Restoration of dystrophin expression in mdx mice by intravascular injection of naked DNA containing full-length dystrophin cDNA

Duchenne muscular dystrophy (DMD) is a lethal, X-linked, recessive disease caused by a defect in the dystrophin gene. No effective therapy is available. Dystrophin gene transfer to skeletal muscle has been proposed as a treatment for DMD. However, successful treatment for DMD requires restoration of dystrophin in the affected muscle fibers to at least 20% of the normal level. Current gene transfer methods such as intramuscular injection of viral vector or naked DNA can only transfect a small area of muscle, and therefore is of little clinical utility. We have developed a semisystemic method for gene transfer into skeletal muscle of mdx mice, an animal model for DMD. Naked DNA was injected through the tail artery or vein of mice, in which the aorta and the vena cava were clamped at the location just below the kidneys. The DNA solution was thus forced into the blood vessels of both legs. Luciferase gene expression was detected in all muscle groups in both legs. The effects of injection speed, injection volume, and ischemia time on gene expression were also optimized. LacZ staining was used to check the spread of gene expression in muscle. Although the percentage of transfected fibers was modest (approximately 10%), beta-galactosidase was found in all muscle groups of both legs. Finally, plasmid DNA encoding full-length dystrophin gene was injected into mdx mice and widespread restoration of dystrophin protein was observed in all muscles of both hind limbs. In conclusion, these results demonstrate that the semisystemic delivery of naked DNA is a potential approach towards the long-term goal of gene therapy for DMD.     

Intraarterial delivery of naked plasmid DNA expressing full-length mouse dystrophin in the mdx mouse model of duchenne muscular dystrophy

Our previous studies have demonstrated that the intraarterial delivery of naked plasmid DNA leads to high levels of foreign gene expression throughout the muscles of the targeted limb. Although the procedure was first developed in rats and then extended to nonhuman primates, the present study has successfully implemented the procedure in normal mice and the mdx mouse model for Duchenne muscular dystrophy. After intraarterial delivery of plasmid DNA expressing the normal, full-length mouse dystrophin from either the cytomegalovirus promoter or a muscle-specific human desmin gene control region, mdx mouse muscle stably expressed dystrophin in 1-5% of the myofibers of the injected hind limb for at least 6 months. This expression generated an antibody response but no apparent cellular response.     

骨髓间充质干细胞移植治疗Duchenne肌营养不良型mdx小鼠后的肌电图和dystrophin蛋白表达改变

目的 研究骨髓间充质干细胞（MSCs）移植治疗Duchenne肌营养不良（DMD）模型鼠mdx鼠后肌电图改变及dystrophin蛋白表达变化。方法 分离培养正常小鼠MSCs局部肌肉注射移植入DMD模型鼠mdx鼠，数周后观察肌电图改变及dystrophin蛋白表达变化。结果 MSCs移植后mdx鼠肌电图明显改善，dystrophin蛋白阳性表达肌纤维增加明显。结论 MSCs局部骨骼肌肌肉内细胞移植治疗mdx鼠有一定效果。     

Immune rejection of human dystrophin following intramuscular injections of naked DNA in mdx mice

Intramuscular administration of plasmid expressing full-length human dystrophin in dystrophin-deficient adult mdx mice resulted in humoral and weak specific T cell responses against the human dystrophin protein. Following plasmid injection, human dystrophin was detected in the injected muscles at 7 days, but decreased thereafter. Anti-dystrophin antibodies were found 21 days following plasmid injection, which coincided with transient myositis. This immune rejection prevented the mice from expressing human dystrophin after a second plasmid injection. No anti-DNA antibodies were found. Anti-dystrophin antibodies were seen in a smaller proportion of plasmid-injected dystrophin-competent C57BL/10 mice, suggesting that the immune rejection of dystrophin may be explained partially by species differences in the dystrophin protein.     

Sustained improvement of muscle function one year after full-length dystrophin gene transfer into mdx mice by a gutted helper-dependent adenoviral vector

Dystrophin gene transfer using helper-dependent adenoviral vectors (HDAd) deleted of all viral genes is a promising option to treat muscles in Duchenne muscular dystrophy (DMD). Previously, we reported high-level dystrophin expression and functional correction of dystrophin-deficient (mdx) mouse muscle 60 days after gene transfer with an HDAd encoding two full-length murine dystrophin cDNAs (referred to as HDCBDysM). In the present study, we tested the long-term efficacy of HDCBDysM by examining muscle contractility parameters and the stability of dystrophin expression 1 year after injection into neonatal mdx muscles. At this point, HDCBDysM-treated muscles averaged 52% dystrophin-positive fibers. Treated muscles also displayed significantly greater isometric force production as well as greater resistance to the force deficits and damage caused by eccentric contractions. The level of protection against eccentric contraction-induced force deficits correlated with the percentage of dystrophin-positive fibers. Furthermore, HDCBDysM treatment restored the dystrophin-glycoprotein complex (DGC) to the sarcolemma and improved other aspects of mdx muscle histopathology examined (central nucleation, muscle hypertrophy, and mononuclear [phagocytic] cell infiltration). These improvements occurred despite the induction of a humoral response against murine dystrophin. Our results indicate that major therapeutic benefits of HDCBDysM are maintained for a long period of the animals' lifespan and suggest that HDCBDys holds promise for treating DMD by gene therapy.     

Aminoglycoside antibiotics restore dystrophin function to skeletal muscles of mdx mice

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene, leading to the absence of the dystrophin protein in striated muscle. A significant number of these mutations are premature stop codons. On the basis of the observation that aminoglycoside treatment can suppress stop codons in cultured cells, we tested the effect of gentamicin on cultured muscle cells from the mdx mouse - an animal model for DMD that possesses a premature stop codon in the dystrophin gene. Exposure of mdx myotubes to gentamicin led to the expression and localization of dystrophin to the cell membrane. We then evaluated the effects of differing dosages of gentamicin on expression and functional protection of the muscles of mdx mice. We identified a treatment regimen that resulted in the presence of dystrophin in the cell membrane in all striated muscles examined and that provided functional protection against muscular injury. To our knowledge, our results are the first to demonstrate that aminoglycosides can suppress stop codons not only in vitro but also in vivo. Furthermore, these results raise the possibility of a novel treatment regimen for muscular dystrophy and other diseases caused by premature stop codon mutations. This treatment could prove effective in up to 15% of patients with DMD.     

Transplantation of a multipotent cell population from human adipose tissue induces dystrophin expression in the immunocompetent mdx mouse

Here, we report the isolation of a human multipotent adipose-derived stem (hMADS) cell population from adipose tissue of young donors. hMADS cells display normal karyotype; have active telomerase; proliferate >200 population doublings; and differentiate into adipocytes, osteoblasts, and myoblasts. Flow cytometry analysis indicates that hMADS cells are CD44+, CD49b+, CD105+, CD90+, CD13+, Stro-1(-), CD34-, CD15-, CD117-, Flk-1(-), gly-A(-), CD133-, HLA-DR(-), and HLA-I(low). Transplantation of hMADS cells into the mdx mouse, an animal model of Duchenne muscular dystrophy, results in substantial expression of human dystrophin in the injected tibialis anterior and the adjacent gastrocnemius muscle. Long-term engraftment of hMADS cells takes place in nonimmunocompromised animals. Based on the small amounts of an easily available tissue source, their strong capacity for expansion ex vivo, their multipotent differentiation, and their immune-privileged behavior, our results suggest that hMADS cells will be an important tool for muscle cell-mediated therapy.     

Eosinophilia in transgenic mice expressing interleukin 5

Experiments in vitro suggest that although interleukin 5 (IL-5) stimulates the late stages of eosinophil differentiation, other cytokines are required for the generation of eosinophil progenitor cells. In this study transgenic mice constitutively expressing the IL-5 gene were established using a genomic fragment of the IL-5 gene coupled to the dominant control region from the gene encoding human CD2. Four independent eosinophilic transgenic lines have thus far been established, two of which with 8 and 49 transgene copies, are described in detail. These mice appeared macroscopically normal apart from splenomegaly. Eosinophils were at least 65- and 265-fold higher in blood from transgenics, relative to normal littermates, and approximately two- or sevenfold more numerous relative to blood from mice infected with the helminth Mesocestoides corti. Much more modest increases in blood neutrophil, lymphocyte, and monocyte numbers were noted in transgenics, relative to normal littermates (less than threefold). Thus IL-5 in vivo is relatively specific for the eosinophil lineage. Large numbers of eosinophils were present in spleen, bone marrow, and peritoneal exudate, and were highest in the line with the greatest transgene copy number. Eosinophilia was also noted in histological sections of transgenic lungs, Peyer's patches, mesenteric lymph nodes, and gut lamina propria but not in other tissues examined. IL-5 was detected in the sera of transgenics at levels comparable to those seen in sera from parasite-infected animals. IL-3 and granulocyte/macrophage colony-stimulating factor (GM-CSF) were not found. IL-5 mRNA was detected in transgenic thymus, Peyer's patches, and superficial lymph nodes, but not in heart, liver, brain, or skeletal muscle or in any tissues from nontransgenics. Bone marrow from transgenic mice was rich in IL-5-dependent eosinophil precursors. These data indicate that induction of the IL-5 gene is sufficient for production of eosinophilia, and that IL-5 can induce the full pathway of eosinophil differentiation. IL-5 may therefore not be restricted in action to the later stages of eosinophil differentiation, as suggested by earlier in vitro studies.     

An inbred line of transgenic mice expressing an internally deleted gene for type II procollagen (COL2A1). Young mice have a variable phenotype of a chondrodysplasia and older mice have osteoarthritic changes in joints.

Studies were carried out on a line of transgenic mice that expressed an internally deleted COL2A1 gene and developed a phenotype resembling human chondrodysplasias (Vandenberg et al. 1991. Proc. Natl. Acad. Sci. USA. 88:7640-7644. Marked differences in phenotype were observed with propagation of the mutated gene in an inbred strain of mice in that approximately 15% of the transgenic mice had a cleft palate and a lethal phenotype, whereas the remaining mice were difficult to distinguish from normal littermates. 1-d- and 3-mo-old transgenic mice that were viable showed microscopic signs of chondrodysplasia with reduced amounts of collagen fibrils in the cartilage matrix, dilatation of the rough surfaced endoplasmic reticulum in the chondrocytes, and decrease of optical path difference in polarized light microscopy. The transgenic mice also showed signs of disturbed growth as evidenced by lower body weight, lower length and weight of the femur, decreased bone collagen, decreased bone mineral, and decreased resistance of bone to breakage. Comparisons of mice ranging in age from 1 d to 15 mo demonstrated that there was decreasing evidence of a chondrodysplasia as the mice grew older. Instead, the most striking feature in the 15-mo-old mice were degenerative changes of articular cartilage similar to osteoarthritis.     

Induction of dystrophin expression by exon skipping in mdx mice following intramuscular injection of antisense oligonucleotides complexed with PEG-PEI copolymers.

Antisense oligonucleotides (AOs) with 2-O-methyl modifications can circumvent dystrophin mutations via exon skipping and, it is hoped, can become drugs for treatment of Duchenne muscular dystrophy (DMD). However, AO-based approaches are hindered by a lack of effective carriers to facilitate delivery of AOs to myonuclei. We examined whether copolymers composed of cationic poly(ethylene imine) (PEI) and polyethylene glycol (PEG) can enhance AO transfection in skeletal muscle of mdx mice. Single intramuscular injections of AO complexed with low Mw PEI2000(PEG550) copolymers into TA muscles of mdx mice resulted in widespread distribution of dystrophin-positive fibers at 3 weeks after injection, with no apparent cytotoxicity. Overall, injections of these low Mw polyplexes, which formed 250-nm aggregate particles, resulted in about sixfold more dystrophin-positive fibers than AO alone. Western analysis confirmed the dystrophin expression in these muscles. Surprisingly, injections of AO complexed with high Mw PEI25000(PEG5000) copolymers, which formed smaller nonaggregated particles, produced about threefold fewer dystrophin-positive fibers than injections of the low Mw polyplexes. We conclude that low Mw PEI2000(PEG550) copolymers function as high-capacity, nontoxic AO carriers suitable for in vivo transfection of skeletal muscle and are promising compounds for potential use in molecular therapy of DMD.     

Enhanced expression of recombinant dystrophin following intramuscular injection of Epstein-Barr virus (EBV)-based mini-chromosome vectors in mdx mice.

Gene transfer by direct intramuscular injection of naked plasmid DNA has been shown to be a safe, simple but relatively inefficient method for gene delivery in vivo. Eukaryotic plasmid expression vectors incorporating the Epstein-Barr virus (EBV) origin of replication (oriP) and EBNA1 gene have been shown to act as autonomous episomally replicating gene transfer vectors which additionally provide nuclear matrix retention functions. Prolonged expression of a LacZ reporter gene and recombinant human dystrophin was shown using EBV-based plasmid vectors transfected into C2C12 mouse myoblast and myotube cultures. Intramuscular injection of EBV-based dystrophin expression plasmids into nude/mdx mice resulted in significant enhancement in the number of muscle fibres expressing recombinant dystrophin compared with a conventional vector. This effect was observed for over 10 weeks after a single administration. These results indicate the potential advantage of EBV-based expression vectors for focal plasmid-mediated gene augmentation therapy in Duchenne muscular dystrophy (DMD) and a range of other gene therapeutic applications.     

Tolerance induced by physiological levels of secreted proteins in transgenic mice expressing human insulin.

We have used transgenic mice to study immune tolerance to autologous, non-MHC encoded proteins that are expressed at physiological levels in the circulation. The transgenic mice used in these studies express the human preproinsulin gene and synthesize human proinsulin. Human and mouse insulin are secreted from the pancreatic islets of transgenic mice in response to normal physiological stimuli, such as glucose. Our data demonstrate that the transgenic mice have acquired tolerance to human insulin. The repertoire of T cells specific for exogenous antigens is shaped by the acquired tolerance to autologous proteins since pork but not beef or sheep insulin is also nonimmunogenic in the transgenic mice. We also found that the transgenic mice were tolerant to human proinsulin, the intracellular precursor of insulin. Unresponsiveness to human proinsulin most likely results from tolerance of insulin-specific and proinsulin-specific T cells that recognize the secreted enzymatic cleavage products of proinsulin, insulin and C-peptide.     

Function of B cells expressing a human immunoglobulin M rheumatoid factor autoantibody in transgenic mice

We have generated transgenic mice that express the immunoglobulin (Ig)M heavy chain and kappa light chain genes coding for a human IgM rheumatoid factor (RF), Les. Transgenic B cells expressing human IgM RF show striking similarities to their counterparts in normal humans. They comprise a significant proportion of the adult B cell population, but secrete only low levels of RF into the serum. The RF transgene- expressing B cells localize to primary B cell follicles and the mantle zone regions of secondary follicles in the spleen. Using these mice we have been able to show that one of the central functions of normal RF- expressing B cells may be to act as highly efficient antigen-presenting cells for low concentrations of immune-complexed antigen. High levels of secretion of IgM RF can not be induced under normal circumstances, although RF-expressing B cells proliferate well in vitro to both aggregated human IgG and anti-human IgM antibodies. However, these mice are not intrinsically secretion deficient. By crossing the RF transgenic mice with the autoimmune MRL/lpr background, we find a dramatic increase, > 200-fold, in levels of serum RF. The results strongly suggest that a major function of normal resting RF B cells is unrelated to antibody secretion. Rather, the RF B cells in the follicles may play a role in antigen presentation and regulation of immune responses to antibody-bound nonself-, and possibly self- antigens. This physiologic role of RF B cells may be disrupted in RF- associated autoimmune disease.     

CTLA4Ig delivered by high-capacity adenoviral vector induces stable expression of dystrophin in mdx mouse muscle

Adenoviral (Ad) vector-mediated gene delivery of normal, full-length dystrophin to skeletal muscle provides a promising strategy for the treatment of Duchenne muscular dystrophy (DMD), an X-linked recessive, dystrophin-deficient muscle disease. Studies in animal models suggest that successful DMD gene therapy by Ad vector-mediated gene transfer would be precluded by cellular and humoral immune responses induced by vector capsid and transgene proteins. To address the immunity induced by Ad vector-mediated dystrophin gene delivery to dystrophic muscle, we developed high-capacity adenoviral (HC-Ad) vectors expressing mouse dystrophin driven by the muscle creatine kinase promoter (AdmDys) and mCTLA4Ig (AdmCTLA4Ig) individually, or together from one vector (AdmCTLA4Ig/mDys). We found stable expression of dystrophin protein in the tibialis anterior muscles of mdx mice, coinjected with AdmCTLA4Ig and AdmDys, or injected alone with AdmCTLA4Ig/mDys, whereas the expression of dystrophin protein in the control group coinjected with AdmDys and an empty vector decreased by at least 50% between 2 and 8 weeks after administration. Additionally, we observed reductions in Ad vector-induced Th1 and Th2 cytokines, Ad vector-specific cytotoxic T lymphocyte activation and neutralizing anti-Ad antibodies in both experimental groups that received a mCTLA4Ig-expressing vector as compared to the control group. This study demonstrates that the coexpression of mCTLA4Ig and dystrophin in skeletal muscle provided by HC-Ad vector-mediated gene transfer can provide stable expression of dystrophin in immunocompetent, adult mdx mouse muscle and applies a potentially powerful strategy to overcome adaptive immunity induced by Ad vector-mediated dystrophin gene delivery toward the ultimate goal of treatment for DMD.     

Impaired glucose homeostasis in transgenic mice expressing the human transient neonatal diabetes mellitus locus, TNDM

Transient neonatal diabetes mellitus (TNDM) is a rare inherited diabetic syndrome apparent in the first weeks of life and again during early adulthood. The relative contributions of reduced islet beta cell number and impaired beta cell function to the observed hypoinsulinemia are unclear. The inheritance pattern of this imprinted disorder implicates overexpression of one or both genes within the TNDM locus: ZAC, which encodes a proapoptotic zinc finger protein, and HYMAI, which encodes an untranslated mRNA. To investigate the consequences for pancreatic function, we have developed a high-copy transgenic mouse line, TNDM29, carrying the human TNDM locus. TNDM29 neonates display hyperglycemia, and older adults, impaired glucose tolerance. Neonatal hyperglycemia occurs only on paternal transmission, analogous to paternal dependence of TNDM in humans. Embryonic pancreata of TNDM29 mice showed reductions in expression of endocrine differentiation factors and numbers of insulin-staining structures. By contrast, beta cell mass was normal or elevated at all postnatal stages, whereas pancreatic insulin content in neonates and peak serum insulin levels after glucose infusion in adults were reduced. Expression of human ZAC and HYMAI in these transgenic mice thus recapitulates key features of TNDM and implicates impaired development of the endocrine pancreas and beta cell function in disease pathogenesis.     

Crohn's disease adherent-invasive Escherichia coli colonize and induce strong gut inflammation in transgenic mice expressing human CEACAM

Abnormal expression of CEACAM6 is observed at the apical surface of the ileal epithelium in Crohn''s disease (CD) patients, and CD ileal lesions are colonized by pathogenic adherent-invasive Escherichia coli (AIEC). We investigated the ability of AIEC reference strain LF82 to colonize the intestinal mucosa and to induce inflammation in CEABAC10 transgenic mice expressing human CEACAMs. AIEC LF82 virulent bacteria, but not nonpathogenic E. coli K-12, were able to persist in the gut of CEABAC10 transgenic mice and to induce severe colitis with reduced survival rate, marked weight loss, increased rectal bleeding, presence of erosive lesions, mucosal inflammation, and increased proinflammatory cytokine expression. The colitis depended on type 1 pili expression by AIEC bacteria and on intestinal CEACAM expression because no sign of colitis was observed in transgenic mice infected with type 1 pili–negative LF82-ΔfimH isogenic mutant or in wild-type mice infected with AIEC LF82 bacteria. These findings strongly support the hypothesis that in CD patients having an abnormal intestinal expression of CEACAM6, AIEC bacteria via type 1 pili expression can colonize the intestinal mucosa and induce gut inflammation. Thus, targeting AIEC adhesion to gut mucosa represents a new strategy for clinicians to prevent and/or to treat ileal CD.Inflammatory bowel disease (IBD) mainly consists of two disorders, ulcerative colitis and Crohn''s disease (CD), with a combined prevalence of ∼150–200 cases per 100,000 in Western countries (Shanahan, 2002; Loftus, 2004). The abnormal inflammatory response observed in IBD requires interplay between host genetic factors and the intestinal microbiota (Podolsky, 2002; for review see Strober et al., 2007). The role of the microbiota in IBD development is highlighted with the following observations: in CD patients, postsurgical exposure of the terminal ileum to luminal contents is associated with increased inflammation, and diversion of the fecal stream is associated with improvement (Rutgeerts et al., 1991); some IBD patients improve upon antibiotic treatment (Sartor, 2004; Sands, 2007); the severity of colitis in multiple animal models is decreased by the administration of antibiotics; and no sign of colitis is observed when those animals are in germ-free conditions (for review see Sartor, 2008).Two broad hypotheses have arisen regarding the role of the intestinal microbiota in the pathogenesis of IBD. Several lines of evidence support the notion that IBD results from an excessive immune response to gut commensal organisms (for review see Strober et al., 2007). However, the disease could result from a problem in the composition of the microflora leading to generalized or localized dysbiosis. Thus, a breakdown in the balance between putative species of “protective” versus “harmful” intestinal bacteria has been reported and may promote inflammation. A low proportion of Faecalibacterium prausnitzii on resected ileal Crohn mucosa is associated with endoscopic recurrence at 6 mo, and this bacteria has antiinflammatory properties (Tamboli et al., 2004; Sokol et al., 2008). In addition, host-mediated inflammation in response to a pathogen infection can disrupt the intestinal microbiota and shift the balance between the protective microbiota and the pathogen in favor of the pathogen, as seen with Citrobacter rodentium infection promoting the overgrowth of Enterobacteriaceae (Lupp et al., 2007) and with Salmonella Typhimurium infection (Stecher et al., 2007). In patients with CD and ulcerative colitis, high concentrations of bacteria forming a biofilm on the surface of the gut mucosa (Swidsinski et al., 2002) and increased numbers of mucosa-associated Escherichia coli are observed (Darfeuille-Michaud et al., 1998; Martin et al., 2004; Conte et al., 2006; Baumgart et al., 2007; Kotlowski et al., 2007; Sasaki et al., 2007). This overgrowth of E. coli can result from host-mediated inflammation or abnormal expression of molecules acting as receptors for bacterial adhesion. In CD patients with ileal involvement of the disease, we recently reported an abnormal ileal expression of carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) 5 and 6 (Barnich et al., 2007) and showed that only CEACAM6 acts as a receptor for pathogenic E. coli. These bacteria, called adherent-invasive E. coli (AIEC), colonize the ileal mucosa of CD patients (Darfeuille-Michaud et al., 2004). They are able to adhere to and invade intestinal epithelial cells and to survive and highly replicate within macrophages, leading to the secretion of high amounts of TNF (Boudeau et al., 1999; Glasser et al., 2001). Interestingly, in vitro studies demonstrated that CEACAM6 expression is increased in cultured intestinal epithelial cells not only after IFN-γ or TNF stimulation (Fahlgren et al., 2003) but also after infection with AIEC bacteria, indicating that AIEC could promote their own colonization in CD patients (Barnich et al., 2007). In the present paper, using transgenic CEABAC10 mice harboring a bacterial artificial chromosome that contains part of the human CEA family gene cluster, including complete human CEACAM3, CEACAM5 (CEA), CEACAM6, and CEACAM7 genes (Chan and Stanners, 2004), we investigated whether LF82 bacteria isolated from a CD patient can colonize the intestinal mucosa as a result of CEACAM expression and whether AIEC LF82 colonization could lead to the development of gut inflammation.     

Hypoplasia of pancreatic islets in transgenic mice expressing activin receptor mutants.

Activin, a member of the TGF-beta superfamily, regulates the growth and differentiation of a variety of cell types. Based on the expression of activin in pancreatic rudiments of rat embryos and stimulation of insulin secretion from adult rat pancreatic islets by activin, activin is implicated in the development and function of islets. To examine the significance of activin signaling in the fetal and postnatal development of islets, transgenic mice expressing a dominant negative form of activin receptor (dn-ActR) or a constitutively active form of activin receptor (ActR-T206D) in islets were generated together with the transgenic mice expressing intact activin receptor (intact ActR) as a negative control. Transgenic mice with both dn-ActR and ActR-T206D showed lower survival rates, smaller islet area, and lower insulin content in the whole pancreas with impaired glucose tolerance when compared with transgenic mice with intact ActR or littermates, but they showed the same alpha cell/beta cell ratios as their littermates. In addition to islet hypoplasia, the insulin response to glucose was severely impaired in dn-ActR transgenic mice. It is suggested that a precisely regulated intensity of activin signaling is necessary for the normal development of islets at the stage before differentiation into alpha and beta cells, and that activin plays a role in the postnatal functional maturation of islet beta cells.     

鞘脂激活蛋白原在mdx小鼠发病早期心肌的表达

目的 检测鞘脂激活蛋白原(prosaposin)mRNA和蛋白在mdx小鼠发病早期心肌内的表达情况.方法 取3～4周mdx鼠和C57BL/6J对照鼠心肌组织,4%多聚甲醛固定,石蜡切片,HE染色检测形态学改变,免疫组织化学染色检测prosaposin蛋白表达.新鲜取材做冰冻切片,原位杂交检测prosaposin mRNA表达情况并进行统计学分析.结果 mdx鼠心肌细胞在形态上无明显改变,原位杂交结果显示prosaposin mRNA在C57BL/6J对照鼠心肌主要以Ps+0形式存在,且在mdx鼠心肌组织表达明显减少(P<0.01),免疫组织化学结果亦提示prosaposin在mdx鼠心肌组织表达量减少.结论 在mdx小鼠发病早期,pmsaposin mRNA和蛋白在心肌表达量减少.