人骨髓间充质干细胞的体外多向分化潜能

目的体外观察人骨髓间充质干细胞(hMSCs)向成骨细胞、脂肪细胞及心肌细胞的分化。方法体外培养扩增hMSCs,流式细胞仪分析其免疫表型。取稳定传代的hMSCs,分别在体外向成骨细胞、脂肪细胞及心肌细胞分化,并采用碱性磷酸酶染色、油红O染色及PTAH染色鉴定3种诱导分化后的细胞。结果 hMSCs传代后形态上为典型的成纤维细胞样结构;流式细胞仪检测表明,hMSCs表达CD44、CD105,不表达CD31、CD34和CD45;hMSCs诱导分化后,细胞化学染色显示呈阳性表达的成骨细胞、脂肪细胞和心肌细胞。结论 hMSCs具有多向分化潜能,可向成骨细胞、脂肪细胞和心肌细胞分化。     

尿酸下调人骨髓间充质干细胞体外诱导为成骨细胞过程中11β-HSD1的表达

目的探讨体外诱导人骨髓间充质干细胞(hBMSCs)向成骨细胞分化过程中,尿酸对其成骨能力以及对11β-羟化类固醇脱氢酶1(11β-HSD1)表达及功能的影响。方法全骨髓体外分离培养健康成年人骨髓间充质干细胞,用细胞形态学及细胞表面标志物对其进行鉴定,培养hBMSCs至第3代后分别以完全培养基(含体积分数10%FBS、1%双抗、89%低糖DMEM)为空白对照组、以成骨培养基(含10-8mol/L地塞米松、50mg/L维生素C、10-2mol/Lβ-甘油磷酸钠的完全培养基)和尿酸干预成骨培养基(分别含0.2、0.4、0.8mmol/L尿酸的成骨培养基)为条件对照组进行培养和诱导。培养诱导14d后,倒置显微镜下观察细胞形态,用碱性磷酸酶染色和茜素红染色进行成骨细胞鉴定,用11β-HSD1免疫细胞化学染色、RT-PCR技术检测各组11β-HSD1 mRNA的表达。结果分离培养的细胞表面标志物CD44表达阳性,CD34表达阴性。成骨培养基和各尿酸干预成骨培养基诱导的细胞碱性磷酸酶染色和茜素红染色均为阳性,钙结节数量随尿酸浓度增高逐渐增多(P0.05)。免疫细胞化学染色结果显示各组细胞胞质均可见棕黄色阳性染色颗粒.病理图象分析软件测定11β-HSD1含量结果显示随尿酸浓度增高,成骨能力增加,光密度值逐渐减少(P0.05)。RT-PCR结果显示各组细胞均有11β-HSD1 mRNA表达,随尿酸浓度增高和成骨能力的增加,11β-HSD1 mRNA表达逐渐减少(P0.05)。结论尿酸能促进hBMSCs向成骨细胞增生和分化,具有浓度依赖性和时间依赖性,尿酸通过下调11β-HSD1 mRNA的表达促进hBMSCs向成骨细胞分化。     

人前脂肪细胞的培养及分化研究

目的建立人前脂肪细胞培养及其在药物诱导下向脂肪细胞分化的细胞模型。方法取中国人腹部皮下脂肪组织,经胶原酶消化后在含胎牛血清的DMEM-F12培养基中培养出梭形细胞,以胰岛素、地塞米松及吡咯列酮诱导该梭形细胞向脂肪细胞分化。结果经油红O脂肪染色可见梭形细胞内脂滴的积累。人前脂肪细胞经诱导并继续培养后在旋涡状生长汇合的细胞中心形成一个高密度的细胞岛,该细胞岛由部分可以贴壁的细胞和悬浮的已分化的脂肪细胞组成。结论成熟脂肪组织中存在着具有能分化为脂肪细胞的前脂肪细胞,经适当诱导可定向分化为脂肪细胞。     

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.     

利用共培养法诱导人胎盘间充质干细胞向成骨细胞分化

目的观察体外人足月胎盘间充质干细胞(h PMSCs)和成骨细胞共培养体系条件下成骨细胞对h PMSCs分化的影响。方法采用胶原酶消化法从人足月胎盘中分离纯化间充质干细胞(MSCs),检测细胞表面标志物、生长曲线、细胞超微结构及成骨能力并对h PMSCs进行鉴定。共培养组将成骨细胞接种于Transwell双层培养皿底层,h PMSCs接种于上层;对照组上层与底层均接种h PMSCs。对诱导后细胞进行碱性磷酸酶染色鉴定。结果胎盘分离细胞经形态、生长速度、细胞表面标志物(CD44和CD29阳性表达为99%,CD34和CD106为1%),确定为胎盘间充质干细胞;头盖骨分离细胞经碱性磷酸酶染色确定为成骨细胞。采用Transwell共培养h PMSCs和成骨细胞组碱性磷酸酶活性染色阳性率为(21.7±5.3)%,表现成骨细胞特性,对照组染色呈阴性。结论人足月胎盘含MSCs,与其他来源MSCs生物学特性相似,成骨细胞生长过程提供的微环境对h PMSCs分化为成骨细胞具有诱导促进作用。     

体外培养人骨髓基质细胞表面抗原检测及分化能力观察

目的检测体外培养人骨髓基质细胞（BMSCs）表面抗原,并观察其分化能力。方法取人颅骨板骨髓组织,以BMSCs专用培养基接种培养,经多次换液得到较纯的BMSCs;在倒置显微镜下观察细胞形态,应用流式细胞仪对细胞表面抗原进行检测;体外脂肪细胞、成骨细胞定向诱导,分别进行油红0染色及yonKossa染色。结果培养细胞表面抗原检测显示,CD14阴性、CD45阴性、CD29阳性、CD90阳性、CD105。阳性;脂肪细胞定向诱导后,油红0染色见胞质内有红色脂肪颗粒;成骨细胞定向诱导后,yonKossa染色见有钙结节形成。结论培养细胞表型特征符合BMSCs,并具有自我更新特性和多向分化潜能。     

大鼠原代前脂肪细胞及诱导分化方法的探讨

目的探寻大鼠原代培养前脂肪细胞及诱导分化的最佳方法。方法采用胶原酶消化法体外培养大鼠前脂肪细胞。显微镜下观察细胞形态改变,流式细胞仪检测细胞增殖活性;以分化培养基诱导分化后,油红O染色法进行细胞鉴定。结果成功培养大鼠前脂肪细胞。前脂肪细胞24 h贴壁时为类圆形,3 d后渐成梭形或多角梭形,6 d左右进入指数增长期;流式细胞仪检测证明细胞具有增殖分化能力;油红O染色可见细胞内出现红染颗粒。结论成功建立高效原代培养前脂肪细胞及诱导分化的方法,为脂肪细胞的相关研究提供了良好的细胞模型。     

再生障碍性贫血患者骨髓间充质干细胞成脂和成骨分化能力的变化

目的 通过观察再生障碍性贫血(再障)患者间充质干细胞(MSCs)成脂肪和成骨能力的变化,研究骨髓MSCs成脂分化的异常在再障患者红髓脂肪化中的作用.方法 分离培养再障患者及正常人的骨髓,观察其一般生物学特性,并在体外诱导其向脂肪、成骨细胞分化,同时用RT-PCR方法检测成脂肪、成骨特异基因的表达时间,比较再障患者和正常对照MSCs向脂肪细胞、成骨分化能力的不同.结果 原代培养7 d,再障组贴壁细胞克隆形成率为(19.30±4.77)/(5×105 MNCs),较正常对照组明显降低(47.72±3.46)/(5×105 MSCs)(P＜0.05).在培养初期,再障组MSCs与正常对照MSCs增殖能力相似,但在连续传8代后,其增殖能力降低.再障组MSCs体外诱导成脂滴早,诱导分化的脂肪细胞leptin(瘦素)基因表达早.再障组MSCs体外诱导成骨形成钙化结节少,碱性磷酸酶活性低,诱导的成骨细胞osteocalcin(骨钙素)基因表达晚.结论 再障患者MSCs成脂分化能力增强而成骨分化能力降低,可能在再障的病程中起一定作用.     

骨髓间充质干细胞体外诱导和端粒酶活性的研究

目的:建立骨髓间充质干细胞(MSCs)体外分离、培养、纯化的方法,探讨其体外诱导条件和端粒酶活性。方法:取正常成人骨髓用含10%小牛血清的LG-DMEM培养液培养、扩增。流式细胞仪行细胞表面抗原检测,细胞化学染色鉴定其生物学特性,在特定培养条件下检测其向成骨和脂肪细胞分化的能力,采用TRAP(Telomerase repeat amplification protocol assay)-银染法测定其端粒酶的活性。结果:分离培养获得的贴壁细胞,碱性磷酸酶染色阳性。流式细胞仪检测CD34、CD45表达阴性,CD29、CD44、CD115、CD166表达阳性。经向成骨和脂肪细胞诱导3周后,可得到成骨和脂肪细胞,经茜素红染色、油红O染色得到证实。TRAP-银染法证实MSCs表达一定的端粒酶活性。结论:体外分离培养的MSCs可以向成骨、脂肪诱导分化,表达一定的端粒酶活性,具有干细胞的生物学特性。     

橘红素对大鼠成骨细胞增殖、分化和成骨功能的影响

目的 研究橘红素(TG)对体外培养大鼠成骨细胞增殖、分化、矿化和成骨功能指标基因蛋白表达的影响。方法 选取出生24 h内的SD大鼠乳鼠颅骨，通过酶消化法培养原代成骨细胞，倒置显微镜下观察细胞形态和状态，并以碱性磷酸酶(ALP)染色和茜素红(ARS)染色法进行细胞鉴定；分别采用噻唑蓝(MTT)法、ALP染色法、ARS染色法检测TG对原代成骨细胞的存活率、评价成骨细胞分化能力和矿化能力的影响；采用Western印迹检测TG对原代成骨细胞成骨相关转录因子(OSX)、ALP、Ⅰ型胶原蛋白(CollagenⅠ)和骨桥蛋白(OPN)等成骨功能指标基因的蛋白表达。结果 与Control组相比，TG作用浓度为5、10、15、20μmol/L时可明显促进成骨细胞的增殖(P<0.01);TG共培养7 d后，与Control组比较，TG作用浓度为5、10、15、20μmol/L时ALP染色明显增加，说明TG能明显促进成骨细胞分化(P<0.05);TG共培养21 d后，与Control组比较，TG浓度为5、10、15、20μmol/L时ARS染色明显增加，说明TG能明显促进成骨细胞的矿化功能(P&...     

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.