Effect of Zibu Shenjing Fang(滋补肾精方) on Growth and Development of the Mouse with Kidney-essence Insufficiency and Study on the Mechanism

Objective:To investigate effects of Zibu Shenjing Fang(滋补肾精方) on growth and development of the mouse with insufficiency of kidney-essence and the mechanism.Methods:Total 50 mice were randomly divided into a normal group,a model group,a Jingui Shenqi Wan(金匮肾气丸) group,a Zibu Shenjing Fang high dose group and a Zibu Shenjing Fang low dose group,10 mice in each group.The kidney-essence insufficiency mouse model was established by use of threat-injuring the kidney combined with over-fatigue.At the same time of modeling,the mice in the model group were intragastrically administrated with saline 20 mL·kg-1·d-1,in the Jingui Shenqi Wan group with suspension of the Jingui Shenqi Wan 2.7 g·kg-1·d-1,in the Zibu Shenjing Fang high dose group with Zibu Shenjing Fang 20 g·kg-1·d-1 and in the Zibu Shenjing Fang low dose group with Zibu Shenjing Fang 10 g·kg-1·d-1,for 21 consecutive days.The general state was observed,the body weight was weighted,and serum growth hormone(GH) and insulin-like growth factor-1(IGF-1) contents were detected.Results:Compared with model group,Zibu Shenjing Fang groups and Jingui Shenqi Wan group could improve manifestation of the mouse with kidney-essence insufficiency,increase body weight of the mouse and serum GH and IGF-1 contents,especially in the high dose group.Conclusion:Zibu Shenjing Fang gives play to the function of tonifying the kidney and replenishing essence through regulating GH and IGF-1 levels,so as to influence growth and development of the mouse.     

Effects of Suxiao Jiuxin Pill (速效救心丸) on Oxidative Stress and Inflammatory Response in Rats with Experimental Atherosclerosis

Objective: To observe the preventive role of Suxiao Jiuxin Pill (SX速效救心丸) on atherosclerosis (AS) and to probe into the mechanism in the atherosclerosis rat model. Methods: The AS rat model was established by a high fat diet and a large dose of calcium (vitamin D3, 0.6 million U/kg, i.p, once). Sixty healthy male adult Sprague-Dawlay (SD) rats were randomly divided into 6 groups, a normal control group (N), a model group (M), a SX low dose group (SXL), a SX middle dose group (SXM), a SX high dose group (SXH...     

Preventive Effects of Nitroglycerine on Glucocorticoid-induced Osteoporosis in Growing Rats

The preventive effects of nitroglycerine （NG） on glucocorticoid-induced osteoporosis in growing rats were studied. Three-month-old female Wistar rats were randomly divided into control group （CON）, dexamethasone group （DXM）, DXM plus a low dose NG group （NG-L）, DXM plus a middle dose NG group （NG-M） and DXM plus a high dose NG group （NG-H）, 8 rats in each group. The rat model of osteoporosis was developed by intramuscular injection of dexamethasone twice a week. NG 0.2, 0.4 and 1.0 mg/kg was administered by oral gavages to the treatment groups every day for 12 weeks. Rats in CON group and DXM group were treated with normal saline of the same amount. After the treatment, the bone mineral density （BMD） and bone metabolism-associated biochemical markers were determined. Compared with CON group, BMD of lumbar spine and femur in DXM group was decreased significantly （P〈0.05 and P〈0.01 respectively）, blood BGP levels and NO levels reduced （both P〈0.01）, and TRAP level increased （P〈0.05）. As compared with DXM group, BMD, serum BGP and NO were increased, and TRAP decreased in NG-L group and NG-M group, but had no significant difference in comparison to CON group. All the markers other than serum NO and TRAP levels had no significant difference between NG-H group and DXM group. It was concluded that low or middle doses of NG could prevent glucocorticoid-induced bone loss in growing rats, but high dose of NG could not. Supplement with NO donor could be considered as a preventive treatment for glucocorticoid-induced osteoporosis in a developing skeleton.     

Effect of Trace Elements on Retinopathy of Prematurity

The effect of the trace elements on retinopathy of prematurity(ROP) were studied.Thirty preterm infants who had potential high risk factors of ROP were selected as observation group and 18 normal infants as control groups.By using atom spectrophotometer,the contents of serum trace elements(Mg,Cu,Zn,Mn,Se) were measured and analyzed statistically.The contents of serum Zn,Cu and Se in observation group were 0.75±0.22,0.41±0.20 and(134.07±71.57)×10-3 mg/L respectively,and 0.55±0.12,0.65±0.194 and(202.92±44.71)×10-3 mg/L in control group respectively(P<0.01).The contents of Cu and Se were obviously lower and that of Zn higher in observation group than those in control group.The same results were obtained between the infants with ROP and controls(P<0.01).However,there was no significant difference in the contents of serum Mg and Mn between two groups(P>0.05).It was concluded that the contents of serum Cu and Se in preterm infants who had high risk factors of ROP were obviously lower than in the controls.The contents of serum Cu and Se in the ROP infants were also much lower while contents of Zn much higher.Attention should be paid to the detection of the trace elements in preterm infants in order to prevent the deficiencies of Cu and Se.Only in this way can we prevent the deficiencies of Cu and Se,so as to decrease the ROP risk factors and prevent the disease.     

Effects of Wei Chang An pill(胃肠安丸) on enzyme activity and levels of vasoactive peptide and substance P in the small intestine of rats with compound diarrhea

OBJECTIVE:To investigate the regulatory effects of Wei Chang An Pill(WCAP) on enzyme activity and gastrointestinal hormones in the small intestine of rats with compound diarrhea.METHODS:Forty Wistar rats were randomly divided into a control,diarrhea model,and WCAP high,medium,and low dose groups.The control group was not treated,and the model group was administered intragastric distilled water.The WCAP groups were given WCAP suspension,80,60 or 40 mg · kg-1 · d-1,for 4 days.Stool properties were observed.After the experiment,thymus and spleen indices were measured,and the activities of lactate dehydrogenase(LDH),malate dehydrogenase(MDH),and disaccharidase(lactase) in the small intestinal mucous membrane,and levels of substance P(SP) and vasoactive peptide(VIP) in the colon were determined.RESULTS:Compared with the control group,thymus and spleen indices were significantly decreased,LDH,MDH,and disaccharidase activity in the small intestine was decreased,and SP and VIP levels in the colon were significantly increased inthe diarrhea model group.Compared with the model group,thymus and spleen indices were significantly increased,and LDH,MDH,and disaccharidase activity in the small intestine and SP and VIP levels in the colon were significantly decreased in theWCAP medium dose group.CONCLUSION:The diarrhea model rats exhibited pathological changes including atrophy of the thymus and spleen,decreased enzyme activity in the small intestine,and gastrointestinal hormone disturbance.WCAP can increase the activity of intestinal digestive enzymes and regulate gastrointestinal hormones,thereby relieving diarrhea.     

Effect and Mechanism of Epidermal Growth Factor on Proliferation of GL15 Gliomas Cell Line

The effects of epidermal growth factor (EGF) on proliferation of G15 glioma cells and the possible mechanisms were investigated. GFAP and EGFR expression was detected by immunohisto-chemical method. After the cells were treated with EGF at different concentrations, cell count method was used to determine the proliferation of glioma cells, cell cycle and apoptosis were analyzed by flow cytometry (FCM), and laser scan confocal microscope (LSCM) was used to measure the cyto-plasmic free calcium. The results showed that GFAP was diffusedly expressed in GL15 cells and EGFR was over-expressed. EGF at doses of≤1 ng/mL could significantly stimulate cell proliferation, cells in phase G0/G1 decreased, and those in phase S increased. EGF at doses of 10 and 100ng/ml could inhibit the cell proliferation significantly, and the apoptosis ratio in high dose of EGF group was higher than in control group. EGF could significantly induce a quick rise of intracellular free calcium, but the peak value of intracellular free calcium activated by high dose of EGF was higher than by low dose of EGF. It was suggested that EGF had a dual effect on gliomas: low dose of EGF could stimulate the cell proliferation of gliomas, but high dose of EGF could induce the cell apoptosis and inhibit the proliferation of gliomas, which might be contributed to the difference of intracellular free calcium.     

Effects of the Drug(BSZGC)--containing Serum on Proliferation of Rat''s Osteoclasts and TRACP Activity in vitro

Objective:To observe effects of the drug-containing serum of Bu Shen Zhuang Gu Capsule(BSZGC 补肾壮骨胶囊 Capsule for Tonilying the Kidney to Strengthen the Bones)on proliferation of the rat's osteoclasts and tartrate-resistant acid phosphatase (TRACP)activity in vitro SO as to delve into the mechanisms of its preventive and therapeutic actions on osteoporosis.Methods:Forty female Sprague.Dawley rats aged three months were randomly divided into high dosage BSZGC group,medium dosage BSZGC group,low dosage BSZGC group,and the control group.BSZGC was orally administered into the rats of high,medium,and low dosage groups at difierent dosages for 12 days.and isometric normal saline was orally administered to the rats of the Control group.The drug-containing serum and control serum were prepared.Osteoclasts isolated mechanically from the femur and tibia of Sprague-Dawley rats aged one week were cultured wim medium added with different drug-containing sera and control serum.The growth of osteoclasts was observed under an inverted phase-contrast microscope,and optic density(OD)value of osteoclasts was determined by MTT colorimetric assay.TRACP activity was measured by the diazol method.Results:OD value of osteoclasts in the high dosage drug-containing serum group,medium dosage drug-containing serum group,and low dosage drug-containing serum group was significantly lower than that in the control serum group(P＜0.05)with a dose-effect correlation.TRACP activity in high dosage drug-containing serum group,medium dosage drug-containing serum group,low dosage drug-containing serum group was significantly lower than that of the control serum group(P＜0.01),and no significant differences in TRACP activity were not found among the difierent dosages drug-containing serum groups.Conclusions:BSZGC can inhibit the proliferation of the osteoclasts and reduce TRACP activity,which may be one of the mechanisms of its preventive and therapeutic actions on osteoporosis.     

Effects of Acupuncture at Baihui(GV 20) and Zusanli(ST 36) on Peripheral Serum Expression of MicroRNA 124,Laminin and Integrin β1 in Rats with Cerebral Ischemia Reperfusion Injury

Objective:To explore the effects of acupuncture at Baihui(GV 20) and Zusanli(ST 36) on the peripheral serum expression of microRNA 124(miRNA 124),laminin and integrin β 1 in rats with cerebral ischemia reperfusion injury(CIRI).Methods:Seventy-two healthy male Sprague-Dawley rats were randomized into a model group,an acupuncture group,and a sham-operated group using a random digits table,with 24 rats per group.Each group was further randomly divided into 1-,3-,5-,and 7-day subgroups based on the reperfusion time according to a random digits table,with 6 rats in each subgroup.In the model and acupuncture groups,CIRI was induced using the thread occlusion method.Electroacupuncture stimulation was applied daily to GV 20 and left ST 36 for 20 min at the indicated time points after successful operations.Serum was sampled for detecting laminin and integrin β1 protein via enzyme-linked immunosorbent assay,and serum miRNA 124 was examined using quantitative polymerase chain reaction.Results:The serum level of miRNA 124 in the cerebral ischemia rats increased significantly,and the peak expression of miRNA 124 in both the model and acupuncture groups occurred at 3 days.The expression of miRNA 124 in the acupuncture group was higher than in the model group at the same time point(5.96 ±0.01 vs.3.11 ±0.04,P0.05).Laminin expression in serum from the cerebral ischemia group was higher than that in the sham-operated group.Compared with the model group,the level of laminin in the serum of the acupuncture group was significantly lower at each time point,especially at the 3-day,and 7-day time points(589.12±3.57 vs.793.05 ± 5.28,and 600.53 ±3.05 vs.899.06 ±5.74,P0.05).The level of integrin β 1 in the serum from the acupuncture group was lower than that in the model group particularly at the 3-day and 7-day time points(208.66 ±0.95 vs.280.83 ±1.77,and212.36 ±0.95 vs.316.77±2.42,P0.05).Additionally,the model group and the acupuncture group showed dual peaks of integrin β 1 and laminin expression at 3-day and 7-day.Conclusions:Acupuncture at GV 20 and ST 36 in rats alleviated CIRI and was associated with upregulated expression of miRNA 124 and with downregulated expression of integrin β 1 and laminin in peripheral serum.These changes may represent one of the mechanisms underlying acupuncture's attenuation of CIRI.     

Protective effects and mechanism of total coptis alkaloids on A β25–35 induced learning and memory dysfunction in rats

Objective: To observe the effect of total coptis alkaloids (TCA) on β-amyloid peptide (Aβ25-35) induced learning and memory dysfunction in rats, and to explore its mechanism. Methods: Forty male Wistar rats were randomly divided into four groups: the control group, the model group, the TCA low dose (60 mg/kg) group and the TCA high dose (120 mg/kg) group, 10 in each. Aβ25-35 (5μl, 2μg/μl) was injected into bilateral hippocampi of each rat to induce learning and memory dysfunction. TCA were administered through intragavage for consecutive 15 days. Morris Water Maze test was used to assess the impairment of learning and memory; concentration of malondialdehyde (MDA) in cerebral cortex was determined by thiobarbituric acid reactive substance to indicate the level of lipid peroxidation in brain tissues; activity of manganese-superoxide dismutase (Mn-SOD) in cerebral cortex was determined by xanthine-oxidase to indicate the activity of the enzyme; and NF-κB protein expression in cerebral cortex was measured by SP immunohistochemistry. Results: (1) Morris Water Maze test showed that, during the 4 consecutive days of acquisition trials, the rats in the model group took longer latency and searching distance than those in the control group (P<0.01), which could be shortened by high dose TCA (P<0.05); during the spatial probe trial on the fifth day, the rats in the model group took shorter searching time and distance on the previous flat area than those in the control group (P<0.01), which could be prolonged after TCA treatment (for low dose group, P<0.05; for high dose group, P<0.01). (2) Analysis of cerebral cortical tissues showed that, compared with the control group, MDA level got significantly increased and Mn-SOD activity decreased in the model group (both P<0.01). After having been treated with TCA, the MDA level got significantly decreased (P<0.05 and P<0.01 respectively for low and high dose group), while relative increase of Mn-SOD activity only appeared in high dose group (P<0.05). (3) Immunohistochemistry analysis showed the protein expression of NF-κB got significantly increased after modeling, while high dose TCA can significantly inhibit it. Conclusion: TCA could improve Aβ25-35 induced dysfunction of learning and memory in rats, and its protective mechanism is associated with its actions in decreasing MDA level, increasing Mn-SOD activity and inhibiting the expression of NF-κB in cerebral cortex.     

An Experimental Study of the Protective Effects of Chinese Medicine Compound Eye-Patch on Asthenopia

Objective:To observe the therapeutic efficacy and the mechanism of Chinese medicine eye-patch with invigorating blood circulation and detoxification on asthenopia.Methods:A total of 180 rabbits were subjected to three tests,namely the skin microcirculation,the microvascular regeneration in the skin and the skin temperature change,with 60 rabbits for each test.The rabbits in each test were randomly and equally divided into three groups: the normal control group was treated with physiological saline on naked back once per day,the low dose group was treated with the eye-patch on naked back for 15 min once per day,while the high dose group was treated for three times per day.Forty Hartley guinea pigs were randomly and equally divided into four groups for the muscle tone test:the normal control group was treated with physiological saline on naked back,the model group was treated without any medication,the low dose group was treated with the eye-patch on naked back for 10 min once per day, while the high dose group was treated for three times per day.All treatments were continued for 14 days until the termination of the test.The microcirculatory blood flow was observed by using a video-microscopy system.The histological sections were used to detect the microvascular regeneration by observing the expression of factorⅧ. The temperature changes on the skin surface were measured by using infrared thermometer,and the muscle tone was tested by the electromyography.Results:In compare with the normal or the model group,the improvement in the skin microcirculation and the blood vascular regeneration,and the decreasing in the muscle tone in low dose and high dose groups were statistically significant with confident level at P<0.05.Conclusions:The eye-patch with invigorating blood circulation and detoxification has great enhancement in blood vascular regeneration and skin microcirculation,and great improvement in the indexes for muscle tone.The study explains certain therapeutic efficacy and mechanism of the eye-patch and shows that it could reduce the symptoms for patients with asthenopia.     

甲基强的松龙对成骨细胞钙通道电流的影响

目的:观察甲基强的松龙(mPSL)对MC3T3-E1成骨细胞L-钙通道电流(L-type voltage-sensi-tive calciumchannels current,L-VSCCsC)的影响。方法:设A组(正常对照组)、B组(10-6mol/L)、C组(10-5mol/L)和D组(10-4mol/L),各浓度的mPSL作用24h后,膜片钳技术记录每组细胞的L-VSCCsC(nA)。结果:正常对照组峰值电流为(-0.2284±0.0209,nA);10-6mol/L组为(-0.1991±0.0158,nA)下降12.8%;10-5mol/L和10-4mol/L组分别为(-0.1839±0.0179,nA)、(-0.1610±0.0033,nA)分别下降19.5%和29.5%,与正常对照组比较有显著性差异(P<0.01)。结论:mPSL剂量依赖性抑制成骨细胞L-VSCCsC。     

伤科接骨片对MC3T3-E1在小鼠体内成骨能力的作用研究

目的：观察伤科接骨片对小鼠体内MC3T3-E1细胞成骨能力的作用。 方法：30只小鼠随机分为3组（每组10只）：(1)药物+细胞组：伤科接骨片及饲料喂养+MC3T3-E1+明胶海绵;(2)细胞组：单纯饲料+MC3T3-E1+明胶海绵;(3)阴性对照组：单纯饲料+生理盐水+明胶海绵。手术在小鼠右侧股部臀大肌下方制成肌袋,用无菌明胶海绵吸附MC3T3-E1细胞悬液（药物+细胞组和细胞组）,或者生理盐水（阴性对照组）后植入肌袋内。术后常规饲料喂养,3天后开始按组分别灌胃给药,方法如下：药物+细胞组将伤科接骨片(0.35g/片)用蒸馏水配制成浓度为20mg/ml的混悬液,按每日2.0mg/g的剂量灌胃给药,细胞组和对照组均灌胃给予每日2.0mg/g的蒸馏水。分别于建模后第4、8周每组麻醉后处死5只小鼠,取材后分别从大体,切片HE染色光镜,以及扫描电镜下观察。 结果：术后4周,药物+细胞组和细胞组肌袋内均可见有新骨出现,术后8周两组均形成松质骨样结构的板层骨,但是药物+细胞组较细胞组形成的板层骨结构更为成熟,成骨细胞数量更多,胶原纤维分泌量也更多,并有更多的钙化结节形成。阴性对照组内未见有明显骨化现象。 结论：伤科接骨片可以促进MC3T3-E1细胞在动物体内的成骨作用。     

木犀草素对小鼠体外培养前成骨细胞MC3 T3-E1增殖、分化、矿化和Wnt通路的影响

目的:探讨木犀草素对小鼠体外培养前成骨细胞MC3T3-E1增殖、分化和矿化及Wnt通路的影响.方法:在体外培养的MC3T3-E1s培养基中加入木犀草素0.25~8.00μmol/L培养12、24和48 h,以洛伐他汀0.04μmol/L作为阳性对照,采用细胞计数试剂盒法检测细胞增殖情况;用碱性磷酸酶(ALP)试剂盒检测细胞内ALP的活性;茜素红S染色法检测细胞矿化能力;采用实时荧光定量反转录PCR法检测细胞Ⅰ型胶原、骨钙素、低密度脂蛋白受体相关蛋白5(Lrp5)、β-连环素、护骨素/细胞核因子κB受体活化因子配体mRNA表达水平.结果:木犀草素可以剂量依赖性和时间依赖性地促进成骨细胞MC3T3-E1增殖(P<0.05~P<0.01);木犀草素(2.00、4.00和8.00μmol/L)可以剂量依赖性地促进MC3T3-E1成骨细胞ALP活性,增强MC3T3-E1成骨细胞矿化能力并上调MC3T3-E1成骨细胞中Ⅰ型胶原、骨钙素、Lrp5、β-连环素和护骨素/细胞核因子κB受体活化因子配体mRNA表达.结论:木犀草素2.00、4.00和8.00μmol/L可以促进MC3T3-E1细胞的增殖、分化与矿化及Wnt通路中Lrp5和β-连环素mRNA的表达.     

活性氧与铁死亡通路相互作用在丙酮醛引起的小鼠胚胎成骨细胞损伤中起重要作用

目的 探讨活性氧和铁死亡在丙酮醛诱导小鼠胚胎成骨细胞（MC3T3-E1）损伤中是否存在相互作用。方法 应用丙酮醛损伤MC3T3-E1细胞建立模拟糖尿病骨质损伤的细胞模型。应用细胞计数试剂盒-8（CCK-8）测定MC3T3-E1细胞的存活率;罗丹明123染色荧光显微镜照相法测定线粒体膜电位;双氯荧光素（DCFH-DA）荧光显微镜照相法检测胞内活性氧水平;碱性磷酸酶试剂盒检测定碱性磷酸酶活性;茜素红染色观察成骨细胞晚期标志物-矿化结节的形成;铁离子试剂盒检测铁离子水平;Western blot检测成骨细胞的抑制铁死亡的标志蛋白谷胱甘肽过氧化物酶4（GPX4）的表达水平。结果 0.6 mmol/L 丙酮醛处理MC3T3-E1细胞24 h可明显减少GPX4的表达（P<0.001）,同时可使胞内铁离子浓度升高,细胞存活率降低,线粒体膜电位丢失,胞内活性氧水平升高,碱性磷酸酶活性降低和矿化结节减少（P<0.001）。应用2 mmol/L 活性氧清除剂N-乙酰半胱氨酸和丙酮醛共处理MC3T3-E1细胞24 h可增加GPX4的表达（P<0.01）,应用4 μmol/L铁死亡抑制剂铁抑素-1与丙酮醛共处理成骨细胞24 h可使胞内活性氧水平降低（P<0.001）;应用N-乙酰半胱氨酸或铁抑素-1与丙酮醛共处理成骨细胞24 h均能对抗丙酮醛引起的上述其他细胞损伤（P<0.001）。结论 活性氧与铁死亡通路相互作用在丙酮醛引起MC3T3-E1成骨细胞损伤中起重要的作用。     

含枸杞多糖血清对MC3T3-E1成骨细胞增殖、分化和矿化的影响

目的研究含枸杞多糖血清对MC3T3-E1成骨细胞细胞增殖、分化和矿化的影响。方法含枸杞多糖血清分别加入MC3T3-E1成骨细胞培养液中,使其终浓度为5%、10%、20%3种体积浓度,通过MTT法,碱性磷酸酶活性测定,骨钙素检测和Von kossa矿化染色法观察含枸杞多糖血清的促细胞增殖、分化、矿化作用。结果含枸杞多糖血清在5%、10%浓度促进MC3T3-E1成骨细胞增殖,差异有统计学意义(P<0.05);10%、20%浓度提高MC3T3-E1成骨细胞内碱性磷酸酶的活性,差异有统计学意义(P<0.01)。含枸杞多糖血清在20%浓度培养7及11 d时能明显促进MC3T3-E1成骨细胞骨钙素合成和分泌,20%浓度培养18 d时MC3T3-E1成骨细胞的矿化结节数目增多,差异有统计学意义(P<0.01)。结论含枸杞多糖血清可以促进MC3T3-E1成骨细胞增殖、分化和矿化能力。     

大黄素通过BMP-9途径促进前体成骨细胞的分化

目的 研究大黄素对前体成骨细胞MC3T3-E1增殖与分化的影响及其作用机制。方法 采用不同浓度（0.05、0.1、0.5、2.0和5.0 μmol/L）大黄素处理MC3T3-E1细胞（给药组）,以不加大黄素为对照组。分别采用MTT法检测细胞的增殖率;对硝基苯基磷酸二钠（PNPP）法定量检测碱性磷酸酶（ALP）活性;茜素红染色观察矿化结节数目;RT-PCR法检测骨形态发生蛋白（BMPs）途径相关信号分子的表达。采用BMPs抑制剂（Noggin）处理细胞后检测ALP活性,观察BMP-9在大黄素促成骨分化中的作用。结果 大黄素对MC3T3-E1细胞的增殖无影响,大黄素浓度为0.5~5.0 μmol/L时可促进细胞ALP表达及矿化结节形成,其中浓度为5 μmol/L时效果最显著。大黄素可增强细胞BMP-9的表达,与BMP-9信号途径相关的上下游信号分子mRNA的表达亦有相应改变;Noggin预处理后,大黄素促成骨分化的功能受到明显抑制。结论 大黄素可通过激活BMP-9信号通路促进前体成骨细胞的分化。     

葡萄糖酸锌碳点在细胞成像和体外促进成骨分化中的作用

目的：通过一步水热法合成葡萄糖酸锌碳点（Zn-CDs）,探讨Zn-CDs的细胞成像及其对小鼠前成骨细胞向成骨方向分化的促进作用。方法：一步水热法合成Zn-CDs,采用透射电子显微镜（TEM）、荧光光谱仪和傅里叶变换红外光谱仪（FT-IR）观察检测Zn-CDs的表征。将不同浓度（0.01、0.10、1.00、10.00、100.00和1 000.00 mg· L^-1） Zn-CDs浸提液与小鼠前成骨细胞系MC3T3-E1共培养作为实验组,空白对照组仅加入细胞培养液。采用MTT法检测各组MC3T-E1细胞相对增殖率（RGR）;采用激光共聚焦成像观察MC3T3-E1细胞的成像特点;采用qRT-PCR法检测各组MC3T3-E1细胞中Runt相关转录因子2基因（Runx2）、碱性磷酸酶基因（ALP）和骨钙素（OC） mRNA相对表达水平;茜素红染色检测各组细胞中钙化结节数。结果：TEM检测,成功合成粒径为5.25 nm的Zn-CDs。荧光光谱,Zn-CDs具有360 nm紫外光激发和450 nm蓝光发射的荧光性质,并表现激发波长依赖特性。FT-IR检测,Zn-CDs表面主要由羧基和羟基基团构成。与空白对照组比较,共培养24 h时1 000.00 mg·L^-1Zn-CDs组MC3T3-E1细胞的RGR明显降低（P<0.01）。荧光成像,Zn-CDs与MC3T3-E1细胞共培养后,细胞呈现蓝色、绿色和红色的荧光图像,形态轮廓清楚且荧光强度细胞质比细胞核强。qRT-PCR检测,随着Zn-CDs浓度的增加,MC3T3-E1细胞中Runx2、ALP和OCmRNA相对表达水平逐渐升高。茜素红染色,诱导21 d后不同浓度Zn-CDs组MC3T3-E1细胞中钙结节数多于空白对照组。结论：Zn-CDs可以有效地进行MC3T3-E1细胞荧光成像,且Zn-CDs具有一定的促MC3T3-E1细胞向成骨方向分化的作用。     

柚皮苷对H_2O_2处理小鼠前成骨细胞MC3T3-E1骨形成的影响

目的 :研究柚皮苷对体外培养过氧化氢(H_2O_2)处理的小鼠前成骨细胞MC3T3-E1骨形成的作用及抗氧化机制。方法:在体外培养的MC3T3-E1s细胞培养基中分别加入不同浓度(0.01 mmol·L-1、0.02 mmol·L-1、0.04 mmol·L-1)的柚皮苷作用48 h,接着加入0.5 mmol·L-1H_2O_2作用4 h,采用CCK-8法检测柚皮苷对成骨细胞活力的影响;采用碱性磷酸酶(ALP)、细胞丙二醛(MDA)、超氧化物歧化酶(SOD)、乳酸脱氢酶(LDH)和谷胱甘肽(GSH)试剂盒检测MC3T3-E1成骨细胞内ALP、SOD活性和MDA、GSH含量及细胞上清LDH活性;用茜素红S染色法检测矿化结节数目;用Real time RT-PCR的方法检测成骨细胞内I型胶原(COL-I)、骨钙素(OCN)、转录因子Osterix和骨形态发生蛋白2(BMP-2)m RNA表达。结果:柚皮苷可增加H_2O_2损伤后MC3T3-E1细胞ALP活力(P0.05,P0.01)和矿化结节数目(P0.05,P0.01),促进骨形成相关指标COL-I、Osterix、BMP-2和OCN m RNA表达(P0.01);柚皮苷提高H_2O_2损伤后MC3T3-E1细胞活力(P0.05,P0.01),上调细胞内SOD活性和GSH含量并降低细胞中MDA含量和上清中LDH活性(P0.01)。结论:一定浓度(0.01,0.02,0.04 mmol·L-1)的柚皮苷可以促进H_2O_2处理后MC3T3-E1细胞的骨形成,这种促进作用可能是通过降低氧化应激发挥抗氧化作用实现的。     

Tenascin-C表达降低抑制成骨细胞分化并促进骨质疏松的机制

目的探讨Tenascin-C在成骨细胞分化中的作用及机制,并分析其表达变化对骨质疏松的调控。方法以免疫印迹法检测 去卵巢致骨质疏松的小鼠股骨松质骨的Tenascin-C 表达变化。体外诱导成骨前体细胞MC3T3-E1 分化,检测瞬时转染 Tenascin-C的siRNA后其碱性磷酸酶的活性。以蛋白质相互作用预测软件STRING筛选与Tenascin-C可能存在相互作用的蛋 白质。在上述候选蛋白中,以免疫沉淀法验证了Tenascin-C与Wnt通路内源性抑制蛋白Dickkopf-1（Dkk-1）的相互作用,并以 免疫印迹检测Tenascin-C对DKK-1的调控。此后进一步采用荧光素酶报告基因系统检测了Tenascin-C对Wnt信号通路转录活 性的影响。结果去卵巢骨质疏松的小鼠股骨松质中Tenascin-C 蛋白表达急剧下降。降低Tenascin-C 的表达能够阻遏 MC3T3-E1向成熟成骨细胞分化,反之,在此基础上过表达β-Catenin可逆转前者导致的成骨细胞分化阻滞。Tenascin-C结合于 DKK-1,敲低Tenascin-C可上调DKK-1的表达并降低Wnt通路的转录活性。结论Tenascin-C的下调可能通过减弱对DKK-1 的负调控从而抑制Wnt通路的转录活性,进而抑制成骨细胞的分化并介导骨质疏松。     

氯离子／钾离子通道阻滞剂对成骨细胞增殖的影响

目的 观察氯离子和钾离子转运体阻滞剂及氯离子／钾离子通道阻滞剂是否影响MC3T3-E1成骨细胞的增殖。方法 采用体外培养MC3T3-E1成骨细胞,分别加入DIOA（氯离子和钾离子转运体阻滞剂,100和500μM）、NPPB（10和300μM,氯离子通道阻滞剂）和喹啉（10和500μM,钾离子通道阻滞剂）,检测细胞增殖情况。结果 DIOA抑制MC3T3-E1成骨细胞的增殖,不同浓度的NPPB和喹啉同样也能够减少MC3T3-E1成骨细胞的增殖。结论 细胞内氯离子的浓度升高或降低都会抑制MC3T3-E1成骨细胞的增殖。