小鼠皮膚傷口內(nèi)源性電場及離子流研究
發(fā)布時間：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)濟負擔。據(jù)統(tǒng)計,美國2008年有650萬慢性傷口患者,醫(yī)療費用超過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。促進傷口愈合是橫跨醫(yī)學眾多領域的關鍵課題,相關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,亟待在基礎理論和臨床應用方面有新的突破。電信號是促進傷口愈合的主導信號,電信號可以刺激并指導修復細胞(如上皮細胞、成纖維細胞、干細胞等皮膚相關細胞)定向生長和遷移。然而,當傷口自然愈合時,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,指導傷口愈合、內(nèi)源性電場形成的規(guī)律、離子機理的研究尚不清楚。本研究以小鼠皮膚傷口為模型,結(jié)合掃描振動電極技術和選擇性離子電極技術對小鼠皮膚傷口的內(nèi)源性電場分布及其離子組成機理進行了系統(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軸方向為內(nèi)流(-28.38±7.69μA/cm2),Y軸方向為外流(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小時有顯著電流產(chǎn)生,且主要在前6小時有較大的電流,24小時后,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)生的離子流動機理及時間空間變化研究,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小時達到最大值,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細胞電場響應的驗證,得到如下結(jié)果:1、Ha Cat細胞對外加電場有趨電反應,單個細胞和成片細胞均向電場的正極遷移;2、Ha Cat成片細胞較單個細胞對電場的反應靈敏;3、HaCat單個細胞在不同電壓強度下表現(xiàn)出逐漸增強的趨電性,在200 m V/mm、400 m V/mm、600 m V/mm的電場強度下,趨電方向性分別為0.13±0.07、0.36±0.06、0.41±0.05;4、HaCat成片細胞則在各個電場強度下的趨電方向性均顯著大于單個細胞,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和選擇性離子電極技術,并應用于組織水平內(nèi)源性電場的研究。首次揭示了小鼠皮膚傷口內(nèi)源性電場的時空分布及變化規(guī)律,深入研究了其離子機理。研究發(fā)現(xiàn)皮膚傷口內(nèi)源性電場分布復雜,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)生的主要貢獻者,且產(chǎn)生的電流方向與實測內(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,在對皮膚細胞(HaCat)的趨電性研究發(fā)現(xiàn),與大多數(shù)細胞對電場方向的響應相反,Ha Cat細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細胞的趨電響應從細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,為生物電促進傷口愈合提供了新的證據(jù),對進一步深入研究傷口內(nèi)源性電場具有重要的指導意義。
[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.
【學位授予單位】：云南師范大學
【學位級別】：博士
【學位授予年份】：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.
【學位授予單位】：云南師范大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R641
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