【作者】 郑翠红；

【导师】 黄光英；

【作者基本信息】 华中科技大学 ， 中西医结合临床， 2007， 博士

【摘要】 针灸,是中国传统医学的重要组成部分,应用于临床实践已有几千年的历史。由于其独特的治疗方法、神奇的治疗效果,针灸逐渐得到了世界的认可和接纳,尤其是针刺镇痛效应更是被全世界所关注。但是,穴位、经络的物质基础是什么?针刺效应是如何产生的?经穴信号到底是如何传导的?这些问题至今没有获得令人满意的回答。缝隙连接(gap junction,GJ)是动物细胞间最普遍存在的亲水性低电阻通道。由缝隙连接介导的细胞间通讯(gap junctional intercellular communication,GJIC)是细胞间直接传递信号的重要方式之一,在细胞生长发育、多细胞器官间的协调及机体自我稳定控制方面有着重要作用。缝隙连接蛋白(connexin,Cx)是构成缝隙连接通道的基本结构和功能蛋白。前人的研究结果表明,针刺能引起与传统意义的经络一致的线性现象,同时测得该线上表现为低电阻。那么,针刺,以及由此引起的循经感传现象,针刺镇痛等针刺效应、缝隙连接细胞间通讯之间是否存在内在的联系?因此,我们提出了如下假说:具有低电阻特性的皮肤、皮下组织细胞间缝隙连接是经穴之间信号传导的主要通道,由缝隙连接介导的细胞间通讯为经穴信号传递的主要方式,细胞间液、组织间液为通道缓冲系统。一旦经穴接受刺激,缝隙连接和缝隙连接通讯将被激活,在神经-体液因素的共同作用下信号将沿着体表低电阻区域细胞间的缝隙连接直接传递,而大脑对经穴接受的刺激信号具有整合作用,经穴、大脑、效应靶器官之间可能产生对话。基于以上假说,我们设计了如下研究方案:(1)首先着重研究了皮肤中主要的缝隙连接蛋白Cx43在大鼠穴位、经脉上的表达及针刺对该表达的影响(第一部分),探讨了穴位、经脉与缝隙连接通讯联系的物质基础;(2)确定了穴位、经脉存在丰富的缝隙连接蛋白Cx43的表达并存在一定的循经特异性后,应用RNA干扰技术沉默大鼠穴位局部缝隙连接蛋白的表达,观察对针刺效应的影响。此部分分为两部分:①针对大、小鼠Cx43 mRNA序列,构建Cx43特异性shRNA真核表达载体并进行体外干扰效率的鉴定(第二部分);②将沉默效果较好的Cx43特异性shRNA真核表达载体通过穴位局部注射观察对针刺效应的影响(第三部分)。第一部分Cx43在大鼠穴位、经脉上的表达及针刺对该表达的影响第一节Cx43在大鼠经脉线上表达的实验研究目的:研究缝隙连接蛋白Cx43在正常大鼠经脉皮肤肌肉组织中的表达,初步探讨Cx43与经脉的可能关系。方法:清洁级健康成年雌性SD大鼠10只,应用二步法免疫酶技术和ASIAS-2000自动扫描图像分析系统对膀胱经背部段、肾经足底段皮肤肌肉组织中Cx43的表达进行定位分析;应用免疫印迹法半定量分析Cx43在各组大鼠膀胱经背部段、肾经足底段与邻近对照线上表达的差异。结果:大鼠皮肤组织中Cx43主要在表皮棘细胞层角质形成细胞、真皮及皮下筋膜层的部分细胞胞浆胞膜中表达,且经脉线下可以看到较明显的Cx43免疫阳性细胞聚集现象。统计学分析免疫印迹结果表明,肾经足底段、膀胱经背部段Cx43的表达均高于邻近对照线,两者相比差异有显著性意义(P<0.05)。结论:大鼠经脉处存在着较邻近对照线更为丰富的Cx43表达,初步提示Cx43与经脉存在一定的相关性,由缝隙连接介导的细胞间通讯可能在经络活动中起重要作用。第二节针刺对大鼠“后三里”Cx43水平的影响目的:研究大鼠“后三里”穴与穴旁对照点缝隙连接蛋白Cx43的表达,并观察针刺对该表达的影响,探讨Cx43、缝隙连接与穴位、经络及针刺的可能关系。方法:清洁级健康成年雌性SD大鼠20只,随机分为非针刺组和针刺组,每组10只。应用二步法免疫酶技术和ASIAS-2000自动扫描图像分析系统对各组“后三里”穴位处Cx43的表达进行定位分析;应用RT-PCR和免疫印迹法定量检测各组穴位、穴旁Cx43的mRNA和蛋白水平。结果:大鼠“后三里”处皮肤组织中Cx43主要在表皮棘细胞层角质形成细胞,真皮、皮下筋膜层的部分细胞胞浆胞膜中表达;穴位处有Cx43免疫阳性细胞聚集现象(尤其是皮下筋膜层),针刺后此聚集现象更明显。RT-PCR、Western-Blot印迹法半定量检测发现,非针刺组中穴位处Cx43的mRNA和蛋白表达水平均高于穴旁(P<0.01),穴位针刺后Cx43表达显著增加(P<0.05),穴旁对照点针刺后表达增加不明显。结论:大鼠“后三里”处存在着较穴旁对照点更为丰富的Cx43表达,并且针刺能引起“后三里”处Cx43 mRNA和蛋白水平的增加。第二部分构建Cx43特异性shRNA表达载体及体外干扰效率的鉴定目的:构建缝隙连接蛋白Cx43基因特异性shRNA真核表达载体,观察其对NIH /3T3细胞Cx43基因的沉默效果。方法:针对大、小鼠Cx43 mRNA同源序列的两个靶点,体外合成两对互补的寡核苷酸链。经退火连接形成双链,插入到Pgenesil-1真核表达载体(分别命名为P-Cx43-shRNA(1)、P-Cx43-shRNA(2))。选择与人、大鼠、小鼠基因组均无明显同源性的序列作为对照质粒(P-con-shRNA)的靶点。经转化、涂布LB平板、挑单克隆、摇菌、质粒小量提取、酶切鉴定及测序等步骤后,将构建好的表达载体分别转染NIH/3T3细胞,G418筛选后应用Western Blot、RT-PCR观察shRNA对Cx43 mRNA水平和蛋白水平的影响。结果:酶切鉴定及测序结果表明,成功构建了两个针对Cx43基因的shRNA真核表达载体及对照质粒(P-con-shRNA);RT-PCR结果显示两个真核表达载体P-Cx43-shRNA(1)、P-Cx43-shRNA(2)分别使NIH/3T3细胞Cx43 mRNA水平下降了68.6%(P<0.01)和16.3%;Western Blot结果说明两个载体分别使Cx43蛋白水平下降了73.5%(P<0.01)和10.8%。阴性对照质粒(P-con-shRNA)对NIH/3T3细胞Cx43 mRNA及蛋白水平无明显影响。结论:重组质粒P-Cx43-shRNA(1)能较好的特异性抑制Cx43基因在NIH/3T3细胞中的表达。第三部分穴位局部沉默Cx43的表达对针刺效应的影响目的:观察沉默穴位局部Cx43的表达对针刺效应的影响。方法:以米非司酮造成大鼠胚泡着床障碍模型,应用RNA干扰技术沉默穴位局部Cx43的表达。将孕鼠随机分为正常组(对照)、模型组(米非司酮)、针刺组(米非司酮+针刺)、干扰组(米非司酮+针刺+P-Cx43-shRNA(1))、干扰对照组(米非司酮+针刺+P-con-shRNA),每组8只。从妊娠d1开始每日下午予以针刺治疗,连续7天,穴位选取双侧“后三里”、“三阴交”及“太冲”穴。干扰组于妊娠d1、d3、d6上午9:00在双侧各针刺穴位局部均皮内皮下注射P-Cx43-shRNA(1)质粒;干扰对照组以同样方法注射P-con-shRNA对照质粒。妊娠d8观察各组大鼠着床胚泡质量及其发育、妊娠率、平均着床胚泡数,以及血清E2、P4、PRL激素水平。结果:①干扰组“后三里”穴位处Cx43 mRNA及蛋白水平均低于针刺组,两者相比差异有显著性统计学意义(P<0.05,P<0.01)。干扰对照组“后三里”穴位处Cx43 mRNA及蛋白水平与针刺组相比,差异无统计学意义。②与正常组相比,模型组子宫着床胚泡数较少,着床胚泡质量差,胚泡明显较小,且大小不均一,分布不均匀,血供较差,针刺组大鼠胚泡着床情况较模型组明显改善,胚泡大小一致,且分布较均匀,血供较好,与正常组相比没有明显区别。干扰组胚泡着床情况与模型组相似,胚泡着床质量差,干扰对照组胚泡着床情况与针刺组及正常组相似。③模型组妊娠率50%,与正常组(100%)相比有下降趋势,针刺组妊娠率(75%)较模型组相比有增高趋势,干扰组妊娠率与模型组相似,干扰对照组与针刺组相似。但各组间的差异还未达到统计学意义。④模型组、干扰组平均着床胚泡数均较正常组低,差异有极显著性统计学意义(P<0.01);与模型组相比,针刺组平均着床胚泡数有升高趋势,但差异还未达到统计学意义;与针刺组相比,干扰组平均着床胚泡数较少,差异具有显著性统计学意义(P<0.05),干扰对照组与针刺组相比无统计学差异。⑤与正常组相比,模型组与干扰组血清PRL水平均较低,差异具有显著性统计学意义(P<0.05);与干扰组相比,干扰对照组血清PRL水平较高,差异具有显著性统计学意义(P<0.05)。各组血清E2、P4水平无显著性差异。结论:针刺能明显地改善胚泡着床障碍大鼠的胚泡着床质量及其发育,并有提高其平均着床胚泡数的趋势。穴位局部注射Cx43特异性shRNA真核表达载体有效地沉默了Cx43的表达,并显著地影响了针刺促胚泡着床效应,说明Cx43在针刺效应中发挥着重要的作用。更多还原

【Abstract】 Acupuncture and moxibustion, which are important ingredients of Chinese traditional medicine, have been used empirically in clinical practice in China for several millennia. Owing to its distinct operation method and marvelous therapeutic efficacy, acupuncture and moxibustion, especially acupuncture analgesia, are approved and accepted gradually by the whole world. Although a large number of previous clinical studies also support the effectiveness of acupuncture, little is known about the intercellular transfer pattern of acupoint signal and the mechanism of the effect created by acupuncture. And the specific material in acupuncture points and meridians still remains mysterious.Gap junctions, which provide low resistance pathways and a possibility for direct coupling of cells to be organized into functional units, are ubiquitous specialized plasma membrane domains composed of collections of the only channels that directly connect neighboring cells. These pathways are responsible for the cell-to-cell transfer of ions and small hydrophilic molecules, including amino acids, nucleotides and second messengers (e.g. Ca2+, cAMP, cGMP, IP3). Gap junctional intercellular communication plays an important role in embryonic development, cell differentiation, proliferation and growth control etc., which is essential for proper development and health in animals and humans. And transfer of electric signal and mechanical stimulation signal have also been attributed to gap junctional communication. Structural studies have demonstrated that each gap junctional channel is composed of a connexon (hemichannel) in the plasma membrane of one cell and a connexon in the apposing cell membrane. Each connexon, which provides a hydrophilic channel, 2 nm in diameter, is an oligomer of 6 protein subunits, termed connexins belonging to the connexins gene family. Although there are more than 20 members in vertebrates, the major components of human keratinocyte gap junctions are Cx43 and Cx26. Immunostaining for Cx43 was observed in epidermal keratinocytes, sebaceous glands, eccrine sweat ducts, and hairs, but Cx26 was only detectable in the two latter epidermal adnexae.On the basis of above facts and our previous research work, we put forward a hypothesis: gap junctions between adjacent cells in skin and subcutaneous layer, which provide low resistance pathways, are the major channels for acupoint signal transfer; gap junctional intercellular communication are the main transfer pattern; intercellular and interstitial fluid are the buffer system. Gap junctions and gap junctional intercellular communication may be activated when the acupoints received stimulation, and signal might be transferred directly through gap junctions along the low resistance area in the co-effect of nerve-humoral factor. The cerebrum might integrate the signal, and there is an intercom among the acupoint, cerebrum and target organ. Therefore, we designed a program to research the correlation between acupoint signal transfer and Cx43. First, to ascertain the structure foundation of connexins in acupoints and meridian, we studied the expression of Cx43 in rat acupoints and meridians, and observed the change after acupuncture (Part I). Secondly, we employed RNA interference technology to construct shRNA plasmid expression vectors to silence the expression of Cx43 (Part II). Third, we injected the vector targeting Cx43 with a better silence effect locally into the acupoint and observed the different acupuncture effect of each group. Part I: Experimental Study of Expression of Connexin 43 in Rat Acupoint and Meridian and the Change after Acupuncture1. Experimental Study of Expression of Connexin 43 in Rat Meridian Objective:To study the expression of connexin 43 in rat Kidney and Bladder meridians and in control lines, and approach the possible relationship between connexin 43 and meridians.Methods:Young adult female Sprague-dawley (SD) rats (210～230g, n=10) were employed. PowerVisionTM two step immunoenzyme technic and ASIAS-2000 automatic image-scan analyzing system was used to detect the distribution and Western Blot was performed to detect the quantity of Cx43 in rat Kidney and Bladder meridians and in control lines.Results:Cx43 was expressed mainly in keratinocytes in spinous layer and some unknown cells in subcutaneous layer and in the dermis. Statistical analysis showed that the expression of Cx43 in Kidney and Bladder meridians was distinctly more than in the controls respectively (P<0.05).Conclusion:Cx43 expression was much abundant in meridians than in the controls, implying there is a relationship between Cx43 and meridians, and gap junctional intercellular communication might be involved in meridian action. 2. The Effect of Acupuncture on the Expression of Cx43 at Rat Acupoint ST36Objective:To probe into the expression of connexin 43 in the effect of acupuncture at rat ST36 acupoint, and discuss the possible relationship among Cx43, acupoint and acupuncture.Methods:Young adult female Sprague-dawley (SD) rats (210～230g, n=20) were divided randomly into two groups: non-acupuncture group (n=10) and acupuncture group (n=10). PowerVisionTM two step immunoenzyme technic and ASIAS-2000 automatic image-scan analyzing system was used to detect the distribution in ST36, RT-PCR and Western Blot was performed to detect Cx43 mRNA and protein level in the acupoints of each group.Results:Cx43 was expressed mainly in keratinocytes in spinous layer and some unknown cells in subcutaneous layer and in the dermis. There were aggregated positive cells for Cx43 immunostaining in rat ST36, especially in the subcutaneous layer, and even more so after acupuncture. In non-acupuncture group, the expression of Cx43 mRNA and protein level in acupoints was significantly higher than in the non-acupoints respectively (P<0.01). After acupuncture, the expression of Cx43 in acupoint was obviously increased, which formed a striking contrast to the acupoint in the non-acupuncture group (P<0.05).Conclusion:Cx43 expression was much abundant in acupoints than in the controls, and acupuncture could significantly increase the Cx43 mRNA and protein level at acupoint. Part II:Construct an shRNA Expression Vector Targeting Cx43 and Identify the Efficiency of RNA Interference in vitroObjective:To construct an shRNA expression vector targeting Cx43 and identify the efficiency of RNA interference in NIH/3T3 cell lines.Methods:Aiming directly at the two target of Cx43 mRNA sequence of rat and mouse homology region, we synthetize two pair complementary oligonucleotide strands in vitro. Double strands were formed after annealing, which were then inserted into Pgenesil-1 plasmid expression vector. After identification by enzyme cutting and sequencing, the recombined plasmids named P-Cx43-shRNA (1), P-Cx43-shRNA (2) and P-con-shRNA were transfected into NIH/3T3 cells. RT-PCR and Western Blot were used to detect the effect of the plasmid on the mRNA and protein level of Cx43 after screened by G418.Results: The results of enzyme cutting and sequencing showed that we successfully construct two shRNA expression vectors targeting Cx43 and a control expression vector for rat and mouse. The Cx43 mRNA level was decreased 68.6%(P<0.01)and 16.3% by P-Cx43-shRNA (1) and P-Cx43-shRNA (2) respectively in NIH/3T3 cells, and the Cx43 protein level was decreased 73.5%(P<0.01)and 10.8% accordingly. The Cx43 mRNA and protein level were not influenced by transfection of P-con-shRNA.Conclusion : The plasmid P-Cx43-shRNA (1) can specificly silence better the expression of Cx43 in NIH/3T3 cells. Part III: The Influence on Acupuncture Effect of Silencing the Expression of Cx43 in AcupointObjective:To investigate the influence on acupuncture effect of silencing the expression of Cx43 in acupoint.Methods:We used mifepristone to result in the model of nidation disorder in rat, and RNA interference technology to silence the expression of Cx43 in acupoint. The mated female rats were randomly divided into five groups (n=8 of each group): Normal (control), Model (mifepristone), Acupuncture (Mife+Acupunc.), Interference (Mife+P-Cx43-shRNA (1)+Acupunc.) and Interference-con (Mife+P-con-shRNA+Acupunc.). Acupuncture was performed in the acupoints“Housanli”,“Sanyinjiao”and“Taichong”in Acupuncture, Interference and Interference-con group from the first day of pregnancy for seven consecutive days. P-Cx43-shRNA (1) was injected locally into the acupoints in Interference group, and P-con-shRNA was used in Interference-con group. On the d8 of pregnancy, the quality of blastocyst, pregnancy rate, average amount of blastocyst and hormone level (E2, P4, PRL) in serum of each group were observed.Results:①The mRNA and protein level of Cx43 in acupoints in Interference group were significantly lower than in the Acupuncture Group (P<0.05,P<0.01), there was no significant difference between Interference-con and Acupuncture group.②Compared with Normal group, the quality of blastocyst in Model group was bad,the size was comparatively small and inhomogeneous with maldistribution and poor blood supply. The results in acupuncture group and Interference-con group were similar to the Normal. The results in Interference group were similar to the Model group.③The pregnancy rate in Normal, Model, Acupuncture, Interference and Interference-con group was 100%, 50%, 75%, 50%, 75% respectively, but the difference was not statistically significant.④The average amount of blastocyst in Model and Interference group were significantly lesser than in Normal group(P<0.01). Compared with Acupuncture group, the average amount of blastocyst in Interference group was lower (P<0.05).⑤The serum PRL level in Model and Interference group was significantly lower than in Normal group (P<0.05) ; Compared with Interference group, the serum PRL level in Interference-con group was higher (P<0.05). The difference of the serum E2 and P4 level (E2, P4) in each group was not statistically significant.Conclusion:Acupuncture could improve the blastocyst quality in the model of nidation disorder of rat, and have the tendency of raising the average amount of blastocyst. Local injection of Cx43 shRNA expression vetor could silence the expression of Cx43 in acupoint and markedly influence acupuncture effect, demonstrating Cx43 is involved in acupuncture effect.更多还原