缺锌对大鼠骺板软骨细胞增殖和凋亡的影响及其机制
发布时间：2018-08-08 18:47
【摘要】： 目的 锌是人体中200余种酶的组成成分,并是许多酶的催化剂,故能促进DNA、蛋白质、胶原的合成,促进生长发育。锌缺乏会使机体的代谢平衡受到破坏,从而影响其他营养素(包括微量元素)在机体中的作用,可使骨密度发生改变,引起骨生长迟缓,对少年儿童生长发育有严重的影响。近年来的研究表明,哺乳动物细胞内有一类类似ATP酶的蛋白质—锌转运蛋白家族(Zinc transporters, ZnTs),可转运锌离子,并调节质膜内外锌离子浓度。ZnT-1(锌转移蛋白1)是ZnTs家族中重要的组成部分,它定位于细胞膜表面上,将锌离子从细胞外聚集到细胞体内,用于细胞内各种含锌蛋白的合成和加工,其中包括很多参与调节细胞基因表达的DNA结合蛋白。本文从缺锌状态下骺板软骨细胞的增殖和凋亡入手,并探讨缺锌与ZnT-1表达的关系。 材料与方法 1.大鼠生长板软骨细胞原代培养：雄性vista大鼠,断颈法处死,进行软骨细胞培养并进行鉴定。 2.细胞缺锌模型的制备：向无血清培养液内加入不同浓度锌螯合齐(?)TPEN(5μM、10μM、20μM作用24h,造成软骨细胞锌缺乏。 3.MTT法检测细胞增殖倍数：将不同浓度的培养液加入96孔培养板中,加入培养的软骨细胞,同时设空白对照。培养24 h后,每孔加入溴化二甲噻唑二苯四氮唑(MTT),继续培养4-6 h后弃上清,加入盐酸异丙醇充分溶解,测量570nm和630nm的OD值,通过曲线测得产物浓度,分析细胞增殖情况。 4.流式细胞学检测凋亡：不含EDTA的胰酶常规消化细胞,PBS洗两遍,加入200μl Binding buffer,重悬后加入10μl Annexin V反应30 min,再加入5μlPI和300μl Binding buffer,混匀反应5 min后上机检测。 5.定位、定量检测ZnT1在对照组和实验组大鼠骺板软骨细胞的分布和变化。Real time-PCR和Western Blotting方法。 6.统计学分析实验重复3次,数据以均数±标准差表示,两组对照采用t检验比较,三组以上采用方差分析比较实验各组均数与对照组均数之间的差异,以P0.05为差异有统计学意义。 实验结果 1.细胞培养：原代培养的生长板软骨细胞中95%以上的细胞Ⅰ、Ⅱ型胶原阳性染色,细胞核周围和细胞膜周边都有Ⅱ型胶原的阳性染色,阳性染色的纤维包裹着细胞核,保持圆形的细胞呈强阳性染色。而X型胶原染色没有阳性反应。 2.MTT检测结果：通过MTT检测随TPEN的浓度的增高,软骨细胞后24 h细胞相对存活率逐渐降低。 3.流式细胞学检测结果：用Annexin V PI染色,检测7 u M TPEN处理细胞时细胞凋亡情况,可见细胞凋亡增加正常组9.93% TPEN组94.78%。 4. Western Blot:检测ZnT-1表达变化,5μmol/L (TPEN)组与对照组相比略有增高,而10μmol/L、20μmol/L组与对照组相比逐渐降低。 5. Real time-PCR检测ZnT-1表达量的变化,可知随TPEN浓度的增加ZnT-1的量逐渐减少。 结论 ZnTl对骺板的生理活动起重要的调节作用,是促进软骨细胞增殖分化的重要因子。
[Abstract]:objective
Zinc is a component of more than 200 enzymes in the human body, and it is a catalyst for many enzymes, so it can promote the synthesis of DNA, protein and collagen, and promote the growth and development. Zinc deficiency will destroy the metabolic balance of the body, thus affecting the role of other nutrients (including trace elements) in the body, which can cause bone density to change and cause bone growth retardation. It has a serious effect on the growth and development of children. Recent studies have shown that there is a protein zinc transporter family (Zinc transporters, ZnTs) similar to ATP enzyme in mammalian cells. It can transport zinc ions and regulate the concentration of zinc ions in the plasma membrane,.ZnT-1 (zinc transfer protein 1), which is an important component of the ZnTs family. On the surface of the cell membrane, the zinc ions are gathered from the cell to the cell, which is used for the synthesis and processing of various zinc containing proteins in the cells, including a lot of DNA binding proteins involved in the regulation of cell gene expression. This article begins with the proliferation and withering of the epiphyseal plate cartilage cells under the state of zinc deficiency, and discusses the relationship between the zinc deficiency and the expression of ZnT-1.
Materials and methods
1. primary culture of rat growth plate chondrocytes: male Vista rats were killed by neck breaking, and chondrocytes were cultured and identified.
2. Preparation of cell zinc deficiency model: Different concentrations of zinc chelate homozygous (?) TPEN (5_ M, 10_ M, 20_ M) were added into serum-free medium for 24 hours, resulting in zinc deficiency of chondrocytes.
3.MTT method was used to detect the proliferation of cell proliferation: the culture solution of different concentration was added to 96 hole culture plate, the cultured chondrocytes were added, and the blank control was set up at the same time. After 24 h, brominated two methothiazole, two benzotetrazoles (MTT) were added to each hole, and after 4-6 h, the supernatant was discarded and the OD value of 570nm and 630nm was measured. The concentration of the product was measured by the curve, and the cell proliferation was analyzed.
4. flow cytology was used to detect apoptosis: non EDTA trypsin routine digestive cells, PBS washed two times, 200 mu L Binding buffer, 10 mu Annexin V and 30 min after suspension, then 5 mu lPI and 300 micron L Binding, and the mixing reaction 5 after the test.
5. Localization and quantification of ZnT1 in epiphyseal chondrocytes of control and experimental groups. Real time-PCR and Western Blotting methods.
6. the statistical analysis experiment was repeated 3 times. The data were expressed in mean number mean difference. The two groups were compared with the t test. The difference between the three groups was compared with the control group. The difference between the two groups was statistically significant with the difference of P0.05.
experimental result
1. cell culture: more than 95% of the cells in the primary cultured growth plate chondrocytes were stained positive with type II collagen. The positive staining of type II collagen was found around the nucleus and the periphery of the cell membrane. The positive staining fibers wrapped the nucleus and maintained the strong positive color of the circular cells, but the X type collagen staining did not react positively.
2.MTT test results: through MTT detection, the relative survival rate of 24 h cells decreased gradually with the increase of TPEN concentration.
3. flow cytology test results: Annexin V PI staining was used to detect the apoptosis of 7 u M TPEN cells, and apoptosis was increased to the normal group of 9.93% TPEN group 94.78%.
4. Western Blot: The expression of ZnT-1 in the 5-micromol/L (TPEN) group was slightly higher than that in the control group, but decreased gradually in the 10-micromol/L and 20-micromol/L groups.
5. Real time-PCR detected the change of ZnT-1 expression. It is known that the ZnT-1 content decreases with the increase of TPEN concentration.
conclusion
ZnTl plays an important role in regulating physiological activities of epiphyseal plates, and is an important factor to promote chondrocyte proliferation and differentiation.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363
本文编号：2172693
[Abstract]:objective
Zinc is a component of more than 200 enzymes in the human body, and it is a catalyst for many enzymes, so it can promote the synthesis of DNA, protein and collagen, and promote the growth and development. Zinc deficiency will destroy the metabolic balance of the body, thus affecting the role of other nutrients (including trace elements) in the body, which can cause bone density to change and cause bone growth retardation. It has a serious effect on the growth and development of children. Recent studies have shown that there is a protein zinc transporter family (Zinc transporters, ZnTs) similar to ATP enzyme in mammalian cells. It can transport zinc ions and regulate the concentration of zinc ions in the plasma membrane,.ZnT-1 (zinc transfer protein 1), which is an important component of the ZnTs family. On the surface of the cell membrane, the zinc ions are gathered from the cell to the cell, which is used for the synthesis and processing of various zinc containing proteins in the cells, including a lot of DNA binding proteins involved in the regulation of cell gene expression. This article begins with the proliferation and withering of the epiphyseal plate cartilage cells under the state of zinc deficiency, and discusses the relationship between the zinc deficiency and the expression of ZnT-1.
Materials and methods
1. primary culture of rat growth plate chondrocytes: male Vista rats were killed by neck breaking, and chondrocytes were cultured and identified.
2. Preparation of cell zinc deficiency model: Different concentrations of zinc chelate homozygous (?) TPEN (5_ M, 10_ M, 20_ M) were added into serum-free medium for 24 hours, resulting in zinc deficiency of chondrocytes.
3.MTT method was used to detect the proliferation of cell proliferation: the culture solution of different concentration was added to 96 hole culture plate, the cultured chondrocytes were added, and the blank control was set up at the same time. After 24 h, brominated two methothiazole, two benzotetrazoles (MTT) were added to each hole, and after 4-6 h, the supernatant was discarded and the OD value of 570nm and 630nm was measured. The concentration of the product was measured by the curve, and the cell proliferation was analyzed.
4. flow cytology was used to detect apoptosis: non EDTA trypsin routine digestive cells, PBS washed two times, 200 mu L Binding buffer, 10 mu Annexin V and 30 min after suspension, then 5 mu lPI and 300 micron L Binding, and the mixing reaction 5 after the test.
5. Localization and quantification of ZnT1 in epiphyseal chondrocytes of control and experimental groups. Real time-PCR and Western Blotting methods.
6. the statistical analysis experiment was repeated 3 times. The data were expressed in mean number mean difference. The two groups were compared with the t test. The difference between the three groups was compared with the control group. The difference between the two groups was statistically significant with the difference of P0.05.
experimental result
1. cell culture: more than 95% of the cells in the primary cultured growth plate chondrocytes were stained positive with type II collagen. The positive staining of type II collagen was found around the nucleus and the periphery of the cell membrane. The positive staining fibers wrapped the nucleus and maintained the strong positive color of the circular cells, but the X type collagen staining did not react positively.
2.MTT test results: through MTT detection, the relative survival rate of 24 h cells decreased gradually with the increase of TPEN concentration.
3. flow cytology test results: Annexin V PI staining was used to detect the apoptosis of 7 u M TPEN cells, and apoptosis was increased to the normal group of 9.93% TPEN group 94.78%.
4. Western Blot: The expression of ZnT-1 in the 5-micromol/L (TPEN) group was slightly higher than that in the control group, but decreased gradually in the 10-micromol/L and 20-micromol/L groups.
5. Real time-PCR detected the change of ZnT-1 expression. It is known that the ZnT-1 content decreases with the increase of TPEN concentration.
conclusion
ZnTl plays an important role in regulating physiological activities of epiphyseal plates, and is an important factor to promote chondrocyte proliferation and differentiation.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363
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相关期刊论文 前3条
1 余晓丹,颜崇淮,沈晓明,薛敏波,余晓刚;0～6岁儿童骨密度与血清锌、铜、血铅关系的研究[J];中国儿童保健杂志;2004年01期
2 张月红,程义勇,洪燕,李树田,王冬兰;大鼠缺锌时股骨骺生长板的病理形态学改变[J];卫生研究;2003年01期
3 余晓丹,颜崇淮,余晓刚,高宇,徐健,沈晓明;生长期大鼠缺锌时股骨病理形态学的改变[J];卫生研究;2005年02期
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