次声对骨髓间充质干细胞生长情况的影响

发布时间：2018-05-19 09:18

  本文选题：次声 + 骨髓间充质干细胞(BMSCs)增殖　； 参考：《南方医科大学》2012年硕士论文

【摘要】：研究背景 干细胞的研究和应用是重大疾病再生性修复和治疗的新途径和新希望。目前关于干细胞的分离、鉴定、培养、扩增、保存、复苏等关键技术得到了各个研究机构及个人的高度关注,而BMSCs因其来源充足、可实现自体移植等优点倍受青睐。 次声波是频率在0.0001～20Hz范围内的机械振动波,可能有机械效应、温热效应,及化学效应等生物学效应,次声对人体各系统均有影响,可引起体内细胞的生物学变化,还可对多种体外培养的细胞产生影响。次声在脑缺血再灌注损伤等神经系统疾病的防护上,有一定效应,还可影响神经前体细胞的增殖。而BMSCs也已经广泛用于神经系统疾病,其机制可能是通过在体内的神经分化实现的,所以开展次声对BMSCs的生物学效应研究就是一项非常有必要的课题。 目前,国内学者多研究次声对成骨细胞、神经前体细胞、小角质细胞、角膜细胞、癌细胞等细胞的超微结构及增殖能力等方面的影响。对BMSCs的研究还鲜有报道,但是已有人做过低强度超声对BMSCs所产生的生物学效应的研究,发现低强度超声可以促进BMSCs的增殖及软骨定向分化,次声同样作为一种机械波,在这方面可能会有共通之处。国外方面：近年来,国外学者对次声在生物医学界的研究较少,多为次声对机体产生的损伤效应。在细胞培养方面,大多数人认为某些细胞因子及氨基酸对细胞的培养及分化有一定的作用,但也有人已经提出力学因素在这一方面的作用。 次声波的生物学效应主要由频率、时间、强度等参数来决定,本课题主要观察低压级次声不同作用时间对BMSCs所产生的生物学效应。 研究目的 观察次声对骨髓间充质干细胞(Bone Marrow Stromal Stem Cells, BMSCs)的生物学效应,包括增殖、凋亡和周期分布,以及超微结构的影响,探讨次声合理应用的时间参数。材料与方法： 1、细胞的培养和鉴定：用颈椎脱位法处死SD大鼠,分离获得股骨和胫骨,用DMEM/F12培养基反复冲洗骨髓腔,将冲洗后的骨髓悬液室温状态下离心1000r.p.m,5min。离心结束后,倒掉上清液,用含10%胎牛血清和90%DMEM/F12培养基的细胞全培液重悬细胞,在37℃、5%CO2孵箱中培养,2天后换液,之后每隔2天换液1次。在原代细胞充分融合后,用含0.02%EDTA的25%的胰酶和PBS液(1：1)消化细胞后,传代纯化细胞,所有实验都采用p3代细胞,采用流式细胞术检测间充质干细胞的表面抗原标志CD29, CD90和CD45的表达情况,用台盼蓝检测细胞活力。 2、试验方法：取P3代细胞分为：实验组(次声处理10min,30min,60min)和对照组(空气暴露相同时间),细胞处理结束后,迅速转入37℃、5%CO2孵箱中培养。采用CCK8法检测细胞的增殖活性,流式细胞术进行细胞凋亡和细胞周期分析,以及采用扫描电镜和透射电镜观察细胞的超微结构变化。 增殖实验在96孔板进行,每组1板共6板。实验组和对照组细胞数均调整为4000个/100ul/孔,每组12孔。细胞铺板后放于孵箱中培养3个小时基本贴壁后,对细胞进行次声和空气暴露的处理,之后在每天的相同时间段处理细胞1次,连续处理3天,处理结束后,迅速将细胞转入孵箱中培养。细胞铺板后48小时进行CCK8检测,每天一次,共观察3天,每天每培养板取4个复孔。 凋亡实验和周期分析实验时,将细胞用培养基重悬后,移至5ml冻存管中进行次声和对照组的处理,处理结束后将细胞吹打重悬、转入25cm2培养瓶中,用含10%胎牛血清的DMEM/F12培养基在孵箱中培养3天。 电镜超微结构观察实验时,将细胞用培养基重悬后,移至5ml冻存管中进行次声和对照组的处理,处理时间为60min。用于扫描电镜观察的细胞在处理完后,用全培液重悬并且转入放有消毒盖玻片的培养皿中,迅速移入孵箱中培养,过夜。用于透射电镜观察的细胞在处理完后,转入25cm2培养瓶中,用含10%胎牛血清的DMEM/F12培养基在孵箱中培养4天。 各组数据以(X±S)表示,采用Spss13.0软件处理。对OD值的处理用析因设计资料的方差分析,对凋亡率和细胞周期分布率用t检验分析,P0.05为差异有统计学意义。 结果 1、台盼蓝结果显示,细胞活力在95%以上,可以进行下一步实验。 2、表面标志物检测显示：CD29和CD90阳性,CD45阴性。 3、采用CCK8法检测的结果：采用析因设计的方差分析显示,经次声处理10min、30min和60mmin的BMSCs,培养48h后,OD值分别为(0.929±0.042；1.094±0.013；1.410±0.016),培养72h后,OD值分别为(1.480±0.001；1.348±0.027；1.493±0.017),培养96h后,OD值分别为(1.774±0.127；1.731±0.062；1.833±0.054),对照组三个培养时间的OD值分别为(1.148±0.088；1.147±0.030；1.112±0.051),(1.479±0.051；1.267±0.006；1.227±0.126)和(1.567±0.032；1.563±0.043；1.632±0.071),可见培养72h后次声组细胞OD值均大于对照组,且随着处理时间的延长,细胞OD值呈现递增或先减后增的趋势,在各观察点次声处理60min的BMSCs OD值都最高。采用t检验进行单独效应分析,可见两组间差异有统计学意义(P0.05)。 4、细胞凋亡结果显示：次声处理10mmin和60min时,BMSCs的早期凋亡率分别为(1.07%±0.12%和0.97%±0.21%),相应对照组的早期凋亡率分别为(1.43%±0.06%和3.33%±0.15%),可见次声处理10min和60min后细胞早期凋亡率降低(P0.01),处理60min还可降低BMSCs的中晚期凋亡率、提高正常细胞百分率(P0.01),处理30min时,对BMSCs的细胞凋亡没有影响。 5、细胞周期结果显示：次声处理10min和30min相比,可对BMSCs的细胞周期产生相反的影响,次声处理10min时,静止期细胞多于对照组(P0.05),处理30min时静止期细胞少于对照组(P0.01),而次声处理60min对BMSCs的细胞周期无影响(P0.05) 6、扫描电镜下显示：次声处理60min培养1天的细胞在细胞大小、形状、表面微绒毛程度与空气暴露对照组有较大差异,次声组细胞多舒展开,为长条状,细胞较长,表面微绒毛较多；对照组含有大量圆形透亮细胞,表面微绒毛较少,提示细胞死亡或活力低下。 7、透射电镜下显示：次声处理60min培养4天的细胞与对照组细胞相比,细胞状态良好,细胞器大量存在,细胞核没有明显改变,核膜清晰；对照组有大量细胞出现核破裂、核溶解,可见大量溶酶体出现,提示细胞大量死亡。 结论 1.次声可以促进BMSCs的增殖,没有发现次声引起BMSCs的凋亡,次声处理细胞60mmin后可抑制细胞凋亡；次声作用3天后大多数BMSCs停留在静止期,但次声可以干扰BMSCs的周期分布。次声可对BMSCs的超微结构产生影响,提示次声可以改善细胞活力。 2.次声作用60min后,可以稳定的促进细胞增殖,抑制细胞凋亡,不改变细胞的正常周期分布,对细胞超微结构有积极作用,比较适合在骨髓间充质干细胞培养中应用。
[Abstract]:Research background
The research and application of stem cells is a new way and new hope for regenerative repair and treatment of major diseases. At present, the key technologies such as isolation, identification, culture, expansion, preservation and resuscitation of stem cells have been highly concerned by various research institutions and individuals, and the advantages of BMSCs because of their abundant sources are popular.
The infrasonic wave is a mechanical vibration wave in the range of 0.0001 ~ 20Hz, which may have biological effects such as mechanical effect, thermothermal effect and chemical effect. Infrasound has influence on all systems of human body, can cause biological changes of cells in the body, and can also affect a variety of cultured cells in vitro. Infrasound in cerebral ischemia reperfusion injury and so on. The protection of systemic diseases has a certain effect and can affect the proliferation of neural precursor cells. And BMSCs has also been widely used in nervous system diseases. The mechanism may be realized through the differentiation of nerve in the body, so it is a very necessary subject to carry out the study of the biological effects of infrasound on BMSCs.
At present, domestic scholars have studied the effects of infrasound on the ultrastructure and proliferation of osteoblasts, neural precursor cells, small keratinocytes, corneal cells, cancer cells and so on. There are few reports on the study of BMSCs. However, there has been a study on the biological effects of low intensity ultrasound on BMSCs. Sound can promote the proliferation of BMSCs and the directional differentiation of cartilage. The infrasound is also a mechanical wave, and there may be a common place in this area. In recent years, foreign scholars have studied the infrasound in the biomedical field less, mostly the damage effect of infrasound to the body. In the cell culture, most people think some cells. Factors and amino acids play a certain role in cell culture and differentiation, but some have suggested the role of mechanical factors in this aspect.
The biological effects of infrasonic wave are mainly determined by the parameters of frequency, time, intensity and so on. This subject is mainly to observe the biological effects of the different action time of the infrasound on the BMSCs.
research objective
To observe the biological effects of infrasound on bone marrow mesenchymal stem cells (Bone Marrow Stromal Stem Cells, BMSCs), including proliferation, apoptosis and periodic distribution, and the influence of ultrastructure, and discuss the time parameters of rational use of infrasound. Materials and methods:
1, cell culture and identification: the SD rats were killed by the cervical dislocation method, the femur and tibia were isolated and the bone marrow cavity was washed with DMEM/F12 medium. The bone marrow suspension after the rinse was centrifuged for 1000r.p.m at room temperature. After the 5min. centrifugation, the supernatant was dropped and the full culture of the cells containing 10% fetal bovine serum and 90%DMEM/F12 medium was suspended. The cells were cultured in the 5%CO2 incubator at 37 degrees C, then changed liquid after 2 days, and then changed 1 times every 2 days. After the primary cells were fully fused, the cells were purified with 0.02%EDTA 25% trypsin and PBS solution (1:1), and all the cells were purified. All the experiments were P3 cells, and the surface antigen marker CD29, CD90 and CD90 were detected by flow cytometry. The expression of CD45 was detected by trypan blue.
2, the test method: the P3 generation cells were divided into: the experimental group (infrasound treatment 10min, 30min, 60min) and the control group (air exposure for the same time). After the cell treatment ended, the cells were quickly transferred to 37 C and incubated in 5%CO2 incubator. The cell proliferation activity was detected by CCK8 method, cell apoptosis and cell cycle analysis were carried out by flow cytometry, and the scanning electricity was used. The ultrastructural changes of the cells were observed by microscope and transmission electron microscope.
The proliferation experiment was carried out in 96 orifice plates with 1 plates in each group of 6 plates. The number of cells in the experimental group and the control group were adjusted to 4000 /100ul/ holes, with 12 holes in each group. After the cell planks were placed in the incubator for 3 hours and basically adhered to the wall, the cells were treated with infrasound and air exposure, and the cells were treated for 1 times in the same time each day for 3 days. At the end of the treatment, the cells were transferred to incubator rapidly. CCK8 test was carried out 48 hours after cell planking. Once a day, 3 days were observed, and 4 duplicate holes were taken per culture plate every day.
In the apoptosis and cycle analysis experiments, the cells were suspended in the medium of suspension and transferred to the 5ml cryopreservation tube for the treatment of the infrasound and the control group. After the treatment, the cells were overhung and transferred into the 25cm2 culture bottle, and the DMEM/F12 medium containing 10% fetal bovine serum was cultured for 3 days in the incubator.
When the ultrastructure of the electron microscope was observed, the cells were suspended in the culture medium and transferred to the 5ml cryopreservation tube for the infrasound and the control group. The cells treated with the scanning electron microscope were treated with the scanning electron microscope after the treatment. The cells were suspended in full culture and transferred into the culture dish with disinfectant glass slide. The cells were quickly moved into the incubator and used for the night. The cells observed after transmission electron microscope were transferred to 25cm2 culture bottle after treatment, and incubated in DMEM/F12 incubator containing 10% fetal bovine serum for 4 days.
The data of each group were expressed by (X + S) and treated with Spss13.0 software. The variance analysis of the design data was used for the treatment of OD values. The apoptosis rate and the cell cycle distribution rate were analyzed by t test. The difference of P0.05 was statistically significant.
Result
1, trypan blue showed that the cell viability was above 95%, and the next experiment could be carried out.
2, surface marker detection showed that CD29 and CD90 were positive and CD45 negative.
3, the results of CCK8 test: the analysis of variance in the factorial design showed that after the secondary sound treatment of 10min, 30min and 60mmin BMSCs, after cultivating 48h, the OD values were respectively (0.929 + 0.042; 1.094 + 0.013; 1.410 + 0.016). After cultivating 72h, the OD values were respectively (1.480 + 0.001; 1.348 + 0.027; 1.493 + 0.017). After developing 96h, the OD values were respectively (0.042) 27, 1.731 + 0.062; 1.833 + 0.054), the three cultures of the control group were (1.148 + 0.088; 1.147 + 0.030; 1.112 + 0.051). On the other hand, the BMSCs o value of 60min was the highest at all observation points, and the difference between the two groups was statistically significant (P0.05).
4, the apoptosis results showed that the early apoptosis rate of BMSCs was (1.07% + 0.12% and 0.97% + 0.21%), respectively (1.43% + 0.06% and 3.33% + 0.15%) in the corresponding control group, respectively (1.43% + 0.06% and 3.33% + 0.15%) in the corresponding control group, and the early apoptosis rate decreased (P0.01) after the infrasound treatment of 10min and 60min (P0.01). The treatment of 60min could also reduce the middle and late BMSCs of the BMSCs. The apoptosis rate increased and the percentage of normal cells (P0.01) increased. The treatment of 30min had no effect on the apoptosis of BMSCs.
5, the cell cycle results showed that the infrasound treatment compared with 10min and 30min could have the opposite effect on the cell cycle of BMSCs. When the infrasound treatment was 10min, the resting cells were more than the control group (P0.05), and the stationary phase cells were less than the control group (P0.01) when treating 30min, while the infrasound processing 60min had no effect on the cell cycle of BMSCs (P0.05).
6, the scanning electron microscope showed that the cell size, shape, surface microvilli degree and air exposure control group were different in the cell size, shape and surface microvilli in the secondary sound treatment for 1 days. The cells in the infrasonic group were long, long and with more surface microvilli in the infrasonic group. The control group contained a large number of round bright cells and less surface microvilli, suggesting cells. Death or low vitality.
7, the transmission electron microscope showed that the cells in the secondary sound treatment for 4 days of 60min culture were in good condition, there were a large number of organelles, the nuclei had no obvious changes and the nuclear membrane was clear, and a large number of cells in the control group had nuclear rupture, nuclear dissolution, and a large number of lysosomes appeared, suggesting a large number of cells died.
conclusion
1. infrasound can promote the proliferation of BMSCs. No infrasound causes apoptosis of BMSCs. Infrasound treatment cells 60mmin can inhibit cell apoptosis. Most of BMSCs stays at rest after 3 days of infrasound action, but infrasound can interfere with the periodic distribution of BMSCs. Infrasound can affect the ultrastructure of BMSCs, suggesting that infrasound can improve cell survival. Power.
After the 2. infrasound action of 60min, it can promote cell proliferation, inhibit cell apoptosis, do not change the normal distribution of cell cycle, have positive effect on cell ultrastructure, and be suitable for the application of bone marrow mesenchymal stem cell culture.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

【参考文献】

相关期刊论文 前10条

1 路艳蒙,傅文玉,朴英杰,乔东访,安连兵;人骨髓间充质干细胞的超微结构[J];电子显微学报;2002年04期

2 赵志刚;次声与应激[J];国外医学(物理医学与康复学分册);2000年03期

3 李玉峰;沈加;吴伟岚;陈同辛;陈慧敏;林芊;董瑜;金晶;卫敏江;;MML-1细胞G_1期阻断对Fas诱导细胞凋亡的抑制作用[J];上海交通大学学报(医学版);2011年04期

4 徐晓菲;杜芳;张嘉靖;赵钢;;次声介导大鼠小胶质细胞活化的体外模型[J];神经解剖学杂志;2008年06期

5 林甜;赵钢;江文;刘娟芳;冯冬蕴;史明;;次声对成年大鼠脑室下区神经前体细胞增殖的影响[J];中华神经外科疾病研究杂志;2009年05期

6 王冰水,陈景藻,李玲,任冬青,方恒虎,刘静,陈丹;16Hz次声暴露对人脐静脉血管内皮细胞内钙离子浓度的影响[J];中国临床康复;2005年11期

7 王斌;陈景藻;刘静;郭国珍;;次声波对成骨样细胞生物学特性的影响[J];中国临床康复;2006年25期

8 王斌;陈景藻;刘静;郭国珍;;低强度次声对成骨样细胞骨桥素和骨粘连蛋白mRNA表达的作用[J];中国组织工程研究与临床康复;2007年02期

9 蒋雪清;邓芳;李瑞满;;低声压级水平次声对初产妇盆底损伤治疗的研究[J];中国医药导报;2011年14期

10 王斌;陈景藻;牟翔;刘静;;低强度次声对体外培养的骨样细胞骨架蛋白F-actin表达的动态影响[J];中国康复医学杂志;2007年03期

相关硕士学位论文 前1条

1 鲍勇;次声对人外周血单个核细胞的影响研究[D];南方医科大学;2008年

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