Dissertation
Dissertation > Medicine, health > Pediatrics > Newborns, premature children disease > Neonatal disease

Experiment Study about the Commensal Enteric Bacteria Effection on Monocytes and Dendritic Cells in Neonatal Cord Blood

Author ZhangYing
Tutor ZhangLin
School Hebei Medical University
Course Pediatrics
Keywords newborn cord blood bifidobacterium E.coli monocyte DC infectious disease allergic disease
CLC R722.1
Type Master's thesis
Year 2007
Downloads 69
Quotes 0
Download Dissertation

Objective:Innate immunity exists in all multicellular organism, and it is the first defense line to protect body from the pathogen infection. As antigen present cells(APC), monocytes and dendritic cells(DC) are important effector cells in our body. They have the ability to probe microbiology, produce cytokine and regulate the body’s immune response. The newborns have immature immune system. Especially, their innate immune system is not healthy, the function of their regulatory T cells is not perfect, the propotion of Th1/Th2 cells is not in balance and the immune response is skewed to Th2. They are highly susceptible to severe bacterial infections and allergic diseases. There is accumulating evidence that commensal bacteria has the ability to drive the maturation of the immune system, to activate the immune cell and to induce the cytokine production. So, it can keep the Th1/Th2 cell response in balance and induce immunologic response or tolerance. Adopting cell culture technique, we used commensal enteric bacteria ( bifidobacteria and E.coli ) as stimulants to observe the morphology and immune change of monocytes and monocytes-derived DC in neonate cord blood. 1 To observe the morphology change and the production of cytokines IL-10 and IL-12 after bifidobacteria and E.coli stimulating monocytes and to discuss the systemic immune effect of commensal enteric bacteria on the newborn.2 To observe the morphology and function change and the production of cytokines IL-10 and IL-12 after bifidobacteria and E.coli stimulating DC and to discuss the local immune effect of commensal enteric bacteria on the newborn. This study could provide the theory basis for reasonable application probiotics to prevent and treat the infectious diseases and allergic diseases on clinical.Method: Cord blood was gathered from 20 normal term newborns including 10 delivered by vagina and 10 by uterus. The first step was to sediment the RBC of cord blood by Methylcellulose. Then we used Ficoll to seperate cord blood mononuclear cells from the supernatants. Monocytes could be obtained after plastic-adherent culture. The experiment was divided into two parts:1 A part of monocytes was divided into 4 groups and cocultured with heat-killed bifidobacterrium, heat-killed E.coli, heat-killed bifidobacterrium and E.coli and PBS(control group) respectively for 24h. we observed the morphology changes of the monocytes by phase-contrast microscope and analyzed the level of IL-10 and IL-12p70 in the supernatants by ELISA.2 GM-CSF and IL-4 were put into a part of monocytes to induce it to differeniate into immature DC (imDC) for 7 day. Then the imDC was divided into 4 groups and cocultered with the heat-killed bifidobacterrium, heat-killed E.coli, heat-killed bifidobacterrium and E.coli and PBS(control group) respectively for 24h. we observed the morphology changes of DC by phase-contrast microscope every day and analyzed the level of IL-10 and IL-12p70 in the supernatants by ELISA.Groups were compared by two-sample t test and paired t test. Statistical analysis was performed using the SAS6.12 statistic software. Data are presented as mean±SD. A probability of p< 0.05 was considered statistically significant.Result1 The morphology of monocytes stimulated by probiotics has not changed.2 There was no statistical difference in the survival percentages of monocytes between stimulus groups and control groups. The survival percentage of each group > 90%.3 Different reaction of monocytes was appeared after stimulated by probiotics. Lower level of IL-10 and IL-12p70 was secreted in control group. After the stimulation of the heat-killed bifidobacterium, the level of IL-10 and IL-12p70 was remarkably increased, especially the level of IL-12p70. There was statistical difference in comparision with control group(p<0.05). After the stimulation of E.coli , the level of IL-10 was considerably increased compared with control group and bifi. group(p<0.05). But the level of IL-12p70 was lower than that in bifi. group(p<0.05).Stimulated by the combination of the two kinds of bacterium, monocytes secreted the level of IL-12p70 similar to bifi. group(p>0.05), but higher than E.coli group. Comparisions with the two independent stimulus groups showed statistical difference(p<0.05).So, combination group could secrete IL-10and IL-12 in relatively high level.4 Immature DC could be gathered on 7th day in vitro culture. Cells became suspended, enlarged and polymorphic. The edge of the cell appeared piliform. Small convexs could be seen. After stimulation of bifidobacterium and E.coli , the amounts of the convexs of the cells became increased and spine-like convexs could be seen. we could get typical mature cells that like the rays of sunlight and the roots of the trees.5 There was no statistical difference in the survival percentages of DC between stimulus groups and control groups. The survival percentage of each group > 90%.6 Lower level of IL-10 and IL-12p70 was secreted in control group of immature DC. After the stimulation of the heat-killed bifidobacterium, the level of IL-10 and IL-12p70 was increased, especially the level of IL-10. There was statistical difference in comparision with control group(p<0.05). After the stimulation of E.coli , the level of IL-10 was considerably increased, higher than that in control group and bifi. group(p<0.05). The level of IL-12p70 was also higher than that in control group(p<0.05), but similar to that in bifi. group(p>0.05). Stimulated by the combination of the two kinds of bacterium, DC secreted the level of IL-12p70 similar to bifi. group(p>0.05), but lower than control group(p<0.05). The level of IL-10 was remarkably increased, higher than that in the two independent stimulus groups(p<0.05).Conclusion1 We demonstrated that the technology of seperating and culturing the monocytes and DCs of cord blood in vitro is mature .2 Making the monocytes of newborns secrete IL-12, bifido- bacterium has the ability to induce immune response to Th1, regulate the balance of Th1/Th2 reponse, activate the immune system of newborns to strengthen the anti-infectious ability and decrease allergic disease.3 Making the DCs of newborns secrete IL-10, bifidobacterium and E.coli has the ability to induce oral tolance, maintain the stable state of the intestine mucosa, produce the specific antibodies such as sIgA, IgM, IgG and so on to decrease allergic disease.4 As the commensal bacteria, bifidobacterium and E.coli do not induce the rejection response of the body, and not affect the survival percentages of monocytes and DCs. They could induce oral tolance as probiotics.5 Commensal bacteria has the ability to regulate the innate and acquired immunity of newborns, regulate the balance of the immune response, enhancing the function of systemic and local immunity. So, they can prevent and intervene the infectious and the allergic diseases of newborns. This study provides the theory basis for using probitics clinically to prevent and intervene the infectious and the allergic diseases of newborns.

Related Dissertations
More Dissertations