从鼠粪中分离纯化微小隐孢子虫卵囊方法的研究

目的 探索一种实用、纯度和回收率高且卵囊活力强的分离纯化微小隐孢子虫卵囊的方法。 方法 分别采用经典的不连续蔗糖密度梯度离心法、改良饱和盐水漂浮法、氯化铯（CsCl）密度梯度离心法和Percoll密度梯度离心法分离纯化C57BL/6小鼠粪中隐孢子虫卵囊。并将4种方法分离纯化获得的卵囊各10⁵体外感染牛输卵管上皮细胞（BFTE）， 48 h和72 h后油镜下观察各分离法卵囊的发育情况，以比较卵囊的纯度和活性。 结果 经典的不连续蔗糖密度梯度离心法分离获得的卵囊数[（2.86±0.08）×10⁷］与改良饱和盐水漂浮法[（2.88±0.15）×10⁷］无明显差异（P>0.05）， 但高于Percoll密度梯度离心法[（1.52±0.08）×10⁷］（P<0.01）和CsCl密度梯度离心法[（2.46±0.13）×10⁷］（P<0.05）。4种方法分离纯化的卵囊体外感染BFTE 48 h和72 h后隐孢子虫数无明显差异（P>0.05）。但CsCl密度梯度离心法纯化的隐孢子虫卵囊纯度明显高于不连续蔗糖密度梯度离心法。 结论 改良饱和盐水漂浮法较经典的不连续蔗糖密度梯度离心法操作简单、快速；CsCl密度梯度离心法分离的隐孢子虫卵囊纯度较高。     

乙型肝炎病毒L蛋白颗粒的真核表达与纯化

目的:真核细胞中表达和纯化乙肝病毒L蛋白颗粒,并初步分析其物理特性与抗原性.方法:真核表达质粒pSVsigLM~ S~-瞬时转染COS-7细胞,收集48h后的培养上清采用CsCl等密度平衡离心、蔗糖密度梯度超速离心和酶联免疫吸附试验(ELISA)3者结合的方法纯化L蛋白,获得的纯化产物行Western blotting鉴定和透射电镜(TEM)观察.结果:采用超速离心结合ELISA法可以纯化L蛋白,CsCl等密度平衡离心显示在密度约1.21 g/cm~3处富含S抗原性.SDS-PAGE证实产物主要由一种42 kDa的蛋白分子组成,Western blotting证实这种蛋白为同时具有S与PreSl抗原性的L蛋白.透射电镜显示分泌的L蛋白自行组装成约23 nm的球形颗粒.结论:融合外源性信号肽能有效的引导L蛋白的组装和分泌.CsCl和蔗糖超速离心能高效纯化L颗粒并保持其完整的抗原性及颗粒形态.     

大鼠骨髓间充质干细胞的分离培养及生物学特性

用密度梯度离心结合贴壁培养法分离纯化大鼠骨髓间充质干细胞(MSCs),体外培养传代扩增,观察细胞生长特性,对细胞进行免疫组化染色鉴定,电镜观察细胞形态。动态观察示培养细胞具有不断增殖的干细胞特性,并保持细胞活性。证实密度梯度离心结合贴壁法以及消化传代能有效分离纯化和扩增骨髓MSCs,培养的细胞具有骨髓MSCs的基本表型和特性。本研究可为临床进行细胞移植奠定基础。     

犬贾第虫携病毒株体外纯培养的建立

目的 培养一携带犬贾第虫病毒的犬贾第虫（Giardia canis）细胞株。 方法 用蔗糖密度梯度离心-G1耐酸漏斗过滤法纯化犬贾第虫包囊，经口接种5日龄长爪沙鼠（Meriones unguiculata），8 d后于其十二指肠无菌收集犬贾第虫滋养体，置改良的 TYI-S-33 培养基中培养，待滋养体在培养管壁上形成细胞单层后进行传代。同时进行冻存和复苏实验，以及纯度、稳定性、细胞生物学特性、微生物污染等4项指标检测。滋养体经液氮冻融3次后3 000 × g离心15 min，取上清，用磷钨酸负染，透射电镜观察病毒粒子。 结果 犬贾第虫滋养体接种14 d 后虫体逐渐适应了培养环境，在培养管壁上形成细胞单层，经上述4项指标检测，证明形成了稳定的犬贾第虫细胞株。电镜观察，见滋养体内有外观球形呈20面体结构、直径约为36 nm的病毒样粒子。 结论 建立了携带GCV的犬贾第虫细胞株的体外纯培养。     

人骨髓间充质干细胞体外培养及其生物学特性分析

目的探讨人骨髓间充质干细胞（MSC）的体外分离培养方法,并分析其生物学特性。方法无菌条件下抽取健康志愿者骨髓,采用密度梯度离心法分离骨髓单个核细胞,培养在含10%胎牛血清的DMEM培养基中,用贴壁筛选法纯化获得MSC。相差显微镜下观察细胞形态学变化,细胞计数法绘制生长曲线,流式细胞仪检测细胞表面抗原及细胞周期。结果原代和传代培养的细胞呈梭形,具有较强的生长增殖能力。细胞表面CD90、CD105表达阳性,CD34、CD11b阴性。第1、3、5代MSC生长曲线呈S形,均经历潜伏期、对数生长期和平台期。第3代MSC中,G0/G1期细胞约占77.42%,S＋G2/M期细胞约占22.58%。结论采用密度梯度离心结合贴壁筛选培养法可获得纯度较高的MSC,且该细胞增殖活性较强。密度梯度离心结合贴壁筛选培养法是一种简单、有效的分离纯化MSC的方法。     

自外周血中获取内皮前细胞的方法

目的探讨自大鼠外周血中获取内皮前细胞（EPC）的方法。方法SD大鼠皮下注射粒细胞集落刺激因子（G-CSF）后抽取外周动脉血，密度梯度离心法获取单个核细胞，用完全培养液在体外培养后进行CD31、CD34、FLK-1和vWF免疫荧光染色，并检测其结合UEA-1、摄取acLDL的能力。结果将此方法收获的细胞进行CD31、CD34、FLK-1和vWF免疫荧光染色并检测其结合UEA-1、摄取ac-LDL的能力，证实为EPC。结论G-CSF刺激、密度梯度离心、完全培养液法可白大鼠外周血中获取较多EPC。     

小鼠肝癌细胞H22源exosomes的制备及其免疫相关蛋白的初步研究

目的 分离提取和电镜观察小鼠肝癌细胞H22分泌的exosomes,并检测其含有的蛋白成分,为exosomes作为肿瘤疫苗提供理论依据。方法 超速分级离心和蔗糖密度梯度离心纯化得到exosomes,并经透射电镜观察鉴定,运用肽质量指纹谱质谱分析和Western blot检测exosomes含有的蛋白成分。结果 从小鼠肝癌细胞H22中提取出exosomes,直径约为20-90 nm的膜性囊泡,呈圆形或椭圆形。该exosomes含有热休克蛋白（HSP）70、ICAM-1、延伸因子G2、动力蛋白轻链A、C-myc和Vav-2蛋白。结论 超速分级离心和蔗糖密度梯度离心提取纯化exosomes的方法具有可行性,小鼠肝癌细胞H22源exosomes表达有与抗原呈递相关蛋白（HSP70、ICAM-1）,迁移相关蛋白（动力蛋白轻链A）,粘附相关蛋白（ICAM-1）,细胞骨架相关蛋白（延伸因子G2）,肿瘤相关蛋白（C-myC蛋白、Vav-2蛋白）,具有免疫原性。     

维拉帕米抑制犬在体心脏早期后除极及室性心动过速的研究

用心外膜接触电极记录犬左室外膜单相动作电位（MAP），观察维拉帕米（异搏定）对氯化铯（CsCl）诱发的早期后除极（EADs）、QT间期延长及室性心动过速（VT）的影响。11只大，CsCl按0．5mg·kg-1次（首剂加倍），每15分钟一次静脉注射，直至诱发出VT。然后在给下一剂CsCl前，先给予维拉帕米0．1～0．2mg／kg静脉注射，后再给予此剂CsCl，以后仍每15分钟静脉注射CsCl一次直至VT再被诱发。结果维拉帕米给药前及给药后的15～60分钟CsCl诱发的EAD振幅占相应MAP振幅的29．2±7．0％和24．8±8．1％、QTc间期分别从对照组386±33ms延长至443±66ms（P＜0．01）和从441±107ms延长至516±93ms（P＜0．01），但给予维拉帕米后即刻，CsCl诱发的EADs仅为16．7±7．6％（与前述二值比较P均＜0．01）、QTc间期仅从对照的418±56ms延长至425±49ms（P＞0．05）。给维拉帕米前CsCl分别在7只和4只诱发出持续性和非持续性室速，给予维拉帕米后即刻仅在4只诱发出非持续性室速。结果示维拉帕米可以抑制CsCl诱发的EADS、QT间期延长及VT。     

溶栓素样品精纯方法的研究

目的建立一种合适的纯化方法，以利于进一步研究溶栓素的结构分析和酶化学性质。方法利用电泳技术提纯，用S2251作为底物验证纯化前后样品的活性变化，用高效液相色谱（HPLC）技术检验纯化效果。结果纯化前后样品活性无变化，纯度得到进一步提高。结论这是一种切实可行的纯化方法，解决了普通实验窀样品需要高度纯化的难题。     

抗柯萨奇病毒A16型单克隆抗体的制备和应用

目的 制备抗CA16病毒的特异性单克隆抗体,评价单克隆抗体在ELISA和细胞免疫化学染色中的作用,并利用单抗建立CA16快速检测胶体金免疫层析法并对其进行初步评价方法 CA16病毒接种RD细胞大量培养,氯化铯密度梯度超速离心纯化病毒颗粒,透射电镜鉴定纯化产物。福尔马林灭活病毒颗粒后免疫Balb/c小鼠,利用细胞融合技术制备稳定分泌抗CA16单抗的杂交瘤细胞株。ELISA和免疫荧光试验分别对单抗特性进行分析。根据单抗反应特性,分析单抗在细胞ELISA、细胞免疫组化中的应用,并利用特异性抗CA16单抗建立CA16胶体金免疫层析快速检测法结果 氯化铯密度梯度离心纯化病毒颗粒电镜观察显示,病毒颗粒为二十面体立体对称球形结构,大小均匀。经免疫小鼠,获得1株稳定分泌抗CA16单克隆抗体的杂交瘤细胞株(mAb CA16-19),该单抗为IgG2a亚型,细胞免疫荧光表明,单抗与RD宿主细胞内的CA16病毒颗粒结合。细胞ELISA显示,随着病毒滴度增加吸光度也增加。细胞免疫化学染色分析表明,单抗可与CA16病毒感染的RD结合,而不与EV71感染RD细胞和正常RD细胞反应。因此,该单抗可用于细胞ELISA和细胞免疫化学染色。同时,依据前期制备的抗CA16单抗(mAb CA16-10),并结合此次制备的mAb CA16-19共同建立胶体金免疫层析试纸条,该试纸条不与EV71和柯萨奇病毒B型交叉反应,最低检测限为6.25×10^6.25TCID₅₀/0.1 mL结论 利用氯化铯密度梯度离心纯化出CA16病毒颗粒,筛选出1株特异性抗CA16单抗,此株单抗可用于细胞免疫荧光试验、细胞ELISA和细胞免疫化学染色试验。并以单抗为原材料建立了CA16快速检测胶体金免疫层析法,为CA16病毒感染的快速诊断提供帮助。     

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.