正常大鼠植入高血压大鼠肾脏后动脉血压的变化

本实验对12周龄的自发性高血压大鼠(spontaneouslyhypertensiverat,SHR)及其对照组WistarKyoto(WKY)大鼠进行了肾脏移植的研究,并观察受肾移植大鼠动脉血压的变化以及免疫抑制剂对动脉血压的影响。用尾套法对接受同窝另一同胞WKY大鼠肾脏移植且存活5周的6只WKY大鼠(A组)及接受SHR肾脏移植且存活5周的6只WKY大鼠(B组)的尾动脉收缩压进行检测,移植前A、B两组受肾移植大鼠的尾动脉收缩压分别为180±093和183±068kPa,无统计学显著差异(P>005);移植后3、4、5周时,B组大鼠的尾动脉收缩压显著高于A组大鼠,移植后5周时,A,B两组大鼠的收缩压分别为190±071和230±069kPa(P<0001);所用剂量的免疫抑制剂CsA对双侧肾脏完整以及右侧肾脏切除的SHR、WKY大鼠的动脉血压无显著影响。以上结果表明,SHR的肾脏在高血压的形成中可能起重要作用     

Applied tissue engineering in the closure of severe burns and chronic wounds using cultured human autologous keratinocytes in a natural fibrin matrix

Whereas in severe burns cultured human epithelial cells may well serve as a life saving method, the true value of tissue-engineered skin products in chronic wound care has yet to be clearly defined. Among other well-known clinical problems, the engraftment rate of commercially available multilayered "sheet grafts" has been shown to vary extremely. Adherence of transplanted cells to the wound bed--especially in the presence of potential wound contamination-- is one of the crucial aspects of this technique. Keratinocyte suspensions in a natural fibrin sealant matrix can potentially treat a variety of skin defects. In acute burn wounds, as well as in chronic wounds the clinical application of this type of tissue-engineered skin substitute demonstrates the capacity of cultured human autologous keratinocytes in a fibrin sealant matrix to adhere to wound beds, attach and spread over the wound resulting in reepithelialization of both acute and chronic wounds. In full thickness burns the combination of this new tool with allogenic dermis is a promising option to achieve complete dermal-epidermal reconstitution by means of tissue engineering and guided tissue repair. When transferring this technique into the treatment of chronic wounds we found an optimal preparation of such recipient wound beds to be crucial to the success. The additional application of continuous negative pressure (vacuum therapy) and preliminary chip skin grafting to optimally prepare the recipient site may be helpful tools to achieve such well-prepared and graftable surfaces. Prospective controlled comparative studies should be designed to further assess the clinical efficacy of this technique.     

Portal hemodynamic changes after hepatocyte transplantation in acute hepatic failure

Hepatocyte transplantation has been proposed as an alternative for rescuing patients with acute hepatic failure. However, portal hemodynamic changes and issues of safety after hepatocyte transplantation in acute hepatic failure have not been systemically evaluated because of the lack of a suitable experimentation system. In this study, we created a novel spring-guidewire introducer needle to simplify the technique for long-term portal cannulation in F-344 rats. The portal cannula was capable of being used for blood sampling, infusion of hepatocytes, and measurement of portal hemodynamic changes. One week after portal cannulation, rats were injected withD-galactosamine (1.35 g/kg, i.p.) to induce hepatic failure. Hepatocytes (2×10⁷) were infused intraportally 24–26 h after induction of liver injury. Portal pressures were recorded for up to 60 min after hepatocyte transplantation. Intraportal infusion of 2×10⁷ hepatocytes caused an instantaneous onset of portal hypertension. The magnitude of the rise in portal pressure was similar in both normal rats and rats with acute hepatic failure (33.0±7.1 vs. 37.7±0.5 mm Hg; p=0.23). However, the resolution rate of portal hypertension was remarkably delayed in rats with acute hepatic failure, and the portal pressure was significantly higher than that in normal rats 60 min after hepatocyte transplantation (25.0±2.8 vs. 14.5±2.4 mm Hg; p=0.007). In conclusion, we have established a simple new technique for long-term portal cannulation of rats. Our studies provide critical insights into the delayed resolution of portal hemodynamics after hepatocyte transplantation in subjects with acute hepatic failure.     

NK cells in allogeneic bone marrow transplantation

NK cells, until recently an ignored subset of lymphocytes, have begun to emerge as important cytotoxic effectors. It is now accepted that NK cells together with T cells constitute major actors in graft-versus-leukemia reaction after allogeneic bone marrow transplantation (BMT). Over the last several years the mechanisms regulating the activation of NK cells have been the subject of intense investigations encouraged by the clinical implications that these studies will have. This article provides a general overview of NK-cells biology and regulation pertinent to their function in allogeneic BMT, followed by a review of the in vivo preclinical and clinical evidence for the beneficial effect of NK cells in the adoptive immunotherapy of leukemia.     

Effects of Septal Grafts on Acetylcholine Release from Rat Hippocampus After 192 IgG-Saporin Lesion

The cholinergic inputs to the rat hippocampus were lesioned by intraseptal injections of 192 IgG-saporin. After 15 days, fetal septal cells were grafted into the hippocampus. Thirteen months later, hippocampal acetylcholine (ACh) release was studied by microdialysis. Lesioning reduced basal ACh release (100%) to 20% of normal, which was compensated for by the graft (71%). Infusion of the serotonin uptake inhibitor citalopram (100 M) enhanced ACh release to the same extent (% of basal release) in all rat groups. Systemic injection of 8-OH-DPAT (0.5 mg/kg, SC), an agonist of 5-HT_1A receptors, caused a smaller ACh release than citalopram. Acetylcholinesterase (AChE) staining and densitometric quantification revealed that the lesion-induced reduction of the AChE-staining density was compensated for by septal grafting. In conclusion, both histochemical and biochemical methods showed that cholinergic hippocampal parameters were drastically impaired by 192 IgG-saporin lesions, but were almost completely restored by septal grafting. The graft responded to intrinsic serotonergic regulation.     

Skeletal muscle repair by adult human mesenchymal stem cells from synovial membrane

We have demonstrated previously that adult human synovial membrane-derived mesenchymal stem cells (hSM-MSCs) have myogenic potential in vitro (De Bari, C., F. Dell'Accio, P. Tylzanowski, and F.P. Luyten. 2001. Arthritis Rheum. 44:1928-1942). In the present study, we have characterized their myogenic differentiation in a nude mouse model of skeletal muscle regeneration and provide proof of principle of their potential use for muscle repair in the mdx mouse model of Duchenne muscular dystrophy. When implanted into regenerating nude mouse muscle, hSM-MSCs contributed to myofibers and to long term persisting functional satellite cells. No nuclear fusion hybrids were observed between donor human cells and host mouse muscle cells. Myogenic differentiation proceeded through a molecular cascade resembling embryonic muscle development. Differentiation was sensitive to environmental cues, since hSM-MSCs injected into the bloodstream engrafted in several tissues, but acquired the muscle phenotype only within skeletal muscle. When administered into dystrophic muscles of immunosuppressed mdx mice, hSM-MSCs restored sarcolemmal expression of dystrophin, reduced central nucleation, and rescued the expression of mouse mechano growth factor.     

Effects of prolonged delivery of brain-derived neurotrophic factor on the fate of neural stem cells transplanted into the developing rat retina

One of the major roles of brain-derived neurotrophic factor (BDNF) is to promote the differentiation and support the survival of neurons in the central nervous system. The objective of the present study was to evaluate the effect of BDNF on the fate of adult rat hippocampus-derived neural stem cells (AHPCs) transplanted into the developing rat retina. Immunohistochemical analysis showed a significant increase in the ratio of grafted AHPCs stained for MAP2ab (P<0.05) and a marked decrease in the ratio of nestin-positive grafted cells in the slow-releasing BDNF group compared with the control group. The respective changes in the ratios of MAP5 and GFAP-positive grafted cells were comparable for the two groups. The results reported here suggest a potentially beneficial role for extended delivery of BDNF in the differentiation of grafted neural stem cells, which may lead to a novel modification of stem cell transplantation.     

Development and retroviral transduction of porcine neonatal pancreatic islet cells in monolayer culture

To learn more about the potential of neonatal porcine pancreatic duct and islet cells for xenotransplantation, the development of these cells when cultured as monolayers was evaluated. Immunostaining for islet hormones and cytokeratin-7 revealed that day eight monolayers consisted of approximately 70% duct cells and less than 10% beta cells. Using Ki-67 immunostaining as a proliferation marker, the fraction of beta cells in the cell cycle was shown to decrease from 20% at day three to 10% at day eight, and for duct cells from 36 to 19%. Insulin secretion increased 2.4-fold upon glucose stimulation, and 38-fold when 10 mm theophylline was added, showing the responsiveness of the neonatal beta cells. Reaggregated monolayers consisted mostly of duct cells, but 4 weeks after transplantation, grafts contained predominantly endocrine cells, with duct cells being almost absent, suggesting in vivo differentiation of duct cells to endocrine cells. Monolayer susceptibility to retroviral transduction was also investigated using a Moloney Murine Leukemia Virus-based vector. Approximately 60% of duct cells but less than 5% of beta cells expressed the transgene, indicating that precursor duct cells are better targets for transgene expression. These results show that porcine neonatal pancreatic cells can be cultured as monolayers in preparation for transplantation. Furthermore, in such a culture setting, precursor duct cells have a high rate of proliferation and are more efficiently transduced with a retrovirus-based reporter gene than are beta cells.     

In vitro induction and transplantation of eye during early Xenopus development

A vertebrate eye was induced via a series of coordinated inductive interactions. Here, we describe a novel in vitro system to induce eye formation at high frequency using Xenopus early gastrulae. The eye formed in vitro is morphologically similar to the normal eye. When the in vitro eye was transplanted into a stage-33 tadpole, the optic nerve was seen extending from the grafted eye to the tectum of the host brain and the in vitro eye graft was retained after metamorphosis. In addition, we transplanted the eye formed in vitro into a tadpole with both eyes removed. The resultant juvenile frogs could perceive brightness using the grafted eye and thereby control their skin color, suggesting that the eye formed in vitro could function normally.     

Articular Cartilage Chondrocytes are more Advantageous for Generating Hyaline-like Cartilage than Mesenchymal Cells Isolated from Microfracture Repairs

Articular cartilage lacks self-repair capacity. Currently, two methods employing autologous cells are used to stimulate repair of articular cartilage. Micro-fracture induced repair induces autologous mesenchymal cell migration from bone marrow. Autologous chondrocytes' transplantation involves in vitro expansion of chondrocytes, and later implantation. In 15 patients de-differentiated chondrocytes obtained by cartilage biopsy were compared to cells derived from repair tissue induced by micro-fracture. These patients all underwent micro-fracture during the cartilage biopsy procedure. Autologous chondrocytes' transplantation was performed at least two months later then the biopsy. Tissue bits from articular cartilage and micro-fracture repair tissue were incubated in-vitro and explant cell cultures established. The cell cultures were assessed by immunohistochemistry and induced to differentiate. Differentiation into bone tissue was stimulated by addition of basic fibroblast growth factor, ascorbate and dexamethasone. High density (micro-mass) culture was used to stimulate chondrogenesis. Both cell cultures consist of mesenchymal progenitors as indicated by fibroblast growth factor receptor 3 expression and anti-CD-34+ antibodies. However, the micro-fracture generated repair tissue consists of osteocalcin-expressing cells destined to become bone. Collagen type II expression does not occur in these cells compared to autologous chondrocytes. Inducible nitric oxide synthase expression by microfracture cells is likely to damage surrounding articular cartilage in vivo. In conclusion, cells recruited by micro-fracture are inferior for cartilage regeneration purposes to those from cartilage biopsies.