中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2010年
3期
273-280
,共8页
翁益云%夏君慧%鲍剑虹%张国勇%张旭
翁益雲%夏君慧%鮑劍虹%張國勇%張旭
옹익운%하군혜%포검홍%장국용%장욱
实验性自身免疫性脑脊髓炎%髓鞘少突胶质细胞糖蛋白%水通道蛋白-4%Foxp3%CD4~+CD25~+调节性T细胞
實驗性自身免疫性腦脊髓炎%髓鞘少突膠質細胞糖蛋白%水通道蛋白-4%Foxp3%CD4~+CD25~+調節性T細胞
실험성자신면역성뇌척수염%수초소돌효질세포당단백%수통도단백-4%Foxp3%CD4~+CD25~+조절성T세포
Experimental allergic encephalomyelitis (EAE)%Myelin oligodendroglia glycoprotein (MOG)%Aquaporin-4%Foxp3%CD4~+ CD25~+ regulatory T cell
目的 以髓鞘少突胶质细胞糖蛋白细胞外免疫球蛋白样结构域(MOG~(Igd))为抗原免疫C57BL/6小鼠,建立实验性自身免疫性脑脊髓炎(EAE)模型,并检测CD4~+CD25~+T细胞、Foxp3 mRNA等指标的表达.方法 采用分子克隆技术诱导表达MOG~(Igd)-TrxA融合蛋白,纯化后以此融合蛋白为抗原于侧腹壁皮下多点注射免疫C57BL/6小鼠作为实验组(MOG组);同时以硫氧还蛋白(TrxA)、豚鼠脊髓匀浆(GPSCH)、生理盐水免疫小鼠分别作为阴性、阳性及正常对照组.双盲法连续早晚观察30 d,通过评估动物的临床神经功能、组织病理(HE染色和Kluver-Barrera法髓鞘染色、免疫组化法)情况,评价模型的质量.流式细胞仪(FACS)检测小鼠脾脏中CD4~+CD25~+调节性T细胞;real-time PCR检测Foxp3 mRNA的相对表达水平,并分析两者相关性.结果 MOG组及GPSCH组小鼠均呈慢性非缓解型病程,两组在发病时间、达峰时间、临床神经功能评分等方面差异均无统计学意义(P>0.05).TrxA组及正常对照组无一动物发病.模型组(包括MOG组和GPSCH组)动物HE染色可见血管周围炎性细胞浸润,胶质结节形成,髓鞘染色可见斑片状髓鞘脱失,大脑、小脑、脑干和脊髓均有不同程度受累.免疫组化定性和半定量分析显示MOG组和GPSCH组病灶周围炎症浸润处AQP-4表达与TrxA组相比明显增高(P<0.05);MOG组CD4~+CD25~+T细胞占CD4~+T细胞比例为(4.71±1.61)%,GPSCH组为(1.44±0.65)%,均明显低于TrxA组和正常对照组(P<0.01),且MOG组和GPSCH组之间的差异亦具有统计学意义(P<0.01).MOG组标准化Foxp3 mRNA的表达量为2.26±1.97,GPSCH组为1.44±1.20,均低于TrxA组的8.58±3.34(P<0.01);但MOG组和GPSCH组间的差异无统计学意义(P>0.05).相关性分析显示,模型组小鼠CD4~+ CD25~+调节性T细胞与Foxp3 mRNA表达水平间存在相关性(r=0.849,P<0.05).结论 用MOG~(Igd)免疫C57BL/6小鼠诱导EAE模型稳定,发病率高,为今后进一步研究多发性硬化的免疫发病机制和采取有效治疗措施打下基础.
目的 以髓鞘少突膠質細胞糖蛋白細胞外免疫毬蛋白樣結構域(MOG~(Igd))為抗原免疫C57BL/6小鼠,建立實驗性自身免疫性腦脊髓炎(EAE)模型,併檢測CD4~+CD25~+T細胞、Foxp3 mRNA等指標的錶達.方法 採用分子剋隆技術誘導錶達MOG~(Igd)-TrxA融閤蛋白,純化後以此融閤蛋白為抗原于側腹壁皮下多點註射免疫C57BL/6小鼠作為實驗組(MOG組);同時以硫氧還蛋白(TrxA)、豚鼠脊髓勻漿(GPSCH)、生理鹽水免疫小鼠分彆作為陰性、暘性及正常對照組.雙盲法連續早晚觀察30 d,通過評估動物的臨床神經功能、組織病理(HE染色和Kluver-Barrera法髓鞘染色、免疫組化法)情況,評價模型的質量.流式細胞儀(FACS)檢測小鼠脾髒中CD4~+CD25~+調節性T細胞;real-time PCR檢測Foxp3 mRNA的相對錶達水平,併分析兩者相關性.結果 MOG組及GPSCH組小鼠均呈慢性非緩解型病程,兩組在髮病時間、達峰時間、臨床神經功能評分等方麵差異均無統計學意義(P>0.05).TrxA組及正常對照組無一動物髮病.模型組(包括MOG組和GPSCH組)動物HE染色可見血管週圍炎性細胞浸潤,膠質結節形成,髓鞘染色可見斑片狀髓鞘脫失,大腦、小腦、腦榦和脊髓均有不同程度受纍.免疫組化定性和半定量分析顯示MOG組和GPSCH組病竈週圍炎癥浸潤處AQP-4錶達與TrxA組相比明顯增高(P<0.05);MOG組CD4~+CD25~+T細胞佔CD4~+T細胞比例為(4.71±1.61)%,GPSCH組為(1.44±0.65)%,均明顯低于TrxA組和正常對照組(P<0.01),且MOG組和GPSCH組之間的差異亦具有統計學意義(P<0.01).MOG組標準化Foxp3 mRNA的錶達量為2.26±1.97,GPSCH組為1.44±1.20,均低于TrxA組的8.58±3.34(P<0.01);但MOG組和GPSCH組間的差異無統計學意義(P>0.05).相關性分析顯示,模型組小鼠CD4~+ CD25~+調節性T細胞與Foxp3 mRNA錶達水平間存在相關性(r=0.849,P<0.05).結論 用MOG~(Igd)免疫C57BL/6小鼠誘導EAE模型穩定,髮病率高,為今後進一步研究多髮性硬化的免疫髮病機製和採取有效治療措施打下基礎.
목적 이수초소돌효질세포당단백세포외면역구단백양결구역(MOG~(Igd))위항원면역C57BL/6소서,건립실험성자신면역성뇌척수염(EAE)모형,병검측CD4~+CD25~+T세포、Foxp3 mRNA등지표적표체.방법 채용분자극륭기술유도표체MOG~(Igd)-TrxA융합단백,순화후이차융합단백위항원우측복벽피하다점주사면역C57BL/6소서작위실험조(MOG조);동시이류양환단백(TrxA)、돈서척수균장(GPSCH)、생리염수면역소서분별작위음성、양성급정상대조조.쌍맹법련속조만관찰30 d,통과평고동물적림상신경공능、조직병리(HE염색화Kluver-Barrera법수초염색、면역조화법)정황,평개모형적질량.류식세포의(FACS)검측소서비장중CD4~+CD25~+조절성T세포;real-time PCR검측Foxp3 mRNA적상대표체수평,병분석량자상관성.결과 MOG조급GPSCH조소서균정만성비완해형병정,량조재발병시간、체봉시간、림상신경공능평분등방면차이균무통계학의의(P>0.05).TrxA조급정상대조조무일동물발병.모형조(포괄MOG조화GPSCH조)동물HE염색가견혈관주위염성세포침윤,효질결절형성,수초염색가견반편상수초탈실,대뇌、소뇌、뇌간화척수균유불동정도수루.면역조화정성화반정량분석현시MOG조화GPSCH조병조주위염증침윤처AQP-4표체여TrxA조상비명현증고(P<0.05);MOG조CD4~+CD25~+T세포점CD4~+T세포비례위(4.71±1.61)%,GPSCH조위(1.44±0.65)%,균명현저우TrxA조화정상대조조(P<0.01),차MOG조화GPSCH조지간적차이역구유통계학의의(P<0.01).MOG조표준화Foxp3 mRNA적표체량위2.26±1.97,GPSCH조위1.44±1.20,균저우TrxA조적8.58±3.34(P<0.01);단MOG조화GPSCH조간적차이무통계학의의(P>0.05).상관성분석현시,모형조소서CD4~+ CD25~+조절성T세포여Foxp3 mRNA표체수평간존재상관성(r=0.849,P<0.05).결론 용MOG~(Igd)면역C57BL/6소서유도EAE모형은정,발병솔고,위금후진일보연구다발성경화적면역발병궤제화채취유효치료조시타하기출.
Objective To induce experimental allergic encephalomyelitis (EAE) in female C57BL/6 mice with the extracellular domain of myelin oligedendroglia glycoprotein(MOG~(Igd)). Percentages of CD4~+ CD25~+ T cell (Tr) were tested , and also normalized expressions of Foxp3. Methods Molecular cloning technology was used to produce MOG~(Igd) fusion protein. The MOG~(Igd)-TrxA fusion protein and TrxA protein were purified by metal chelate affinity chromatography (MCAC). Mice were injected s. c. in the flank with 300 μg MOG~(Igd) in complete Frcund's adjuvant (CFA) supplemented with 4 μg/μl Mycobacterium tuberculosis. H37Rv. Mice received 0.4 ml emulsion of spinal cord homogenate of guinea pigs (GPSCH) in positive control group, and the same volume emulsiom of TrxA in negative control group, while mice served as normal control received only saline/adjuvant. Mice were monitored two times a day for continuously 30 days by double bind. Clinical scores and histopathology were evaluated. Then, mice were sacrificed. The spinal cord and brain were removed and fixed in buffered formalin. Horizontal sections taken from the central nervous system(CNS) were stained with haematoxylin and eosin (HE), and Kluver-Barrera staining. Also, immunohistochemistry was performed. Percentages of CD4~+ CD25~+ T cells were tested through flow cytometric analysis, and real-time PCR was performed to test normalized expressions of Foxp3 mRNA. Then, correlations between the two were performanced. Results Mice in both MOG group and GPSCH group shew chronic non-remitting course. The onset of disease, time when the most severe clinical symptoms happened and the clinical score between the two groups shew no significant differnces (P>0.05). However, neither in TrxA treated group nor in normal control group did animals exhibit clinical signs of EAE. Histologic sections of the brain and spinal cord taken from affected animals shew perivascular infiltration of mononuclear cells, gliosis, and multifocal demyelination. Lesions scattered throughout the CNS including brainstem, spinal cord, cerebellum, and penventricular white matter. There were significant differences between MOG group and TrxA group in the level of lesion-ceutric AQP-4 expression showing up by immunohistochemistry (P<0.05). Percentages of CD4~+ CD25~+ T cells in MOG group and GPSCH group were (4.71±1.61) % and (1.44±0.65) %, respectively, both of which were significantly lower than those in the normal control group or TrxA treated group (P<0.01). And the difference between MOG group and GPSCH group also reached statistics meaning (P<0.01). Normalized expression of Foxp3 mRNA in MOG group was 2.26± 1.97, and was not significantly higher than the 1.44±1.20 level in GPSCH group (P>0.05). However, they beth were statistically lower than that in the negative control group, namely 8.58±3.34 (P<0.01). Percentages of CD4~+ CD25~+ T cells was statistically correlated with expressions of Foxp3 mRNA (P< 0.05). Conclusion EAE induced in C57BL/6 mice with MOG~(Igd) is reproduceable. It shares the similar clinial signs and pathologic features with human multiple sclerosis(MS). Thus, we find a good way to further study the immune mechanisms of MS and also to search for the effective treatments.
