本文選題：大黃酸 + 破骨細胞�。� 參考：《河北醫(yī)科大學》2012年碩士論文

【摘要】：目的：利用除去間質(zhì)細胞的骨髓細胞培養(yǎng)系(前破骨細胞形成系)及小鼠單核細胞RAW264.7細胞培養(yǎng)系分別誘導培養(yǎng)破骨前驅(qū)細胞及成熟破骨細胞，研究大黃酸對兩種體外培養(yǎng)的破骨細胞形成及分化的影響，觀察破骨細胞早期階段及成熟階段的形態(tài)學變化，為預防和治療破骨細胞性骨吸收性疾病提供一種新制劑。 方法：選用兩種細胞培養(yǎng)系：除去間質(zhì)細胞的骨髓細胞培養(yǎng)系(前破骨細胞形成系)及小鼠單核細胞RAW264.7細胞培養(yǎng)系，進行體外破骨細胞誘導培養(yǎng)，分別觀察大黃酸對破骨前驅(qū)細胞及成熟破骨細胞形成、分化的影響。 1細胞培養(yǎng)及藥物添加： 1.1前破骨細胞形成系：取4周齡SD雄性大鼠一只(80-120g),用異氟烷麻醉后,在無菌條件下取其脛骨和股骨，去掉骨垢端暴露骨髓腔,然后用10ml無菌注射器抽取不含血清的α-MEM培養(yǎng)液沖洗骨髓腔，制取全骨髓細胞,1500轉(zhuǎn)/分離心5分鐘后,棄去上清液,加入含15%胎牛血清的α-MEM培養(yǎng)液20ml，充分吹打混勻后形成單細胞懸液，此為全骨髓細胞懸液，經(jīng)過離心濃縮成2ml,然后注入葡聚糖凝膠柱G10(Sephadex G10)中過濾，除去骨髓間質(zhì)細胞，搜集非附著性骨髓細胞10-12ml,利用細胞計數(shù)板,計算細胞數(shù)，然后配制濃度為2×106cells/ml的細胞懸液13ml，添加破骨細胞分化因子(sRANKL)和活化型雙羥維生素D3(1α，25(OH)2D3)，使其終濃度分別為40ng/ml和10-8Mol/ml，將細胞懸液播種于24孔培養(yǎng)板中，每孔加入細胞懸液0.5ml(1×106cells)。然后將培養(yǎng)板放入37℃、5%CO2培養(yǎng)箱中培養(yǎng)，培養(yǎng)至第4天時更換培養(yǎng)液一次，共計培養(yǎng)7天。 1.2小鼠單核細胞RAW264.7細胞培養(yǎng)系：從液氮罐中取出RAW264.7細胞株，放入37℃水浴箱中快速復蘇，用無菌吸管將細胞液移至15ml離心管內(nèi)，吸取生理鹽水洗滌凍存管兩次，一并將其移至上述離心管中，上離心機，1000轉(zhuǎn)/分離心5分鐘后，棄去上清液,加入含10%胎牛血清的1640培養(yǎng)液約10ml，充分吹打混勻形成單細胞懸液，根據(jù)實驗要求培養(yǎng)細胞。當細胞生長狀態(tài)良好，長滿瓶底壁約90%時進行消化，收集細胞，1000轉(zhuǎn)/分離心5分鐘后，棄去上清液,加入含10%胎牛血清的1640培養(yǎng)液約10ml，充分吹打混勻形成單細胞懸液，利用細胞計數(shù)板，計算細胞數(shù)，然后配制成濃度為4.5×104cells/ml的細胞懸液5ml，添加破骨細胞分化因子(sRANKL)50ng/ml，，播種細胞于96孔培養(yǎng)板中，利用24孔，共4行6列，每孔加入細胞懸液150μl，然后將培養(yǎng)板放入37℃、5%CO2培養(yǎng)箱中培養(yǎng)，培養(yǎng)4~5天。 1.3藥物添加：在4行6列的24孔培養(yǎng)板中添加藥物大黃酸,使其終濃度分別為0、10-3、10-4、10-5、10-6、10-7mol/L,每個濃度每列4孔(n＝4)，第一列為對照組。在96孔培養(yǎng)板中選擇24個孔，使其呈4行6列，添加藥物大黃酸，使其終濃度分別為0、10-3、10-4、10-5、10-6、10-7mol/L,每個濃度每列4孔(n＝4)，第一列為對照組。 2抗酒石酸酸性磷酸酶(TRAP)染色：破骨前驅(qū)細胞培養(yǎng)系培養(yǎng)至第7天及RAW264.7細胞培養(yǎng)系培養(yǎng)至第5天后行TRAP染色，倒置光鏡下觀察，計算并記錄染色陽性細胞數(shù)。 3統(tǒng)計學分析：使用SPSS13.0軟件進行統(tǒng)計學分析。數(shù)據(jù)處理采用mean±SD檢驗分析，數(shù)據(jù)資料采用Student’s t-test和非參數(shù)檢驗的方差分析，各不同濃度的加藥組與對照組之間相比較。 結(jié)果： 1前破骨細胞形成系： 1.1破骨前驅(qū)細胞的形態(tài)學特征經(jīng)TRAP染色后，可見到大量染色陽性的破骨前驅(qū)細胞，其形態(tài)學特征與成熟破骨細胞相似，但是細胞體積較小，形態(tài)多樣(見Fig.1)，可呈橢圓形、類圓形、四邊形、條索狀和不規(guī)則形等。細胞大多為單核，也可見到2核者。 1.2大黃酸對破骨前驅(qū)細胞形成的抑制在前破骨細胞形成系中，10-3、10-4、10-5、10-6、10-7mol/L組與對照組相比，TRAP染色陽性細胞(單核或2核)數(shù)僅為對照組的4.98%、12.27%、18.57%、34.14%、78.57%。經(jīng)統(tǒng)計學分析，加藥組對破骨前驅(qū)細胞的形成有抑制作用，當藥物濃度為10-3mol/L時，與對照組相比,對破骨前驅(qū)細胞的形成有顯著抑制(p㩳0.05)(見Fig.3和Table1)。2RAW264.7細胞培養(yǎng)系： 2.1破骨細胞樣細胞(成熟破骨細胞)的形態(tài)學特征經(jīng)TRAP染色后，可見到大量破骨細胞樣細胞，其形態(tài)學特征與成熟破骨細胞相似，細胞體積較大，形態(tài)多樣(見Fig.5)，可呈星形、圓形、類圓形、梭形、條索狀及不規(guī)則形。細胞核為多個。 2.2大黃酸對破骨細胞樣細胞形成的抑制在RAW264.7細胞培養(yǎng)系中,10-3、10-4、10-5、10-6、10-7mol/L組與對照組相比，TRAP染色陽性細胞數(shù)僅為對照組的4.36%、63.39%、72.73%、81.94%、92.73%。經(jīng)統(tǒng)計學分析，加藥組對破骨細胞樣細胞的形成有抑制作用，當藥物濃度為10-3mol/L時，與對照組相比,對破骨細胞樣細胞的形成有顯著抑制(p㩳0.05)(見Fig.6和Table2)。 結(jié)論： 1大黃酸能抑制破骨前驅(qū)細胞及成熟破骨細胞的形成與分化。 2大黃酸對破骨細胞的抑制作用在一定濃度范圍內(nèi)具有濃度依賴性。
[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錛

本文編號：1859100