Regulation of proliferation of peritoneal tissue repair cells by peritoneal macrophages

Macrophages produce soluble mediators which modulate fibroblast growth during tissue repair. Interaction between tissue repair fibroblasts (TRC) and regulatory proteins from surgically elicited macrophages is important for peritoneal reepithelialization. In this study, we compared the effects of an extract from postsurgical macrophage spent medium with those of known growth factors on TRC collected from injured peritoneum to evaluate certain characteristics of macrophage secretory products on peritoneal healing. Rabbits underwent a midline laparotomy followed by resection and reanastomosis of the ileum or abrasion of the abdominal wall. TRC were then collected at various times after surgery. Peritoneal macrophages recovered from nonsurgical or postsurgical rabbits were cultured for 2 days in vitro. The peak of thymidine incorporation by TRC occurred on day 5 after surgery; this gradually decreased with extended postsurgical times. Fibroblast growth factor and epidermal growth factor stimulated, whereas TGF-beta inhibited, [3H]thymidine incorporation into TRC. Maximal thymidine incorporation occurred when TRC from Postsurgical Day 5 were cultured with an extract from postsurgical macrophage spent medium. However, when TRC recovered from Postsurgical Days 2 and 10 were cultured with an extract of postsurgical macrophage spent medium, they showed greater stimulation than Day 5 TRC. These data suggest that postsurgical macrophages may produce an array of factors that stimulate fibroblast growth and differentiation and may in turn affect tissue repair throughout the wound healing process.     

Mitogenic and protein synthetic activity of tissue repair cells: control by the postsurgical macrophage

It is well known that fibroblasts are a main source of extracellular matrix synthesis necessary for tissue repair. In addition, macrophages secrete products that are known to modulate synthesis of extracellular matrix. Accordingly, we studied the incorporation of [3H]thymidine, [3H]proline, and [35S]sulfate into macromolecules produced by fibroblasts recovered from the site of peritoneal tissue repair cultured with and without spent media from postsurgical peritoneal macrophages. Rabbits underwent resection and reanastomosis of their small intestines. Peritoneal exudative cells (PEC) were then collected on postsurgical day 5 and day 10 as well as from nonsurgical controls, separated by discontinuous Percoll gradient centrifugation, and cultured for 48 h. A second group of rabbits underwent peritoneal wall abrasion from which fibroblast tissue repair cells (TRC) were collected from the site of injury at postsurgical day 7 and maintained in culture for varying times. Incorporation of radiolabeled precursors into DNA, collagen, and sulfated proteoglycans was determined. Incorporation of [3H]thymidine and [3H]proline into untreated TRC gradually decreased with culture duration. Conversely, [35S]sulfate incorporation gradually increased during prolonged culture. Macrophage spent media increased the levels of [3H]thymidine incorporation by the TRC. [3H]Proline and [35S]sulfate incorporation into TRC were also stimulated by macrophage spent media. However, this stimulation may be due to the enhanced proliferation of TRC by macrophage spent media. In conclusion, tissue repair fibroblasts are activated for postsurgical repair at the site of injury by many factors including secretory products from postsurgical macrophages.     

Modulation of proline and glucosamine incorporation into tissue repair cells by peritoneal macrophages

The purpose of this study was to determine the patterns of [14C]proline and [14C]glucosamine incorporation by tissue repair cells (TRC) as modulated by postsurgical macrophages. Rabbits underwent a midline laparotomy followed by resection (2.0 cm) and reanastomosis of their ileum. Another group of rabbits underwent peritoneal wall abrasion with sterile gauze until punctate bleeding developed. Postoperative (1-28 days) exudate cells (PEC) were recovered from the peritoneal cavity after reanastomosis, and (TRC) were obtained directly from the injured peritoneal surface after abrasion. Since the postsurgical exudate was composed mainly of macrophages, we examined the effect of postsurgical macrophage-spent media on the incorporation of [14C]proline, [14C]glucosamine, and [3H]thymidine by TRC. After 7 days of culture, Postsurgical Day 7 TRC were incubated with spent media from postsurgical PEC (greater than 90% macrophages). When TRC were cultured with macrophage-spent media, the number of TRC increased significantly compared to that of fresh medium-treated controls. The incorporation of [3H]thymidine by TRC was also enhanced by macrophage-spent media. The incorporation of [14C]proline and [14C]glucosamine by TRC was also enhanced when incubated with macrophage-spent medium. However, when data were expressed on a per cell basis, incorporation of [14C]proline and [14C]glucosamine by TRC cultured with macrophage-spent media was the same or less than that by cells incubated with fresh medium. These data suggest that the increase in incorporation of glucosamine and proline into connective tissue protein by postsurgical repair cells may be directly modulated by macrophages recruited in response to surgical injury and that this increase is due to the fibroproliferative effect of postsurgical macrophages.     

Kinetic analysis of experimental post-operative peritoneal healing: The incorporation of proline and glucosamine by exudative and tissue repair cells

The purpose of this study was to experimentally investigate the cellular composition of post-surgical peritoneal fluid and peritoneal tissue and determine the patterns of¹⁴C-proline and¹⁴C-glucosamine incorporation by the peritoneal exudative cells and peritoneal tissue repair cells (PEC and PTRC). One group of rabbits underwent resection (2.0 cm) and reanastomosis of their ileum, and another group underwent peritoneal wall abrasion. Postoperatively (1–28 days), the PEC and PTRC were collected and incubated for 5 days with 0.5 μCi¹⁴C-glucosamine or¹⁴C-proline, and the specific activity thereafter determined by beta counting. On the 1st postoperative day, the total cell number (TCN) had increased (7.7×10⁷ cells/rabbit) to 770 per cent of the control values primarily as a result of the PMN influx (89.9 per cent). On day 3, the TCN was 6.1×10⁷, 58.5 per cent of which comprised macrophages, which had become the principle cell type by day 5. The incorporation of proline and glucosamine into the PEC increased significantly peaking on day 7 then decreasing to the control value by day 21. Proline incorporation into the PTRC increased significantly, reaching a peak value on day 5, which decreased by day 10. Glucosamine incorporation reached a peak value on day 7 then decreased by day 10. In conclusion, the increase in glucosamine and proline incorporation into the PTRC paralles the increase in PEC, comprised principally of macrophages. These findings suggest that analysis of the metabolic activities in peritoneal activated macrophages may provide a useful tool to dissect the central mediation of postsurgical peritoneal re-epithelialization.     

Kinetic analysis of experimental post-operative peritoneal healing: the incorporation of proline and glucosamine by exudative and tissue repair cells.

The purpose of this study was to experimentally investigate the cellular composition of post-surgical peritoneal fluid and peritoneal tissue and determine the patterns of 14C-proline and 14C-glucosamine incorporation by the peritoneal exudative cells and peritoneal tissue repair cells (PEC and PTRC). One group of rabbits underwent resection (2.0 cm) and reanastomosis of their ileum, and another group underwent peritoneal wall abrasion. Postoperatively (1-28 days), the PEC and PTRC were collected and incubated for 5 days with 0.5 mu Ci 14C-glucosamine or 14C-proline, and the specific activity thereafter determined by beta counting. On the 1st postoperative day, the total cell number (TCN) has increased (7.7 x 10(7) cells/rabbit) to 770 per cent of the control values primarily as a result of the PMN influx (89.9 per cent). On day 3, the TCN was 6.1 x 10(7), 58.5 per cent of which comprised macrophages, which had become the principle cell type by day 5. The incorporation of proline and glucosamine into the PEC increased significantly peaking on day 7 then decreasing to the control value by day 21. Proline incorporation into the PTRC increased significantly, reaching a peak value on day 5, which decreased by day 10. Glucosamine incorporation reached a peak value on day 7 then decreased by day 10. In conclusion, the increase in glucosamine and proline incorporation into the PTRC parallels the increase in PEC, comprised principally of macrophages. These findings suggest that analysis of the metabolic activities in peritoneal activated macrophages may provide a useful tool to dissect the central mediation of postsurgical peritoneal re-epithelialization.     

生长因子对间皮细胞增殖及合成细胞外基质的影响

目的　探讨血小板源生长因子ｂｂ（ＰＤＧＦｂｂ） 、转化生长因子β（ＴＧＦβ） 对人腹膜间皮细胞（ＨＰＭＣ）增殖及合成细胞外基质（ＥＣＭ） 的影响。方法　建立ＨＰＭＣ体外培养体系。３ＨＴｄＲ 掺入法测定细胞增殖；放射免疫法检测细胞上清中Ⅲ型前胶原（ ⅢＰＣ） 。结果　ＰＤＧＦｂｂ 促进、但ＴＧＦβ抑制ＨＰＭＣ增殖，均呈剂量依赖性（ Ｐ＜ ０－０１） 。２ 者均能刺激ＨＰＭＣ 产生ⅢＰＣ（ Ｐ＜ ０－０１）。结论　ＣＡＰＤ相关性腹膜炎时腹腔中高浓度的ＰＤＧＦｂｂ 、ＴＧＦβ能调节间皮层的修复和重塑，刺激其合成分泌ＥＣＭ，后者可能是腹膜炎（ＰＩ）反复发生最终导致腹膜硬化的重要机制之一。     

Production of hemopoietic growth factors by bone tissue and bone cells in culture

This study was carried out to determine whether bone might be a source of hemopoietic growth factors. Both neonatal murine calvaria and primary cultures of cells isolated from calvaria released, upon stimulation with lipopolysaccharide, an activity that stimulated the growth of the interleukin (IL) 3-dependent cell lines, 32D cl, 123, and NSF 60. Upon gel filtration, this activity eluted with a molecular weight of 30,000 kDa. Further characterization, however, revealed that the major activity in conditioned medium was not IL 3. Activity was absorbed by DEAE-Sephacel at low salt concentration, whereas IL 3 does not adhere. Furthermore, an IL 3-specific antiserum did not neutralize the activity from cells and only partly neutralized the activity generated by whole calvaria. After gel filtration, the 30-kDa activity stimulated the growth of very large colonies in semisolid medium consisting mainly of granulocytes with the remainder being macrophages. No colony types belonging to other hemopoietic lineages were found, indicating, again, that the activity was not identical to IL 3. Subsequently, conditioned medium was fractionated by hydrophobic chromatography on Phenyl-Sepharose CL-4B, yielding two peaks of activity. Neutralization of activity with antisera to granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL 3 and use of colony assays showed that medium conditioned by whole calvaria contained GM-CSF and granulocyte CSF (G-CSF) in similar amounts together with a little IL 3, and medium conditioned with calvaria cells contained GM-CSF and little G-CSF. We conclude that bone releases hemopoietic growth factors that could contribute both to hemopoiesis and to the recruitment of osteoclasts from progenitors resident in the adjacent marrow.     

The use of growth factors in cartilage repair

Articular cartilage, which enables smooth gliding of joints during skeletal motion, is vulnerable to injuries and degenerative diseases over time. Bone growth factors have a role in the preservation of the cartilage matrix. This article reviews the potential to treat cartilage damage for bone morphogenetic proteins, insulin-like growth factors, hepatocyte growth factor, basic fibroblast growth factor, and transforming growth factor beta.     

浓缩生长因子在骨组织再生和修复上的作用研究进展

骨组织再生过程发生异常会导致骨量或骨质结构的改变。而临床中涉及骨组织再生的领域甚多,如:骨折的修复重建、骨肿瘤术后骨缺损的修复,尤其在种植外科领域中齿槽嵴的增高、上颌窦提升术、种植体植入后周围支持骨的修复所涉及到的骨组织再生和修复问题是近年研究的热点之一。因此,深入研究骨组织再生和修复的过程,对于临床应用具     

腹膜透析液诱导结缔组织生长因子在大鼠腹膜间皮细胞表达

目的 研究应用不同腹膜透析液对大鼠腹膜间皮细胞(RPMCs)结缔组织生长因子(CTGF)合成的影响。方法 分离培养的RPMC分为6组，分别以不同腹膜透析液[1.5% Dextrose(低糖组)、2.5% Dextrose(中糖组)、4.25% Dextrose(高糖组)、7.5% Icodextrin(糊精组) ]进行刺激培养，而无血清DMEM为阴性对照(对照组)， TGF-β1(2.5 ng/ml)为阳性对照(阳性对照组)。刺激培养24 h后，RT-PCR法检测RPMCs的CTGF mRNA、胶原Ⅰ mRNA、α-SMA mRNA表达。Western印迹法检测RPMCs的CTGF、胶原Ⅰ、α-SMA蛋白表达以及培养上清中的CTGF蛋白表达。结果 各组均见CTGF mRNA表达，高糖组、阳性对照组CTGF mRNA 表达水平显著高于中糖组、低糖组及对照组(P < 0.05)；中糖组与糊精组CTGF mRNA 表达亦显著上调(P < 0.05)。各组细胞均检测到CTGF蛋白质表达，为相对分子质量38 000及 25 000的2种亚型，与RT-PCR结果一致。高糖组、阳性对照组CTGF 蛋白表达水平显著高于糊精组、中糖组、低糖组及对照组(P < 0.05)。RPMCs培养上清液中检测出CTGF 38 000亚型的表达，其表达强弱趋势与CTGF在细胞中表达一致。高糖组、阳性对照组胶原I mRNA、蛋白质的表达水平显著高于对照组(P < 0.05)，其余各组间无显著性差异。各组α-SMA mRNA、蛋白质表达未见显著性差异(P > 0.05)。结论 正常培养的RPMCs表达低水平的CTGF。腹膜透析液、尤其是高浓度葡萄糖透析液，能明显上调CTGF表达的水平，这可能是导致长期腹膜透析过程中腹膜结构改变的机制之一。糊精腹膜透析液生物相容性可能优于高浓度葡萄糖透析液。     

Effects of growth factors on proliferation of cultured human peritoneal mesothelial cells]

Loss of ultrafiltration during continuous ambulatory peritoneal dialysis (CAPD) is often caused by the structural peritoneal membrane alteration, namely the disappearance of mesothelial cells and the proliferation of peritoneal collagen fibers. The interleukin hypothesis has been proposed to explain the etiology of peritoneal fibrosis. The CAPD procedure has been shown to induce macrophages and lymphocytes in the peritoneum, resulting in the production of interleukin-1 (IL-1) and interferon-gamma (IFN-gamma), which may be promote to the development of peritoneal fibrosis. On the other hand, the mesothelial defect can be rapidly restored by proliferation of mesothelial cells implanted on the wound surface. In this study, we demonstrated that IL-1 beta, IFN-gamma, epidermal growth factor (EGF) and platelet derived growth factor (PDGF) enhance to the growth of cultured human peritoneal mesothelial (CHPM) cells. The cell cultures were derived from surgically removed omentum using the enzymatic disaggregation method. CHPM cells were cultured with Ham's F-12 medium containing 10% FCS up to third generation. At a concentration of 1x10(4) cells/well were cultured with various concentrations of IL-1 beta, IFN-gamma, EGF, PDGF and IL-6. [3H] TdR (37MBq/well) was added to the cultures during the last 12hr of the 48hr culture period and then radioactivity was measured to determine the uptake of [3H] TdR. It was shown that IL-1 beta, IFN-gamma, EGF and PDGF induced the proliferation of CHPM cells in a dose dependent manner when cultured in medium containing 3% FCS.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of cytokines, coagulation, and fibrinolysis in peritoneal tissue repair.

Peritoneal tissue repair is a distinct entity. Regardless of the type of injury, a common series of events follows, culminating in inflammation and restoration. Molecular actors interact in a series of events in which the balance of fibrin deposition and degradation is vital. Although the complexity of the repair is illustrated by the multitude of effects and the overlap of molecular mediators involved, a framework is emerging. In this context, the overall role of cytokines is to shift the balance of fibrin deposition and degradation in favour of fibrin residues. Coagulation, as well as generating fibrin, is probably of importance in stimulating remesothelialisation, and fibrinolysis is instrumental in the degradation of fibrin deposits. As far as wound healing in concerned, we propose that the ultimate goal may not be to prevent adhesions, but rather to control their formation. To attain this, site-specific modulation of the repair process is essential. The new insights in mediators and modulators reviewed in this paper may provide means for site-specific modulation of peritoneal tissue repair as well as constituting molecular markers of the repair process.     

The mitogenic activity of human breast milk

There are constituents of breast milk that make it superior to cows milk for the nutrition, health, and development of human infants. We have discovered a new activity in breast milk. We have found in human breast milk a mitogen that stimulates DNA synthesis and induces division in cells grown in culture. This mitogenic activity is concentration dependent and also depends on time since lactation. No similar mitogenic activity is found in commercially available formulas or cows milk 1 week after birthing. The human milk mitogen might be involved in the growth and development of cells in the neonate or in the growth of cells in the mammary gland.     

Chondrocytes or adult stem cells for cartilage repair: the indisputable role of growth factors

Articular cartilage is easily injured but difficult to repair and cell therapies are proposed as tools to regenerate the defects in the tissue. Both differentiated chondrocytes and adult mesenchymal stem cells (MSCs) are regarded as cells potentially able to restore a functional cartilage. However, it is a complex process from the cell level to the tissue end product, during which growth factors play important roles from cell proliferation, extracellular matrix synthesis, maintenance of the phenotype to induction of MSCs towards chondrogenesis. Members of the TGF-β superfamily, are especially important in fulfilling these roles. Depending on the cell type chosen to restore cartilage, the effect of growth factors will vary. In this review, the roles of these factors in the maintenance of the chondrocyte phenotype are discussed and compared with those of factors involved in the repair of cartilage defects, using chondrocytes or adult mesenchymal stem cells.     

转化生长因子β1对人腹膜间皮细胞结缔组织生长因子的影响

目的 观察转化生长因子(TGF)β1对人腹膜间皮细胞(HPMCs)结缔组织生长因子(CTGF)的mRNA和蛋白表达影响并探讨其可能的机制。方法 原代培养HPMCs，用5ng／ml TGF-β1刺激第3代细胞，采用免疫组织化学染色、Western印迹、ELISA和RT-PCR等方法，观察CTGF的mRNA和蛋白表达、纤连蛋白(FN)和Ⅰ型胶原(ColⅠ)的mRNA和蛋白表达，细胞内磷酸化Smad2／3(p-Smad2／3)的蛋白表达以及在细胞内的迁移。结果 (1)刺激组CTGF的mRNA表达与对照组比均显著增加，其中48h为峰值；对照组细胞内仅有少量CTGF的蛋白表达，在TGF-β1刺激后24h表达明显增加，48h达峰值。(2)刺激组FN和ColⅠ的mRNA表达与对照组比均呈时间依赖性显著增加，上清液FN和细胞内ColⅠ的蛋白表达与对照组比也呈时间依赖性显著增加。(3)对照组细胞内几乎不表达p-Smad2／3(阳性细胞率3％)，在刺激后15min表达增加(29％)，细胞着色主要分散在胞质中；1h增加最明显(84％)，细胞着色加深且集中在胞核及周边；2h明显回落(37％)，细胞着色转淡并又分散至胞质中。结论TGF-β1在致腹膜纤维化过程中诱导了HPMCs内CTGF的转录和蛋白表达，可能与TGF-β1激活了HPMcs内Smad信号通路有关。     

Transforming growth factor-β enhances connective tissue repair in perforated rat mesentery but not peritoneal macrophage chemotaxis

The perforated rat mesentery model was used to study the effect of transforming growth factor-beta (TGF-beta) on connective tissue repair and influx of macrophages into the peritoneal cavity during such repair. Sprague-Dawley rats were laparotomized, and mesenteric wounds were made with a scalpel. A daily intraperitoneal injection of 0.5 microg TGF-beta was given for either 2 or 4 days. After 1 to 10 days, the animals received an intravenous injection of tritium-labeled thymidine before decapitation. Macrophages were collected by peritoneal washing, and the number of closed perforations was counted. Peritoneal cells were quantitated and a labeling index was determined by autoradiography. TGF-beta given for either 2 (p < 0.001) or 4 (p < 0.004) days accelerated closure of perforations on days 3 to 7 after injury. Laparotomy as such significantly increased leukocyte influx (p < 0.004), as well as macrophage-labeling index (p < 0.02). However, TGF-beta did not significantly influence either leukocyte influx or macrophage-labeling index. We concluded that TGF-beta significantly enhances connective tissue repair in this perforated rat mesentery model and that TGF-beta-induced stimulation of repair is not caused by an increased influx of macrophages into the peritoneal cavity.