本文選題：針刺 + 經(jīng)脈　； 參考：《貴陽中醫(yī)學院》2013年碩士論文

【摘要】：目的：探討體外壓力、摩擦力、張力刺激對來源于經(jīng)脈相關(guān)筋膜結(jié)締組織成纖維細胞的整合素-細胞骨架力學信號傳導通路的影響,為針推基本機械力學作用的細胞生物物理學作用原理提供新的實驗和理論依據(jù)。 方法：體外利用酶消化法獲取經(jīng)脈(督脈)相關(guān)筋膜結(jié)締組織成纖維細胞,鑒定后培養(yǎng)建立來源于經(jīng)脈相關(guān)筋膜結(jié)締組織成纖維細胞的細胞生物學實驗平臺,并運用不同壓力、摩擦力、張力對細胞進行刺激,應用熒光顯微鏡技術(shù)觀察成纖維細胞整合素β1分布情況并進行定性、半定量分析；觀察成纖維細胞在不同機械力刺激后細胞骨架-微絲形態(tài)學變化。 結(jié)果： (1)不同體外壓力、摩擦力、張力刺激對經(jīng)脈相關(guān)筋膜結(jié)締組織成纖維細胞整合素β1表達均有影響,差異有以下不同： ①壓力刺激：單次刺激各組中,50Kpa組較空白對照組熒光亮度變化不明顯,100Kpa和200Kpa組的熒光亮度均較空白組強；多次刺激各組中,50Kpa組的熒光亮度最亮,100Kpa組的亮度次之,200Kpa組的熒光仍然較空白組強； ②摩擦力刺激：單次刺激各組中,0.5Hz組較空白對照組熒光亮度變化不明顯,1Hz和2Hz組的熒光亮度較空白對照組要亮,以1Hz組的亮度最亮；多次刺激各組中,0.5Hz組的熒光亮度最亮,1Hz組與空白對照組的亮度無明顯區(qū)別,2Hz組的熒光較空白對照組要暗； ③張力刺激：單次刺激各組中,隨著刺激強度的增加,熒光亮度逐漸變亮,3000μ strain組的熒光亮度最亮；多次刺激各組中,1000μ strain組的熒光較空白組亮,2000μ strain組的熒光亮度最亮,3000μ strain組與空白對照組的亮度無明顯區(qū)別。 (2)不同體外壓力、摩擦力、張力刺激對經(jīng)脈相關(guān)筋膜結(jié)締組織成纖維細胞微絲形態(tài)結(jié)構(gòu)及染色有影響,其影響有： ①壓力刺激：單次刺激各組中,50Kpa和100Kpa組可見微絲與細胞核染色加深,微絲排列較空白對照組無明顯區(qū)別,200Kpa組可見微絲與細胞核染色明顯加深,微絲排列較空白對照組仍無明顯區(qū)別；多次刺激各組中,50Kpa組微絲及細胞核染色加深,微絲分布較為散亂,少有貫穿細胞全長,100Kpa組較空白對照組微絲及細胞核染色明顯深染,微絲明顯解聚變短,排列無規(guī)則,出現(xiàn)少許點狀分布于胞內(nèi)為解聚后的近肌球蛋白,偶可見細胞膜絲狀偽足；200Kpa多次加力組細胞核與微絲更加深染,微絲明顯解聚,排列無規(guī)則,出現(xiàn)大量點狀分布于胞內(nèi)為解聚后的近肌球蛋白,細胞膜周緣極不規(guī)則,可見大量絲狀偽足。 ②摩擦力刺激：單次刺激各組中,0.5Hz和1Hz組較空白對照組變化不大,可見微絲與細胞核染色加深,微絲排列較空白對照組無明顯區(qū)別,2Hz組可見微絲與細胞核染色明顯加深,微絲排列較空白對照組散亂；多次刺激各組中,0.5Hz組較空白對照組微絲及細胞核染色加深微絲分布較為散亂,微絲解聚變短,出現(xiàn)點狀分布于胞內(nèi),未見絲狀偽足,1Hz組細胞核與微絲明顯深染,微絲明顯解聚變短,排列無規(guī)則,出現(xiàn)點狀分布于胞內(nèi)為解聚后的近肌球蛋白,偶可見細胞膜絲狀偽足,2Hz組細胞核與微絲更加深染,微絲明顯解聚,排列無規(guī)則,出現(xiàn)大量點狀分布于胞內(nèi)。 ③張力刺激：單次刺激各組中,1000μ strain組可見細胞核及微絲染色加深,微絲排列較空白對照組稍亂；2000μ strain組可見細胞核及微絲染色加深,微絲排列較亂,明顯解聚變短,排列無規(guī)則；3000μ strain組可見細胞核及微絲染色加深,微絲排列較亂,微絲明顯解聚變短,排列無規(guī)則,偶可見點狀顆粒分布于胞內(nèi)；多次刺激各組中,1000μ strain組可見細胞核及微絲染色加深,微絲明顯解聚變短,排列無規(guī)則,出現(xiàn)少許點狀分布于胞內(nèi)為解聚后的近肌球蛋白；2000μstrain組可見細胞核及微絲染色加深,微絲明顯解聚變短,排列無規(guī)則,出現(xiàn)少許點狀分布于胞內(nèi),出現(xiàn)沿力學方向細胞微絲與細胞核“脫離”；3000μ strain組可見細胞核及微絲染色加深,微絲明顯解聚變短,排列無規(guī)則,出現(xiàn)少許點狀分布于胞內(nèi),出現(xiàn)沿力學方向細胞微絲與細胞核“脫離”。 結(jié)論：體外壓力、摩擦力和張力刺激過程中,經(jīng)脈相關(guān)筋膜結(jié)締組織成纖維細胞整合素—細胞骨架系統(tǒng)中整合素表達量和微絲形態(tài)學能發(fā)生相應改變。 (1)經(jīng)脈相關(guān)筋膜結(jié)締組織成纖維細胞力感受分子整合素β1在感受不同體外壓力、摩擦力、張力刺激的共同規(guī)律和差異特征是： ①共同規(guī)律：單次刺激情況下,整合素β1在中重度刺激強度下表達量高,多次刺激情況下,整合素β1在輕中度刺激強度下表達量高。 ②差異特征：無論單次刺激還是多次刺激情況下,整合素β1在外力刺激下表達量總體雖成增高態(tài)勢,但多次重度摩擦力刺激情況下,整合素β1表達相對空白狀態(tài)呈現(xiàn)“翻轉(zhuǎn)”情形,整合素β1表達受到抑制,表達量下降。 (2)經(jīng)脈相關(guān)筋膜結(jié)締組織成纖維細胞力傳遞通路細胞骨架微絲在傳導不同體外壓力、摩擦力、張力刺激的共同規(guī)律和差異特征是： ①共同規(guī)律：無論單次刺激還是多次刺激情況下,微絲形態(tài)結(jié)構(gòu)的改變均呈現(xiàn)漸變特征,并且多次刺激的漸變特征比單次刺激的漸變特征變化明顯。 ②差異特征：微絲在傳導張力刺激時,其形態(tài)結(jié)構(gòu)的改變比在傳導壓力和摩擦力刺激時更明顯。 (3)體外壓力、摩擦力和張力刺激過程中,經(jīng)脈相關(guān)筋膜結(jié)締組織成纖維細胞整合素表達量和微絲形態(tài)學改變的規(guī)律從細胞力學角度可能揭示了針推基本機械力刺激在經(jīng)脈相關(guān)筋膜結(jié)締組織成纖維細胞的物理學信號感受和傳導的細胞生物學原理,其針灸推拿學的臨床意義可能是： ①從信號感受的角度看：在臨床治療上,單次或短時間周期的治療,可能適宜采用較重手法,使信號感受更為強烈；多次或長時間周期的治療可能則適宜使用較輕的手法,使經(jīng)穴的感受保持在較高水平。 ②從信號傳遞的角度看：能使經(jīng)絡(luò)腧穴受到張力刺激的手法,其信號傳遞效率更高。
[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 .

【學位授予單位】：貴陽中醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R686.3

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