采用1.068 g/ml分离液对犬骨髓间充质干细胞的分离纯化与体外培养方法的探讨

目的探讨更为可靠、纯净度高的犬骨髓间充质于细胞分离、培养方法。方法将犬骨髓抽取液分别以1．068g／ml及1．073g／ml分离液进行密度梯度离心，采用贴壁培养法获得骨髓间充质干细胞（MSCs）；以LG-DMEM加15％FBS培养；应用于细胞表面标志蛋白，多向诱导分化等方法进行细胞鉴定。结果采用1．068g／ml分离液可获得纯度较高的单核细胞，接种细胞生长良好，孵育24h即可见细胞贴壁生长，平均倍增周期为1d，细胞呈纺锤状，螺旋梳状排列。连续培育8代以上，未见细胞形态、增殖特性改变。MSCs表面标志SH2阳性，CD45阴性，可定向分化为心肌细胞、成骨细胞及脂肪细胞。结论采用1．068g／m1分离液能够较好地进行犬MSCs的体外分离、培养与扩增，分离得到的细胞具备MSCs的特性。     

犬骨髓间叶干细胞的多向分化潜力

目的观察犬骨髓间叶干细胞的体外多向分化潜力,为损伤组织的干细胞移植修复提供理论基础.方法利用梯度-贴壁筛选法分离、培养与扩增骨髓间叶干细胞.利用化学诱导剂5-氮胞苷(20μmol/L),血管内皮细胞生长因子(VEGF 10ng/ml),成骨细胞诱导剂(地塞米松10nMol/L,抗坏血酸0.05mMol,β甘油磷酸钠10mMol/L)以及成脂肪细胞诱导剂(1-甲基3-异丁基黄嘌呤0.5mmol/L,地塞米松1μmol/L,胰岛素10mg/L,消炎痛100mmol/L),分别定向诱导骨髓间叶干细胞分化为心肌细胞、血管内皮细胞、成骨细胞与脂肪细胞.再应用细胞形态观察、免疫细胞化学染色及电镜对分化细胞进行鉴定.结果利用梯度-贴壁筛选法可以分离培养与扩增骨髓间叶干细胞.5-氮胞苷可诱导干细胞呈现肌管、肌丝、心房颗粒,且肌细胞特异性蛋白α-actinin,Myosin,α-Actin,Troponin Ⅰ免疫染色阳性;VEGF可诱导骨髓间叶干细胞出现管网状、血管样及鹅卵石样结构,vWF免疫染色阳性;成骨细胞诱导剂可诱导分化细胞碱性磷酸酶阳性;成脂肪细胞诱导剂使分化细胞内出现脂肪滴,油红O染色示脂滴为橙红色.结论成年犬骨髓间叶干细胞在体外具备多向分化潜力.在化学或生物诱导剂的作用下,犬骨髓间叶干细胞能够定向分化为心肌细胞、血管内皮细胞、成骨细胞、脂肪细胞等多种间叶组织类型细胞.     

自体骨髓间叶干细胞诱导分化移植治疗房室阻滞的初步观察水

利用诱导分化的骨髓间叶干细胞自体移植治疗房室阻滞,探讨生物介入方法治疗缓慢型心律失常的新途径。11只实验犬随机分为实验组(n=6)和对照组 (n=5)。应用射频技术消融His束,制备永久Ⅲ度房室阻滞动物模型;每只犬抽取 10ml犬骨髓液,应用密度梯度离心法及贴壁培养法分离、培养和扩增骨髓间叶干细胞, 5 氮胞苷对骨髓间叶干细胞进行诱导分化;房室阻滞 4周后,开胸心脏直视下将BrdU标记的分化干细胞 (1ml, 1. 5×107细胞)多点注射至该犬消融的His束部位,而对照组仅将 1mlDMEM培养液替代干细胞悬液多点注射至相同部位。术后 1~12周,应用心电图观察两组活体动物房室功能恢复情况,应用组织病理、免疫组化等技术对移植干细胞的存活、增殖、分化与缝隙连接功能进行评价。结果:在动物房室阻滞模型中,实验组犬在自体骨髓干细胞移植后 12周, 2 /6只犬的房室传导功能得以改善;病理与免疫组化示诱导分化的骨髓干细胞在His束区存活、增殖和分化为心肌细胞、血管内皮细胞,并与宿主细胞建立缝隙连接;而对照组 5只犬未见上述变化。结论:自体移植诱导分化的骨髓间叶干细胞有可能改善His束传导功能。     

骨髓间叶干细胞与成心肌细胞分化研究进展

1 骨髓间叶干细胞的特点间叶干细胞 (mesenchymalstemcells)的基本特点是贴壁生长的纤维细胞样形态 ,巨大的培养增殖潜力 ,独特的细胞表型 ,多系间叶组织细胞分化能力。尽管对许多动物的骨髓间叶干细胞进行过研究 ,但以人类骨髓间叶干细胞的研究较为深入。人类骨髓间叶干细胞约占骨髓分离单个有核细胞的 0 0 0 1 %～ 0 0 1 % [1 ] ;体外培养呈贴壁生长 ,1～ 2周后 ,95 %～ 99%的细胞呈单一纯系细胞 ,其形态类似成纤维细胞[1 ] ;细胞倍增时间 33h ,细胞周期分析显示约 1 0 %的细胞处于S期[2 ] ;与骨髓造血干细胞明显不同 ,间叶干细胞具有…     

自体骨髓间叶干细胞诱导分化移植治疗房室阻滞的初步观察

利用诱导分化的骨髓间叶干细胞自体移植治疗房室阻滞,探讨生物介入方法治疗缓慢型心律失常的新途径。11只实验犬随机分为实验组(n=6)和对照组 (n=5)。应用射频技术消融His束,制备永久Ⅲ度房室阻滞动物模型;每只犬抽取 10ml犬骨髓液,应用密度梯度离心法及贴壁培养法分离、培养和扩增骨髓间叶干细胞, 5 氮胞苷对骨髓间叶干细胞进行诱导分化;房室阻滞 4周后,开胸心脏直视下将BrdU标记的分化干细胞 (1ml, 1. 5×107细胞)多点注射至该犬消融的His束部位,而对照组仅将 1mlDMEM培养液替代干细胞悬液多点注射至相同部位。术后 1~12周,应用心电图观察两组活体动物房室功能恢复情况,应用组织病理、免疫组化等技术对移植干细胞的存活、增殖、分化与缝隙连接功能进行评价。结果:在动物房室阻滞模型中,实验组犬在自体骨髓干细胞移植后 12周, 2 /6只犬的房室传导功能得以改善;病理与免疫组化示诱导分化的骨髓干细胞在His束区存活、增殖和分化为心肌细胞、血管内皮细胞,并与宿主细胞建立缝隙连接;而对照组 5只犬未见上述变化。结论:自体移植诱导分化的骨髓间叶干细胞有可能改善His束传导功能。     

小鼠骨髓基质细胞的生物学特性及血管新生能力

目的 探讨体外培养的骨髓基质细胞的一些生物学特性及体内移植后在缺血区新血管生成中的作用。方法分离5—6周龄的小鼠胫骨、股骨，用预冷的DMEM／F12培养基冲洗出骨髓，经密度梯度离心分离出骨髓单个核细胞，接种后12～16d形成单层贴壁的成纤维样细胞。体外诱导分化鉴定分离的细胞，用传代的细胞进行生长曲线测定，观察其接种贴壁率、分裂指数，检测细胞周期和超微结构，并建立下肢缺血模型。荧光标记的体外扩增的骨髓单个核细胞被移植入缺血组织。移植后2周，荧光显微镜及内皮细胞碱性磷酸酶染色，检查荧光阳性细胞与染色阳性细胞的时空关系。结果体外传代培养的单个核细胞倍增时间约为42h。传代10h贴壁率达90％以上。分裂指数曲线与生长曲线相似。细胞周期显示约83％的细胞处于G1期。结论 体外培养的骨髓基质细胞生长稳定，传代后的细胞适应性强，增殖较快，表现出较早期细胞特点，在体外及移植入体内缺血区能分化为血管内皮细胞，有望用于改善组织缺血。     

大鼠骨髓间充质干细胞体外诱导分化为神经元样细胞的初步研究

目的探讨体外培养、扩增骨髓间充质干细胞(MSCs)的方法,诱导MSCs向神经元样细胞定向分化。方法取大鼠骨髓,采用贴壁培养筛选法分离MSCs,进行培养扩增,观察其生物学特性;用IBMX诱导MSCs向神经元样细胞分化,并通过细胞免疫化学法鉴定分化细胞。结果大鼠MSCs可通过贴壁法成功分离,并可在体外大量扩增,经IBMX诱导,MSCs可向神经元样细胞分化,出现胞体和突起,细胞免疫化学染色神经元特异性烯醇化酶和巢蛋白呈阳性。结论MSCs易分离和培养,体外培养条件下生长良好,可连续传代,诱导剂作用下可分化出神经元样细胞。     

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。     

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。     

大鼠骨髓间充质干细胞体外分离培养及生物学特性研究

目的建立大鼠骨髓间质干细胞（mesenchymal stem cells，MSCs）体外分离、纯化及扩增的方法及对大鼠MSCs的生物学特性进行研究。方法采用贴壁筛选法分离大鼠MSCs，并通过不断传代进行纯化和扩增培养，检测大鼠MSCs生长周期，绘制细胞生长曲线。结果大鼠MSCs在体外分离培养扩增，大鼠MSCs形态呈长梭形，细胞周期显示第3代MSCs约有81．48％的细胞处于G0／G1期，细胞生长曲线呈S形。结论所建立的分离和培养方法获取的大鼠MSCs具有生物学特性。     

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.