慢性氟中毒大鼠软骨的损害及COLIXA3蛋白、表达的实验研究

目的 探讨不同程度的氟中毒是否影响染氟大鼠软骨组织中COLIXA3蛋白的表达.方法 选用3～4周龄健康雄性Wistar大鼠40只,按体质量随机分为5组,每组8只,分笼饲养,期间分别自由饮用含氟化钠0(对照)、25、50、100、150mg/L的蒸馏水,饲喂6月后建立氟中毒大鼠模型.应用光学显微镜分析实验大鼠骨组织的病理形态学变化过程.并采用免疫组织化学技术检测大鼠股骨干骺端COLIXA3蛋白的表达情况.结果 大鼠骨组织HE染色显示,各染氟组股骨干骺端出现不同程度软骨骨化,骨密度增加,具有硬化性氟骨症病变.对照组软骨组织未见明显异常.大鼠软骨细胞COLIXA3免疫组织化学染色结果为阳性,胞浆内可见有棕黄色颗粒,25、50、100 mg/L组在软骨组织中COLIXA3蛋白表达(23.3±4.5、41.2±5.6、26.4±7.5)增强.其中50、100 mg/L组表达与对照组(6.1±3.5)相比明显增加,组间比较差异有统计学意义(P均<0.05).150 mg/L组COLIXA3蛋白(13.3±4.2)较前面3组表达减弱,仍高于对照组,组问比较差异无统计学意义(P>0.05).结论 动物模型中,大鼠病理学为单纯性骨硬化表现.低剂量氟促进,高剂量抑制大鼠软骨细胞的增生.随着染氟时间的延长,外环境中氟浓度过高时,对软骨细胞就表现为氟离子的直接毒性作用.氟化物影响染氟大鼠软骨组织中COLIXA3蛋白的表达,低剂量氟可以促进COLIXA3蛋白的表达,随剂量增加氟的促进作用减弱.

Abstract：

Objective To explore whether different degrees of fluorosis influence the expression of cartilage COLIXA3 protein in fluorosis model rats. Methods Forty male Wistar rats 3 to 4 weeks old were randomly divided into 5 groups according to body mass, and these rats were fed with distilled water containing sodium fluoride(NaF) of 0(control), 25, 50, 100 and 150 mg/L for 6 months, respectively, in order to establish the animal model of drinking water type fluorosis. Pathomorphologieal changes of the osseous tissues of rats were analyzed under light microscope and transmission electron microscope, and the expression of COLIXA3 protein of femur metaphysis was examined by immunohistochemistry. Results HE staining showed different degrees of femoral metaphyseal ossification of cartilage in each experimental group, bone density increased, with sclerotic lesions of skeletal fluorosis. The control group showed no abnormal cartilage. Electron microscopy showed that the experimental groups with varying degrees of cartilage cell swelling, cell matrix fades, 50 mg/L group .showed hyperplasia, and 100,150 mg/L groups were observed with organelles decreased, part of the disintegration of the cartilage cell lacunae, lmmunohistochemical staining of rat chondrocytes COLIXA3 was positive, cytoplasm with brown granules, cartilage COLIXA3 protein expression(23.3 ± 4.5, 41.2 ± 5.6, 26.4 ~ 7.5) in the 25, 50 and 100 mg/L groups enhanced. Compared to the control group (6.1 ± 3.5), the expression of 50 and 100 mg/L groups was significantly increased, and the differences were statistically significant(all P < 0.05). The expression(13.3 ± 4.2)of COLIXA3 protein in 150 mg/L group was decreased compared with the previous three, but is still higher than that of control, and the difference was not statistically significant(P > 0.05). Conclusions There has pathological changes of sclerosing skeletal fluorosis in animal model. Low-dose fluoride promotes while high-dose inhibits cartilage cell proliferation. When fluorine concentration in external environment is too high and with extended exposure to fluoride, direct toxic effects of fluoride on cartilage cells is observed. Fluorine affects and promotes the expression of COLIXA3 protein in cartilage. Low-dose fluoride can promote COLIXA3 protein expression, as the dose increases (over 100 mg/L), the effect decreases.     

Effects of fluoride exposure on microRNA-200c expression and its target in human osteoblast Saos-2 cells

正Objective To investigate the effect of fluoride exposure on expression of mi RNA (miR)-200c and its target in human osteoblast Saos-2 cells.Methods Saos-2 cells were cultured in DMEM/F-12 medium and treated with fluoride (sodium fluoride, NaF). There were two groups including:control group (0 mg/L) and fluoride group(4 mg/L). Cells were harvested after 48 hours of culture with fluoride. The expression of mi R-200c, the m RNA of alkaline phosphatase (ALP), osteocalcin (BGP), the target phosphatase and tensin homolog deleted on chro-     

慢性氟中毒大鼠脑组织中c-Jun氨基末端激酶表达水平观察

Objective To investigate the expression of c-Jun-N-terminal kinase(JNK) in rat brains with chronic fluorosis and try to reveal the molecular mechanism for the neural impairment induced by the disease.Methods The rats were randomly divided into 3 groups, normal control group(drinking water containing less than 0.5 mg/L of sodium fluoride, NaF), lower fluoride exposed group(drinking water containing 5 mg/L NaF) and higher fluoride exposed group(drinking water containing 50 mg/L NaF), 24 in every group. The rats were examined at the sixth month after feeding. The concentration of fluorine in urine and blood was detected by F-ion selective electrode. The expression of JNK in brains was investigated by using Western blotting and immunohitochemistry staining, and analyze the correlation between activating of JNK and the concentration of fluorine in blood. Results The increased concentration of fluorine in urine(control: 0.92 ± 0.30, lower fluoride exposed group: 2.56 ± 0.91,higher fluoride exposed group: 5.73 ± 3.14, P ＜ 0.05) were observed when 6 months after the beginning of the experiment, and the amount of fluorine in blood was also higher in rats with fluorosis(control: 0.12 ± 0.07, lower fluoride exposed group: 0.36 ± 0.14, higher fluoride exposed group: 0.50 ± 0.18, P ＜ 0.05). The expression of phospho-JNK at protein levels were higher in the brains of rats with fluorosis than that of controls (control: 1.00 ± 0.37, lower fluoride exposed group: 1.20 ± 0.28, higher fluoride exposed group: 1.74 ± 0.69, P ＜ 0.05), whereas no change of total-JNK was found(F = 0.046, P ＞ 0.05). Furthermore, the expression of phospho-JNK in the parietal cortex(119.3 ± 14.1), occipital cortex(112.7 ± 5.4), hippocampus CA3(100.6 ± 8.9), dorsal thalamus (117.8 ± 10.4) and olivary nucleus( 112.6 ± 5.9) of rats in higher fluoride exposed group were higher than that in control( 104.1 ± 8.9,106.6 ± 9.6,106.6 ± 9.7,108.9 ± 6.4,100.3 ± 8.4, all P ＜ 0.05) and lower fluoride exposed group(96.7 ± 17.1,102.5 ± 8.3,106.4 ± 6.5,110.2 ± 9.3,102.4 ± 4.7,102.5 ± 9.8, all P＜ 0.05). The positive stained neurons of total-JNK also distributed in the same brain regions of rats, but no difference was detected between the rats with fluorosis and controls(all P ＞ 0.05). The increased level of phospho-JNK was positively correlated with the fluoride contents in blood of the rats with fluorosis (r = 0.677). Conclusions The expression of phospho-JNK in brains of rats with fluorosis was significantly increased with a correlation to fluoride content in blood, which might be connected to the mechanism of neural impairment induced by chronic fluorosis.     

Correlation between expression of gastrin, somatostatin and cell apoptosis regulation gene bcl-2／bax in large intestine carcinoma

AIM: To explore the correlation between expression of somatostatin (SS), gastrin (GAS) and cell apoptosis regulation gene bcl-2/bax in large intestine carcinoma. METHODS: Sixty-two large intestine cancer tissue samples were randomly and retrospectively selected from patients with large intestine carcinoma. Immunohistochemical staining for bcl-2, bax, GAS, SS was performed according to the standard streptavidin-biotin-peroxidase (S-P) method. According to the semi-quantitative integral evaluation, SS and GAS were divided into three groups as follows. Scores 1-3 were defined as the low expression group, 4-8 as the intermediate expression group, 9-16 as the high expression group. Bax and bcl-2 protein expressions in different GAS and SS expression groups of large intestine carcinoma were assessed. RESULTS: The positive expression rate of bax had a prominent difference between SS and GAS high, intermediate and low expression groups (P<0.05, x2SS = 9.246; P<0.05, x2GAs = 6.981). The positive expression rate of bax in SS high (80.0%, 8/10) and intermediate (76.5%, 13/17) expression groups was higher than that in low expression group (40.0%, 14/35) (P<0.05, X2high vslow = 5.242; P<0.05, x2middle vs low = 6.097). The positive expression rate of bax in GAS high expression group (27.3%, 3/8) was lower than that in low expression group (69.4%, 25/36) (P<0.05, x2 = 4.594). However, bax expression in GAS intermediate expression group (46.7%, 7/15) was lower than that in low expression group, but not statistically significant. The positive expression rate of bcl-2 had a prominent difference between SS and GAS high, intermediate and low expression groups (P<0.05, x2ss = 7.178; P<0.05, x2GAS = 13.831). The positive expression rate of bcl-2 in GAS high (90.9%, 10/11) and intermediate (86.7%, 13/15) expression groups was higher than that in low expression group (44.4%, 16/36) (P<0.05, x2high,vslow = 5.600; P<0.05, x2middle vs low = 7.695). However, the positive expression rate of bcl-2 in SS high (40. 0%, 4/10) and intermediate (47.1%, 8/9) expression groups was lower than that in low expression group (77.1%, 27/35) (P<0.05, x2high vs low = 4.710; P<0.05, x2middle vs low = 4.706). There was a significant positive correlation between the integral ratio of GAS to SS and the integral of bcl-2 (P<0.01, r = 0.340). However, there was a negative correlation between the integral ratio of GAS to the SS and bax the integral of (P<0.05, r= -0.299). CONCLUSION: The regulation and control of gastrin, somatostatin in cell apoptosis of large intestine carcinoma may be directly related to the abnormal expression of bcl-2, bax.     

过量氟暴露对成骨细胞微小染色体维系蛋白3及成骨相关基因表达的影响

Objective To study the effects of fluoride on minichromosone maintenance(MCM)3 mRNA and the bone formation-related gene:bone sialoprotein(BSP),osteocalcin(OC),osteopontin(OP)mRNA expression on human osteoblast cells.The expression of MCM3 was tested for diagnosis and surveillance value on osteoblast treated with excess fluoride.Methods Human osteoblast cell(Saos-2)was cultured in McCoy5A medium and treated with fluoride(sodium fluoride,NaF).There were eight groups including:0(control),0.625,1.250,2.500,5.000,10.000,20.000,40.000 mg/L groups.Expression of MCM3,BSP,OC,OP mRNA were detected by real-time PCR.Dual-standard curve method was used for analysis.ALPase was determined by measuring the absorbance using a micro titer plate reader. Results Expression of MCM3 mRNA was lower in the 0.625,1.250,2.500,5.000,20.000, 40.000 mg/L groups(0.059 ± 0.003,0.027 ± 0.001,0.272 ± 0.004,0.115 ± 0.002,0.137 ± 0.004,0.754 ±0.002, all P > 0.05) and was higher in10.000 mg/L group(21.300 ± 1.200, P < 0.01 ) than control group( 1.000 ±0.020), especially 10.000 mg/L group was higher than groups treated with fluoride(all P < 0.01 ), the differences among groups were significant(F = 305.842, P < 0.01 ). Expression of BSP mRNA was significantly higher in 0.625,1.250,2.500,5.000,10.000 mg/L groups(71.80 ± 3.60,133.00 ± 7.20,85.50 ± 0.60,80.90 ± 1.20,304.00 ± 21.00)than the control group( 1.00 ± 0.04), especially 10.000 mg/L group was higher than others groups treated with fluoride(all P < 0.01 ), the differences among groups were signifieant(F = 159.531, P < 0.01 ). Expressions of OC mRNA were higher in 0.625,1.250,2.500,5.000 mg/L groups(110.00 ± 12.00,143.00 ± 2.10,90.60 ± 4.10,23.70±1.20) than control group(1.00 ± 0.01, all P < 0.01), and the differences among groups were significant (F = 158.734, P < 0.01 ). Expression of OP mRNA were higher in 0.625,1.250,2.500,5.000,10.000,20.000 mg/L groups(167.00 ± 11.20, 111.00 ± 12.10,72.50 ± 3.50,134.00 ± 14.00,42.30 ± 2.40,45.20 ± 3.30) than the control group(1.00 ± 0.04, all P < 0.05 or < 0.01 ), the differences among groups were significant(F = 60.226, P < 0.01 ).Compared with control group(4.2 ± 1.2), the ALPase activity was increased in all groups treated with fluoride (6.0 ± 0.4,5.8 ± 0.1,5.7 ± 0.4,7.7 ± 1.1,19.2 ± 2.4,8.5 ± 3.0,18.1 ± 4.2), but only 10.000 mg/L and 40.000 mg/L groups were higher than control group and other groups treated with fluoride(all P < 0.01 ), the differences among groups were signifieant(F = 7.806, P < 0.01 ). Conclusions Irregular expression of MCM3 mRNA is not suitable as a diagnostic and monitoring biomarker of osteoblasts exposed to excessive fluoride. Fluoride may affect the osteoblast-related gene expression and to promote osteogenic differentiation.     

过量氟暴露对成骨细胞微小染色体维系蛋白3及成骨相关基因表达的影响

Objective To study the effects of fluoride on minichromosone maintenance(MCM)3 mRNA and the bone formation-related gene:bone sialoprotein(BSP),osteocalcin(OC),osteopontin(OP)mRNA expression on human osteoblast cells.The expression of MCM3 was tested for diagnosis and surveillance value on osteoblast treated with excess fluoride.Methods Human osteoblast cell(Saos-2)was cultured in McCoy5A medium and treated with fluoride(sodium fluoride,NaF).There were eight groups including:0(control),0.625,1.250,2.500,5.000,10.000,20.000,40.000 mg/L groups.Expression of MCM3,BSP,OC,OP mRNA were detected by real-time PCR.Dual-standard curve method was used for analysis.ALPase was determined by measuring the absorbance using a micro titer plate reader. Results Expression of MCM3 mRNA was lower in the 0.625,1.250,2.500,5.000,20.000, 40.000 mg/L groups(0.059 ± 0.003,0.027 ± 0.001,0.272 ± 0.004,0.115 ± 0.002,0.137 ± 0.004,0.754 ±0.002, all P > 0.05) and was higher in10.000 mg/L group(21.300 ± 1.200, P < 0.01 ) than control group( 1.000 ±0.020), especially 10.000 mg/L group was higher than groups treated with fluoride(all P < 0.01 ), the differences among groups were significant(F = 305.842, P < 0.01 ). Expression of BSP mRNA was significantly higher in 0.625,1.250,2.500,5.000,10.000 mg/L groups(71.80 ± 3.60,133.00 ± 7.20,85.50 ± 0.60,80.90 ± 1.20,304.00 ± 21.00)than the control group( 1.00 ± 0.04), especially 10.000 mg/L group was higher than others groups treated with fluoride(all P < 0.01 ), the differences among groups were signifieant(F = 159.531, P < 0.01 ). Expressions of OC mRNA were higher in 0.625,1.250,2.500,5.000 mg/L groups(110.00 ± 12.00,143.00 ± 2.10,90.60 ± 4.10,23.70±1.20) than control group(1.00 ± 0.01, all P < 0.01), and the differences among groups were significant (F = 158.734, P < 0.01 ). Expression of OP mRNA were higher in 0.625,1.250,2.500,5.000,10.000,20.000 mg/L groups(167.00 ± 11.20, 111.00 ± 12.10,72.50 ± 3.50,134.00 ± 14.00,42.30 ± 2.40,45.20 ± 3.30) than the control group(1.00 ± 0.04, all P < 0.05 or < 0.01 ), the differences among groups were significant(F = 60.226, P < 0.01 ).Compared with control group(4.2 ± 1.2), the ALPase activity was increased in all groups treated with fluoride (6.0 ± 0.4,5.8 ± 0.1,5.7 ± 0.4,7.7 ± 1.1,19.2 ± 2.4,8.5 ± 3.0,18.1 ± 4.2), but only 10.000 mg/L and 40.000 mg/L groups were higher than control group and other groups treated with fluoride(all P < 0.01 ), the differences among groups were signifieant(F = 7.806, P < 0.01 ). Conclusions Irregular expression of MCM3 mRNA is not suitable as a diagnostic and monitoring biomarker of osteoblasts exposed to excessive fluoride. Fluoride may affect the osteoblast-related gene expression and to promote osteogenic differentiation.     

Correlation between expression of peroxisome proliferator-activated receptor γ and oxidative stress in brains of rats with chronic fluorosis

正Objective To detect the expression of peroxisome proliferator-activated receptorγ(PPARγ) in the brains of rats with chronic fluorosis and elucidate the relationship between PPARγand oxidative stress in chronic fluorosis.Methods According to body weight (100-120 g),sixty healthy SD rats were divided into control group(less than 0. 5 mg/L fluoride in drinking water), low fluoride group (5. 0 mg/L fluoride in drinking water,prepared by NaF), and high fluoride group (50. 0 mg/L fluoride in drinking water) via the random number table     

DNA damage, apoptosis and cell cycle changes induced by fluoride in rat oral mucosal cells and hepatocytes

AIM:To study the effect of fluoride on oxidative stress,DNA damage and apoptosis as well as cell cycle of ratoral mucosal cells and hepatocytes.METHODS:Ten male SD rats weighing 80～120 g wererandomly divided into control group and fluoride group,5 animals each group.The animals in fluoride group hadfree access to deionized water containing 150 mg/L so-dium fluoride(NaF).The animals in control group weregiven distilled water.Four weeks later,the animals werekilled.Reactive oxygen species(ROS)in oral mucosa andliver were measured by Fenton reaction,lipid peroxida-tion product,malondialdehyde(MDA),was detected bythiobarbituric acid(TBA)reaction,reduced glutathione(GSH)was assayed by dithionitrobenzoic acid(DTNB)reaction.DNA damage in oral mucosal cells and hepa-tocytes was determined by single cell gel(SCG)electro-phoresis or comet assay.Apoptosis and cell cycle in oralmucosal cells and hepatocytes were detected by flowcytometry.RESULTS:The contents of ROS and MDA in oral mucosaand liver tissue of fluoride group were significantly high-er than those of control group(P<0.01),but the level ofGSH was markedly decreased(P<0.01).The contents ofROS,MDA and GSH were(134.73±12.63) U/mg protein,(1.48±0.13)mmol/mg protein and(76.38±6.71)mmol/mg protein in oral mucosa respectively,and(143.45±11.76)U/mg protein,(1.444-0.12)mmol/mg protein and(78.83±7.72)mmol/mg protein in liver tissue respective-ly.The DNA damage rate in fluoride group was 50.20%in oral mucosal cells and 44.80% in hepatocytes,higherthan those in the control group(P<0.01).The apop-tosis rate in oral mucosal cells was(13.63±1.81)% influoride group,and(12.76±1.67)% in hepatocytes,higher than those in control group.Excess fluoride coulddifferently lower the number of oral mucosal cells andhepatocytes at G_0/G_1 and S G_2/M phases(P<0.05).CONCLUSION:Excess fluoride can induce oxidative stress and DNA damage and lead to apoptosis and cellcycle change in rat oral mucosal cells and hepatocytes.     

Effects of ivabradine on cardiomyocyte apoptosis after rabbit acute myocardial infarction

Epidemiological studies have confirmed that high heart rate means high risk to patients with coronary heart disease.The research investigates the viability and the effects of ivabradine versus atenolol in early phases of acute myocardial infarction(AMI) on rabbits after 28 days of follow-up.The ratio of bcl-2 protein to Bax protein determines survival or death after an apoptotic stimulus.We forecast that bcl-2 or Bax expression places a premium on ischemia and that it may be linked to myocyte death in human hearts.Methods Forty-three New Zealand white rabbits(male or female) were used to build AMI mode through ligating left anterior descending coronary artery.Survived rabbis were randomly divided into four groups:group S,group M,group A and group I.Drugs were provided 12 hours post myocardial infarction induction.The myocardium in ischemic necrosis tissue was sampled 28 days post dose.AI and bcl-2 /bax protein expression were detected.The heart rates of rabbits before operation and 28 days after operation were recorded by electrocardiography and analyzed.Results On 28th day post-operation,the heart rate of rabbits in groups A and I significantly became slower compared with that in group M(P < 0.01).The proportion of myocardial cell apoptosis in groups I and A was significantly lower than that in group M and higher than that in group S(P < 0.01) on 28th day post-operation On 28th day post-operation,compared with group M,the level of Bcl2 protein in rabbits of groups I and A significantly increased(P < 0.01),the level of Bax protein significantly decreased(P < 0.01),and No statistical difference was found between group I and group A.Conclusion reating myocardial infarction rabbits with ivabradine for 28 days could effectively reduce the incidence of myocardial cell apoptosis and increase bcl-2 /bax ratio.     

氟对体外培养乳鼠软骨细胞增殖的影响

目的 探讨氟对体外培养乳鼠软骨细胞增殖的影响.方法 采用细胞培养的方法,原代培养昆明乳鼠软骨细胞,传第3代后按染氟剂量不同分为O(对照)、5、10、20、40 ms/L组,10 d后光、电镜观察软骨细胞形态学变化;采用生长曲线、噻唑蓝(MTT)方法,在染氟24、48、72 h测定细胞数量变化及细胞增殖率.结果 染氟10 d后,镜下0、5、10mg/L组软骨细胞表现为增殖,细胞生长旺盛,均可见部分细胞核中核仁数增加:40mg/L组部分软骨细胞中有染色质固缩或凝结成块状,可见到凋亡细胞.在染氟24 h,细胞增殖活力各染氟组组间比较差异无统计学意义(F=2.313,P＞0.05).在染氟48、72 h,0 mg/L组[(23.5±4.6)%、(29.9±1.7)%]、5mg/L组[(34.6±4.7)%、(45.3±5.9)%]、10mg/L组[(39.9±4.8)%、(56.8±5.5)%]、20mg/L组[(31.8±4.1)%、(38.3±6.5)%]、40 mg/L组[(28.3±4.3)%、(33.4±4.8)%]组问比较差异有统计学意义(F值分别为11.401、25.671,P＜0.05);各染氟组细胞增殖活力与对照组比较差异均有统计学意义(P＜0.05),其中5、10ms/L组明显高于40mg/L组(P＜0.05).结论 低剂量氟在较短作用时间内可以促进体外培养小鼠软骨细胞的增殖,剂量升高时表现为抑制.     

T-2毒素和硒对人软骨蛋白聚糖酶的影响

目的 研究T-2毒素和硒对人软骨蛋白聚糖代谢的影响.方法 体外培养胎儿软骨细胞,根据施加的实验措施将其分为8组,对照组(T-2毒素:0 mg/L,硒:0 mg/L)、1、10、20μg/L T-2毒素组、加硒(0.1mg/L)组、1、10、20 μg/L T-2毒素加硒(0.1 mg/L)组,每组设3个平行样.在T-2毒素和(或)硒作用5d后,采用免疫蛋白印迹法检测软骨细胞蛋白聚糖的蛋白表达水平,用RT-PCR方法检测软骨细胞蛋白聚糖酶-1和蛋白聚糖酶-2的mRNA表达水平.结果 硒、T-2毒素对蛋白聚糖的蛋白表达量、蛋白聚糖酶-1 mRNA表达水平、蛋白聚糖酶-2的mRNA表达水平有影响(F=0.294、27.71,107.45、362.25,34.68、22.26,P均＜0.05),硒与T-2毒素存在交互作用(F=79.99、230.76、388.33,P均＜0.05),表现为拮抗作用.在硒水平为0 mg/L时,1、10、20 μg/L的T-2毒素组蛋白聚糖的蛋白表达(0.278±0.015、0.235±0.029、0.195±0.028)均低于0 mg/L的T-2毒素组(0.472±0.0358,P均＜0.05);在硒水平为0.1 mg/L时,1、10、20 μg/L的T-2毒素组的蛋白聚糖的蛋白表达(0.399±0.028、0.299±0.020、0.263±0.019)均高于0 mg/L的T-2毒素组(0.197±0.018,P均＜0.05).在T-2毒素分别为1、10、20μg/L时,0.1 mg/L硒组的蛋白聚糖的蛋白表达均高于0 mg/L硒组(P均＜ 0.05).在硒水平为0 mg/L时,1、10、20μg/L的T-2毒素组蛋白聚糖酶-1 mRNA表达(0.535±0.033、1.071±0.043、1.454±0.058)均高于0 mg/L的T-2毒素组(0.481±0.023,P均＜0.05);在硒水平为0.1 mg/L时,1、10、20μg/L的T-2毒素组的蛋白聚糖酶-1 mRNA表达(1.057±0.048、0.555±0.036、0.902±0.045)均高于0 mg/L的T-2毒素组(0.346±0.020,P均＜0.05).在硒水平为0 mg/L时,1、10、20μg/L的T-2毒素组蛋白聚糖酶-2 mRNA表达(0.596±0.038、0.656±0.033、0.949±0.049)均高于0 mg/L的T-2毒素组(0.387±0.020,P均＜0.05);在硒水平为0.1 mg/L时,1、10、20μg/L的T-2毒素组的蛋白聚糖酶-2 mRNA表达(0.600±0.040、0.453±0.031、0.164±0.011)均低于0 mg/L的T-2毒素组(1.021±0.046,P均＜0.05);在T-2毒素分别为10、20μg/L时,0.1 mg/L硒组的蛋白聚糖酶-1和蛋白聚糖酶-2 mRNA表达均低于0 mg/L硒组(P均＜ 0.05).结论 T-2毒素通过上调蛋白聚糖的主要代谢酶蛋白聚糖酶-1和蛋白聚糖酶-2达到降解蛋白聚糖的作用,微量元素硒对T-2毒索引起的软骨细胞蛋白聚糖降解有一定保护作用.     

硒对体外培养大骨节病软骨细胞生长及凋亡的影响

目的 观察硒对体外培养大骨节病(KBD)患者和正常人关节软骨细胞增殖和凋亡的影响,探索补硒防治KBD的作用,并为硒对正常软骨细胞生长的影响提供依据.方法 依据<大骨节病临床诊断标准>(GB 16003-1995),选择Ⅱ度和Ⅲ度KBD患者5例和非病区正常人意外事故者5例的关节软骨进行体外分离、培养.KBD组和对照组分别给予不同剂量的硒(0、0.0125、0.0250、0.0500、0.1000、0.2500、0.5000、1.0000 mg/L)进行干预,采用四氮唑蓝(MTT)法、流式细胞仪和免疫组化法观察细胞生长和凋亡情况.结果 对照组第6天时各剂量组的细胞增殖率(0.086±0.025、0.077±0.012、0.073±0.027、0.071±0.017、0.058±0.028、0.052±0.028和0.046±0.037)比0 mg/L组(0.138±0.026)明显降低(P均＜0.05);0.1000～1.0000 mg/L剂量组的平均细胞增殖率为负值(-0.001±0.001、-0.003±0.000、-0.003±0.001和-0.004±0.001),显著低于0 mg/L组(0.025±0.003,P均＜0.05);KBD组,与0 mg/L组(0.115±0.011)比较,0.2500 mg/L剂量组促进细胞增殖(0.128±0.037,P＜0.05),1.0000 mg/L剂量组细胞生长受到抑制(0.071±0.019,P＜0.05).对照组0.0500～1.0000mg/L剂量组的细胞凋亡率[(18.88±0.02)%、(17.58±0.01)%、(17.09±0.04)%、(56.00±0.02)%、(57.85±0.03)%]比0 mg/L组[(13.51±0.01)%]增高(P均＜0.05);KBD组,与0 mg/L组[(25.84±0.02)%]比较,0.0250～0.2500 mg/L剂量组的细胞凋亡率[(13.69±0.02)%、(15.96±0.03)%、(16.68±0.03)%、(16.67±0.02)%]降低,0.5000、1.0000 mg/L剂量组的细胞凋亡率[(59.58±0.03)%、(73.48±0.04)%]明显增高(P均＜0.05).KBD组0.0500～0.2500 mg/L剂量组的Fas表达[(41.2±1.5)%、(40.3±2.0)%、(50.2±2.5)%]低于同剂量硒干预的对照组[(52.4±1.0)%、(67.2±4.0)%、(75.1±5.0)%,P均＜0.05],0.0500、0.1000 mg/L剂量组的Caspase-3表达[(40.8±1.1)%、(45.1±2.1)%]低于同剂量硒干预的对照组[(68.0±3.0)%、(70.6±3.5)%,P均＜0.05].结论 适宜的补硒剂量(0.1000～0.2500 mg/L)具有促进KBD软骨细胞生长的作用,降低细胞凋亡率,但补硒剂量＞0.5000 mg/L时具有损伤作用;促进KBD软骨细胞生长的硒剂量并非也能促进正常人活体软骨细胞的生长.     

硒对高氟所致兔血管内皮损伤及动脉硬化影响的病理形态学研究

目的 研究硒对高氟所致兔动脉血管内皮细胞损伤和动脉硬化病理形态学变化的影响作用.方法 20只健康雄性新西兰白兔,体质量(2.0±0.5)kg,按体质量随机分对照组(饮去离子水,饲基础饲料)、加氟组(饮含氟离子100mg/L去离子水,饲基础饲料)、加硒组(饮含硒1 mg/L去离子水,饲基础饲料)、加氟加硒组(饮含氟离子100 mg/L、含硒1 mg/L去离子水,饲基础饲料),每组5只,实验期6个月.于实验第0、3、6个月取血测定血清含氟量和含硒量;实验终末取胸主动脉,观察主动脉病理及超微结构变化.结果实验第3、6个月时,加氟组和加氟加硒组血清氟[(0.589±0.146)、(0.772±0.175)mg/L和(0.502±0.094)、(0.693±0.158)mg/L]显著高于对照组[(0.174±0.002)、(0.208±0.031)mg/L,P均＜0.01];加氟组第6个月血清氟显著高于第3个月(P＜0.05).实验第3、6个月时,加硒组和加氟加硒组血清硒[(0.252±0.022)、(0.319±0.052)mg/L和(0.239±0.016)、(0.294±0.018)mg/L]显著高于对照组[(0.135±0.014)、(0.167±0.019)mg/L,P均＜0.01];加硒组第6个月血清硒显著高于第3个月(P＜0.05).对照组、加氟组、加硒组、加氟加硒组内皮细胞凋亡指数分别为(4.92±1.32)%、(30.30±6.80)%、(6.57±2.14)%和(14.29±2.99)%,氟与硒各自的主效应有统计学意义(F值分别为106.833、20.082,P均＜0.01),高氟与适硒之间存在显著的拮抗作用(F=30.402,P＜0.01).病理观察加氟组主动脉内皮有红细胞及纤维蛋白沉着,细胞走向及结构发生改变,血管受损严重;加氟加硒组减少内皮细胞凋亡,附着的纤维蛋白以及红细胞数量减少,内皮细胞结构基本正常,血管受损程度和范围明显减轻.结论适量硒抑制高氟引起的内皮细胞凋亡,减轻高氟所致主动脉结构破坏,保持内皮细胞的完整性,以此拮抗高氟对血管的损伤和促动脉粥样硬化作用.     

过量氟暴露对成骨细胞微小染色体维系蛋白3及成骨相关基因表达的影响

目的 观察氟对体外培养人成骨肉瘤细胞内微小染色体维系蛋白(minichromosone maintenance,MCM)3和成骨相关基因表达的影响,探讨MCM3基因能否作为诊断及监测成骨细胞染氟过量的生物学标志.方法 采用McCoy5A培养液,体外培养人成骨肉瘤细胞(Saos-2).按染氟(NaF)剂量将骨肉瘤细胞分为O(对照)、0.625、1.250、2.500、5.000、10.000、20.000、40.000 mg/L组,染氟培养24 h后收集细胞,实时荧光定量(real-time.RT-PCR)PCR测定成骨细胞MCM3和骨涎蛋白(bone sialoprotein,BSP)、骨钙素(osteocalcin,OC)、骨桥蛋白(osteopontin,OP)3种成骨相关基因mRNA的表达,化学比色法测定碱性磷酸酶(alkaline phosphatase,ALP)活性,用双标准曲线法计算基因表达的相对比值.结果 成骨肉瘤细胞MCM3 mRNA表达在0.625、1.250、2.500、5.000、20.000、40.000 mg/L组(0.059±0.003、0.027±0.001、0.272±0.004、0.115±0.002、0.137±0.004、0.754±0.002)低于对照组(1.000±0.020,P均>0.05),但10.000 mg/L组(21.300±1.200)显著高于对照组(P<0.01),也高于其他染氟组(P均<0.01),组间比较差异有统计学意义(F=305.842,P<0.01).BSPmRNA表达在0.625、1.250、2.500、5.000、10.000 mg/L组(71.80±3.60、133.00±7.20、85.50±0.60、80.90±1.20、304.00±21.00)显著高于对照组(1.00±0.04,P均<0.01),尤其10.000 mg/L组明显高于其他染氟组(P均<0.01),组问比较差异有统计学意义(F=159.531,P<0.01).OC mRNA表达在0.625、1.250、2.500、5.000 mg/L组(110.00±12.00、143.00±2.10、90.60±4.10、23.70±1.20)高于对照组(1.00±0.01,P均<0.01),组间比较差异有统计学意义(F=158.734,P<0.01).OP mRNA表达,在0.625、1.250、2.500、5.000、10.000、20.000 mg/L组(167.00±11.20、111.00±12.10、72.50±3.50、134.00±14.00、42.30±2.40、45.20±3.30)高于对照组(1.00±0.04,P均<0.05或<0.01),组间比较差异有统计学意义(F=60.226,P<0.01).ALP的活性虽然在0.625～40.000mg/L组(6.0±0.4、5.8±0.1、5.7±0.4、7.7±1.1、19.2±2.4、8.5±3.0、18.1±4.2)与对照组(4.2±1.2)比较是增加的,但仅10.000、40.000 mg/L组明显高于对照组和其他染氟组(P均<0.01),组间比较差异有统计学意义(F=7.806,P<0.01).结论 MCM3表达不规律,不适合作为诊断及监测成骨细胞在过量氟作用下的生物标志;氟可能通过影响各成骨相关基因的表达而促进成骨分化.     

白藜芦醇对实验性兔骨关节炎软骨细胞凋亡及bcl-2 bax表达的影响

目的 研究白藜芦醇对兔实验性骨关节炎(OA)软骨细胞凋亡及凋亡调控基因bcl-2、bax表达的影响,探讨其治疗OA的机制.方法 30只新西兰大白兔随机平分为5组,A组(健康对照组)、B组(模型对照组)、C组(白藜芦醇高剂量干预组)、D组(白藜芦醇中剂量干预组)、E组(白藜芦醇低剂量干预组).除A组外,其他各组均以Hulth法复制膝OA模型.术后第4周开始,A组、B组每天以5 ml含0.1%二甲基亚砜的蒸馏水灌胃;白藜芦醇干预各组每天以相应剂量白藜芦醇溶液(浓度为60 mg/ml)灌胃,C、D、E组日剂量分别为120、60、30mg·kg-1·d-1,连续6周.第10周处死大白兔,取右膝关节股骨内髁内侧软骨,软骨组织切片用脱氧核糖核苷酸末端转移酶介导的缺口末端标记(TUNEL)法观察软骨细胞凋亡,免疫组织化学法观察软骨细胞bcl-2和bax的表达.结果 ①模型对照组关节软骨细胞凋亡率明显高于健康对照组;白藜芦醇干预各组可不同程度地降低OA软骨细胞凋亡率,差异有统计学意义(P＜0.05),且呈剂量依赖性.②与健康对照组相比,模型对照组关节软骨细胞bcl-2和bax阳性率均升高(P＜0.01),bcl-2/bax的比值降低;白藜芦醇干预后,bcl-2阳性率升高更为明显(P＜0.01);而bax阳性率有不同程度降低(P＜0.01);bcl-2/bax的比值均不同程度提高(P＜0.01).结论 白藜芦醇通过上调bcl-2的表达,下调bax的表达,提高bcl-2/bax的比值,以抑制兔实验性OA中软骨细胞的过度凋亡,达到保护软骨,防治OA的目的 .     

氟对大鼠骨组织Runx2 mRNA及其蛋白表达的影响

目的 观察氟对大鼠骨组织Runx2 mRNA和蛋白表达水平的影响.方法 14只SD大鼠按体质量随机分为对照组[饮用自来水,含氟(F-)＜0.06 mg/L]、染氟组(饮水含F-50 mg/L),饲养4个月后股动脉放血处死.采用蛋白质印迹法(Western blot)检测Runx2蛋白表达水平;采用RT-PCR法检测Runx2 mRNA的表达水平.结果 与对照组相比,染氟组大鼠骨组织Runx2 mRNA及蛋白表达水平(2.287±0.261、0.929±0.229)均高于对照组(0.995±0.123、0.317±0.068,t值分别为11.85、6.78,P均＜0.05).结论 氟可导致大鼠骨组织Runx2 mRNA及蛋白表达水平增高,Runx2可能参与了氟引起的骨骼损伤的发生机制.     

小干扰RNA特异性沉默CD44基因对羧甲基壳聚糖保护大鼠软骨细胞凋亡的影响

目的 探讨小干扰RNA(siRNA)沉默大鼠软骨细胞的CD44基因后对细胞CD44基因表达的抑制作用及对软骨细胞在羧甲基壳聚糖(CMCS)作用下细胞生物学特性变化及其作用机制.方法 构建针对CD44基因特异性的siRNA(siRNA-1、siRNA-2、siRNA-3),LipofectamineTM 2000转染软骨细胞.通过免疫荧光鉴定CD44基因特异性siRNA(CD44-siRNA)的转染情况,通过反转录-聚合酶链反应(RT-PCR)及蛋白印迹法检测CD44-siRNA转染细胞中CD44基因及蛋白的表达情况;通过硝普钠诱导软骨细胞凋亡并通过流式细胞技术检测CMCS对硝普钠诱导正常及经CD44-siRNA转染软骨细胞凋亡的影响.采用单因素方差分析及SNK-q检验对结果进行统计学分析.结果 免疫荧光检测发现CD44-siRNA成功转染进入软骨细胞,转染率在60％左右.RT-PCR检测结果表明,转染siRNA-1后的24、48及72 h,与空白对照组(0.429±0.053,0.501±0.037,0.341±0.009)相比,CD44的mRNA表达明显减弱(分别为0.198±0.007,0.211±0.016,0.153±0.005;q=5.93,7.01,11.23; P＜0.01).蛋白印迹法检测表明转染siRNA-1后24 h,与空白对照组相比,CD44的蛋白表达明显减弱(0.231±0.064与0.675±0.113,q=13.09,P＜0.01).流式细胞检测结果表明3 mmol/L硝普钠可以成功诱导软骨细胞发生早期凋亡[(70±6)％];50、100、200μg/ml CMCS对硝普钠诱导的凋亡有一定的抑制作用[凋亡率分别为(51±7)％,(30±4)％,(15±4)％;q=5.08,6.97,9.73;P＜0.01];但CMCS对硝普钠诱导的CD44-siRNA-1转染的软骨细胞的凋亡抑制作用比未转染组明显减弱[凋亡率分别为(34±6)％和(15±4)％,q=6.95,P＜0.01 ].结论 CD44特异性siRNA-1转染体外培养的大鼠软骨细胞能显著下调CD44基因的表达;CD44基因在CMCS保护硝普钠诱导软骨细胞凋亡中发挥重要的作用.     

吡格列酮对成骨细胞Bax、Bcl-2蛋白表达的影响

目的 研究吡格列酮对成骨细胞增殖及凋亡的影响,并进一步了解其凋亡发生机制.方法 以MC3T3-E1成骨细胞株为实验对象,分别用0、5、10、20、30、40 μmol/L吡格列酮干预,观察细胞活性、细胞周期及凋亡率的变化,同时检测细胞Bcl-2,Bax蛋白表达.结果 随着浓度增加,成骨细胞的活性逐渐降低;与对照组相比,G0/G1,G2/M期细胞增多,S期细胞明显减少;凋亡率在5、10 μmol/L时低于对照组,30、40 μmol/L较对照组显著增高;Bax表达在10 μmol/L时明显减弱,20 μmol/L时回复到正常,30、40μmol/L较对照组显著增强;Bcl-2表达在浓度≤20 μmol/L时显著增强;对于Bax/Bcl-2相对表达强度与细胞凋亡率做相关性分析,两者呈显著性正相关(n=15,r=0.796,P＜0.01).结论 吡格列酮在低浓度抑制成骨细胞凋亡,对细胞起保护作用,较高浓度则促进细胞凋亡,Bax/Bcl-2参与其凋亡机制,并可能起关键调控作用.吡格列酮抑制成骨细胞DNA合成,抑制细胞增殖,导致细胞活性下降.

Abstract：

Objective To investigate the effects of pioglitazone on osteoblast proliferation and apoptosis.Methods MC3T3-E1 mouse osteoblastic cells were treated with 0, 5, 10, 20, 30, and 40 μmol/L pioglitazone for 24 h. Cell viability was measured by MTT, cell cycle and apoptosis were inspected with flow cytometry, the expressions of Bcl-2 and Bax proteins were examined via immuno-chemical staining. Results Survival of osteoblasts decreased in a dose-dependent manner. Compared with the control group, the cells in the G0/G1 and G2/M stages increased, while the cells in S stage decreased significantly. The percentage of apoptosis at 5 and 10 μmol/L were lower than that of the control group(P ＜ 0.05), While it was increased significantly at 30 and 40 μmol/L(P ＜0.01). Bax expression was attenuated at 10 μmol/L(P＜0. 01), returned to normal by 20 μmol/L, and was increased by 30 and 40 μmol/L(P ＜ 0. 01). Bcl-2 expression was enhanced at the dose ≤ 20 μmol/L(P ＜0.01). Positive correlation was found between the death rate and the expression intensity of Bax/Bcl-2(n = 15, r=0.796, P＜0.01). Conclusions Pioglitazone inhibits apoptosis of osteoblasts at low concentrations and protects the cells, but promotes their apoptosis at higher concentration, Bax/Bcl-2 may play an important role in mediating the piglitazone-induced apoptosis of osteoblasts. It inhibits DNA synthesis and cell proliferation.