小鼠皮膚傷口內(nèi)源性電場及離子流研究
發(fā)布時(shí)間：2018-08-27 17:16
【摘要】：現(xiàn)代社會,老齡和各種代謝性疾病(例如,糖尿病)引起的一系列難愈合或慢性傷口(潰瘍)是較大的健康和經(jīng)濟(jì)負(fù)擔(dān)。據(jù)統(tǒng)計(jì),美國2008年有650萬慢性傷口患者,醫(yī)療費(fèi)用超過250億美元,中國難愈創(chuàng)面患者發(fā)病率為1.5%~3.0%,以創(chuàng)傷感染為主,60歲以上的老人居多,多見糖尿病足和各種壓迫性、靜脈性潰瘍。促進(jìn)傷口愈合是橫跨醫(yī)學(xué)眾多領(lǐng)域的關(guān)鍵課題,相關(guān)研究主要集中在藥物(例如,生長因子)以及各種仿生敷料,但真正用于臨床的有效方法非常有限,亟待在基礎(chǔ)理論和臨床應(yīng)用方面有新的突破。電信號是促進(jìn)傷口愈合的主導(dǎo)信號,電信號可以刺激并指導(dǎo)修復(fù)細(xì)胞(如上皮細(xì)胞、成纖維細(xì)胞、干細(xì)胞等皮膚相關(guān)細(xì)胞)定向生長和遷移。然而,當(dāng)傷口自然愈合時(shí),內(nèi)源性電場的特性、變化規(guī)律和產(chǎn)生原理研究方法極為有限,指導(dǎo)傷口愈合、內(nèi)源性電場形成的規(guī)律、離子機(jī)理的研究尚不清楚。本研究以小鼠皮膚傷口為模型,結(jié)合掃描振動電極技術(shù)和選擇性離子電極技術(shù)對小鼠皮膚傷口的內(nèi)源性電場分布及其離子組成機(jī)理進(jìn)行了系統(tǒng)的研究,結(jié)果如下:皮膚傷口內(nèi)源性電場的時(shí)間空間變化研究,得到如下結(jié)果:A、小鼠皮膚傷口邊緣在Z軸方向和Y軸方向均有顯著高于完整皮膚(-2.81±0.78μA/cm2)的電流,Z軸方向?yàn)閮?nèi)流(-28.38±7.69μA/cm2),Y軸方向?yàn)橥饬?18.48±1.54μA/cm2)。小鼠皮膚傷口電流在水平面呈傷口邊緣較大,傷口中心(-9.18±3.55μA/cm2)較小的趨勢;在縱切面,靠近表皮為(15.61±2.17μA/cm2)外流,靠近真皮為內(nèi)流(-9.90±2.25μA/cm2);B、小鼠皮膚傷口從制造傷口到傷口愈合的過程中,僅在前24小時(shí)有顯著電流產(chǎn)生,且主要在前6小時(shí)有較大的電流,24小時(shí)后,電流降為-3.66±1.32μA/cm2,與完整皮膚相比無顯著差異。皮膚傷口內(nèi)源性電流產(chǎn)生的離子流動機(jī)理及時(shí)間空間變化研究,發(fā)現(xiàn):A、鈉、鉀、鈣、氯四種離子在小鼠皮膚傷口處的離子流均在傷口形成后的前2小時(shí)達(dá)到最大值,在新鮮傷口邊緣的鈉、鉀、鈣離子流均為內(nèi)流,分別為-76.66±48.59 nmol·cm-2·s-1、-4.734±0.73 nmol·cm-2·s-1、-1.48±0.35 nmol·cm-2·s-1,氯離子流為外流,大小為224.08±29.38 nmol·cm-2·s-1,而傷口中心的鈉、鉀、鈣、氯離子流則均小于傷口邊緣,分別為-36.46±15.91 nmol·cm-2·s-1、-0.98±0.09 nmol·cm-2·s-1、-0.54±0.27 nmol·cm-2·s-1和84.08±16.80 nmol·cm-2·s-1,四種離子流產(chǎn)生的電流方向與實(shí)際測量的電流方向一致。B、鈉離子和氯離子是構(gòu)成傷口內(nèi)源性電場的主要離子,鈉離子流和氯離子流在水平面的分布與電流一致,且方向一致,而在縱切面的分布則在靠近真皮的位置一致。C、用4μM的ANO1抑制劑處理傷口后,小鼠皮膚傷口電流降為-7.86±1.51μA/cm2,較對照有極顯著差異(p0.001,n=3),在50μM的Furosemide處理傷口之后,小鼠皮膚傷口電流降為-12.98±3.50μA/cm2,較對照有顯著差異(p0.01,n=4)。對Ha Cat細(xì)胞電場響應(yīng)的驗(yàn)證,得到如下結(jié)果:1、Ha Cat細(xì)胞對外加電場有趨電反應(yīng),單個(gè)細(xì)胞和成片細(xì)胞均向電場的正極遷移;2、Ha Cat成片細(xì)胞較單個(gè)細(xì)胞對電場的反應(yīng)靈敏;3、HaCat單個(gè)細(xì)胞在不同電壓強(qiáng)度下表現(xiàn)出逐漸增強(qiáng)的趨電性,在200 m V/mm、400 m V/mm、600 m V/mm的電場強(qiáng)度下,趨電方向性分別為0.13±0.07、0.36±0.06、0.41±0.05;4、HaCat成片細(xì)胞則在各個(gè)電場強(qiáng)度下的趨電方向性均顯著大于單個(gè)細(xì)胞,分別為0.68±0.06、0.82±0.04、0.78±0.04。本研究建立了掃描振動電極技術(shù)和選擇性離子電極技術(shù),并應(yīng)用于組織水平內(nèi)源性電場的研究。首次揭示了小鼠皮膚傷口內(nèi)源性電場的時(shí)空分布及變化規(guī)律,深入研究了其離子機(jī)理。研究發(fā)現(xiàn)皮膚傷口內(nèi)源性電場分布復(fù)雜,與角膜傷口的內(nèi)源性電場分布有較大區(qū)別,且與理論推測的皮膚傷口內(nèi)源性電場分布也有一定差異。通過對小鼠皮膚傷口離子流的研究發(fā)現(xiàn),氯離子和鈉離子流是傷口內(nèi)源性電流產(chǎn)生的主要貢獻(xiàn)者,且產(chǎn)生的電流方向與實(shí)測內(nèi)源性電場方向一致,印證了對傷口內(nèi)源性電場分布的研究結(jié)果。此外,在對皮膚細(xì)胞(HaCat)的趨電性研究發(fā)現(xiàn),與大多數(shù)細(xì)胞對電場方向的響應(yīng)相反,Ha Cat細(xì)胞在電場中向電場正極遷移。對傷口內(nèi)源性電場的研究也表明傷口中心是內(nèi)源性電場的正極,HaCat細(xì)胞的趨電響應(yīng)從細(xì)胞層面解釋了小鼠皮膚傷口內(nèi)源性電場的分布狀況與角膜傷口不同的原因,為生物電促進(jìn)傷口愈合提供了新的證據(jù),對進(jìn)一步深入研究傷口內(nèi)源性電場具有重要的指導(dǎo)意義。
[Abstract]:In modern society, a series of hard-to-heal or chronic wounds (ulcers) caused by aging and various metabolic diseases (e.g. diabetes) are a great health and economic burden. According to statistics, there were 6.5 million patients with chronic wounds in the United States in 2008, with medical costs exceeding 25 billion US dollars. The incidence of hard-to-heal wounds in China ranged from 1.5% to 3.0%. Promoting wound healing is a key issue across many fields of medicine. Relevant research mainly focuses on drugs (e.g., growth factors) and various bionic dressings, but the effective methods for clinical use are very limited and need to be in basic theory and clinical practice urgently. New breakthroughs have been made in applications. Electrical signals are the leading signals for wound healing. Electrical signals can stimulate and guide the directional growth and migration of repair cells (such as epithelial cells, fibroblasts, stem cells and other skin-related cells). In this study, the endogenous electric field distribution and ionic composition mechanism of skin wounds in mice were systematically studied by scanning vibrating electrode and selective ion electrode techniques. The results are as follows: A, the skin wound edge of mice in Z-axis direction and Y-axis direction were significantly higher than that of intact skin (-2.81 (-0.78 mu A/cm2)), Z-axis direction was internal (-28.38 (-7.69 mu A/cm2), Y-axis direction was outflow (-18.48 (-1.54 mu A/cm2). In the vertical section, the outflow near the epidermis was (15.61 [2.17] A / cm2) and the outflow near the dermis was (- 9.90 [2.25] A / cm2). In B, there was a significant current generation in the first 24 hours of wound healing, mainly in the first 6 hours. After 24 hours, the current decreased to - 3.66 6550 The maximum values of sodium, potassium and calcium ion currents at the edge of fresh wounds were - 76.66 (+ 48.59) nmol (+) - cm - 2 (+) - S - 1, - 4.734 (+) - 0.73 nmol (+) - cm - 2 (+) - S - 1, - 1.48 (+) - 0.35 nmol (+) - cm - 2 (+) - S - 1), and chloride ion currents at the center of the wound were smaller than those at the edge of the wound, respectively, and the values of sodium, potassium, calcium and chloride ion were 224.08 (+) - 29.38 nmol (+) - 2 ( The current direction produced by the four ionic currents is consistent with the measured current direction. B. Sodium and chloride ions are the main ions in the wound endogenous electric field. Sodium and chloride ion currents and chloride ion currents are at the level. C. After treating the wound with 4 mu M of A NO1 inhibitor, the skin wound current of the mice decreased to - 7.86 (+ 1.51 mu A / cm2), which was significantly different from that of the control group (p0.001, n = 3). After treating the wound with 50 mu M of Furosemide, the skin wound current of the mice decreased. The response of HaCat cells to the electric field was verified as follows: 1. HaCat cells showed electrotaxis to the applied electric field, and single cells and slice cells migrated to the positive electrode of the electric field; 2. HaCat slice cells were more sensitive to the electric field than single cells; 3. HaCat single cells were in the presence of a single cell. At 200 m V/m m, 400 m V/m m and 600 m V/m m, the electrotaxis of HaCat patch cells were 0.13 (+ 0.07), 0.36 (+ 0.06), 0.41 (+ 0.05) and 0.68 (+ 0.06), 0.82 (+ 0.04), 0.78 (+ 0.04) respectively. Scanning vibrating electrode technique and selective ion electrode technique were established and applied to the study of endogenous electric field at tissue level. The temporal and spatial distribution and variation of endogenous electric field in skin wounds of mice were revealed for the first time, and the ionic mechanism was studied. The distribution of the endogenous electric field in the mouth is quite different from that in the skin wound, and it is also different from that in the skin wound. In addition, the electrotaxis of skin cells (HaCat) showed that, contrary to the response of most cells to the electric field, HaCat cells migrated to the positive electrode of the electric field. Cellular electrotaxis can explain the difference between the distribution of endogenous electric field in skin wounds and corneal wounds on the cellular level, and provide new evidence for wound healing promoted by bioelectricity.
【學(xué)位授予單位】：云南師范大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R641
[Abstract]:In modern society, a series of hard-to-heal or chronic wounds (ulcers) caused by aging and various metabolic diseases (e.g. diabetes) are a great health and economic burden. According to statistics, there were 6.5 million patients with chronic wounds in the United States in 2008, with medical costs exceeding 25 billion US dollars. The incidence of hard-to-heal wounds in China ranged from 1.5% to 3.0%. Promoting wound healing is a key issue across many fields of medicine. Relevant research mainly focuses on drugs (e.g., growth factors) and various bionic dressings, but the effective methods for clinical use are very limited and need to be in basic theory and clinical practice urgently. New breakthroughs have been made in applications. Electrical signals are the leading signals for wound healing. Electrical signals can stimulate and guide the directional growth and migration of repair cells (such as epithelial cells, fibroblasts, stem cells and other skin-related cells). In this study, the endogenous electric field distribution and ionic composition mechanism of skin wounds in mice were systematically studied by scanning vibrating electrode and selective ion electrode techniques. The results are as follows: A, the skin wound edge of mice in Z-axis direction and Y-axis direction were significantly higher than that of intact skin (-2.81 (-0.78 mu A/cm2)), Z-axis direction was internal (-28.38 (-7.69 mu A/cm2), Y-axis direction was outflow (-18.48 (-1.54 mu A/cm2). In the vertical section, the outflow near the epidermis was (15.61 [2.17] A / cm2) and the outflow near the dermis was (- 9.90 [2.25] A / cm2). In B, there was a significant current generation in the first 24 hours of wound healing, mainly in the first 6 hours. After 24 hours, the current decreased to - 3.66 6550 The maximum values of sodium, potassium and calcium ion currents at the edge of fresh wounds were - 76.66 (+ 48.59) nmol (+) - cm - 2 (+) - S - 1, - 4.734 (+) - 0.73 nmol (+) - cm - 2 (+) - S - 1, - 1.48 (+) - 0.35 nmol (+) - cm - 2 (+) - S - 1), and chloride ion currents at the center of the wound were smaller than those at the edge of the wound, respectively, and the values of sodium, potassium, calcium and chloride ion were 224.08 (+) - 29.38 nmol (+) - 2 ( The current direction produced by the four ionic currents is consistent with the measured current direction. B. Sodium and chloride ions are the main ions in the wound endogenous electric field. Sodium and chloride ion currents and chloride ion currents are at the level. C. After treating the wound with 4 mu M of A NO1 inhibitor, the skin wound current of the mice decreased to - 7.86 (+ 1.51 mu A / cm2), which was significantly different from that of the control group (p0.001, n = 3). After treating the wound with 50 mu M of Furosemide, the skin wound current of the mice decreased. The response of HaCat cells to the electric field was verified as follows: 1. HaCat cells showed electrotaxis to the applied electric field, and single cells and slice cells migrated to the positive electrode of the electric field; 2. HaCat slice cells were more sensitive to the electric field than single cells; 3. HaCat single cells were in the presence of a single cell. At 200 m V/m m, 400 m V/m m and 600 m V/m m, the electrotaxis of HaCat patch cells were 0.13 (+ 0.07), 0.36 (+ 0.06), 0.41 (+ 0.05) and 0.68 (+ 0.06), 0.82 (+ 0.04), 0.78 (+ 0.04) respectively. Scanning vibrating electrode technique and selective ion electrode technique were established and applied to the study of endogenous electric field at tissue level. The temporal and spatial distribution and variation of endogenous electric field in skin wounds of mice were revealed for the first time, and the ionic mechanism was studied. The distribution of the endogenous electric field in the mouth is quite different from that in the skin wound, and it is also different from that in the skin wound. In addition, the electrotaxis of skin cells (HaCat) showed that, contrary to the response of most cells to the electric field, HaCat cells migrated to the positive electrode of the electric field. Cellular electrotaxis can explain the difference between the distribution of endogenous electric field in skin wounds and corneal wounds on the cellular level, and provide new evidence for wound healing promoted by bioelectricity.
【學(xué)位授予單位】：云南師范大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R641
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