零下非结冰UW液、Celsior液和HTK液保存C3A细胞的效果比较

目的 比较UW液、Celsior液和HTK液零下非结冰(-0.8℃)保存生物人工肝用C3A细胞的效果.方法 制备好的C3A细胞悬液分以下3组:UW液保存组(UW液组);Celsior液保存组(CS液组);HTK液保存组(HTK液组).各组细胞于-0.8℃低温保存72 h后,分别测定细胞存活率及死亡率(流式细胞术)、LDH释放、尿素合成功能及白蛋白分泌功能.结果 UW液及Celsior液比HTK液显著提高了零下非结冰保存72 h的C3A细胞的存活率[(84.34±4.25)%,(83.53±3.73)% vs (75.65±3.01)%,P<0.001]和细胞内ATP含量[(5.54±1.44)μg/106 个细胞,(5.51±1.31)μg/106个细胞 vs (2.75±1.03)μg/106 个细胞,P<0.001];抑制了LDH释放(P<0.001);更好地维持了细胞尿素合成功能[(0.63±0.10)mmol/L,(0.62±0.06)mmol/L vs (0.39±0.04)mmol/L,P<0.001]和白蛋白分泌功能[(1.99±0.38)g/L,(1.96±0.24)g/L vs (1.50±0.18)g/L,P<0.05].UW液与Celsior液零下非结冰保存C3A细胞的效果无差异.结论 同HTK液相比,使用UW液或者Celsior液零下非结冰(-0.8℃)保存C3A细胞可以明显地提高复温后细胞存活率和细胞内ATP含量,降低低温损伤引起的LDH释放,有效地保护肝细胞尿素合成功能和白蛋白分泌功能.UW液同Celsior液零下非结冰保存C3A细胞的效果无差异,保存时间不宜超过72 h.     

零下非结冰保存L-02细胞的效果及与细胞凋亡的关系

目的 采用UW液零下非结冰(-0.8℃)保存生物人工肝用L-02细胞,与常规低温(4℃及0℃)比较,探索零下非结冰保存肝细胞的效果以及同细胞凋亡的关系.方法 制备好的UW液保存的L-02细胞悬液分为3组:-0.8℃组(零下非结冰组),0℃组(0℃非结冰组),4℃组(对照组).低温保存24 h、48 h及72 h后,分别测定细胞存活率及凋亡率(流式细胞术)、LDH释放、尿素合成功能、白蛋白分泌功能.结果 零下非结冰(-0.8℃)较常规低温(0℃及4℃)明显提高了低温保存L-02细胞的存活率[72 h:-0.8℃(70.17±2.82)% vs 4℃(60.05±3.17)%],降低了细胞凋亡率[72 h:-0.8℃(5.73±1.68)% vs 4℃(9.20±2.35)%].零下非结冰明显抑制了LDH的释放[72 h:-0.8℃(113.88±5.64)U/L vs 4℃(170.47±11.80)U/L],更好地维持了L-02细胞的尿素合成功能[72 h:-0.8℃(1.01±0.14)mmol/L vs 4℃(0.66±0.09)mmol/L]及白蛋白分泌功能[72 h:-0.8℃(9.04±0.53)μg/ml vs 4℃(7.70±0.52)μg/ml].结论 与0℃及4℃相比,零下非结冰(-0.8℃)可明显提高L-02细胞存活率,降低低温损伤引起的细胞凋亡,有效的保护肝细胞尿素合成功能和白蛋白分泌功能.     

零下非结冰抑制线粒体途径相关的细胞凋亡成功保存生物人工肝用L02细胞

目的 比较常规低温(4℃及0℃)及零下非结冰温度(-0.8℃)保存L-02肝细胞时Bcl-2/Bax基因和蛋白表达的差异及与低温保存引起细胞凋亡的关系.方法 L-02细胞分为3组:-0.8C组(零下非结冰组),0°C组(0℃非结冰组),4℃组(对照组).低温保存72 h后,分别测定细胞存活率及凋亡率(流式细胞术),检测LDH、ALT释放及凋亡基因Bcl-2与Bax的mRNA及蛋白定量表达并计算Bcl-2/Bax比值.结果 零下非结冰组较0℃组及4℃组显著提高了低温保存72 h的L-02细胞存活率[(70.95％±3.33％)vs(65.9％±3.22％)、(61.02％±3.37％),均P＜0.05];降低了细胞凋亡率[(5.82％±1.68％)vs(8.53％±1.67％)、(9.40％±2.57％),均P＜0.05];抑制了LDH[(101.50±6.58) U/L vs(127.67±12.09)U/L、(150.13±11.38) U/L,均P＜0.05]与ALT释放[(6.07±0.63) U/L vs (7.00 ±0.60) U/L、(8.63±1.25) U/L,P ＜0.05];提高了L-02细胞Bcl-2基因mRNA及蛋白表达(均P＜0.05),降低了Bax基因mRNA及蛋白表达(均P＜0.05),Bcl-2/Bax指数上升(均P＜0.05).结论 同常规低温保存相比,零下非结冰可显著提高肝细胞低温保存后细胞存活率,降低低温损伤引起的细胞凋亡.零下非结冰降低了低温源性细胞凋亡的机制可能同Bcl-2基因表达增加、Bax基因表达降低即Bcl-2/Bax比值上升有关.     

生物人工肝用肝细胞低温保存的现状与进展

大量功能好的肝细胞是生物人工肝支持系统(bio-artificial liver support system,BLASS)的核心,是制约BLASS临床广泛应用的瓶颈.探索出一种实用的肝细胞低温保存方法,建立一个随时可用(ready to use)的肝细胞库是BALSS普遍推广的基础.目前肝细胞低温保存分为4℃或零下非结冰保存和-80℃或-196℃深低温冻存两大类,两大类保存方法各有优缺点.影响肝细胞低温保存的因素很多,如保存(冻存)液、(冻存)保护剂等.有关肝细胞在低温保存过程中死亡的机制尚未完全阐明,但大量研究发现细胞凋亡是除了坏死之外低温保存肝细胞死亡的另一个重要的途径.     

葡萄糖酸镁对离体大鼠心肌缺血/再灌注细胞凋亡的影响及机制

目的:观察葡萄糖酸镁对离体大鼠心肌缺血/再灌注(I/R)损伤的保护作用及可能机制。方法:将48只大鼠等分为对照组、I/R组、葡萄糖酸镁组。实验1:每组取8只大鼠,对照组用改良的K-H液持续灌注110min;I/R组用改良的K-H液灌流20min后,停灌30min,再灌注60min;葡萄糖酸镁组在改良的K-H液中加入葡萄糖酸镁2.4mmol/L,余同I/R组。检测心肌灌流液中肌酸激酶(CK),乳酸脱氢酶(LDH)含量,心肌组织中总超氧化物歧化酶(T-SOD)、丙二醛(MDA)含量。实验2:每组8只大鼠,实验过程基本与实验1相同,仅将再灌注时间改为120min,检测心肌细胞凋亡指数(AI)。观察实验1、2合组再灌注心律失常发生情况。结果:葡萄糖酸镁组与I/R组相比,室性心律失常发生率显著下降(VT:43.8%对100.0%,VF:12.5%对75.0%,P<0.01);再灌流出液中CK、LDH含量显著降低[CK:(121.76±0.75)U/L对(132.33±1.73)U/L,LDH:(51.94±1.93)U/L对(62.73±2.18)U/L,P<0.01];心肌组织T-SOD活性显著升高[(49.12±0.54)NU/mg对(40.09±1.64)NU/mg,P<0.01];MDA含量、细胞凋亡指数显著降低[MDA:(3.05±0.19)μmol/g对(3.94±0.16)μmol/g,AI:(27.79±1.59)%对(33.61±2.10)%,P<0.01]。结论:葡萄糖酸镁对离体大鼠心肌I/R损伤具有保护作用,其机制可能与清除氧自由基、抗细胞凋亡有关。     

葡萄糖酸镁对离体大鼠心肌缺血／再灌注细胞凋亡的影响及机制

目的:观察葡萄糖酸镁对离体大鼠心肌缺血/再灌注(I/R)损伤的保护作用及可能机制.方法:将48只大鼠等分为对照组、I/R组、葡萄糖酸镁组.实验1:每组取8只大鼠,对照组用改良的K-H液持续灌注110 min;I/R组用改良的K-H液灌流20 min后,停灌30 min,再灌注60 min;葡萄糖酸镁组在改良的K-H液中加入葡萄糖酸镁2.4 mmol/L,余同I/R组.检测心肌灌流液中肌酸激酶(CK),乳酸脱氢酶(LDH)含量,心肌组织中总超氧化物歧化酶(T-SOD)、丙二醛(MDA)含量.实验2:每组8只大鼠,实验过程基本与实验1相同,仅将再灌注时间改为120 min,检测心肌细胞凋亡指数(AI).观察实验1、2合组再灌注心律失常发生情况.结果:葡萄糖酸镁组与I/R组相比,室性心律失常发生率显著下降(VT:43.8%对100.0%,VF:12.5 %对75.0%,P<0.01);再灌流出液中CK、LDH含量显著降低[CK:(121.76±0.75)U/L对(132.33±1.73)U/L,LDH:(51.94±1.93)U/L对(62.73±2.18)U/L,P<0.01];心肌组织T-SOD活性显著升高[(49.12±0.54)NU/mg对(40.09±1.64)NU/mg,P<0.01];MDA含量、细胞凋亡指数显著降低[MDA:(3.05±0.19)μmol/g对(3.94±0.16)μmol/g,AI:(27.79±1.59)%对(33.61±2.10)%,P<0.01].结论:葡萄糖酸镁对离体大鼠心肌I/R损伤具有保护作用,其机制可能与清除氧自由基、抗细胞凋亡有关.     

促肝细胞生长素联合不同DMSO浓度冻存小鼠肝细胞的质量研究

目的使用自行配制组方含有促肝细胞生长素联合不同浓度DMSO的冻存液冻存小鼠肝细胞,以探索一种效果较好的冻存液从而改善冻存肝细胞质量。方法以体重20~30 g的昆明小鼠为肝细胞供体,用改良的胶原酶灌注法分离肝细胞,将分离好的肝细胞分别以10%、20%、30%浓度的DMSO联合促肝细胞生长素的冻存液(试验组1、2、3组)和标准冻存液(对照组)进行逐级降温缓慢冻存,置液氮中。2个月后快速复苏肝细胞,观察并测定肝细胞活率、功能和形态学。结果冻存小鼠肝细胞2个月复苏检测,试验1组、2组、3组的肝细胞活率台盼蓝(TB)染色结果为80.18±2.44%、86.20±2.33%、78.50±3.43%,而对照组肝细胞活率TB染色49.71±3.51%,试验组和对照组差异具有统计学意义(P0.05),试验2组与其他试验组比较差异具有统计学意义(P0.05);血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)和乳酸脱氢酶(LDH)漏出率的值,试验1组、2组、3组分别为14.03±2.21 U/L、8.05±1.25 U/L、18.40±3.25 U/L;15.14±3.03 U/L、9.12±1.56 U/L、20.51±3.56 U/L;15.11±2.10 U/L、8.26±1.23 U/L、20.31±3.21 U/L,而对照组分别为24.28±1.96 U/L、25.44±2.06 U/L、26.22±3.23 U/L,两组差异具有统计学意义(P0.05),试验2组与其他试验组比较差异具有统计学意义(P0.05);合成血清白蛋白的值,试验1组、2组、3组为3.24±0.18 g/L、4.12±0.23 g/L、3.01±0.45 g/L,对照组为2.56±0.32 g/L,两组差异具有统计学意义(P0.05),试验2组与其他组比较差异具有统计学意义(P0.05)。结论在本研究中,冻存小鼠肝细胞2个月,促肝细胞生长素联合20%DMSO冻存液较常规冻存液能有效减轻小鼠肝细胞的损伤,发挥良好保护作用。     

卡巴胆碱对肠上皮细胞氧化损伤的保护作用

目的:探讨卡巴胆碱对大鼠肠上皮细胞(intes- tinal epiIhelia cell,IEC)过氧化损伤的影响.方法:将H_2O_2加入IEC培养液中制成IEC过氧化损伤模型.实验设对照组、H_2O_2组(2.5 mmol/L)、卡巴胆碱组(卡巴胆碱100μmol/L).用四甲基偶氮唑盐(MTT)实验检测IEC的存活率,测定培养液中测乳酸脱氢酶(LDH)和细胞中丙二醛(MDA)含量.结果:与对照组相比,H_2O_2组LDH(7.40±2.10 vs 0.81±0.12,P<0.01)、MDA水平显著增高(P<0.0 1),细胞活力明显降低(37.25%±0.80%vs 100%±0.13%,P<0.01).卡巴胆碱组与H_2O_2组相比,LDH漏出MDA形成明显减少(4.64±1.31 vs 7.40±2.10,P<0.01),细胞的细胞活力显著增加(78.70%±2.80%vs 37.25%±0.80%,P<0.01).结论:卡巴胆碱对大鼠IEC过氧化损伤具有保护作用.     

马尾松花粉多糖硫酸酯对肝癌HepG2细胞的诱导分化

目的:观察马尾松花粉多糖硫酸酯(SPPM60)对人肝癌细胞株HepG2的诱导分化作用, 并对其机制进行初步探讨.方法:MTT法检测HepG2细胞增殖活力; 倒置显微镜、HE染色及透射电镜观察细胞形态;流式细胞技术检测细胞周期; 荧光分光光度计检测细胞内钙离子浓度的变化; 免疫细胞化学方法检测甲胎蛋白的表达, 溴甲酚绿法测定白蛋白含量; 重氮反应比色法测定γ-谷氨酰转肽酶活力, 比色法测定碱性磷酸酶活力, 考马斯亮蓝法测定总蛋白含量.结果:经SPPM60处理后, HepG2细胞的增殖受到抑制; 细胞形态趋于正常; 与对照组相比, SPPM60组G0/G1期细胞数增加(76.97%±0.91% vs 64.62%±0.18%, P<0.05), S期细胞数减少(17.21%±0.71% vs 24.26%±0.15%,P<0.05), G2/M细胞数减少(5.82%±0.20% vs11.13%±0.34%, P<0.01); [Ca2+]i降低; 甲胎蛋白表达降低, 白蛋白分泌量上升(6.77±0.33 mg/106细胞vs 4.87±0.30 mg/106细胞,P<0.05), γ-谷氨酰转肽酶与碱性磷酸酶活力降低(16.3±0.7 U/g vs 22.3±1.2 U/g; 223.3±15.7 U/g vs 311.1±13.4 U/g P<0.05或0.01), 胎盘型碱性磷酸酶的比例降低(46.4%±1.5% vs62.5%±2.3%, P<0.05). PPM60组细胞没有明显变化.结论:硫酸酯化赋予了SPPM60抑制HepG2细胞的活性, 其作用机理可能是降低细胞内钙离子浓度, 阻滞细胞周期于G0/G1期, 诱导HepG2细胞分化逆转其恶性.     

经门静脉骨髓基质细胞移植治疗肝纤维化

目的: 探讨移植骨髓基质细胞(BMSCs)减轻小鼠肝纤维化的作用.方法: BALB/c小鼠BMscs分离培养及经门静脉移植到BALB/c小鼠肝脏,二乙基亚硝胺诱导肝纤维化.60只小鼠随机分为对照组.模型组及治疗组.3 mo后测定ALT、AST、透明质酸酶(HA)和层黏连蛋白(LN)浓度,及肝脏羟基脯氨酸(Hyp)含量.免疫组化检测肝脏a.平滑肌肌动蛋白(α-SMA)表达,及荧光原位杂交鉴定移植的BMSCs向肝细胞的分化.结果: BMsCs在添加肝细胞生长因子(HGF)的培养基中体外培养能分化为肝细胞样细胞.与模型组相比.移植BMscs能显著降低血清ALT、AST、HA和LN的水平以及肝脏Hyp含量和α-SMA的表达(208±44 U/L 341±66 U/L,372±84 U/L vs 506±81 U/L,289±74μg/L vs 362±83 μg/L,178±48 μg/L vs 232±63 ug/L,900±141 mg/g liver vs1255±205mg/g liver,,均p<0.01).荧光原位杂交显示DEN诱导的损伤肝脏中有骨髓来源的肝细胞,3 mo后10%的肝细胞来源于BMSCs.结论: 在肝纤维化模型中,经门静脉移植的BMscs能分化为肝细胞,有效地恢复肝功能和减轻肝纤维化.     

The immunoneuroendocrine role of melatonin

Abstract: A tight, physiological link between the pineal gland and the immune system is emerging from a series of experimental studies. This link might reflect the evolutionary connection between self-recognition and reproduction. Pinealectomy or other experimental methods which inhibit melatonin synthesis and secretion induce a state of immunodepression which is counteracted by melatonin. In general, melatonin seems to have an immunoenhancing effect that is particularly apparent in immunodepressive states. The negative effect of acute stress or immunosuppressive pharmacological treatments on various immune parameters are counteracted by melatonin. It seems important to note that one of the main targets of melatonin is the thymus, i.e., the central organ of the immune system. The clinical use of melatonin as an immunotherapeutic agent seems promising in primary and secondary immunodeficiencies as well as in cancer immunotherapy. The immunoenhancing action of melatonin seems to be mediated by T-helper cell-derived opioid peptides as well as by lymphokines and, perhaps, by pituitary hormones. Melatonin-induced-immuno-opioids (MHO) and lymphokines imply the presence of specific binding sites or melatonin receptors on cells of the immune system. On the other hand, lymphokines such as -γ-interferon and interleukin-2 as well as thymic hormones can modulate the synthesis of melatonin in the pineal gland. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ.     

Changes in connexin43, the gap junction protein of astrocytes, during development of the rat pineal gland

Abstract: The abundance of gap junctions between rat pineal astrocytes formed by connexin43 (Cx43) was studied during development. Levels and distribution of Cx43 were measured by immunoblotting and indirect immunofluorescence, respectively. The amount of Cx43 in cells located within the gland was low until about the 7th postnatal day and increased to adult values between the 14th and 21st days postpartum. Although astrocytes, recognized by their vimentin immunoreactivity, were scarce before birth, they were abundant by the 7th postnatal day suggesting that the low levels of Cx43 found at this age corresponded to a low expression of this protein. Localization of the immunoreactivity to Cx43 and vimentin showed a close correlation, indicating that mature or immature pineal astrocytes form gap junctions made of Cx43. Since Cx43 levels attained their adult values at about the time the innervation and the functional state of the gland reached maturity (2–3 weeks after birth), it is proposed that astrocyte gap junctions are involved in the function of the adult rat pineal gland.     

Response of rat pineal melatonin to calcium, magnesium, and lithium is circadian stage dependent

Abstract: Herein we documented the response of pineal melatonin production to electrolytes known to be effective on pineal function in view of a possible circadian stage dependence. We studied the release of melatonin by perifused rat pineal glands at 2 different circadian stages corresponding to the middle of the light and dark periods, i.e., respectively, 7 and 19 HALO (Hours After Light Onset, L:D = 12:12). The initial efflux rates were, as expected, much higher in the perifusates of glands removed from rats sacrificed during the dark phase than of those removed during the light phase. After 3 hr of perifusion, melatonin release reached similar levels which were found constant up to the 8th hr of perifusion, whatever the circadian stage. Perifusion of the glands with physiological concentrations for the rat of calcium (5.2 mmol/1) and magnesium (1.34 mmol/1) resulted in a stimulatory effect on the pineal glands removed from rats sacrificed in the middle of the dark period (19 HALO), whereas no effects were observed on the pineal glands removed from rats sacrificed during the light (7 HALO). Lithium (0.28 and 0.55 mmol/1) was ineffective on melatonin release in pineal glands removed 7 and 19 HALO. Our results show differences in the initial efflux rates of melatonin and in the response of perifused pineal glands to calcium and magnesium according to the circadian stage.     

Conservative management of perforated duodenal diverticulum： A case report and review of the literature

Duodenal diverticula are a relatively common condition. They are asymptomatic, unless they become complicated, with perforation being the rarest but most severe complication. Surgical treatment is the most frequently performed approach. We report the case of a patient with a perforated duodenal diverticulum, which was diagnosed early and treated conservatively with antibiotics and percutaneous drainage of secondary retroperitoneal abscesses. We suggest this method could be an acceptable option for the management of similar cases, provided that the patient is in good general condition and without septic signs.     

Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland

Abstract: The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M_r range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [¹²⁵I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (K_a of 4.7 ± 0.2 × 10⁹ M^-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M_r 24,000. The functional status of PRL-and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.     

Microbiology of human immunodeficiency virus anorectal disease

PURPOSE: Individuals who are seropositive for the human immunodeficiency virus are at high risk for opportunistic infection and anorectal disorders. Little prospective information is available regarding anorectal pathogens in these patients. METHODS: One hundred sixty-three HIV-seropositive patients presented to the colorectal clinic between 1989 and 1992. Forty-seven (29 percent) patients were thought to have an infectious process and were prospectively studied using a standardized multiculture protocol. RESULTS: Mean age was 33 (range, 19–59) years. All were male; high-risk behavior accounted for 87 percent of HIV transmissions. Presenting complaints included anorectal pain (79 percent), pus per anum (28 percent), and blood per anum (26 percent). Examination revealed perianal tenderness (60 percent), condyloma (38 percent), perianal ulcers (38 percent), and anal fissures (34 percent). Sixty-six sets of cultures were performed; 28 patients had one set, 15 had two sets, and 4 had three sets. Thirty-two of these 47 patients (68 percent) had positive cultures including herpes (50 percent), cytomegalovirus (25 percent),Neisseria gonorrhoeae (16 percent), chlamydia (16 percent), acidfast bacilli (2 percent), and others (9 percent). Six of 32 patients with positive cultures had more than one organism cultured. Sixteen (50 percent) patients with positive cultures were treated medically, 8 (25 percent) were treated surgically and 8 (25 percent) were treated with both modalities. Sixty-one procedures were performed on 17 patients for condylomata. Eighteen patients had 20 procedures for abscesses, 50 percent of whom had positive cultures for other than common bowel flora; all improved. Fourteen patients underwent 33 procedures for perianal fistulas.Mycobacterium fortuitum was cultured from one patient who required 13 procedures for abscesses and fistulas. Forty-five (96 percent) patients were followed for an average of 12.5 months ±2.9 SEM (range, 1–94 months). Symptoms were improved or resolved in 22 of 32 (69 percent) patients with positive cultures and in 11 of 13 (84 percent) with negative cultures. CONCLUSIONS: Specific pathogens may often be identified in human immunodeficiency virus-seropositive patients with anorectal disorders if aggressively sought. Although patients without specific pathogens identified may be expected to improve with planned empiric treatment, positive identification allows more directed therapy.