铅对SD乳鼠成骨细胞毒性机理的研究
摘要
许多研究表明,铅对全身各系统和器官均有毒性作用,主要是神经、心血管系统、骨代谢。铅可以通过对钙磷代谢、骨钙素、内分泌系统等的影响,干扰了骨形成,造成功能性的衰退,引起独特的疾病类型或病理性的改变。成骨细胞是骨形成细胞,负责骨基质的合成、分泌和矿化,对骨组织的生长发育、骨代谢平衡、骨量维持和损伤修复起关键作用。体外分离培养高纯度、活力强的成骨细胞是毒理学研究的基础。本试验在建立体外分离培养新生SD大鼠颅盖骨成骨细胞基础上,研究铅对成骨细胞毒性的机理,为探讨铅对正常骨生理功能的影响,提供了一定的理论依据。
1.成骨细胞的分离培养与鉴定
采用改良的二次酶消化法获得大鼠颅盖骨成骨细胞,待细胞融合成单层后传代。倒置显微镜下观察成骨细胞的形态及生长情况,BCIP/NBT酶底物显色试剂盒和茜素红矿化结节染色鉴定成骨细胞。结果,此法分离的成骨细胞生长状态良好,纯度较高。
2.铅对成骨细胞形态与增殖及分化的影响
为观察铅对成骨细胞增殖及分化的影响,添加了不同浓度的铅溶液(0、20、40、80μmol/L)作用成骨细胞24h。倒置显微镜观察成骨细胞形态结构的变化,MTT测定成骨细胞增殖,PNPP法检测细胞内碱性磷酸酶(ALP)活性。结果显示,对照组成骨细胞生长旺盛,结构特征典型;与对照组比较,铅溶液浓度80μmol/L时,细胞毒性加重,细胞多崩解。成骨细胞增殖和ALP活性随着铅浓度的增加逐渐降低。各染毒组成骨细胞的增殖和ALP活性,均差异显著或极显著(P<0.05或P<0.01);说明,一定量的铅可以导致成骨细胞的形态结构受损,降低成骨能力。
3.铅对成骨细胞钙调蛋白含量的影响
为了解铅对成骨细胞钙调蛋白含量的影响,通过添加不同浓度的铅溶液(0、20、40、80μmol/L)作用成骨细胞24h。结果显示,与对照组比较,铅的染毒浓度20μmol/L时,成骨细胞钙调蛋白的mRNA和蛋白表达减少;铅的染毒浓度≥40μmol/L时,成骨细胞钙调蛋白的mRNA和蛋白表达量明显增加。总体趋势表明铅在一定浓度范围内能引起成骨细胞的钙调蛋白的mRNA和蛋白表达增加。
4.铅对成骨细胞磷脂酰胆碱特异性磷脂酶C(PC-PLC)的影响
通过加入不同浓度的铅溶液(0、20、40、80μmol/L)培养成骨细胞24h,研究铅对成骨细胞PC-PLC的影响。结果表明,各不同浓度的铅溶液导致成骨细胞PC-PLC活性下降和磷脂酰胆碱特异性磷脂酶C蛋白量表达降低,与对照组比较,差异极显著(P<0.01)。表明一定浓度的铅会导致成骨细胞PC-PLC活性降低,对成骨细胞的PC-PLC蛋白表达量表现为不同程度的抑制作用。
Many studies have shown that lead have toxic in body systems and organs,such as neurological, cardiovascular system and bone metabolism. Lead has impacted on calcium metabolism, BGP, the endocrine system. It not only has interfered with bone formation and resulted in functional decline, but aslo caused by a unique type of disease or pathological changes. Osteoblasts are bone-forming cells and responsible for the synthesis, secretion, and mineralization of bone matrix. It plays a very important role in bone tissue growth and development, the balance of bone metabolism, bone repair and maintain. Isolating and culturing osteoblasts in vitro provides conditions for toxicology. High purity and vitality osteoblasts are the premise for study. To study the effects of lead toxicity on osteoblasts, osteoblasts were isolated from novo-rat cranial bones and cultured in vitro. These results provided rationale for the normal physiological function of bone.
1.Isolation and identification of rat osteoblasts
To establish the skill of isolation osteoblasts in order to study the effect of lead injection on the level of cell of bone metabolism diseases. Mouse primary osteoblasts were obtained from calvaria of SD novo-rat that digested by trypsin and collagenase and identified by staining of alkaline phosphatase (ALP) and mineralized matrix (alizarin red). It was concluded that the modified twice-enzyme isolation method was an ideal technique to obtain and culture osteoblasts with typical characteristics and high purity for the experiment.
2.Effects of lead on the osteoblasts cultured in vitro
The mOBs were divided into four groups: [Pb 0], [Pb 20], [Pb 40], and [Pb 80] to receive 0μM, 20μM, 40μM and 80μM of Pb2+ as Pb acetate solution respectively. Then shape and growth of OB were observed under inverted microscope. Cell proliferation was observed by microtitration (MTT), ALP activity was detected through PNPP method. The results indicated that, among three treated groups, Pb exposure induced gap of the cells was observed to scale-up in the group [Pb 20]. Moreover, most of cells collapsed and died in group [Pb 80] when compared to control. In addition, Pb exposure caused marked the inhibited proliferation of osteobals in the group [Pb 80] (P<0.01). Among three treated groups there was descrease in ALP activity (P<0.05). It was concluded that lead could resulte in the damage patterns and reduce the ability of bone formation.
3.Effects of lead on the osteoblasts CaM
In order to understand the effects of lead on the osteoblasts CaM, the mOBs were divided into four groups: [Pb 0], [Pb 20], [Pb 40] and [Pb 80] to receive 0μM, 20μM, 40μM and 80μM of Pb2+ as Pb acetate solution respectively. After 24h, the results show that Pb exposure caused marked increase in CaM protein level and CaM mRNA level in group [Pb 80] and in group [Pb 40]. It is concluded that there were increase in CaM protein levels and CaM mRNA level.
4.Effects of lead on the osteoblasts Phosphatidylcholine Phospholipase C(PC-PLC)
To study effects of lead on the osteoblasts PC-PLC through different concentrations of lead (0, 20, 40, 80μmol/L) were added into culture solution. After 24h, results showed the decline in PC-PLC protein level. PC-PLC activities also significantly decreased (P<0.01). It is concluded that Pb lead to a decrease in PC-PLC activity and protein level in a dose-dependent manner.
In conclusion, we isolated and identificated of rat osteoblasts, and found the effects of lead on cell proliferation and ALP activities of the osteoblasts cultured in vitro. Our studies first indicated that lead may play an important role in the mOBs through the osteoblasts CaM and PC-PLC.
1.成骨细胞的分离培养与鉴定
采用改良的二次酶消化法获得大鼠颅盖骨成骨细胞,待细胞融合成单层后传代。倒置显微镜下观察成骨细胞的形态及生长情况,BCIP/NBT酶底物显色试剂盒和茜素红矿化结节染色鉴定成骨细胞。结果,此法分离的成骨细胞生长状态良好,纯度较高。
2.铅对成骨细胞形态与增殖及分化的影响
为观察铅对成骨细胞增殖及分化的影响,添加了不同浓度的铅溶液(0、20、40、80μmol/L)作用成骨细胞24h。倒置显微镜观察成骨细胞形态结构的变化,MTT测定成骨细胞增殖,PNPP法检测细胞内碱性磷酸酶(ALP)活性。结果显示,对照组成骨细胞生长旺盛,结构特征典型;与对照组比较,铅溶液浓度80μmol/L时,细胞毒性加重,细胞多崩解。成骨细胞增殖和ALP活性随着铅浓度的增加逐渐降低。各染毒组成骨细胞的增殖和ALP活性,均差异显著或极显著(P<0.05或P<0.01);说明,一定量的铅可以导致成骨细胞的形态结构受损,降低成骨能力。
3.铅对成骨细胞钙调蛋白含量的影响
为了解铅对成骨细胞钙调蛋白含量的影响,通过添加不同浓度的铅溶液(0、20、40、80μmol/L)作用成骨细胞24h。结果显示,与对照组比较,铅的染毒浓度20μmol/L时,成骨细胞钙调蛋白的mRNA和蛋白表达减少;铅的染毒浓度≥40μmol/L时,成骨细胞钙调蛋白的mRNA和蛋白表达量明显增加。总体趋势表明铅在一定浓度范围内能引起成骨细胞的钙调蛋白的mRNA和蛋白表达增加。
4.铅对成骨细胞磷脂酰胆碱特异性磷脂酶C(PC-PLC)的影响
通过加入不同浓度的铅溶液(0、20、40、80μmol/L)培养成骨细胞24h,研究铅对成骨细胞PC-PLC的影响。结果表明,各不同浓度的铅溶液导致成骨细胞PC-PLC活性下降和磷脂酰胆碱特异性磷脂酶C蛋白量表达降低,与对照组比较,差异极显著(P<0.01)。表明一定浓度的铅会导致成骨细胞PC-PLC活性降低,对成骨细胞的PC-PLC蛋白表达量表现为不同程度的抑制作用。
Many studies have shown that lead have toxic in body systems and organs,such as neurological, cardiovascular system and bone metabolism. Lead has impacted on calcium metabolism, BGP, the endocrine system. It not only has interfered with bone formation and resulted in functional decline, but aslo caused by a unique type of disease or pathological changes. Osteoblasts are bone-forming cells and responsible for the synthesis, secretion, and mineralization of bone matrix. It plays a very important role in bone tissue growth and development, the balance of bone metabolism, bone repair and maintain. Isolating and culturing osteoblasts in vitro provides conditions for toxicology. High purity and vitality osteoblasts are the premise for study. To study the effects of lead toxicity on osteoblasts, osteoblasts were isolated from novo-rat cranial bones and cultured in vitro. These results provided rationale for the normal physiological function of bone.
1.Isolation and identification of rat osteoblasts
To establish the skill of isolation osteoblasts in order to study the effect of lead injection on the level of cell of bone metabolism diseases. Mouse primary osteoblasts were obtained from calvaria of SD novo-rat that digested by trypsin and collagenase and identified by staining of alkaline phosphatase (ALP) and mineralized matrix (alizarin red). It was concluded that the modified twice-enzyme isolation method was an ideal technique to obtain and culture osteoblasts with typical characteristics and high purity for the experiment.
2.Effects of lead on the osteoblasts cultured in vitro
The mOBs were divided into four groups: [Pb 0], [Pb 20], [Pb 40], and [Pb 80] to receive 0μM, 20μM, 40μM and 80μM of Pb2+ as Pb acetate solution respectively. Then shape and growth of OB were observed under inverted microscope. Cell proliferation was observed by microtitration (MTT), ALP activity was detected through PNPP method. The results indicated that, among three treated groups, Pb exposure induced gap of the cells was observed to scale-up in the group [Pb 20]. Moreover, most of cells collapsed and died in group [Pb 80] when compared to control. In addition, Pb exposure caused marked the inhibited proliferation of osteobals in the group [Pb 80] (P<0.01). Among three treated groups there was descrease in ALP activity (P<0.05). It was concluded that lead could resulte in the damage patterns and reduce the ability of bone formation.
3.Effects of lead on the osteoblasts CaM
In order to understand the effects of lead on the osteoblasts CaM, the mOBs were divided into four groups: [Pb 0], [Pb 20], [Pb 40] and [Pb 80] to receive 0μM, 20μM, 40μM and 80μM of Pb2+ as Pb acetate solution respectively. After 24h, the results show that Pb exposure caused marked increase in CaM protein level and CaM mRNA level in group [Pb 80] and in group [Pb 40]. It is concluded that there were increase in CaM protein levels and CaM mRNA level.
4.Effects of lead on the osteoblasts Phosphatidylcholine Phospholipase C(PC-PLC)
To study effects of lead on the osteoblasts PC-PLC through different concentrations of lead (0, 20, 40, 80μmol/L) were added into culture solution. After 24h, results showed the decline in PC-PLC protein level. PC-PLC activities also significantly decreased (P<0.01). It is concluded that Pb lead to a decrease in PC-PLC activity and protein level in a dose-dependent manner.
In conclusion, we isolated and identificated of rat osteoblasts, and found the effects of lead on cell proliferation and ALP activities of the osteoblasts cultured in vitro. Our studies first indicated that lead may play an important role in the mOBs through the osteoblasts CaM and PC-PLC.
引文
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[1] Samn L H, Gonzalez, Cojo T, et a1. Accumulation of lead in bone and its effects on health [J]. Salud Publica Mex, 1998, 40 (4): 359-368.
[2]石凯丽.铅对儿童骨代谢的影响机制[J]..国外医学儿科学分册,2002,29(6): 289-291.
[3]陈迎风,吴兴中.磷脂酰胆碱特异性磷脂酶C的研究[J].生命的化学,1999, 19 (4):173-175.
[4] Kolesnick, R. Signal transduction through the sphingomyelin pathway [J]. Mol Chem.Neuropathol, 1994, 21(2): 287-297.
[5] Ferretti A, Podo F, Carpinelli G, et al. Detection of neutral active phosphatidyl- choline-specific phospholipase C in Friend leukemia cells before and after erythroiddifferentiation [J]. Anticancer Res, 1993, 13(6): 2309-2317.
[6] Goering PL. Lead-protein interactions as a basis for lead toxicity [J]. Neurotoxicol, 1993, 14(2-3): 45-60.
[7]周桂凤,刘栋,蒋云生.铅结合蛋白研究进展[J].国外医学卫生学分册,2006,33(2):100-105.
[8]卢虹,陈迎风,吴兴中.肝细胞内两种磷脂酰胆碱特异性磷脂酶C的活性[J].复旦学报(医学科学版) ,2001, 28 (3):198-201.
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