Visualization of Bonghan Microcells by Electron and Atomic Force Microscopy

ObjectivesThe origin of adult stem cells remains an open question. If they derive from embryos, it is difficult to determine the mechanism which interrupts their differentiation during tissue formation. In the 1960s, the Bonghan microcell was suggested as one possible, yet to be described, route of stem cell production, such that they have the potential to proliferate to produce normal cells.Materials and MethodsIn this study, Bonghan microcells were isolated from Bonghan tissues on rat organ surfaces, and their detailed morphology examined by electron and atomic force microscopy.ResultsThe ultrastructure observed distinguished them from apoptotic bodies and other microorganisms, and their unique, possible proliferation feature, as protruding threads, was imaged by atomic force microscopy.ConclusionsThe unique threadlike structure of the Bonghan microcell is consistent with Prof. Kim's observation in the first step of making a cell. Understanding of the functions of this threadlike structure may give a clue to understand the origin or the differentiation cue of adult stem cells.     

The Origin of Endothelial Cells in Novel Structures,Bonghan Ducts and Bonghan Corpuscles Determined Using Immunofluorescence

Bonghan ducts (BHDs), and their associated Bonghan corpuscles (BHCs), which are novel threadlike structures, were recently observed in rats and rabbits by using various methods. As further support for the putative circulatory function of the novel threadlike structures (NTS), we investigated the presence and the origin of the endothelial cells within these structures. We immunostained the NTS with anti-CD146, an endothelial cell marker, and with anti-podoplanin, a lymphatic cell marker. Positive expression of CD146 in the BHDs was obtained, and the distribution of endothelial cells showed that the inner boundaries of the channels in the subducts branched from the BHDs and curled around, in a complicated manner, inside a BHCs. The negative expression of podoplanin implies that the endothelial cells in the BHDs are likely to be of vascular and not of lymphatic origin.     

Visualization of Acupuncture Meridians in the Hypodermis of Rat Using Trypan Blue

Bonghan Kim claimed that the primo-vessel (Bonghan duct) and the primo-node (Bonghan corpuscle) are anatomical structures corresponding to acupuncture meridians and acupoints, respectively, but this claim has not been confirmed. Recently we found that Trypan blue preferentially stained primo-vessels and primo-nodes on the surfaces of internal organs, not staining other structures like blood or lymph vessels or nerves. In this work, we applied Trypan blue to visualize the putative acupuncture meridian along skin skeletal muscles in the hypodermal layer of a rat. The Trypan blue stained-structures are morphologically similar to the meridian primo-vessels, as claimed by Bonghan Kim. Further study is needed to investigate the network of these Trypan blue-stained structures in order to establish them as acupuncture meridians.     

Novel Anatomic Structures in the Brain and Spinal Cord of Rabbit That May Belong to the Bonghan System of Potential Acupuncture Meridians

Novel threadlike structures of 20 to 40 μm in diameter were observed running afloat in the cerebrospinal fluid of the brain ventricles and the spinal central canal of a rabbit. We developed an effective in situ staining technique using hematoxylin to visualize the threadlike structure. The presence of the rod-shaped nuclei in the threadlike structure was confirmed by various nucleus specific staining dyes such as 4′,6′-diamidino-2-phenylindole, propidium iodide and yoyo-1. The threadlike structure was surrounded by a cellular membrane, whose presence was visualized by using 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate staining. The location, diameter, optical transparency and the presence of rod-shaped nuclei in and the surrounding membranes of the threadlike structure were consistent with a nerve Bonghan duct. The Bonghan duct was claimed to be the extension of the physical substance of acupuncture meridians and to be a distinct type of circulatory system present in mammals. Although Bonghan theory has not been reproduced for a long time, recently, some portions of the Bonghan duct network were confirmed in various organs in mammals including blood vessels, lymphatic vessels and enteric organs. The novel threadlike structure in the central nervous system, more specifically in brain ventricles, is one in a series of findings in an attempt to rediscover the Bonghan duct network.     

Bonghan Ducts as Possible Pathways for Cancer Metastasis

ObjectiveThe present study has been designed to find a possible new route for the metastasis of cancer cells on the fascia surrounding tumor tissue using a novel technique of trypan blue staining.Materials and MethodsTumor tissues were grown in the skin of nude mice after sub-cutaneous inoculation with human lung cancer cells. Trypan blue was recently identified as a dye with specificity for Bonghan ducts (BHDs) and not other tissues, such as blood or lymph vessels or nerves.ResultsWe demonstrate that the trypan blue staining technique allows the first visualization of BHDs which are connected to tumor tissues.ConclusionSince BHDs are known to make up a circulatory system corresponding to acu puncture meridians or collaterals, we propose that, in addition to the currently known blood or lymph vessels, BHDs on tumor tissue fascia may be a novel pathway for metastasis.     

肿瘤微环境新生血管的病络机制

肿瘤属新生血管依赖性疾病，肿瘤微环境中不完整新生血管，具有升高肿瘤间质液压，加快肿瘤细胞侵袭及转移速度，加重肿瘤细胞恶化程度，抑制免疫细胞免疫应答等多重损害作用。肿瘤微环境新生血管生成及增殖，形态结构病理改变，血管内皮细胞功能异常，与络病学说中的病络过程十分相似，即“络虚”引起“毒损络脉”，络脉生长亢进无度；“结络”“盛络”“横络”“络弛”等病络改变，使新生血管形态结构异常；“络破”引起新生血管功能失常，导致血管高通透性和淋巴管液体滞留。文章通过探讨肿瘤微环境新生血管的中医病络机制，对于明确中药干预肿瘤新生血管的药效方向及机制，提高活血化瘀中药抑制血管生成疗效，具有重要理论指导价值。     

Characterization of DNA-containing granules flowing through the meridian-like system on the internal organs of rabbits

It is an essential question for acupuncture whether the acupuncture meridians form an anatomically distinctive system of threadlike ducts that spread throughout an animal body. We observed the threadlike structures on the surface of internal organs of animals. This threadlike structure may be the physical basis of the meridian-like network of internal organs as detected by the Bi-Digital O-Ring test imaging method. One of the characteristics of the threadlike structures is the presence of DNA containing granules or Bonghan granules that flow through the ducts. The aim of this study was to separate and characterize their morphology and ultrastructural features. Using confocal laser-scanning microscopy (CLSM) and transmission electron microscopy (TEM), we found that Bonghan granules were round and measure 1.7-2.5 microm in diameter. They had a small nucleus surrounded by a layer of cytoplasm and a trilaminar plasma membrane. TEM also showed that they had cytoplasmic protrusions like pseudopodia. They contained fragmented DNA with a significant amount of 3'-OH ends. We compared their properties with the cell structure and general morphology of apoptotic bodies, bacteria, and the microcells. The motion of these granules was influenced by light, which may be related to the photo-therapy on the acupuncture points.     

Development of a Laparoscopic System for In Vivo Observation of the Bonghan Structure

Bonghan structures composed of corpuscles and ducts have been observed in numerous animal experiments. The structure has been suggested to be the physical substrate for acupuncture points and meridians used in traditional Chinese medicine. The corpuscle-duct structure on the organ surface has been extensively studied. However, it was only observed after the abdomen of a rat was fully exposed. It may be desirable to observe these structures under minimally invasive conditions. Our laparoscope is specifically designed for in situ, in vivo observation of the Bonghan structure on the surface of internal organs in rats. We made a laparoscope consisting of a borescope combined with an illumination module and an imaging data acquisition part. The Bonghan structure is transparent and is hard to see, even with the borescope. Spraying a specific dye, trypan blue and washing with saline is a necessary process to visualize the structure. For this reason, we devised a staining and washing system attached in parallel with the borescope in the laparoscopic system. The system operated successfully to reveal a Bonghan structure without surgery. This tool opens a host of possible applications such as observation of flow of stained liquid or drug in the Bonghan duct flowing from the skin to internal organs, and long-term observation of the physiological changes in Bonghan corpuscles.     

肺一丸抑制肺癌细胞增殖的实验研究

目的研究中药复方肺一丸对肿瘤细胞增殖和凋亡的影响.方法应用人类小细胞肺癌裸小鼠模型观察中药复方肺一丸对癌细胞的抑制作用;利用透射电镜观察肺一丸诱导癌细胞凋亡的影响.结果中药复方肺一丸能明显抑制肺癌细胞的增殖,同时,透射电镜观察结果表明有明显的诱导肺癌细胞凋亡的作用,出现凋亡小体.结论中药复方肺一丸对肺癌细胞有明显的抑制增殖和诱导调亡的作用,中药复方治疗肺癌的分子生物学机制需要进一步研究.     

电针对血管性痴呆模型大鼠海马神经胶质细胞及微血管的影响

目的 :观察电针对实验性血管性痴呆 (VD)大鼠海马神经胶质细胞和毛细血管的影响。方法 :实验用 4 血管阻断模型 ,用Morris水迷宫测定大鼠学习记忆能力 ,并用电镜观察鼠脑海马神经胶质细胞和毛细血管的超微结构变化。结果 :模型组海马神经组织超微结构的形态损害明显 ,胶质细胞变性、水肿 ,毛细血管内皮细胞受损。电针能明显减轻或抑制缺血的大鼠海马神经胶质细胞及毛细血管的损害。结论 :电针明显改善痴呆大鼠的学习记忆能力 ,与其对脑组织神经元、神经胶质细胞及微血管的全脑保护作用密切相关     

益气活血解毒方对小鼠卵巢癌ID-8细胞株增殖及血管生成过程的影响

目的:研究益气活血解毒方抑制鼠卵巢上皮癌ID-8细胞株增殖的作用,并观察本方对于脐静脉内皮细胞(HUVECs)形成血管过程的作用,为本方发挥疗效提供实验依据。方法:采用细胞增殖与检测方法(cell counting kit-8,CCK-8)检测本方对于ID-8细胞增殖的抑制作用;将含中药血清、磁珠分选的调节性T细胞(regulatory T cells,Tregs)与抗血管内皮生长因子(VEGF)抗体分组共培养,观察血管生成情况差异。结果:与空白组比较,10%,20%含药血清均能够抑制卵巢上皮癌ID-8细胞株的增殖,此抑制作用在培养96 h时最明显;在72,96 h时,20%含药血清组的抑制率明显高于10%含药血清组(P0.05,P0.01)。与Tregs(-)各组相比,Tregs(+)各组HUVECs细胞呈现明显的血管网状结构,中药含药血清组血管成网不明显,VEGF抗体联合含药血清组血管成网情况最差。结论:益气活血解毒方对小鼠卵巢上皮癌ID-8细胞株的增殖过程有显著的抑制作用,同时能够干预血管的形成,这也许与本方影响Tregs促进肿瘤新生血管的作用相关。     

金黄膏外敷治疗糖尿病溃疡的实验研究

[目的]从动物实验探讨金黄膏对糖尿病溃疡的治疗作用机制。[方法]SD大鼠,诱发糖尿病皮肤溃疡模型,随机分为A金黄膏组、B普通胰岛素组、C凡士林组,分时段观察肉芽组织中成纤维细胞数、血管内皮细胞生长因子(VEGF)、CD34、新生毛细血管数。[结果]A组在纤维细胞数、新生毛细血管数及VEGF、CD34等指标与B组、C组比较均有显著性差异。[结论]金黄膏具有明显的促进糖尿病溃疡愈合作用,可能是通过促进成纤维细胞和新生毛细血管的增殖、加快肉芽组织的形成来体现。     

终末期肾病患者血清对内皮细胞凋亡的影响及黄芪总黄酮的干预作用

目的:探讨终末期肾病患者血清对内皮细胞凋亡的影响及黄芪总黄酮的干预作用。方法:收集22例健康志愿者和25例终末期肾病规律血液透析患者的血清,在体外模拟终末期肾病患者内环境,以人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)为对象,分为空白对照组(10%胎牛血清),正常对照组(10%健康人血清),终末期肾病组(10%终末期肾病患者血清),低剂量(TFA1),中剂量(TFA2),高剂量(TFA3)组(在终末期肾病组基础上分别加入0.5,1.0,2.0 g·L-1黄芪总黄酮),培养24 h后光镜下观察血管内皮细胞形态,MTT法检测细胞生存率,免疫组化法检测C/EBP同源蛋白(CHOP)的表达,ELISA法检测4-羟基壬烯醛(4-HNE)的含量,TUNEL法检测细胞凋亡。结果:与正常对照组相比,终末期肾病患者组细胞生存率降低(P<0.01),CHOP和4-HNE升高(P<0.05),凋亡细胞数增高(P<0.01)。黄芪总黄酮干预后,各组HUVECs生存率提高(P<0.05);CHOP的表达降低(P<0.05),中剂量剂量组优于低剂量组(P<0.01),但高剂量组与中剂量组差异无统计学意义;4-HNE含量降低(P<0.05),细胞凋亡减少(P<0.05),呈剂量依赖性(P<0.05)。相关性分析显示:CHOP与4-HNE的含量呈正相关(P<0.01)。结论:终末期肾病患者血清可诱导HUNFCs凋亡,其机制部分与内质网应激诱导的细胞凋亡通路相关,黄芪总黄酮可通过减轻内质网应激从而抑制终末期肾病患者血清诱导的内皮细胞凋亡。     

Induction of apoptosis by a synergistic lignan composition from Cedrus deodara in human cancer cells

AP9‐cd, a synergistic lignan mixture from Cedrus deodara (Pinaceae) consisting of (?)‐wikstromal, (?)‐matairesinol and dibenzyl butyrolactol, depicted cytotoxic effects against numerous human cancer cell lines reported previously. The aim of this study was to investigate the mechanism of cell death in human cancer cells. The viability, morphological and ultrastructural changes in Molt‐4 cells were investigated. Using the trypan blue exclusion assay, we demonstrated that AP9‐cd significantly reduced the viability of Molt‐4 cells in a time‐ and dose‐dependent manner. Apoptotic assays using light microscopy revealed that this agent induced Molt‐4 cell apoptosis at varied concentrations. The treatment causes a loss in cell viability by activating the apoptotic process as identified by light and electron microscopy. The morphological changes of intracellular organelles in Molt‐4 cells treated with 30 µg/ml of AP9‐cd revealed the disruption of mitochondrial cristae. Other features included the vacuolization, chromatin condensation and formation of micronuclei. Surface ultrastructural studies of four different tumor cell lines (Molt‐4, HL‐60, PC‐3 and A‐549) treated with AP9‐cd depicted loss of surface projections, condensation and formation of apoptotic bodies. AP9‐cd treatment to transgenic fruit fly, Drosophila, carrying human adenomatous polpyposis coli (hAPC) gene enhanced eye phenotypes and therefore may inhibit Wnt/Wg pathway which is important in the aetiology of a number of human cancers. Copyright © 2008 John Wiley & Sons, Ltd.     

四妙勇安汤抑制中性粒细胞-内皮细胞黏附的实验研究

目的： 探讨四妙勇安汤对于凝血酶处理的内皮细胞与中性粒细胞黏附及内皮细胞P-选择素表达的影响。方法： MTT法检测四妙勇安汤以生药22.5 g·kg^-1·d^-1,ig 7 d制备含药血清对于内皮细胞活力的影响,EA.hy926细胞加入0.2 U·mL^-1凝血酶、中性粒细胞作为模型组,2.5%,5%,10%,20%四妙勇安汤含药血清预处理内皮细胞24 h后造模为干预组。虎红法检测细胞黏附率,流式细胞术、ELISA检测内皮细胞P-选择素的表达。结果： 空白组与空白对照组在各项检测中均无显著性差异;10%,20%含药血清组对黏附率有抑制作用,10%组效果较好;ELISA结果显示10%含药血清组预处理组能抑制P-选择素的表达,其他3个干预组则无差异;流式细胞术检测10%,20%含药血清组对于细胞表面P-选择素有抑制作用,10%含药血清组效果最好。结论： 四妙勇安汤能抑制中性粒细胞、内皮细胞的黏附,这一作用可能是通过抑制内皮细胞P-选择素的表达来实现的。     

当归补血汤调控缺氧血管内皮细胞增殖及其分子机制研究

目的: 通过当归补血汤对缺氧血管内皮细胞增殖及分子表达的研究,探讨当归补血汤调控缺氧血管内皮细胞增殖的可能分子机制。 方法: 应用连二亚硫酸钠溶液建立缺氧血管内皮细胞(EA.hy926)模型(以下简称缺氧模型),不同剂量当归补血汤干预24 h后,CCK-8法观察缺氧血管内皮细胞的增殖;ELISA法检测血管内皮生长因子(VEGF)及其受体(VEGFR1,VEGFR2,sVEGFR1,sVEGFR2)的表达。 结果: 与正常血管内皮细胞比较,缺氧后血管内皮细胞增殖能力显著下降(P<0.01);当归补血汤能够促进正常血管内皮细胞及缺氧血管内皮细胞的增殖,其中高、中剂量组促进缺氧血管内皮细胞增殖的幅度明显大于促正常血管内皮细胞增殖的幅度(P<0.01)。当归补血汤各剂量组均能促进缺氧血管内皮细胞VEGF,VEGFR1,VEGFR2的表达,且与剂量呈正相关,抑制缺氧血管内皮细胞sVEGFR1,sVEGFR2的表达。 结论: 当归补血汤能够促进缺氧血管内皮细胞的增殖,其机制可能与其调节VEGF与VEGFR和sVEGFR两种受体的结合有关。     

黄芪注射液对宫颈永生化上皮细胞生长的作用

目的:观察黄芪注射液对人宫颈永生化上皮细胞(H8细胞)的生长抑制作用,分析其细胞周期调控机制。方法:采用MTT法检测黄芪注射液对H8细胞生长的作用,光学显微镜观察药物作用后细胞形态,利用流式细胞仪(FCM)分析黄芪注射液作用后H8细胞的周期。免疫荧光分析的第一抗体为细胞周期蛋白(cyclin)Dl,B1,细胞周期蛋白依赖性激酶(CDK)1,4和细胞周期蛋白依赖性激酶抑制因子(CDKI)p21蛋白,第二抗体采用荧光素标记,应用FCM检测黄芪注射液作用后H8细胞荧光强度。结果:黄芪注射液对H8细胞的生长有抑制作用,其中20 g·L-1作用最明显。黄芪注射液处理组可见细胞生长稀疏、死亡或凋亡,细胞固缩成深染圆形小体。黄芪注射液作用于H8细胞时,S,G2期细胞减少,使细胞阻滞于G1期。H8细胞cyelin D1蛋白表达降低极显著;cyclin B1,CDK4蛋白,p21蛋白表达略降低,无统计学意义;CDK1蛋白表达显著降低。结论:黄芪注射液有可能作为一种细胞周期调控剂来预防或辅助治疗宫颈癌前病变。     

食物中脂质对葛根素微乳经淋巴转运的影响

目的以实验室前期制备的葛根素微乳为基础,采用乳糜微粒阻断法研究食物中脂质对葛根素微乳经淋巴转运的影响。方法以环己酰亚胺为乳糜微粒阻滞剂阻断药物经淋巴转运,HPLC法测定大鼠血浆中葛根素浓度,比较阻断组与未阻断组生物利用度来计算淋巴转运的比例。结果葛根素微乳在脂质处理组的淋巴转运比例与水处理组不存在显著性差异,但证实了微乳对促进葛根素淋巴转运及提高其生物利用度的重要贡献。ig给予脂质不能进一步促进葛根素微乳的经淋巴转运,但证实了葛根素以微乳为载体能降低食物中脂质对其口服生物利用度的影响。结论食物中的脂质并未进一步促进葛根素微乳淋巴转运,且对生物利用度影响不大。     

蕤仁的生药学鉴别研究

目的对蕤仁药材Prinsepiae Nux的性状、微性状及显微鉴别特征进行系统研究,为其鉴定以及《中国药典》2020年版的制定提供科学依据。方法采用性状鉴别法、微性状鉴别法、常规显微鉴别法及偏振光显微鉴别法,对12批蕤核Prinsepia uniflora和9批齿叶扁核木P. uniflora Batal. var. serrata Rehd.果核进行系统的生药学研究。结果从形状、大小、颜色、表面特征、质地、断面、气、味等方面,对蕤核及齿叶扁核木果核的性状特征进行观察。针对质地坚硬、经软化处理后仍不适宜制备横切片以观察完整植物组织构造并确定组织部位的药材,采用定位取材徒手切片技术,可准确获取特定部位的植物组织显微特征;针对木化组织集成较大群束,经粉碎后难以呈现单个细胞完整形态特征的药材,采用解离组织制片技术,可获取清晰、完整无重叠的单个细胞全貌及特征信息。结论首次获取了蕤仁显微鉴别特征(内果皮石细胞、种皮表皮细胞、中果皮纤维、内胚乳细胞、子叶细胞)及微性状鉴别特征(内果皮、子叶、种皮)的全息彩色影像数据。显微特征中内果皮石细胞可作为蕤仁的专属性显微鉴别标志物,显微和微性状鉴别研究结果填补了彩色影像信息的空白。     

冬凌草甲素通过线粒体途径诱导人黑色素瘤A375-S2细胞凋亡

目的　研究冬凌草甲素诱导人黑色素瘤 A375 - S2细胞凋亡的作用机制。方法　 MTT法检测冬凌草甲素对 A375 - S2细胞生长的影响及各种 Caspase抑制剂、佛波酯 (PMA)、PKC抑制剂 H- 7对冬凌草甲素作用的影响 ;DNA凝胶电泳法检测细胞凋亡 ;L DH法测定乳酸脱氢酶 (L DH)活力。结果　冬凌草甲素对 A375 - S2细胞生长有显著抑制作用 ,并能显著诱导 A375 - S2细胞发生凋亡 ,其作用呈明显的量效关系和时间依赖性。形态学观察可见凋亡小体的形成 ,琼脂糖凝胶电泳可见凋亡典型的 DNA梯带 ;Caspase家族抑制剂 ,Caspase- 9抑制剂能显著抑制冬凌草甲素诱导 A375 - S2细胞的凋亡。大剂量 PMA(4 0 0 ng/m L)显著抑制 34.3μmol/L 冬凌草甲素诱导 A375 - S2细胞凋亡 ,而小剂量 PMA(10 ng/m L)与冬凌草甲素有协同杀死细胞的作用 ,且 PKC抑制剂 H- 7也可下调这种凋亡作用。 Caspase- 3的抑制剂可抑制 Caspase- 3的活力升高。结论　适宜浓度的冬凌草甲素 (34.3μm ol/L )诱导A375 - S2细胞凋亡 ,这种作用是通过线粒体调节 Caspase的激活来实现的。