微直流电刺激对成骨细胞生物学性能影响的体外实验研究
发布时间：2018-07-18 12:29
【摘要】：目的:通过对体外培养的成骨细胞刺激不同强度的直流电刺激,探讨微量直流电对成骨细胞增殖和功能的影响,为口腔修复临床应用直流电促进牙槽骨生长提供体外细胞学依据。方法:自行设计研制体外成骨细胞直流电刺激装置:将电源、滑动变阻器、开关、六孔培养板固定在一块300mm×200mm的有机玻璃板上,并把电路串联起来,在六孔培养板每孔所对应的板盖上打两个小孔,将镍钛弓丝一端与电路连接,另一端穿过培养板盖浸入培养液内,将电路接通,用万用表测定电流强度和稳定性。通过组织块法原代分离培养新生Sprague-Dawley大鼠颅顶骨的成骨细胞,传代培养至第三代进行碱性磷酸酶(alkaline phosphatase,ALP)染色鉴定。纯化后扩增成骨细胞,传代培养至第四代或第五代备用。实验第一部分,将细胞按1.0x105个/孔的浓度接种于六孔培养板中,每板接种四孔。实验分为三组,微电流对三组细胞刺激的强度分别为0μA、20μA和100μA,每天刺激三次,于微电流刺激的第1天,第3天,第5天,第7天时分别收集三组细胞,用MTT法测定三组成骨细胞的数量,之后重复实验两次,用Spss16.0软件将所得数据进行统计学分析并绘制细胞增殖曲线;实验第二部分,细胞分组及接种的方法同第一部分,之后于微直流电刺激的第1天,第3天,第5天,第7天时收集三个实验组的成骨细胞测定其ALP活性,重复该实验两次,将实验所得数据用Spss16.0软件进行统计学分析并绘制成骨细胞ALP活性直方图。结果:本实验自行研制的成骨细胞直流电刺激装置通电过程中电流稳定,绝对误差在3μA内;经强度为20μA和100μA的直流电刺激后的细胞其增殖和对照组无统计学差异,三组细胞均在第3～5天时增殖速度最快,第5～7天时增殖速度减慢;经强度为20μA和100μA的直流电刺激后的成骨细胞和对照组成骨细胞的ALP活性均有统计学差异;强度为20μA的直流电刺激组与强度为100μA的直流电刺激组比较,成骨细胞ALP活性也有统计学差异(P0.01),且直流电强度为20μA的刺激组成骨细胞ALP活性增加较明显。结论:直流电强度为20μA和100μA时对细胞的增殖无促进作用,但可以增加成骨细胞的ALP活性,且经20μA的直流电刺激的成骨细胞ALP活性明显优于经100μA直流电刺激的成骨细胞,证明微量直流电可以促进成骨细胞的活性,特别是直流电强度为20μA时对成骨细胞活性的促进作用更为显著,这为微量直流电用于口腔修复临床促进牙槽骨的增长提供了体外细胞学依据。
[Abstract]:Objective: to investigate the effects of direct current stimulation on proliferation and function of osteoblasts by stimulating osteoblasts with different intensities in vitro. To provide in vitro cytological basis for dental repair clinical application of direct current to promote alveolar bone growth. Methods: a direct current electric stimulation device for osteoblast in vitro was designed and developed. The electric source, sliding rheostat, switch and six hole culture plate were fixed on a 300mm 脳 200mm plate, and the circuit was connected in series. Two holes were made on the plate cover corresponding to each hole of the six-hole culture plate, one end of the nickel titanium bow wire was connected to the circuit, the other end was immersed in the culture medium through the culture plate cover, the circuit was connected, and the current intensity and stability were measured by multimeter. Osteoblasts of newborn Sprague-Dawley rat cranioparietal bone were isolated and cultured by tissue block method. The osteoblasts were subcultured to the third generation and identified by alkaline phosphatase staining. After purification, osteoblasts were amplified and cultured to the fourth or fifth generation. In the first part of the experiment, the cells were inoculated into the six hole culture plate at the concentration of 1.0x105 per well, each plate was inoculated with four holes. The experiment was divided into three groups. The intensity of microcurrent stimulation was 0 渭 A 20 渭 A and 100 渭 A, respectively. The cells were collected on the 1st, 3rd, 5th and 7th day of microcurrent stimulation. MTT assay was used to determine the number of osteoblasts in the three groups, and then repeated experiments twice. The data were analyzed statistically and the cell proliferation curve was plotted by Spss16.0 software. The second part of the experiment, the methods of cell grouping and inoculation were the same as those of the first part. The ALP activity of osteoblasts of the three experimental groups was collected on day 1, day 3, day 5 and day 7 after microdirect current stimulation, and the activity of ALP was repeated twice. The experimental data were analyzed by SPSS 16.0 software and the ALP activity histogram of osteoblasts was drawn. Results: the current of osteoblast direct current stimulation device was stable and the absolute error was within 3 渭 A, and the proliferation of osteoblasts stimulated by 20 渭 A and 100 渭 A was not significantly different from that of the control group. The proliferation rate of the three groups was the fastest at the 3rd day and the slower at the 5th day, and the ALP activity of osteoblasts stimulated with 20 渭 A and 100 渭 A was significantly different from that of the control group. The ALP activity of osteoblasts in 20 渭 A group was significantly higher than that in 100 渭 A group (P0.01), and the ALP activity of osteoblast in 20 渭 A group was significantly higher than that in 100 渭 A group (P0.01). Conclusion: the direct current intensity of 20 渭 A and 100 渭 A can not promote the proliferation of osteoblasts, but can increase the ALP activity of osteoblasts, and the ALP activity of osteoblasts stimulated by 20 渭 A DC is better than that of osteoblasts stimulated with 100 渭 A. The results showed that micro DC could promote the activity of osteoblasts, especially when the DC intensity was 20 渭 A, the activity of osteoblasts was more obvious. This provides in vitro cytological evidence for the application of micro direct current in dental repair to promote the growth of alveolar bone.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
[Abstract]:Objective: to investigate the effects of direct current stimulation on proliferation and function of osteoblasts by stimulating osteoblasts with different intensities in vitro. To provide in vitro cytological basis for dental repair clinical application of direct current to promote alveolar bone growth. Methods: a direct current electric stimulation device for osteoblast in vitro was designed and developed. The electric source, sliding rheostat, switch and six hole culture plate were fixed on a 300mm 脳 200mm plate, and the circuit was connected in series. Two holes were made on the plate cover corresponding to each hole of the six-hole culture plate, one end of the nickel titanium bow wire was connected to the circuit, the other end was immersed in the culture medium through the culture plate cover, the circuit was connected, and the current intensity and stability were measured by multimeter. Osteoblasts of newborn Sprague-Dawley rat cranioparietal bone were isolated and cultured by tissue block method. The osteoblasts were subcultured to the third generation and identified by alkaline phosphatase staining. After purification, osteoblasts were amplified and cultured to the fourth or fifth generation. In the first part of the experiment, the cells were inoculated into the six hole culture plate at the concentration of 1.0x105 per well, each plate was inoculated with four holes. The experiment was divided into three groups. The intensity of microcurrent stimulation was 0 渭 A 20 渭 A and 100 渭 A, respectively. The cells were collected on the 1st, 3rd, 5th and 7th day of microcurrent stimulation. MTT assay was used to determine the number of osteoblasts in the three groups, and then repeated experiments twice. The data were analyzed statistically and the cell proliferation curve was plotted by Spss16.0 software. The second part of the experiment, the methods of cell grouping and inoculation were the same as those of the first part. The ALP activity of osteoblasts of the three experimental groups was collected on day 1, day 3, day 5 and day 7 after microdirect current stimulation, and the activity of ALP was repeated twice. The experimental data were analyzed by SPSS 16.0 software and the ALP activity histogram of osteoblasts was drawn. Results: the current of osteoblast direct current stimulation device was stable and the absolute error was within 3 渭 A, and the proliferation of osteoblasts stimulated by 20 渭 A and 100 渭 A was not significantly different from that of the control group. The proliferation rate of the three groups was the fastest at the 3rd day and the slower at the 5th day, and the ALP activity of osteoblasts stimulated with 20 渭 A and 100 渭 A was significantly different from that of the control group. The ALP activity of osteoblasts in 20 渭 A group was significantly higher than that in 100 渭 A group (P0.01), and the ALP activity of osteoblast in 20 渭 A group was significantly higher than that in 100 渭 A group (P0.01). Conclusion: the direct current intensity of 20 渭 A and 100 渭 A can not promote the proliferation of osteoblasts, but can increase the ALP activity of osteoblasts, and the ALP activity of osteoblasts stimulated by 20 渭 A DC is better than that of osteoblasts stimulated with 100 渭 A. The results showed that micro DC could promote the activity of osteoblasts, especially when the DC intensity was 20 渭 A, the activity of osteoblasts was more obvious. This provides in vitro cytological evidence for the application of micro direct current in dental repair to promote the growth of alveolar bone.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
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1 杨军,董为伟,贾延R，
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