皮肤组织对红外激光辐照热响应的细胞机理研究
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摘要
光热响应是红外激光与生物组织相互作用的主要形式之一,但其产生、传输和作用机理尚不十分清晰,这导致在临床应用中还主要以经验为主,不能更为有效地控制激光治疗的过程和效果。为了明确激光辐照过程中的作用机理,以期达到优化治疗效果,研究者开始从细胞层面着手,来认识这一作用。本文主要采用膜片钳技术与激光辐照相同步技术,对激光辐照生物组织过程中细胞外液在光吸收诱导的热响应中所起的媒介作用进行了理论和实验两方而的探究。
皮肤疾病是目前应用激光治疗最有效的一个领域,红外激光又大多以热作用的形式和标准来确定其相关参数。基于这样一个前提,在组织层面上,本文从理论上研究了激光辐照皮肤的光热响应情况。研究中采用目前研究光皮肤组织热响应过程时常用的两层模型,理论计算了激光辐照过程中不同部位皮肤组织热响应的温度分布,对比了不同部位皮肤表皮层开始汽化的时间和探讨了能量累积问题,结果表明不同部位的皮肤层厚度、皮肤组织各层中水、脂肪、血红蛋白等色团的光学性质和热学性质、以及所选取激光的参数包括波长、能量密度、脉冲宽度等在作用过程中都起到了至关重要的作用,但要具体辨识色团的作用就需要进行细胞方面的实验。
为了明确水在红外光热响应过程中介导方面的作用,本文选取980nm和845nm两个波长的近红外激光进行了理论和实验两方面的研究,选择两个波长的依据是：由于水是生物组织中含量最高的色团(按重量),在生物组织的生命活动中起着至关重要的作用,另外,两个波长的激光在临床上较为常用,且在水中的光吸收系数分别为0.502cm-1(980nm)和0.0378cm-1(845nm),其比值大约10倍。
在研究过程中把激光辐照的作用过程分为温升建立和耗散两个阶段,在温升建立阶段,理论方面充分考虑激光辐照的脉冲时程(500ms)长于水介质的热弛豫时间(17.6ms),且空间作用长度远远短于两个波长的光在水中的穿透深度等因素。主要考虑轴向吸收和径向热扩散两方面的作用。实验上是基于膜片钳系统来测量细胞外溶液中,已进行温度标定的、充灌溶液的玻璃微电极(Open-pipette method)电导变化,根据这个电导变化来定量研究溶液的光热响应与其吸收特性的关联。实验结果显示温度场建立过程中双波长辐照产生的温升比大约为10倍。在耗散阶段(关闭激光后),理论方面主要是运用热扩散理论来分析温度场的衰逝进程,实验方面使用膜片钳监测神经细胞电生理功能在温度场衰逝过程中的变化,这样可以最大限度的排除其它光学效应(比如光化学、光力学等)的影响。理论和实验结果都证明了Na通道的电流强度在双波长激光辐照后都得到了较明显的增强,目.980nm激光辐照后的电流强度增加值明显高于845nm激光辐照后的电流强度的增加值。又由于在实验过程中只改变激光波长,其它实验条件保持一致,因此这两个结果具有波长依赖的可比性。理论和实验两方面的结果都表明,溶液在低强度近红外激光的吸收特性决定了光热响应,从而对细胞的电生理功能进行调制。这一结果,可以直接用于生物组织光热响应特性相关的机理研究。
由于第四章中理论分析电流峰值的变化时所采用的近似方法使得实验和理论计算有一定的差异,在接下来的工作中对其理论进行了修正。文中采用离子热迁移理论分析了激光辐照溶液中的细胞Na离子通道电流的峰值变化。通过理论分析发现：由于局部温升造成的离子迁移,致使细胞周围的离子浓度减小,而Na离子浓度的减小减弱了电压依赖的Na离子通道的电流强度的增强程度。
最后,由于血液中的主要吸收色团为水(约90%)和血红蛋白,那么对于近红外光,血液吸收的能量明显强于周围的组织。基于该事实本文进行了近红外激光的静脉观测实验,发现使用近红外激光辅助可以使手背静脉较清晰的显现出来,这对临床上静脉穿刺等临床医学应用特别是对血管比较难以辨认的患者提供了帮助,同明证明了红外光辐照皮肤过程中水的吸收起到了决定性的作用。
Photothermal response is the main form in infrared laser with biological tissue, however, the mechanism of generation, transmission and function is not very clear. The clinical application so far has to be counting on the experiences which make the process of laser treatment and therapeutic effect is difficult to manage and control. In order to clarify its mechanism, the photothermal effect has been studied in cellular level. In this present study, we employed the patch clamp to examine the photothermal effect induced by optical absorption in process of laser-tissue interaction in theory and experiment study.
The most effective field in laser physiotherapy is skin disease. The related parameters, used infrared laser physiotherapy, are determined by the form of thermal. Based on such a premise, the two-layer model of skin is adoptive to calculate the temperature distribution, to compare the time of the beginning of the gasification of epidermis, and to study the cumulative of heat of different body part in photothemal effect according the optical properties of skin, The results showed that the optical properties of water, fat and hemoglobin, and thermal properties of skin played an important role in laser irradiation skin as well as the wavelength, energy density, and duration time of pulse, however, the experiments in cellular level are needed to do for know the role of chromophores.
In order to confirm the mediated effect of water in photothermal response of laser-tissue interaction, the dual-wavelength laser (980nm and845nm) were chosen to study the mechanism in laser irradiation the cell. It is well known that water is the main chromophores (by weight) in biological tissue, it plays an important role in life; and the dual-wavelength laser is widely applied in study of laser medicine; the magnitude of absorption coefficients (0.502cm-1at980nm and0.0378cm-1at845nm) of this two wavelength lasers is10times difference, they can produce a correspondingly proportional absorption-driven temperature rises. Thus,980nm and845nm as two probe wavelengths have been chosen to assess the photofhermal effect during the activation of neurons by a continuous near-infrared laser irradiation.
The photothermal effect can be assessed in two stages:the establishment and the dissipation of the absorption-driven temperature rises:In the establishment stage, because the duration time of laser irradiation (500ms) is longer than thermal relaxation time of water (17.6ms), and the spatial distance is far shorter than penetration depth of this two wavelength laser in water, the role of absoiption in axial direction and thermal diffusion in radial direction were take into account in theoretical study. According to the temperature-dependence of conductivity of solution, an open pipette method was employed to measure the absorption-driven temperature rises by fabricating a glass pipette which was filled with electrolyte solution, formed a several mega-ohm resistance and calibrated as a temperature sensor by using a pico-ampere current amplifier technique (patch clamp). The experiment results showed that ratio of temperature rises is about10times in absorption-driven temperature rises. In the dissipation stage (turn off the laser source), we used the theory of thermal diffusion to describe the dissipative process. The electrophysiological function of a living neuron cell in dissipative process was studied based on a patch clamp. This method can decrease the influence of photochemistry and photomechanics. Because all experimental conditions are kept as the same, while the wavelengths are switched, the results have the comparability of wavelength-dependence. The theoretical calculation and the experimental results showed that the optical properties of solution determined the photothermal effect in laser-tissue interaction, and then modulated the electrophysiological function of a living neuron cell. The results can be used to study the mechanism of photothermal effect in laser-tissue interaction.
However, there is some difference between experiment results and theoretical results for the adoptive approximation method when we use the theoretical method to analyze the changes of current peak. The theoretical results are corrected in the following work. We analyzed the current change of Na ion channel with thermophoresis theory in the process of theoretical analysis. The ions displace along the gradient of temperature for laser irradiation, it will decrease the concentration of Na ions in local cell, then it will change the current intensity, and the physiological function is modulated.
Finally, because the chromophores are the water (90%) and hemoglobin in blood, according to the optical properties of hemoglobin in vein, we made the vein imaging experiment with near infrared laser irradiation. This method can make the vein of opisthenar clearly, and may provide a lot of help for the clinical venipuncture, especially to the patients, whose blood vessels are not difficult to identify.
皮肤疾病是目前应用激光治疗最有效的一个领域,红外激光又大多以热作用的形式和标准来确定其相关参数。基于这样一个前提,在组织层面上,本文从理论上研究了激光辐照皮肤的光热响应情况。研究中采用目前研究光皮肤组织热响应过程时常用的两层模型,理论计算了激光辐照过程中不同部位皮肤组织热响应的温度分布,对比了不同部位皮肤表皮层开始汽化的时间和探讨了能量累积问题,结果表明不同部位的皮肤层厚度、皮肤组织各层中水、脂肪、血红蛋白等色团的光学性质和热学性质、以及所选取激光的参数包括波长、能量密度、脉冲宽度等在作用过程中都起到了至关重要的作用,但要具体辨识色团的作用就需要进行细胞方面的实验。
为了明确水在红外光热响应过程中介导方面的作用,本文选取980nm和845nm两个波长的近红外激光进行了理论和实验两方面的研究,选择两个波长的依据是：由于水是生物组织中含量最高的色团(按重量),在生物组织的生命活动中起着至关重要的作用,另外,两个波长的激光在临床上较为常用,且在水中的光吸收系数分别为0.502cm-1(980nm)和0.0378cm-1(845nm),其比值大约10倍。
在研究过程中把激光辐照的作用过程分为温升建立和耗散两个阶段,在温升建立阶段,理论方面充分考虑激光辐照的脉冲时程(500ms)长于水介质的热弛豫时间(17.6ms),且空间作用长度远远短于两个波长的光在水中的穿透深度等因素。主要考虑轴向吸收和径向热扩散两方面的作用。实验上是基于膜片钳系统来测量细胞外溶液中,已进行温度标定的、充灌溶液的玻璃微电极(Open-pipette method)电导变化,根据这个电导变化来定量研究溶液的光热响应与其吸收特性的关联。实验结果显示温度场建立过程中双波长辐照产生的温升比大约为10倍。在耗散阶段(关闭激光后),理论方面主要是运用热扩散理论来分析温度场的衰逝进程,实验方面使用膜片钳监测神经细胞电生理功能在温度场衰逝过程中的变化,这样可以最大限度的排除其它光学效应(比如光化学、光力学等)的影响。理论和实验结果都证明了Na通道的电流强度在双波长激光辐照后都得到了较明显的增强,目.980nm激光辐照后的电流强度增加值明显高于845nm激光辐照后的电流强度的增加值。又由于在实验过程中只改变激光波长,其它实验条件保持一致,因此这两个结果具有波长依赖的可比性。理论和实验两方面的结果都表明,溶液在低强度近红外激光的吸收特性决定了光热响应,从而对细胞的电生理功能进行调制。这一结果,可以直接用于生物组织光热响应特性相关的机理研究。
由于第四章中理论分析电流峰值的变化时所采用的近似方法使得实验和理论计算有一定的差异,在接下来的工作中对其理论进行了修正。文中采用离子热迁移理论分析了激光辐照溶液中的细胞Na离子通道电流的峰值变化。通过理论分析发现：由于局部温升造成的离子迁移,致使细胞周围的离子浓度减小,而Na离子浓度的减小减弱了电压依赖的Na离子通道的电流强度的增强程度。
最后,由于血液中的主要吸收色团为水(约90%)和血红蛋白,那么对于近红外光,血液吸收的能量明显强于周围的组织。基于该事实本文进行了近红外激光的静脉观测实验,发现使用近红外激光辅助可以使手背静脉较清晰的显现出来,这对临床上静脉穿刺等临床医学应用特别是对血管比较难以辨认的患者提供了帮助,同明证明了红外光辐照皮肤过程中水的吸收起到了决定性的作用。
Photothermal response is the main form in infrared laser with biological tissue, however, the mechanism of generation, transmission and function is not very clear. The clinical application so far has to be counting on the experiences which make the process of laser treatment and therapeutic effect is difficult to manage and control. In order to clarify its mechanism, the photothermal effect has been studied in cellular level. In this present study, we employed the patch clamp to examine the photothermal effect induced by optical absorption in process of laser-tissue interaction in theory and experiment study.
The most effective field in laser physiotherapy is skin disease. The related parameters, used infrared laser physiotherapy, are determined by the form of thermal. Based on such a premise, the two-layer model of skin is adoptive to calculate the temperature distribution, to compare the time of the beginning of the gasification of epidermis, and to study the cumulative of heat of different body part in photothemal effect according the optical properties of skin, The results showed that the optical properties of water, fat and hemoglobin, and thermal properties of skin played an important role in laser irradiation skin as well as the wavelength, energy density, and duration time of pulse, however, the experiments in cellular level are needed to do for know the role of chromophores.
In order to confirm the mediated effect of water in photothermal response of laser-tissue interaction, the dual-wavelength laser (980nm and845nm) were chosen to study the mechanism in laser irradiation the cell. It is well known that water is the main chromophores (by weight) in biological tissue, it plays an important role in life; and the dual-wavelength laser is widely applied in study of laser medicine; the magnitude of absorption coefficients (0.502cm-1at980nm and0.0378cm-1at845nm) of this two wavelength lasers is10times difference, they can produce a correspondingly proportional absorption-driven temperature rises. Thus,980nm and845nm as two probe wavelengths have been chosen to assess the photofhermal effect during the activation of neurons by a continuous near-infrared laser irradiation.
The photothermal effect can be assessed in two stages:the establishment and the dissipation of the absorption-driven temperature rises:In the establishment stage, because the duration time of laser irradiation (500ms) is longer than thermal relaxation time of water (17.6ms), and the spatial distance is far shorter than penetration depth of this two wavelength laser in water, the role of absoiption in axial direction and thermal diffusion in radial direction were take into account in theoretical study. According to the temperature-dependence of conductivity of solution, an open pipette method was employed to measure the absorption-driven temperature rises by fabricating a glass pipette which was filled with electrolyte solution, formed a several mega-ohm resistance and calibrated as a temperature sensor by using a pico-ampere current amplifier technique (patch clamp). The experiment results showed that ratio of temperature rises is about10times in absorption-driven temperature rises. In the dissipation stage (turn off the laser source), we used the theory of thermal diffusion to describe the dissipative process. The electrophysiological function of a living neuron cell in dissipative process was studied based on a patch clamp. This method can decrease the influence of photochemistry and photomechanics. Because all experimental conditions are kept as the same, while the wavelengths are switched, the results have the comparability of wavelength-dependence. The theoretical calculation and the experimental results showed that the optical properties of solution determined the photothermal effect in laser-tissue interaction, and then modulated the electrophysiological function of a living neuron cell. The results can be used to study the mechanism of photothermal effect in laser-tissue interaction.
However, there is some difference between experiment results and theoretical results for the adoptive approximation method when we use the theoretical method to analyze the changes of current peak. The theoretical results are corrected in the following work. We analyzed the current change of Na ion channel with thermophoresis theory in the process of theoretical analysis. The ions displace along the gradient of temperature for laser irradiation, it will decrease the concentration of Na ions in local cell, then it will change the current intensity, and the physiological function is modulated.
Finally, because the chromophores are the water (90%) and hemoglobin in blood, according to the optical properties of hemoglobin in vein, we made the vein imaging experiment with near infrared laser irradiation. This method can make the vein of opisthenar clearly, and may provide a lot of help for the clinical venipuncture, especially to the patients, whose blood vessels are not difficult to identify.
引文
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