Research on the development of peritoneal lymphatic stomata in mouse diaphragm～*

Background and Purpose Von Recklinghausen found under an optical microscope, every peritoneal surface adjacent mesothelial cells connected at the holes, he first named these holes lymphatic stomata (lymphatic stomata, LS), and LS is the peritoneal cavity and peritoneal a direct channel between the lower lymphatic. 1977 Tsilibary first electron microscopy applied to the study of LS and LS mice under the electron microscope confirmed, some scholars have observed in different animal LS. 1990 Li Jicheng first reported human peritoneal LS and LS is a relatively constant structure. LS is opening peritoneal lymphatic capillaries in the peritoneal mesothelial cells. Membership LS, the peritoneal cavity and the vascular system directly connected. LS initiative absorbing function. Leak bacteria and some drug particles injected into the peritoneal cavity and found that these particles are rapidly through the LS. Li Jicheng respectively trypan blue solution and erythrocytes injected into the peritoneal cavity of mice and rabbits, observed under the electron microscope, trypan blue particles and erythrocytes LS absorption. The Nanba research also found that tumor cells can also be transferred by the LS. Therefore, in the case of physiological or pathological, LS are considered to be the most important outcome of the peritoneal cavity of liquids, particulate matter and cellular components. Absorption with ascites, peritoneal dialysis closely related to loss of ultrafiltration, transfer and proliferation of tumor cells. As the body of a constant structure, morphology and function of the LS of concern by many scholars, but so far little information about LS phylogeny. Domestic scholars have not yet done research in this area. In this study, using scanning electron microscopy and computer image processing, transmission electron microscopy and enzyme histochemistry of all ages in the embryonic mouse and postnatal mice peritoneal barrier LS and every lymphatic research. And described the LS and every lymphatic dynamic and developmental, phylogenetic data for further LS function of research and clinical applications. Materials and Methods 1, animal grouping: NIH mice a total of 150, three per cage, to Raising a mixture of male and female ratio of 2: l. Female vaginal suppository for pregnancy (Embryonc day, EDI). Take the embryo 13 (Embryonc days13, ED13), E15 (Embryonic days 15, ED15), 18 (ED18) after birth (Postnatal day, PDI), 5 (PDS). 10 (PD10) and adult mice across the peritoneal tissue of the 30 cases. 2, TEM sample preparation and observation: the candied peritoneal trimmed to IX lmm 'small tissue blocks, 2.5% glutaraldehyde and 1% acid bis hunger! Liu Ding, 2% uranyl acetate block staining, graded ethanol and acetone dehydration, EPON SLZ embedded positioning of light microscopy, ultra-thin sections, uranyl acetate and configurations in addition to acid lead double staining, P hi ip SEM the 4I0-type transmission electron microscopy observation, speed Tun pressure 60kV. 3, scanning electron microscopy sample preparation, observed: 2.5% glutaraldehyde Lee 1% acid hungry dual fixed conductive treatment of 2% tannic acid gradient dehydration of ethanol and 100% acetone, 100% isoamyl acetate replacement. HCP-2 critical point drying instrument dry, E-1020-type gold-plated membrane ion sputtering instrument, observe the Stereoscan 260 scan Tuen mirror,, acceleration voltage of 20kV. 4 Elescope computer image processing software: a scanning electron microscopy image of the analog video signal by the image sampling the card (FlyVideo98) is converted into a digital image stored in the computer hard disk, and using Elescope knife image processing software for analysis and processing. The software according to the gray value of the image, the white Activity and semi-automatic analysis of the sampled digital image. 5 Statistical analysis: calculation of all ages mice CMC area of ??the region and accounted for every area of ??the peritoneum white than the average size of every LS and the average distribution density. X S. 6, enzyme histochemistry: (1) frozen sections: Take fresh diaphragm samples, trim into a rectangle, with 0. Flush IM PSS, the Leca CM3050 type slicer for frozen section 2; (2) 5'-nuclear Governor acid enzyme (Nase) - alkaline phosphatase (ALPase) double staining: reaction solution (0.IMol / L Tris * turns Ding ene acid buffer 42m!, 5'-AMP20mg, 2.5% magnesium sulfate sin! sucrose 3g, 2% lead nitrate 3ml, pH 7.2) were incubated for 1 hour, water bath temperature was controlled at 37-38oC, double distilled rinse with water by 1% amine disulfide color; reaction solution (naphthol AS-BI 20mg the DMF 0.sml, 0.! Mol / LTR BU - 50 ml of HCl buffer, fast blue B salt 50mg PHg.2) incubated for two hours, water bath temperature control at 37-38oC, rinsed with distilled water, dried, neutral resin Fengpian, optical microscope and photographed. And as blank control group, 5-Nase single-stained control group and AIPase single-stained control group. 7, axillary muscle lymphatic tube development of dynamic observation: according to J \u0026 muscle lymphatic tube development morphological characteristics and enzyme tissue chemical reaction of strength, on the development of lymphatic vessels of the mouse compartment semi-quantitative analysis results l cubic cells of the embryonic period (cuboidal mesothelial cell, CMC) and early peritoneal lymphatic stomata (nonage lymphatic stomata, NLS) N Health: E DI3 across the peritoneal surface covered with a layer of flat mesothelial cells (fi attene dmesotheia eel, FMC); ED15 across the peritoneal ribs began to appear the scattered distribution CMC and during the NLS, peritoneal Bu no lymphatic capillaries: the BD18 across the peritoneal sternum and spine Ministry tufted the cuboidal cells regional NLS distribution every peritoneal began to appear lymphatic capillaries. trypan blue test the embryonic period NLS do not have the absorptive function; born mature lymphatic stomata (matured lymphatic cited m team, MLS ) occurs: the P01 of intestinal peritoneal muscle Ministry banded structure of the cubic cell area the muscle - CMC regional shy at the junction and the center of the chest tufted distribution between the CMC many LS LS, protrusion of the lymphatic endothelial cells. observation of ultrathin sections, found LS the CMC basement membrane Lee its deep surface of the connective tissue disconnect (dsrupted), two adjacent lymphatic endothelial cell cytoplasm processes to LS, close to the CMC, constitutes a small tube in the peritoneum (hannel) the peritoneum under tubules communicate with LS, the peritoneal cavity substances drainage?