Ⅰ.低强度激光照射对离体血液流变学指标的影响及其机制探讨　Ⅱ.胞质内段亚基间相互作用是决定内向整流钾通道激活的结构因素

论文题目: Ⅰ.低强度激光照射对离体血液流变学指标的影响及其机制探讨　Ⅱ.胞质内段亚基间相互作用是决定内向整流钾通道激活的结构因素

论文类型: 博士论文

论文专业: 凝聚态物理

作者: 宓现强

导师: 周鲁卫

关键词: 激光照射,粘度,红细胞,变形能力,电泳率,血红蛋白,胞质内段,亚基间作用,通道激活,结构

文献来源: 复旦大学

发表年度: 2005

论文摘要: 激光具有高方向性、高亮度、高单色性和高度相干性等特征。激光的主要生物效应包括热效应、压强效应、光化效应和电磁场效应。大量生物试验和临床治疗研究发现采用低强度激光照射疗法,生物组织不仅不会受到损伤,而且还能促进病灶组织恢复正常状态,这被称为低强度激光的“生物刺激效应”。临床上常用的低强度激光波长有632.8 nm、820 nm、830 nm及904 nm,目前532 nm波长激光用的也越来越多。低强度激光疗法采用的照射功率一般在10—90 mW之间;照射方式一般有单点照射,散焦照射等,一般直接照射在病变部位区,有时和针灸,光敏剂等配合治疗。研究发现,低强度激光能够对下列细胞功能产生影响:蛋白合成、细胞生长分裂、细胞运动、神经递质释放、膜电位、ATP合成等。临床发现对如下的疾病低强度激光治疗具有一定的疗效:心血管疾病、神经系统疾病、免疫系统疾病、口腔疾病、炎症性疾病、各种疼痛等。对低强度激光照射治疗的光化学基础和分子机制,许多学者提出不同的学说。有的人认为是生物组织中的光受体分子吸收光能后,将能量转移给其它的分子,受激活的分子使得周围的组织产生化学变化。这种光化学变化可以成功的用在PDT治疗上。还有人认为光子必须被生物细胞体内的发色团吸收才能发挥作用,在可见及近红外光处存在吸收峰,内源性卟啉,线粒体细胞色素,黄素蛋白被认为是可能的发色团。譬如Karu提出当红光及近红外光照时,细胞色素氧化酶是可能的光受体。他提出的主要四个机制为:光激发呼吸链各成分电子态后呼吸链上各成分的氧化还原特性的改变;单分子氧产生;吸光发色团的局部短暂升温;超氧阴离子及其歧化产物H2O2的产生。与细胞动态平衡有关参数变化相关的一系列反应被认为是细胞内的光信号传导和扩增链。Lubart认为在非血细胞中的NADPH氧化酶（含一种黄素蛋白）也可能是光的作用靶位,光的效应通过氧自由基（ROS）和Ca2+介导产生。 低强度激光疗法的应用之一是低强度激光血管内照射,该疗法自90年代引入我国后,已有数万名患者接受过治疗,对脑梗塞、高血压等很多心脑血管病取得了较为满意的控制或治疗效果。但此类研究大多以疗效观察和临床现象总结为主,虽已开展了不少基础研究,增加了不少认识,但由于其复杂性,在作用机制上,仍众说纷纭,至今尚无定论。原因之一是由于血液的实验样品大多取自患者,病人的病因各异使样品本身存在很大差异,给统计比较带来很大困难,导致许多不同的结论。 激光血管内照射的全身效应应该是通过血液循环实现的,其直接的靶位是可能是血管内皮细胞和血液成分。低强度激光对血管内皮细胞的生物刺激作用国外已经有较为肯定的报道,但仍不能完全解释其全身效应。红细胞占血液中有形元素总体

论文目录:

Part I 中文摘要

Part II 中文摘要

Part I Abstract

Part II Abstract

Part I The In Vitro Effects and Mechanism of Low Power LaserIrradiation on Hemortheological Parameters

Chapter 1 Review

Review 1 Low power laser therapy

1 Introduction

2 Biophysics of laser therapy

3 Laboratory research and clinica treatment

3.1. Cardiac and vascular conditions

3.2. Neurological conditions

3.3. Wound healing

3.4. Pain

3.4.1. Myofascial pain syndrome

3.4.2. Low back pain

3.4.3. Patellofemoral pain

3.4.4. Lateral epicondylitis (Tennis elbow)

3.4.5. Trigger points

3.4.6. Rotator cuff tendinitis

4 Mechanism discussion

5 Conclusions

Review 2: Intravascular low power laser irradiation: clinical use and possible mechanism

Chapter 2 Materials and methods

1 The in vitro effects of low power laser irradiation on hermorheological parameters

1.1 Blood samples

1.2 Laser irradiation

1.2.1 For the in vitro effects of Helium Neon laser irradiation on human blood: blood viscosity and deformability of erythrocyte

1.2.2 For the in vitro effects of low power laser irradiation on animal erythrocyte rheology

1.2.3 For the comparative study of 632.8 and 532 nm laser irradiation on some rheological factors in human blood in vitro

1.3 Measurements of rheological factors

1.3.1 Measurements of hematocrit (HCT) and erythrocyte sedimentation rate

1.3.2. Measurement of blood viscosity

1.3.3. Measurement of erythrocyte deformability

1.3.4. Measurement of electrophoretic mobility

2 The in vitro effect of low power laser irradiation on membrane attached hemoglobin (Hbm)

2.1. Blood preparation

2.2 Laser irradiation

2.3. Sample preparation

2.3.1 Hbm induced in erythrocyte ghost

2.3.2 Hbm induced in intact erythrocytes

2.4. Measurements of Hbm

3. Statistical analysis

Chapter 3 Results and discussion

1. The in vitro effects of Helium Neon laser irradiation on human blood: blood viscosity and deformability of erythrocytes

1.1. Effect of He-Ne laser irradiation on ESR and HCT

1.2. Effect of He-Ne laser irradiation on blood reduced viscosity

1.3. Effect of He-Ne laser irradiation on erythrocyte deformability

1.4. Effect of He-Ne laser irradiation on erythrocyte deformability (CaCl_2 treated samples)

2. In vitro effects of low power laser irradiation on animal erythrocyte's rheology's property

2.1. The changes of erythrocyte's deformability after laser irradiation with different intensity (650nm)

2.2. Comparative improvements of erythrocyte's deformability after 650nm and 632.8 nm laser irradiation

2.3. Changes of mouse erythrocyte's electrophoresis mobility

3 A comparative study of 632.8 and 532 nm laser irradiation on some rheological factors in human blood in vitro

3.1. Effects of laser irradiation on HCT and ESR

3.2. Effects of laser irradiation on blood visosity

3.3. Effects of laser irradiation on electrophoretic mobility of erythrocytes

3.4. Effects of laser irradiation on deformability of erythrocytes

4 Mechanism of low power laser irradiation on red blood cell deformability

4.1 Effects of laser irradiation on Hbm of erythrocyte ghost

4.2 Effects of laser irradiation on Hbm of intact erythrocytes

Chapter 4 Conclusion

Part II Intersubunit Interaction within the Cytoplasmic Domain of Inwardly Rectifying Potassium Channels Is a Structural Determinant for Channel Activation

Chapter 1.Review

Review one: Ion channel, potassium channel and inwardly rectifying potassium channel

1. Ion channel

2. Potassium channel

3. Inward Rectifying Potassium K+ channel

3.1. Structure of Kir channels

3.1.1. Kir channels comprise a large superfamily

3.1.2. Primary structure of Kir channels

3.2. Chromosomal location

Review two: Structure and function of ATP-sensitive potassium channels

1 Introduction

2 Function research of KATP channel

2.1.ATPandKir6.2

2.1.1. Where does Kir6.2 bind ATP?

2.1.2. Mutations affecting ATP sensitivity

2.1.3. Effects of thiol reagents affecting ATP sensitivity

2.1.4. Direct evidence of ATP binding to Kir6.2

2.1.5. Mechanism of channel inhibition by ATP

2.2. Phosphatidylinositol and Kir6.2

2.2.1. Physiological coupling between phosphatidylinositol cycle and KATP

2.2.2 The effects of PPIs on channel function of KATP

2.2.3 Regulation of KATP by PPIs exists in cells: change of membrane PIP2 levels correlates with KATP activity

2.2.4 Mutations affecting PPIs regulation of KATP channel

2.2.5 Electrophysiological characterization of the specificity: Effect of phosphoinsitides varies with headgroup structure

2.2.6 Mechanism of PPI regulation studied by electrophysiological method

2.2.6.1 Evidence for direct interaction of phosphoinosidies with the channel-forming subunit, Kir6.2

2.2.6.2 Cross-interaction between PPI effect and nucleotide2inhibition of KATP: a partial competition hypothesis

2.2.6.3 The primary site of PPI's modulation effect is on Kir6.2while incorporation of SUR potentiates the modulation

2.2.6.4. Implication for specific interaction of PPIs and Kir6.2

2.3 PH and KATP channel

3. Structural research of KATP channel

3.1. KATP channels are the heteromultimers of SUR and Kir6.2 subunits in which Kir 6.2 forms the pores

3.2. Gating of the Kir channels: channel gating kinetics represent two independent gating mechanisms controlling the pore

3.2.1. Intrinsic gating

3.2.2. Controlled gating

3.3. Location of the controllable gate

3.4. A Hypothetical gating mechanism for Kir channels

Chapter 2.Materials and methods

1. Cell biology techniques:

1.1. Cell line and cell culture:

1.2. Protocol for passage of the COS-1 cells

1.3.Protocol for transient tranfection COS-1 cells with mBir and eGFP103

2. Molecular biology techniques

2.1. cDNA clones:

2.2. Chimera and fusion protein construction:

2.3. Site-directed mutation, insertion, and deletion:

2.4. Heterologous expression in cultured cells:

2.5. Reporting and analysis of expression and subcellular distribution:

2.5.1 GFP Florescence

2.5.2 Double Immunoflorescencestaining and Confocal Microscopy Observation

3. Functional assays

3.1. Physiological buffers used in the in vitro experiments:

3.2 Patch-clamp recording and analysis of single- or multiple- channel currents:

3.3. Monitoring subcellular distribution of PLC-GFP with confocal microscopy:

Chapter 3 Results and discussion

1. Structural models developed describing molecular structure and channel gate

1.1. A structural model of the Kir6.2 channel based on the known structures of KirBac 1.1 and Kir3.1

1.2. Involvement of the cytoplasmic domain of Kir6.2 channels in the control of gating

1.3. Hypothesi s development

2. Functional experiments assessing changes in channel currents in response to structural perturbation

2.1. Mutations on channel rundown

2.1.1 Effects of mutations at the selected residues on channel activity in inside-out patches

2.1.2 Statistical summary of the effects of mutations at the selected residues on the rundown process

2.1.3 Statistical summary of the effects of mutational substitutions of V236 on the rundown process in mutant V236 channels

2.2. Channel stimulation by ATP

2.2.1 The stimulatory effect is observed upon removal of ATP (inhibition)

2.2.2 Statistical summary comparison of channel stimulation by ATP in mutations in the area of H234 - G243

3. Structural model: possible interpretation

3.1. A close-up view of the structural details at the intersubunit interface around Val-236

3.2. A kinetic model interpreting the effects of mutation and ATP

4. Mechanisms for channel regulation by cytoplasmic domain interacting signaling molecules:

4.1 The stimulatory effect of phosphoinositides is absent in 236S mutant channels, but potentiated in the double mutant

4.2 Channel stimulation by acidosis is abbreviated in V236S mutant channel

4.2.1 Cellular acidosis on channel currents of control and mutant channels

4.2.2 Statistical comparison of the effect of cellular acidosis on channel currents in control and mutant channels

Chapter 4 Summary and Conclusion

Part I REFERENCES:

Part II REFERENCES

PUBLICATIONS

GRANT FUNDING

CONFERENCES

VITA

ACKNOWLEDGEMENTS

论文独创性声明

论文使用授权声明

发布时间: 2005-09-19

参考文献

• [1].低强度激光辐照对大鼠背根神经节神经元和软骨细胞的影响及机制研究[D]. 郑莉琴.福建师范大学2015

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