小鼠皮肤伤口内源性电场及离子流研究
发布时间：2018-08-27 17:16
【摘要】：现代社会,老龄和各种代谢性疾病(例如,糖尿病)引起的一系列难愈合或慢性伤口(溃疡)是较大的健康和经济负担。据统计,美国2008年有650万慢性伤口患者,医疗费用超过250亿美元,中国难愈创面患者发病率为1.5%~3.0%,以创伤感染为主,60岁以上的老人居多,多见糖尿病足和各种压迫性、静脉性溃疡。促进伤口愈合是横跨医学众多领域的关键课题,相关研究主要集中在药物(例如,生长因子)以及各种仿生敷料,但真正用于临床的有效方法非常有限,亟待在基础理论和临床应用方面有新的突破。电信号是促进伤口愈合的主导信号,电信号可以刺激并指导修复细胞(如上皮细胞、成纤维细胞、干细胞等皮肤相关细胞)定向生长和迁移。然而,当伤口自然愈合时,内源性电场的特性、变化规律和产生原理研究方法极为有限,指导伤口愈合、内源性电场形成的规律、离子机理的研究尚不清楚。本研究以小鼠皮肤伤口为模型,结合扫描振动电极技术和选择性离子电极技术对小鼠皮肤伤口的内源性电场分布及其离子组成机理进行了系统的研究,结果如下:皮肤伤口内源性电场的时间空间变化研究,得到如下结果:A、小鼠皮肤伤口边缘在Z轴方向和Y轴方向均有显著高于完整皮肤(-2.81±0.78μA/cm2)的电流,Z轴方向为内流(-28.38±7.69μA/cm2),Y轴方向为外流(18.48±1.54μA/cm2)。小鼠皮肤伤口电流在水平面呈伤口边缘较大,伤口中心(-9.18±3.55μA/cm2)较小的趋势;在纵切面,靠近表皮为(15.61±2.17μA/cm2)外流,靠近真皮为内流(-9.90±2.25μA/cm2);B、小鼠皮肤伤口从制造伤口到伤口愈合的过程中,仅在前24小时有显著电流产生,且主要在前6小时有较大的电流,24小时后,电流降为-3.66±1.32μA/cm2,与完整皮肤相比无显著差异。皮肤伤口内源性电流产生的离子流动机理及时间空间变化研究,发现:A、钠、钾、钙、氯四种离子在小鼠皮肤伤口处的离子流均在伤口形成后的前2小时达到最大值,在新鲜伤口边缘的钠、钾、钙离子流均为内流,分别为-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,氯离子流为外流,大小为224.08±29.38 nmol·cm-2·s-1,而伤口中心的钠、钾、钙、氯离子流则均小于伤口边缘,分别为-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-1,四种离子流产生的电流方向与实际测量的电流方向一致。B、钠离子和氯离子是构成伤口内源性电场的主要离子,钠离子流和氯离子流在水平面的分布与电流一致,且方向一致,而在纵切面的分布则在靠近真皮的位置一致。C、用4μM的ANO1抑制剂处理伤口后,小鼠皮肤伤口电流降为-7.86±1.51μA/cm2,较对照有极显著差异(p0.001,n=3),在50μM的Furosemide处理伤口之后,小鼠皮肤伤口电流降为-12.98±3.50μA/cm2,较对照有显著差异(p0.01,n=4)。对Ha Cat细胞电场响应的验证,得到如下结果:1、Ha Cat细胞对外加电场有趋电反应,单个细胞和成片细胞均向电场的正极迁移;2、Ha Cat成片细胞较单个细胞对电场的反应灵敏;3、HaCat单个细胞在不同电压强度下表现出逐渐增强的趋电性,在200 m V/mm、400 m V/mm、600 m V/mm的电场强度下,趋电方向性分别为0.13±0.07、0.36±0.06、0.41±0.05;4、HaCat成片细胞则在各个电场强度下的趋电方向性均显著大于单个细胞,分别为0.68±0.06、0.82±0.04、0.78±0.04。本研究建立了扫描振动电极技术和选择性离子电极技术,并应用于组织水平内源性电场的研究。首次揭示了小鼠皮肤伤口内源性电场的时空分布及变化规律,深入研究了其离子机理。研究发现皮肤伤口内源性电场分布复杂,与角膜伤口的内源性电场分布有较大区别,且与理论推测的皮肤伤口内源性电场分布也有一定差异。通过对小鼠皮肤伤口离子流的研究发现,氯离子和钠离子流是伤口内源性电流产生的主要贡献者,且产生的电流方向与实测内源性电场方向一致,印证了对伤口内源性电场分布的研究结果。此外,在对皮肤细胞(HaCat)的趋电性研究发现,与大多数细胞对电场方向的响应相反,Ha Cat细胞在电场中向电场正极迁移。对伤口内源性电场的研究也表明伤口中心是内源性电场的正极,HaCat细胞的趋电响应从细胞层面解释了小鼠皮肤伤口内源性电场的分布状况与角膜伤口不同的原因,为生物电促进伤口愈合提供了新的证据,对进一步深入研究伤口内源性电场具有重要的指导意义。
[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.
【学位授予单位】：云南师范大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：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.
【学位授予单位】：云南师范大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R641
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