【作者】 许自川；

【导师】 易著文；

【作者基本信息】 中南大学 ， 儿科学， 2010， 博士

【摘要】 目的分离、培养大鼠脾脏T细胞、胚胎后肾间充质细胞,体外诱导培养骨髓源性树突细胞,为后续体外模拟胚胎后肾间充质细胞的免疫调节作用、细胞移植研究提供细胞来源及依据。方法(1)健康SD雌性大鼠,10%水合氯醛麻醉后无菌条件下取出股骨和胫骨,RPM I 1640基本培养基冲洗骨髓腔获骨髓腔冲洗液,密度梯度分离后将单个核细胞用含GM-CSF、TNF-α和IL 4完全营养液混悬后与37℃、体积分数为0.05的CO2孵箱中培养,并进行免疫表型鉴定。(2)取SD大鼠,麻醉后无菌条件下取脾,研磨成脾细胞悬液,密度梯度离心取单核细胞层,以RPMI 1640基本培养基制成2×106／ml淋巴细胞悬液,置37℃、体积分数为0.05的CO2孵箱中培养,并进行流式细胞检测。(3)孕14天大鼠胚胎,分离胚胎肾脏,去除输尿管芽,消化后置37℃、5%CO2、饱和湿度的培养箱中培养。取传至第3代的MMS倒置相差显微镜下观察培养细胞,并行HE染色、电镜观察、生长曲线测定；ABC免疫酶染色法检测波形蛋白、角蛋白、nestin、CD133、CD34。另取传12代细胞及冻存复苏后细胞(传5代)培养,形态观察,并波形蛋白、角蛋白检测。结果(1)骨髓单个核细胞诱导后呈现典型树突细胞形态,表现为毛刺状突起、半贴壁生长,并表达CD11C、CD86、MHC-Ⅱ等树突细胞表面标志。(2)由脾脏分离的T淋巴细胞,经流式细胞仪检测,CD3阳性率为75%,CD4阳性率为48%,CD8阳性率为31%(3)MMS形态为成纤维细胞样,贴壁生长,48-72小时达生长高峰；波形蛋白、nestin、CD133表达阳性；角蛋白、CD34、DB表达阴性。传12代细胞及冻存复苏后细胞形态观察及波形蛋白、角蛋白检测较前无改变。结论体外培养的树突细胞具有典型树突细胞的形态学表现,脾源性T培养细胞纯度达到试验要求,后肾间充质细胞,纯度、稳定性较高并符合干细胞特征。目的观察胚胎后肾间充质细胞和抗IL-10抗体对T细胞、树突细胞增殖以及对IL-4、INF-γ、穿孔素以及颗粒酶B表达的影响,探讨其可能机制方法(1)体外培养大鼠骨髓源性树突细胞、脾源性T细胞和胚胎后肾间充质细胞；(2)建立混合培养体系,观察胚胎后肾间充质细胞和抗IL-10抗体对脾源性T细胞、树突细胞以及树突细胞诱导的T细胞增殖的影响；(3)建立Transwell培养体系,胚胎后肾间充质细胞和抗IL-10抗体对脾源性T细胞、树突细胞以及树突细胞诱导的T细胞增殖的影响；(4)建立混合培养体系,观察胚胎后肾间充质细胞和抗IL-10抗体对树突细胞诱导的T细胞IL-4、INF-γ、穿孔素以及颗粒酶BmRNA和蛋白表达的影响；(5)建立Transwell培养体系,观察胚胎后肾间充质细胞和抗IL-10抗体对树突细胞诱导的T细胞IL-4、INF-γ、穿孔素以及颗粒酶BmRNA和蛋白表达的影响。结果(1)混合培养体系中,与对照组相比,试验组T淋巴细胞增殖活性、树突细胞增殖活性、树突细胞诱导的T细胞增殖活性均降低,在R：S比值1：80范围内成剂量依赖关系,(P值均<0.05)。细胞增殖抑制率在1：80、1：160两组之间没有显著性差异,(P>0.05)。与R：S(1：80)组相比,Anti-IL-10组T细胞增殖率明显增加,(P<0.05),与阳性对照组相比,增殖率明显降低(P<0.05)；(2)Transwell培养体系中,与对照组相比,试验组T淋巴细胞增殖活性、树突细胞增殖活性、树突细胞诱导的T细胞增殖活性均降低,与R：S比值成剂量依赖关系,随R：S的减小,T细胞的增殖率逐渐下降,(P值均<0.05)。细胞增殖抑制率在1：80、1：160两组之间没有显著性差异,(P>0.05)。与R：S(1：80)组相比,Anti-IL-10组T细胞增殖率明显增加,(P<0.05),与阳性对照组相比,增殖率明显降低(P<0.05)；(3)混合培养体系中,与对照组相比,试验组树突细胞诱导的T细胞IL-4、INF-γ、穿孔素以及颗粒酶B mRNA和蛋白表达均明显下降,在R：S比值1：80范围内成剂量依赖关系。细胞增殖抑制率在1：80、1：160两组之间没有显著性差异,(P>0.05)。与R：S(1：80)组相比,Anti-IL-10组T细胞增殖率明显增加,(P<0.05),与阳性对照组相比,增殖率明显降低(P<0.05)；(4) Transwell培养体系中,与对照组相比,试验组树突细胞诱导的T细胞IL-4、INF-γ、穿孔素以及颗粒酶B mRNA和蛋白表达均明显下降,在R：S比值1：20-1：80范围内成剂量依赖关系。细胞增殖抑制率在1：80、1：160两组之间没有显著性差异,(P>0.05)。与R：S(1：80)组相比,Anti-IL-10组T细胞增殖率明显增加,(P<0.05),与阳性对照组相比,增殖率明显降低(P<0.05)。结论胚胎后肾间充质细胞可能通过直接接触和旁分泌作用抑制树突细胞、T细胞以及树突细胞诱导的T细胞的增殖,抑制树突细胞诱导的T细胞IL-4、INF-γ、穿孔素以及颗粒酶B mRNA和蛋白的表达。目的探讨胚胎后肾间充质细胞对慢性血清病肾炎大鼠免疫调节作用以及对肾脏损伤的治疗作用。方法(1)体外原代培养胚胎后肾间充质细胞,GFP标记；(2)建立慢性血清病肾炎模型大鼠；(3)大鼠分为5组：空白对照组、阴性对照组、模型组、MMS预处理组和MMS移植干预组；(4)荧光显微镜定位GFP标记的胚胎后肾间充质细胞在大鼠体内的分布；(5)常规病理染色观察五组大鼠肾脏病理变化;(6)流式细胞术检测五组大鼠外周血T细胞亚群；(7)ELISA法检测五组大鼠血清IL-4、INF-γ水平；(8)RT-PCR法检测五组大鼠外周血单个核细胞IL-4、INF-γ、穿孔素和颗粒酶B mRNA的表达。结果(1)模型复制结束时,与空白对照组和阴性对照组相比,模型组、MMS预处理组大鼠尿蛋白明显升高,总蛋白水平、白蛋白水平明显降低,总胆固醇水平明显增加(P＜0.05); MMS预处理组尿蛋白水平较模型组明显降低(P<0.05)。(2)实验结束时,与模型组相比,MMS移植组尿蛋白水平均较模型组明显下降(P<0.05),预处理组下降更明显(P<0.01)。与移植组相比,预处理组尿蛋白水平明显降低(P<0.05)。(3)GFP标记的MMS向肝脏、脾脏和肾脏归巢,但不能向脑组织以及心脏归巢。(4)与模型组比较,MMS移植组肾脏病理损伤减轻；与MMS移植组相比,MMS预处理组肾脏病理损伤减轻。(5)与空白对照组和阴性对照组相比,模型组、MMS预处理组和MMS移植组大鼠外周血Th细胞比例下降、Ts细胞比例升高,Th／Ts比例下降,差别具有显著性意义(P<0.05)。与模型组比较,MMS预处理组和MMS移植组大鼠外周血Th细胞比例下降、Ts细胞比例升高,差别无统计学意义(P>0.05),Th／Ts比例下降,差别具有显著性意义(P<0.05)。与MMS预处理相比,MMS移植组大鼠Th／Ts比例下降,差别具有显著性意义(P<0.05)。(6)与空白对照组和阴性对照组相比,模型组大鼠外周血IL-4水平明显增高,INF-γ水平明显降低,差别具有显著性意义(P＜0.01), MMS预处理组和MMS移植组大鼠外周血IL-4水平明显增高,INF-γ水平明显降低,差别具有显著性意义(P<0.05)。与模型组比较,MMS预处理组和MMS移植组大鼠外周血IL-4水平降低,INF-γ水平明显升高,差别有统计学意义(P<0.05)。与MMS预处理相比,MMS移植组大鼠大鼠外周血IL-4水平增高,INF-γ水平明显降低,差别具有显著性意义(P<0.05)。(7)与空白对照组和阴性对照组相比,模型组大鼠外周血单个核细胞IL-4、穿孔素、颗粒酶BmRNA表达增高,INF-γmRNA表达明显减低,差别具有非常显著性意义(P<0.01), MMS预处理组和MMS移植组大鼠外周血IL-4、穿孔素、颗粒酶BmRNA表达增高,INF-γmRNA表达明显减低,差别具有显著性意义(P<0.05)。与模型组比较,MMS预处理组和MMS移植组大鼠外周血IL-4、穿孔素、颗粒酶B mRNA表达表达水平降低,INF-γmRNA表达明显增高,差别有统计学意义(P<0.05)。与MMS预处理相比,MMS移植组大鼠大鼠外周血IL-4、穿孔素、颗粒酶B mRNA表达水平增高,INF-γmRNA表达水平降低,差别具有显著性意义(P<0.05)。结论胚胎后肾间充质细胞可以调节慢性血清病肾炎大鼠免疫状态,减轻肾脏损伤。结论胚胎后肾间充质细胞可以一定程度上纠正慢性血清病肾炎大鼠免疫紊乱,减轻肾脏损伤。更多还原

【Abstract】 Part 1 Isolation, cultivation and identification of bone marrowme derived dentritic cells, spleen derived T cells and metanephric mesenchymal cellsAbstract Objective To isolate and culture rat spleen T cells, embryonic metanep-hric mesenchymal cells, and bone marrow-derived dendritic cells in vitro as the cell source for the following research including the immune regulation and transplantation.Methods (1)The mononuclear cells obtained from the bone marrow of SD rats after anesthetized by 10% chloral hydrate, were cultured in with complete RPMI1640 medium containing GM-CSF, IL-4 and TNF-a in 37℃and 5% CO2 incubator and cellular immunochemistry was performed to test the special antigens. (2) The spleen was obtained from SD rats under sterile conditions after anesthesia and was grinded into the suspension followed by density gradient centrifugation to get the T cells which were cultured at 37℃,0.05% CO2 incubator. The flow cytometry was performed to determine the cells antigen expression. (3) Rats were anaesthetized on gestation day 14 and metanephric balstemata were separated from ureteric buds by microdissection. Cells were grown in DMEM containing 10% fetal calf serum at 37℃in a 5% CO2 atmosphere. The morphology of MMS were observed by inverted phase contrast microscope, HE staining, and electronic microscope. The surface antigen phenotypes were identified by immunocytochemistry, and the purification was identified by flow cytometry. A growth curve to be used for studying the characteristics of growing. The morphology and the surface antigen phenotypes (vimentin, cytometry) of MMS at passage 12 and resused MMS at passage 5 were also observed.Results (1) the bone marrow mononuclear cells showed a typical dendritic cell morphology, such as burr-like protrusions, semi-adherent growth, and expressed CD11C, CD86, MHC-II after the induction (2) T-lymphocytes isolated from the spleen showed that CD3-positive rate was 75%, CD4-positive rate was 48%, CD8-positive rate was 31% by flow cytometry,(3) MMS grew adherencely as fibroblast-like, positive for vimentin, nestin, and CD 133. Instead, they were negative for keratin, CD34 and Dolichos Biflorus (DB). The flow cytometry showed a single peak of MMS. The morphology and the surface antigen phenotypes (vimentin, cytometry) of MMS at passage 12 and resused MMS at passage 5 had no changes.Conclusion dendritic cells cultured showed a typical morphology of dendritic cells in vitro, spleen-derived T cell got a considerate purity to meets the test requirements, matenephric mesenchymal cells hold a qualified purity, stability and showed stem cell characteristics. Objective To observe the effect of metanephric mesenchymal stem cells and anti-IL-10 antibody on the proliferation of T cells, dendritic cells and T cells induced by dentric cells and the expression of IL-4, INF-γ, perforin and granzyme B in T cells. To explore the mechanism.Methods (1) Culture the bone marrow derived dendritic cells, spleen derived T cells and the metanephric mesenchymal stem cells in vitro. (2) The establishment of mixed-culture system was performed and the influence of metanephric mesenchymal cells and anti-IL-10 antibody to the proliferation rate of spleen-derived T cells, dendritic cells and T cells induced after dendritic cell was recorded. (3) The establishment of Transwell culture system was performed and the influence of metanephric mesenchymal cells and anti-IL-10 antibody to the proliferation rate of spleen-derived T cells, dendritic cells and T cells induced after dendritic cell was recorded. (4) The establishment of mixed-culture system was performed and the influence of metanephric mesenchymal cells and anti-IL-10 antibody to the expression of IL-4, INF-γ, perforin and granzyme B in T cells after induced by dendritic cells were recorded. (5) The establishment of mixed-culture system was performed and the influence of metanephric mesenchymal cells and anti-IL-10 antibody to the expression of IL-4, INF-γ, perforin and granzyme B in T cells after induced by dendritic cells were recorded.Results (1) Compared with that in control, the proliferation rate of T lymphocytes, dendritic cells, T cells after dendritic cells stimulation in mixed culture system decreased in the intervention group with a dose-dependent pattern within the R:S ratio1:20-1:80(P<0.05). The difference of cell proliferation rate in the 1:80 andl:160 groups held no obvious significance (P> 0.05). Compared with R:S (1:80) group, the cell proliferation rate in anti-IL-10 group increased significantly (P＜0.05), while decreased significantly when compared with that in control group (P<0.05);(2) Compared with that in control, the proliferation rate of T lymphocytes, dendritic cells, T cells after dendritic cells stimulation in Transwell culture system decreased in the intervention group with a dose-dependent pattern within the R:S ratio1:20-1:80(P<0.05). The difference of cell proliferation rate in the 1:80 and1:160 groups were comparable(P> 0.05). Compared with R:S (1:80) group, the cell proliferation rate in anti-IL-10 group increased significantly (P<0.05), while decreased significantly when compared with that in control group (P<0.05);(3) Compared with that in control, the expression of IL-4, INF-y, perforin and granzyme B mRNA and protein T lymphocytes after dendritic cells stimulation in mixed culture system decreased in the intervention group with a dose-dependent pattern within the R:S ratio1:20-1:80(P＜0.05). The difference of cell proliferation rate in the 1:80 andl:160 groups held no obvious significance (P> 0.05). Compared with R:S (1:80) group, the cell proliferation rate in anti-IL-10 group increased significantly (P<0.05), while decreased significantly when compared with that in control group (P<0.05);(4) Compared with that in control, the expression of IL-4, INF-y, perforin and granzyme B mRNA and protein T lymphocytes after dendritic cells stimulation in transwell culture system decreased in the intervention group with a dose-dependent pattern within the R:S ratio1:20-1:80(P<0.05). The difference of cell proliferation rate in the 1:80 andl:160 groups held no obvious significance (P> 0.05). Compared with R:S (1:80) group, the cell proliferation rate in anti-IL-10 group increased significantly (P<0.05), while decreased significantly when compared with that in control group (P <0.05).Conclusion Metanephric mesenchymal stem cells inhibited the proliferation rate of dendritic cells, T lymphocytes and T lymphocytes after dendritic cell stimulation, inhibited IL-4, INF-y, Perforin and granzyme B mRNA and protein expression in T lymphocytes after dendritic cells stimulation. And the direct contact depression and paracrine could be responsible for the inhibition influence. Objective To investigate the immunomodulation of metanephric mesenchymal stem cells in chronic serum sickness nephritis rats as well as the therapeutic effects on renal injury.Methods (1) The metanephric mesenchymal stem cells were cultured and labelled by GFP in vitro, (2) The chronic serum sickness nephritis establishment in rats was performed, (3) Rats were divided into 5 groups: blank control, negative control, model, MMS preconditioning group and MMS transplantation group; (4) The localization of MMS labeled by GFP in rats was determined by fluorescence microscope, (5) Renal pathological changes in rats was observed by conventional pathological staining, (6) T cell subsets of rats from five groups were detected by flow cytometry, (7) ELISA assay was employed to detect serum IL-4, INF-y concentration; (8) RT-PCR assay was employed to detected IL-4, INF-y, perforin and granzyme B mRNA expression of mononuclear cells from rats perpheral blood of five groups.Results (1) Compared with that from blank control and negative control rats, rats from model and MMS preconditioning group showed increased urinary protein and cholesterol level and decreased total protein and albumin concentration when the chronic serum sickess nrphritis model was finished. Compared wth the model rats, MMS preconditioning rats displayed a lower urinary protein(P<0.05). (2) Compared with that in the model rats, a decreased urinary protein level was found in MMS transplantation rats showed (P＜0.05) and MMS preconditioning rats(P<0.01). Compared with the MMS transplantion rats, MMS preconditioning rats showed a decreased urinary protein levels(P<0.05). (3) GFP labeled MMS were found in the liver, spleen and kidney, without heart and the brain tissue. (4) Compared with model rats, MMS transplantation rats displayed a slight kidney damage and even slighter in MMS preconditioning rats. (5) Compared with the blank control and negative control rats, model rats, MMS preconditioning rats and MMS transplantation rats showed a decreased Th cells and Th/Ts ratio, increased Ts cell ratio in peripheral blood(P<0.05). Compared with model rats, the decreased Th cells and increased Ts cell ratio in MMS and MMS preconditioning rats did not holded significance(P＞0.05), while Th/Ts ratio decreased significantly(P<0.05). Compared with the MMS preconditioning rats, MMS transplantion rats held a decreased Th/Ts ratio(P<0.05). (6) Compared with that in the blank control rats and and negative control rats, an increased IL-4 and decreased INF-γconcentration in peripheral blood were found in model rats (P＜0.01) and MMS preconditioning rats and MMS transplantation rats(P<0.05). Compared with the model rats, MMS preconditioning rats and MMS transplantation rats got a reduced level of IL-4 and increased INF-y levels in peripheral blood(P<0.05). Compared with MMS preconditioning rats, MMS transplantation rats held an higher IL-4 and lower INF-γconcentration in peripheral blood(P<0.05). (7) Compared with that in the blank control rats and and negative control rats, an increased IL-4, perforin, granzyme B mRNA expression and reduced INF-γmRNA expression was abtained in mononuclear cells of peripheral blood were found in model rats(P<0.01) and in MMS preconditioning rats and MMS transplantation rats(P<0.05). Compared with the model rats, MMS preconditioning rats and MMS transplantation rats got a reduced level of IL-4 perforin, granzyme B mRNA expression and increased INF-ymRNA expression in peripheral blood(P<0.05). Compared with MMS preconditioning rats, MMS transplantation rats held an higher IL-4, perforin, granzyme B mRNA expression in a lower level of expression,and lower INF-γconcentration in peripheral blood(P<0.05).Conclusion Metanephric mesenchymal cells could perform the immunomodulation in serum sickness nephritis rats and reduce the renal damage.更多还原