【作者】 李燕园；

【导师】 李继承；

【作者基本信息】 浙江大学 ， 病理学与病理生理学， 2004， 博士

【摘要】 研究背景：腹膜淋巴孔（peritoneal lymphatic stomata）是腹膜毛细淋巴管在腹膜间皮上的开口。籍腹膜淋巴孔，使腹膜腔与脉管系沟通。所以，腹膜淋巴孔是腹膜腔内物质转归的主要部位，有重要的理论研究与临床应用价值。 在1863年von Recklinghausen首先报道了腹膜淋巴孔。他认为，通过腹膜淋巴孔，腹膜腔与腹膜淋巴管系直接相通。Recklinghausen的结论曾引起许多学者的争论。Tsilibary和Wissig首先在电子显微镜下证实了小鼠膈腹膜淋巴孔的存在。其后许多学者在不同动物及人体内对腹膜淋巴孔的形态和功能、分布、发育和调控等进行了研究，结果显示腹膜淋巴孔是毛细淋巴管的微小开口，是相对恒定的结构。腹膜腔内的液体、颗粒、细胞、甚至感染性微生物和肿瘤细胞等成分，均可以通过淋巴孔快速吸收。 腹膜淋巴孔分布的广泛，除分布在膈腹膜上，Mironov等在大鼠的大网膜，Shinohara等在小鼠卵巢表面的脏层腹膜，Tesch在人肝镰状韧带，李继承等在人盆壁腹膜，隋梅花和李继承在豚鼠卵巢囊和人卵巢表面均发现了腹膜淋巴孔的存在。 李继承和俞寿民最早报道了人隔腹膜上的腹膜淋巴孔于妊娠20-32周时就可在立方形间皮细胞上被观察到。Nakatani等进一步把腹膜淋巴孔的形态发生分为四个连续的阶段：即①腹膜下淋巴管出现；②淋巴内皮细胞的胞质突起伸入结缔 浙江人学博I:学位论文组织中:③内皮细胞的胞质突起伸入基膜和间皮细胞之间;④细胞间连接的分离形成淋巴孔。李慧和李继承最近的研究表明，胚胎13天时，小鼠隔腹膜仅由扁平形I’AJ皮细胞（flattened mesothelial eell）覆盖:胚胎15天时，开始出现立方形间皮细胞（euboidal mesothelial eell）和淋巴孔，但出生后的腹膜淋巴孔刁‘具有物质吸收功能。 对淋巴孔调控的研究是该领域最活跃的一个课题。Tsilibary和wissig用唬拍胆碱和卡巴哇诱导隔肌的舒张与收缩，发现当隔肌收缩时，腹膜淋巴孔数量大为减少;而在隔肌松弛时，腹膜淋巴孔数量显著增加。腹内压的变化也是调控腹膜淋巴孔开放的重要因素，腹腔内液体增加到引起腹内压增高时，腹膜淋巴孔开放的数量会增加。 隋梅花和李继承用妊娠和激素干预豚鼠的卵巢，发现妊娠可促使卵巢囊淋巴孔的开放，导致吸收功能增强。而雄激素干预组的卵巢囊淋巴孔开放和吸收功能均较妊娠组、促排卵组降低。 李继承等通过大量实验，证实一些中药如健脾益气类药物白术、党参，活血化癖类药物丹参，利水消肿类药物泽泻等，具有较强的腹膜淋巴孔调控作用，均可开放腹膜淋巴孔，导致腹膜淋巴孔对腹水的吸收作用加强，并在实验性肝硬化腹水研究时，发现有显著的腹水转归作用。另外，李继承等发现在长期腹膜透析时，NO产量增高，与腹膜淋巴孔孔径和分布密度成正比，NO有扩大腹膜淋巴孔，促使腹膜淋巴孔吸收的作用。Doboszynska等还证实猪的子宫阔韧带淋巴孔聚集区立方型间皮细胞有NOS活性，并推测NO对淋巴孔可能有调节作用。而丁世萍和李继承最近的研究则进一步表明，NO是中药调控淋巴孔的关键所在。但NO调控淋巴孔，促进淋巴吸收的细胞信号转导机制尚不清楚。 但目前对胸膜淋巴孔的研究较少，更无对胸膜淋巴孔和淋巴吸收调控的报道。本实验就胸膜淋巴孔的超微结构及其淋巴引流途径和NO调控进行探讨，以期为胸腔积液的形成和治疗、胸内炎症与肿瘤的转移扩散及局部免疫等方面的研究，提供可靠的形态学依据。同时，我们利用体内外实验研究了NO调控淋巴孔的细胞信号转导机制，为药物的淋巴孔调控，促进肝硬化腹水转归和提高腹膜透析疗效等课题提供重要的理论依据，并为淋巴孔调控的分子生物学研究奠定基 浙江大学博₁:学位论文础。研究目的（1）研究胸膜淋巴孔的超微结构以揭示其作为胸膜腔物质引流主要通路的结构基础;（2）研究NO是否对胸膜淋巴孔和淋巴吸收具有调控作用;（3）探讨NO调节淋巴孔，促进淋巴吸收的细胞内信号转导途径，为药物的淋巴孔调控和临床应用提供实验依据。研究方法（l）通过扫描电镜、透射电镜和Na0H消化一SEM研究胸膜淋巴孔的超微结构。（2） NO供体和一氧化氮合酶（Nos）抑制剂分别腹膜腔给药，示踪剂（台盼蓝）胸膜腔注射后，测血清N0和台盼蓝浓度;扫描电镜下观察各组胸膜淋巴孔，并用Elesc叩e计算机图像处理软件对胸膜淋巴孔做定量处理。（3）在培养间皮细胞上，利用cGMP检测试剂盒和激光共聚焦扫描显微镜，研究NO对间皮细胞内cGMP和游离【ca2+]i的影响，研究腹膜淋巴孔调控，促淋巴吸收的NO-eGMP一CaZ+信号转导途径。研究结果（l）脚膜淋巴孔的超微结构和胸膜腔淋巴引流通路:家兔肋胸膜的内表面被覆有扁平和立方形间皮细胞，后者的胞质突起和胞体共同形成圆形或椭圆形的淋巴孔。其平均面积和在立方形间皮细胞区的平均密度分别为:7.加士3.69p mZ和1.21士0.72个/0 .olmmZ。在淋巴孔分布的区域更多还原

【Abstract】 Background Peritoneal lymphatic stomata are openings of the peritoneal lymphatic vessels into the peritoneal cavity. The peritoneal cavity is connected with the vascular system by the lymphatic stomata. It is the most important pathway to drainage of substance in the peritoneal cavity, and plays an extremely important role in physiological and pathological conditions.In 1863, von Recklinghausen firstly described that there are some pores existing among the adjoining peritoneal mesothelium, and named them as the peritoneal lymphatic stomata. He considered that the peritoneal cavity was connected with the lymph system by the stomata. But at that time his standpoint was controversial. In 1977, Tsilibary and Wissig confirmed the lymphatic stomata in mouse diaphragm by electron microscopy. Later a number of researchers made detailed studies about the peritoneal lymphatic stomata in different animals and human being including the structure and function, the distribution, the development, and the regulations etc.It is showed that the lymphatic stomata are surrounded by cuboidal mesothelial cells. They are relatively stable structure, and drained of fluid, particulate material and especially cells from the serious cavity. The lymphatic stomata distribute not only in the diaphragm but also in the pelvic peritoneum, in the omentum, in the pericardium and in the ovarian bursaand etc.Li Jicheng and Yu Shouming reported human peritoneal lymphatic stomata in fetuses aging from 20-32 weeks. Nakatani et al. studied the detailed process of lymphatic stomata formation. Li Hui and Li Jicheng found that lymphatic stomata firstly appeared on embryonic day 15 in the mice, but only those postnatal lymphatic stomata have absorptive function.The regulation of the lymphatic stomata is the most noticeable topic in the field. It has been known that the peritoneal are adjustable by some factors, i.e. intraabdominal pressure, some traditional Chinese medicine, nitric oxide (NO) and vascular endothelial growth factor (VEGF), etc. Tsilibary and Wissig found that stomata open when fluid pressure within the peritoneal cavity is elevated. The stomata also open when the diaphragm is stimulated to relax with succinylcholine and closed when treated with carbachol. Sui Meihua and Li Jicheng reported recently that both the openings and absorptive functions of the lymphatic stomata of ovary bursa in guinea pig could be enhanced by pregnancy and decreased by exogenous homone. Li Jicheng et al. suggested that Chinese meteria medica could regulate the patency of the lymphatic stomata. He also found that the increased NO production in long-term peritoneal dialysis could relax lymphatic stomata leading to the enhanced lymph absorption or ultrafiltration failure. Ding Shiping and Li Jicheng lately proved that NO is the key factor of Chinese meteria medica regulate lymphatic stomta and removing ascites. But, there are no reports on the cell signal transduction mechanism of NO regulating lymphatic stomata and lymph drainage.However, the material about the structure of pleural lymphatic stomata is limited, and even no reports on regulation of the pleural lymphatic stomata. In the present experiment, we studied detailed ultrastructure of pleural lymphatic stomata and the effect of NO on the pleural lymphatic stomata and lymph absorption from the pleural cavity. These study has great significant in the explaination of hydrothorax formation, expression of infection and tumor metastasis in the pleural cavity. We also studied the cell signal transduction mechanism of NO on lymphatic stomata and lymph absorption expecting to provide some evidence for the Chinese meteria medica regulating lymphatic stomata and promoting curative effect of peritoneal dialysis.Objective (1) To study the ultrastructural structure of costal lymphatic stomata in the rabbit. (2) To study the effect of NO on the drainage capacity of pleural lymphatic stomata in the rat. (3) To study the cell signal mechanism of NO on the lymphatic stomata and lymph drainage capacity in th更多还原