青少年特发性脊柱侧凸患者褪黑素信号传导通路的初步研究

目的:了解青少年特发性脊柱侧凸(adolescentid iopathic scoliosis,AIS)患者褪黑素信号传导通路是否存在异常。方法:7例女性AIS患者,年龄13～17岁,平均14.7岁;最大Cobb角40°～80°,平均59.3°,行后路手术时取髂骨组织进行成骨细胞培养,并用碱性磷酸酶染色、骨钙素免疫荧光检测方法对培养细胞进行成骨细胞鉴定。取原代培养的成骨细胞,先采用福斯可林刺激成骨细胞内cAMP升高,然后用不同浓度褪黑素刺激细胞,检测细胞内cAMP水平。结果:碱性磷酸酶染色、骨钙素免疫荧光检测结果均证实所培养细胞表现为成骨细胞特性,所有AIS患者成骨细胞的cAMP的基础水平都很低,在给予福斯可林刺激以后cAMP的水平明显升高,此后再给予生理剂量的褪黑素后,cAMP的水平未见明显下降;给予药理剂量的褪黑素后同样没有观察到cAMP水平明显的下降。结论:AIS患者的褪黑素信号传导通路存在异常。     

人骨髓间充质干细胞褪黑素受体的表达及其意义

目的:检测体外培养的人骨髓间充质干细胞(bone mesenchymals stromal cells;BMSCs)褪黑素受体的表达;并探讨其意义.方法:10例12～18岁志愿者;分别从髂前上棘处穿刺抽取10ml骨髓;肝素抗凝.采用密度梯度离心法分离BMSCs.体外培养并传至P3代进行表型鉴定.抽提细胞总RNA;采用逆转录-聚合酶链式反应(RT-PCR)方法检测褪黑素受体MT1、MT2 mRNA的表达;同时应用免疫组化染色方法检测细胞MT1、MT2蛋白的表达.结果:分离的单个核细胞经培养至P3代;使用流式细胞仪进行细胞表型鉴定;体外培养细胞的表型与BMSCs的表面标志相符.细胞抽提总RNA以MT1和MT2两种受体的RT-PCR扩增产物电泳呈阳性条带;免疫组化结果显示细胞存在MT1和MT2蛋白的表达.主要分布在细胞膜和细胞浆;呈棕褐色阳性颗粒;而细胞核上未见阳性颗粒.结论:人BMSCs中存在褪黑素受体MT1、MT2的表达;它是褪黑素作用的靶细胞.这为进一步研究BMSCs褪黑索信号通路提供了实验基础.     

不同浓度麝香含药血清对骨髓间充质干细胞迁移影响的实验研究

目的探讨麝香对外源性骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)增殖和迁移的影响。方法将60只SD大鼠随机分为麝香高、中、低剂量组及空白对照组,制备麝香含药血清及生理盐水血清。15只SD大鼠利用全骨髓贴壁法分离BMSCs,培养至P3代,通过形态学观察、表型鉴定、成骨成脂诱导鉴定BMSCs,鉴定认为培养成功后通过麝香含药血清干预BMSCs,检测细胞增殖率,利用Transwell实验检测麝香含药血清对BMSCs迁移的影响。结果外源性大鼠BMSCs呈梭形贴壁生长,生长状态良好;表型鉴定:CD45、CD34阴性表达,CD44、CD90阳性表达;细胞成骨、成脂诱导后可定向成骨、成脂分化;不同浓度麝香组与对照组比较均能提高BMSCs增殖率(P0.05);与对照组比较,不同浓度麝香在24 h、48 h、72 h均增加BMSCs迁移(P0.05),以低浓度组效果最佳。结论麝香含药血清可以促进BMSCs增殖,促进BMSCs的体外迁移。     

Runx2在青少年特发性脊柱侧凸患者骨髓间质干细胞中的表达及意义

目的：从骨髓间质干细胞（MSCs）水平探讨青少年特发性脊柱侧凸（AIS）发病的机制。方法：40例12～18岁志愿者，AIS患者20例，先天性脊柱侧凸（CS）患者10例，正常对照组10例。分别从髂前上棘处穿刺抽取10ml骨髓，肝素抗凝。采用密度梯度离心法分离MSCs，体外培养并传至P3代时行表型鉴定，采用RT-PCR法检测3组MSCs中核心结合因子α1（Runx2）mRNA的表达强度。结果：分离所得单个核细胞传至P3代经流式细胞仪鉴定表型与MSCs表面标记相符。AIS组Runx2的mRNA表达强度与CS组及正常对照组相比有显著统计学差异（P〈0．01），CS组与正常对照组相比无统计学差异（P〉0．05）。结论：转录因子Runx2在MSCs水平表达强度的异常可能与AIS发病有关。     

高截面纵横比容器和微载体技术在胎儿骨髓基质干细胞向软骨细胞转化中的应用

目的利用体外细胞培养方法,进行胎儿骨髓基质干细胞(BMSCs)向软骨细胞的诱导分化,以有效获得表型正常的软骨组织工程功能细胞。方法从胎儿骨髓血中提取BMSCs,体外扩增培养3代,流式细胞仪鉴定胎儿BMSCs的纯度后,1.2×107个细胞与Cytodex-3微载体充分混匀,放入含无血清完全培养基的高截面纵横比容器(HARV,旋转式细胞培养系统的一种)中诱导培养,7、14d取材,用RT-PCR检测细胞内Ⅱ型前胶原mRNA的表达,同时以培养瓶诱导培养作为对照。结果HARV诱导培养组在培养7、14d检测出细胞内Ⅱ型前胶原mRNA的表达,而培养瓶诱导培养组则未检出。结论HARV诱导培养具有使胎儿BMSCs向软骨细胞转化的能力。     

左、右归丸及其拆方含药血清对骨髓间充质干细胞增殖和成骨诱导的影响

目的 观察左、右归丸及其拆方含药血清对体外培养的大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)增殖和向成骨细胞(osteoblast,OB)诱导的影响.方法 运用全骨髓贴壁法分离和培养大鼠BMSCs;分别以左归丸、右归丸、两方共同药、滋肾阴药、补肾阳药、阳性对照药补佳乐制备的大鼠含药血清加诱导剂(地塞米松、维生素C、β-甘油磷酸钠)、诱导剂和空白含药血清组共8组对BMSCs进行干预,采用MTT法检测BMSCs增殖和左、右归丸及其拆方含药血清对BMSCs增殖的影响;流式细胞仪对BMSCs进行鉴定;用PNPP法检测左、右归丸及其拆方含药血清对BMSCs向OB诱导的影响.结果 P4代以后细胞细胞表型CD90呈阳性表达,CD11 b/c、CD45呈阴性表达.左、右归丸及其拆方含药血清对BMSCs增殖和成骨诱导都具有促进作用,左归丸、右归丸、滋肾阴药组与其他组比较效果显著(P＜0.05).结论 左、右归丸及其拆方含药血清可以协同诱导剂对BMSCs增殖和成骨诱导具有显著的促进作用.     

青少年特发性脊柱侧凸患者软骨细胞中Sox9的表达及意义

目的:检测转录因子Sox9在青少年特发性脊柱侧凸(AIS)患者软骨细胞中的表达,探讨其在AIS患者生长发育异常中的可能作用.方法:14例(男1例,女13例)年龄10～16岁(平均13.1岁)的AIS患者(AIS组),Cobb角41°～88°,平均51.4°;8例(男1例,女7例)非AIS患者(对照组),年龄10～15岁,平均12.9岁;腰椎骨折1例,脊髓室管膜瘤1例,腰椎间盘突出症1例,脊柱骨样骨瘤1例,先天性髋关节脱位4例.在行手术治疗时获取髂骨生长板软骨,采用酶消化法体外分离、培养、传代,并观察细胞形态;采用Ⅱ型胶原细胞免疫组织化学法对传至P2代的细胞行表型鉴定;采用逆转录-聚合酶链反应(RT-PCR)、蛋白免疫印迹(Western blotting)法检测两组软骨细胞中Sox9 mRNA及蛋白表达情况.结果:酶消化法体外单层培养软骨细胞成功;细胞传至P2代时具有软骨细胞的典型形态特征,呈多角状;Ⅱ型胶原细胞免疫组化染色呈阳性,细胞胞浆内可见棕褐色颗粒,很好地保持了软骨细胞的表型特征;AIS组患者软骨细胞中Sox9核酸表达强度为1.08±0.14,蛋白表达强度为0.38±0.14,均较对照组高(P<0.05).结论:转录因子SOx9在软骨细胞水平表达强度的异常可能与AIS患者生长发育异常有关.     

骨碎补促进骨髓间充质干细胞增殖及成骨分化

[目的]研究骨碎补对骨髓间充质干细胞(BMSCs)增殖与成骨分化的影响。[方法]40只SD大鼠随机分为4组,包括空白对照组和3个处理组,空白对照组给予生理盐水,而3个处理组分别给予高、中、低浓度骨碎补水煎液连续灌胃3个月。给药期满后,取各组动物骨髓体外培养,全骨髓贴壁法纯化培养BMSCs,第三代BMSCs作为检测细胞。CCK-8法分析不同浓度的骨碎补水煎液对BMSCs增殖的作用。采用ALP试剂盒测定BMSCs成骨诱导后ALP活性,茜素红染色观察钙化结节情况,并测定成骨细胞相关RUNX2和Osterix及OCN mRNA的表达情况,以评估骨碎补对BMSCs成骨分化的影响。[结果]CD90和CD45细胞免疫荧光显色证实所培养细胞为BMSCs。CCK-8法检测显示骨碎补能够促进BMSCs增殖,并呈剂量依赖性,3个处理组与空白对照组间的差异具有统计学意义(P0.05)。在成骨诱导培养基中培养后,3个处理组间培养液上清ALP活性呈剂量依赖性增高,并伴有培养皿茜素红染色观察钙化结节的相应增多,与空白对照组间的差异具有统计学意义(P0.05)。此外,相应的RUNX2和Osterix及OCN的表达也呈骨碎补剂量依赖性增加,3个处理组与空白对照组间的差异具有统计学意义(P0.05)。[结论]骨碎补能够促进大鼠骨髓间充质干细胞的增殖和成骨分化。     

Smad1在青少年特发性脊柱侧凸患者骨髓间质干细胞中的表达及意义

目的:探讨Smad1在青少年特发性脊柱侧凸(AIS)患者骨髓间质干细胞(MSCs)中的表达及其在发病机制中的作用。方法:30例12～18岁志愿者分为两组:AIS组20例,同年龄正常对照组10例。分别从髂前上棘处骨穿刺抽取10ml骨髓,肝素抗凝。采用密度梯度离心法分离MSCs,体外培养并传至P2代行表型鉴定,分别采用RT-PCR及免疫蛋白印记法检测两组患者MSCs中Smad1的表达情况。结果:AIS组与对照组Smad1mRNA水平的平均表达率分别为4.48±0.58和1.03±0.22,蛋白水平的平均表达率分别为2.62±0.55和0.84±0.22。不论是核酸水平还是蛋白水平AIS组Smad1的表达均高于对照组(P<0.01)。结论:Smad1在AIS患者MSCs中表达强度异常,可能与AIS发病的分子机制相关。     

SOX9在青少年特发性脊柱侧凸患者骨髓间质干细胞的表达及意义

目的：探讨SOX9在青少年特发性脊柱侧凸（AIS）发病机制中的作用。方法：30例12～18岁志愿者分为两组，AIS组20例，同年龄对照组10例。分别从髂前上棘处穿刺抽取10ml骨髓，肝素抗凝。采用密度梯度离心法分离骨髓间质干细胞（MSCs），体外培养并传至P2代行表型鉴定，分别采用RT—PCR法、Western blotting法及免疫荧光法检测两组MSCs中SOX9的表达情况。结果：不论是核酸水平还是蛋白水平，AIS组患者的SOX9表达均高于对照组（P〈0．01）。结论：转录因子SOX9在MSCs水平表达强度的异常可能与AIS发病相关。     

Melatonin reduces prostate cancer cell growth leading to neuroendocrine differentiation via a receptor and PKA independent mechanism

BACKGROUND: Melatonin, the main secretory product of the pineal gland, inhibits the growth of several types of cancer cells. Melatonin limits human prostate cancer cell growth by a mechanism which involves the regulation of androgen receptor function but it is not clear whether other mechanisms may also be involved. METHODS: Time-course and dose-dependent studies were performed using androgen-dependent (LNCaP) and independent (PC3) prostate cancer cells. Cell number, cell viability, and cell cycle progression were studied. Neuroendocrine differentiation of these cells was evaluated by studying morphological and biochemical markers. Finally, molecular mechanisms including the participation of melatonin membrane receptors, intracellular cAMP levels, and the PKA signal transduction pathway were also analyzed. RESULTS: Melatonin treatment dramatically reduced the number of prostate cancer cells and stopped cell cycle progression in both LNCaP and PC3 cells. In addition, it induced cellular differentiation as indicated by obvious morphological changes and neuroendocrine biochemical parameters. The role of melatonin in cellular proliferation and differentiation of prostate cancer cells is not mediated by its membrane receptors nor related to PKA activation. CONCLUSIONS: The treatment of prostate cancer cells with pharmacological concentrations of melatonin influences not only androgen-sensitive but also androgen-insensitive epithelial prostate cancer cells. Cell differentiation promoted by melatonin is not mediated by PKA activation although it increases, in a transitory manner, intracellular cAMP levels. Melatonin markedly influences the proliferative status of prostate cancer cells. These effects should be evaluated thoroughly since melatonin levels are diminished in aged individuals when prostate cancer typically occurs.     

Divergent effects of forskolin on 3′,5′ cyclic adenosine monophosphate production and parathyroid hormone secretion

Summary Forskolin, a diterpene which directly stimulates adenylate cyclase, markedly stimulated cAMP production in intact rat parathyroid glands and dispersed cells from hyperplastic and adenomatous human parathyroid tissues. Stimulation of cAMP production in human parathyroid adenomas occurred as early as 2 min and continued for at least 2 h; furthermore, a dose-response relationship was observed, with a maximal 80-fold cAMP response occurring at 100 μM forskolin. When PTH secretion by rat or human parathyroid tissues was studied at low (0.5 mM) and high (2.5 mM) extracellular Ca²⁺ in either the presence or absence of forskolin, no significant stimulation by forskolin was observed at 15 min, 1 h, and 2 h. When 10 human parathyroid specimens were studied with varying concentrations of forskolin at 1 mM Ca²⁺, 6 failed to show stimulation of PTH secretion and 4 showed modest but detectable increases in PTH that did not appear dose-related. We conclude that (1) at low and high Ca²⁺ levels, marked stimulation of cAMP production by forskolin can occur without a corresponding increase in PTH secretion; (2) inhibition of PTH secretion by high extracellular Ca²⁺ levels continues unchanged despite stimulation of cAMP production by forskolin; and (3) at intermediate Ca²⁺ levels (1.0 mM), PTH secretion is affected either minimally or not at all by forskolin in human hyperparathyroid tissue preparations. The marked stimulation of parathyroid adenylate cyclase by forskolin without concomitant increases in PTH secretion in the majority of tissues suggests that the level of cAMP production is not a primary or sufficient determinant of hormone secretion.     

Notch信号通路在骨髓间充质干细胞及骨代谢疾病中的功能研究进展

骨髓间充质干细胞是存在于骨髓基质中的一种多能干细胞,具有多向分化的潜能,其天然再生能力对骨骼的生长代谢和骨转换起着至关重要的作用。Notch信号通路是一条在进化中高度保守的信号转导通路,与骨髓间充质干细胞的增殖、分化与凋亡密切相关,影响人体骨骼发育,也是多种骨代谢疾病的重要调节通路。以往对Notch信号通路的研究主要集中在神经干细胞,对骨髓间充质干细胞的研究较少。本文通过查阅文献,阐述不同的影响因素介导Notch信号通路对骨髓间充质干细胞分化的调节,并总结了Notch信号通路在骨代谢疾病如Alagille综合征、Adams Oliver综合征、脊椎肋骨发育不良、HajduCheney综合征、骨折愈合中的研究近况。     

水蛭素通过上调VEGF/Notch1信号通路促进人骨髓间充质干细胞成骨分化

摘要：目的 观察水蛭素对人骨髓间充质干细胞（bone marrow mesenchymal stem cell,BMSC）成骨分化的影响。方法 BMSCs细胞分为正常培养的对照组、成骨诱导的诱导组以及加入不同浓度（1、10、20 ATU/mL）处理的水蛭素组。MTT检测细胞增值并筛选水蛭素最适作用浓度。流式细胞仪检测细胞凋亡。RT-PCR和Western blot分别检测成骨基因Runx2、Osterix、COL1A1的mRNA和蛋白表达。BCIP/NBT染色法检测细胞中的碱性磷酸酶水平。茜素红染色检测矿化结节。检测VEGF、Notch1、Jagged1和CBF1的mRNA和蛋白表达。结果 骨髓间充质干细胞经成骨诱导细胞增殖显著增加,中高浓度的水蛭素可以不同程度促进成骨诱导的BMSCs细胞增殖（P＜0.05）,并筛选出20 ATU/mL作为水蛭素的使用浓度。水蛭素抑制成骨诱导的BMSCs细胞凋亡,上调Runx2、Osterix、COL1A1的mRNA和蛋白表达,增加碱性磷酸酶水平,促进细胞中矿化结节的生成,并提升BMSCs细胞中VEGF、Notch1、Jagged1和CBF1的表达（P＜0.05）。结论 水蛭素可能通过上调VEGF/Notch1信号通路促进人骨髓间充质干细胞成骨分化。     

beta-adrenergic desensitization after burn excision not affected by the use of epinephrine to limit blood loss

BACKGROUND: Burn patients have impaired myocardial function and decreased beta-adrenergic responsiveness. Further beta-adrenergic dysfunction from systemic absorption of topically administered epinephrine that is given to limit blood loss during burn excision could affect perioperative management. The authors evaluated the effect of topical epinephrine administration to patients during burn excision on the lymphocytic beta-adrenergic response. METHODS: Fifty-five patients (age, 2-18 yr) with 20-90% body surface area burns received a standardized anesthetic for a burn excision procedure. Lymphocyte samples were taken at baseline and 1 and 3 h after the initial use of epinephrine (n = 43) or thrombin (controls, n = 12). Plasma epinephrine levels were measured by high-performance liquid chromatography. Lymphocyte beta-adrenergic responsiveness was assessed by measuring production of cyclic adenosine monophosphate (cAMP) after stimulation with isoproterenol, prostaglandin E1 (PGE1), and forskolin. beta-adrenergic receptor binding assays using iodopindolol and CGP12177 yielded beta-adrenergic receptor density. RESULTS: Epinephrine levels were elevated at 1 h (P < 0.01) and 3 h (P < 0.01) after epinephrine use but not in control patients. Production of cAMP in lymphocytes 1 h after epinephrine was greater in patients receiving epinephrine than in control patients on stimulation with isoproterenol (P < 0.05) and PGE1 (P < 0.05). Three hours after epinephrine administration, production of cAMP decreased when compared with baseline in both control patients and those receiving epinephrine after stimulation with isoproterenol (P < 0. 05), PGE1(P < 0.05), and forskolin (P < 0.05). Lymphocytic beta-adrenergic receptor content was not changed. CONCLUSIONS: Topical epinephrine to limit blood loss during burn excision resulted in significant systemic absorption and increased plasma epinephrine levels. Acute sensitization of the lymphocytic beta-adrenergic cascade was induced by the administration of epinephrine reflected by increased cAMP production after stimulation with isoproterenol and PGE1. The lymphocytic beta-adrenergic cascade exhibited homologous and heterologous desensitization 3 h after the use of epinephrine or thrombin, indicating that epinephrine administration was not a causative factor.     

脂联素通过调节BMP信号通路促进骨髓干细胞成骨分化

目的 研究脂联素（adiponectin, ApN）对骨髓间充质干细胞（bone marrow mesenchymal stem cells, BMSCs）成骨分化的作用,并探讨其可能的机制。方法 体外培养BMSCs,构建ApN过表达质粒及干扰质粒,转染至BMSCs中。将BMSCs随机分为5组：对照组、过表达组、过表达空载组、干扰组和干扰空载组。茜素红染色观察各组细胞钙化沉积。碱性磷酸酶（alkaline phosphatase, ALP）染色观察各组细胞成骨分化能力。qRT-PCR检测ApN受体、骨形态发生蛋白（bone morphogenetic protein, BMP）信号通路及成骨相关基因表达情况。结果 与对照组相比,过表达组BMSCs中钙化沉积和ALP阳性表达增多,AdipoR1、AdipoR2、BMP2、RUNX2、Smad1和Smad5 mRNA表达量显著升高（P<0.05）;干扰组BMSCs中钙化沉积和ALP阳性表达减少,AdipoR1、AdipoR2、BMP2、RUNX2、Smad1和Smad5 mRNA表达量显著降低（P<0.05）。结论 ApN可能通过上调BMP信号通路促进BMSCs成骨分化。     

β-Adrenergic Desensitization after Burn Excision Not Affected by the Use of Epinephrine to Limit Blood Loss

Background: Burn patients have impaired myocardial function and decreased [beta]-adrenergic responsiveness. Further [beta]-adrenergic dysfunction from systemic absorption of topically administered epinephrine that is given to limit blood loss during burn excision could affect perioperative management. The authors evaluated the effect of topical epinephrine administration to patients during burn excision on the lymphocytic [beta]-adrenergic response.

Methods: Fifty-five patients (age, 2-18 yr) with 20-90% body surface area burns received a standardized anesthetic for a burn excision procedure. Lymphocyte samples were taken at baseline and 1 and 3 h after the initial use of epinephrine (n = 43) or thrombin (controls, n = 12). Plasma epinephrine levels were measured by high-performance liquid chromatography. Lymphocyte [beta]-adrenergic responsiveness was assessed by measuring production of cyclic adenosine monophosphate (cAMP) after stimulation with isoproterenol, prostaglandin E1 (PGE1), and forskolin. [beta]-adrenergic receptor binding assays using iodopindolol and CGP12177 yielded [beta]-adrenergic receptor density.

Results: Epinephrine levels were elevated at 1 h (P < 0.01) and 3 h (P < 0.01) after epinephrine use but not in control patients. Production of cAMP in lymphocytes 1 h after epinephrine was greater in patients receiving epinephrine than in control patients on stimulation with isoproterenol (P < 0.05) and PGE1 (P < 0.05). Three hours after epinephrine administration, production of cAMP decreased when compared with baseline in both control patients and those receiving epinephrine after stimulation with isoproterenol (P < 0.05), PGE1(P < 0.05), and forskolin (P < 0.05). Lymphocytic [beta]-adrenergic receptor content was not changed.