胰岛素样生长因子-1预处理对大鼠骨髓间充质干细胞移植效率及心肌修复的影响

目的:探讨胰岛素样生长因子-1(IGF-1)预处理对大鼠骨髓间充质干细胞(MSCs)移植效率及心肌修复的影响.方法:不同浓度的IGF-1(0、2.5、5、10 ns/m1)以不同时间(4、12、24、48 h)预处理MSCs,流式细胞术分析趋化因子受体4的表达.建立大鼠心肌梗死模型,尾静脉分别注射不含细胞的100μl 培养液(对照组),无预处理的MSCs(MSCs组)和含经IGF-1预处理的MSCs(IGF-1-MSCs组),每组8只大鼠.MSCs移植4周后分析血流动力学和心肌梗死面积,移植细胞的归巢情况,Ⅷ因子相关抗原的表达,心肌特异性蛋白肌钙蛋白T的表达.结果:流式细胞术显示10 ns/mi IGF-1预处理48 h后MSCs趋化因子受体4的表达较0、2.5、5 ng/ml IGF-1预处理48 h后显著增高(P0.05).结论:IGF-1通过提高MSCs的归巢情况来提高细胞移植治疗心肌梗死的效果.     

体外预处理骨髓间充质干细胞移植对心肌梗死作用的实验研究

目的初步研究基质细胞衍生因子-1(SDF-1)预处理后骨髓间充质干细胞(MSCs)产生的系列效应变化;并将预处理后的MSCs移植入大鼠心肌梗死模型,观察细胞归巢、增殖,及对心肌缺血区细胞凋亡、新生毛细血管生成、心室重构和心功能的影响,进一步探讨MSCs作用于缺血性心脏病的机理。方法干细胞部分:取SD大鼠胫骨及股骨分离骨髓,用全骨髓差异贴壁法培养MSCs,并进行表面标志鉴定;用5-溴脱氧尿核苷(Brd U)标记MSCs细胞,制作细胞爬片,检测其标记率,以确保标记能满足移植示踪及观察需要;将MSCs分别在不含(对照组)或含(SDF-1预处理组)SDF-1的无血清培养基中孵育60分钟,检测各上清液中血管内皮生长因子(VEGF)含量,及处理后细胞表面CXCR-4受体表达的变化。动物实验部分:建立大鼠心梗模型48只,随机平均分为三组:空白组(A),干细胞对照组(B),SDF-1预处理组(C);分别在其心梗后1天经尾静脉注射生理盐水(A组)、未处理的MSCs(B组)或SDF-1预处理后的MSCs(C组);移植后24小时检测梗死周边区心肌细胞凋亡;一周后检测梗死周边区归巢增殖的Brd U阳性细胞;四周后检测心功能,计算梗死面积,并通过血管荧光灌注显像检测梗死周边区新生血管密度。结果利用全骨髓差异贴壁法可以获取稳定分裂大鼠MSCs,经表型鉴定证明其为CD29~+CD90~+CD34~-细胞:用10μmol/L Brd U标记MSCs24小时,标记率>98%,能满足移植示踪需要;用SDF-1预处理MSCs可显著增加其释放VEGF能力[(68.92±5.03)pg/mL vs.(38.79±3.16)pg/mL,P<0.05],并使CXCR-4受体发生内摄作用,表现为膜外表达减少(P<0.05)。结论 (1)SDF-1预处理MSCs可显著增加其释放VEGF的能力,并使CXCR-4受体发生内摄作用,增强归巢能力,促进细胞旁分泌作用。(2)SDF-1预处理MSCs后移植,可减少大鼠梗死周边区心肌细胞的凋亡,增加缺血区的灌注,从而抑制心室重构,改善心功能。     

单核细胞趋化蛋白-1在骨髓间质干细胞归巢到大鼠急性梗死心肌中的作用

目的探讨单核细胞趋化蛋白-1(MCP-1)在骨髓间质干细胞(MSCs)归巢到急性梗死心肌中的作用。方法129只大鼠随机分为检测组(65只)和处理组(64只),检测组分为心肌梗死组(35只)和非心肌梗死组(30只),处理组则分为Ⅰ组、Ⅱ组、Ⅲ组、Ⅳ组4个亚组(各16只)。检测组以免疫组织化学检测MCP-1在2个亚组大鼠心肌中表达的差异。处理组大鼠心肌梗死4天后予以MCP-1、抗MCP-1抗体干预,同时经尾静脉注射BrdU标记的MSCs,7天后检测心肌梗死区MSCs归巢量,28天后检测大鼠心功能状况、心肌梗死区及其周围的血管密度。结果MCP-1于心肌梗死后第1天即升高,第7天到达峰值,28天时恢复至正常水平。心肌梗死组MSCs归巢量大于非心肌梗死组(P=0.000)。Ⅰ组大鼠心肌梗死区MSCs归巢量、心功能水平及血管密度大于Ⅲ组及Ⅱ组(P<0.01)。结论大鼠心肌梗死后早期梗死区MCP-1表达水平升高;MCP-1能够促进MSCs归巢并改善心功能。     

重建心脏生物起搏器模型的体外研究

目的研究超极化激活环核苷酸门控阳离子通道基因亚型4(HCN4)改造的大鼠骨髓间充质干细胞(MSCs)与心室肌细胞共培养,构建具有自律性的心脏生物起搏器模型的可行性;并研究MSC中过表达连接蛋白45(CX45)对以上生物起搏器模型自律性的影响。方法 (1)体外分离培养新生大鼠的心室肌细胞,鉴定纯度及活力;并通过膜片钳方法记录心肌细胞的自发性动作电位。(2)基因克隆的方法构建包含HCN4基因的穿梭质粒pCDH1-GFP-HCN4,并通过四质粒系统包装成慢病毒颗粒,转染大鼠的MSCs,构建成HCN4+MSCs;将仅转染有GFP基因的慢病毒颗粒的MSCs设为HCN4-MSCs。通过RT-PCR,以及荧光观察鉴定HCN4基因在MSCs中的表达;电压钳记录阳性转染细胞的起搏电流If。(3)将HCN~4+MSCs与乳鼠心室肌细胞共培养构建心脏生物起搏器模型,同时将HCN4-MSCs与心肌细胞共培养设为"HCN4-MSCs对照组"。为了监测生物起搏器的自律性,初步分析自律性变化的原因,计数心室肌细胞自发搏动频率、记录自发性动作电位;并通过免疫荧光标记等方法鉴定共培养的两类细胞间连接蛋白43(CX43)的表达;加入缝隙连接阻断剂甘珀酸验证缝隙连接在模型中的价值。(4)为了构建CX45基因稳定表达的MSCs细胞株,首先通过基因克隆的手段构建含有CX45基因的表达质粒pDsRED2-N1-RFP/Gja7,通过脂质体2000的介导将pDsRED2-N1-RFP/Gja7转染MSCs;G418筛选获得稳定转染的细胞株CX45+MSCs;同时将pDsRED2-N1-RFP空质粒转染的MSCs设为CX45-MSCs。为了鉴定MSCs中CX45基因的转录,进行了RT-PCR试验;CX43与CX45单克隆抗体免疫双标MSCs,鉴定CX45~+MSCs中连接蛋白类型的变化。(5)将CX45~+MSCs作为生物起搏的载体细胞,转染HCN4基因,构建HCN4~+CX45+MSCs;将HCN4~+CX45~+MSCs与乳鼠心室肌细胞共培养重新构建心脏生物起搏器;同时将CX45-MSCs中转染HCN4基因,与心肌细胞共培养后构建的生物起搏器模型设为"HCN4~+CX4     

扶正化瘀胶囊对心肌梗死大鼠心肌缝隙连接蛋白43表达的影响

目的研究扶正化瘀胶囊对心肌梗死大鼠心肌缝隙连接蛋白43(connexin43,CX43)表达的影响。方法SD雄性大鼠随机分为假手术组、模型组以及扶正化瘀胶囊组和卡托普利组,每组各6只,采用结扎左冠状动脉前降支造成心肌梗死。术后10d开始灌胃给药,持续8周。用免疫组织化学方法观察心肌CX43表达的变化。结果在心肌梗死区域内,模型组与假手术组比较,CX43排列紊乱,阳性表达面积、平均光密度值和积分光密度均显著低于假手术组(P<0.01)。在梗死边缘区,扶正化瘀胶囊组CX43平均光密度值高于模型组(P<0.01),积分光密度高于模型组(P<0.05),CX43排列紊乱有所减轻,阳性表达面积与模型组无统计学意义。卡托普利组CX43平均光密度值高于模型组(P<0.01),积分光密度和阳性表达面积均高于模型组(P<0.05),CX43排列紊乱也有减轻。结论扶正化瘀胶囊能够部分改善心肌梗死大鼠心肌的CX43重构。     

同种异体骨髓间充质干细胞心内膜移植治疗小型猪急性心肌梗死

目的:探讨同种异体骨髓间充质干细胞(MSCs)心内膜移植治疗小型猪急性心肌梗死的疗效。方法:分离、纯化、扩增培养小型猪MSCs;开胸结扎第1与第2对角支间前降支中段形成急性前壁心肌梗死的动物模型,心肌梗死后2~3周经心内膜注射移植DAPI标记的同种异体MSCs,心肌梗死前、后及移植后3个月行超声心动图及生化检查,并取梗死周围心肌组织冰冻切片及苏木精-伊红染色,免疫荧光法鉴定结蛋白(desmin)和心肌肌钙蛋白I(cTnI)的表达。结果:每40ml骨髓液经2~3周传3代平均获得(3.81±0.09)×107个细胞,移植后3个月左室射血分数较心肌梗死后及对照组改善明显,分别为(56.7±0.8)%,(40.1±1.2)%,(34.9±0.9)%(均P<0.05),肝肾功能、血糖及血气分析正常,3个月冰冻切片可见坏死心肌周围细胞核蓝染的移植细胞,免疫组化提示desmin及cTnI表达阳性。结论:同种异体MSCs经心内膜移植安全有效,心功能明显改善。     

骨髓间充质干细胞静脉移植对心肌梗死大鼠心功能的影响

目的探讨静脉途径移植骨髓间充质干细胞(MSCs)对急性心肌梗死(AMI)大鼠心功能的影响。方法雄性SD大鼠20只,分为干细胞移植组(10只)和对照组(10只)。结扎大鼠左冠状动脉建立心肌梗死模型。同种异体大鼠MSCs体外分离、纯化、扩增,4’,6-二脒-2-苯基吲哚(4’,6-diamidino-2-phenylindole,DAPI)标记,并通过尾静脉于心肌梗死后1周移植入AMI大鼠体内,对照组则注射等量培养液。4周后测定心功能,并行免疫组织化学检测。结果心肌梗死4周后,干细胞移植组心肌组织冷冻切片中可以观察到DAPI标记细胞存在,免疫组织化学显示阳性标记细胞a-Actin肌动蛋白检测阳性。干细胞移植组心功能较对照组有明显改善(P<0.05)。结论MSCs经静脉移植可以归巢至梗死心肌处,并能改善受损的心功能。     

经静脉途径移植骨髓间充质干细胞修复梗死心肌的可行性与安全性研究

目的探讨经静脉注射移植同种异体骨髓来源间充质干细胞(MSC)修复损伤心肌是否可行和安全,观察移植MSC在宿主的归巢与组织学分布。方法雄性Wistar大鼠30只,分为正常大鼠MSC移植组,急性心肌梗死MSC移植组,假手术组,每组10只。结扎冠状动脉左前降支,建立大鼠急性心肌梗死模型。体外分离纯化、扩增同种大鼠骨髓MSC,于建立心肌梗死模型24h给各组大鼠经静脉输注4’6’二乙酰基2苯基吲哚(DAPI)标记的MSC,4周后处死、摘取心脏等脏器,行组织病理切片和免疫组织化学染色。结果(1)在急性心肌梗死MSC移植组,梗死区及其周边部位可见到DAPI标记的MSC;(2)在梗死心肌周边区,移植的MSC胞浆心肌特异性蛋白肌钙蛋白I和转录因子4免疫组织化学染色阳性;(3)在各组大鼠其他脏器,移植的MSC主要分布在肺脏、脾脏和肝脏;(4)细胞移植大鼠心肌组织切片未见淋巴细胞增殖,各脏器没有肿瘤形成。结论经静脉移植的MSC可归巢至大鼠梗死心肌部位,并分化为心肌细胞表型,该方法治疗缺血性心脏病安全、可行。     

静脉移植骨髓间充质干细胞对心衰大鼠心肌细胞cx-43表达的影响

目的探讨骨髓间充质干细胞移植对阿霉素诱导的心衰大鼠模型心功能及细胞缝隙连接蛋白-43(cx-43)的影响。方法选取雌性Wistar大鼠(n=38),先随机选取8只作为正常对照组,其余30只通过腹腔注射阿霉素(2.5mg/kg,每周1次,连续6周)建立心衰模型。6周后存活大鼠(n=21)再随机分为细胞移植组(n=11)和移植对照组(n=10)。正常对照组大鼠腹腔注射等量的生理盐水。进行体外分离,纯化,增殖骨髓间充质干细胞(MSCs)并用含有12%胎牛血清的DMEM培养基培养,采用流式细胞仪对体外培养的MSCs表型CD44进行鉴定。取第三代MSCs进行移植,移植前用5-溴-2’脱氧尿苷(BrdU)进行标记。末次阿霉素注射后1周,将5×106个MSCs通过鼠尾静脉注射的方法移植到心衰细胞移植组大鼠。移植对照组大鼠注射等量培养基。细胞移植后4周,采用超声测量心功能及心室重塑,心脏切片行病理免疫组织化学了解移植细胞在受体心脏的存活及cx-43表达情况。结果体外培养的第三代MSCs流式细胞仪鉴定结果显示:CD44阳性细胞数占(99.7±0.9)%,心衰细胞移植组心肌组织中可以见5-溴-2’脱氧尿苷标记的骨髓间充质干细胞(MSCs)归巢并表达cx-43阳性;左室射血分数细胞移植心衰组、对照组与正常对照组分别为(89.36±3.74)%、(72.93±3.08)%和(97.54±2.30)%(P<0.05),心衰细胞移植相对于心衰对照组左室心功能有统计学意义(P<0.05)。结论经静脉移植骨髓间充质干细胞可以归巢心衰大鼠心肌组织而且改善心功能并影响受体心肌组织细胞表达cx-43。     

缺血预处理间充质干细胞移植治疗心肌梗死实验研究

目的探讨静脉移植缺血预处理的间充质干细胞（MSCs）对梗死心肌的修复作用。方法选择纯种大白兔46只制作心肌梗死模型,分为缺氧预处理MSCs移植组（A组）、非缺氧预处理MSCs移植组（B组）和对照组,于心肌梗死后30d分别检测心功能、梗死心肌的形态学变化以及新生毛细血管密度。结果与对照组比较,A、B组左心室收缩末期内径、舒张末期内径降低,心功能提高,室壁厚度增加,心肌梗死面积缩小,梗死区和梗死边缘区新生毛细血管密度增加（P〈0．05）。结论MSCs移植治疗兔心肌梗死可改善心功能、缩小梗死面积、增加梗死心肌新生毛细血管密度,经缺氧预处理的MSCs移植治疗更有优势。     

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.