tyrosine hydroxylase, and calcitonin gene-related peptide was significantly larger than numbers found for the MG and BMSC-LacZ groups. Rats from BMSC-BDNF and BMSC-LacZ groups showed significant recovery of hind limb function compared with MG rats; however, there was no significant difference between groups in degree of functional recovery. These findings demonstrate that adenovirus vector-mediated ex vivo gene transfer of BDNF enhances the capacity of BMSC to promote axonal regeneration in this completely transected spinal cord model; however, BDNF failed to enhance hind limb functional recovery. Further investigation is needed to establish an optimal combination of cell therapy and neurotrophin gene transfer for cases of spinal cord injury.     

A novel method for gene delivery and expression in esophageal epithelium with fibrin glues containing replication-deficient adenovirus vector

Background: Gene transfer to the esophageal epithelium holds the potential for the therapy of malignant as well as premalignant lesions in the upper gastrointestinal tract. Replication-deficient recombinant adenoviruses represent an efficient means of introducing genes in vivo into cells in a variety of organs. The majority of in vivo studies utilize direct submucosal injection for delivery of the viral vectors into the locoregional area of the gut; transferring genes into epithelial cells, however, is difficult because viruses are retained in the subepithelial space. To establish the efficient method for gene transfer into the epithelial cells, we have developed a multiluminal spray catheter that can be passed through the accessory channel of an endoscope, and we have evaluated the feasibility of fibrin glues as a vehicle of recombinant adenoviruses in a porcine model. Methods: The fibrinogen solution and the thrombion solution containing an E1/E3 deleted recombinant adenovirus expressing the bacterial lacZ gene (Ad-lacZ) were endoscopically sprayed on the porcine esophagus through the catheter attached to the dual-barrel syringe. Twenty-four hours after gene delivery, -galactosidase activity of the esophagus was determined under the microscope following X-gal staining. Results: The fibrin glue could be locally sprayed on the porcine esophagus by using the multichannel catheter through the endoscope. Attachment of the fibrin glue comtining Ad-lacZ caused strong -galactosidase staining on epithelial cells in the mucosal surface, but not in the basal cell layer. Conclusion: Endoscopic local delivery of recombinant adenoviruses in aerosolized fibrin glues through a multiluminal catheter could provide an optimal technique for gene transfer into epithelial cells in the mucosal surfece, which may have important implications for the treatment of human esophageal premalignant diseases.     

Maintaining the permanence principle for death during in situ normothermic regional perfusion for donation after circulatory death organ recovery: A United Kingdom and Canadian proposal

There is international variability in the determination of death. Death in donation after circulatory death (DCD) can be defined by the permanent cessation of brain circulation. Post‐mortem interventions that restore brain perfusion should be prohibited as they invalidate the diagnosis of death. Retrieval teams should develop protocols that ensure the continued absence of brain perfusion during DCD organ recovery. In situ normothermic regional perfusion (NRP) or restarting the heart in the donor's body may interrupt the permanent cessation of brain perfusion because, theoretically, collateral circulations may restore it. We propose refinements to current protocols to monitor and exclude brain reperfusion during in situ NRP. In abdominal NRP, complete occlusion of the descending aorta prevents brain perfusion in most cases. Inserting a cannula in the ascending aorta identifies inadequate occlusion of the descending aorta or any collateral flow and diverts flow away from the brain. In thoracoabdominal NRP opening the aortic arch vessels to atmosphere allows collateral flow to be diverted away from the brain, maintaining the permanence standard for death and respecting the dead donor rule. We propose that these hypotheses are correct when using techniques that simultaneously occlude the descending aorta and open the aortic arch vessels to atmosphere.     

Prevalence Estimates of Predicted Pathogenic COL4A3–COL4A5 Variants in a Population Sequencing Database and Their Implications for Alport Syndrome

Open in a separate window