Development Changes Of Gut Peptide In Macaque Small Intestine Mucosa And Gut Associated Lymphocytic Tissue

Background and aimGut mucosal immune play important role in the mucosal immune system. Knowledge of physiology and pathophysiology increased greatly in recent 10 yesars.But study focused on adult animals. The rules of structure and function in gut associated lymphocytic tissue were not reported.in physiological conditions.during development. Intestine which produce gut peptide more than 50, was regarded as the largest endocrine organ. More was emphasized on structure, function and the role in many diseases of gut peptide by resercheres, however less was noticed on the changes in the development, Little konweledge of basic rule of gut immune make it diffcult in learn the course of development.Life science reveal that the system of immune ,nerve and endocrine can not work independently, but exchange information and interetation through the common subtance in the methord of " immune-nereve- endocrine" net. while intestine is the crucial place for this. The question about whether " immune-nereve- endocrine" net can interetation was not seen. It which konweledge from rodent is used to deduct changes of physiology and pathophysiology about human gut mucosa immunity could result in prejudice and misunderstanding.From embryo, juvenile to adult, the morphay and functions of immuneorgans have a great variety. But whats the rule? Little about the basic musocal immune rule make it diffucult to recognize the course of pathophysiology. so we should explore the rule on production, distribution, corrpending receptors expression and binding ability, and physiology changes about gut musocal immunine in the development, gut peptide which comprose of microenviromenal, and whether the intestine immune cells in development were interfered by gut peptide .the results help to understand the development, recession and tumor formation of digestion system.Methods1. The experiment animals-macaque are from wild-life animals world of Chengdu. Four helthy macaque age are 6-month fetus and 2-day neonate. 45-day neonate, adult..All animals are qualification in quarantine.2. The tissue samples of small intestine and liver or blood samples from peripheral and portal vein of 4 macaques in 6-month fetus, 2-day neonate, 45-day neonate and adult were obtained after anesthetizing.3. Lymphocytes from intestine were digested in collagenase and Percoll. incontinuity density gradient centrifugation4. Purification of lymphocytes were cultured with SST or VIP in vitro5. The morphological characteristic of the development stages of gut associated lymphocytic tissue were visualized by HE; The lymphocyte ultrastructure of the development stages were observed through electron microscope.6. The concentrations of SST and VIP in blood or tissues from macaques were measured by radioimmunoassay.7. The distribution of SST, VIP, CD4+ T lymphocytes, CD8+ T lymphocytes, B lymphocytes in small intestinal ,NK cell, oc4.p7and MadCAM-1 were visualized by immunohistochemical staining.8. The expression of a4p7 in lymphocyte was detected by FCM.9. The expression of SSTR2 and VIPR1 were detected by situ hybridization.10. The area and cytoskeleton of lymphocyte from intestine tissue and peripheral blood were measured by LCAM.Results1. SST and VIP concentration of intestinal tissue in 6-month fetus of macaque were27.3±16.6 ng/mg protein and 514.8 + 492.4 ng/mg protein, respectively in adult. It is significantly higher than that in fetus, p<0.01.2. SST and VIP levels of portal vein in the developing stages kept significantly higher than those of peripheral blood, p<0.013. Strong positive staining of SST could be found in both mucosal crypts and myenteric nerve plexus of adult animal. SSTR-2 was strongly visualized in intestinal epithelium of fetus but became greatly down expressed in epithelium and was shifted to mucosal crypts when entering into adult. Negative staining of SSTR2 in muscle layer of fetal or neonatal macaque turned to be positive in myenteric nerve plexus of adult.4. Expression of VIPR-1 in intestinal epithelium was gradually up-regulated from embryo to adult. Expression of VIP or VIPR-1 in intestinal muscle layer was strongly visualized between annular muscle and indulge muscle.5. The levels of SST^ SSTR-2 or VH\ VIPR-1 in liver were decreased gradually during development. Positive-staining of VIPR-1 in liver vessel was higher, After birth, positive-staining of VIPR-1 was gradually decreased.6. Matural PP located at subepithelium of ileum is not be seen until inadult which contain germinal center.7. The size of intestinal villus get larger and the finger-like villus became branch-like during the development. Also intestinal gland became more developed.8. Ultrastructure of the lymphocyte which entered to intestine from bone marrow not been changed during the development.9. In the development stages, CD4+ T lymphocytes exist in intraepithelium and lamina propria. The former are more than that of the latter. CD4+ T lymphocytes are absent in immatural PP during 6-month fetus and 2-day neonate, in 45-day neonate, CD4+ T lymphocytes exist diffusively in immatural PP.In adult, CD4+ T lymphocytes aggregate at marginal area of PP base with the present of germinal center.10. CD8+T lymphocytes are absent in lamina propria and immatural PP in embryo and new born which are low positive-staining in intraepithelium.in adult. CD8+ T lymphocytes scatterly and lessly distribute in intraepithelium and lamina propria .11. NK cells(CD57+) always exist in lamina propria and submucosa that do not change in the development period, and which do not exist in intraepithelium and PP.12. B lymphocytes (CD20+) do not exist in gut associated lymphocytic tissue in 6-month fetus and 2-day neonate. 45-day neonate, a large of CD20+ locate at PP. In adult, CD20+ lymphocytes aggregate at marginal area of PP lamina aside.13. In 6-month fetus and 2-day neonate, IEL was positive-staining of a4p7. In 45-day neonate, expression of IEL was decreased gradually, expression of LPL is gradually increase. In adult, expression of LPL is stronger than that of IEL. Positive-staining of a4p7 in lamina proprialocate at basal area of intraepithelium.14. Strong positive-staining of MAdCAM-1 is detected in intraepithelium and lamina propria from embryo to adult. MAdCAM-1 could be detected in blood capillary of intraepithelium and lamina propria but not be detected in PP.15. Compared to control group, the volum of lymphocytes from intestine tissue was larger in SST or VIP group. Also, increased mitochondria, membrane system and ribosome were visualized within cytoplasm. However expression of a4(37 in SST group (6.0%±0.82%) and in VIP ( 8.0 %±0.74%) was no significantly difference than that in control group (3.5%±0.46%) ,/X0.01.Conclusions1. Most of SST or VIP endocrined by intestine have been degraded in portal vein before entering in liver, not been degraded in liver.2. SST, SSTR2 and VIP positive nerve fiber locate in mucosal crypt, and VIPR-1 scatterly distribute whole intestinal villus.3. The inhibitive effect of SST on the motility of small intestine became possible through else nerve fiber because SST positive nerve fiber locating in myenteric nerve do not directly contral muscle cell.4. From embryo to adult, VIP in intestine was firstly from nerves of submucusa, when VIP nerve fiber became thicker, and it gradually developed to mucosal crypt and the junction of annular muscle and indulge muscle. VIPR-1 distribute mainly intestinal annular muscle.5. In 6-month fetus, Peyer patch having primary lymphoid follicle formed, With food increased, In 45-day neonate, the quantitiesand volum of PP became larger which it did not maturate structure of PP.Matural PP located at subepithelium of ileum is not be seen until in adult which contain germinal center. cc4|37 and MadCAM-1 are not key molecule in the formation and development of PP .These results revealed chemokines are difference greatly among macaque and rodent in the formation and development of PP.6. B lymphocytes (CD20+) do not exist in gut associated lymphocytic tissue in 6-month fetus and 2-day neonate. A large of B lymphocytes and CD4+ T lymphocyte exist in PP of adult macaque intestine, CD20+ lymphocytes aggregate at marginal area of PP lamina aside, CD4+ T lymphocytes aggregate at marginal area of PP base. The view on action site agree with Thl,Th2 and B lymphocytes can influence one another physiological function.7. Ultrastructure of the lymphocyte which enter to intestine from bone marrow were none of changes during the development stages, In adult, intraepithelial lymphocytes are CD4+ T lymphocytes. CD4+T lymphocytes are more than CD8+T lymphocytes . exist most of lamina propria lymphocytes are NK cells(CD57+) and CD4+ T lymphocytes, not B lymphocytes.these results are significant difference from the study about rodent.8. Compared to control group, the volum of lymphocytes from intestine tissue was larger in SST or VIP group., increased mitochondria, membrane system and ribosome were visualized within cytoplasm with the condition of antigen stimulation in vitro, thease results show lymphocytes are in the state of function activity. SST or VIP can promote lymphocytes function activity.