Protein adsorption on ex vivo catheters and polymers exposed to peritoneal dialysis effluent.

BACKGROUND: Deposition of proteins on surfaces of medical devices has been recognized to putatively relate to the process of regulation of biomaterial-associated complications by attachment of fibrin clots, eukaryotic cells, and microbes. The molecules adsorb to a varying extent, depending not only on the physicochemical properties of the biomaterial, but also on the composition of the host fluid. OBJECTIVE: Adsorption of proteins on catheters exposed both ex vivo and in vitro to dialysate of patients on peritoneal dialysis (PD) was studied. METHODS: Peritoneal dialysis effluent was collected from 5 patients with end-stage renal disease on continuous ambulatory PD. Tenckhoff catheters were obtained from 16 patients. Deposition of proteins on excised Tenckhoff catheters and tubing of different materials exposed to PD effluent in vitro was studied using 125iodine-labeled antibodies. Adhesion of Staphylococcus aureus and Staphylococcus epidermidis strains was quantified on tubing exposed to PD effluent in vitro. RESULTS: The presence of albumin, transferrin, immunoglobulin G, fibrinogen, fibronectin, von Willebrand factor, vitronectin, and thrombospondin was determined at various concentrations in PD effluent. All proteins analyzed were detected on PD catheters removed from patients. The extent of protein deposition on Tenckhoff catheters exposed to PD effluent, in vitro, rapidly reached a plateau and remained constant, as it did on polyvinyl chloride and polyethylene tubing. Adhesion of staphylococci was enhanced on Tenckhoff catheters exposed to PD effluent compared to unused PD solution. CONCLUSIONS: The data identify surface exposed proteins that may serve as adhesion sites for microbes on peritoneal catheters indwelled in patients undergoing PD.     

Electrophysiology and glucose transport of human peritoneal mesothelial cells: implications for peritoneal dialysis.

OBJECTIVE: To elucidate ionic and glucose transport across human peritoneal mesothelium, we utilized an Ussing chamber setup and studied the electrophysiological characteristics and tissue permeabilities of human peritoneal mesothelial cells (HPMC) to L- and D-glucose. METHODS: Human mesothelial cells were grown on polyester filters (snapwell; Costar, Cambridge, MA, U.S.A.) that, upon confluence, were fitted into Ussing chambers. Transmesothelial resistance and resting potential were determined using electrophysiological techniques. Radiolabeled glucose was added to one side of the chamber and the permeabilities determined by serial sampling in the receptive compartment. RESULTS: The transmesothelial potential and resistance were 0.54 +/- 0.07 mV (apical positive) and 20.4 +/- 3.2 ohms x cm2 respectively (mean +/- SEM, n = 36). The course of overall transfer of D- and L-glucose was examined using L-glucose as a positive diffusion-plus-leak marker. The permeabilities of HPMC to D-glucose were 3.00 +/- 0.26 cm/sec (apical-to-basolateral) and 3.25 +/- 0.27 cm/sec (basolateral-to-apical) [n = 6 experiments, p = not significant (NS)], which were not different from those of L-glucose: 3.00 +/- 0.30 cm/sec (apical-to-basolateral) and 2.71 +/- 0.24 (basolateral-to-apical) (n = 6 experiments, p = NS). CONCLUSIONS: The transepithelial resistance of HPMC is low and the ionic gradient, although it exists, is small and inconsequential. Passive paracellular flow accounts for the majority of transmesothelial glucose transport. The existence of a large paracellular shunt precludes the mesothelial membrane as a clinically relevant osmotic barrier.     

Non-viral ex vivo transduction of human hepatocyte cells to express factor VIII using a human ribosomal DNA-targeting vector

BACKGROUND: In gene therapy, one of the most important issues is the choice of the vectors. pHrneo is a human-derived vector previously constructed by our group, which can target a foreign gene into a human ribosomal DNA (hrDNA) locus. METHODS AND RESULTS: In this study, we inserted an expression cassette of reconstructive hFVIII (hFVIII-BDDAK39) to pHrneo to construct a targeting vector: pHrneo-BDDAK39. Through electroporation of pHrneo-BDDAK39 into HL7702 cells (human hepatocyte), we identified the homologous recombinants using polymerase chain reaction, and tested the expression of hFVIII-BDDAK39 located at the hrDNA locus. The hFVIII-BDDAK39 was successfully targeted into the hrDNA locus of HL7702 by pHrneo-BDDAK39, and the efficiency of site-specific integration was 1.1 x 10(-5). The hFVIII-BDDAK39 at the hrDNA locus of HL7702 was found to be able to express efficiently (4.3 +/- 0.9 ng 10(-6) cells 24 h(-1)). CONCLUSION: It has been indicated that the targeting vector pHrneo-BDDAK39 can be used in gene therapy for hemophilia A.     

胰岛素对人腹膜间皮细胞增殖、损伤及凋亡的影响

目的明确胰岛素是否对人腹膜间皮细胞具有毒性或者保护作用。方法使用人腹膜间皮细胞细胞株，传代培养。用四甲基偶氮唑盐比色法(MTT)测定HPMC的增殖程度。采用全自动生化分析仪测定培养液中乳酸脱氢酶LDH水平示细胞损伤，Hoechst33258染色观察细胞凋亡形态学变化及计算凋亡细胞百分比。分为单纯培养基对照组，4．25％葡萄糖组，不同浓度胰岛素组和不同浓度的胰岛素加4．25％葡萄糖组。结果与对照组相比，4．25％葡萄糖致LDH水平明显增高，并显著降低NIT渗入量，明显增加凋亡细胞百分比；不同浓度的胰岛素对HPMC增殖无影响，不同浓度胰岛素24h后可明显升高LDH水平(P＜0．05)，不同浓度胰岛素作用12h均后导致凋亡细胞百分比显著升高(P＜0．05)；不同浓度葡萄糖胰岛素组12h、24hMMT渗入量较葡萄糖组明显升高(P＜0．05)，组间差异无显著性(P＞0．05)，浓度大于25U／L胰岛素加葡萄糖组作用24h后与葡萄糖组相比显著降低LDH水平(P＜0．05)；不同浓度胰岛素加葡萄糖组作用12h后与葡萄糖组相比凋亡细胞百分比明显下降(P＜0．05)。结论胰岛素能导致HPMC损伤、诱导凋亡，同时在一定范围内可缓解高糖对HPMC的毒性作用。     

A novel approach to ex vivo gene therapy for familial hypercholesterolemia using human amniotic epithelial cells as a transgene carrier.

This study has demonstrated the potential of human amniotic epithelial cells (HAEC) as a transgene carrier to treat patients with familial hypercholesterolemia (FH). One approach to liver-directed gene therapy is represented by transplantation of autologous hepatocytes that have been genetically modified in vitro. However, the hepatocytes must be isolated from surgically resected tissue and it is difficult to expand the hepatocytes in culture. In contrast, the advantages for using HAEC are the higher availability and the nonimmunogenicity after allotransplantation. Our strategy involved isolating HAEC from an amnion, transducing a human low-density lipoprotein receptor (LDLR) gene into these cells with a recombinant adenovirus, and transplanting the genetically modified cells into the liver of an animal model of FH. Each animal, treated with the LDLR-transduced HAEC, exhibited a substantial decrease in serum cholesterol with an eventual return to pretreatment level. Moreover, the transplanted HAEC migrated out of the sinusoids into the hepatic parenchyma and expressed the LDLRs until at least 20 days after transplantation. However, the transplanted HAEC markedly decreased in number after 10 days post-transplant with an increase of inflammatory cells. The temporary nature of the metabolic improvement may be associated with xenograft rejection and transient function of the adenoviral vector.     

Pharmacokinetic characterization of amrubicin cardiac safety in an ex vivo human myocardial strip model. I. Amrubicin accumulates to a lower level than doxorubicin or epirubicin

Antitumor anthracyclines such as doxorubicin and epirubicin are known to cause cardiotoxicity that correlates with anthracycline accumulation in the heart. The anthracycline amrubicin [(7S,9S)-9-acetyl-9-amino-7-[(2-deoxy-β-d-erythro-pentopyranosyl)oxy]-7,8,9,10-tetrahydro-6,11-dihydroxy-5,12-napthacenedione hydrochloride] has not shown cardiotoxicity in laboratory animals or patients in approved or investigational settings; therefore, we conducted preclinical work to characterize whether amrubicin attained lower levels than doxorubicin or epirubicin in the heart. Anthracyclines were evaluated in ex vivo human myocardial strips incubated in plasma to which anthracycline concentrations of 3 or 10 μM were added. Four-hour incubations were performed to characterize myocardial anthracycline accumulation derived from anthracycline uptake in equilibrium with anthracycline clearance. Short-term incubations followed by multiple washouts were performed to obtain independent measurements of anthracycline uptake or clearance. In comparison with doxorubicin or epirubicin, amrubicin attained very low levels in the soluble and membrane fractions of human myocardial strips. This occurred at both 3 and 10 μM anthracycline concentrations and was caused primarily by a highly favorable clearance of amrubicin. Amrubicin clearance was facilitated by formation and elimination of sizeable levels of 9-deaminoamrubicin and 9-deaminoamrubicinol. Amrubicin clearance was not mediated by P glycoprotein or other drug efflux pumps, as judged from the lack of effect of verapamil on the partitioning of amrubicin and its deaminated metabolites across myocardial strips and plasma. Limited accumulation of amrubicin in an ex vivo human myocardial strip model may therefore correlate with the improved cardiac tolerability observed with the use of amrubicin in preclinical or clinical settings.     

大黄素对家兔离体主动脉平滑肌细胞增殖影响的可能途径

目的：探讨大黄素干预对家兔离体血管平滑肌细胞增殖的作用，并观察其浓度效应剂量。方法：实验于2004—06／12在第三军医大学西南医院药学部完成。选择雄性家兔1只作为动脉平滑肌细胞的动物来源，采用组织贴块法进行主动脉平滑肌细胞的培养。细胞培养成功后分2组：对照组和大黄素组。对照组不加药，大黄素组按剂量分为5个亚组即1．25，2、5，5，10，20mg／L组，每组6孔。选用生长良好的第三四代细胞，加^3H-标记的胸腺脱氧核苷酸行液闪计数以表征细胞增殖的程度，细胞增殖测定采用酶标仪上比色（波长为570nm）用吸光度值表示。超过氧化物歧化酶测定采用邻苯三酚自氧化抑制法，直接以每毫升培养液中超过氧化物歧化酶kat表示。脂质过氧化物测定采用改良TBA微量法，以脂质过氧化物的稳定终产物丙二醛浓度（nmol／L）表示。实验结果采用单因素方差分析（方差齐性检验），用SNK法进行均数之间的显著性检验。结果：①大黄素对细胞增殖的影响：随着大黄素剂量的增加，吸光度值逐渐减少，抑制率逐渐增高。大黄素5，10，20mg／L剂量组的吸光度值明显低于对照组（0．594&;#177;0．037，0．410&;#177;0．014，0．301&;#177;0．024，0．730&;#177;0.041，P〈0.05）。②大黄素对胸腺嘧啶脱氧核苷酸掺人量的影响：随着大黄素剂量的增加，胸腺嘧啶脱氧核苷酸掺人量逐渐减少，抑制率逐渐增高。大黄素5，10，20mg／L剂量组每分液闪计数值明显低于对照组大黄素（33537&;#177;3713．29304&;#177;4336，21155&;#177;1938，73958&;#177;2276，t=5．862，7．330．18．688，P〈0．05）。③大黄素对超过氧化物歧化酶的影响：大黄素剂量1．25，2．5mg／L剂量组，超过氧化物歧化酶高于对照组，但差异不显著（P〉0．05）。5，10，20mg／L剂量组明显高于对照组[（118．19&;#177;3．56），（127．48&;#177;4．59）．（140．23&;#177;4．96），（100．16&;#177;9．83）kat／L，t=4．224．6．170．8．914，P〈0．051。④对丙二醛含量变化的影响：随着大黄素剂量的增加，其水平逐渐减少，当大黄素剂量为5，10，20mg／L时与对照组相比显著减低[（2．336&;#177;0．113），（1．945&;#177;0．135），（1．381&;#177;0．193），（3．110&;#177;0．256）nmol／L，t=6．774，9．857，13．187．P〈0．051。结论：随着培养液中大黄素浓度的增加，超过氧化物歧化酶活性逐渐增强，同时丙二醛水平逐渐降低，从而抑制血管平滑肌细胞的增殖此，此作用均从5mg／L剂量组开始，并具有浓度依赖性。     

大黄素对家兔离体主动脉平滑肌细胞增殖影响的可能途径

目的:探讨大黄素干预对家兔离体血管平滑肌细胞增殖的作用,并观察其浓度效应剂量。方法:实验于2004-06/12在第三军医大学西南医院药学部完成。选择雄性家兔1只作为动脉平滑肌细胞的动物来源,采用组织贴块法进行主动脉平滑肌细胞的培养。细胞培养成功后分2组:对照组和大黄素组。对照组不加药,大黄素组按剂量分为5个亚组即1.25,2.5,5,10,20mg/L组,每组6孔。选用生长良好的第三四代细胞,加3H-标记的胸腺脱氧核苷酸行液闪计数以表征细胞增殖的程度,细胞增殖测定采用酶标仪上比色(波长为570nm)用吸光度值表示。超过氧化物歧化酶测定采用邻苯三酚自氧化抑制法,直接以每毫升培养液中超过氧化物歧化酶kat表示。脂质过氧化物测定采用改良TBA微量法,以脂质过氧化物的稳定终产物丙二醛浓度(nmol/L)表示。实验结果采用单因素方差分析(方差齐性检验),用SNK法进行均数之间的显著性检验。结果:①大黄素对细胞增殖的影响:随着大黄素剂量的增加,吸光度值逐渐减少,抑制率逐渐增高。大黄素5,10,20mg/L剂量组的吸光度值明显低于对照组(0.594±0.037,0.410±0.014,0.301±0.024,0.730±0.041,P<0.05)。②大黄素对胸腺嘧啶脱氧核苷酸掺入量的影响:随着大黄素剂量的增加,胸腺嘧啶脱氧核苷酸掺入量逐渐减少,抑制率逐渐增高。大黄素5,10,20mg/L剂量组每分液闪计数值明显低于对照组大黄素(33537±3713,29304±4336,21155±1938,73958±2276,t=5.862,7.330,18.688,P<0.05)。③大黄素对超过氧化物歧化酶的影响:大黄素剂量1.25,2.5mg/L剂量组,超过氧化物歧化酶高于对照组,但差异不显著(P>0.05)。5,10,20mg/L剂量组明显高于对照组犤(118.19±3.56),(127.48±4.59),(140.23±4.96),(100.16±9.83)kat/L,t=4.224,6.170,8.914,P<0.05犦。④对丙二醛含量变化的影响:随着大黄素剂量的增加,其水平逐渐减少,当大黄素剂量为5,10,20mg/L时与对照组相比显著减低犤(2.336±0.113),(1.945±0.135),(1.381±0.193),(3.110±0.256)nmol/L,t=6.774,9.857,13.187,P<0.05犦。结论:随着培养液中大黄素浓度的增加,超过氧化物歧化酶活性逐渐增强,同时丙二醛水平逐渐降低,从而抑制血管平滑肌细胞的增殖此,此作用均从5mg/L剂量组开始,并具有浓度依赖性。     

钙对人腹膜间皮细胞损伤、增殖及纤维连接蛋白合成的影响

目的研究不同浓度钙对体内外人腹膜间皮增殖、损伤和间质纤维化的影响。方法体外试验:以人腹膜间皮细胞永生化细胞株(HMrSV5)为研究对象,予不同浓度钙(0、0.25、0.75、1.25、1.75、2.25mmol/L)处理该细胞12、24h。采用四甲基偶氮唑盐(methyl thiazolyl tetrazolium,MTT)比色法和乳酸脱氢酶(lactate dehydrogenase,LDH)评估细胞的增殖能力和损伤。蛋白免疫印迹法检测纤维连接蛋白(fibronectin,FN)的表达。临床试验:随机选择47例既往使用低钙透析液(Baxter PD4,含钙1.25mmol/L)的稳定维持性腹膜透析患者,平均透析时间为(18.5±11.7)个月。采用前后自身对照法进行研究。以患者刚入选本研究时作为对照组,入选后换用含钙1.75mmol/L的PD2进行透析(其他成份均与PD4相同)4周,其他治疗(降压、降磷、活性维生素D3等)不变。患者使用PD2透析后的每一周末作为一组(分别为第1、2、3、4周组)。酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测腹膜透析引流液中的FN和LDH,电化学发光法(electrochemiluminescence assay,ECLA)检测CA125。同时检测血清钙、磷及全段甲状旁腺激素(intact parathyroid hormone,iPTH)水平。结果体外试验:钙呈时间及浓度依赖性促进HMrSV5增殖,含钙1.75mmol/L组最高;不同浓度钙呈时间依赖性显著诱导LDH上调,其中钙1.25mmol/L组LDH最低(P<0.05);FN亦呈钙浓度及时间依赖性表达上调。临床试验:4个试验组腹膜透析引流液中LDH值均与对照组有显著差异且呈时间依赖性升高。第4周组的FN和LDH水平显著高于其他组,CA125水平明显低于其他组,差异均有统计学意义(均P<0.01)。第4周组的血清钙高于对照组,血磷低于对照组差异均有统计学意义(均P<0.05),iPTH与对照组差异无统计学意义(P>0.05)。结论在体内外试验中,高钙(1.75mmol/L)可促进人腹膜间皮细胞增殖,同时加重细胞损伤,促进FN合成,而生理浓度钙(1.25mmol/L)对人腹膜间皮细胞有一定保护作用并可能预防腹膜纤维化。     

TNF-alpha and TLR agonists increase susceptibility to HIV-1 transmission by human Langerhans cells ex vivo

Genital coinfections increase an individual’s risk of becoming infected with HIV-1 by sexual contact. Several mechanisms have been proposed to explain this, such as the presence of ulceration and bleeding caused by the coinfecting pathogen. Here we demonstrate that Langerhans cells (LCs) are involved in the increased susceptibility to HIV-1 in the presence of genital coinfections. Although LCs are a target for HIV-1 infection in genital tissues, we found that immature LCs did not efficiently mediate HIV-1 transmission in an ex vivo human skin explant model. However, the inflammatory stimuli TNF-α and Pam3CysSerLys4 (Pam3CSK4), the ligand for the TLR1/TLR2 heterodimer, strongly increased HIV-1 transmission by LCs through distinct mechanisms. TNF-α enhanced transmission by increasing HIV-1 replication in LCs, whereas Pam3CSK4 acted by increasing LC capture of HIV-1 and subsequent trans-infection of T cells. Genital infections such as Candida albicans and Neisseria gonorrhea not only triggered TLRs but also induced TNF-α production in vaginal and skin explants. Thus, during coinfection, LCs could be directly activated by pathogenic structures and indirectly activated by inflammatory factors, thereby increasing the risk of acquiring HIV-1. Our data demonstrate a decisive role for LCs in HIV-1 transmission during genital coinfections and suggest antiinflammatory therapies as potential strategies to prevent HIV-1 transmission.     

血必净对高糖作用下大鼠腹膜间皮细胞增殖及细胞因子表达的影响

目的观察血必净对高糖刺激后腹膜间皮细胞(peritoneal mesothelial cells,PMC)增殖、高迁移率族蛋白-1(HMGB-1)及转化生长因子-β1(TGF-β1)表达的影响。方法原代培养PMC;并用MTT和ELISA法测定血必净对高糖(2.5%葡萄糖)作用下PMC增殖及其分泌HMGB-1、TGF-β1的影响。结果高糖能显著抑制PMC的增殖(P<0.05),而2～20mg/mL的血必净能部分逆转高糖对PMC增殖的抑制作用(P<0.05);高糖能显著增加PMC表达HMGB-1和TGF-β1(均P<0.05),2～20mg/mL的血必净能明显减弱高糖上调HMGB-1和TGF-β1的作用(均P<0.05)。结论血必净可能通过逆转高糖对PMC的增殖抑制作用,抑制高糖上调PMC表达炎症介质HMGB-1和致纤维化因子TGF-β1的作用,进而修复高糖所致的PMC的损伤,延缓腹膜纤维化,为改善间皮细胞损伤和防治腹膜纤维化提供实验依据。     

Influence of bicarbonate/low-GDP peritoneal dialysis fluid (BicaVera) on in vitro and ex vivo epithelial-to-mesenchymal transition of mesothelial cells

♦ Background: Peritoneal membrane damage induced by peritoneal dialysis (PD) is largely associated with epithelial-to-mesenchymal transition (EMT) of mesothelial cells (MCs), which is believed to be a result mainly of the glucose degradation products (GDPs) present in PD solutions.♦ Objectives: This study investigated the impact of bicarbonate-buffered, low-GDP PD solution (BicaVera: Fresenius Medical Care, Bad Homburg, Germany) on EMT of MCs in vitro and ex vivo.♦ Methods: In vitro studies: Omentum-derived MCs were incubated with lactate-buffered standard PD fluid or BicaVera fluid diluted 1:1 with culture medium.Ex vivo studies: From 31 patients randomly distributed to either standard or BicaVera solution and followed for 24 months, effluents were collected every 6 months for determination of EMT markers in effluent MCs.♦ Results: Culturing of MCs with standard fluid in vitro resulted in morphology change to a non-epithelioid shape, with downregulation of E-cadherin (indicative of EMT) and strong induction of vascular endothelial growth factor (VEGF) expression. By contrast, in vitro exposure of MCs to bicarbonate/low-GDP solution had less impact on both EMT parameters.Ex vivo studies partially confirmed the foregoing results. The BicaVera group, with a higher prevalence of the non-epithelioid MC phenotype at baseline (for unknown reasons), showed a clear and significant trend to gain and maintain an epithelioid phenotype at medium- and longer-term and to show fewer fibrogenic characteristics. By contrast, the standard solution group demonstrated a progressive and significantly higher presence of the non-epithelioid phenotype. Compared with effluent MCs having an epithelioid phenotype, MCs with non-epithelioid morphology showed significantly lower levels of E-cadherin and greater levels of fibronectin and VEGF. In comparing the BicaVera and standard solution groups, MCs from the standard solution group showed significantly higher secretion of interleukin 8 and lower secretion of collagen I, but no differences in the levels of other EMT-associated molecules, including fibronectin, VEGF, E-cadherin, and transforming growth factor β1.Peritonitis incidence was similar in both groups. Functionally, the use of BicaVera fluid was associated with higher transport of small molecules and lower ultrafiltration capacity.♦ Conclusions: Effluent MCs grown ex vivo from patients treated with bicarbonate/low-GDP BicaVera fluid showed a trend to acquire an epithelial phenotype, with lower production of proinflammatory cytokines and chemokines (such as interleukin 8) than was seen with MCs from patients treated with a lactate-buffered standard PD solution.     

Dynamic effects of acid on Barrett's esophagus. An ex vivo proliferation and differentiation model.

Barrett's esophagus (BE), or specialized intestinal metaplasia, is a premalignant heterogeneous epithelium associated with reflux and an increased risk for adenocarcinoma. Since acid is a major component of refluxate, we investigated its effects ex vivo on cell differentiation as determined by villin expression; and on cell proliferation, as determined by tritiated thymidine incorporation and proliferating cell nuclear antigen expression. To mimic known physiological conditions, endoscopic biopsies of normal esophagus, BE, and duodenum were exposed, in organ culture, to acidified media (pH 3-5) either continuously, or as a 1-h pulse and compared with exposure to pH 7.4 for up to 24 h. Before culture, villin expression was noted in 25% of BE samples, and increased after 6 or 24 h of continuous acid to 50% or 83% of BE samples, respectively. Increased villin expression correlated with ultrastructural maturation of the brush border. In contrast, an acid-pulse followed by culture at pH 7.4, did not alter villin expression in BE. Moreover, continuous acid exposure blocked cell proliferation in BE, whereas, an acid-pulse enhanced cell proliferation, as compared to pH 7.4. Based on our ex vivo findings, we propose a model in which the diverse patterns of acid exposure in vivo may contribute to the observed heterogeneity and unpredictable progression to neoplasia of BE.     

人与家兔脂肪来源干细胞体外培养特性的比较

背景：不同种属来源的脂肪来源干细胞在体外培养时特性是否存在差异目前尚未定论。目的：观察在相同培养条件下,人与家兔脂肪来源干细胞体外培养特性的异同。方法：体外分离人腹部取皮植皮术来源的脂肪来源干细胞、家兔背部皮下脂肪来源的脂肪来源干细胞,体外培养并传代,观察各自生长形态,取第3代脂肪来源干细胞,比较二者生长及增殖能力、表面CD分子鉴定情况及成脂、成骨分化能力。结果与结论：人和家兔皮下脂肪均能在体外分离出＂成纤维细胞样＂贴壁生长呈长梭形的细胞;人脂肪来源干细胞一般6～8d可传代,兔脂肪来源干细胞则需要四五天传代。四唑盐结果显示兔、人脂肪来源干细胞分别在第4,6天达到生长高峰。表面标记流式鉴定二者均显示CD29＋CD31-。体外分离培养的人脂肪来源干细胞和兔脂肪来源干细胞均具有干细胞的培养特性。与人脂肪来源干细胞相比,兔脂肪来源干细胞具有更强的增殖和诱导成脂能力,但诱导成骨能力较差,家兔是做脂肪移植研究实验动物不错的选择。     

Comparison of ex vivo expansion culture conditions of mesenchymal stem cells for human cell therapy

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent stem cells. Based on their properties, several clinical trials have been designed to explore their potential therapeutic effect. Fetal calf serum (FCS, commonly used for in vitro expansion) is an undesirable source of xenogeneic antigens and bears the risk of transmitting contaminations. As an alternative for FCS, platelet lysate (PL) and both autologous and allogeneic human serum have been proposed. The aim of this study is to compare the culture of bone marrow (BM)-derived MSCs in the presence of different serum supplements to determine the effect on cell growth, differentiation potential, and immunologic function.
STUDY DESIGN AND METHODS: MSCs from BM of healthy volunteer donors were grown in the presence of 10% FCS supplemented with 1 ng/mL basic fibroblast growth factor (bFGF), 10% human serum supplemented with 1 ng/mL bFGF, 5% PL, and PL 5% supplemented with 1 ng/mL bFGF (PL plus bFGF).
RESULTS: MSCs that expanded in either medium showed a comparable morphology, phenotype, and proliferative and differentiation capacity. While the presence of MSCs in vitro significantly decreased CD3/CD28-mediated T-cell activation, this effect was significantly higher in MSCs cultured with human serum. Production of interferon-γ was inhibited by cocultured media with MSCs while MSCs also induced a significant inhibition of cell cycle in T cells.
DISCUSSION: In conclusion, PL or autologous serum could offer an alternative to the use of FCS in MSC expansion for clinical use maintaining the same growing potential, phenotype, immunomodulatory properties, and differentiation potential.     

Catabolism of newly synthesized decorin in vitro by human peritoneal mesothelial cells.

OBJECTIVE: Previous studies have shown that decorin and biglycan account for over 70% of the proteoglycans (PGs) synthesized by human peritoneal mesothelial cells (HPMCs). Since these PGs are involved in the control of cell growth, cell differentiation, and matrix assembly, we investigated their turnover in cultured HPMCs. METHODS: Confluent HPMCs were metabolically labeled with [35S]-sulfate and the labeled products isolated from the cell medium and the cell layer characterized by sensitivity to bacterial eliminases. Experiments were undertaken with exogenous labeled decorin, and its metabolic state was studied. RESULTS: In a 24-hour labeling period, 75% of the newly synthesized chondroitin sulfate/dermatan sulfate (CS/DS) PGs appeared in the culture medium, the majority of which (90%) was decorin. In the cell layer, protein-free glycosaminoglycan (GAG) chains accounted for 21% of the total CS/DS at 24 hours and exhibited constant specific activity at 12-16 hours. The latter material was turned over with a half-life of approximately 2.5 hours. Exogenous decorin underwent receptor-mediated endocytosis and subsequent intracellular degradation. Uptake but not degradation could be inhibited by heparin. CONCLUSIONS: HPMCs are distinguished by a rapid turnover of decorin. A characteristic metabolic feature is the existence of a large intracellular pool of protein-free DS-GAGs. Understanding the control of decorin turnover in HPMCs might lead to delineation of its potential role in both the physiology and pathophysiology of the membrane in PD patients.