【摘要】： 目的 鋅是人體中200余種酶的組成成分,并是許多酶的催化劑,故能促進(jìn)DNA、蛋白質(zhì)、膠原的合成,促進(jìn)生長(zhǎng)發(fā)育。鋅缺乏會(huì)使機(jī)體的代謝平衡受到破壞,從而影響其他營(yíng)養(yǎng)素(包括微量元素)在機(jī)體中的作用,可使骨密度發(fā)生改變,引起骨生長(zhǎng)遲緩,對(duì)少年兒童生長(zhǎng)發(fā)育有嚴(yán)重的影響。近年來(lái)的研究表明,哺乳動(dòng)物細(xì)胞內(nèi)有一類(lèi)類(lèi)似ATP酶的蛋白質(zhì)—鋅轉(zhuǎn)運(yùn)蛋白家族(Zinc transporters, ZnTs),可轉(zhuǎn)運(yùn)鋅離子,并調(diào)節(jié)質(zhì)膜內(nèi)外鋅離子濃度。ZnT-1(鋅轉(zhuǎn)移蛋白1)是ZnTs家族中重要的組成部分,它定位于細(xì)胞膜表面上,將鋅離子從細(xì)胞外聚集到細(xì)胞體內(nèi),用于細(xì)胞內(nèi)各種含鋅蛋白的合成和加工,其中包括很多參與調(diào)節(jié)細(xì)胞基因表達(dá)的DNA結(jié)合蛋白。本文從缺鋅狀態(tài)下骺板軟骨細(xì)胞的增殖和凋亡入手,并探討缺鋅與ZnT-1表達(dá)的關(guān)系。 材料與方法 1.大鼠生長(zhǎng)板軟骨細(xì)胞原代培養(yǎng)：雄性vista大鼠,斷頸法處死,進(jìn)行軟骨細(xì)胞培養(yǎng)并進(jìn)行鑒定。 2.細(xì)胞缺鋅模型的制備：向無(wú)血清培養(yǎng)液內(nèi)加入不同濃度鋅螯合齊(?)TPEN(5μM、10μM、20μM作用24h,造成軟骨細(xì)胞鋅缺乏。 3.MTT法檢測(cè)細(xì)胞增殖倍數(shù)：將不同濃度的培養(yǎng)液加入96孔培養(yǎng)板中,加入培養(yǎng)的軟骨細(xì)胞,同時(shí)設(shè)空白對(duì)照。培養(yǎng)24 h后,每孔加入溴化二甲噻唑二苯四氮唑(MTT),繼續(xù)培養(yǎng)4-6 h后棄上清,加入鹽酸異丙醇充分溶解,測(cè)量570nm和630nm的OD值,通過(guò)曲線測(cè)得產(chǎn)物濃度,分析細(xì)胞增殖情況。 4.流式細(xì)胞學(xué)檢測(cè)凋亡：不含EDTA的胰酶常規(guī)消化細(xì)胞,PBS洗兩遍,加入200μl Binding buffer,重懸后加入10μl Annexin V反應(yīng)30 min,再加入5μlPI和300μl Binding buffer,混勻反應(yīng)5 min后上機(jī)檢測(cè)。 5.定位、定量檢測(cè)ZnT1在對(duì)照組和實(shí)驗(yàn)組大鼠骺板軟骨細(xì)胞的分布和變化。Real time-PCR和Western Blotting方法。 6.統(tǒng)計(jì)學(xué)分析實(shí)驗(yàn)重復(fù)3次,數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差表示,兩組對(duì)照采用t檢驗(yàn)比較,三組以上采用方差分析比較實(shí)驗(yàn)各組均數(shù)與對(duì)照組均數(shù)之間的差異,以P0.05為差異有統(tǒng)計(jì)學(xué)意義。 實(shí)驗(yàn)結(jié)果 1.細(xì)胞培養(yǎng)：原代培養(yǎng)的生長(zhǎng)板軟骨細(xì)胞中95%以上的細(xì)胞Ⅰ、Ⅱ型膠原陽(yáng)性染色,細(xì)胞核周?chē)图?xì)胞膜周邊都有Ⅱ型膠原的陽(yáng)性染色,陽(yáng)性染色的纖維包裹著細(xì)胞核,保持圓形的細(xì)胞呈強(qiáng)陽(yáng)性染色。而X型膠原染色沒(méi)有陽(yáng)性反應(yīng)。 2.MTT檢測(cè)結(jié)果：通過(guò)MTT檢測(cè)隨TPEN的濃度的增高,軟骨細(xì)胞后24 h細(xì)胞相對(duì)存活率逐漸降低。 3.流式細(xì)胞學(xué)檢測(cè)結(jié)果：用Annexin V PI染色,檢測(cè)7 u M TPEN處理細(xì)胞時(shí)細(xì)胞凋亡情況,可見(jiàn)細(xì)胞凋亡增加正常組9.93% TPEN組94.78%。 4. Western Blot:檢測(cè)ZnT-1表達(dá)變化,5μmol/L (TPEN)組與對(duì)照組相比略有增高,而10μmol/L、20μmol/L組與對(duì)照組相比逐漸降低。 5. Real time-PCR檢測(cè)ZnT-1表達(dá)量的變化,可知隨TPEN濃度的增加ZnT-1的量逐漸減少。 結(jié)論 ZnTl對(duì)骺板的生理活動(dòng)起重要的調(diào)節(jié)作用,是促進(jìn)軟骨細(xì)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.
【學(xué)位授予單位】：中國(guó)醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R363

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