Studies On The Main Functioning Cytology In The Intestine, Spleen And Pituitary Gland Of Chinese Soft-Shelled Turtle, Pelodiscus Sinensis

Soft-shelled turtle, Pelodiseus Sinensis distributed widely in China and this species was famous for its important value in economy and in pharmacy. In the biology aspect, Pelodiseus Sinensis were belonged to Reptilia, they were ectothermic animal and had hibernation. The physiological action and metabolic process of the digest, immunity and endocrine secretion were different from other kinds of animals. With the scale of the turtle hand-feeding has rapid development, the communicable diseases became more and more serious. Most of the scholars were focused on the researches of turtle disease, there was almost no studies on the special structural feature of turtle. The present study was used Pelodiseus Sinensis as research target. We selected three typical tissues in the turtle, they were intestine of the digestive system, the spleen of the immune system and the pituitary gland of the endocrine system. The text included three parts, in the first part, we examined the structural feature and cell component of mechanical barrier and immunologic barrier, the distribution and ultrastructure of interstitial cells of Cajal in the turtle gut. In the second part, the ellipsoid high endothelial capillary and lymphocytes migration cytologic basement in the spleen were studied. In the third part, the ultrastructure and seasonal change of endocrine cell were investigated in the adenohypophysis. All the aboved three parts, the turtle cytological feature and their significance were expounded in detail. The studies provided theoretical foundation for the further research on the physiologic and immunologic function, physiological aspection during the hibernation and non-hibernation of the turtle. At the same time, they also provided sciencific basis for the hand-feeding, disease prevention and cure in the Pelodiseus Sinensis.Experiment I The fine structure and cell character of barrier in the intestine mucosal epithelium of Chinese soft-shelled turtle, Pelodiseus Sinensis. Light and transmission electron microscopy were used to study the structural feature of barrier and the distribution pattern of intestinal intraepithelial lymphocyte (IELs) in different month in the normal turtles intestine mucosal epithelium. IELs was one of the most important part of the immunologic barrier. The results showed that in the small intestine, the mucosal epithelium was single layered or pseudostratified columnar epithelium. In the apical parts of the epithelium, enterocytes were arranged tightly. There have cell junctional complex of tight junction, intermediate junction and desmosomes. In the middle and basal parts of mucosal epithelium, enterocytes were connected by desmosomes and interdigitations. In the small intestinal epithelium, most of enterocytes contained several mitochondria in the cytoplasm. A few enterocytes were loaded with numerous mitochondria and lipid droplets. In the large intestine, the epithelium was pseudostratified. All the enterocytes did not contain lipid droplets. Enterocytes were arraged tight in the apical parts of the epithelium. They were arranged loosely and formed the obvious intercellular spaces in the middle and basal parts of mucosal epithelium. Numerous mucous cells were scattered between the enterocytes of the entire mucosal epithelium. Some of them contained mucus granules of low electron density in the cytoplasm, others had the high electron density. The mucus was secreted by mucous cells and covered the surface of intestinal mucosa. No microfold cells (M cells) were observed throughout intestinal epithelium. In the whole intestine mucosal epithelium, channels with a diameter of 1-7μm were found between the enterocytes. Sometimes, a lymphocyte resided in the channel. IELs (4-9μm in diameter) located in the whole intestinal epithelium, some of them had protruded pseudopodium between the enterocytes, showing the migration tendency, and others were found in the lumen of the intestine. In the nuclear layer and basal layer of the small and large intestinal epithelium, there were two types of plasma cells, one had the dilated rough endoplasmic reticulum and the other the parallel. The amounts of IELs decreased gradually from May to September, up to December. It was therefore suggested that the intestine mucosal epithelium have the mucosal barrier function in the normal turtle. Enterocytes and their tight junction complex, the mucus of mucous cell were composed of the mechanical barrier. Immunologic barrier contained plasma cell, IEL and channel. The distribution of mucosal immune cell in the turtle intestine was affected by the season.Experiment II Distribution of CD3+T、CD22+B lymphocytes in the intestine mucosal immunologic barrier of Chinese soft-shelled turtle, Pelodiseus Sinensis. Using immunocytochemistry SABC, we studied the distribution feature of CD3+T lymphocytes、CD22+B lymphocytes in the intestine mucosal immunologic barrier of Pelodiscus sinensis. At the same time, the distribution regularity of the CD3+T、CD22+B lymphocytes have been statisticed and analyzed in the different segment of turtle intestine. The results showed that CD3+T lymphocytes predominanted in the lamina propria and fewer in the epithelium. By contrast, great numbers of CD22+B lymphocytes were present in the epithelium and small numbers in the lamina propria. In the epithelium, the density of CD3+T lymphocytes in the distal small intestine was much greater than in any region of the small intestine. They increased from proximal to distal small intestine, decreased from proximal to distal large intestine. In the lamina propria, the highest density of CD3+T lymphocytes occurred in the middle small intestine. They reduced from proximal to distal large intestine. In the epithelium and lamina propria, CD3+T lymphocytes showed significant difference among the most segment of small and large intestine. Throughout the intestine, the total quantity of CD3+T lymphocytes in the lamina propria were 3.11 times of these cells in the epithelium. The number of CD3+T lymphocytes in the small intestine were 2 times of these positive cells in the large intestine. With regard to CD22+B lymphocytes, in the epithelium, the highest numbers of these cells were found in the distal small intestine, and they decreased from proximal to distal large intestine. In the lamina propria, CD22+B lymphocytes decreased in turn from proximal to distal small intestine, reduced from proximal to distal large intestine. In the epithelium, the amount of CD22+B lymphocytes showed no obvious difference during the most segment of small and large intestine. In the lamina propria, there was no remarkable difference among the each segment of small and large intestine. The numbers of CD22+B lymphocytes in the small intestine were 1.75 times of in the large intestine, and the total numbers of these cells in the epithelium were 7.99 times of these positive cells in the lamina propria of the whole intestine. The distributional difference of T and B lymphocytes in the turtle intestinal immunologic barrier suggested that they may play the different biologic function in the immune response of the intestinal mucosa.ExperimentⅢThe immunocytochemistry reaction of S-100 protein in the intestine of Chinese soft-shelled turtle, Pelodiseus Sinensis. By using immunohistochemistry, this paper studied the distribution of S-100 protein positive cells in the Pelodiscus sinensis intestinal tract. S-100 protein positive cells were classified into two groups. S-100 protein positive cells in the first group had irregular shape and processes with different thickness, length. Most of these cells were located in the lamina propria and submucosa of small intestine. Fewer cells were found in the large intestine. Cells in the second group included bipolar and stellate S-100 protein positive cells. Bipolar S-100 protein positive cells had long and short processes. They were observed in the interspace between the submucosa and circular muscle layer, within the circular muscle layer, as well between the longitudinal and circular muscle layers of whole intesinte and submucosa of large intestine. Stellate S-100 protein positive cells were only found in the large intestine lamina propria, their irregular processes were connected each other forming a network. S-100 protein was regarded as specific antibody for dendritic cell, it also expressed in the interstitial cells of Cajal and glial cells. According to the morphology and distribution of S-100 protein positive cells in the intestine. Cells in the first group were regard as dendritic cells. Cells in the second group were either interstitial cells of Cajal or glial cells. Previous researches suggested that dendritic cells could paly the function of antigen presentation. Interstitial cells of Cajal were regarded as pacemaker and glial cells were maintained the intestinal mucosal epithelium integrity. Base on the findings, we presumed that the mucosal immunologic barrier was correlated with dendritic cells in the turtle small intestine. Interstitial cells of Cajal and glial cells could regulate intestinal movement and barrier function, respectively.Experiment IV The distributional difference and ultrastructure of interstitial cells of Cajal in the intestinal tract of Chinese soft-shelled turtle, Pelodiseus Sinensis. By using c-Kit immuocytochemistry and transmission electron microscopy, this paper investigated the distribution and ultrastructure of interstitial cells of Cajal (ICCs) in the intestinal tract of Pelodiscus sinensis. The c-Kit immuocytochemistry results showed that ICCs were bipolar and stellate cells. The processes of bipolar c-Kit+ICCs some were long, some were short or no obvious. Bipolar c-Kit+ICCs were present in the large intestine submucosa, in the interspace between the submucosa and circular muscle layer, the circular muscle layer, between circular and longitudinal muscle layer, serosa of small and large intestine. In the submucosa, bipolar c-Kit+ICCs were found arrounding the blood vessel. In the interspace between the submucosa and circular muscle layer, between circular and longitudinal muscle layer, bipolar c-Kit+ICCs showed as shape of line or cluster. Sometimes, these cells were surrounded the blood vessel. In the circular muscle layer, bipolar c-Kit+ICCs were appeared surrounding the blood vessel or parallel to the adjacent smooth muscle cell. Bipolar c-Kit+ICCs scattered in the serosa. Stellate c-Kit+ICCs had numerous long and short processes that formed a network in the villus. These cells were found only in the large intestine lamina propria, they were surrounded the blood vessel. Bipolar and stellate ICCs were identified in the turtle intestine by transmission electron microscope which contain many characteristics; viz., numerous mitochondria, caveolae, thin cytoplasmic extensions or processes, rough endoplasmic reticulum. Gap junctions were formed between ICCs and ICCs, ICCs and nerve, ICCs and smooth muscle cells. All the finding suggested that ICCs have unique cell networks and ultrastructure characterwhich were suitable with its functions.Experiment V The discovering of ellipsoid high endothelial capillary and lymphocytes migration cytologic base in the spleen of Chinese soft-shelled turtle, Pelodiseus Sinensis. By using microscopy and electron microscopy, the architecture feature of spleen white plup of the Chinese soft-shelled turtle, Pelodiseus Sinensis were examined in detail. The results showed that the ellipsoid capillary was different from capillary in the other kinds of animals, it belonged to high endothelial capillary. The endothelial cells of the ellipsoid capillary were cube or cylindroid. So, it was equaled to the mammalian high endothelial venule. There had blood-spleen barrier (BSB) in the turtle spleen. BSB could prevent the foreign antigen (carbon particles) enter the periellipsoidal lymphocyte sheath (PELS). At the same time, they could form special and temporary channel structure, allow lymphocyte movement and arrive at the PELS. The channel structure was composed of reticular cells and fibers, migrating cells (lymphocytes, macrophages, blood cells), supporting cells, ellipsoid associated cells (EACs), channels. All the components formed a microenvironment specific for lymphocytes migration. The processes of lymphocyte migration was showed following. First, channels were formed between the endothelial cells of ellipsoid high endothelial capillary. Lymphocytes were along the channel and passed the basement membrane of ellipsoid high endothelial capillary, moved in the ellipsoid. Then, by the supporting cells and their processes, lymphocytes along the channel, arrived outside of PELS. At last, lymphocytes were moved from external to the inner PELS by connecting with the EACs and their processes. This study was provided cytologic groundwork for further researching on the reptile lymphocyte homing. Experiment VI The ultrastructure and seasonal change of endocrine cell in the Pelodiscus sinensis’s adenohypophysis. The ultrastructure of endocrine cells in the Pelodiscus sinensis’s adenohypophysis were studied by the transmission electron microscope, during the hibernation and non-hibernation period. The results showed that it contained 5 types of endocrine cells in the adenohypophysis.They were growth hormone (GH) cells, prolactin (PRL) cells, thyrotropin (TSH) cells, adrenocorticotrophic hormone (ACTH) cells, gonadotrophin (GTH) cells. Their ultrastructural features appeared characteristically. GH cells were rounded or elliptic in shape, which about 10-12μm in diameter. Numerous round secretory granules were found in the cytoplasm. PRL cells were ovoid or irregular in shape, contained irregular nucleus. The cytoplasm had high electron density. Their diameter was about 7-9μm. TSH cells were ovoid or irregular polygon in shape, contained elliptic nucleus. The diameter was about 8-12μm. A few secretory granules which were occurred in cytoplasmic periphery. ACTH cells had irregular shape and elliptic nucleus. The diameter was reached 8-10μm. Small numbers of secretory granules were observed in the cytoplasm. GTH cells had rounded or elliptic shape, contained round or elliptic nucleus. The diameter was about 12-15μm. They contained two types of secretory granules, the large secretory granules and small ones. In the different seasons, the structural feature and secretory granules of endocrine cell in the turtle adenohypophysis were changed. The organelle of GH cells in the hibernation period was decreased than in the non-hibernation period. TSH cells had the higher numbers in the non-hibernation period. The several secretory granules of GTH cells were fused and became large in the hibernation period, their diameter were larger than in the non-hibernation period, the largest one was reached at 2μm. The ultrastructure of PRL cells and ACTH cells had no obvious differences during the different season.