经冠脉移植骨髓单个核细胞和间充质干细胞治疗急性心肌梗死的对比实验研究

目的 对比研究经冠脉骨髓单个核细胞（BM-MNC）和间充质干细胞（MSC）移植对实验性急性心肌梗死（AMI）心功能的影响及其机制.方法 选用12只雄性冀中白猪随机分为：正常对照组、AMI模型组、BM-MNC组、MSC组各3只,经导管球囊封闭前降支制作AMI的动物模型,于梗死后1 h直接冠脉球囊成型术后经OTW球囊注入骨髓干细胞.分别于术前及术后4 w经心脏超声检测心功能,4 w后取材行光、电镜病理学检查,实时定量RT-PCR检测心肌血管内皮生长因子（VEGF）和碱性成纤维细胞生长因子（bFGF）mRNA表达.结果 4 w时干细胞组比AMI模型组室壁运动异常指数显著减轻（P＜0.05）、射血分数显著提高（P＜0.01）.与AMI模型组相比：BM-M;NC和MSC组梗死区及梗死边缘区血管数显著增多、BM-MNC组增加比MSC组显著（P＜0.01）,心肌细胞凋亡指数显著降低,BM-MNC组及MSC组间无明显差异（P＜0.01）.干细胞移植组梗死边缘区冠脉血管周围可见异常细胞生长,有毛细血管＂芽生＂现象,可见不成熟的心肌细胞和细胞凋亡.4 w时左室射血分数（LVEF）与心肌血管数成正相关（r=0.694 9,P=0.037 7）,LVEF与心肌细胞凋亡指数成负相关（r=0.913 3,P=0.000 6）.BM-MNC组,心肌梗死区及梗死边缘区VEGF基因表达比其他三组均明显增加（梗死区F=4.23,P=0.045 6,边缘区F=5.66,P=0.022 3）.BM-MNC及MSC组心肌梗死区bFGF基因表达比梗死模型组显著增加（梗死区F=7.49,P=0.010 4）.结论 经冠脉骨髓单个核细胞和间充质干细胞移植均可改善实验性AMI心功能;改善心功能的机理与梗死区及梗死边缘区VEGF及bFGF表达增加,血管密度增加,心肌细胞凋亡减少有关;骨髓单个核细胞移植的促血管增生作用优于间充质干细胞移植.     

骨髓间充质干细胞移植对心肌缺血/再灌注大鼠心肌胶原与血管新生的影响

目的通过心肌缺血/再灌注模型观察大鼠骨髓间充质干细胞心肌移植对心肌细胞外胶原、小血管新生和心功能变化的影响,并探讨其机制。方法分离、原代培养Wistar大鼠骨髓间充质干细胞,建立缺血/再灌注动物模型,分为假手术组、心肌梗死 骨髓间充质干细胞移植组、心肌梗死对照组,观察骨髓间充质干细胞移植后动物模型血流动力学指标、心肌梗死面积和心肌细胞形态、心肌间质、血管密度、心室膨展指数、心肌细胞横截面积和心肌胶原变化。结果(1)骨髓间充质干细胞心肌移植可缩小60min缺血/再灌注大鼠28d心肌梗死面积;(2)骨髓间充质干细胞移植可减轻心肌梗死动物左心室重塑,但心功能改善作用不明显;(3)骨髓间充质干细胞移植发挥细胞外基质抑制、促血管生成作用,心肌间质胶原含量、胶原分数下降,心肌毛细血管、小动脉密度增加。结论骨髓间充质干细胞移植可改善心肌梗死后心室重塑、促进血管新生和降低心肌胶原作用。     

骨髓动员与骨髓细胞移植对急性心肌梗死猪再灌注后心肌修复及血管再生作用的比较研究

目的比较骨髓间充质干细胞移植和骨髓动员对急性心肌梗死再通模型中心功能改善作用,并比较两者的心肌及血管再生作用.方法将模型分为3组,对照组(7头),骨髓动员组(5头),细胞移植组(8头).心梗再通模型制作后,骨髓动员组予G-CSF静脉注射1天,皮下注射连续6天,细胞移植组予骨髓间充质干细胞4～8×106经梗死相关冠脉注入.3周后观察以下指标1超声心动图检测心功能及局部室壁运动.2免疫组织化学染色,行不同区域血管计数,并观察增殖期内皮细胞数和新生心肌细胞数.结果(1)骨髓动员组和细胞移植组心功能及局部室壁运动均见明显改善.(2)细胞移植组在心梗周围区可见分散分布的BrdU标记移植细胞,骨髓动员组中未观察到处于增殖期的心肌细胞;细胞移植组和骨髓动员组均可见大量新生血管,但二者分布区域不同.结论骨髓动员及骨髓间充质干细胞移植均可明显改善心肌梗死后心功能,而骨髓间充质干细胞可通过补充可收缩细胞而使心功能改善更加明显,二者对不同区域的血管再生作用不同.     

骨髓动员与细胞移植对直接经皮腔内冠状动脉成形术后急性心肌梗死治疗作用

目的比较骨髓间充质干细胞移植和骨髓动员对急性心肌梗死再通模型的心脏修复作用.方法人重组粒细胞集落刺激因子静注1d,皮下注射连续5d行骨髓动员,骨髓间充质干细胞(4～8)×106经梗死相关冠状动脉注入行细胞移植.3周后观察超声心动图指标;免疫组织化学染色行不同区域血管计数,并观察增殖期内皮细胞和心肌细胞或再生心肌细胞.结果骨髓动员组和细胞移植组心功能明显改善;二组均可见心肌细胞再生;细胞移植组和骨髓动员组均可见大量新生血管,但二者分布区域不同.结论骨髓动员及骨髓间充质干细胞移植均可明显改善心肌梗死后心功能,二者可促进心肌细胞再生且对不同区域的血管再生作用不同.     

骨髓间充质干细胞治疗心肌梗死后心功能衰竭的远期疗效

目的采用2’脱氧5-氮杂胞苷诱导的骨髓间充质干细胞移植于心肌梗死后心功能衰竭大鼠心肌中,评价其存活、分化及对心功能的保护作用,并研究其慢性演变。方法体外培养的Wistar大鼠骨髓间充质干细胞,用0.3μmol/L的2’脱氧5-氮杂胞苷体外两次诱导第二代骨髓间充质干细胞,并用溴氮胞苷标记后植入心肌梗死10天心功能衰竭大鼠的心肌疤痕中,同时以注射无血清培养基的实验动物为对照。移植前及移植后1、2、3个月通过心脏射血分数等指标评价其对心功能的保护作用,同时通过双重免疫组织化学及电镜观察对移植细胞的存活和分化进行3个月的慢性动态观察。结果移植后1个月,骨髓间充质干细胞移植组大鼠射血分数(83.3%±8.1%,n=13)高于无血清培养基移植组(47.2%±11.8%,n=12)(P<0.01);并且细胞移植组移植后较移植前心功能(64.3%±10.4%)也有改善(P<0.01)。细胞移植组移植后第2个月(射血分数为83.1%±14.4%,n=7)、第3个月(射血分数为86.3%±3.7%,n=6)心功能改善情况继续保持,与无血清培养基移植组第2个月(射血分数为51.6%±11.2%,n=6)、第3个月(射血分数为49.1%±7.7%,n=6)差异均有显著性(P<0.01)。移植的骨髓间充质干细胞在心肌及疤痕中存活,双重免疫组织化学证实其溴氮胞苷及肌钙蛋白T均阳性。电镜观察提示移植后1个月其在疤痕中具心肌样细胞部分特点,但分化细胞核大浆少,未形成明显肌小节及Z带;移植后3个月,可见明显Z带样结构。结论经2’脱氧5-氮杂胞苷诱导的骨髓间充质干细胞可改善心肌梗死后心功能衰竭大鼠的心功能,并能长期保持(观察3个月);其能在心肌梗死后心功能衰竭大鼠心肌及疤痕中存活,并能逐渐向心肌细胞分化。     

移植冠心病患者骨髓间充质干细胞治疗心肌梗死大鼠的机制探讨

目的:在大鼠心肌梗死模型上移植冠心病患者来源的骨髓间充质干细胞,探讨人骨髓间充质干细胞改善心肌梗死后心功能的作用机制。方法:抽取冠心病患者骨髓,用人骨髓间质细胞专用培养液进行体外培养、扩增、标记。结扎大鼠冠状动脉左前降支制作心肌梗死模型,2周后使用超声筛选出合格的动物模型。然后动物随机分为细胞移植组(n=14)和培养液注射组(对照组,n=13)。在移植前后对心脏的收缩、舒张功能和心室重构指标进行超声评价和对比,取材后进行病理学检查和分子生物学检测。结果:移植后进行超声评价,治疗组较对照组射血分数值和缩短分数值显著改善。免疫组化显示移植细胞在宿主体内可以向肌源性细胞分化。逆转录—聚合酶链反应(RT-PCR)显示细胞治疗组梗死区心肌Ⅰ型、Ⅲ型胶原基因表达水平明显高于对照组,而在非梗死区低于对照组。细胞治疗组梗死区间质细胞源因子、血管内皮生长因子、Bcl-2基因表达水平均明显高于对照组。结论:冠心病患者骨髓间质细胞移植后可以提高心肌梗死后心脏收缩功能,减轻左心室重塑。作用机制可能为新血管生成增加、细胞外基质改变、旁分泌、抗凋亡和心肌再生等共同作用的结果。     

骨髓间充质干细胞移植对大鼠梗死心脏核因子κB活性的影响

目的 观察骨髓间充质干细胞对心肌梗死的治疗效果,以及心肌梗死后炎症因子表达的影响,探讨其治疗心肌梗死的可能机制.方法 提取SD大鼠骨髓间充质干细胞,体外培养、纯化扩增.采用结扎冠状动脉前降支方法复制大鼠心肌梗死模型,将建立的模型随机分为:1)假手术组(仅穿线不结扎血管,n=8);2)PBS溶液注射组(n=8);3)干细胞移植组(n=8).模型制作成功即刻,将骨髓间充质干细胞通过心外膜分4点注射到梗死周边部位.4周后测定血流动力学指标.随后通过Western blot方法测定核因子κB活性,RT-PCR和westernblot法测定肿瘤坏死因子α、白介素6 mRNA和蛋白表达.结果 1)4周后,假手术组大鼠死亡率20%(2/10),治疗阴性对照组大鼠死亡率33.3%(4/12),干细胞移植组大鼠死亡率20%(2/10),3组死亡率没有统计学差异(P=0.646);2)与注射PBS溶液相比,骨髓间充质干细胞移植可以改善心肌梗死大鼠血流动力学指标;3)骨髓间充质干细胞可以抑制大鼠梗死心肌核因子出活性,下调肿瘤坏死因子α和白介素6 mRNA和蛋白表达水平.结论 骨髓间充质干细胞可以改善心肌梗死大鼠的心功能,同时可调节大鼠梗死心脏炎症反应.     

微泡破坏及骨髓间充质干细胞移植治疗犬急性心肌梗死模型的有效性

目的探讨超声介导的微泡破坏联合骨髓间充质干细胞移植到梗死区治疗犬急性心肌梗死的有效性。方法造模组犬随机分为磷酸盐缓冲液(PBS)组、微泡破坏+PBS组、骨髓间充质干细胞(BMSCs)组、微泡破坏+BMSCs组4组。术后28 d,用心肌声学造影(MCE)检测心功能和灌注缺损面积占左室总灌注面积的百分比(DA%),NBT染色检测心肌梗死面积,用免疫组化检查BrdU染色阳性的细胞数来测定BMSCs梗死区归巢数,用免疫组化检测α-肌动蛋白评估心肌细胞的收缩功能。结果 BMSCs的移植能够显著改善心功能,缩小心肌梗死面积。其中,超声+微泡+BMSCs组心功能的改善最显著(P<0.05),梗死区域BrdU染色阳性的细胞数最多(P<0.05),α-肌动蛋白阳性细胞数目最多,心肌梗死面积小于其他各组(P<0.05)。结论诊断超声介导微泡破坏同BMSCs的移植能够使更多的BMSCs归巢,使心肌梗死面积缩小,改善心功能。     

高表达整合素连接激酶对大鼠骨髓间充质干细胞旁分泌功能的影响

目的 研究转染腺病毒介导的整合素连接激酶(ILK)对大鼠骨髓间充质干细胞旁分泌作用的影响.方法 构建携带人野生型整合素连接激酶质粒cDNA和人重组绿色荧光蛋白的腺病毒载体(adeno-ILK)和空载腺病毒(MSC),通过全骨髓贴壁法分离培养骨髓间充质干细胞.采用无血清培养转染adeno-ILK(MSC-ILK组)或空载腺病毒(MSC组),通过流式细胞术检测人重组绿色荧光蛋白从而测定转染效率,确定最佳的病毒感染复数.提取骨髓间充质干细胞mRNA,用Real-time PCR测定血管内皮生长因子、成纤维细胞生长因子2和胰岛素样生长因子1的基因表达量.结果 MSC-ILK组旁分泌因子血管内皮生长因子、成纤维细胞生长因子2和胰岛素样生长因子1的基因表达量分别是MSC组的 8.2±0.4、2.6±0.2及2.4±0.2倍(P＜0.05或P＜0.01).结论 整合素连接激酶基因转染可明显促进骨髓间充质干细胞表达旁分泌因子血管内皮生长因子、成纤维细胞生长因子2和胰岛素样生长因子1.     

骨髓间充质干细胞移植对心肌梗死后心室肌细胞钾离子通道Ito Kv4.2的影响

目的 研究心肌梗死后移植骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSCs)对心室肌细胞钾离子通道Ito亚单位Kv4.2基因表达的影响方法 40只SD(Sprague-Dawley)大鼠随机分成4组(10只/组):假手术组、心肌梗死组、心肌梗死+干细胞组和心肌梗死+细胞培养基组.开胸结扎冠状动脉前降支建立心肌梗死模型,建模成功后,在梗死周围分别注入BMMSCs和细胞培养基,心肌梗死组仅建立心肌梗死模型,假手术组仅开胸子以前降支穿线但不结扎.2周后行心肌组织HE染色和荧光显微镜观察移植细胞,逆转录聚合酶链反应(RT-PCR)和Western blot分别检测钾离子通道Ito亚单位Kv4.2基因mRNA和蛋白水平.结果 (1)心肌组织免疫荧光检测发现,BMMSCs集中分布于梗死区和梗死心肌周围;(2)心肌梗死组和心肌梗死+细胞培养基组Kv4.2 mRNA量(0.39±0.02,0.41±0.04)和蛋白量(0.47±0.02,0.50±0.05)明显下降,与假手术组(0.76±0.05,0.74±0.06)相比差异有统计学意义(P＜0.01);心肌梗死+干细胞组Kv4.2 mRNA量和蛋白表达量(0.57±0.05,0.64±0.03)较心肌梗死组(0.39±0.02,0.47±0.02)明显升高(P＜0.01).结论 骨髓间充质干细胞移植后心肌梗死大鼠钾离子通道Ito亚单位Kv4.2基因表达上升,可能减少心律失常发生.     

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.     

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.     

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.     

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.