微直流電刺激對(duì)成骨細(xì)胞生物學(xué)性能影響的體外實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-07-18 12:29
【摘要】：目的:通過對(duì)體外培養(yǎng)的成骨細(xì)胞刺激不同強(qiáng)度的直流電刺激,探討微量直流電對(duì)成骨細(xì)胞增殖和功能的影響,為口腔修復(fù)臨床應(yīng)用直流電促進(jìn)牙槽骨生長(zhǎng)提供體外細(xì)胞學(xué)依據(jù)。方法:自行設(shè)計(jì)研制體外成骨細(xì)胞直流電刺激裝置:將電源、滑動(dòng)變阻器、開關(guān)、六孔培養(yǎng)板固定在一塊300mm×200mm的有機(jī)玻璃板上,并把電路串聯(lián)起來,在六孔培養(yǎng)板每孔所對(duì)應(yīng)的板蓋上打兩個(gè)小孔,將鎳鈦弓絲一端與電路連接,另一端穿過培養(yǎng)板蓋浸入培養(yǎng)液內(nèi),將電路接通,用萬用表測(cè)定電流強(qiáng)度和穩(wěn)定性。通過組織塊法原代分離培養(yǎng)新生Sprague-Dawley大鼠顱頂骨的成骨細(xì)胞,傳代培養(yǎng)至第三代進(jìn)行堿性磷酸酶(alkaline phosphatase,ALP)染色鑒定。純化后擴(kuò)增成骨細(xì)胞,傳代培養(yǎng)至第四代或第五代備用。實(shí)驗(yàn)第一部分,將細(xì)胞按1.0x105個(gè)/孔的濃度接種于六孔培養(yǎng)板中,每板接種四孔。實(shí)驗(yàn)分為三組,微電流對(duì)三組細(xì)胞刺激的強(qiáng)度分別為0μA、20μA和100μA,每天刺激三次,于微電流刺激的第1天,第3天,第5天,第7天時(shí)分別收集三組細(xì)胞,用MTT法測(cè)定三組成骨細(xì)胞的數(shù)量,之后重復(fù)實(shí)驗(yàn)兩次,用Spss16.0軟件將所得數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析并繪制細(xì)胞增殖曲線;實(shí)驗(yàn)第二部分,細(xì)胞分組及接種的方法同第一部分,之后于微直流電刺激的第1天,第3天,第5天,第7天時(shí)收集三個(gè)實(shí)驗(yàn)組的成骨細(xì)胞測(cè)定其ALP活性,重復(fù)該實(shí)驗(yàn)兩次,將實(shí)驗(yàn)所得數(shù)據(jù)用Spss16.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析并繪制成骨細(xì)胞ALP活性直方圖。結(jié)果:本實(shí)驗(yàn)自行研制的成骨細(xì)胞直流電刺激裝置通電過程中電流穩(wěn)定,絕對(duì)誤差在3μA內(nèi);經(jīng)強(qiáng)度為20μA和100μA的直流電刺激后的細(xì)胞其增殖和對(duì)照組無統(tǒng)計(jì)學(xué)差異,三組細(xì)胞均在第3～5天時(shí)增殖速度最快,第5～7天時(shí)增殖速度減慢;經(jīng)強(qiáng)度為20μA和100μA的直流電刺激后的成骨細(xì)胞和對(duì)照組成骨細(xì)胞的ALP活性均有統(tǒng)計(jì)學(xué)差異;強(qiáng)度為20μA的直流電刺激組與強(qiáng)度為100μA的直流電刺激組比較,成骨細(xì)胞ALP活性也有統(tǒng)計(jì)學(xué)差異(P0.01),且直流電強(qiáng)度為20μA的刺激組成骨細(xì)胞ALP活性增加較明顯。結(jié)論:直流電強(qiáng)度為20μA和100μA時(shí)對(duì)細(xì)胞的增殖無促進(jìn)作用,但可以增加成骨細(xì)胞的ALP活性,且經(jīng)20μA的直流電刺激的成骨細(xì)胞ALP活性明顯優(yōu)于經(jīng)100μA直流電刺激的成骨細(xì)胞,證明微量直流電可以促進(jìn)成骨細(xì)胞的活性,特別是直流電強(qiáng)度為20μA時(shí)對(duì)成骨細(xì)胞活性的促進(jìn)作用更為顯著,這為微量直流電用于口腔修復(fù)臨床促進(jìn)牙槽骨的增長(zhǎng)提供了體外細(xì)胞學(xué)依據(jù)。
[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.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：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.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 楊軍,董為偉,賈延R，
本文編號(hào)：2131908
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