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Study of Underling Mechanisms about Experimental Autoimmune Encephalomyelitis Intervened by MSCs

Author: ChenLianFeng
Tutor: LuoXiangYang
School: Sun Yat-sen University
Course: Pediatrics
Keywords: Multiple Sclerosis Experimental Autoimmune Encephalomyelitis StromalInteraction Molecule1 Mesenchymal Stem Cell Interleukin-17
CLC: R744.3
Type: Master's thesis
Year: 2012
Downloads: 44
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Abstract


BACKGROUNDWith improvement in medical science, nowadays pediatric multiple sclerosis (MS)gets more attention than before.There are some clinical manifestations in pediatric MSsuch as: optic neuritis, movement disorder, abnormal gait, paraesthesia, and so on, thesame as adult MS. While a higher rate of disability and relapse happens to pediatricMS.What’s worse, there is unsatisfied therapy of MS, and long-term prognosis is poor.These badly influence physical and psychological health of children, and bring heavyburden to families and society. There is still something about pathogenesis of MS toexplore, among which whiter matter demyelinating caused by autoimmune responsesvia CD4+helper T cell is accepted. Ideal animal model of MS is experimentalautoimmune encephalomyelitis (EAE).Recently some research reported that stromalinteraction molecule1(STIM-1) may take a role in activation of T cell andimmunologic function. Mice which were deleted gene of STIM-1have a resistance toEAE.STIM-1presumably has a relation to EAE. Meanwhile, nowadays biologicaltreatment via stem cells is MS research topic. Bone marrow mesenchymal stem cells(MSCs) are multi-potent stem cells. MSCs have some potentialities such asimmunomodulation, differentiation into neurons-glial cells.Studies reported MSCshave protective treatment to EAE.However,mechanisms have not yet fully understood, so further study is necessary.OBJECTIVE1.To observe clinical manifestations and pathological changes in central nervoussystem (CNS) of EAE intervened by MSCs, and to compare expression of STIM-1inspleen and central nervous system, as well as to detect interleukin-17and anti-bodiesto myelin basic protein(anti-MBP)of peripheral blood.2.To discuss whether treatment of MSCs has a positive influences on EAE andexplore possible mechanisms, in order to offer experimental theories about lookingfor a new treatment to MS.RESEARCH DESIGN AND METHODS1.Bone marrow cells of young female Wistar rats’ femurs were cultured to get someprimary MSCs, and proliferation of MSCs were done. The phenotype of MSCs wasverified by detecting expressions of CD29, CD44, CD34and CD45with flowcytometry.2.The animal model of EAE was established in female Wistar rats by injecting ratswith guinea pig spinal cord and brain homogenate, complete freund’s adjuvant andpertussis toxin, followed by intervening of bone MSCs at two different times.3.Sixty-six healthy female Wistar rats were randomly divided into four groups:groupA(group of EAE model:GPSCH+CFA+PTX),which was divided into subgroup A1,A2and A3according to seventh day, sixteenth day and twenty-fifth day whenexperiments terminate;group B(group intervened by MSCs:GPSCH+CFA+PTX+MSCs transfused on third day), which was divided into subgroupB1,B2and B3asthe same with group A divided into subgroup; group C(group treated by MSCs:GPSCH+CFA+PTX+MSCs transfused on eleventh day), which was divided intosubgroup C2and C3according to sixteenth day and twenty-fifth day whenexperiments terminate; group D(experimental control group,NS+CFA+PTX), whichwas divided into subgroup D1,D2and D3as the same with group A divided intosubgroup. There were eleven subgroups in all, and six rats in each subgroup. 4.1.0×106MSCs were transfused into each rat in group B on the third day afterimmunization, the same as rats in group C on the eleventh day. Rats’ weight, clinicalmanifestations were observed everyday from immunization to sacrifice, while theseverity of EAE was scored according to nervous signs and symptoms. Rats in eachsubgroup were sacrificed at respective times. Peripheral blood was obtained to detectinterleukin-17and anti-MBP by ELISA. Spinal cord, brain and spleen were got toembed and make paraffin sections. Hematoxylin-eosin (HE) staining was performedon some paraffin sections of rats’ spinal cord and brain. And the number ofinflammation focus in central nervous system was counted under the opticalmicroscope. Expression of STIM-1in spleen, spinal cord and brain tissue wasdetected by immunohistochemistry technique. Experimental results were analyzedwith SPSS16.0.RESULTS1.Clinical manifestations of rats in each group: No disease phenotype was observed ingroup D. Each rat in group A caught morbidity. After incubation period, it happenedto rats that was poor appetite, loss of weight, reduction of activity, irritation, tailparalysis, limb paralysis, gatism, and so on. Two rats died at peak time of disease andone at the period of remission. Comparing to group A, incubation period and peaktime of disease in group B delayed, the same as group C; rats in both groups had lessclinical manifestations and lower average scores of nervous signs and symptoms(P<0.05).2. Pathological changes: There were lots of inflammatory cells in the vicinity of somevessels which look like vascular sleeves in central nervous system of rats in subgroupA2, especially at regions of grey matter and junctions between grey matter and whitematter in intumescentia lumbalis, as well as at the vicinity of lateral ventricle;infiltration of inflammatory cells alleviated at the period of remission, less vascularsleeves in central nervous system in subgroup A3(P<0.05). Comparing to group A,there were less inflammatory infiltration into central nervous system in both Groupsof A and B (P<0.05). 3. Expression of interleukin-17in peripheral blood: Expression of interleukin-17wasextremely low in group D. Comparing to group D, there was much more expression ofinterleukin-17in group A (P<0.05). With most serious clinical manifestation insubgroup A2, expression of interleukin-17was the most among group A, whichweakened less along with remission in subgroup A3(P<0.05). There was lowerexpression of interleukin-17in both subgroups C2and B2than that in subgroup A2(P<0.05), respectively. It was the same as in both subgroups of B3and C3than insubgroup A3(P<0.05).4. Expression of anti-MBP in peripheral blood: Expression of anti-MBP in group Aincreased much more than in group D (P<0.5). Among group A, there was the mostexpression of anti-MBP in subgroup A3. No significant difference happened toexpression of anti-MBP Among group A, B and C (P>0.05).5. Expression of STIM-1in central nervous system: Positive expression of STIM-1incentral nervous system was weak in group D.In group A, there was intensive positiveexpression of STIM-1in central nervous system, which was more intensive insubgroup A2than A1(P<0.05). Positive expression of STIM-1in both subgroups A2and A3was more intensive than in corresponding subgroups among group D (P<0.05).Comparing to subgroup A2, positive expression of STIM-1weakened markedly insubgroup B2and C2, respectively (P<0.05)6. Expression of STIM-1in spleen: Expression of STIM-1in spleen was weak ingroup D. Positive expression of STIM-1in spleen was intensive in both subgroups A1and A2. Positive expression of STIM-1in subgroup A2was more intensive than insubgroup A3(P<0.05). Comparing to group A2, positive expression of STIM-1weakened dramatically in subgroup C2(P<0.05)..CONCLUSIONS1.STIM-1plays a role in the pathogenesis of EAE. Expression of STIM-1in spleenand central nervous system was more intensive in EAE. Positive expression ofSTIM-1was much too intensive in serious inflammatory infiltration in central nervous system.2. There was much intensive expression of STIM-1at peak time of EAE,accompanying with increasing expression of IL-17. Intensive expression of STIM-1was weaker with remission, as well as expression of IL-17.Clinical manifestation ofEAE alleviated markedly when MSCs were transfused into rats, and Intensiveexpression of STIM-1weakened, followed by expression of IL-17reducing. One ofunderlying mechanisms which MSCs have a positive effect on treatment of Wistarrats’ EAE is to weaken expression of interleukin-17and alleviate CNS’sinflammatory response by down-regulating expression of STIM-1.3. Anti-MBP exacerbated EAE. There was a higher expression of Anti-MBP in EAE.

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CLC: > Medicine, health > Neurology and psychiatry > Neurology > Spinal cord disease > Myelitis
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