大黄酸对破骨细胞形成及分化的影响
发布时间：2018-05-08 00:17
本文选题：大黄酸 + 破骨细胞 ； 参考：《河北医科大学》2012年硕士论文
【摘要】：目的：利用除去间质细胞的骨髓细胞培养系(前破骨细胞形成系)及小鼠单核细胞RAW264.7细胞培养系分别诱导培养破骨前驱细胞及成熟破骨细胞，研究大黄酸对两种体外培养的破骨细胞形成及分化的影响，观察破骨细胞早期阶段及成熟阶段的形态学变化，为预防和治疗破骨细胞性骨吸收性疾病提供一种新制剂。 方法：选用两种细胞培养系：除去间质细胞的骨髓细胞培养系(前破骨细胞形成系)及小鼠单核细胞RAW264.7细胞培养系，进行体外破骨细胞诱导培养，分别观察大黄酸对破骨前驱细胞及成熟破骨细胞形成、分化的影响。 1细胞培养及药物添加： 1.1前破骨细胞形成系：取4周龄SD雄性大鼠一只(80-120g),用异氟烷麻醉后,在无菌条件下取其胫骨和股骨，去掉骨垢端暴露骨髓腔,然后用10ml无菌注射器抽取不含血清的α-MEM培养液冲洗骨髓腔，制取全骨髓细胞,1500转/分离心5分钟后,弃去上清液,加入含15%胎牛血清的α-MEM培养液20ml，充分吹打混匀后形成单细胞悬液，此为全骨髓细胞悬液，经过离心浓缩成2ml,然后注入葡聚糖凝胶柱G10(Sephadex G10)中过滤，除去骨髓间质细胞，搜集非附着性骨髓细胞10-12ml,利用细胞计数板,计算细胞数，然后配制浓度为2×106cells/ml的细胞悬液13ml，添加破骨细胞分化因子(sRANKL)和活化型双羟维生素D3(1α，25(OH)2D3)，使其终浓度分别为40ng/ml和10-8Mol/ml，将细胞悬液播种于24孔培养板中，每孔加入细胞悬液0.5ml(1×106cells)。然后将培养板放入37℃、5%CO2培养箱中培养，培养至第4天时更换培养液一次，共计培养7天。 1.2小鼠单核细胞RAW264.7细胞培养系：从液氮罐中取出RAW264.7细胞株，放入37℃水浴箱中快速复苏，用无菌吸管将细胞液移至15ml离心管内，吸取生理盐水洗涤冻存管两次，一并将其移至上述离心管中，上离心机，1000转/分离心5分钟后，弃去上清液,加入含10%胎牛血清的1640培养液约10ml，充分吹打混匀形成单细胞悬液，根据实验要求培养细胞。当细胞生长状态良好，长满瓶底壁约90%时进行消化，收集细胞，1000转/分离心5分钟后，弃去上清液,加入含10%胎牛血清的1640培养液约10ml，充分吹打混匀形成单细胞悬液，利用细胞计数板，计算细胞数，然后配制成浓度为4.5×104cells/ml的细胞悬液5ml，添加破骨细胞分化因子(sRANKL)50ng/ml，，播种细胞于96孔培养板中，利用24孔，共4行6列，每孔加入细胞悬液150μl，然后将培养板放入37℃、5%CO2培养箱中培养，培养4~5天。 1.3药物添加：在4行6列的24孔培养板中添加药物大黄酸,使其终浓度分别为0、10-3、10-4、10-5、10-6、10-7mol/L,每个浓度每列4孔(n＝4)，第一列为对照组。在96孔培养板中选择24个孔，使其呈4行6列，添加药物大黄酸，使其终浓度分别为0、10-3、10-4、10-5、10-6、10-7mol/L,每个浓度每列4孔(n＝4)，第一列为对照组。 2抗酒石酸酸性磷酸酶(TRAP)染色：破骨前驱细胞培养系培养至第7天及RAW264.7细胞培养系培养至第5天后行TRAP染色，倒置光镜下观察，计算并记录染色阳性细胞数。 3统计学分析：使用SPSS13.0软件进行统计学分析。数据处理采用mean±SD检验分析，数据资料采用Student’s t-test和非参数检验的方差分析，各不同浓度的加药组与对照组之间相比较。 结果： 1前破骨细胞形成系： 1.1破骨前驱细胞的形态学特征经TRAP染色后，可见到大量染色阳性的破骨前驱细胞，其形态学特征与成熟破骨细胞相似，但是细胞体积较小，形态多样(见Fig.1)，可呈椭圆形、类圆形、四边形、条索状和不规则形等。细胞大多为单核，也可见到2核者。 1.2大黄酸对破骨前驱细胞形成的抑制在前破骨细胞形成系中，10-3、10-4、10-5、10-6、10-7mol/L组与对照组相比，TRAP染色阳性细胞(单核或2核)数仅为对照组的4.98%、12.27%、18.57%、34.14%、78.57%。经统计学分析，加药组对破骨前驱细胞的形成有抑制作用，当药物浓度为10-3mol/L时，与对照组相比,对破骨前驱细胞的形成有显著抑制(p㩳0.05)(见Fig.3和Table1)。2RAW264.7细胞培养系： 2.1破骨细胞样细胞(成熟破骨细胞)的形态学特征经TRAP染色后，可见到大量破骨细胞样细胞，其形态学特征与成熟破骨细胞相似，细胞体积较大，形态多样(见Fig.5)，可呈星形、圆形、类圆形、梭形、条索状及不规则形。细胞核为多个。 2.2大黄酸对破骨细胞样细胞形成的抑制在RAW264.7细胞培养系中,10-3、10-4、10-5、10-6、10-7mol/L组与对照组相比，TRAP染色阳性细胞数仅为对照组的4.36%、63.39%、72.73%、81.94%、92.73%。经统计学分析，加药组对破骨细胞样细胞的形成有抑制作用，当药物浓度为10-3mol/L时，与对照组相比,对破骨细胞样细胞的形成有显著抑制(p㩳0.05)(见Fig.6和Table2)。 结论： 1大黄酸能抑制破骨前驱细胞及成熟破骨细胞的形成与分化。 2大黄酸对破骨细胞的抑制作用在一定浓度范围内具有浓度依赖性。
[Abstract]:Objective: To study the effect of rhubarb on the formation and differentiation of osteoclasts of two cultured osteoclasts by using the bone marrow cell culture line (anterior osteoclast line) and mouse monocyte culture line (RAW264.7 cell culture line) to study the effect of rhubarb on the formation and differentiation of osteoclasts in vitro, and to observe the early stage and formation of osteoclast. Morphological changes at maturity stage provide a new preparation for prevention and treatment of osteoclastic bone resorption diseases.
Methods: the effects of rhubarb on the formation and differentiation of osteoclasts of osteoclast and mature osteoclasts were observed by removing two kinds of cell culture lines: bone marrow cell culture line (anterior osteoclast line) and mouse monocyte RAW264.7 cell culture line.
1 cell culture and drug addition:
1.1 anterior osteoclast formation system: a 4 week old SD male rat (80-120G). After anaesthesia, the tibia and femur were taken under aseptic condition. The bone marrow cavity was removed from the end of bone scale and the bone marrow cavity was removed. Then the bone marrow cavity was washed with 10ml sterile syringe without serum containing alpha -MEM, and the whole bone marrow cells were produced. After 1500 turn / separation heart 5 minutes, the bone marrow cells were abandoned. The supernatant, adding an alpha -MEM culture medium containing 15% fetal bovine serum 20ml, is fully blown and mixed to form a single cell suspension. This is the whole bone marrow cell suspension, which is concentrated into 2ml by centrifugation and then filtered into the dextran gel column G10 (Sephadex G10) to remove the marrow stromal cells and collect non adherent bone marrow cells 10-12ml and use cell counting board. Count the cell number, then prepare the cell suspension 13ml with a concentration of 2 x 106cells/ml, add the osteoclast differentiation factor (sRANKL) and the activated dihydroxyvitamin D3 (1 alpha, 25 (OH) 2D3), make the final concentration 40ng/ml and 10-8Mol/ml respectively, sow the cell suspension in the 24 hole culture plate, and add the cell suspension 0.5ml (1 * 106cells) per pore. Then the culture plate is made. They were incubate in 5%CO2 incubator at 37 C for fourth days to replace the culture medium for 7 days.
1.2 RAW264.7 cell culture system of monocyte in mice: remove RAW264.7 cell line from liquid nitrogen tank and quickly resuscitation in 37 centigrade water bath, transfer cell liquid to 15ml centrifuge tube with aseptic sucker, draw two times of saline washing cryopreservation tube, and move it into the above centrifuge tube, on centrifuge, 1000 turn / separation heart for 5 minutes and discard. In the clear liquid, 1640 culture solution containing 10% fetal bovine serum was added to about 10ml, which was fully blown and mixed to form a single cell suspension. The cells were cultured according to the experimental requirements. When the cells grew well, the cells were digested at about 90% of the bottom wall of the bottle, and the cells were collected. After 5 minutes of 1000 turn / separation heart, 1640 culture containing 10% fetal bovine serum was added to about 10ml. A single cell suspension was formed by mixing and mixing, and cell count was used to calculate cell number. Then a cell suspension 5ml with a concentration of 4.5 x 104cells/ml was prepared, and the osteoclast differentiation factor (sRANKL) 50ng/ml was added. The seeding cells were in the 96 hole culture plate. The 24 holes were used in a total of 4 rows and 6 columns. The cell suspension was added to 150 mu L for each hole, and the culture plate was placed in 37, 5. %CO2 culture box is cultivated for 4~5 days.
1.3 medications added: the drug rhubarb acid was added to the 24 hole culture plate of 4 rows and 6 columns, the final concentration was 0,10-3,10-4,10-5,10-6,10-7mol/L, each concentration was 4 holes (n = 4), the first was the control group, and 24 holes were selected in the 96 hole culture plate to make it 4 rows 6 columns, and the drug rhubarb acid was added, and the final concentration was 0,10-3,10-4,10-5,10-6, respectively. 10-7mol/L, each concentration of 4 holes per column (n = 4), the first column was the control group.
2 anti tartaric acid acid phosphatase (TRAP) staining: the culture line of the osteoclast precursor cells was cultured to seventh days and the culture line of RAW264.7 cells was cultured to fifth days after TRAP staining. The number of positive cells was calculated and recorded by inverted light microscope.
3 statistical analysis: SPSS13.0 software was used for statistical analysis. The data processing was analyzed by mean + SD test, and the data were analyzed by Student 's t-test and non parametric test, and the different concentration groups were compared with those of the control group.
Result锛
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