【作者】 张睿；

【导师】 唐全勇；

【作者基本信息】 河北医科大学 ， 口腔临床医学， 2011， 硕士

【摘要】 目的:利用全骨髓细胞培养系,研究M-CSF、RANKL和1,25-（OH）2D3对体外诱导培养的破骨细胞形成及分化的影响,探讨三种细胞因子间相互作用及协同作用,为进一步研究破骨细胞及合理选用细胞因子提供依据。方法:选用4周龄SD雄性大鼠（体重80-120g）,利用全骨髓细胞培养系,进行体外破骨细胞诱导培养,观察M-CSF、RANKL和1,25-（OH）2D3对破骨细胞形成及分化的影响,同时比较三种细胞因子的作用。1细胞培养及细胞因子添加:1.1全骨髓细胞培养:取4周龄SD雄性大鼠一只（80—120g）,异氟烷麻醉后,无菌条件下取其股骨和胫骨,剪掉骨垢端暴露骨髓腔,用10ml无菌注射器抽取不含胎牛血清的α-MEM培养液冲洗骨髓腔,收集全骨髓细胞,将所提取的细胞悬液充分吹打至没有细胞团块后,置于离心机内离心5分钟（1500转/分）,弃去上清液,加入含15%胎牛血清的α-MEM培养液,充分吹打混匀后形成单细胞悬液,用细胞计数板计算细胞数,计算出细胞原液浓度,然后配制浓度为2×10⁶cells/ml的细胞悬液,加入青霉素100单位/ml和链霉素100ug/ml。将细胞播种于24孔培养板（4行6列）中,每孔加入浓度为2×10⁶cells/ml的细胞悬液0.5ml。1.2细胞因子的添加:24孔培养板第一列为对照组,不添加任何药物;第二列加入1,25-（OH）₂D₃(1×10^-8Mol/ml);第三列加入1,25-（OH）₂D₃+RANKL( 1,25-（OH）₂D₃ 1×10^-8Mol/ml +RANKL 20ng/ml );第四列加入1,25-（OH）₂D₃+M-CSF(1,25-（OH）₂D₃ 1×10^-8Mol/ml +M-CSF 20ng/ml);第五列加入1,25-（OH）₂D₃+M-CSF+RANKL(1,25-（OH）₂D₃ 1×10^-8 Mol/ml +M-CSF 20ng/ml+ RANKL 20ng/ml);第六列加入RANKL+M-CSF（RANKL 20ng/ml+M-CSF 20ng/ml）。1.3细胞培养:细胞因子添加完毕后将培养板置于37℃、5% CO₂培养箱中培养,培养至第4天时更换培养液一次,共培养7天。2抗酒石酸酸性磷酸酶（TRAP）染色:培养至第7天后行TRAP染色,倒置光镜下观察,计算3核以上染色阳性细胞数。3统计学分析:使用SPSS13.0软件进行统计学分析。数据处理采用非参数检验的方差分析和S-N-K检验。结果:1破骨细胞的形态学特征培养前三天各组均未见破骨细胞形成,培养第五天开始实验组可见少量破骨细胞形成,培养至第七天实验组破骨细胞已经分化成熟,而对照组始终没有破骨细胞的形成。对全骨髓细胞培养进行TRAP染色后,光镜下可见到大量成熟的破骨细胞,其形态学特征为:细胞体积较大,呈椭圆形、漏斗形、油煎蛋样、条索状或不规则形。细胞浆丰富呈紫红色,细胞核较大,数目达几个甚至多达几十个,核仁清晰,细胞浆内可见空泡结构（见图1-5）。2 M-CSF、RANKL和1,25-（OH）₂D₃对破骨细胞的形成和分化均具有促进作用,多因子比单因子和双因子在相同条件下形成的破骨细胞数目多。全骨髓培养系中,培养板第一列（对照组）没有添加细胞因子,最终没有破骨细胞的形成;第二列加入1,25-（OH）₂D₃形成了破骨细胞,证明1,25-（OH）₂D₃对破骨细胞的形成和分化具有促进作用;第三列、第四列除加入1,25-（OH）₂D₃外,还分别加入了RANKL和M-CSF,最终都有破骨细胞的形成,并且形成破骨细胞的数目较第二列多,与第二列相比统计学有显著性差异（P<0.05）,说明RANKL和M-CSF对破骨细胞的形成和分化也具有促进作用,但第三、四列间无显著性差异（P>0.05）;第五列同时加入M-CSF、RANKL和1,25-（OH）₂D₃后形成的破骨细胞数目最多,说明三种细胞因子对破骨细胞形成和分化具有协同促进作用,与第二、三、四、六列比较,有显著性差异（P<0.05）。第六列只加入RANKL和M-CSF后也形成了破骨细胞,但数量和第三、四列相似（见表1、直方图）。结论:1 M-CSF、RANKL和1,25-（OH）₂D₃对破骨细胞的形成和分化均具有促进作用。2单因子和双因子间对破骨细胞形成和分化有显著性差异。3三种细胞因子共存在对破骨细胞形成和分化具有协同促进作用。更多还原

【Abstract】 Objective: To study the influence of M-CSF,RANKL and 1,25-（OH）₂D₃ on formation and differentiation of cultured osteoclast in the whole bone marrow cell culture system and explore the interaction and synergy of these three cytokines, which will provide the theory evidence for the futher study on osteoclast and screening of cytokines.Method:A 4-week-old SD male rat（weight 80-120g） was selected to obtain the whole marrow culture system.The influence of M-CSF,RANKL and 1,25-（OH）₂D₃ on the formation and differentiation of osteoclasts were observed and the effect of three cytokines were compared.1 Cell culture and cytokines adds;1.1 The whole marrow culture system:Under ether anaesthesia,the femur and tibia were gained from a 4-week-old SD male rat（weight 80-120g） with sterilization.Cut away epiphysis and expose bone cavity, then flush the cavity by 10ml a-MEM culture fluid to extract the whole marrow cells,Placed in centrifuge 5 minutes （1500 turn/min）after blow and mix enough to form cell suspension.Discarded the supernatant liquid,then added the a-MEM culture fluid containing 15% FBS（fetal bovine serum）, blow and mix enough to form cell suspenion. Prepare the 2×10⁶cells/ml cell suspension after compute a cell number by FACS and calculating the concentration of this original cell liquid. Added 100 units of Penicillin per milliliter and 100 micrograms of Streptomycin per milliliter. The cells were seeded in 24-well culture plates （4 line 6）, each well by adding the cell suspension 0.5ml of 2×10⁶cells/ml.1.2 Cytokines adds: The first column of 24-well cell culture plate as the control group,without adding any drug;The second column by adding 1,25-（OH）2D(31×10^-8Mol/ml); The third column by adding 1,25-（OH）₂D₃ and RANKL(1,25-（OH）₂D₃ 1×10^-8Mol/ml and RANKL 20ng/ml);The fourth column by adding 1,25-（OH）₂D₃ and M-CSF(1,25-（OH）₂D₃ 1×10^-8Mol/ml and M-CSF 20ng/ml);The fifth column by adding 1,25-（OH）₂D₃,M-CSF and RANKL(1,25-（OH）₂D₃ 1×10^-8 Mol/ml,M-CSF 20ng/ml and RANKL 20ng/ml);The sixth column by adding RANKL and M-CSF（RANKL 20ng/ml and M-CSF 20ng/ml）。1.3 Cell culture: Put the 24-well cell plate into CO₂ incubator after adding cytokines,culture cells under the 37℃, 5% CO₂ condition.Changed culture fluid on the 4^th day for one time,and totally culture procedure is 7 days.2 Tartrate Resistant Acid Phosphatase （TRAP） stainings: The whole marrow culture system was stained by TRAP after being culture for 7 days respectively. TRAP positive cells were observed and calculated under the microscope （more than 3 nucleus in the whole marrow cell culture system）.3 SPSS13.0 software used for statistical analysis. Data processing using non-parametric analysis of variance and S-N-K test.Result:1 Morphological characteristics of osteoclast:In the first three days culturing,no osteoclast was formed in each group,few osteoclast were formed in experimental group from the fifth day of culture.At the seventh day,the osteoclasts had been mature in the experimental groups, while in the control group,osteoclast formation was always not found.A large number of mature ostelclasts were found after the TRAP staining, which were characteristiced by larger cell size , oval form, funnel form, fry in oil egg form, linear form or irregular form.Osteoclast contained large number of red cytoplasm with clear vacuole struvture, several to two or three dozens of cell nucleuses with significant nucleoli, and reachs out many lamellipodia（Figure 1-5）.2 M-CSF, RANKL and 1,25-（OH）₂D₃ played a facilitating role on osteoclast formation and differentiation.Under the same conditions, multi-factor formed more osteoclasts than single factor and two-factor.In the whole bone marrow cell culture system, the first column （control group）of culture plate did not add cytokines,finaly,there was no formation of osteoclasts;in the second column by adding 1,25-（OH）₂D₃, there was formation of osteoclasts,which proved that 1,25-（OH）₂D₃ has a promoting role on osteoclast formation and differentiation; In the third column and the fourth column,except adding 1,25-（OH）₂D₃, also added RANKL and M-CSF, Eventually had the formation of osteoclasts, and the number of osteoclasts was larger than the second column,and compared with the second column had statistically significant difference（P<0.05）, RANKL and M-CSF also play a promoting role in ostelclast formation and differentiation, but the third and fourth column showed no significant difference（P>0.05）; In the fifth column, the largest number of osteoclasts were formed after added M-CSF,RANKL and 1,25-（OH）₂D₃,which indicated that three cytokines had synergistic effects on osteoclast formation and differentiation, and compared with the second, third, fourth and sixth column there was significant difference （P<0.05）. In the sixth column also formed osteoclasts after added RANKL and M-CSF, but the number of osteoclasts were similar compared with the third and fourth column （Table 1,histogram）.Conclusion:1 M-CSF, RANKL and 1,25-（OH）₂D₃ have promoting role on osteoclast formation and differentiation.2 There is significant difference on osteoclast formation and differentiation between the single factor and two-factor.3 There is a synergistic effect on osteoclast formation and differentiation,when three cytokines are together exist.更多还原