雷洛昔芬对小鼠成骨细胞中护骨素核因子-κB受体活化因子配体表达的影响

目的 雷洛昔芬对小鼠成骨细胞中护骨素(OPG)、核因子-κB(NF-κB)受体活化因子配体(RANKL)表达的影响. 方法 取出生24 h内的小鼠30只,无菌的条件下取出颅骨,应用酶消化法进行成骨细胞的培养,在培养成骨细胞的培养液中加入不同浓度的雷洛昔芬(0、1012、1010、109mol/L),应用反转录聚合酶链扩增(RT-PCR)法检测其对OPG/RANKL mRNA的表达及酶联免疫吸附法(ELISA)法检测OPG蛋白分泌的影响. 结果 OPG mRNA在实验组中表达较对照组强,并且不同浓度组间的比较,差异有统计学意义(均为P<0.05),1010.mol/L组较10～mol/L、1012mol/L组的表达明显增强;RANKL mRNA在实验组的表达均较对照组弱,其组间的差异亦有统计学意义(P<0.01).并且随着雷洛营芬浓度的增加,RANKL mRNA的表达明显减弱,呈现明显的浓度依赖性.在试验组小鼠成骨细胞培养液中OPG浓度(10-9mol/L组为3.017±0.459,1010mol/L组为3.981±0.762,1012mol/L组为2.864±0.416)较对照组(2.106±0.316)增高,差异有统计学意义(P<0.05). 结论 雷洛昔芬能促进成骨细胞的中OPG的mRNA的表达及其蛋白的分泌,同时抑制RANKL的mRNA的表达.     

雌二醇对成骨细胞OPG和RANKL的影响

目的探讨不同浓度17β-雌二醇(E2)作用下体外培养大鼠成骨细胞护骨素(OPG)、细胞核因子-κB受体活化因子配体(RANKL)的表达及雌激素治疗骨质疏松的机制。方法采用不同浓度(10-10、10-9、10-8、10-7mol/L)17β-E2刺激培养的大鼠成骨细胞(OB),RT-PCR方法检测OPG、RANKL mRNA的表达。结果原代OB呈多角形、梭形,传代OB胞核较大,细胞质内见黑色颗粒,细胞形态为多角形或梭形,见突起,三代细胞碱性磷酸酶染色阳性,胞质见大量灰黑色颗粒,细胞鉴定符合成骨细胞特征。不同浓度(10-9、10-8、10-7mol/L)17β-E2对OB OPG mRNA表达具有显著的增强作用(P<0.05),其中10-8mol/L 17β-E2对OB OPG mRNA表达最高,与对照组比较差异有统计学意义(P<0.01),而10-10mol/L 17β-E2对成骨细胞OPG mRNA表达几乎无影响,与对照组比较无显著差异(P>0.05),10-10、10-9、10-7mol/L 17β-E2对成OB RANKL mRNA表达几乎无影响,与对照组比较无明显差异(P>0.05),其中10-8mol/L17β-E2对OB RANKL mRNA的表达具有明显的抑制作用,其与对照组比较差异明显(P<0.05)。结论雌激素治疗骨质疏松的作用可能与其促进成骨细胞OPG表达、抑制RANKL表达相关。     

Interleukin-1 beta up-regulates tissue inhibitor of matrix metalloproteinase-1 mRNA and phosphorylation of c-jun N-terminal kinase and p38 in hepatic stellate cells

AIM: To study the relationship between interleu-kin-1beta (IL-1β) up-regulating tissue inhibitor of matrix metalloproteinase-1 (TIMMP-1) mRNA expression and phosphorylation of both c-jun N-terminal kinase (JNK) and p38 in rat hepatic stellate cells (HSC). METHODS: RT-PCR was performed to measure the expression of TIMMP-1 mRNA in rat HSC. Western blot was performed to measure IL-1β-induced JNK and p38 activities in rat HSC. RESULTS: TIMMP-1 mRNA expression (1.191±0.079) was much higher after treatment with IL-1β(10 ng/mL) for 24 h than in control group (0.545±0.091) (P<0.01). IL-1βactivated JNK and p38 in a time-dependent manner. After stimulation with IL-1βfor 0, 5, 15, 30, 60 and 120 min, the JNK activity was 0.982±0.299, 1.501±0.720, 2.133±0.882, 3.360±0.452, 2.181±0.789, and 1.385±0.368, respectively. There was a significant difference in JNK activity at 15 min (P<0.01), 30 min (P<0.01) and 60 min (P<0.01) in comparison to that at 0 min. The p38 activity was 1.061±0.310, 2.050±0.863, 2.380±0.573, 2.973±0.953, 2.421±0.793, and 1.755±0.433 at the 6 time points (0, 5, 15, 30, 60 and 120 min) respectively. There was a significant difference in p38 activity at 5 min (P<0.05), 15 min (P<0.01), 30 min (P<0.01) and 60 min (P<0.01) compared to that at 0 min. TIMMP-1 mRNA expression trended to decrease in 3 groups pretreated with different concentrations of SP600125 (10μmol/L, 1.022±0.113; 20μmol/L, 0.869±0.070; 40μmol/L, 0.666±0.123). Their decreases were all significant (P<0.05, P<0.01,P<0.01) in comparison to control group (without SP600125 treatment, 1.163±0.107). In the other 3 groups pretreated with different concentrations of SB203580 (10μmol/L, 1.507±0.099; 20μmol/L, 1.698±0.107; 40μmol/L, 1.857±0.054), the expression of TIMMP-1 mRNA increased. Their levels were higher than those in the control group (without SB203580 treatment, 1.027±0.061) with a significant statistical significance CONCLUSION: IL-1βhas a direct action on hepatic fi-brosis by up-regulating TIMMP-1 mRNA expression in rat HSC. JNK and p38 mitogen-activated protein kinases (MAPKs) are involved in IL-1β-induced TIMMP-1 gene expression, and play a distinct role in this process, indicating that p38 and JNK pathways cooperatively mediate TIMP-1 mRNA expression in rat HSC.     

甲状旁腺激素对成骨肉瘤细胞系SaOS-2细胞成骨活性的影响

目的 观察不同浓度重组甲状旁腺激素1～34(简称hPTH)对成骨肉瘤细胞系SaOS-2细胞(简称SaOS-2细胞)骨碱性磷酸酶(ALP)和骨钙素(BGP)mRNA表达的影响.方法 SaOS-2细胞正常传代培养,用0、1、10、100 nmol/L hPTH处理细胞,分别在12、24、48 h提取细胞总RNA,反转录合成cDNA,采用实时荧光定量PCR的方法测定ALP和BGP mRNA的表达.结果 ①hPTH的不同剂量、不同的作用时间以及二者的交互作用对ALP的表达量均有影响(F值分别为29.32、2.92、7.64,P均<0.05).干预48 h,0、1、10、100 nmol/L组ALP mRNA的表达量(0.78±0.43、0.71±0.05、0.75±0.19、0.76±0.14)均低于同浓度组的12、24 h(12 h:1.01±0.16、1.37±0.38、1149±0.16、2.52±0.70,24 h:1.80±0.47、1.30±0.36、1.27±0.17、1.17±0.11,P均<0.05).干预12 h,100 nmol/L组ALP mRNA的表达量高于0 nmol/L组(P<0.05);干预24 h,1、10、100nmol/L组SaOS-2细胞ALP mRNA的表达量均低于0 nmol/L组(P均<0.05).②hPTH的不同剂量、不同的作用时间以及二者的交互作用对SaOS-2细胞BGP mRNA的表达量均有影响(F值分别为8.26、10.33、5.51,P均<0.05).干预48 h,0、1、10、100 nmol/L组BGP mRNA的表达量(1.17±0.28、0.98±0.08、0.92±0.17、0.84±0.59)均低于同浓度组的12、24 h(12 h:1.01±0.14、1.21±0.18、1.34±0.30、1.68±0.62,24 h:1.71±0.35、1141±0.47、1.28±0.31、1.01±0.18,P均<0.05).干预12 h,100 nmol/L组BGP mRNA的表达量高于0、1nmol/L组(P均<0.05);干预24 h,10、100 nmol/L组SaOS-2细胞BGP mRNA的表达量均低于0 nmol/L组(P均<0.05),100 nmol/L组SaOS-2细胞BGP mRNA的表达量低于1 nmoL/L组(P<0.05);干预48 h,10、100 nmol/L组SaOS-2细胞BGP mRNA的表达量均低于0 nmol/L组(P均<0.05).结论 在体外培养条件下,hPTH在短时间作用下可以显著增强SaOS-2细胞的成骨活性,随着刺激时间的延长,可使成骨活性呈现下降的趋势.

Abstract：

Objective To observe the effects of recombinant human parathyroid hormone 1 to 34(referred to as hPTH) on the expression level of alkaline phosphatase(ALP) and bone gla protein(BCP) in human osteosarcoma cell line SaOS-2(referred to as SaOS-2 cells). Methods SaOS-2 cells were subcultured and treated with 1, 10 and 100 nmol/L hPTH for 12, 24 and 48 h. Total cellular RNA was extracted, cDNA was synthesized by reverse doses of hPTH, different duration of action, and their interaction on the expression level of ALP mRNA of SaOS-2 cells was significantly different(F = 29.32, 2.92, 7.64, all P < 0.05). The expression level of ALP mRNA(0.78 ± 0.43, 0.71 ± 0.05, 0.75 ± 0.19, 0.76 ± 0.14) of SaOS-2 cells after treatment with 0, 1, 10 and 100 nmol/L hPTH for 48 h was lower than those of treated for 12 h(1.01 ± 0.16, 1.37 ± 0.38, 1.49 ± 0.16, 2.52 ± 0.70, all P< 0.05) and 24 h (1.80 ± 0.47, 1.30 ± 0.36, 1.27 ± 0.17, 1.17 ± 0.11, all P< 0.05). The expression level of ALP mRNA of SaOS-2 cells after treatment with 100 nmol/L hPTH for 12 hours was higher than that of the control(P < 0.05); the expression level of ALP mRNA of SaOS-2 cells after treatment with 1, 10 and 100 nmol/L hPTH for 24 h interaction on the expression level of BGP mRNA of SaOS-2 were significantly different (F = 8.26, 10.33, 5.51, all P< 0.05). The expression level of BGP mRNA(1.17 ± 0.28, 0.98 ± 0.08, 0.92 ± 0.17 and 0.84 ± 0.59) of SaOS2 cells after treatment with 0, 1, 10 and 100 nmol/L hPTH for 48 h was lower than those of treated for 12 h( 1.01 ± 0.14, 1.21 ± 0.18, 1.34 ± 0.30, 1.68 ± 0.62, all P< 0.05), and 24 h(1.71 ± 0.35, 1.41 ± 0.47, 1.28 ± 0.31 and 1.01 ± 0.18, all P < 0.05). The expression level of BGP mRNA of SaOS-2 cells after treatment with 100 nmol/L hPTH for 12 h was higher than that of those groups treated with 0 and 1 nmol/L hPTH(all P< 0.05). The expression level of BGP mRNA of SaOS-2 cells after treatment with 10 and 100 nmol/L hPTH for 24 h and 48 h was lower than those of the control(all P < 0.05). The expression level of BGP mRNA of SaOS-2 cells after treatment with 100 nmol/L hPTH for 24 hours was lower than that the group treated with 1 nmol/L hPTH(P < 0.05). Conclusions In vitro, hPTH significantly enhances osteogenic activities of human osteoblast in a short time, however, with prolonged stimulation time, osteogenic activity can show a downward trend.     

Protective effects of recombinant human growth hormone on cirrhotic rats

AIM:To investigate the effects and molecular mechanismsof recombinant human growth hormone(rhGH)onprotecting liver function and alleviating portal hypertensionof liver cirrhotic rats.METHODS:Liver cirrhosis of male Sprague-Dawley ratswas induced by administration of thioacetamide.The ratswith or without liver cirrhosis were randomly divided intofour groups.Group A consisted of the normal rats wastreated with normal saline(NS),group B consisted of thenormal rats was treated with rhGH,group C consisted ofcirrhotic rats was treated with NS,and group D consistedof cirrhotic rats was treated with rhGH.The rats of differentgroups were subcutaneously injected with 0.5 mL of NS or333 ng/kg of rhGH daily for 7 d.After treatments,thefollowing parameters were examined,including GH-bindingcapacity(R_T)by ~(125)I-hGH binding,growth hormone receptormRNA(GHR mRNA)expression by RT-PCR,relative contentof collagen(RCC)by histomorphomertry,and level ofmalon-dialdehyde(MDA)and superoxide dismutase(SOD)in liver tissue by thiobarbituric acid reaction and pyrogallicacid self-oxidation,respectively.Serum albumin(ALB),alanine transaminase(ALT)and portal vein pressure(PVP)were also examined.RESULTS:rhGH up-regulated both the GH-binding capacity(R_T)and the expression of GHR mRNA in vivo.RT in groupA(72±12 fmol/mg protein)was significantly higher thanthat in group C(31±4 fmol/mg protein)(P<0.05).R_T ingroup B (80±9 fmol/mg protein)increased markedlycompared to group A(P<0.05).R_T in group D(40±7 fmol/mgprotein)raised remarkably compared with group C(P<0.05),but less than that in group A,and there was no significantGH binding affinity contrast(Kd)change.The GHR mRNAlevel(iOD,pixel)in group A(29±3)was significantly higherthan that in group C(23±3)(P<0.05).GHR mRNA levelswere significantly raised in group B(56±4)and group D(42±8)compared with groups A and C(29±3 and 23±3,respectively)(P<0.05).Compared with the normal liver, MDA level was higher and SOD level was lower in cirrhoticlivers.After rhGH treatment,MDA level was significantlydeclined to 12.0±2.2 nmol/mg protein and SOD was raisedto 1029±76 U/mg protein in group D(P<0.05).ALB levelsin groups B and D(42±7 g/L and 37±7 g/L,respectively)were significantly raised compared with those in groups Aand C(35±5 g/L and 29±4 g/L,respectively)(P<0.05).ALT level was markedly lower in group D(69±7 U/L)compared to group C(89±15 U/L)(P<0.05),and close togroup A(61±10 U/L).RCC in group C(22.30±3.86%)wassignificantly higher than that in group A(1.14±0.21%)andgroup D(14.70±2.07%)(P<0.05).In addition,rhGHmarkedly alleviated portal hypertension in liver cirrhoticrats(group D vs C,9.3±1.5 cmH_2O vs 14.4±2.0 cmH_2O)(P<0.05).CONCLUSION:Pharmacological doses of rhGH canincrease R_T and GHR mRNA expression,ameliorate liverfunctions,repress fibrosis and decline portal hypertension,suggesting it has potentially clinical usage as a hepatotropicfactor.     

氟中毒对体外培养破骨细胞数量及骨吸收功能的影响

目的 观察氟中毒对体外培养破骨细胞数量及骨吸收功能的影响,探讨其作用机制.方法 机械分离法作用于新生SD大鼠四肢长骨,于TC199培养液(含10%胎牛血清)中获得破骨细胞和骨髓基质细胞.将破骨细胞接种于96孔培养板和象牙片培养,而骨髓基质细胞接种于6孔培养板培养,分别于2 h后换液并染氟(氟化钠),对照组、低氟组、中氟组、高氟组染氟剂量分别为0、2.5×10-5、5.0×10-5、10.0×10-5mol/L.培养2、5d后对培养板中破骨细胞进行抗酒石酸酸性磷酸酶(TRAP)染色,光镜下计数破骨细胞数量;培养5 d后象牙片经1%甲苯胺蓝染色,光镜下分析破骨细胞骨吸收陷窝面积.骨髓基质细胞染氟作用8 h后提取总RNA,实时荧光定量PCR法检测细胞核因子κβ受体活化因子配体(RANKL)和骨保护素(0PG)mRNA表达水平.结果 ①体外培养2 d时,对照组、低氟组、中氟组、高氟组破骨细胞数量分别为(337.5 4-70.5)、(447.5 ±43.4)、(472.9±34.8)、(475.3±24-3)个/孔,各染氟组明显高于对照组(P均<0.05);体外培养5 d时,对照组、低氟组、中氟组、高氟组破骨细胞数量分别为(92.5±22.1)、(123.0±26.4)、(135.5 ±22.2)、(136.9 ±23.0)个/孔,各染氟组明显高于对照组(P均<0.05).②体外培养5 d时,对照组、低氟组、中氟组、高氟组破骨细胞骨吸收陷窝面积分别为(0.088±0.030)、(0.100 ±0.018)、(0.152±0.015)、(0.242±0.031)mm2/片,中氟组和高氟组明显高于对照组(P均<0.05).③对照组、低氟组、中氟组、高氟组骨髓基质细胞RANKL/OPG mRNA表达比值分别为100.00±56.02、144.95±97.21、223.25 ±184.48、193.98 ±137.93,中氟组和高氟组明显高于对照组(P均<0.05).结论 氟中毒可引起体外培养破骨细胞数量增多,促进其细胞分化及骨吸收活性,该作用可能与其上调 RANKL/OPC,mRNA表达比值有关.

Abstract：

Objective To determine the effects of fluoride on osteoclasts's quantity and bone resorption function in vitro and its mechanisms. Methods The osteoclasts and bone marrow stromal cells(BMSCs) isolated from long bone of new born rats were cultured respectively in TC199 medium (containing 10% fetal bovine serum) with fluoride. The osteoclasts were inoculated in 96-well culture plate and ivory slice, BMSCs were inoculated in 6- well culture plate, respectively, medium were changed after 2 hours incubation. They were divided into control group, low-dose fluoride, medium-dose fluoride and high-dose fluoride groups, the doses of sodium fluoride were 0,2.5 × 10-5,5.0 × 10-5,10.0 × 10-5 mol/L, respectively. Tartrate-resistant acid phosphatase(TRAP) staining positive cells were counted under light microscope after TRAP staining on the 2nd and the 5th day and the pit formed in ivory slices were measured by histomorphometry after staining with toludine blue. The expression of receptor activator of NK-κβ ligand(RANKL) and osteoprotegerin(OPC) was detected by real-time fluorescence quantitative (337.5 ± 70.5), (447.5 ± 43.4), (472.9 ± 34.8), (475.3 ± 24.3)/well in the control group, the low-dose, mediumdose and high-dose fluoride groups, respectively. The differences were statistically significant between these groups and the control group (all P < 0.05). After in vitro culture for 5 days, the numbers of osteoclasts were (92.5 ± 22.1), (123.0 ± 26.4), (135.5 ± 22.2), (136.9 ± 23.0) per well in the control group, the low-dose, medium-dose and high-dose fluoride groups, respectively. The differences were statistically significant between these groups and the (0.088 ± 0.030), (0.100 ± 0.018), (0.152 ± 0.015), (0.242 ± 0.031 )mm2 per piece in the control group, the lowdose, medium-dose and high-dose fluoride groups, respectively. The values of medium-dose and high-dose fluoride BMSCs in the control group, the low-dose, medium-dose and high-dose fluoride groups were 100.00 ± 56.02, 144.95 ± 97.21,223.25 ± 184.48,193.98 ± 137.93, respectively. The values of medium-dose and high-dose fluoride groups were significantly higher than that of control group (all P < 0.05). Conclusions Fluoride can cause increase in the number of osteoclasts in vitro and promote their cell differentiation and bone resorption activity, which may be related to increased expression ratio of RANKL/OPG mRNA in BMSCs.     

补骨脂素对成骨细胞核因子-κB受体激活配体和骨保护素表达的影响

目的研究补骨脂素(psoralen,PSO)对体外培养的小鼠成骨细胞(OB)分化及核因子-κB受体激活配体(receptor activator of nuclearfactor-κB ligand,RANKL)和骨保护素(osteoprotegerin,OPG)表达的影响。方法取第1代BALB/c小鼠颅盖骨成骨细胞,将PSO以0.1、1、10μmol/L3种浓度分别加入新生大鼠颅骨成骨细胞培养液中,MTT法观察各组药物对成骨细胞的增殖作用并绘制细胞生长曲线;用PNPP法测定成骨细胞内碱性磷酸酶(alkaline phosphatase,ALP)活性;RT-PCR法检测成骨细胞OPG和RANKL的转录水平。结果细胞生长曲线显示各组成骨细胞数量均随时间延长而增加,中、高浓度PSO能显著提高成骨细胞的ALP活性,促进OPG、RANKL的表达(P<0.05),OPG/RANKL升高(P<0.05)。结论低浓度PSO(0.1μmol/L)对骨更新作用不明显,而中、高浓度PSO(1、10μmol/L)能通过上调OPG、RANKL mRNA表达及OPG/RANKL比例,促进成骨细胞的生成功能,增强骨更新。     

Effects of estradiol on liver estrogen receptor-α and its mRNA expression in hepatic fibrosis in rats

AIM:Estradiol treatment regulates estrogen receptor (ER)level in normal rat liver.However,little information is availableconcerning the role of estrogen in regulating liver ER inhepatic fibrosis in rats.The present study was conducted todetermine whether estradiol treatment in CCl_4-induced liverfibrosis of female and ovariectomized rats altered liver ERαand its mRNA expression,and to investigate the possiblemechanisms.METHODS:Seventy female rats were divided into sevengroups with ten rats in each.The ovariectomy groups wereinitiated with ovariectomies and the sham operation groupswere initiated with just sham operations.The CCl_4 toxicfibrosis groups received 400 mL/L CCl_4 subcutaneously at adose of 2 mL/kg twice weekly.Estrogen groups were treatedsubcutaneously with estradiol 1 mg/kg,the normal controlgroup and an ovariectomy group received injection of peanutoil vehicle twice weekly.At the end of 8 weeks,all the ratswere killed to detect their serum and hepatic indicators,their hepatic collagen content,and liver ER and ER mRNAexpression.RESULTS:Estradiol treatment in both ovariectomy andsham ovariectomy groups reduced liver levels of ALT (from658~220 nkat/L to 311±146 nkat/L and 540±252 nkat/L to314±163 nkat/L,P<0.05) and AST (from 697±240 nkat/L to321±121 nkat/L and 631±268 nkat/L to 302±153 nkat/L,P<0.05),increased serum nitric oxide (NO) level (from53.7±17.1 μmol/L to 93.3±24.2 μmol/L and 553±23.1 μmol/Lto 87.5±23.6 Hmol/L,P<0.05) and hepatic nitric oxide synthase(NOS) activity (from 1.73±0.71 KU/g to 2.49±1.20 KU/g and1.65±0.46 KU/g to 2.68±1.17 KU/g,P<0.05),diminishedthe accumulation of hepatic collagen,decreased centrolobularnecrotic areas as well as the inflammatory reaction in ratssubjected to CCl_4.The positive signal of ER and ER mRNAdistributed in parenchymal and non-parenchymal hepaticcells,especially near the hepatic centrolobular and periportalareas.Ovariectomy decreased ER level (from 10.2±3.2 to4.3±1.3) and ER mRNA expression (from 12.8±2.1 to 10.9±1.3)significantly (P<0.05).Hepatic ER and ER mRNA concentrationswere elevated after treatment with estradiol in bothovariectomy (15.8±2.4,20.8±3.1) and sham ovariectomy (18.7±3.8,23.1±3.7) fibrotic groups (P<0.05).CONCLUSION:The increase in hepatic ER and mRNAexpression may be part of the molecular mechanismsunderlying the suppressive effect of estradiol on liver fibrosisinduced by CCl_4 administration.     

强直性脊柱炎外周血破骨细胞前体细胞活性的研究

Objective To investigate the number of osteoclast (OC) precursor in the peripheral blood of patients with ankylosing spondylitis (AS) and its relationship with serum receptor activator of nuclear factor KB-ligand (RANKL) and Osteoprotegerin (OPG) concentration as well as the disease activity. Methods The peripheral blood mononuclear cells from 8 cases of AS patients and 5 healthy controls were cultured in the medium containing macrophage colony-stimulating factor (M-CSF) (25 ng/ml) and RANKL (40 ng/ml). After being cultured for 14 days, cytochemistry was applied to detect tartrate-resistant acid phosphatase (TRAP) expression and the cells with TRAP expression and ≥3 nuclei were counted and defined as OC. Bone resorption assay was used to demonstrate OC function. ELISA was used to measure serum RANKL and OPG concentration in 23 cases of AS and 17 healthy controls. The relationship was analyzed in AS patients between the number of OC precursors and serum RANKL and OPG concentration as well as the disease activity. The indicators of disease activity were Bath ankylosing spondylitis disease activity index (BASDAI), Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). T test, t' test and Spearman correlation were selec-ted. Results ① Significantly higher OC production was observed in the peripheral blood of AS patients than that of healthy control group. The OC number per ten fields was 10.9±3.4 and 6.2±1.3 respectively (P<0.05); ② There was significant difference between AS patients and healthy controls in serum concentration of OPG and RANKL and the ratio of RANKL/OPG. OPG was significantly higher in AS patients [(157±49) pg/ml] than in healthy controls [(105±20) pg/ml] (P<0.05). RANKL was significantly higher in AS patients [(5.4± 3.8) pg/ml] than in healthy controls [(1.6±0.8) pg/ml] (P<0.05). The ratio of RANKL/OPG was significantly higher in AS patients (0.037±0.026) than in healthy controls (0.016±0.008) (P<0.01 );③Significantly positive correlation was observed between the OC number and the serum concentration of RANKL (r=0.692, P=0.009), the ratio of RANKL/OPG (r=0.813, P=0.001);④ In AS patients, serum concentration of OPG was found to have significantly negative correlation with BASDAI (r=-0.444, P=0.044). Serum RANKL concentration was found to have significantly positive correlation with BASDAI (r=0.543, P=0.011). The ratio of RANKL/OPG was found to have significantly positive correlation with BASDAI (r=0.672, P=0.001). Conclusion ① More OC precursors exist in the peripheral blood of AS patients. These cells may differentiate into osteoclasts, which might play a role in joints destructions in AS;② The mechanism of high OC production is likely to be due to high RANKL concentration which is caused by inflammatory reaction.     

强直性脊柱炎外周血破骨细胞前体细胞活性的研究

Objective To investigate the number of osteoclast (OC) precursor in the peripheral blood of patients with ankylosing spondylitis (AS) and its relationship with serum receptor activator of nuclear factor KB-ligand (RANKL) and Osteoprotegerin (OPG) concentration as well as the disease activity. Methods The peripheral blood mononuclear cells from 8 cases of AS patients and 5 healthy controls were cultured in the medium containing macrophage colony-stimulating factor (M-CSF) (25 ng/ml) and RANKL (40 ng/ml). After being cultured for 14 days, cytochemistry was applied to detect tartrate-resistant acid phosphatase (TRAP) expression and the cells with TRAP expression and ≥3 nuclei were counted and defined as OC. Bone resorption assay was used to demonstrate OC function. ELISA was used to measure serum RANKL and OPG concentration in 23 cases of AS and 17 healthy controls. The relationship was analyzed in AS patients between the number of OC precursors and serum RANKL and OPG concentration as well as the disease activity. The indicators of disease activity were Bath ankylosing spondylitis disease activity index (BASDAI), Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). T test, t' test and Spearman correlation were selec-ted. Results ① Significantly higher OC production was observed in the peripheral blood of AS patients than that of healthy control group. The OC number per ten fields was 10.9±3.4 and 6.2±1.3 respectively (P<0.05); ② There was significant difference between AS patients and healthy controls in serum concentration of OPG and RANKL and the ratio of RANKL/OPG. OPG was significantly higher in AS patients [(157±49) pg/ml] than in healthy controls [(105±20) pg/ml] (P<0.05). RANKL was significantly higher in AS patients [(5.4± 3.8) pg/ml] than in healthy controls [(1.6±0.8) pg/ml] (P<0.05). The ratio of RANKL/OPG was significantly higher in AS patients (0.037±0.026) than in healthy controls (0.016±0.008) (P<0.01 );③Significantly positive correlation was observed between the OC number and the serum concentration of RANKL (r=0.692, P=0.009), the ratio of RANKL/OPG (r=0.813, P=0.001);④ In AS patients, serum concentration of OPG was found to have significantly negative correlation with BASDAI (r=-0.444, P=0.044). Serum RANKL concentration was found to have significantly positive correlation with BASDAI (r=0.543, P=0.011). The ratio of RANKL/OPG was found to have significantly positive correlation with BASDAI (r=0.672, P=0.001). Conclusion ① More OC precursors exist in the peripheral blood of AS patients. These cells may differentiate into osteoclasts, which might play a role in joints destructions in AS;② The mechanism of high OC production is likely to be due to high RANKL concentration which is caused by inflammatory reaction.     

肾素血管紧张素拮抗剂对血管紧张素Ⅱ诱导的基质金属蛋白酶高表达的抑制作用

目的 研究血管紧张素转换酶抑制剂卡托普利和血管紧张素Ⅱ(AngⅡ)受体拮抗剂氯沙坦对AngⅡ诱导的血管平滑肌细胞基质金属蛋白酶-1(MMP-1)和MMP-9 mRNA表达的干预作用.方法 体外原代培养雄性Wistar大鼠胸主动脉血管平滑肌细胞,分为对照组、AngⅡ组、卡托普利组、氯沙坦组和卡托普利与氯沙坦联合应用组.收集各组培养终点细胞,以反转录聚合酶链反应(RT-PCR)检测所收集标本中MMP-1和MMP-9 mRNA的表达,观察不同浓度AngⅡ和作用时间对血管平滑肌细胞MMP-1、MMP-9 mRNA表达的影响及卡托普利、氯沙坦的干预作用.结果 MMP-1 mRNA表达随AngⅡ浓度增加及作用时间延长而增加(P＜0.05),AngⅡ浓度为10-6mol/L时最为显著(P＜0.01).一定浓度的卡托普利(5×10-6mol/L)和氯沙坦(5×10-6mmol/L)可抑制AngⅡ的作用(P＜0.05,P＜0.01);AngⅡ为10-7、10-6、10-5、10-4mol/L时,MMP-9 mRNA表达量分别为0.47±0.03、0.86±0.04、0.94±0.14和1.12±0.19,与对照组0.10±0.04比较,差异有统计学意义(P＜0.05或P＜0.01).卡托普利和氯沙坦可抑制AngⅡ对血管平滑肌细胞分泌的MMP-9 mRNA的促进表达作用,以卡托普利和氯沙坦联合应用时抑制作用最强;MMP-9 mRNA的表达随AngⅡ作用时间延长而增加;MMP-9 mRNA较MMP-1表达早.结论 AngⅡ可以诱导血管平滑肌细胞分泌的MMP-1和MMP-9 mRNA高表达,且存在剂量和时间依赖性.卡托普利、氯沙坦可抑制AngⅡ诱导的血管平滑肌细胞MMP-1和MMP-9 mRNA高表达;卡托普利和氯沙坦联合应用时抑制作用最强;这种抑制作用与作用时间相关.

Abstract：

Objective To investigate the effect of angiotensin converting enzyme inhibitor (ACEI) captopril and angiotensin Ⅱ receptor antagonist losartan on the mRNA expression of matrix metalloproteinase (MMP)-1 and MMP-9 in vascular smooth muscle cells induced by angiotensin Ⅱ (Ang Ⅱ ).Methods Male Wistar rats' thoracic aortic vascular smooth muscle cells were cultured in vitro.The cultured cells were divided in to control group,Ang Ⅱ group,captopril group,losartan group,and captopril plus losartan group.Cells in all groups were collected at the culture end-point.MMP-1 and MMP-9 mRNA expressions were detected by RT-PCR method in the collected specimens,and the effects of Ang Ⅱ on MMP-1 and MMP-9 mRNA expression and the intervention effects of captopril and losartan were observed in different Ang Ⅱ concentrations and different action times to vascular smooth muscle cells.Results ( 1 ) MMP-1 mRNA expression gradually increased along with the increments of Ang Ⅱ concentration and the action time (P＜0.05),and the most significant concentration was 10-6 mol/L (P＜0.01).(2)Captopril (5 × 10-6 mol/L) and losartan (5 × 10-6mol/L) inhibited the action of AngⅡ (P＜0.05,P＜0.01).MMP-9 mRNA expression was 0.47±0.03 ,0.86 ± 0.04,0.94±0.14 and 1.12±0.19 vs.0.10±0.04 (P＜0.05,P＜0.01) respectively when Ang Ⅱ concentration was 10-7 ,10-6 ,10-5 and 10-4 mol/L respectively.Captopril (5 × 10-6mol/L) and losartan (5 × 10-6 mol/I) significantly inhibited the MMP-9 mRNA expression which was stimulated by Ang Ⅱ (P＜0.05,P＜0.01),especially in captopril plus losartan group.The MMP-9 mRNA expression increased with the prolonging of stimulating time of Ang Ⅱ,MMP-9 mRNA expression was earlier than that of MMP-1.Conclusions AngⅡ increases the expression of MMP-1 and MMP-9 of vascular smooth muscle cells in a dose-and time-dependence manner.Captopril and losartan inhibit the MMP-1 and MMP-9 mRNA expression of vascular smooth muscle cells induced by Ang Ⅱ ,and the inhibition is the strongest when losartan was combined with captopril.The inhibitive effects is positively correlated to action time.     

蛇床子素骨靶向药物对体外培养大鼠成骨细胞护骨素、核因子-κB受体活化因子配基表达的影响

目的观察蛇床子素骨靶向药物(NSC-OST)对体外培养的大鼠成骨细胞(OB)护骨素(OPG)及核因子κB受体活化因子配基(RANKL)表达的影响。方法不同浓度的NSC-OST作用于成骨细胞7 d,采用ELISA法检测成骨细胞OPG、RANKL的表达。结果终浓度为10-6mmol/L和10-5mmol/L组促进成骨细胞OPG的表达作用高于空白对照组(P<0.01);10-7、10-6mmol/L和10-5mmol/L组具有抑制大鼠成骨细胞表达RANKL的作用(P<0.01);终浓度为10-6、10-5mmol/L的NSC-OST可显著上调OPG/RANKL的比值(P<0.01)。结论适当浓度的NSC-OST可促进成骨细胞OPG的表达,抑制RANKL分泌,上调OPG/RANKL的比值。     

罗格列酮对经诱导的骨髓间充质干细胞ALP、BMP-2及TGF-β1分泌的影响

目的 体外观察罗格列酮对骨髓间充质干细胞(BMSCs)向成骨细胞分化的影响,并观察对碱性磷酸酶(ALP)、骨形成蛋白-2(BMP-2)、转化生长因子β1(TGF-β1)分泌的影响,探讨其对骨代谢的作用机制.方法 无菌条件下从大鼠长骨骨髓中分离获取BMSCs,采用全骨髓贴壁培养法对BMSCs进行纯化、传代扩增,随后在1、2、5、10 μmol/L罗格列酮干预下诱导成骨细胞分化培养21 d,进行茜素红染色观察矿化结节,并测定成骨细胞标记物ALP、BMP-2及TGF-β1的分泌.结果 1、2、5、10 μmol/L罗格列酮干预组与成骨经典组对比,成骨细胞的钙结节形成比例显著降低(P＜0.05),ALP、BMP-2、TGF-β1水平呈剂量依赖性地下降(均P＜0.05).结论 罗格列酮可剂量依赖性地抑制BMSCs向成骨细胞分化,这可能是罗格列酮致骨质疏松的重要机制.

Abstract：

Objective To observe the effects of rosiglitazone on differentiation of rat bone-marrow stromal cells (BMSCs) into osteoblasts (OB) and on secretion of alkaline phosphatase (ALP), bone morphogenetic protein2 (BMP-2), and transforming growth factor-β1 (TGF-β1) in order to investigate its mechanism of the impact on the bone metabolism.Methods BMSCs from long bone were bred by using differential time adherent culture method,and then were interfered with 1,2,5,10 μmol/L rosiglitazone to differentiate into osteoblasts in the presence of an osteogenic medium.The rate of mineralization was examined by staining mineralized nodules with Alizarin red S,and the secretion of ALP, BMP-2, and TGF-β1 was examined by enzyme linked immunosorbent assay (ELISA)after 21 d of culture.Results Compared with the classic group, the rate of mineralization was significantly decreased by 1,2,5 and 10 μmoL/L rosiglitazone (P＜0.05), the levels of ALP, BMP-2, and TGF-β1 decreased in a dose-depedent manner (P＜0.05).Conclusion Rosiglitazone dose-dependently inhibits differentiation of BMSCs into osteoblasts, which may be an important mechanism in causing osteoporosis.     

铁离子对人成骨细胞RANKL/OPG基因及蛋白表达的影响

目的研究人成骨细胞(hFOB1.19)在不同铁离子状态下RANKL/OPG基因及蛋白的表达。方法成骨细胞株(hFOB1.19)在DMEM/F-12培养基培养传代至第3代后,用不同终浓度枸橼酸铁铵(50、100、200μmol/L)加入细胞培养基中干预24h,用RT-PCR方法和免疫印迹法(WesternBlot)检测干预后成骨细胞的RANKL、OPGmRNA和蛋白表达并计算RANKL/OPG比率。结果RT-PCR检测结果显示,对照组、50、100、200μmol/L组RANKL/OPGmRNA表达比分别为0.56±0.13、0.58±0.01、0.69±0.01、1.84±0.92;Westernblot结果显示,对照组、50、100、200μmol/L组RANKL/OPG蛋白表达比分别为0.82±0.66、0.82±0.64、1.09±0.11、1.25±0.14。统计学分析显示,在mRNA和蛋白水平,100和200μmol/L浓度的枸橼酸铁铵干预后RANKL/OPG比值明显高于对照组(P0.05),50μmol/L枸橼酸铁干预后与对照组差异无统计学意义。结论不同浓度枸橼酸铁铵可以影响人成骨细胞RANKL/OPG基因及蛋白的表达,进而可能影响骨形成和骨吸收。