Objective:To investigate the immunoregulatory effects of allogeneic BMSCs on T and B cells from BXSB mice with SLE.Methods:In this work,3H-TdR incorporation assay,ELISA,FACS were performed to evaluate the effects of BMSCs on immunocompetent cells of BXSB mice.Results:BMSCs from BALB/c had the inhibitory effects on BXSB mice T lymphocytes proliferation without affecting their activation;Significant inhibition on ConA-induced CD4+ IL-4+ producing cells and enhancing effects on CD4+ IFN-γ+ producing cells;As to B lymphocytes of BXSB mice,BMSCs had inhibitory effects on their proliferation,activation and IgG secretion;In addition,BALB/c BMSCs had inhibitory effects on CD40 ligand (CD40L) ectopic hyper-expression on B cells of BXSB mice.Conclusion:Allogeneic BMSCs had some immuno-regulatory effects on T and B cells of BXSB mice with SLE.

BMSCs not only can enter the immunoorgans of allogeneic mouse, but also can exist there for at last 30 days. Ex vivo tests show: (1)BALB/C BMSCs can suppress the T cells proliferation of BALB/C, B6 and BXSB mice stimulated by ConA. (2)BALB/C BMSCs can promote the B cells proliferation and Ig secretion of both BALB/C and B6 mice stimulated by LPS. But they decreased the B cells proliferation and Ig secretion of BXSB mice. (3)BALB/C BMSCs can reduce the IFN#gamma#-producing cell count of BALB/C and B6 mice stimulated by ConA. But showed no affect on their IL-4-producing cell count. For BXSB mice, BALB/C BMSC can cut down the IL-4-producing cell count, but can augment the IFN#gamma#-producing cell count. And all above effects appear as a dose-dependant fashion. Allogeneic BMSC can enter the immunoorgans of recipient, and can exist there for relatively long time (at last 30 days). In addition, BMSC shows some immunoregulatory effects on normal syngeneic, normal allogeneic and allogeneic mice with autoimmune disease.

Objective To investigate the possibility of immune tole rance induced by bone marrow-derived mesenchymal stem cells in allogeneic organ transplantation. Methods Allogeneic bone marrow-derived mesenchymal stem cells and syngeneic bone marrow cells were cotransplanted to lethally irradiated female C57BL/6 recipient mice. FACS was used to analyze the chimerism 150 days later. Mixed lymphocyte reaction and ConA induced proliferation test were performed to evaluate proliferative activity of mice spleen cells in cell-transplanted group. Skin transplantation test was done to observe immune response of cell-transplanted group mice against organ graft from donor mice. Results About 5 97% donor T cells were detected in splenocytes of cell-transplanted group mice. MLR showed that mean SI of cell-transplanted group mice was 1.79, and that of untreated group mice was 7 28. ConA induced proliferation test showed that mean SI of cell-transplanted group mice was 31 92; and that of untreated group mice was 34 99. Mean survival time of donor-derived skin graft in cell-transplanted group mice was more than 90 days; and that in untreated group mice was 8 days. Conclusion Our results showed for the first time that induction of stable mixed hematopoietic chimerism after allogeneic bone marrow derived mesenchymal stem cells transplantation lead to stable donor-specific tolerance in allogeneic host and skin graft survival from donor mice.

Recently stem cell plasticity has arouse great interests in stem cell research for its potential therapeutic application in degenerative or inherited diseases. Transplantation of bone marrow-derived stem cells in vivo has been shown to give rise to cells of muscle,liver,nerve,endothelium,epithelium etc. But there are still disputes as to stem cell plasticity, as is in the case of contribution of bone marrow-derived cell to skin cells. Here we show that cells co-purified from bone marrow mesenchymal stem cells can evidently give rise to skin tissue in vivo. In the present study, we first isolated and expanded bone marrow-derived multipotentianl mesenchymal stem cells from BALB/C mice(H-2K~(d), white hair), then co-transplanted them with certain bone marrow cells from C57BL/6 mice via tail vein into lethally-irradiated 12 weeks old C57BL/6 mice(H-2K~(b),black hair).The recipient mice grew out white hair on the back about forty days later.

Aim To explore the possibility of the application of self-made anti-BrdU mAb in TUNEL assay for detecting apoptotic cells. Methods TUNEL-based immunoflourescent and immunohistochemical assays were used respectively to examine apoptotic cells in Dex-induced thymocytes and γ-ray irradiated rat skin tissue. Results It was found that the nuclear of apoptotic cells exhibited bright flourescence or brown color,while non-apoptotic cells did not. Conclusion This anti-BrdU mAb can be used to detect apoptotic cells in single cell samples and in tissue section with immunoflourescence or immunohistochemical technique, thus providing a new mefhcd for detection of apoptotic cells at home.

为了比较全面准确地了解系统性红斑狼疮(SLE)BXSB小鼠的发病过程中,淋巴细胞增殖与凋亡的动力学变化及其机制.采用细胞双色荧光染色的标记技术,检测了脾脏淋巴细胞中的增殖细胞和凋亡细胞的百分率,并且测定了巨噬细胞吞噬凋亡细胞的能力.结果发现,发病的雄性BXSB小鼠和雌性BXSB小鼠脾脏中增殖的CD4+T淋巴细胞和B淋巴细胞百分率显著高于对照C57小鼠,而凋亡的B淋巴细胞的百分率显著低于对照C57小鼠;但是,雌雄BXSB小鼠和对照C57小鼠巨噬细胞吞噬凋亡细胞的吞噬指数相同.本研究结果表明,在BXSB小鼠的SLE发病过程中,淋巴细胞的增殖速度异常升高、而凋亡速度下降,可能与其脾脏肿大有关;而且淋巴细胞的增殖与凋亡的失衡与巨噬细胞的功能无关,可能与淋巴细胞内在的异常有关.

目的观察胰岛素样生长因子-1(IGF-1)所致小鼠肺纤维化的病理变化.方法应用电脉冲裸基因肌肉注射法,观察注射截短型IGF1后2,3,4及8周组小鼠肺组织病理学变化.结果 2周组小鼠肺间质充血水肿.3周组肺泡隔明显增厚,大量肺泡上皮细胞,巨噬细胞及成纤维细胞增生.4周组肺间质大量胶原沉积,肺纤维化样改变.8周组肺纤维化病变明显减轻,肺泡结构渐趋恢复.结论 IGF-1可直接导致肺纤维化发生.IGF-1介导的肺纤维化早期病变可以逆转.

Objective To probe into roles of hCKLF-1 in the development of systemic lupus erythematosus in BXSB mice in vivo.Methods The recombinant eukaryotic expression plasmid for hCKLF-1 (pCDI-hCKLF-1) was transferred and expressed in lupus-prone BXSB mice with the technique of muscle-mediated transgene by electric pulse;the levels of serum IgM and IgG anti-DNA antibodies as well as serum BUN and urine protein were detected.Results hCKLF-1 expression in vivo did not make any effects on the levels of IgM and IgG anti-DNA antibodies in female and male BXSB mice,but markedly enabled abnormal elevation of urine protein in male BXSB mice in short time,suggesting its ability for accelerating glomerulonephritis.Conclusion hCKLF-1 may be one of inflammation mediators,playing an important role in the lupus glomerulonephritis of male BXSB mice,and its target cells may be monocyte and macrophage.

Objective To construct eukaryotic expression plasmid for humaninsulin-like growth factor 1(IGF-1) . Methods IGF-1 cDNA was cloned by PCR from the human liver cDNA library， and inserted into eukaryotic expression vector pcDNA3 with gene recombinant technique， forming recombinant pcDNA3-IGF-1 plasmid. The latter was transfected into COS7 cells with liposome-mediated method．Concentration and activity of IGF-1 in the supernatant were detected by ELISA and fibro-blast proliferation assays respectively. Results IGF-1cDNA sequence and its insertion direction in recombinant pcDNA3-IGF-1 plasmid were are correct. The COS7 cells transfected with the pcDNA3-IGF-1 plasmid secreted high concentration of IGF-1，which markedly induced the proliferation of human fibroblast and NIH3T3 cells. Conclusion The recombinant pcDNA3-IGF-1 plasmid can express active IGF-1 with high efficiency， which facilitates the study of its physical and pathological roles in vivo.

Objective To study the in vivo effects of human insulin like growth factor 1 (hIGF 1) on the development of systemic lupus erythematosus of BXSB mouse. Methods Recombinant eukaryotic expression plasmid for hIGF 1 (pcDNA3 IGF 1) was transferred and expressed in lupus prone BXSB mice with the technique of muscle mediated transgene of electric pulse. Autoimmune related assays were carried out and analyzed systemically. Results hIGF 1 increased the production of IgG anti DNA antibody in male BXSB mouse, but strikingly retarded the continueous elevation of proteinuric concentration as aging, followed by the suppression of macrophage secretion for IL 1 and the weight decrease of spleen. Conclusion IGF 1 may have the ability of alleviating severity of lupus glomerulonephritis of male BXSB mouse, which is possibly associated with the inhibition of inflammation mediators secreted by macrophage.

The cDNA of Des(1-3)IGF1 was cloned into an fusion protein expression plasmid, pMTY4, using gene recombinant technique. The protein was purified by ion exchange chromatography and identified by SDS-polyacrylamide gel electrophoresis, radioimmunoassay(RIA), N-terminal amino acid sequence and biological activity. A prokaryotic expression vector was constructed and the fusion protein containing MS2 polymerae fragment, thrombin recognition site and human Des(1-3)IGF1 was expressed in E. coli at high level. It was showed that the purified recombinant Des(1-3)IGF1 released from the fusion protein after digestion with thrombin was identical to the native Des(1-3)IGF1.

系统性红斑狼疮(SLE)小鼠模型是研究人SLE发病机制的主要工具.本室曾发现狼疮性BXSB小鼠B细胞增生异常升高,更新速度明显加快[1].

目的 探讨凋亡细胞在系统性红斑狼疮发病中的作用。方法 将体外诱导的凋亡胸腺细胞经腹腔注射给同基因同性别的正常C5 7小鼠和具有狼疮倾向的雌雄BXSB小鼠 ;检测血清中IgM和IgG类型的抗dsDNA抗体和抗ssDNA抗体的水平 ,尿蛋白浓度以及肾脏中IgG类型免疫复合物的沉积。结果 用凋亡胸腺细胞免疫后 ,诱导C5 7小鼠产生IgG类型的抗dsDNA抗体和抗ssDNA抗体 ;相比之下 ,诱导雌性BXSB小鼠产生更高水平的IgG类型抗dsDNA抗体和抗ssDNA抗体 ;并且使雄性BXSB小鼠的尿蛋白浓度显著升高。结论 凋亡细胞具有免疫原性 ,它可能是狼疮性BXSB小鼠体内自身抗原的主要来源 ;狼疮性遗传因素和Yaa基因能促进凋亡细胞的免疫原性和增强凋亡细胞的致肾炎作用

目的:研究Yaa基因与骨髓B细胞系细胞的分化发育是否有相互关系.方法:用Whitlock-Witte法从骨髓细胞中培养纯化出B细胞系细胞,继而检测B细胞系细胞对脂多糖(lipopolysaccharide, LPS)刺激后的表型、增殖反应和抗DNA抗体的产生以及对地塞米松诱导后的凋亡情况.结果:用Whitlock-Witte法成功地从骨髓细胞中培养分离出B细胞系细胞.经LPS刺激后,雌雄BXSB小鼠B细胞系细胞膜表面CD19抗原和膜表面Ig的分布相似,都出现较强的增殖反应和产生IgM类型的抗ssDNA和dsDNA 抗体,但是对地塞米松诱导的凋亡作用不敏感.结论:Yaa基因对BXSB小鼠骨髓中B细胞系细胞的分化发育无影响,提示Yaa基因仅在外周免疫器官的成熟B细胞中表达活性;并且BXSB小鼠骨髓中的自身反应性B细胞系细胞未被删除,可能是产生自身抗体的内在原因之一.

The level of the anti-DNA antibody in BXSB serum and the effect of BXSB serum on lymphocyte proliferation in normal mouse were observed by using enzyme-linked immunosorbent assay(ELISA) and 3H -thymidine incorpora- tion. The level of anti-DNA antibody in male BXSB serum rose from the age of 3 month and reached to the highest value at the age of 4 month. Although there were a little drop in the antibody level at the age of 5 month, it was still higher than the one of control group (P=0.032, BXSB serum vs control serum, ANOVA ). The experiment also showed that there was a dramatic negative correlation between the level of anti-DNA antibody and the effect of the same portion of BXSB serum on normal lymphocyte proliferation(r=0.232, n=53, P0.05 ). It was concluded that the male BXSB mouse may manifest SLE from the 3rd month after birth. There generated in the SLE serum some substances which not only showed the inhibitory effect on normal lymphocyte proliferation but also were parallel, in its effect, with the level of anti-DNA antibody.

目的探讨系统性红斑狼疮性小鼠抗DNA抗体水平的动态变化与疾病严重程度之间的关系.方法测定了不同月龄的狼疮性BXSB小鼠的各种抗DNA抗体,血尿素氮和尿蛋白质浓度.结果BXSB小鼠抗DNA抗体量的动态变化无规律性,但血尿素氮和尿蛋白质的浓度随鼠龄的增加而逐步升高.结论抗DNA抗体水平并不与BXSB小鼠疾病的严重程度相平行,提示在诊断SLE或判断其严重程度时应测定多项实验指标为宜.

利用溴化脱氧尿嘧啶(Brdu)作为胸苷类似物可掺入到新合成的DNA中的特点,通过检测Brdu标记的细胞便能定性、定量地反映细胞的增殖状态[1].我们已筛选出分泌特异性抗Brdu单克隆抗体(mAb)的杂交瘤细胞株[2].为制备荧光素标记的抗BrdumAb用于流式细胞术定量检测增殖细胞,参考有关文献,用亲和层析纯化小鼠IgG亚类的方法,从腹水中纯化了抗BrdumAb,并用FITC荧光素进行标记.经初步用于体外检测Brdu标记细胞的数量,取得较好的结果.

DNA的5-溴,2'-脱氧尿嘧啶核苷(BrdU)标记法近年来作为一种新的、简便、敏感特异的检测方法,在肿瘤生物学、遗传学、分子生物学特别是细胞增殖动力学等领域得到广泛应用,在一定程度推动了相关学科的发展.为了满足广泛开展BrdU标记DNA检测对BrdU特异性单抗的需要,我们制备了一株产生BrdU单抗的杂交瘤细胞株.本文对此细胞株制备过程及单抗鉴定情况作了简单叙述.

目的：利用抑制性底物杂交技术（Ｓｕｐｐｒｅｓｓｉｏｎ ｓｕｂｔｒａｃｔｉｖｅ ｈｙｂｒｉｄｉｚａｔｉｏｎ，ＳＳＨ），以ＢＸＳＢ 狼疮小鼠为模型，研究系统性红斑狼疮（ＳＬＥ） 发病相关基因。方法：分离ＢＸＳＢ和Ｃ５７ＢＬ６ 小鼠骨髓细胞，提取ｍＲＮＡ，反转录构建ｃＤＮＡ 文库，利用ＳＳＨ 技术研究ＢＸＳＢ小鼠差异表达基因。结果：获得了１２ 个阳性克隆，其中有３ 个克隆编码ＴＣＲ、ＭＨＣ Ⅱ类分子及逆转录病毒基因表达产物，均在ＢＸＳＢ狼疮小鼠高表达，表明其与系统性红斑狼疮发病有关；１ 个在ＢＸＳＢ狼疮小鼠高表达的基因可能为新的ｃＤＮＡ片段，已被ＧｅｎＢａｎｋ 收录，登记号码为ＡＦ０９３４５３。结论：ＴＣＲ、ＭＨＣ Ⅱ类分子及逆转录病毒基因表达产物与系统性红斑狼疮发病有关。