Effect Of Lentinan On PPL Proliferation And Its Induction Of M Cell Differentiation

ObjectiveBecause of the complex structure and hard to be aborbed factures, Chinese herb polysaccharides face great difficulties of researches for their immune pharmacological mechanism. In this thesis, effects to intestinal mucosal immunity will be observed, represented by Lentinan. And then, the potential mechanisms for these effects will be investigated based on the activation of PPL and their induction for M cell differentiation. And finally, research foundation for Chinese herb polysaccharise will hope to be established.Methods1、Assay for polysaccharide content:Use sulfuric acid-phenol method to measure polysaccharide content.2、Establish the immunosuppressive model:Different dose of cyclophosphamide and dexamethasone will be chosed as the immunosuppressive angents to search approprite method for immunosuppressive model. Lymphocytes ratio and their activation proportion of gut Peyer’s patches and retroauricular lymph nodes of DTH mice will be observed chiefly, and combined with mouse viscera index and their delyed type hypersensitivity to evaluate immunosuppressive effectes for this two angents.3、Determinate immunomodulate activities for Lentinan via its effects to whole immune system and intestinal mucosal immunity:Differente dose of Lentinan will be chosed to observe their effectes to proliferation of mice spleen lymphocytes and delyed type hypersansitivity ability. Chosing optimal dose, Lentinan effectes on the intestinal mucosal immune based on its whole immune system regulation will be detached, using PPs numbers, gut slgA secretion levels, and lymphocytes ratio and their activation proportion of PPs. 4、Using different administration of Lentinan to study its immune effectes to whole immune system and intestinal mucosal immune system, and then reveal the importance of immunoregulation to intestinal mucosal immunity system of polysaccharides by oral administration. To compare effects of Lentinan on whole immune system and intestinal mucosal immune system, oral administration and intraperitoneal injection will be chosed. And the detection index will include lymphocytes proliferation of spleen, mesenteric lymph nodes and peyers patches; delyed type hypersensitivity and gut secrection slgA levels of mice, combinding with lymphocytes ratio and their activation proportion of peyer’s patches and retroauricular lymph nodes.5、To persum possible mechenisim of immunoregulation effects to intestinal mucosal immunity system by observing M cell number interfered with Lentinan. Using tissue section and immunohistochemistry to observe damage repair of peyer’s patches, as well as its M cell number change, induced by cyclophosphamide. Then PPL inducing Caco-2cell to M-like cell in vitro will be established and the cell transpor function of fluorescent microspheres and its surface structure will be detected to evaluate this conversion system. Finally, inducing differentiation ability of PPL from immunosuppressvie mice, administrated with Lentinan, will be observed using this covering system.6、To persum possible mechenism of PPL activation and its inducing differentiation of M cell of Lentinan by observing lymphocyte activity of spleen, mesenteric lymph node and peyer’s patch in votro under Lentinan. Using defferent dose of Lentinan combined with ConA to inducing PPL proliferation. And MTT methods will be used to compare the stimulated index between different doses. Different dose of Lentinan will be co-culture with lymphocyte of spleen, PP and MLN. And then, MTT method will be used to compare viable count of defferent doses.Results1、The polysaccharide cintent is62～63%measured by sulfuric acid-phenol method.2、Every dose of cyclophosphamide and dexamethasone are all show different level of immunosuppress effects.100mg/kg and120mg/kg dose of cyclophosphamide show significant immunosuppress effects on viscera index, PPs number and delyed type hypersensitivity. As well as lymphocytes ratio and their activation proportion of gut Peyer’s patches and retroauricular lymph nodes of DTH mice are all statistically significant compared with control group. In contrast, dexamethasone shows obvious effects on viscera index and PPs number, but not the delyed type hypersensitivit and activation proportion of lymphocytes under DTH.3、Decreasing of viscera index, dropping ability of lymphocyte proliferation of spleen lymphocytes and delayed type hypersensitivity of mice were up regulated after oral administration of lentinan.600mg/kg and800mg/kg dose show more obvious effects and dose-related trend. The decreasing proportion of PPs number, lymphocyte proliferation of mesenteric lymph node and PPs, gut sIgA secretion level, as well as lymphocyte activation are all modified after oral administration of800mg/kg dose of lentinan. And the differences are statistically significant compared with immunosuppressive model group.4、After oral or intraperitoneal injection of Lentinan, immune suppressed mice show varing degrees of recovery trend. In oral administration group, lymphocytes proliferation of spleen, mesenteric lymph nodes and peyer’s patches, gut secrection sIgA levels of miceand mice delyed type hypersensitivity are all significantly improved. And the differences are statistically significant compared with immunosuppressive model group. In intraperitoneal injection group, only PPL proliferation is significantly improved. The differences are statistically significant compared with immunosuppressive model group. Moreover, lymphocytes ratio and their activation proportion of peyer’s patches and retroauricular lymph nodes are modulated after oral administration of Lentinan, especially lymphocytes ratio and T cell activation of peyer’s patches. The differences are statistically significant compared with immunosuppressive model group. However, this modulation effect is not show in intraperitoneal injection group. Only CD69expression of T cells of retroauricular lymph nodes increase obviously. The differences are statistically significant compared with immunosuppressive model group.5、After immunosuppression by cyclophosphamide, lymphocytes in PPs are decrease and show a loose appearance. And the number of M cell in follicular epithlum is significantlly decreased. The differences are statistically significant compared with control group. After oral administration of Lentinan, number of PPL and M cell are all increased. The differences are statistically significant compared with immunosuppressive model group. In M cell differentiation induction system, microvilli on Caco-2cell surface are reduced or even disappear after inducing by PPL. And the new cells acquire the zbility to transcellular transport of fluorescence microspheres. After oral administration of Lentinan, mice PPL associated with ConA can significantly enhance the efficiency of transcellular transport of fluorescence microspheres of Caco-2cells. The differences are statistically significant compared with immunosuppressive model group. And moreover, the reduction of microvilli on cell surface is more significant compared with immunosuppressive model group.6、Combing with ConA, PPL proliferation in vitro can be significanty promoted by Lentinan. The stimulation index of50、100、200、400μg/ml of Lentinan show a significant defference compared with ConA control group. And this effect shows a positive correlation between50-～00μg/ml. Without ConA, Lentinan can mentain the cell viability of spleen, mesenteric lymph node and PPs. And especially to PPL, Lentinan even shows proliferation effects. Bwteen all different doses, optically density of6.25,12.5,25,200,400μg/ml show significantly differences caompared with only cell control group.Conclusion1、 The content of crude polysaccharide is suitable for this research basically.2、The100mg/ml dose of cyclophosphamide is more suitable for making immunosuppression animal models for this research.3、Lentinan has immune effects of on immunosuppressive mice for their whole immune system at the same time, also show immunoregulation activity for the intextinal mucaosal immunity, which, combinding with hard to be absorbed features, suggest intestinal mucosal immunity system plays an important role in immunoregulation activity of Chinese herb polysaccharides.4、Lentinan can regulate intestinal mucosal immunity and system immunity more obviously after oral administration compared to intraperitoneal injection, which suggest intestinal mucosal may be the pathway of immunoregulation effects of Chinese herb polysaccharides under oral administration.5、The effects of Lentinan on improve morphology of PPs and increase M cell in follicle associated epithelium suggest close relationship between immunoregulation of intestinal mucosal immunity system and maintenance of lymphocyte number for modifying shape of PPs,as well as keeping proportion of lymphocyte activation level. And M cell is likely to be the key cells among these co-effects.6、The effects of maintain lymphocyte activity or promote its proliferation suggest Lentinan has direct immunological activity to PPL. Lentinan hard to be absorbed may bring its immunoregulation effects via inducing intestinal local immune response.