甲氨蝶呤对人骨肉瘤MG-63细胞凋亡及28Livin和Caspase-3表达的影响

目的 观察甲氨蝶呤(MTx)对人骨肉瘤MG-63细胞凋亡及Livin和Caspase-3表达的影响.方法 体外培养骨肉瘤MG-63细胞株,用0、50、100、200和400μmol/L的MTX作用于骨肉瘤MG-63细胞24、48、72 h后,用噻唑蓝(MTT)比色法检测骨肉瘤MG-63细胞的增殖活性,用流式细胞仪测细胞的凋亡率,用Western blot检测不同浓度MTX作用于骨肉瘤MG-63细胞24 h后各组细胞的Livin和Caspase-3蛋白表达水平.结果 随MTX浓度增加骨肉瘤MG-63细胞的增殖活性降低,同时细胞的凋亡率增加(P＜0.05),随MTX浓度增加各组细胞中Livin蛋白的表达降低,Caspase-3蛋白表达增加(P＜0.05).结论 MTX诱导人骨肉瘤MG-63细胞凋亡,其机制可能与下调Livin表达继而上调Caspase-3表达有关.

Abstract：

Objective To observe the effect of Methotrexate (MTX) on apoptosis and expression of Livin and Caspase-3 in human osteosarcoma cell line MG-63. Methods After treatment of MG-63 cells with MTX at different concentrations (0, 50, 100,200,400 μmol/L) for 24, 48 and 72 h, methyl thiazol tetrazolium(MTT) assay was used to observe the growth inhibition of MG-63. The apoptosis was assessed by flow cytometry. The protein expression of Livin and Caspase-3 was detected by Western blotting. Results When MTX was added, growth inhibition and increased apoptosis of MG-63 cells were detected,which was showed in a dose- and time-dependent manner. MTX also down-regulated the level of the protein expression of Livin (P＜0.05), and elevated the protein expression of Caspase-3 (P＜0.05). Conclusion MTX can induce apoptosis of MG-63 cells, by down-regulating Livin expression and subsequently up-regulating Caspase-3 expression.     

二硫代氨基甲酸吡咯烷提高甲氨蝶呤对人骨肉瘤MG-63细胞增殖活性和凋亡的影响

目的 观察二硫代氨基甲酸吡咯烷(PDTC)和甲氨蝶呤(MTX)对人骨肉瘤MG-63细胞凋亡和Livin表达的影响.方法 体外培养骨肉瘤MG-63细胞株,用0、50、100、200和400μmol/L的PDTC和MTX作用于骨肉瘤MG-63细胞24、48和72 h后,用噻唑蓝(MTT)比色法检测骨肉瘤MG-63细胞的增殖活性,用流式细胞仪测细胞的凋亡率,用Western blot检测各组细胞的Livin蛋白表达水平.结果 各组骨肉瘤MG-63细胞的增殖活性随着时间增加降低,同时细胞的凋亡率随着时间增加(P＜0.05),各组细胞中Livin蛋白的表达明显降低(P＜0.05).结论 PDTC能提高MTX对骨肉瘤MG-63细胞的凋亡,其机制可能与下调Livin表达,阻断了Livin介导的抗凋亡途径有关.

Abstract：

Objective To detect the effect of pyirolidine dithiocarbamate (PDTC) and methotrexate (MTX) on apoptosis and expression of livin of human osteosarcoma MG-63 cells. Methods MG-63 cells were cultured in vitro. At 24, 48 and 72 h after treatment of PDTC and MTX at different concentrations (0, 50, 100, 200 and 400 μmol/L) , MTT assay was used to observe the growth inhibition of MG-63 cells. The apoptosis was assessed by flow cytometry. The protein expression of livin was detected by Westem blotting. Results When PDTC and MTX were added, growth inhibition and increased apoptosis of MG-63 cells were detected. The protein expression level of livin was down-regulated obviously ( P ＜0. 05). Conclusion PDTC can promote the apoptosis of MTX-treated MG-63 cells, which may be correlated with down-regulation of the livin expression.     

人参皂苷CK对人骨肉瘤细胞MG-63的凋亡诱导作用

[目的]探讨人参皂苷CK对人骨肉瘤细胞MG-63的凋亡诱导作用及其具体作用机制。[方法]MG-63细胞分为空白对照组和CK药物处理组。采用MTT法检测细胞活性及增殖能力,倒置显微镜下观察细胞凋亡形态。细胞凋亡形态检测、Hoechst细胞核染色及Annexin V/PI细胞凋亡实验检测药物CK对细胞的诱导凋亡作用;Western blotting检测凋亡相关蛋白Caspase-9、Bcl-2及BAX的表达,研究其具体凋亡机制。[结果]CK能够显著降低MG-63的体外活性及生存率(P<0.05);细胞凋亡形态检测、Hoechst细胞核染色及Annexin V/PI细胞凋亡实验证明CK可诱导MG-63凋亡(P<0.05);CK处理后MG-63细胞表达的Caspase-9及BAX表达上调,相反Bcl-2表达下调(P<0.05)。[结论]人参皂苷CK对人骨肉瘤细胞MG-63具有凋亡诱导作用,而以Caspase-9为关键因素的线粒体凋亡途径在此凋亡中起着决定性作用。     

Survivin基因干扰RNA对骨肉瘤MG-63细胞系增殖和凋亡的影响

[目的]观察Survivin基因siRNA表达载体对骨肉瘤细胞增殖和凋亡的影响。[方法]体外构建Survivin shRNA表达载体pSilence2．1-neo-Survivin，转染骨肉瘤细胞系MG-63，筛选稳定表达的克隆，倒置显微镜观察各组细胞形态学变化，RT-PCR、Weston-blot方法检测转染后Survivin mRNA及蛋白的表达，噻唑蓝（MTT）法、克隆形成实验检测细胞的增殖情况，吖啶橙荧光染色法观察细胞凋亡情况。[结果]转染后MG-63细胞Survivin基因mRNA水平和蛋白表达显著下降，其抑制率分别为85．08％和81．14％；细胞增殖受到显著抑制，培养48h后与空白组比较，抑制率达63．4l％；吖啶橙染色显示转染组细胞出现明显核碎裂等凋亡改变，转染组凋亡率为24．54％。[结论]靶向Survivin基因的siRNA表达载体可以显著抑制骨肉瘤细胞增殖，促进细胞凋亡。     

硫化砷对骨肉瘤细胞生长和凋亡的影响

目的 研究硫化砷对骨肉瘤细胞生长和凋亡的影响，并探讨其可能的机制。方法 应用噻唑蓝（MTT）比色法检测细胞生长抑制率、倒置显微镜观察细胞形态、AnnexinV法检测细胞凋亡、逆转录-聚合酶链反应（RT-PCR）法检测bcl-2及bax基因的表达，观察硫化砷对骨肉瘤细胞株MG-63诱导凋亡的作用。结果 硫化砷可明显抑制MG-63细胞的增殖，1．0mg／L浓度的硫化砷作用24h后，MG-63细胞的抑制率可达26．43％。倒置显微镜观察被硫化砷抑制的MG-63细胞呈典型的凋亡改变．流式细胞仪检测凋亡率与硫化砷处理时间、处理浓度呈正相关。在硫化砷的作用下，MG-63细胞的bcl-2表达下调，而bax表达无明显改变．结论 硫化砷可有效地诱导骨肉瘤细胞凋亡，bcl-2表达下调是诱导细胞凋亡的重要机制。     

反义c-myc寡核苷酸对骨肉瘤细胞(MG-63)凋亡的影响

[目的]研究反义c-myc寡核苷酸(c-mycASODN)对人骨肉瘤MG-63细胞凋亡的影响。[方法]设计c-mycASODN片段,将其转入人骨肉瘤MG-63细胞,通过HE染色光镜观察、透射电镜超微结构及流式细胞仪(FCM)分析,观察和分析其对瘤细胞凋亡、细胞周期及c-myc基因蛋白表达的影响。[结果]形态学观察到典型瘤细胞凋亡特征;流式细胞仪分析证实反义c-myc寡核苷酸主要阻止瘤细胞从G1期进入S期,即产生G1/S期阻滞,出现明显的凋亡峰,凋亡率达36.82%,并可抑制c-myc基因蛋白的表达。[结论]反义c-myc寡核苷酸可明显诱导人骨肉瘤MG-63细胞的凋亡。     

凋亡相关因子Smac、Livin与Caspase-3在骨肉瘤中表达及其意义

目的 观察人骨肉瘤组织中凋亡相关因子Smac、Livin和Cagpage-3表达,及其对骨肉瘤生物学行为影响.方法 采用免疫组织化学技术(SABC法)检测46例骨肉瘤组织中Smac、Livin及Cagpage-3蛋白表达,比较Smac、Livin表达与骨肉瘤主要临床病理参数的关系.结果 Smac、Livin及Cagpage-3基因在骨肉瘤中表达分别为29例(63.0%)、30例(65.2%)、32例(72.4%);Smac、Livin表达率与骨肉瘤组织学分级、WHO分型无关,与转移有关(P<0.05);骨肉瘤中Smac和Livin基因表达正相关(r=0.639,P<0.01),两者与Caspase-3表达无关.结论 凋亡相关因子Smac、Livin和Cagpage-3高表达于骨肉瘤中,可能通过影响细胞凋亡共同参与肿瘤发生发展.     

MG-132对肝癌细胞BEL7402凋亡及超微结构影响的实验研究

目的:探讨泛素-蛋白酶体通路阻断剂MG-132对肝癌细胞BEL7402细胞凋亡和超微结构的影响。方法:以5和10μmol／L MG-132分别处理人肝癌BEL7402细胞24和48h,用流式细胞术检测细胞凋亡;DNA片段分析进一步证实凋亡存在,Western印迹检测Bax蛋白表达,比色法测Caspase-3活性变化,用透射电镜观察细胞超微结构的变化。结果:随着MG-132浓度的增加和处理时间延长,细胞凋亡率增加;细胞DNA抽提电泳发现特征性凋亡梯状条带;Bax蛋白表达增加;Caspase-3活化;电镜观察到典型凋亡细胞,线粒体等细胞器的形态变化与MG-132浓度和作用时间有关。结论:蛋白酶体抑制剂MG-132可诱导肝癌细胞BEL7402凋亡;线粒体损伤、Bax蛋白上调、Caspase-3激活可能在MG-132 诱导BEL7402细胞凋亡起重要作用。     

鹰嘴豆芽素A诱导人成骨肉瘤MG63细胞凋亡作用研究

[目的]探讨植物雌激素(phytoestrogen)鹰嘴豆芽素A (biochanin-A, BA)诱导人成骨肉瘤MG-63细胞凋亡及其调控丝裂原活化蛋白激酶(mitogen-altivated protein kinase, MAPK)/凋亡信号通路的作用机制。[方法]用不同浓度的鹰嘴豆芽素A处理MG-63细胞,以MTT法检测细胞活性的抑制情况;以DAPI染色法和Annexin-V/PI流式细胞仪检测细胞的凋亡情况;应用Western blot法检测MG-63细胞中MAPK信号通路相关蛋白磷酸化和主要凋亡通路Bcl-2家族和caspase家族相关蛋白的表达情况。[结果] BA有效抑制MG-63细胞的细胞活性,并呈时间和剂量依赖效应,且可以诱导细胞凋亡。40μmol/L和80μmol/L的BA作用于MG-63细胞48 h,细胞凋亡率分别为(45.28±3.79)%和(82.51±5.23)%,高于正常组(5.31±3.14)%(P0.05)。Western blot结果显示,BA可促进caspase-9、caspase-3和PARP发生剪切(P0.05),并促进p-p38、p-JNK、Bax的表达(P0.05),抑制Bcl-2的表达(P0.05),并呈剂量依赖效应。[结论]鹰嘴豆芽素A可有效抑制人成骨肉瘤MG-63细胞的细胞活性,促进细胞凋亡,作用机制可能是通过激活MAPK/凋亡信号通路来实现。     

Effects of Sevoflurane and Isoflurane on Renal Function and on Possible Markers of Nephrotoxicity

Background: Low-flow sevoflurane anesthesia is associated with increasing circuit concentrations of compound A, which is nephrotoxic in rats, but the effect of compound A and low-flow sevoflurane anesthesia on renal function in humans is unclear. The authors compared the effects of high- and low-flow sevoflurane and isoflurane anesthesia on renal function and on several possible markers of nephrotoxicity in humans.

Methods: Forty-two patients without preexisting renal disease underwent either low-flow isoflurane (1 l/min, n = 14), low-flow sevoflurane (1 l/min, n = 14), or high-flow sevoflurane (6 l/min, n = 14) anesthesia for body-surface-area surgery scheduled to last at least 4 h. Twenty-four-hour urinary excretion of N-acetyl-[small beta, Greek]-glucosaminidase (NAG), [small beta, Greek]2-microglobulin, protein, glucose, blood urea nitrogen (BUN), and serum creatinine concentrations were measured before and after anesthesia.

Results: There were no differences in blood urea nitrogen, creatinine, and creatinine clearance among the three groups after anesthesia. Increased urinary N-acetyl-[small beta, Greek]-glucosaminidase excretions were seen in the low-flow and high-flow sevoflurane groups, but not in the low-flow isoflurane group (P < 0.01). Ten patients in the low-flow sevoflurane group had 24-h urinary excretion of protein that exceeded the normal ranges after anesthesia, but only one patient in the isoflurane and none in the high-flow sevoflurane groups had this.     

Effect of duodenal osmolality on gastrin and secretin release and on gastric and pancreatic secretion

We have measured the osmolality of duodenal contents in 9 dogs after a hypertonic meal, given either by mouth or directly into the stomach or perfused into the duodenum. A test meal of 2,475 mosm/kg, given by mouth, raised the intraduodenal osmolality to 700–1,500 mosm/kg over a 1-hour period. Hypertonic glucose solutions (2,000 and 3,400 mosm/kg), given into the stomach, were found to be diluted to about 700 and 1,100 mosm/kg, respectively, at the level of the mid-duodenum 30 minutes later. Hypertonic saline solutions (1,800 and 2,700 mosm/kg), perfused into the duodenum, created a stable intraluminal osmolality of 800 and 1,200 mosm/kg, respectively, (about 45% that of the perfusate) after 30 minutes. In 6 Heidenhain pouch dogs, gastric secretion and gastrin release stimulated by food were significantly diminished by administration of hypertonic sodium chloride solution (1,800 mosm/kg) into the duodenum. This hyperosmolality caused greater suppression of acid secretion (52%) than of gastrin release (28%). Stimulation of pancreatic water and bicarbonate secretion and of release of radioimmunoassayable secretin by intraduodenal HCl (pH 1.3) were significantly suppressed when the osmolality of the HCl solution was raised to 2,700 mosm/kg. Pancreatic protein secretion remained unchanged with hypertonic solutions. We have confirmed that stimulation of intraduodenal osmoreceptors inhibits gastric acid secretion in dogs, and we suggest that this is due, at least in part, to a suppression of gastrin release. We further suggest that duodenal osmolar inhibition of pancreatic secretion involves suppression of secretin but does not appear to involve cholecystokinin.

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Résumé Nous avons mesuré l'osmolalité du contenu duodénal chez 9 chiens après un repas hypertonique administré soit per os, soit directement dans l'estomac, soit par perfusion dans le duodénum. Un repas à 2,475 mosm/kg, administré per os, élève l'osmolalité duodénale à 700–1,500 mosm/kg pendant une heure. Des solutions de glucose hypertonique à 2,000 et 3,400 mosm/kg, introduites directement dans l'estomac, sont, 30 minutes plus tard, diluées à 700 et 1,100 mosm/kg au niveau de la partie moyenne du duodénum. Des solutions salines hypertoniques à 1,800 et 2,700 mosm/kg, en perfusion intraduodénale, donnent, après 30 minutes, une osmolalité duodénale de 800 et 1,200 mosm/kg (±45% de la solution perfusée).Chez 6 chiens à poche de Heidenhain, la sécrétion gastrique et la libération de gastrine provoquées par un repas sont réduites de façon significative par la perfusion intraduodénale de chlorure de sodium en solution hypertonique (1,800 mosm/ kg). Cette hyperosmolalité réduit plus la sécrétion d'acide (52%) que la libération de gastrine (28%). La perfusion dans le duodénum d'une solution d'HCl (pH 1.3), dont l'osmolalité est portée à 2,700 mosm/kg, diminue la stimulation des sécrétions d'eau et de bicarbonate pancéatiques et la libération de sécrétine mesurable par radio-immunoessai. La sécrétion pancréatique de protéines n'est pas modifiée par les solutions hypertoniques. Nous avons done confirmé l'inhibition de la sécrétion gastrique d'acide chez le chien par stimulation des osmorécepteurs duodénaux; nous pensons que cette inhibition est due, en partie en tous cas, à la suppression de la libération de gastrine. Nous estimons de plus que l'inhibition de la sécrétion pancréatique par l'hyperosmolalité duodénale résulte d'une suppression de la libération de sécrétine, mais est sans effet sur la libération de cholécystokinine.

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Supported by grants from the National Institutes of Health (AM 15241) and the John A. Hartford Foundation, Inc.

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An abstracted preliminary report of a portion of this work has appeared (Physiologist 20:93, 1977).

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Recipient of a grant from the Deutsche Forschungsgemeinschaft (Te 79/1).     

甲状腺素及促甲状腺素对骨转换作用机制的研究进展

甲状腺功能亢进症是引起继发性骨质疏松症的危险因素。目前关于甲亢导致的骨质疏松机制还不完全明了,但过量甲状腺素会对骨转换产生影响,促甲状腺素水平的降低可能也参与其中。本文综述甲状腺素、促甲状腺素对骨转换作用机制的研究,为以后甲亢性骨质疏松病因机制研究提供新思路。     

Effects on calcium and phosphate metabolism and on parathyroid function of acute administration of tricalcium phosphate

The effects of the ingestion of tricalcium phosphate on calcium and phosphate metabolism and on parathyroid function were evaluated in 10 young adults. Each subject was studied during a control period of two hours before and during an experimental period of four hours after ingestion of a single oral dose of tricalcium phosphate containing 1500 mg of calcium and 770 mg of phosphorus. Serum and urinary calcium and phosphate and the nephrogenous cAMP fraction were measured. Significant rises in serum (from 2.32 ± 0.05 to 2.44 ± 0.08 mmol/l) and urinary (from 1.08 ± 0.65 to 3.43 ± 1.38 μmol/l GF) calcium and in serum phosphate (from 1.05 ± 0.18 to 1.28 ± 0.14 mmol/l) occurred. Unexpectedly, the acute supply of calcium in the form of tricalcium phosphate did not provoke significant alteration of nephrogenous cAMP level. In order to assess the respective effects of calcium and of phosphate, similar tests with ingestion of similar amounts either of calcium (as a glucoheptogluconate salt) or of phosphate were subsequently performed in the same subjects. Significant increases in serum total calcium were observed after calcium glucoheptogluconate as after tricalcium phosphate. However, the effects on parathyroid function differed, since a significant (p < 0.001) decrease in nephrogenous cAMP followed the ingestion of calcium glucoheptogluconate. Otherwise, a stimulating effect of phosphate on parathyroid function was observed. These findings suggest that the respective effects of calcium and of phosphate are counterbalanced when administered as tricalcium phosphate, resulting in the absence of parathyroid suppression.     

Early Effects of Parathormone and Calcitonin on the Number of Osteoclasts and on Serum-Calcium in Rats

Using succinic dehydrogenase staining of osteoclasts, the authors have studied the early effects on these cells of parathormone and calcitonin in rats. Thirty minutes after injection of the hormones the number of osteoclasts had increased (parathormone) or decreased (calcitonin), associated with inverse changes in total serum-calcium. The results confirm earlier studies showing the remarkably rapid changes in the number of osteoclasts after substances acting on serum calcium.     

Effect of laryngoscopy and tracheal intubation on pulse pressure and influence of age on this response

The study objective was to measure the change in pulse pressure associated with laryngoscopy and tracheal intubation and to relate these changes to trends in systolic, diastolic and mean blood pressure. The rationale was that the rise in systolic and diastolic blood pressure may be disproportionate and may result in either increase or decrease in pulse pressure. We also looked at the influence of age on this response. This prospective observational study measured the changes in pulse pressure secondary to laryngoscopy and tracheal intubation in eighty adult surgical patients. Two groups of forty patients each were included, young (group A) 18-25 years and middle-aged (group B) 45-55 years. The patients were ASA Class 1 or 2, of either gender and non-hypertensive. Systolic, diastolic, and mean blood pressure, and heart rate were measured preinduction and 1, 2 and 3 minutes after induction. Thereafter they were measured every minute for five minutes after intubation. Pulse pressure was obtained by subtracting the diastolic from the systolic blood pressure. No pulse pressure change occurred in the young group despite of a significant increase in both systolic and diastolic blood pressures. The middle aged group showed an average rise of +18 mm of Hg in pulse pressure (taken at 1 minute post-intubation) compared to the baseline measurement (P<0.0001). These changes in pulse pressure during anaesthesia may indicate an additional pulsatile stress in vulnerable patients in addition to the changes associated with resistance alone and need to be studied further.     

Contrasting effects on bone formation and on fracture healing of cholecalciferol and of 1 α-hydroxycholecalciferol

Summary Experimental fractures were performed on tibiae of vitamin D-depleted chicks. The chicks were divided into three groups; they were treated daily with (a) cholecalciferol or (b) 1α-hydroxycholecalciferol (1α(OH)D₃), or (c) they received no treatment. Microscopic examination of the calluses formed at the fractured sites and of the proximal tibiae of the contralateral legs showed that treatment with 1α(OH)D₃ failed to heal most of the rachitic signs seen in the nontreated chicks, despite normal plasma concentrations of calcium and of inorganic phosphorus. In a second experiment, experimental fractures were performed on tibiae of chicks that were fed a vitamin D-deficient diet, but were dosed continuously with radioactive cholecalciferol. Analysis of cholecalciferol metabolites in the callus tissue showed perferential accumulation of 25-hydroxycholecalciferol (25(OH)D₃) and of 24,25-dihydroxycholecalciferol (24,25(OH)₂D₃). Very little 1α, 25-dihydroxycholecalciferol (1α,25(OH)₂D₃) was detected in bones or in calluses. Based on the data obtained, the following conclusions were drawn: (a) that cholecalciferol is directly involved in bone formation; and (b) that 1α, 25(OH)₂D₃ is not the sole metabolite of cholecalciferol involved in this process.     

常规灌肠辅以指压粪石解除肠梗阻的效果

目的 探讨灌肠辅以指压粪石解除肠梗阻的效果,为粪石梗阻患者找到更易解除梗阻的方法.方法 将48例粪石梗阻患者随机分为对照组与观察组各24例,对照组采用常规灌肠,观察组在常规灌肠的基础上辅以指压粪石解除肠梗阻.结果 观察组肠梗阻缓解效果显著优于对照组(P<0.05).结论 常规灌肠辅以指压粪石,可使粪石被挤压下移,提高了通过灌肠解除肠梗阻的成功率.     

Early Effects of Parathormone and Calcitonin on the Number of Osteoclasts and on Serum-Calcium in Rats

Using succinic dehydrogenase staining of osteoclasts, the authors have studied the early effects on these cells of parathormone and calcitonin in rats. Thirty minutes after injection of the hormones the number of osteoclasts had increased (parathormone) or decreased (calcitonin), associated with inverse changes in total serum-calcium. The results confirm earlier studies showing the remarkably rapid changes in the number of osteoclasts after substances acting on serum calcium.