胰腺干细胞诱导分化为胰岛素分泌细胞的实验研究

目的研究大鼠胰腺组织干细胞向胰岛内分泌细胞分化及其在实验性糖尿病治疗中的应用。方法应用Nestin结合的免疫磁珠从大鼠胰腺导管细胞中分离和纯化干细胞,经体外扩增及诱导分化形成胰岛样结构,进行体外和体内的功能评价。结果大鼠胰腺干细胞经体外扩增和诱导分化后,表达胰岛素mRNA,并形成胰岛样结构。免疫荧光染色显示胰岛结构内含有大量胰岛素阳性细胞和少量胰高糖素阳性细胞。在葡萄糖刺激下干细胞分化出胰岛,有胰岛素释放,其释放量约为正常胰岛的39.4%。将干细胞分化来的胰岛移植到免疫缺陷的糖尿病小鼠后可以明显改善糖代谢的紊乱。结论胰腺组织干细胞可以在体外诱导分化成为具有治疗糖尿病功能的胰岛细胞。     

肝细胞生长因子及葡萄糖对人胰腺干细胞向胰岛素分泌细胞分化的影响

目的 探讨人胰腺干细胞体外扩增及其向胰岛素分泌细胞分化的影响因素.方法 应用剪碎消化法从孕4～6月龄引产的人胎儿胰腺中获得胰岛组织,选用差速贴壁法从胰岛中分离胰腺干细胞,经扩增后定向诱导成具有胰岛素分泌功能和胰岛样结构的胰岛样细胞团,观察肝细胞生长因子和葡萄糖对胰腺干细胞向胰岛素分泌细胞诱导分化以及胰岛样细胞团胰岛素分泌功能的影响.采用流式细胞仪和免疫组织化学法检测扩增前后nestin阳性细胞和ABCG2阳性细胞.行葡萄糖刺激的胰岛素释放试验,以酶联免疫吸附法（ELISA）检测每1×105个扩增细胞经定向诱导后形成的胰岛样细胞团释放的胰岛素量,比较肝细胞生长因子和葡萄糖对定向分化的影响.采用单因素方差分析或多样本均数两两比较进行统计学分析.结果 经过15代连续扩增,nestin阳性细胞和ABCG2阳性细胞分别扩增了27.9倍和37.8倍.扩增后的细胞经15 d定向诱导后形成具有胰岛素分泌功能和胰岛样结构的细胞团.葡萄糖刺激的胰岛素分泌试验显示,当诱导培养基中肝细胞生长因子浓度为0、5、10、15μg/L时,5.6 mmol/L葡萄糖刺激后,胰岛素分泌量分别为5.00、12.93、14.91和11.23μU;25.0 mmol/L葡萄糖刺激后,胰岛素分泌量分别为19.91、35.53、47.43和23.33 μU.诱导培养基中的葡萄糖浓度为0、2.8、5.6和11.2 mmol/L时,5.6 mmol/L葡萄糖刺激后,胰岛素分泌量分别为9.07、14.21、6.91和3.53 μU;25.0 mmol/L葡萄糖刺激后,胰岛素分泌量分别为26.64、48.97、37.63和34.20 μU.结论 人胰腺中nestin和ABCG2阳性胰腺干细胞具有很强的体外扩增能力.扩增后的胰腺干细胞仍然具有形成胰岛素分泌细胞的功能,经体外诱导后能形成胰岛样细胞团.在胰腺干细胞向胰岛素分泌细胞分化诱导过程中,适当浓度的肝细胞生长因子和葡萄糖有利于胰岛样细胞团的形成和胰岛素分泌.     

Conophylline联合Betacellulin-δ4对胎猪导管源胰腺干细胞体外诱导分化的促进作用

目的:研究Conophylline(CnP)联合Betacellulin-δ4(BTCδ4)对胎猪导管源胰腺干细胞的体外诱导分化作用.方法:在培养基中加入CnP、BTCδ4等诱导因子诱导胎猪导管源胰腺干细胞分化,台盼蓝检测CnP对胰岛样细胞团(islet-like cell cluster,ICC)的毒性,ICC行EL...     

胰腺干细胞

引言近年研究发现,成年胰腺中存在干细胞,胰腺干细胞能分化形成胰腺导管、胰岛及胰腺外分泌腺泡等特定的胰腺组织细胞,并具有无限分裂和自我更新能力胰腺干细胞属未分化细胞,可表达干细胞的一些分子     

胎儿胰腺组织中巢蛋白阳性细胞的分离和体外增殖以及诱导分化

目的 研究从胎儿胰腺组织中分离巢蛋白(Nestin)阳性细胞以及该细胞的体外扩增与向胰岛内分泌细胞分化的能力。方法 采用胶原酶消化法，从胎儿胰腺组织中分离获得胰岛样细胞簇(islet-like cell clusters，ICCs)，ICCs经手工挑拣后接种，待形成单层上皮样细胞后，进行传代培养和诱导分化。利用逆转录-聚合酶链反应(RT-PCR)、免疫荧光染色及放射免疫分析(RIA)等方法，检测该细胞中分子标志物的表达，并对其向胰岛内分泌细胞分化的能力进行鉴定。结果 (1)上述单层上皮样细胞具有很强的增殖能力，可至少连续传16代；(2)RT-PCR、免疫荧光染色分析显示，该细胞可表达干细胞的标志分子巢蛋白和ABCG2；(3)RT-PCR分析显示，在多种细胞因子和无血清的条件下，巢蛋白阳性细胞经诱导后可出现胰岛素、胰升糖素和胰十二指肠同源盒基因-1(PDX-1)mRNA的表达，而巢蛋白和Neurogenin3(Ngn3)mRNA表达消失。RIA分析也可检测到诱导后的细胞内有胰岛素产生。结论 从胎儿胰腺中分离得到的巢蛋白阳性细胞具有胰腺前体细胞的特性，在体外具有很强的增殖能力，并可诱导分化为胰岛内分泌细胞。该细胞有望为胰岛移植提供一种新的细胞来源。     

血管内皮细胞与胰岛发育和干细胞分化的关系

血管内皮细胞是构成胰岛微环境的重要组成部分.内皮细胞参与胰腺的发生、胰腺内分泌细胞的分化和增殖、胰腺的空间分布等各个方面,其作用贯穿于胰腺的整个发育过程.不同状态的内皮细胞在不同的作用时间窗所起的作用不同,可影响内皮细胞与胰岛细胞之间的信号交联,进而影响胰腺内分泌细胞的分化、胰岛的形态及其功能.内皮细胞可分泌肝细胞生长因子、1-磷酸-鞘氨醇等细胞因子,并且可产生层黏连蛋白、Ⅳ型胶原等细胞外基质成分,这些分子在胰岛的发育和功能调控中发挥重要作用.此外,内皮细胞还可参与调控干细胞的自我更新和定向分化.内皮细胞在促进干细胞向胰岛细胞分化方面的作用是糖尿病再生医学领域备受关注的研究方向.     

骨髓间充质干细胞转化为胰岛样细胞的研究进展

骨髓间充质干细胞是存在于骨髓中的一群具有自我更新和多向分化潜能的细胞.新近研究表明,骨髓间充质干细胞在体内微环境及体外细胞因子的作用下不仅能够分化为胰岛β样细胞,而且具有一定的β细胞分泌功能,能够降低血糖,克服了胰岛移植治疗糖尿病所面临的供体细胞缺乏和免疫排斥反应问题,从而为临床糖尿病细胞治疗开辟了一条新的途径.     

骨髓间充质干细胞转化为胰岛样细胞的研究进展

骨髓间充质干细胞是存在于骨髓中的一群具有自我更新和多向分化潜能的细胞。新近研究表明，骨髓间充质干细胞在体内微环境及体外细胞因子的作用下不仅能够分化为胰岛β样细胞，而且具有一定的β细胞分泌功能，能够降低血糖，克服了胰岛移植治疗糖尿病所面临的供体细胞缺乏和免疫排斥反应问题，从而为临床糖尿病细胞治疗开辟了一条新的途径。     

骨髓间充质干细胞诱导的胰岛样细胞治疗大鼠胰腺损伤

目的 探讨骨髓间充质干细胞(MSCs)诱导后促进胰腺损伤修复的方法.方法 应用贴壁法分离、纯化大鼠MSCs,经bFGF、EGF、HGF、2%B27、activeA、β巯基乙醇、尼克酰胺诱导12～14 d,RT-PCR鉴定后,以DAPI标记治疗胰腺损伤模型大鼠.治疗1 w后激光共聚焦显微镜观察胰岛样细胞的迁移、定位,RT-PCR鉴定参与胰腺损伤修复的细胞来源.结果 MSCs经诱导12～14 d,分化为可表达Ins1、Ins2的胰岛样细胞,经DAPI标记后治疗胰腺损伤模型大鼠1 w,使炎性细胞浸润明显、细胞结构不清、胰岛结构崩解的损伤区的胰腺组织结构开始恢复,胰岛再建;RT-PCR检测Sry进一步证实损伤区胰腺组织内存在输注的胰岛样细胞.结论 MSCs可诱导分化成胰岛样细胞,该细胞参与损伤胰腺组织的修复.     

microRNA与胰腺发育以及干细胞分化的研究进展

microRNA(miRNA)是一类参与基因转录后调控的非编码小分子RNA,其在胚胎发育、细胞命运决定、生长调控等方面发挥重要作用.业已证实,一些特定的miRNA通过靶向调控某些胰腺发育相关的重要转录因子而参与胰腺胚胎发育过程.通过模拟体内胰岛发育过程,可将干细胞在体外诱导定向分化为胰岛素分泌细胞.此外,miRNA在干细胞的维持和分化中也可能具有调控作用.因此,miRNA在胰腺发育和干细胞分化中的作用及其机制值得深入研究,这将为胰岛功能重建治疗糖尿病策略提供新的思路.     

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.