钙、磷对体外培养SD大鼠成骨细胞增殖、分化及矿化影响的研究
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摘要
随着人们对骨代谢疾病的重视,研究不同因子对成骨细胞(osteoblast,OB)的影响已成为当今热点。OB有形成骨骼、调节细胞外骨基质成分的构成以及骨基质矿化的功能。因此,OB功能状态与骨骼生长塑造、再造及骨质疏松的发展有着极其密切的联系。有些疾病可以通过增加OB的数量或增强OB的活性而引起骨量的增加。钙和磷是骨骼的重要物质成分,骨代谢过程必然涉及动物机体对钙、磷的吸收和代谢。摄入充足的钙、磷对保持动物的骨骼完好至关重要。体内钙、磷代谢的平衡和钙磷在细胞内、外液中浓度的稳定对维持正常骨代谢有重要作用。因此,建立一种快速获得OB的培养方法,研究钙、磷对OB体外增殖、分化及矿化的影响,对骨质疏松症的研究有重要的理论和指导意义。
本试验选用5 d SD大鼠头盖骨作为试验用OB的来源,通过胰蛋白酶预消化后,剪碎,经Ⅱ型胶原酶消化获得细胞,采用碱性磷酸酶(alkaline phosphatase,ALP)、钙化结节染色鉴定,经台盼蓝染色、“反复贴壁法”纯化后,进行了成活率和纯度分析,用MTT法检测了一个生长周期内OB的活性分布,成功建立了SD大鼠头盖骨OB体外培养模型;在体外培养的基础上,在培养液中添加不同浓度的钙、磷及其比例,共分9组,分别为不添加任何成分的对照组、添加1、2、4 mmol/L钙组、添加1、2、4 mmol/L磷组、钙磷比1∶2(添加1 mmol/L钙+2 mmol/L磷)组和钙磷比2∶1(添加2 mmol/L钙+1 mmol/L磷)组。分别从OB的形态学、OB分泌ALP活性、OB分泌蛋白、Ⅰ型胶原(collagen type I,Col-I))、骨桥蛋白(osteopontin,OPN)的含量、OB表达ALP、Col-I、OPN、骨钙素(bone glaprotein,BGP)mRNA的含量及OB内Ca2+沉积及体外钙化等指标进行了分析,详细的研究了钙、磷及其比例对乳鼠头盖骨OB增殖、分化及矿化的影响。结果表明,①本试验培养的头盖骨OB,经ALP染色鉴定,并计数,OB纯度高达98.06%,台盼蓝染色计数分析,原代OB成活率为88.69%,传代的OB成活率高达94.12%,且在一个生长周期内,在第3、4 d进入对数生长期,细胞融合并开始重叠生长,在第7 d达到峰值,并进入钙化期,随后细胞进入衰减期。因此,本试验将选择在细胞融合时定为钙、磷处理OB的时间点,检测处理后第2、5、8 d的各种指标;②与对照组比较,添加钙各组均促进OB增殖,且有剂量效应,钙4 mmol/L从第2 d开始差异显著(P<0.05),钙1、2 mmol/L从第5 d开始差异显著(P<0.05),而钙磷比(2:1、1:2)只在第8 d差异显著(P<0.05)。而添加磷对OB增殖作用影响不明显。③与对照组比较,添加钙使OB胞体饱满,表面针状突起增多,形态结构无破损,细胞膜、核膜完整,线粒体轻度肿胀,添加磷及不同钙磷比使OB胞体扁平,针状突起减少,但形态结构无破损,细胞膜、核膜完整。④与对照组比较,添加钙、磷及其比例在第2、5、8 d均抑制细胞内ALP活性(P<0.05),在第5 d抑制其mRNA的表达(P<0.01),但在第2、8 d却促进其mRNA的表达(P<0.05)。⑤与对照组比较,添加钙、磷及其比例各试验组除1 mmol/L钙外,在第2 d均促进Col-I分泌(P<0.01),钙各组和1 mmol/L磷在第5 d和钙各组和1、2 mmol/L磷和钙磷比(2:1)在第8 d均抑制其分泌(P<0.05或P<0.01),在第2、8 d均促进其mRNA表达(P<0.01),在第5 d除钙磷比(1:2)外均抑制其mRNA表达(P<0.01)。⑥与对照组比较,添加钙、磷及其比例作用后,各试验组均促进OPN mRNA的表达(P<0.01),除第2 d钙1、2 mmol/L组外,均促进其分泌(P<0.05)。⑦与对照组比较,添加钙、磷及其比例作用后,各试验组均促进BGP mRNA的表达(P<0.01)。⑧与对照组比较,添加钙、磷及其比例作用后,各试验组均能促进OB内Ca2+的沉积及体外钙化。表明,钙(1、2、4 mmol/L)及钙磷比(1∶2 ,2∶1 )能促进OB增殖、分化及体外钙化,利于新骨的形成。磷(1、2、4 mmol/L)能促进OB的分化及体外钙化,但对增殖作用不明显。
In recent years, with the importance of the disease of the bone metabolism, study the effects of different factors on osteoblast (OB) are growing up. OBs to form bone there is to regulate extracellular matrix composition of bone as well as the function of bone matrix mineralization. Therefore, the function status of OB and bone growth to mold, reconstruction and the development of osteoporosis have extremely close ties. Some diseases can increase the number of OBs or enhance the activity of OBs caused an increase in bone mass. Calcium (Ca) and phosphorus (P) are important components of the bone, the process of bone metabolism in the body of animals necessarily involves Ca and P absorption and metabolism. Adequate intake of Ca and P to maintain bone intact animals is essential. The body's Ca balance and Ca metabolism in cells, the concentration of extracellular fluid to maintain the stability of the normal has an important role in bone metabolism. Therefore, to establish a cultural method of fast obtained OBs to study the effect of Ca and P on OBs, there is important theory and guiding significance for the study of osteoporosis.
In this study, the experimental parietal bones were obtained from the 4-day-old SD rats, which were subjected to 20min digestion with 2.5 mg/ml trypsin before being cut into fine pieces which were subsequently digested for 1 hour with 1mg/ml collagenase type II to obtain the cell. The cell were identified using alkaline phosphatase (ALP) and calcification nodules staining, trypan blue staining and“repeatedly adherent”cleansing to analysis the survival rate and purity, and using MTT assay with a growth cycle of the activity of OBs distribution, so the model of SD rat parietal bones OBs in vitro is successfully established. OBs were cultured with no added factors (control group), added 1, 2 and 4 mmol/L calcium groups, added 1, 2 and 4 mmol/L phosphorus groups, Ca:P (1:2), i.e. 1 mM Ca and 2 mM P, and Ca:P (2:1), i.e. 2 mM Ca and 1 mM P. We observed morphous, surface structure and fine structure of OBs, measured the proliferation of OBs, ALP activity, content of osteopontin (OPN) and type I collagen (Col- I) and the corresponding mRNA and bone glaprotein (BGP) mRNA transcriptions. Comparing with control group, we found the following: (1) In this study,the cell that we obtained from parietal bones was OBs by identifying with ALP and calcification nodules staining, which purity was reach 98.06%. The survival rate of origin generation OBs was 88.69%, passage OBs was 94.12% by analysis of accounts with trypan blue staining. In a growth cycle, OBs entered into the logarithmic phase on 3-4 day, cells fusion and overlap growth, and reached peak amplitude on day 7, then entered into the calcification phase followed by cells entering the decay phase. Therefore, OBs were treated with Ca and P in cell fusion, to detect variety of indicators on 2nd, 5th and 8th day after treatment with Ca and P. (2) Compared with the control group, Ca in each group and Ca:P (1:2, 2:1) promoted proliferation of OBs, and there is a dose-effects in Ca groups, Ca at 4 mM showed significant effect(P<0.05)on early 2nd day. Ca at 1 and 2 mM showed significant effect(P<0.05)on early 5th day, Ca:P (1:2, 2:1) demonstrated significant effect only on 8th day(P<0.05). P on the proliferation of OBs was not obvious. (3) Compared with the control group, Ca could make the cell body become fuller, needle-like protrusions increased on the surface. The cell morph, architectonic, cell envelope, nuclear membrane was integrity, and mitochondria were mild swelled. P and Ca:P (1:2, 2:1) made the cell body become applanation, needle-like protrusions decreased on the surface. The cell morph, architectonic, cell envelope, nuclear membrane was integrity, and mitochondria cristae fragmentation. (4) Compared with the control group, Ca and P all experiment groups inhibited ALP activity(P<0.05)except Ca at 1 mM on 2nd day, and enhanced the transcription of ALP mRNA on 2nd and 8th days(P<0.05) and inhibited transcription of it on 5th day(P<0.01). (5) Compared with the control group, Ca and P all experiment groups except Ca at 1 mM enhanced the excretion of Col-I on 2nd day(P<0.01), but on 5th and 8th day inhibited its excretion(P<0.05)except P at 2, 4 mM and Ca:P (2:1,1:2)on 5th day and P at 4 mM and Ca:P (1:2) on 8th day; All those enhanced its mRNA transcription on 2nd and 8th day(P<0.01), inhibited its mRNA transcription on 5th day(P<0.01). (6) Compared with the control group, Ca and P all experiment groups on 2nd, 5th and 8th day promoted OPN mRNA transcription (P<0.01)and its excretion(P<0.05) except Ca at 1, 2 mM on 2nd day. (7) Compared with the control group, Ca and P all experiment groups on 2nd, 5th and 8th day promoted BGP mRNA transcription (P<0.01). (8) Compared with the control group, Ca and P all experiment groups enhanced OBs calcification in vitro. All those indicated that Ca at 1, 2 and 4 mM and Ca∶P(1∶2,2∶1)enhanced OBs proliferation, differentiation and calcification in vitro, benefited new bone form. P enhanced OBs differentiation and calcification in vitro, but show not significant effects on OB proliferation.
本试验选用5 d SD大鼠头盖骨作为试验用OB的来源,通过胰蛋白酶预消化后,剪碎,经Ⅱ型胶原酶消化获得细胞,采用碱性磷酸酶(alkaline phosphatase,ALP)、钙化结节染色鉴定,经台盼蓝染色、“反复贴壁法”纯化后,进行了成活率和纯度分析,用MTT法检测了一个生长周期内OB的活性分布,成功建立了SD大鼠头盖骨OB体外培养模型;在体外培养的基础上,在培养液中添加不同浓度的钙、磷及其比例,共分9组,分别为不添加任何成分的对照组、添加1、2、4 mmol/L钙组、添加1、2、4 mmol/L磷组、钙磷比1∶2(添加1 mmol/L钙+2 mmol/L磷)组和钙磷比2∶1(添加2 mmol/L钙+1 mmol/L磷)组。分别从OB的形态学、OB分泌ALP活性、OB分泌蛋白、Ⅰ型胶原(collagen type I,Col-I))、骨桥蛋白(osteopontin,OPN)的含量、OB表达ALP、Col-I、OPN、骨钙素(bone glaprotein,BGP)mRNA的含量及OB内Ca2+沉积及体外钙化等指标进行了分析,详细的研究了钙、磷及其比例对乳鼠头盖骨OB增殖、分化及矿化的影响。结果表明,①本试验培养的头盖骨OB,经ALP染色鉴定,并计数,OB纯度高达98.06%,台盼蓝染色计数分析,原代OB成活率为88.69%,传代的OB成活率高达94.12%,且在一个生长周期内,在第3、4 d进入对数生长期,细胞融合并开始重叠生长,在第7 d达到峰值,并进入钙化期,随后细胞进入衰减期。因此,本试验将选择在细胞融合时定为钙、磷处理OB的时间点,检测处理后第2、5、8 d的各种指标;②与对照组比较,添加钙各组均促进OB增殖,且有剂量效应,钙4 mmol/L从第2 d开始差异显著(P<0.05),钙1、2 mmol/L从第5 d开始差异显著(P<0.05),而钙磷比(2:1、1:2)只在第8 d差异显著(P<0.05)。而添加磷对OB增殖作用影响不明显。③与对照组比较,添加钙使OB胞体饱满,表面针状突起增多,形态结构无破损,细胞膜、核膜完整,线粒体轻度肿胀,添加磷及不同钙磷比使OB胞体扁平,针状突起减少,但形态结构无破损,细胞膜、核膜完整。④与对照组比较,添加钙、磷及其比例在第2、5、8 d均抑制细胞内ALP活性(P<0.05),在第5 d抑制其mRNA的表达(P<0.01),但在第2、8 d却促进其mRNA的表达(P<0.05)。⑤与对照组比较,添加钙、磷及其比例各试验组除1 mmol/L钙外,在第2 d均促进Col-I分泌(P<0.01),钙各组和1 mmol/L磷在第5 d和钙各组和1、2 mmol/L磷和钙磷比(2:1)在第8 d均抑制其分泌(P<0.05或P<0.01),在第2、8 d均促进其mRNA表达(P<0.01),在第5 d除钙磷比(1:2)外均抑制其mRNA表达(P<0.01)。⑥与对照组比较,添加钙、磷及其比例作用后,各试验组均促进OPN mRNA的表达(P<0.01),除第2 d钙1、2 mmol/L组外,均促进其分泌(P<0.05)。⑦与对照组比较,添加钙、磷及其比例作用后,各试验组均促进BGP mRNA的表达(P<0.01)。⑧与对照组比较,添加钙、磷及其比例作用后,各试验组均能促进OB内Ca2+的沉积及体外钙化。表明,钙(1、2、4 mmol/L)及钙磷比(1∶2 ,2∶1 )能促进OB增殖、分化及体外钙化,利于新骨的形成。磷(1、2、4 mmol/L)能促进OB的分化及体外钙化,但对增殖作用不明显。
In recent years, with the importance of the disease of the bone metabolism, study the effects of different factors on osteoblast (OB) are growing up. OBs to form bone there is to regulate extracellular matrix composition of bone as well as the function of bone matrix mineralization. Therefore, the function status of OB and bone growth to mold, reconstruction and the development of osteoporosis have extremely close ties. Some diseases can increase the number of OBs or enhance the activity of OBs caused an increase in bone mass. Calcium (Ca) and phosphorus (P) are important components of the bone, the process of bone metabolism in the body of animals necessarily involves Ca and P absorption and metabolism. Adequate intake of Ca and P to maintain bone intact animals is essential. The body's Ca balance and Ca metabolism in cells, the concentration of extracellular fluid to maintain the stability of the normal has an important role in bone metabolism. Therefore, to establish a cultural method of fast obtained OBs to study the effect of Ca and P on OBs, there is important theory and guiding significance for the study of osteoporosis.
In this study, the experimental parietal bones were obtained from the 4-day-old SD rats, which were subjected to 20min digestion with 2.5 mg/ml trypsin before being cut into fine pieces which were subsequently digested for 1 hour with 1mg/ml collagenase type II to obtain the cell. The cell were identified using alkaline phosphatase (ALP) and calcification nodules staining, trypan blue staining and“repeatedly adherent”cleansing to analysis the survival rate and purity, and using MTT assay with a growth cycle of the activity of OBs distribution, so the model of SD rat parietal bones OBs in vitro is successfully established. OBs were cultured with no added factors (control group), added 1, 2 and 4 mmol/L calcium groups, added 1, 2 and 4 mmol/L phosphorus groups, Ca:P (1:2), i.e. 1 mM Ca and 2 mM P, and Ca:P (2:1), i.e. 2 mM Ca and 1 mM P. We observed morphous, surface structure and fine structure of OBs, measured the proliferation of OBs, ALP activity, content of osteopontin (OPN) and type I collagen (Col- I) and the corresponding mRNA and bone glaprotein (BGP) mRNA transcriptions. Comparing with control group, we found the following: (1) In this study,the cell that we obtained from parietal bones was OBs by identifying with ALP and calcification nodules staining, which purity was reach 98.06%. The survival rate of origin generation OBs was 88.69%, passage OBs was 94.12% by analysis of accounts with trypan blue staining. In a growth cycle, OBs entered into the logarithmic phase on 3-4 day, cells fusion and overlap growth, and reached peak amplitude on day 7, then entered into the calcification phase followed by cells entering the decay phase. Therefore, OBs were treated with Ca and P in cell fusion, to detect variety of indicators on 2nd, 5th and 8th day after treatment with Ca and P. (2) Compared with the control group, Ca in each group and Ca:P (1:2, 2:1) promoted proliferation of OBs, and there is a dose-effects in Ca groups, Ca at 4 mM showed significant effect(P<0.05)on early 2nd day. Ca at 1 and 2 mM showed significant effect(P<0.05)on early 5th day, Ca:P (1:2, 2:1) demonstrated significant effect only on 8th day(P<0.05). P on the proliferation of OBs was not obvious. (3) Compared with the control group, Ca could make the cell body become fuller, needle-like protrusions increased on the surface. The cell morph, architectonic, cell envelope, nuclear membrane was integrity, and mitochondria were mild swelled. P and Ca:P (1:2, 2:1) made the cell body become applanation, needle-like protrusions decreased on the surface. The cell morph, architectonic, cell envelope, nuclear membrane was integrity, and mitochondria cristae fragmentation. (4) Compared with the control group, Ca and P all experiment groups inhibited ALP activity(P<0.05)except Ca at 1 mM on 2nd day, and enhanced the transcription of ALP mRNA on 2nd and 8th days(P<0.05) and inhibited transcription of it on 5th day(P<0.01). (5) Compared with the control group, Ca and P all experiment groups except Ca at 1 mM enhanced the excretion of Col-I on 2nd day(P<0.01), but on 5th and 8th day inhibited its excretion(P<0.05)except P at 2, 4 mM and Ca:P (2:1,1:2)on 5th day and P at 4 mM and Ca:P (1:2) on 8th day; All those enhanced its mRNA transcription on 2nd and 8th day(P<0.01), inhibited its mRNA transcription on 5th day(P<0.01). (6) Compared with the control group, Ca and P all experiment groups on 2nd, 5th and 8th day promoted OPN mRNA transcription (P<0.01)and its excretion(P<0.05) except Ca at 1, 2 mM on 2nd day. (7) Compared with the control group, Ca and P all experiment groups on 2nd, 5th and 8th day promoted BGP mRNA transcription (P<0.01). (8) Compared with the control group, Ca and P all experiment groups enhanced OBs calcification in vitro. All those indicated that Ca at 1, 2 and 4 mM and Ca∶P(1∶2,2∶1)enhanced OBs proliferation, differentiation and calcification in vitro, benefited new bone form. P enhanced OBs differentiation and calcification in vitro, but show not significant effects on OB proliferation.
引文
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