次声对人外周血单个核细胞的影响研究

发布时间：2018-01-03 20:45

本文关键词：次声对人外周血单个核细胞的影响研究　出处：《南方医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 背景次声是频率在10~(-5)～20Hz之间的机械振动波。随着对次声的研究深入,次声波在疾病诊疗方面的巨大开发潜力已日益受到国内外学者的重视。目前有个别学者认为一定强度的次声能加强人的免疫系统功能,但鲜见关于此的实验及机制研究。人的免疫系统在抗感染和适应环境方面起重要作用,人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)是免疫系统的重要组成部分,是机体完成细胞免疫和体液免疫的重要基础,人外周血单个核细胞可作为反应免疫功能的一个指标,其分类技术(分离液密度梯度离心法)已十分成熟,所以人外周血单个核细胞在临床及实验研究得到广泛的应用。研究目的: 本研究拟在探讨治疗用次声对健康人单个核细胞(PBMC)的生物学效应,包括对健康人PBMC的活性和细胞周期及凋亡的影响,对健康人PBMC的超微结构的影响,为次声对人体免疫系统的影响提供实验依据,为次声治疗提供相关参数。研究假设: 本研究采用的次声治疗剂量对健康人PBMC的存在生物学效应,对健康人PBMC的细胞活性有促进作用,可改变细胞的凋亡率;次声处理使PBMC膜超微结构的改变。材料与方法: 每个健康志愿者为1例样本,共20例(n=20),分离后每例分成3份:次声组(次声处理)、对照组(处理同次声组但次声治疗仪处于关闭状态)、空白组(淋巴细胞分离后一直置于5%CO2饱和湿度的培养箱内培养);对次声治疗仪进行消毒后,超净台内实验处理15 min、30 min、60 min、90 min、120 min,每时间段依次取样后放入5%CO2饱和湿度的培养箱内进行培养。培养24 h、48 h,检测相关指标,包括:台盼蓝染色法、噻唑兰染色法(MTT法)、流式细胞术以及扫描电镜和透射电镜的检测。实验数据采用spss13.0进行统计分析,用重复测量的方差分析方法,多组间比较采用LSD方法。结果: 1台盼蓝染色法(重复3次)结果显示,分离后的细胞存活率达到95%以上,说明整个分离过程对PBMC的损伤不大,不会对下面进行的实验或对照处理产生影响,可进行下一步实验。 2采用MTT法检测的结果显示,实验处理后培养24 h、48 h,次声组的OD值与对照组之间的差异均无统计学意义(P＞0.05);但次声组OD值随处理时间呈依次增大趋势,而对照组这种趋势不明显。此外,各组的OD值的变异系数较大。 3流式细胞仪检测结果显示,各次声处理组与相应的对照组比较,凋亡细胞(早期凋亡,晚期凋亡)以及其他各类细胞的比率,其差异性均无统计学意义(P值均＞0.05)。 4扫描电镜下显示:次声处理作用120 min/培养48 h在细胞的大小、形状、表面微绒毛疏密程度与空白组差异不大;而对照组细胞却有细胞表面微绒毛减少、异形细胞增加等变化。 5透射电镜下显示:次声组与空白组相比,细胞状态尚好,细胞核未有明显改变,核膜清晰但不规则,线粒体略肿胀,细胞膜表面突起略减少。而对照组,细胞外形不规则,细胞微绒毛减少,细胞核呈均质化,个别细胞出现核膜模糊甚至破坏,核溶解,胞质未见结构完整清晰的细胞器,可见自噬泡,其中含有变性的胞质成分。结论: 本研究采用的次声治疗剂量对健康人PBMC的细胞活性可能有一定促进作用,但对其凋亡率的影响不大;次声处理可能会使细胞膜性结构产生变化;次声处理过程可能对PBMC的自然老化过程和外界损害因素的影响有延缓作用;次声所致的PBMC膜超微结构的改变或生物膜通透性的变化可能是其对PBMC的生物学效应的机制之一。总结: 本实验采用的次声对健康人PBMC的细胞活性可能有一定促进作用,进而影响人的免疫功能。但次声对PBMC凋亡影响不明显,次声对PBMC的增殖和细胞周期影响不大。次声可导致PBMC膜超微结构的改变;次声处理过程可能对PBMC的自然老化或凋亡过程和外界损害因素的影响有延缓作用。本研究提示,INFRASOUND 8~(TM)次声治疗仪对健康人PBMC的生物学效应可能与次声对细胞膜超微结构的直接作用有关,次声可影响人的免疫系统功能,但次声对免疫反应中的其他过程的影响有待进一步研究。
[Abstract]:background
Infrasound is at the frequency of 10~ (-5) - mechanical vibration wave 20Hz. Along with the study of infrasound depth, infrasound in clinical diagnosis and treatment of the huge potential for development have been paid more and more attention of scholars at home and abroad. At present, some scholars believe that the function of the immune system of a certain intensity of infrasound can enhance people's, but study on experiment and mechanism about this is rare.
The immune system plays an important role in anti infection and adapt to the environment, human peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMC) is an important part of the immune system, is an important basis for the body to complete the cellular immunity and humoral immunity, an index of human peripheral blood mononuclear cells can be used as immune function the reaction, the classification technology (separation medium density gradient centrifugation) has been very mature, so the human peripheral blood mononuclear cells have been widely used in clinical and experimental research.
The purpose of the study is:
This study was to explore the treatment of infrasound on healthy human mononuclear cells (PBMC) of biological effects, including activity and effect on cell cycle and apoptosis in healthy human PBMC, effects on ultrastructure of healthy human PBMC, to provide the experimental basis for the effect of infrasound on the human immune system, provide relevant parameters for infrasound therapy.
Research hypothesis:
The biological effect of infrasound therapy dose on healthy human PBMC is promoted by this study. It can promote the cell activity of PBMC in healthy people, and change the apoptosis rate of cells. Infrasound treatment can make the ultrastructure of PBMC membrane change.
Materials and methods:
Each healthy volunteers were 1 samples, a total of 20 cases (n=20), separated into 3 cases each: infrasound group (infrasound treatment), control group (treatment group for sound treatment instrument is closed but infrasound), control group (culture box has been placed on the 5%CO2 saturated humidity after separation of lymphocytes); infrasound therapy after disinfection, super clean bench experimental treatment for 15 min, 30 min, 60 min, 90 min, 120 min, each time in the incubator after sampling in 5%CO2 saturated humidity were cultured. After 24 h, 48 h, including the related indicators detection: trypan blue staining, thiazole blue staining method (MTT method), flow cytometry and scanning electron microscopy and transmission electron microscopy.
The experimental data were analyzed by SPSS13.0, and the method of repeated measurement was used for the analysis of variance. The LSD method was used in multiple groups.
Result:
1 trypan blue staining (repeated 3 times) showed that the survival rate of the cells after separation was over 95%, indicating that the whole separation process had little damage to PBMC and would not affect the following experiments or control treatments. Further experiments could be carried out.
2 detected by MTT showed that after the experimental treatment and cultured for 24 h, 48 h, the difference between the OD value of the infrasound group and the control group had no statistical significance (P > 0.05); but the infrasound group od with treatment time showed a trend of increasing, while the control group, this trend is not obvious. In addition, the OD value of each the coefficient of variation is larger.
3 flow cytometry results showed that there was no significant difference in the ratio of apoptotic cells (early apoptosis, late apoptosis) and other kinds of cells between the infrasound treatment group and the corresponding control group (P > 0.05).
4 scanning electron microscope showed: infrasound treatment for 120 min/ and culture for 48 h had no significant difference in cell size, shape and surface microvilli density, while in control group, there was a decrease in cell surface microvilli and an increase in heteromorphic cells.
5 transmission electron microscope: compared with infrasound group and blank control group, cell nucleus state is still good, no obvious change, but the nuclear membrane was clear and irregular, mitochondrial slightly swollen, cell membrane surface were reduced slightly. While the control group cells, irregular shape, microvilli, cell nucleus was homogenized, individual cell membrane fuzzy even destruction, nuclear dissolution, cytoplasmic organelles structure is not complete and clear, visible autophagic vacuoles, which contain cytoplasmic components of degeneration.
Conclusion:
Cell activity dose used in this study of infrasound on healthy human PBMC may have a certain role in promoting, but has little effect on the apoptosis rate; infrasound treatment may cause cell membrane structure changes; infrasound process may affect the natural aging process of PBMC and external damage factors delayed changes; or the biological membrane permeability of PBMC membrane ultrastructure caused by the infrasound could be one of the mechanisms of the biological effects of PBMC.
Summary:
In this experiment, the cell activity of infrasound on healthy human PBMC may have a certain role in promoting, thereby affecting the immune function. But the effect of infrasound on the apoptosis of PBMC is not obvious, infrasound proliferation and cell cycle of PBMC is not affected. Infrasound can cause PBMC membrane ultrastructure change; time sound processing may impact on PBMC the natural aging process and external damage or apoptosis factors have delayed effect.
This study suggested that INFRASOUND 8~ (TM) biological effect of infrasound therapy on healthy human PBMC may be related to the direct effects of infrasound on ultrastructure and cell membrane structure, infrasound can affect the human immune system function, but the effect of infrasound on other processes in the immune response remains to be further studied.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

【引证文献】