Noncultured cell transplantation in an ovine model of right ventricular preparation

OBJECTIVE: Beyond the first 2 months of life, pulmonary artery banding is warranted before two-stage arterial switch operation. The aim of this study was to explore whether myogenic cell transplantation could contribute to right ventricular function during pulmonary artery constriction in an ovine model. METHODS: Sixteen rams were assigned to one of the following groups: group 1, simple pulmonary artery banding (n = 5); group 2, pulmonary artery banding and cell implantation in the right ventricle (n = 7); and group 3, pulmonary artery banding and placebo injection in the right ventricle (n = 4). Hemodynamic assessment with pressure-volume loops was performed on days 0 and 60. The pulmonary artery banding and the injections were achieved through a left fourth intercostal thoracotomy. Autologous myogenic cell implantation was carried out with a noncultured cell preparation, as previously described by our group. Implanted sites were processed with monoclonal antibodies to a fast skeletal-specific isoform of myosin heavy chain (MY32). RESULTS: Skeletal myosin heavy chain expression was detected at 2 months after noncultured cell implantation in all grafted animals. Right ventricular training resulted in statistically significant increased signs of contractility in all three groups. There was no observed difference, however, between the cell therapy group and the other two groups with respect to signs of cardiac function. CONCLUSION: Successful engraftment of noncultured cells into right ventricular myocardium did not translate into a functional benefit that we could demonstrate in our ovine model. Cellular therapy thus is probably not useful to strengthen a left ventricle being retrained through pulmonary artery banding before arterial switch operation. However, cell transplantation may affect the outcome of right ventricular failure long term after atrial switch operation. Although preliminary, this investigation paves the way for further research into cellular cardiomyoplasty, right ventricular failure, and congenital heart disease.     

Changes in mass and performance in rabbit muscles after muscle damage with or without transplantation of primary satellite cells

Changes in morphology, metabolism, myosin heavy chain gene expression, and functional performances in damaged rabbit muscles with or without transplantation of primary satellite cells were investigated. For this purpose, we damaged bilaterally the fast muscle tibialis anterior (TA) with either 1.5 or 2.6 ml cardiotoxin 10(-5) M injections. Primary cultures of satellite cells were autotransplanted unilaterally 5 days after muscle degeneration. Two months postoperation, the masses of damaged TAs, with or without transplantation, were significantly larger than those of the controls. Furthermore, damaged transplanted muscles weighed significantly more than damaged muscles only. The increase in muscle mass was essentially due to increased fiber size. These results were independent of the quantity of cardiotoxin injected into the muscles. Maximal forces were similar in control and 2.6 ml damaged TAs with or without satellite cell transfer. In contrast, 1.5 ml damaged TAs showed a significant decrease in maximal forces that reached the level of controls after transplantation of satellite cells. Fatigue resistance was similar in control and 1.5 ml damaged TAs independently of satellite cell transfer. Fatigue index was significantly higher in 2.6 ml damaged muscles with or without cell transplantation. These changes could be explained in part by muscle metabolism, which shifted towards oxidative activities, and by gene expression of myosin heavy chain isoforms, which presented an increase in type IIa and a decrease in type I and IIb in all damaged muscles with or without cell transfer. Under our experimental conditions, these results show that muscle damage rather than satellite cell transplantation changes muscle metabolism, myosin heavy chain isoform gene expression, and, to a lesser extent, muscle contractile properties. In contrast, muscle weight and fiber size are increased both by muscle damage and by satellite cell transfer.     

血管植入变性骨骼肌与自体神经瘤片段联合移植修复周围神经陈旧性缺损

目的 评估血管植入变性骨骼肌与自体神经瘤片段联合移植修复陈旧性神经缺损的效果。方法 用自体神经移植及血管植入变性骨骼肌作为对照组比较，经5个月观察，采用电生理和形态定量学的检测。结果 联合移植组在电生理、再生轴突密度恢复率和再生轴突面积恢复率方面与血管植入肌桥组有显著性差异（P＜0.05）。与自体神经移植组无显著性差异（P＞0.05）。结论 联合移植能获得与自体神经移植相似的效果。     

Effect of heart transplantation on skeletal muscle metabolic enzyme reserve and fiber type in end-stage heart failure patients

BACKGROUND: Skeletal muscle myopathy is a hallmark of chronic heart failure (HF). Phenotypic changes involve shift in myosin heavy chain (MHC) fiber type from oxidative, MHC type I, towards more glycolytic MHC IIx fibers, reductions in oxidative enzyme activity, and increase in glycolytic enzyme activity. However, it is unknown if muscle myopathy is reversed following heart transplantation. The purpose of this study was to determine the effect of heart transplantation on skeletal muscle metabolic enzyme reserve and MHC fiber type in end-stage HF patients. METHODS: Thirteen HF subjects were prospectively studied before and two months after heart transplantation and a subgroup (n = 6) at eight months after transplantation. Skeletal muscle biopsy of the vastus lateralis was performed and relative MHC composition was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lactate dehydrogenase (LDH), citrate synthase (CS), and 3-hydroxyacyl-CoA-dehydrogenase (HACoA) enzyme activity assays were performed to assess glycolytic, oxidative, and beta-oxidative metabolic enzyme reserves, respectively. RESULTS: Lactate dehydrogenase activity (130.5 +/- 13.3 vs. 106.1 +/- 13.2 micromol/g wet wt/min, p < 0.05), CS activity (14.0 +/- 1.2 vs. 9 +/- 0.9 micromol/g wet wt/min, p < 0.05), and HACoA activity (4.5 +/- 0.48 vs. 3.6 +/- 0.3 micromol/g wet wt/min, p < 0.05) decreased two months after heart transplantation. At eight months, LDH activity was restored (139.0 +/- 11 micromol/g wet wt/min), but not CS or HACoA activity compared with before transplantation. There was no significant change in muscle %MHC type I (28.7 +/- 3.5% vs. 25.3 +/- 3.0%, p = NS), %MHC type IIa (33.2 +/- 2.0% vs. 34.6 +/- 1.9%, p = NS), or %MHC type IIx (38.1 +/- 2.8% vs. 40.1 +/- 3.7%, p = NS) fiber type two months after heart transplantation. However, %MHC type I (19.3 +/- 6.6%) was decreased and %MHC type IIx (51.0 +/- 6.5%) was increased at eight months after (p < 0.05) compared with before transplantation. CONCLUSIONS: Skeletal muscle glycolytic, oxidative, and beta-oxidative enzymatic reserves are diminished early after heart transplantation, with reduced oxidative capacity persisting late in the first year. The myopathic MHC phenotype present in end-stage HF persists early in the post-operative state and declines further by eight months.     

Comparison of the effects of fetal cardiomyocyte and skeletal myoblast transplantation on postinfarction left ventricular function

OBJECTIVES: Transplantation of fetal cardiomyocytes improves function of infarcted myocardium but raises availability, immunologic, and ethical issues that justify the investigation of alternate cell types, among which skeletal myoblasts are attractive candidates. METHODS: Myocardial infarction was created in rats by means of coronary artery ligation. One week later, the animals were reoperated on and intramyocardially injected with culture growth medium alone (controls, n = 15), fetal cardiomyocytes (5 x 10(6) cells, n = 11), or neonatal skeletal myoblasts (5 x 10(6) cells, n = 16). The injections consisted of a 150-microL volume and were made in the core of the infarct, and the animals were immunosuppressed. Left ventricular function was assessed by echocardiography immediately before transplantation and 1 month thereafter. Myoblast-transplanted hearts were then immunohistologically processed for the expression of skeletal muscle-specific embryonic myosin heavy chain and cardiac-specific connexin 43. RESULTS: The left ventricular ejection fraction markedly increased in the fetal and myoblast groups from 39.3% +/- 3.9% to 45% +/- 3.4% (P =.086) and from 40.4% +/- 3.6% to 47.3% +/- 4.4% (P =.034), respectively, whereas it decreased in untreated animals from 40.6% +/- 4% to 36.7% +/- 2.7%. Transplanted myoblasts could be identified in all animals by the positive staining for skeletal muscle myosin. Conversely, clusters of connexin 43 were not observed on these skeletal muscle cells. CONCLUSIONS: These results support the hypothesis that skeletal myoblasts are as effective as fetal cardiomyocytes for improving postinfarction left ventricular function. The clinical relevance of these findings is based on the possibility for skeletal myoblasts to be harvested from the patient himself.     

Time course of physical reconditioning during exercise rehabilitation late after heart transplantation.

BACKGROUND: Exercise rehabilitation improves physical capacity in heart transplant recipients. The time course of physical reconditioning and skeletal muscle adaptation late after transplantation are unknown. METHODS: Twenty-one heart transplant recipients, at 5.2 +/- 2.1 years after transplantation, completed 1 year of an individually tailored home ergometer-training program (2.1 +/- 0.7 sessions weekly with matched heart rates, intensity at 10% below anaerobic threshold). We analyzed time course of physical reconditioning data for each home-training session (n = 2,396). Constant-load tests with consistent blood lactate concentrations were performed quarterly (n = 105) to estimate the time course of skeletal muscle adaptation. Nine heart transplant recipients served as a control group (CG). RESULTS: After 12 months, exercise capacity for matched heart rates (112 +/- 11 beats/min; CG, 114 +/- 8 beats/min) increased by 35% +/- 19% (from 43 +/- 14 to 58 +/- 18 W; p < 0.001; CG, 53 +/- 18 to 54 +/- 18 W); 24% of the increase was caused by improved skeletal muscle function and 11% by central functioning. Physical reconditioning showed its greatest increase within the first 3 months (+18%; p < 0.001); 50% of the increase consisted of better skeletal muscle or central functioning. Between the 4(th) and 12(th) months, exercise capacity increased continuously (+15%; p < 0.001), mainly because of better skeletal muscle functioning. CONCLUSIONS: The persistent improvement in exercise capacity along with consistent lactate concentrations during 12 months of training indicates that exercise training could counteract the negative side effects of immunosuppressive treatment on skeletal muscles. Even late after heart transplantation, physical training should be performed regularly to prevent the accelerated decrease in exercise capacity and in skeletal muscle function.     

Myosin and myoglobin as tumor markers in the diagnosis of rhabdomyosarcoma. A comparative study

Antibodies against the myosin heavy chain of adult chicken pectoral muscle and heart muscle which cross-react with myosin of human fast type II fibers ( antifast myosin) and slow type I fibers ( antislow myosin), respectively, and antibodies against human myoglobin have been assessed for their usefulness in diagnosing rhabdomyosarcoma. Formaldehyde-fixed and paraffin-embedded tissue and the avidin-biotinyl-peroxidase complex technique were used. Of 23 rhabdomyosarcomas studied, 20 were positive with antifast myosin and 11 with antimyoglobin . All tumors were negative with antislow myosin. Positive staining was observed in all three types of rhabdomyosarcoma, i.e., embryonal, alveolar, and pleomorphic, regardless of the antiserum used. Staining with antimyoglobin was generally limited to the cytoplasm-rich tumor cells. Besides rhabdomyosarcomas, the only other positive neoplasms were those which contained rhabdomyoblastic differentiation such as malignant Triton tumors and malignant mixed müllerian tumors. Our results indicate that antibodies against the fast myosin heavy chain are a useful tool for diagnosing rhabdomyosarcoma and that they can be used to distinguish that tumor from other small round cell tumors in childhood. The results are discussed in the light of the embryogenesis of skeletal muscle.     

Combined autologous cellular cardiomyoplasty with skeletal myoblasts and bone marrow cells in canine hearts for ischemic cardiomyopathy

OBJECTIVES: Cellular cardiomyoplasty with isolated skeletal myoblasts and bone marrow mononuclear cells is an encouraging therapeutic strategy for heart failure. We investigated the achievements accomplished with combined cell therapy of skeletal myoblast and bone marrow mononuclear cell transplantation to the ischemic canine myocardium. METHODS: Autologous skeletal myoblasts (1 x 10(8)) and autologous bone marrow mononuclear cells (3 x 10(6)) were injected directly into the damaged myocardium of canine hearts that had undergone 2 weeks of left anterior descending coronary artery ligation. Treatment groups were as follows: skeletal myoblasts plus bone marrow mononuclear cells (combined cell therapy, n = 4), myoblasts (n = 4), bone marrow mononuclear cells (n = 4), and medium only (n = 4). In similarly designed supporting experiments, angiogenic factor expression was evaluated by enzyme-linked immunosorbent assay after cell transplantation in rat hearts that had undergone left anterior descending coronary artery ligation. RESULTS: Four weeks after cell implantation, echocardiography demonstrated better cardiac performance with reduced left ventricular dilation and significantly improved ejection fraction in the combined cell therapy group compared with that seen in the other groups (pretreatment, 37.7% +/- 1.1%, vs combined cell therapy, 55.4% +/- 8.6%; myoblasts, 47.4% +/- 7.4%; bone marrow mononuclear cells, 44.4% +/- 6.7%; medium only [control], 34.4% +/- 5.4%; P < .05). A significantly high number of neovessels were observed in the group receiving combined cell therapy only (combined cell therapy, 45.5 +/- 12 x 10(2)/mm2; myoblasts, 26.5 +/- 8 x 10(2)/mm2; bone marrow mononuclear cells, 30.7 +/- 15 x 10(2)/mm2; medium only [control], 7.1 +/- 1 x 10(2)/mm2; P < .05). Immunostained sections expressed the skeletal specific marker myosin heavy chain, although they did not express the cardiac specific marker troponin T. Results of enzyme-linked immunosorbent assay showed the highest expression of vascular endothelial growth factor (combined cell therapy, 2.9 +/- 0.7 ng/g tissue; myoblasts, 0.24 +/- 0.7 ng/g tissue; bone marrow mononuclear cells, 1.9 +/- 0.2 ng/g tissue; medium only [control], 0.19 +/- 0.004 ng/g tissue; P < .05) and hepatocyte growth factor in the combined cell therapy hearts. CONCLUSIONS: Combined autologous cellular therapy induced both myogenesis and angiogenesis with enhancement of cardiac performance and reduction of cardiac remodeling, suggesting a capable strategy for treating severe ischemic cardiomyopathy clinically.     

Cellular therapy reverses myocardial dysfunction

OBJECTIVES: Cellular cardiomyoplasty refers to the implantation of autologous skeletal muscle cells into the myocardium to reinforce its structure and function. In this study a reproducible method for the creation of a myocardial lesion was developed. The functional benefit of cell implantation was evaluated by 2-dimensional echocardiography for global contraction and color kinesis echocardiography, which allows the precise assessment of the regional contraction. METHODS: A left ventricular intramyocardial injection with snake cardiotoxin was carried out on a sheep model to induce a well-delineated transmural lesion. Three weeks later, the lesion was assessed by echocardiography. Thereafter, autologous skeletal muscle cells or culture media (control) were injected into the lesion. Two months after cell implantation, the myocardial contraction was again evaluated by echocardiography and the implanted cells were analyzed by a fast myosin heavy chain antibody. RESULTS: 1. The snake cardiotoxin produced a well-delineated transmural lesion in all animals. 2. Echocardiographic studies showed a significant improvement in global and regional left ventricular function in cell-treated sheep. 3. Histologic analyses demonstrated satellite cell survival at the periphery of the lesions. CONCLUSION: Satellite cells implanted in a cardiotoxin-induced myocardial lesion survived for a 2-month period and were associated with a significant functional improvement of both local and global contraction.     

Transplantation of cryopreserved muscle cells in dilated cardiomyopathy: effects on left ventricular geometry and function

OBJECTIVE: Cell transplantation to prevent congestive heart failure in patients with inherited dilated cardiomyopathy might require the use of noncardiac donor cells unaffected by the genetic defect and cryopreservation to permit cell storage until the time of transplantation. However, the effects of cryopreservation on peripheral muscle cells harvested from a cardiomyopathic recipient and their subsequent ability to restore cardiac structure and function after transplantation are unknown. METHODS: Skeletal myoblasts and vascular smooth muscle cells from cardiomyopathic hamsters (delta-sarcoglycan-deficient BIO 53.58 hamster) and age-matched normal donor hamsters were isolated, expanded in culture, and cryopreserved. After reanimation in culture, cell morphology and growth rate were assessed and compared with values seen in noncryopreserved cells. A total of 4 x 10(6) previously cryopreserved skeletal myoblasts (n = 10) and vascular smooth muscle cells (n = 10) harvested from cardiomyopathic donors were then transplanted into the left ventricles of 17-week-old BIO 53.58 hamsters. Hearts injected with culture medium alone (n = 11) served as controls. Heart function was assessed 5 weeks after transplantation on a Langendorff apparatus, and left ventricular geometry was quantified by means of computerized planimetry. Staining with 5-bromo-2'-deoxyuridine identified the injected cells. RESULTS: Vascular smooth muscle cells from cardiomyopathic donors had an abnormal morphology and diminished growth rates in culture compared with vascular smooth muscle cells from normal donors. These markers of injury were exacerbated by cryopreservation. In contrast, vascular smooth muscle cells from normal donors and skeletal myoblasts from either cardiomyopathic or normal donors appeared normal in culture and were unaffected by cryopreservation. Both cryopreserved vascular smooth muscle cells and skeletal myoblasts from cardiomyopathic donors formed a viable muscle-resembling tissue that prevented wall thinning, limited left ventricular dilatation, and preserved global systolic function in hamsters with a genetic dilated cardiomyopathy. However, attenuation of cardiac remodeling and preservation of global heart function was greater after skeletal myoblast transplantation compared with vascular smooth muscle cell transplantation in parallel to the in vitro morphologic and growth characteristics of these cells. CONCLUSIONS: Cryostorage of healthy donor cells does not prevent the benefits of cell transplantation on limiting remodeling and preserving cardiac function in the failing heart. The health of donor cells in vitro predicts their subsequent benefits on cardiac structure and function after transplantation. Cryopreservation of donor cells might facilitate a clinically applicable and effective approach for ventricular restoration with cell-transplantation therapy for patients with inherited dilated cardiomyopathy.     

Cell transplantation to prevent heart failure: a comparison of cell types

BACKGROUND: Autologous cell transplantation may restore viable muscle after a myocardial infarction. We compared the effect of three cell types or an angiotensin-converting enzyme (ACE) inhibitor on preservation of ventricular function after cardiac injury. METHODS: A uniform transmural myocardial scar was created in adult rats by cryoinjury. Three weeks later the rats were randomly assigned to one of four blinded treatments: transplantation with 5 x 10(6) aortic smooth muscle cells (SMC, n = 12), ventricular heart cells (VHC, n = 13), skeletal muscle cells (SKC, n = 13) or culture medium alone (control, n = 11). The ACE inhibitor group (n = 8) received enalapril (1.0 mg/kg per day), also beginning 3 weeks after cryoinjury. Five and 12 weeks after transplantation, left ventricle (LV) function was assessed in a Langendorff apparatus, and histologic and immunohistological evaluation of the LV scars was performed. RESULTS: At 5 weeks, greater scar elastin content and better LV function was noted with cell transplantation or ACE inhibitor therapy compared with control rats (p < 0.05). Twelve weeks after transplantation, cell-transplanted rats still had greater elastin content and better LV function than control rats, although elastin content and LV function had declined in ACE inhibitor-treated animals to levels below those observed in control rats (p < 0.05). CONCLUSIONS: Transplantation of SMC, VHC, and SKC preserved ventricular function equivalent to the effects of an ACE inhibitor. Muscle cell transplantation, but not ACE inhibitor therapy, continues to be effective later after cryoinjury. No differences were detected between the muscle cells.     

From dynamic to cellular cardiomyoplasty

Dynamic Cardiomyoplasty. Latissimus dorsi dynamic cardiomyoplasty has been used in our institution for heart failure patients refractory to medical therapy; 113 cases were operated at Broussais and Pompidou Hospitals and 75 patients by our team abroad, in the scope of an international cooperative program. Cardiomyoplasty has been associated with better results due to technical improvements, the most significant mini-invasive techniques, the latest the use of growth factors to enhance muscle vascularization. Risk factors have been identified, resulting in more precise indications, a lower hospital mortality, and a wider use of this operation. There has been a new tendency to associate cardiomyoplasty with electrophysiological therapies: implantation of ventricular defibrillators and multisite cardiac pacing (for atrioventricular and interventricular resynchronization). Cellular Cardiomyoplasty. Adult myocardium cannot repair after infarction due to the absence of stem cells. Cell transplantation strategies for heart failure have been designed to replace damaged cells with cells that can perform cardiac work. Current possibilities in cell therapy for heart failure is the transplantation into the infarcted myocardium of autologous myoblasts (satellite cells originated from skeletal muscle), fetal cardiomyocytes, autologous heart cells, cells derived from bone marrow stem cells, and smooth muscle cells. Experimental studies demonstrated that cell transplantation into the myocardium was associated with the recovery of myocardial contractility and compliance, as well as the diastolic pressure-strain relationship in animal models (infarctlike myocardial lesions and dilated cardiomyopathy models). Healthy myoblasts and myotubes were observed 2 months after myocardial implantation. Clinical studies are now in progress.     

Xenogeneic embryonic stem cell-derived cardiomyocyte transplantation

PURPOSE: The purpose of this study was to investigate the survival of xenogeneic embryonic stem cell (ES cell)-derived cardiomyocytes transplanted into the normal myocardium. MATERIAL AND METHODS: Undifferentiated mouse ES cells carrying the enhanced green fluorescent protein (EGFP) were cultured in hanging drops and then plated onto dishes. These cells were identified as cardiomyocytes by the expression of cardiac-specific genes, recording of action potential, and immunostaining with anti-sarcomeric myosin antibody. Donor cells were injected into the normal myocardium, with cyclosporine administered daily. One week after the transplantation, we investigated donor cell survival by examining EGFP expression, hematoxylin and eosin staining, and immunostaining with anti-sarcomeric myosin antibody. RESULTS: In vitro donor cells derived from ES cells expressed myosin light chain-2v and alpha-myosin heavy chain genes, had action potentials of a ventricular myocyte type, and were stained by anti-sarcomeric myosin antibody. In vivo 1 week after transplantation, EGFP-expressed cells were detected in the cell transplanted area. No lymphocytic infiltration was observed around these cells. CONCLUSIONS: ES cell-derived cardiomyocytes survived in the normal myocardium after the transplantation, even in a discordant xenogeneic transplantation model. These results indicate that cell transplantation using cardiomyocytes derived from ES cells, even if xenogeneic represents an attractive strategy for treating heart disease.     

Effectiveness of transient immunosuppression using cyclosporine for xenomyoblast transplantation for cardiac repair

We studied the survival of human myoblast for cellular myocardial reconstruction in a porcine model of chronic myocardial ischemia with immune tolerance using transient immunosuppression. A porcine model of chronic cardiac ischemia was created in 10 pigs (DMEM medium-injected n = 4; myoblast transplanted n = 6) by clamping ameroid ring around left circumflex coronary artery. Three weeks later, 3 x 10(8) human myoblasts carrying lac-z reporter gene were transplanted in multiple sites (0.25 mL each) into the left ventricular wall. Immunosuppression was achieved with 5 mg/kg cyclosporine for 6 weeks after cell transplantation. After animals were euthanized between 6 and 30 weeks after cell transplantation; the heart was removed for histological studies. Discontinuation of immunosuppression after 6 weeks of cell transplantation did not result in donor cell rejection. The lac-z-positive donor cells were detected in porcine host cardiac tissue for up to 30 weeks posttransplantation, expressing human skeletal myosin heavy chain. The results highlight the effectiveness of transient immunosuppression for myoblast transplantation for cardiac repair.     

An ovine model of chronic heart failure: echocardiographic and tissue Doppler imaging characterization

BACKGROUND AND AIM OF THE STUDY: Heart failure in the western world is a major health-care issue. In order to validate novel surgical or pharmacological treatments, reproducible animal models of left ventricular dysfunction are necessary. In the current study, we report our data and experience with a model of toxin-induced heart failure in the sheep. METHODS: Sequential intracoronary injections of doxorubicin (0.75 mg/kg) were carried out every 2 weeks until standard echocardiographic and tissue Doppler imaging detection of myocardial systolic dysfunction. The animals were assessed 1 month later and harvested. Indices of cardiac function from baseline to last day of protocol were recorded and their differences were evaluated by a Wilcoxon rank test for paired data. RESULTS: Ten sheep received 2.5 +/- 0.7 intracoronary injections of a cumulative dose of 88.8 +/- 25 mg/m2 doxorubicin. All available parameters demonstrated signs of severe cardiac dysfunction with statistical significance. All hearts demonstrated severe histological lesions, some of which were consistent with doxorubicin-induced toxicity. CONCLUSIONS: The present study shows that this ovine model is reproducible and stable. It can therefore be relevant to the study of chronic heart failure. It will be incorporated in our future studies concerning novel treatments (such as cell therapy) of nonischemic dilated cardiomyopathy.     

骨髓间质干细胞体外转化为肌细胞的研究

目的 采用骨髓间质干细胞（MSCs)体外转化为肌细胞，为心衰的干细胞移植治疗提供新的细胞材料，方法 抽取贵州香猪骨髓液3ml，按照Wakitani的方法培养骨髓间质干细胞，经5-氮胞苷（5-azacytidine)刺激24h后，进行结蛋白（desmin)，肌球蛋白重链（MHC）免疫组织化学，透射电镜鉴定。结果 骨髓间质干细胞经5-aza刺激后，部分细胞呈梭形，结蛋白，肌球蛋白重链阳性，有多核肌管形成，透射电镜可见明显的肌丝形成，结论 骨髓间质干细胞可在体外的环境下向肌细胞转化。有望成为心衰干细胞移植的较理想细胞材料。     

Nonsustained effect of short-term bisphosphonate therapy on bone turnover three years after renal transplantation

BACKGROUND: We recently showed that two doses of 4 mg of zoledronic acid (ZOL) ameliorated the bone loss and improved bone histology within the first six months after kidney transplantation. The aim of the present study was to evaluate whether this early short-term intervention exhibited a sustained bone-sparing effect. METHODS: A homogenous group of 20 de novo renal transplant recipients were equally randomized to two infusions of 4 mg of ZOL or placebo at two weeks and three months after engraftment. Patients were followed up for three years by sequential determination of bone densitometry and specific biochemical markers. RESULTS: From month six to three years after transplantation, both treatment groups exhibited an improvement of bone mineralization. Femoral neck bone mineral density z-scores increased statistically significantly from -1.3 (2.6) to -0.2 (3.6) in the placebo group and from -1.6 (2.9) to -1.2 (1.9) in the ZOL group (median, range). Biochemical parameters of osteoblast activity such as osteocalcin and bone-specific alkaline phosphatase did not increase significantly in both groups. Osteoprotegerin, a marker of osteoclast inhibition, was significantly elevated over the first six months in the ZOL group, but decreased to similar levels, as in the placebo group, over the next two and a half years. Other markers of osteoclast activity such as c-telopeptide of type 1 collagen, calcitonin, and intact parathyroid hormone were not different between six months and three years in either group. CONCLUSION: The early bone-sparing effect of short-term ZOL therapy confers no sustained benefit versus placebo at three year post-transplantation.     

Single fibers of skeletal muscle as a novel graft for cell transplantation to the heart

OBJECTIVE: Skeletal myoblast transplantation is a promising alternative to treat heart failure. A single fiber, the minimal functional unit of skeletal muscle, retains skeletal myoblasts beneath the basal lamina. When surrounding muscle is injured, myoblasts migrate from the fiber into the damaged area to regenerate muscle. We hypothesized that such isolated fibers could be used as an efficient vehicle to deliver myoblasts into damaged myocardium, resulting in improved cardiac function. METHODS: Living single fibers of rat skeletal muscle were isolated, and their behavior was characterized in vitro. Single fibers were injected into the myocardium (at 4 sites, each receiving a single fiber) of rats in 2 models of heart failure induced either by means of doxorubicin administration or left coronary artery occlusion. RESULTS: Skeletal myoblasts dissociated from an isolated single fiber, proliferated, and differentiated into multinucleated myotubes in vitro. Within 3 days after grafting in vivo, original fibers provided putative myoblasts and disappeared. At 4 weeks, discrete loci consisting of several multinucleated myotubes were observed. Furthermore, single-fiber transplantation significantly improved cardiac function compared with the control treatment in either doxorubicin-treated hearts (maximum dP/dt, 4013.9 +/- 96.1 vs 3603.1 +/- 102.3 mm Hg/s; minimum dP/dt, -2313.7 +/- 75.1 vs. -2057.1 +/- 52.4 mm Hg/s) or ischemic hearts (maximum dP/dt, 3905.6 +/- 103.0 vs 3572.6 +/- 109.7 mm Hg/s; minimum dP/dt, -2336.1 +/- 69.7 vs -2106.4 +/- 74.2 mm Hg/s). CONCLUSION: Single-fiber transplantation acts as a vehicle for delivering putative skeletal myoblasts that appear to differentiate into myotubes within the myocardium. This was associated with improved function of failing hearts, suggesting its efficacy as a novel graft for cellular cardiomyoplasty.     

Pamidronate in the prevention of bone loss after liver transplantation: a randomized controlled trial

Rapid bone loss and high rates of fractures occur following liver transplantation. To analyze the effect of intravenous pamidronate on bone loss after liver transplantation. A randomized, double‐blind, placebo‐controlled study was performed. Seventy‐nine patients were randomized to two groups of treatment: the pamidronate group (n = 38) was treated with 90 mg/IV of pamidronate within the first 2 weeks and at 3 months after transplantation; the placebo group (n = 41) received glucose infusions at the same time points. All patients received calcium and vitamin D. Bone mineral density (BMD) at the lumbar spine (L₂–L₄) and proximal femur using dual energy X‐ray absorptiometry and also spinal X‐rays were performed before, and at 6 and 12 months after liver transplantation. Biochemical and hormonal determinations were performed previous to transplantation, at 24 h before and after treatment, as well as at 6 and 12 months after liver transplantation. At 12 months after transplantation, there were significant differences in lumbar BMD changes (6 months: pamidronate 1.6% vs. placebo 0.8%, P = NS; 12 months: pamidronate 2.9% vs. placebo 1%, P < 0.05). Femoral neck BMD decreased in the pamidronate‐ and placebo groups during the first 6 months (6 months: pamidronate ?3.1% vs. placebo ?2.9%, P = NS; 12 months: pamidronate ?3.2% vs. placebo ?3.1%, P = NS). BMD at the trochanter remained stable in the pamidronate group, whilst a reduction was observed in the placebo group at 6 months (6 months: pamidronate ?0.7% vs. placebo ?3.7%, P < 0.05; 12 months: pamidronate ?0.5% vs. placebo ?1.2%, P = NS). Moreover, no significant differences in the incidence of fractures, serum parathyroid hormone and serum 25‐hydroxyvitamin D values between both groups were found. Pamidronate did not increase the risk of serious adverse events. The results of this study show that 90 mg of intravenous pamidronate within the first 2 weeks and at 3 months following liver transplantation preserve lumbar bone mass during the first year, without significant adverse events. However, pamidronate does not reduce bone loss at the femoral neck and furthermore it does not reduce skeletal fractures.     

Current status and future prospects for cell transplantation to prevent congestive heart failure

Although most cardiac cell therapy trials have focused on patients with acute myocardial infarction, attempts at "regenerating" chronically failing hearts have also been performed. These studies have entailed use of skeletal myoblasts and bone marrow-derived cells. In the case of skeletal myoblasts, the randomized placebo-controlled myoblast autologous grafting in ischemic cardiomyopathy (MAGIC) trial has failed to show that myoblast injections increased ejection fraction beyond that seen in controls but the finding that the highest dose of myoblasts resulted in a significant antiremodeling effect compared with the placebo group provides an encouraging signal. In the case of bone marrow cells, surgical injections of the mononuclear fraction combined with coronary artery bypass surgery have not shown a substantial benefit but positive results have been reported with intraoperative epicardial injections of CD133(+) progenitors. There are three possible reasons for these mixed results. The first is the marked heterogeneity of cell functionality (particularly in the case of bone marrow), which would expectedly translate into variable clinical outcomes. The second reason is the low rate of sustained engraftment. The third possible explanation is a mismatch between the choice of end points and the presumed mechanism of action of the cells. The initial assumption that adult stem cells could effect myocardial tissue regeneration has led to usual focus on ejection fraction as the major surrogate endpoint. It is now increasingly recognized that adult stem cells, in contrast to their embryonic counterparts, have little if any regenerative capacity and that their presumed beneficial effects more likely involve paracrine signaling, in which case infarct size, perfusion, or left ventricular volumes might be more appropriate markers. Altogether, these observations provide a framework for future research, the results of which will then have to be integrated in the protocol design of second-generation clinical trials.