体外不同力学刺激对经脉相关筋膜成纤维细胞整合素—细胞骨架信号传导通路影响的研究

发布时间：2018-04-16 05:07

本文选题：针刺 + 经脉　；参考：《贵阳中医学院》2013年硕士论文

【摘要】：目的：探讨体外压力、摩擦力、张力刺激对来源于经脉相关筋膜结缔组织成纤维细胞的整合素-细胞骨架力学信号传导通路的影响,为针推基本机械力学作用的细胞生物物理学作用原理提供新的实验和理论依据。方法：体外利用酶消化法获取经脉(督脉)相关筋膜结缔组织成纤维细胞,鉴定后培养建立来源于经脉相关筋膜结缔组织成纤维细胞的细胞生物学实验平台,并运用不同压力、摩擦力、张力对细胞进行刺激,应用荧光显微镜技术观察成纤维细胞整合素β1分布情况并进行定性、半定量分析；观察成纤维细胞在不同机械力刺激后细胞骨架-微丝形态学变化。结果： (1)不同体外压力、摩擦力、张力刺激对经脉相关筋膜结缔组织成纤维细胞整合素β1表达均有影响,差异有以下不同： ①压力刺激：单次刺激各组中,50Kpa组较空白对照组荧光亮度变化不明显,100Kpa和200Kpa组的荧光亮度均较空白组强；多次刺激各组中,50Kpa组的荧光亮度最亮,100Kpa组的亮度次之,200Kpa组的荧光仍然较空白组强； ②摩擦力刺激：单次刺激各组中,0.5Hz组较空白对照组荧光亮度变化不明显,1Hz和2Hz组的荧光亮度较空白对照组要亮,以1Hz组的亮度最亮；多次刺激各组中,0.5Hz组的荧光亮度最亮,1Hz组与空白对照组的亮度无明显区别,2Hz组的荧光较空白对照组要暗； ③张力刺激：单次刺激各组中,随着刺激强度的增加,荧光亮度逐渐变亮,3000μ strain组的荧光亮度最亮；多次刺激各组中,1000μ strain组的荧光较空白组亮,2000μ strain组的荧光亮度最亮,3000μ strain组与空白对照组的亮度无明显区别。 (2)不同体外压力、摩擦力、张力刺激对经脉相关筋膜结缔组织成纤维细胞微丝形态结构及染色有影响,其影响有： ①压力刺激：单次刺激各组中,50Kpa和100Kpa组可见微丝与细胞核染色加深,微丝排列较空白对照组无明显区别,200Kpa组可见微丝与细胞核染色明显加深,微丝排列较空白对照组仍无明显区别；多次刺激各组中,50Kpa组微丝及细胞核染色加深,微丝分布较为散乱,少有贯穿细胞全长,100Kpa组较空白对照组微丝及细胞核染色明显深染,微丝明显解聚变短,排列无规则,出现少许点状分布于胞内为解聚后的近肌球蛋白,偶可见细胞膜丝状伪足；200Kpa多次加力组细胞核与微丝更加深染,微丝明显解聚,排列无规则,出现大量点状分布于胞内为解聚后的近肌球蛋白,细胞膜周缘极不规则,可见大量丝状伪足。 ②摩擦力刺激：单次刺激各组中,0.5Hz和1Hz组较空白对照组变化不大,可见微丝与细胞核染色加深,微丝排列较空白对照组无明显区别,2Hz组可见微丝与细胞核染色明显加深,微丝排列较空白对照组散乱；多次刺激各组中,0.5Hz组较空白对照组微丝及细胞核染色加深微丝分布较为散乱,微丝解聚变短,出现点状分布于胞内,未见丝状伪足,1Hz组细胞核与微丝明显深染,微丝明显解聚变短,排列无规则,出现点状分布于胞内为解聚后的近肌球蛋白,偶可见细胞膜丝状伪足,2Hz组细胞核与微丝更加深染,微丝明显解聚,排列无规则,出现大量点状分布于胞内。 ③张力刺激：单次刺激各组中,1000μ strain组可见细胞核及微丝染色加深,微丝排列较空白对照组稍乱；2000μ strain组可见细胞核及微丝染色加深,微丝排列较乱,明显解聚变短,排列无规则；3000μ strain组可见细胞核及微丝染色加深,微丝排列较乱,微丝明显解聚变短,排列无规则,偶可见点状颗粒分布于胞内；多次刺激各组中,1000μ strain组可见细胞核及微丝染色加深,微丝明显解聚变短,排列无规则,出现少许点状分布于胞内为解聚后的近肌球蛋白；2000μstrain组可见细胞核及微丝染色加深,微丝明显解聚变短,排列无规则,出现少许点状分布于胞内,出现沿力学方向细胞微丝与细胞核“脱离”；3000μ strain组可见细胞核及微丝染色加深,微丝明显解聚变短,排列无规则,出现少许点状分布于胞内,出现沿力学方向细胞微丝与细胞核“脱离”。结论：体外压力、摩擦力和张力刺激过程中,经脉相关筋膜结缔组织成纤维细胞整合素—细胞骨架系统中整合素表达量和微丝形态学能发生相应改变。 (1)经脉相关筋膜结缔组织成纤维细胞力感受分子整合素β1在感受不同体外压力、摩擦力、张力刺激的共同规律和差异特征是： ①共同规律：单次刺激情况下,整合素β1在中重度刺激强度下表达量高,多次刺激情况下,整合素β1在轻中度刺激强度下表达量高。 ②差异特征：无论单次刺激还是多次刺激情况下,整合素β1在外力刺激下表达量总体虽成增高态势,但多次重度摩擦力刺激情况下,整合素β1表达相对空白状态呈现“翻转”情形,整合素β1表达受到抑制,表达量下降。 (2)经脉相关筋膜结缔组织成纤维细胞力传递通路细胞骨架微丝在传导不同体外压力、摩擦力、张力刺激的共同规律和差异特征是： ①共同规律：无论单次刺激还是多次刺激情况下,微丝形态结构的改变均呈现渐变特征,并且多次刺激的渐变特征比单次刺激的渐变特征变化明显。 ②差异特征：微丝在传导张力刺激时,其形态结构的改变比在传导压力和摩擦力刺激时更明显。 (3)体外压力、摩擦力和张力刺激过程中,经脉相关筋膜结缔组织成纤维细胞整合素表达量和微丝形态学改变的规律从细胞力学角度可能揭示了针推基本机械力刺激在经脉相关筋膜结缔组织成纤维细胞的物理学信号感受和传导的细胞生物学原理,其针灸推拿学的临床意义可能是： ①从信号感受的角度看：在临床治疗上,单次或短时间周期的治疗,可能适宜采用较重手法,使信号感受更为强烈；多次或长时间周期的治疗可能则适宜使用较轻的手法,使经穴的感受保持在较高水平。 ②从信号传递的角度看：能使经络腧穴受到张力刺激的手法,其信号传递效率更高。
[Abstract]:Objective : To study the effects of external pressure , friction and tension stimulation on the conduction path of integrin - cytoskeletal mechanics signals derived from the connective tissue fibroblasts of the meridians and provide a new experimental and theoretical basis for the principle of cellular biophysics for the action of the basic mechanical mechanism of the needle .

Methods : The cells were cultured in vitro by enzyme digestion method . The cell biological experiment platform derived from the connective tissue fibroblasts was established , and the cells were stimulated with different pressure , friction and tension , and the distribution of integrin 尾 1 was observed by fluorescence microscope .
The cytoskeletal - microfilament morphological changes of fibroblasts after stimulation with different mechanical forces were observed .

Results :

( 1 ) The different in vitro pressure , friction and tension stimulation had an influence on the expression of integrin 尾 1 in the connective tissue fibroblasts . The difference was as follows :

( 1 ) Pressure stimulation : The fluorescence intensity of 50Kpa group was less than that of blank control group , and the fluorescence intensity of 100Kpa and 200Kpa group was higher than that in blank group .
In each group , the brightness of 50 Kpa group was the brightest , and the brightness of the 100 Kpa group was the second , and the fluorescence intensity in 200Kpa group was still higher than that in the blank group .

( 2 ) Friction stimulation : In single stimulation group , the change of fluorescence intensity of the 0 . 5Hz group compared with the blank control group was not obvious , the fluorescence intensity of the 1Hz and 2Hz groups was brighter than that of the blank control group , and the brightness of the 1Hz group was brightest ;
In each group , the brightness of the 0 . 5Hz group was the brightest , the brightness of the 1Hz group was not significantly different from that of the blank control group , and the fluorescence of the 2Hz group was darker than that of the blank control group .

( 3 ) Tension stimulation : With the increase of stimulation intensity , the fluorescence intensity gradually became brighter and the fluorescence intensity of 3000 渭strain group was brightest with the increase of stimulus intensity .
In each group , the fluorescence intensity of the 1000 渭strain group was brighter than that in the blank group , and the brightness of the 2000 渭strain group was the brightest , and the brightness of the 3000 渭strain group was not significantly different from that of the blank control group .

( 2 ) The effects of different external pressure , friction and tension stimulation on the morphology and dyeing of the fibroblasts in the connective tissue fibroblasts were affected .

( 1 ) Pressure stimulation : There was no significant difference between microfilament and nucleus staining in 50Kpa and 100Kpa group , and there was no significant difference between the microfilaments and the blank control group .
The microfilament and the nucleus of 50Kpa group were more extensive than those in the control group . The microfilaments and the nuclei of the 100 Kpa group were significantly darker than those in the blank control group .
In 200Kpa , the nucleus and the microfilament were more deeply stained , the microfilaments were obviously depolymerized , the arrangement was irregular , and there appeared a large number of near - myosin light spots distributed in the cells , and the periphery of the cell membrane was very irregular , and a large number of filiform pseudopods can be seen .

( 2 ) Friction stimulation : In single stimulation group , the changes of the 0 . 5Hz and 1 Hz group were not great than that in the blank control group .
In each group , the microfilament and nucleus staining of 0.5 Hz group were more extensive than that in the control group , and the microfilaments were dispersed in the cytoplasm .

( 3 ) Tension stimulation : In single stimulation group , the nucleus and microfilament staining of 1000 渭strain group were deepened , and the microfilament arrangement was slightly disturbed than the blank control group .
In 200渭strain group , the nucleus and microfilament staining was deepened , the microfilament arrangement was disordered , the obvious defusion was short , and the arrangement was irregular ;
In 3000 渭strain group , the nucleus and microfilament staining were deepened , the microfilament arrangement was disordered , and the microfilaments were obviously defocused , the arrangement was irregular , and even visible dot - like particles were distributed in the cells ;
In each group , the nuclei and microfilament staining of 1000 渭strain group were deepened , and the microfilaments were significantly shorter and the arrangement was irregular , and little dot - like distribution was found in the cells .
In 2000 渭strain group , the nucleus and microfilament staining was deepened , and the microfilaments were significantly shorter and the arrangement was irregular , and a few spots appeared in the cells , and the cells in the mechanical direction were separated from the cell nucleus .
In the 3000 渭strain group , the nucleus and microfilament staining were deepened , and the microfilaments were significantly shorter and the arrangement was irregular , and a little dot - like distribution was found in the cell , and the cell microfilaments in the mechanical direction were separated from the nucleus .

Conclusion : In vitro pressure , frictional force and tension stimulation , the expression of integrin and the morphology of microfilaments can be changed accordingly .

( 1 ) The common law and difference between the stress , friction and tension of the muscle cells of the connective tissue of the warp - related fascia in vitro were :

( 1 ) Under the condition of single stimulus , the expression of integrin 尾 1 under moderate and severe stimulation intensity was high , and the expression of integrin 尾 1 under mild and moderate stimulation intensity was high .

( 2 ) In the case of single stimulus or multiple stimulation , the expression of integrin 尾 1 under external force was generally increased , but in the case of multiple severe friction stimulation , the expression of integrin 尾 1 was inhibited and the expression of integrin 尾 1 was decreased .

( 2 ) The common law and difference between the different in vitro stress , friction and tension stimulation of the cell skeleton microfilaments of the connective tissue fibroblasts of the warp and vein are :

( 1 ) The common law : the change of the morphology of the microfilaments showed gradual change in the case of single stimulus or multiple stimulation , and the gradual change of multiple stimuli was more obvious than that of single stimulus .

( 2 ) The difference feature : when the microfilaments are stimulated by the conduction tension , the change of the morphological structure is more obvious than when the conduction pressure and the frictional force are stimulated .

( 3 ) In the process of external pressure , friction and tension stimulation , the regulation of the expression of integrin expression and morphology of microfilaments in the related fascia connective tissue may reveal the basic mechanical force stimulation of needle pushing on the physical signal sensing and conduction of the connective tissue fibroblasts in the vein . The clinical significance of acupuncture and moxibustion may be as follows :

( 1 ) From the point of view of signal experience : in clinical treatment , single or short period of treatment , it may be appropriate to use a heavy technique to make the signal feel more intense ;
Multiple or long periods of treatment may be appropriate to use a lighter technique to maintain the feel of the acupoints at a higher level .

( 2 ) From the point of view of signal transmission : the meridian points can be stimulated by tension , and the signal transmission efficiency is higher .

【学位授予单位】：贵阳中医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R686.3

【参考文献】