靶向SMAD3基因的shRNA重组慢病毒对大鼠肝再生的作用

目的:应用大鼠肝大部切除肝再生模型,将前期构建好的靶向SMAD3基因的shRNA重组慢病毒注射入大鼠体内,观察SMAD3 shRNA对大鼠肝再生的影响.方法:将60只♂Wistar大鼠随机分为3组:SMAD3 shRNA组(20只)、shRNA对照组(20只)、生理盐水对照组(20只),通过脾脏注射方法给药,慢病毒给药剂量为1.0×108TU/只,生理盐水对照组给予等体积500L生理盐水.给药96h后行2/3肝大部切除,构建大鼠肝再生模型;肝大部切除术后96h各组分别除死大鼠7只,剩余大鼠于144h处死,收集肝脏标本,Real-Time PCR、免疫组织化学检测肝组织SMAD3表达,免疫组织化学检测肝组织Ki67表达,测定大鼠肝质量/体质量,观察SMAD3 shRNA对肝再生的影响.结果:Real-Time PCR检测显示,通过脾脏注射慢病毒,在96、144h处死时间点,SMAD3 shRNA组SMAD3 mRNA分别较shRNA对照组平均下降73%、63%.免疫组织化学检测显示SMAD3蛋白表达明显下降.Ki67免疫组织化学结果显示,肝大部切除术后96、144h,SMAD3 shRNA组Ki67表达阳性细胞数均明显多于生理盐水对照组及shRNA对照组,表明抑制SMAD3表达后肝细胞增殖活跃.大鼠肝质量与体质量比值显示,SMAD3 shRNA组分别较生理盐水对照组及shRNA对照组有增加趋势(96h:4.50±0.43vs3.97±0.55vs3.98±0.40,144h:4.66±0.54vs4.15±0.51vs4.20±0.34),但没有统计学意义(P>0.05).结论:SMAD3 shRNA在大鼠体内可一定程度上促进肝细胞增殖,但对肝再生的促进作用尚弱.     

白藜芦醇对部分肝切除术大鼠肝再生和肝组织NK细胞活性的影响*

目的 探讨白藜芦醇(RES)对肝部分切除大鼠肝组织自然杀伤(NK)细胞活性和肝再生的影响。方法 随机将90只SD大鼠分为对照组、小剂量白藜芦醇和大剂量白藜芦醇处理组,各组30只,分别经尾静脉注射生理盐水、白藜芦醇10 g.kg^-1·d^-1和20 g.kg^-1·d^-1,连续5 d,然后行70%肝脏切除术。检测肝脏再生指数,使用流式细胞术测定肝组织NK细胞百分率,采用⁵¹Cr释放法测定NK细胞杀伤活性,采用蛋白印迹法检测肝组织增殖细胞核抗原(PCNA)和CyclinD1蛋白表达。结果 在24 h和48 h,大剂量白藜芦醇处理组大鼠肝脏再生指数分别为(1.89±0.04)和(2.45±0.07),显著大于小剂量白藜芦醇处理组【(1.59±0.06)和(2.12±0.05),P<0.05】或对照组【(1.43±0.03)和(1.92±0.05),P<0.05】;大剂量白藜芦醇处理组大鼠NK细胞百分率分别为(24.62±1.36)%和(25.47±1.19)%,显著高于小剂量白藜芦醇处理组【(22.21±1.98)%和(22.36±1.78)%,P<0.05】或对照组【(17.36±1.78)%和(18.65±1.69)%,P<0.05】;大剂量白藜芦醇处理组大鼠NK细胞杀伤活力分别为(48.48±2.69)%和(49.01±2.78)%,显著高于小剂量白藜芦醇处理组【(41.88±2.65)%和(42.32±2.58)%,P<0.05】或对照组【(28.32±2.36)%和(30.12±2.36)%,P<0.05】;大剂量白藜芦醇处理组大鼠肝组织PCNA和CyclinD1蛋白相对表达量显著强于小剂量白藜芦醇处理组和对照组,差异有统计学意义(P<0.05)。结论 RES可以促进肝部分切除术大鼠肝再生,可能与增强了NK细胞活性和相关蛋白表达有关。     

粒细胞集落刺激因子促进小鼠肝再生的作用

目的:探讨粒细胞集落刺激因子(granulocyte colony-stimulating factor,G-CSF)促进肝大部切除小鼠肝再生的作用.方法:建立小鼠肝大部分切除(约70%)模型,将肝切除小鼠随机分为3组.单纯肝切除组:肝切除后24 h,腹腔注射生理盐水5 d:G-CSF+肝切除组:rhG-CSF 150 μg/(kg·d)腹腔注射5 d,24 h后进行肝切除:肝切除+G-CSF组:肝切除术后24 h,rhG-CSF 150 μg/(kg·d)腹腔注射5 d.于术后7 d取血清和肝组织,用全自动生化分析仪检测血清肝功能指标,并采用免疫组织化学方法观察肝内的增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)的表达及BrdU阳性细胞.结果:肝切除+G-CSF组和G-CSF+肝切除组Brdu及PCNA表达及BrdU阳性细胞明显升高,与对照组比较差异均具有统计学意义(PCNA:74.08%±8.86%,68.91%±9.64% vs 57.36%±13.37%,均P<0.05);肝组织病理检查发现G-CSF+肝切除组27%(3/11)小鼠肝组织出现了不同程度的炎症反应,肝功生化学也显示血清ALT及AST升高,且其差异有统计学意义(均P<0.05).结论:G-CSF具有明显促进肝大部切除小鼠肝再生的作用,但也容易诱发肝脏炎症反应.     

重组人肝再生增强因子对大鼠肝部分切除后肝再生的影响

目的观察原核表达的重组人肝再生增强因子（rhALR）对大鼠肝再生的影响。方法按Higgins方法进行大鼠34％肝切除。术后4-6h各实验组大鼠经腹腔注射rhALR剂量分别为50μg、100μg、200μg、400μg、800μg,对照组给生理盐水。术后30h杀鼠取肝,增殖核抗原（PCNA）免疫组化染色和HE染色,进行肝细胞核PCNA阳性细胞计数和有丝分裂计数。结果rhALR能促进肝部分切除后大鼠肝细胞的有丝分裂和细胞核PCNA的表达,并呈现一定的剂量依赖关系。结论rhALR能促进在鼠肝再生和肝细胞增殖。     

5-羟色胺2A受体阻断剂对大鼠肝脏再生的影响

目的观察5-羟色胺2A受体阻断剂酮色林对大鼠肝部分切除后肝再生的影响,了解5-羟色胺及其受体在肝脏再生中的作用。方法 80只雄性Wistar大鼠随机分为实验组和对照组。采用肝大部分切除术建立肝再生模型,术后16 h分别给予腹腔内注射酮色林（实验组）和生理盐水（对照组）,采用免疫组化及流式细胞技术动态观察并比较两组大鼠术后24、36、48、72 h肝脏Ki67、增殖细胞核抗原的表达情况。结果大鼠肝大部切除术后24、36 h肝脏表达Ki67、增殖细胞核抗原最为活跃,而后表达逐渐下降。实验组大鼠肝脏表达Ki67、增殖细胞核抗原较对照组显著下降（P〈0.05）。结论 5-羟色胺2A受体阻断剂酮色林显著抑制大鼠肝大部切除术后的肝脏再生,说明5-羟色胺具有一定的促进肝再生的作用,2A受体是其重要的信号传导受体之一。     

外源性血管内皮细胞生长因子对急性肝功能衰竭小鼠肝再生的影响

目的探讨外源性血管内皮细胞生长因子(VEGF)对小鼠急性肝功能衰竭的保护作用。方法雄性昆明种小鼠分为正常对照组、模型组和保护组。模型组和保护组一次性腹腔注射脂多糖/D-氨基半乳糖(LPS/D-GalN),保护组于造模前60min给予VEGF腹腔注射,而模型组、正常对照组分别注射同等体积的0.5%氯化钠溶液作为对照,给药后6h分别留取血清、肝组织标本。检测肝功能变化,HE染色观察肝组织病理改变,ELISA检测血清肝细胞生长因子(HGF)水平,免疫组织化学分析肝组织增殖细胞核抗原(PCNA)表达。结果保护组小鼠血清ALT[(328.44±38.26)U/L比(754.55±89.58)U/L]和AST[(345.94±53.86)U/L比(812.66±72.68)U/L]水平明显低于模型组(均P<0.05)。与模型组比较,保护组肝组织炎性细胞浸润明显减少,肝细胞坏死程度明显减轻。保护组小鼠血清HGF水平[(1.31±0.09)ng/L比(0.56±0.05)ng/L]和肝组织中PCNA表达阳性率[(30.34±2.58)%比(11.63±1.08)%]明显高于模型组(均P<0.05)。结论外源性VEGF对小鼠急性肝功能衰竭具有保护作用,其机制可能与促进肝细胞再生有关。     

门静脉动脉化对肝硬化犬肝切除后肝再生的作用

目的: 探讨门静脉动脉化( portal vein arterialization, PVA)对肝硬化犬肝切除后肝再生的作用, 为扩大PVA临床应用提供依据.方法: 将肝硬化模型犬随机分为3组, A组(手术组): 行肝左外叶切除加门静脉动脉化手术;B组(对照组): 仅行肝左外叶切除手术;C组(假手术组);均于术后4 wk处死, 实验过程中检测肝脏再生率(SE)与动脉血酮体比(AKBR), 免疫组织化学法检测增殖细胞核抗原(PCNA).结果: 肝脏再生率SE值A组显著高于B组, 两者有显著性差异(86.6%±2.9% vs 73.7%±6.9%, P<0.01);术后A、B两组AKBR值均较C组降低(均P<0.01), 术后4 wk A组该值(1.32±0.14)已接近C组(1.33±0.11), 差异无统计学意义P>0.05;术后PCNA表达A组显著快于B组.结论: 门静脉动脉化对肝硬化犬部分肝切除后肝脏再生有明显的促进作用.     

粒细胞集落刺激因子促进大鼠移植肝的再生作用

目的:探讨部分肝移植后,粒细胞集落刺激因子(granulocyte colony-stimulating factor, G-CSF)对部分肝移植物再生的促进作用．方法:采用改良“二袖套法”建立SD大鼠 50％部分肝移植模型,受体鼠随机分为实验组和对照组,术后分别注射(sc)G-CSF和相同体积的生理盐水5 d．观察大鼠移植肝存活时间．术后1,3,5,7和14 d取血清和移植肝脏,测移植物与受体质量比(graft-recipient weight ratio,GRWR),检测血清生化指标,并采用免疫组织化学方法观察肝内的增殖细胞核抗原 (proliferating cell nuclear antigen,PCNA)的表达．结果:实验组移植肝存活率较对照组高(90％ vs 60％,X2=5．03,P<0．05),术后第3-5天,实验组GRWR较对照组明显增加(P<0．05)．两组肝再生均于移植后3 d达高峰．与对照组相比, 实验组肝坏死灶少,AST,ALT水平低(3 d:t= 17．61,P<0．05;t=20．16,P<0．05;5 d:t=15．64, P<0．05;t=23．08,P<0．05),白蛋白水平高(3 d: 36．2±4．7 vs 29．5±3．4,P<0．05;5 d:43．2± 4．1 vs 33．8±3．9,P<0．05),PCNA表达升高(t= 23．08,P<0．05)．结论:大鼠部分肝移植后G-CSF可以促进肝细胞再生,减轻肝损伤．     

脂蛋白脂酶基因缺陷小鼠急性胰腺炎的研究

目的 探讨应用脂蛋白脂酶基因(LPL)缺陷的杂合子小鼠作为制备高脂血症相关性急性胰腺炎的动物模型的可行性.方法 野生型及LPL杂合子小鼠均分为实验组和对照组,每组又分为12、24 h 2个时点.实验组腹腔注射雨蛙肽(50 μg/kg体重)7次,每次间隔1 h.对照组同法注射等量生理盐水.检测各组小鼠血浆三酰甘油(TG)、淀粉酶水平,观察胰腺形态学改变并评分.结果 LPL杂合子小鼠对照组血TG为(3.55±0.27)mmol/L,显著高于野生型小鼠对照组的(0.94±0.18)mmol/L(P＜0.05).杂合子小鼠实验组12 h的血TG、淀粉酶水平分别为(3.55±0.27)mmol/L和(3685±484)U/L,均显著高于野生型小鼠实验组12 h的(0.92±0.11)mmol/L和(2501±410)U/L(P＜0.05).杂合子小鼠实验组12 h的胰腺水肿、坏死、出血和炎细胞浸润的分值分别为3.94±0.21、3.94±0.21、1.84±0.25和1.84±0.25,均显著高于野生型小鼠实验组12 h的3.06±0.01、2.52±0.51、0.46±0.22和0.58±0.38(P＜0.05).结论 LPL杂合子小鼠血TG中度升高且稳定,在雨蛙肽诱导下产生严重的急性胰腺炎,是研究高脂血症相关性急性胰腺炎发病机制的一种理想的动物模型.     

硫代乙酰胺诱导的A型肝性脑病小鼠焦虑样反应观察

目的探讨硫代乙酰胺(TAA)诱导的A型肝性脑病小鼠的精神障碍。方法 30只雄性昆明种小鼠随机分为实验组15只,对照组15只。实验组采用TAA 200 mg·kg-1·d-1,连续腹腔注射3 d至累积计量到600 mg/kg,正常对照组腹腔注射生理盐水,造模24 h后进行脑功能评分、明暗箱、旷场及高架十字实验。行为学检测结束后24 h行血氨及肝脏生化指标检测。两组间比较采用独立样本t检验。结果实验组小鼠在明暗箱实验中的明箱滞留时间(t=-4.006,P0.01)与穿梭次数(t=-2.656,P0.05);旷场实验的中央活动路程(t=-3.639,P0.05)、中央活动时间(t=-2.294,P0.05)、垂直运动次数(t=-2.282,P0.05)、理毛时间(t=5.992,P0.01);高架十字实验的开臂进入次数(t=-3.584,P0.05)、开臂滞留时间(t=-3.992,P0.05)与对照组小鼠相比,差异均具有统计学意义。但旷场实验中两组小鼠活动总路程差异无统计学意义(t=-0.96,P0.05)。且实验组小鼠血氨(t=-3.168,P0.05)、ALT(t=4.316,P0.05)、AST(t=-2.581,P0.05)、TBil(t=-9.127,P0.01)水平明显大于对照组,差异均有统计学意义。结论用TAA腹腔注射建立的A型肝性脑病小鼠模型有焦虑样反应;小剂量TAA诱导A型肝性脑病小鼠的焦虑样反应不伴有自发活动能力下降。     

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.     

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.     

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.     

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.