第三方骨髓间充质干细胞对同种异体皮肤移植的影响

目的 探讨第三方骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)对同种异体皮肤移植的影响.方法 40只雌性C57BL/6和50只雄性BALB/C小鼠分别作为皮肤移植的供体和受体.将50只BALB/C小鼠随机分为5组,每组10只:空白对照组,直接进行皮肤移植;给予受体环磷酰胺组(Cyelophosphamide,CP组);给予受体SD大鼠BMSCs移植组(SD-BMSCs组);给予受体CP+SD-BMSCs移植组(CP+SD-BMSCs组);CM-DiI荧光标记组.CP+SD-BMSCs组给予大剂量CP腹腔注射,200 mg/kg,2 d,移植当天自受体小鼠尾静脉注射1×10~6个SD-BMSCs.检测项目包括:观察皮片存活时间、单向混合淋巴反应、组织HE染色、组织荧光镜检,流式细胞仪检测受体外周血单核细胞中SD-BMSCs的含量等.结果 CP+SD-BMSCs组皮肤移植物存活时间(14.5±1.9)d,空白对照组为(8.O±0.8)d,CP组为(11.1±1.4)d,SD-BMSCs组为(10.9±1.4)d,CP+SD-BMSCs组皮肤移植物存活时间明显比后3组延长(P<0.05).混合淋巴反应结果显示,BMSCs对淋巴细胞的增殖抑制作用存在量效关系.HE染色发现CP+SD-BMSCs组不但有血管生成,淋巴细胞浸润也较少.CM-DiI标记的SD-BMSCs能够在BALB/c小鼠体内存活30 d以上,SD-BMSCs组与CP+SD-BMSCs组外周血流式细胞仪均检测到SD-BMSCs.结论 BMSCs能够在异种受体体内存活较长时间;第三方BMSCs能够延长同种异体移植物的存活时间;BMSCs能够抑制异种T淋巴细胞的活化增殖.     

联合成骨细胞移植促进骨髓移植小鼠的造血功能重建

目的 探讨联合成骨细胞移植对骨髓移植小鼠造血功能重建的影响.方法 Balb/c小鼠60只,取其中18只制备移植用骨髓有核细胞和成骨细胞.将其余42只小鼠分为3组.单纯移植组:小鼠18只,仅进行骨髓移植(BMT);联合移植组:18只,进行BMT的同时每只小鼠输入成骨细胞2×106个;正常对照组:小鼠6只,不做任何处理,仅作为移植前的正常对照.移植组小鼠在全身照射(TBI)预处理后4 h经尾静脉注入骨髓细胞2×106个.移植后第7、14和21天,分别处死移植组小鼠6只,对小鼠的外周血细胞和骨髓单个核细胞(BMMNC)进行计数,采用流式细胞术测定BMMNC中CD34+细胞的百分比,使用HPIAS-1000高清晰度彩色病理图像系统测量骨髓造血组织面积,采用免疫组化染色法测定骨髓组织微血管密度(MVD).结果 移植后第7天,单纯移植组和联合移植组小鼠外周血细胞计数及BMMNC数均明显低于正常对照(P＜0.01).第21天时联合移植组小鼠白细胞、红细胞及BMMNC数明显恢复,血小板数已接近正常对照组;单纯移植组小鼠外周血细胞数和BMMNC数虽然有所恢复,但其恢复程度明显弱于联合移植组.移植后第7、14和21天,联合移植组外周血细胞计数及BMMNC数均明显高于同期单纯移植组(P＜0.01或P＜0.05).移植后第7、14、21天,单纯移植组和联合移植组小鼠骨髓造血组织面积、BMMNC中CD34+细胞百分比及骨髓组织MVD均明显低于正常对照组(P＜0.01),但联合移植组均高于同期单纯移植组(P＜0.01或P＜0.05).结论 联合成骨细胞移植能有效促进骨髓移植小鼠骨髓造血系统的重建.

Abstract：

Objective To explore the effects of cotransplantation with osteoblasts on hematopoietic reconstitution in mice after bone marrow transplantation (BMT). Methods The typical model of syngeneic BMT was established. 18 Balb/c mice were used to prepare the bone marrow nuclear cells and osteoblasts for BMT. The 42 Balb/c mice were randomly divided into 3 group:normal group (6 mice, without any treatment), the single BMT group ( 18 mice, given 2 × 106 bone marrow nuclear cells/each mouse) and the cotransplantation group of HSC with osteoblaats (18 mice,given 2 × 106 bone marrow nuclear cells and osteoblasts/each mouse). The following factors were measured on day 7, 14, 21 after BMT: peripheral blood cells, bone marrow mononuclear cells (BMMNC), the percentage of CD34+ cells in BMMNC (assayed by flow cytometry), the hematopoietic tissue changes (detected by HPIAS-1000 image analysis system) and micro vascular density (MVD) of bone marrow tissue (with immunohistochemistry). Results The levels of periphral WBC, RBC, PLT, BMMNC in the contransplantation group were higher than those in the single BMT group (P＜0. 01 or P＜0. 05). In the contransplantation group, the percentage of CD34+ cells in BMMNC, the hematopoietic tissue area and the MVD of bone marrow were also higher than the single BMT group on the 7th, 14th, 21st day after BMT(P＜0.01 or P＜0.05). Conclusion Cotransplantation with osteoblasts could significantly promote hematopoietic reconstruction in mice after BMT. Cotransplantation may represent a promising means of achieving higher engraftment rate after BMT.     

雷公藤甲素预处理诱导Tregs细胞减轻小鼠肝脏缺血再灌注损伤的实验研究

Objective To investigate the effect and related mechanism of triptolide pretreatment to prevent from ischemia/reperfusion (I/R) injury in mice liver. Methods Sixty male C57BL/6 mouse were randomized into four groups (15/group): A:sham group with saline , B: sham group with triptolide, C: saline I/R group, D: triptolide I/R group. The mice were pretreated with either saline or triptolide (0. 1 mg/kg/d) through intraperitoneal (ip) injection for one week. The mouse partial liver model of I/R injury was established, and samples were collected at 24 h after the I/R injury. Results Serum ALT and AST levels were significantly decreased and histological damage was significantly alleviated in the triptolide I/R group as compared with the saline I/R group (P＜0.05), the concentration of MDA in the triptolide groups was significantly decreased, while SOD activity was significantly increased compared with that of the saline I/R group (P＜0.05). The percentages of CD4+ CD25+ regulatory T cells (Tregs) cells among CD4+ T cells in groups A, B, C, and D were(7. 55 ± 1.87)%, (12. 59±3. 87)%,(7. 85±1.07)%, and(12. 02±3. 16)% in liver tissue, respectively. The expression levels of Foxp3 mRNA were significantly higher in the triptolide I/R group than those of saline I/R group (P＜0. 05). ELISA showed that triptolide could significantly inhibit the levels of IL-6, IL-Iβ and TNF-αand promoted the level of IL-10 in the serum (P＜0.05). Conclusion Pretreatment with triptolide could effectively prevent from liver I/R injury, which may be related to the induction of Treg cells by triptolide, the increase in the level of IL-10 in serum, and the inhibition of IL-6, IL-1β and TNF-α production in serum.     

CD4+CD25+T细胞对同种异体小鼠心脏移植的免疫调节作用

目的 观察CD4+CD25+T细胞(Treg)对小鼠同种异体心脏移植的免疫调节作用.方法 流式细胞仪检测正常小鼠和胸腺切除+PC61小鼠淋巴结、脾脏和外周血的Treg的比例.将供体鼠BALB/C心脏移植到受体鼠B6腹腔内,观察对照组(n=6)、胸腺切除组(THY,n=8)、hCTLA4Ig组(n=8)、DST+hCTLA4Ig组(n=8)和THY+PC61+DST+hCTLA4Ig组(n=6)小鼠心脏移植后生存时间和移植心脏病理学检查.结果 正常B6小鼠淋巴结、脾脏和外周血的Treg的比例分别为5.1%、4.5%和1. 7%,明显高于胸腺切除+PC61处理组(1. 8%、1.7%、0.7%).移植心脏平均存活时间在对照组和胸腺切除组分别为(8.2±2.9)d和(7.6±3.0)d,两组间差异无统计学意义(P＞0.05);而在hCTLA4Ig组和DST+hCTLA4Ig组分别为(43.0±11.8)d和(135.0±29.7)d,均较对照组或胸腺切除组明显延长(P＜0.01);THY+PC61+DST+hCTLA4Ig组移植心脏平均存活时间(25.8±8.9)d,也明显较对照组明显延长,但短于hCTLA4Ig组和DST+hCTLA4Ig组(P＜0.01).DST+hCTLA4Ig组移植的心脏存活时间(135.0±29.7)d明显高于其他各组(P＜0.01),其病理组织学表现为间质内有较多的淋巴细胞浸润,伴毛细血管增生,管壁增厚,间质纤维化.结论 CD4+CD25+T细胞水平对同种异体心脏移植的免疫耐受具有免疫调节作用.

Abstract：

Objective To investigate the immunoregulation effects of CD4 + CD25 + T cells in mice heart allograft transplantation. Methods Flow cytometry was used to analyze the contents of CD4 + CD25 +T regulatory cells (Tregs) of the lymph nodes, spleen and blood in the normal mice group and the thymusectomy (THY) + PC61 group. BALB/C mice served as the donors and C57BL/6 (B6) mice as the recipients. Five groups were established, including control group ( n = 6 ), THY group ( n = 8 ), hCTLA4Ig group ( n = 8 ), DST ( donor-specific T-depleted spleen cells) + hCTLA4Ig group ( n = 8) and THY + PC61+ DST + hCTLA4Ig group (n = 6). The survival time after heart allograft transplantation was observed and pathological examination was done in different groups. Results In control group, the rate of Tregs in lymph nodes, spleen and blood was 5. 1%, 4. 5% and 1.7% respectively, which was significantly higher than in THY + PC61 group ( 1. 8% , 1. 7% and 0. 7% respectively). The average survival time of control and THY groups was 8. 2 ± 2.9 and 7.6 ± 3. 0 days respectively ( P ＞ 0. 05 ). The average survival time of hCTLA4Ig and DST + hCTLA4Ig groups was 43.0 ± 11.8 and 135.0 ± 29. 7 days respectively, which was significantly longer than in control group or THY group ( P ＜0. 01 ). The average survival time of THY +PC61 + DST + hCTLA4Ig group was 25.8 ± 8.9 days, which was significantly longer than in control group,but shorter than in hCTLA4Ig group or DST + hCTLA4Ig group ( P ＜ 0. 01 ). The survival time in DST +hCTLA4Ig group was 135.0 ± 29. 7 days, which was significantly longer than other groups ( P ＜ 0. 01 ).The pathological examination revealed that there were more lymphocytes infiltration and capillary vessel proliferation in the desmohemoblast in the transplanted heart of DST + hCTLA4Ig group. Conclusion CD4 +CD25 +T cells regulate the immune tolerance in the allograft transplantation.     

CD103分子介导CD8+T淋巴细胞对同种胰岛移植物的损伤

目的 检验CD103分子是否介导了CD8+T淋巴细胞对同种移植胰岛的免疫损伤.方法 用流式细胞仪检测野生型C57BL/6小鼠外周血CD8+T淋巴细胞表达CD103的情况.以Balb/c小鼠为供者,C57BL/6小鼠为受者,制作同种胰岛移植模型.受者分为3组:M290-SAP组小鼠注射CD103免疫毒素M290-SAP;M290组小鼠注射抗CD103单克隆抗体M290;另以仅接受胰岛移植、不注射任何药物的小鼠为未处理组.检测移植胰岛CD3、CD8、CD44和CD103阳性细胞的表达,检测肠系膜淋巴结中CD3、CD8和CD103阳性细胞的表达.移植物功能丧失或观察期结束时获取移植胰岛,行HE染色和免疫组织化学染色.结果 野生型C57BL/6小鼠外周血的CD8+T淋巴细胞中有44.06%表达CD103.未处理组移植胰岛浸润的细胞成分中有29%的CD8+T淋巴细胞表达CD103.M290-SAP组小鼠淋巴细胞不仅丧失了CD103的表达,而且CD8+T淋巴细胞的绝对数量也减少,该组小鼠血糖稳定时间超过100 d(未处理组为13 d,P＜0.05),移植胰岛组织学形态良好.结论 CD8+T淋巴细胞免疫损伤同种移植胰岛必须表达CD103,CD103有可能成为胰岛移植抗排斥反应治疗的新靶点.

Abstract：

Objective To test whether the CD103 molecule mediates CD8+ T lymphocytes on allogeneic islet graft immune injury. Methods By using flow cytometry, the expression of CD103 in peripheral CD8+ T lymphocytes in wild-type C57BL/6 mice was detected. Allogenic islet transplantation models were made using Balb/c donor mice and C57BL/6 recipient mice. Recipients were divided into 3 groups: M290-SAP-treated mice were injected with CD103 immunotoxin M290-SAP; M290-treated mice were injected with CD103 monoclonal antibody M290; untreated mice were only transplanted islet without any drug treatment. CD3, CD8, CD44 and CD103 positive cells were counted in islet allograft infiltrative lymphocytes. CD3, CD8, and CD103 positive cells were measured in the mesenteric lymph node. The islet allografts were removed and subjected to HE staining and immunohistochemical staining at the time of graft loss or the end of the observation period. Results 44. 06% peripheral CD8+ T cells expressed CD103 in wild-type C57BL/6 mice. 29 % CD8+ T cells expressed CD103 in the infiltrative lyrnphocytes of islet allografts in the untreated mice. In M290-SAP-treated mice, the lymphocytes had no CD103 expression and the absolute number of CD8+ lymphocytes was decreased as well The blood glucose was maintained stable for more than 100 days (13 days in untreated group, P＜0.05) in the M290-SAP-treated mice. Moreover, the transplanted islets retained intact. Conclusion CD103 expression is required for destruction of pancreatic islet allograft by CD8+ T cells. CD103 might provide a novel target for therapeutic intervention in islet allograft rejection.     

CXC趋化因子受体6在小鼠心脏移植排斥反应中的表达及意义

目的 研究CXC趋化因子受体6(CXCR6)在同种异体小鼠心脏移植中的表达及CXC趋化因子配体16(CXCL16)与CXCR6相互作用对移植物存活时间的影响.方法 以野生型Balb/c小鼠(H-2d)为供者(同种移植组),或以野生型C57BL/6小鼠(H-2b)为供者(同系移植组),以野生型C57BL/6小鼠为受者分别行小鼠腹腔异位心脏移植.测定同系和同种移植组小鼠移植心脏CXCR6mRNA的表达,并测定受者脾脏CD8+T淋巴细胞CXCR6的表达.另制作小鼠同种异位心脏移植模型(Balb/c小鼠为供者,C57BL/6小鼠为受者),将其分为实验组和对照组,实验组受者移植当天至发生排斥反应时腹腔注射抗CXCL16抗体,对照组受者同期注射对照抗体.记录两组移植心脏存活时间.进行CD8+T淋巴细胞的细胞毒试验,即用Balb/c小鼠脾细胞免疫C57BL/6小鼠后,获取C57BL/6小鼠脾脏CD8+T淋巴细胞,将Balb/c小鼠脾细胞与C57BL/6小鼠CD8+T淋巴细胞混合培养,分别加入抗CXCL16抗体、小鼠IgG(对照抗体)和抗CD40L抗体.结果 同种移植组移植心脏中CXCR6 mRNA的表达以及脾脏CD8+T淋巴细胞上CXCR6的表达均高于同系移植组和正常对照组.抗CXCL16抗体对CD8+T淋巴细胞的细胞毒活性无影响.与对照组相比较,实验组小鼠移植心脏存活时间并未明显延长.结论 小鼠心脏移植排斥反应中CD8+T淋巴细胞CXCR6的表达上升,阻断CXCL16/CXCR6相互作用并不能延长移植心脏的存活时间.

Abstract：

Objective To investigate the expression of CXCR6 in allograft rejection and effect of CXCL16/CXCR6 interaction on allograft survival Methods Intra-abdominal heterotopic heart transplantation was performed using wild type (WT) Balb/c mice (H-2d) (allogeneic) as donors or WT C57BL/6 mice (B6, H-2b) (syngeneic) as donors, and using WT B6 mice as recipients. The intragraft expression of CXCR6 and expression of CXCR6 in CD8+ T cells of the spleens from syngeneic and allogeneic recipients were examined. The allogeneic recipients were further divided into the experimental group (n = 5) and control group (n = 6) randomly. The experiment group and control group were injected with anti-CXCL16 mAb or control mAb respectively until rejection occurred. The cardiac allograft survival in experimental group and control group was evaluated. Results Rejected allografts showed higher expression of CXCR6 than syngeneic cardiac grafts. More importantly,expression of CXCR6 in CD8+ T cells was also up-regulated by allograft rejection. However, injection of anti-CXCL16 mAb could not inhibit cytotoxic activity of CD8+ T cells. Moreover, experimental group could not prolong the cardiac graft survival time as compared with control group. Conclusion Expression of CXCR6 in CD8+ T cells is up-regulated in allograft rejection.     

Radioprotective effects of the expression of FLT3 ligand regulated by Egr-1 regulated element on radiation injury of SCID mice

Objective:In order to explore the radioprotective effects of the expression of hematopoietic growth factors regulated by radio-inducible promoter on radiation injury. Methods:The human FL (Flt3 ligand) cDNA and EGFP (enhanced green fluorescent protein) cDNA were linked together with IRES and then inserted into the eukaryotic expression vector pCI-Egr, which was constructed by substituting CMV promoter in pCIneo with the Egr-1 promoter (Egr-EF). The vector was transferred into human bone marrow stromal cell line HFCL by lipofectin. The transduced cell clones (HFCL/EF) had been selected by the addition of G418. The cells were exposed to γ-radiation by 60 Co source for 0.5-20Gy. The expressions of transduced cells were detected with FACS, Northern blot ELISA and CFU assay. The HFCL/EF and CD34+ cells from human umbilical cord blood were one after the other transplanted i.v. into sublethally irradiated severe combined immunodeficient (SCID) mice. The white blood cell amount in peripheral blood and human cell engrafted in recipent mice were detected by flow cytometry and CFU-GM etc. Results:The activity of EGFP in transduced cells increased by 3.1 fold as compared to non-transduced cells at 18h after exposure to 2.5Gy. The amounts of secreted FL in serum-free supernatants of Egr-EF increased by 605.46±107.21pg/ml, which were significantly higher than the control group (214.45±35.61pg/ml). The effects of FL in HFCL/EF cultural supernatants on expansion of CD34+ cells derived from cord blood in the presence of SCF, IL-6 and IL-3 were also studied. The results showed that at day 10 of culture the number of CD34+ cells increased by 173. 09±11.58×103/ml, which was significantly higher than that of non-radiation group(68. 04± 13. 73 × 103/ml). It showed that radiation can enhance the ability of the supernatants containing FL of HFCL/EF to expand early hematopoietic progenitor cells and protect hematopoietic cells from radiation-injury effects. The HFCL/EF and CD34+cells from human umbilical cord blood were one after the other transplanted i. v. into sublethally irradiated severe combined immunodeficient (SCID) mice. In contrast to two control groups (HFCL and HFCL/F), HFCL/EF (the Egr-1 regulatory element-drived expression of FL gene therapy) resulted in a proportionally obvious increase in the number of the white blood cell at early stage after radiation, while no significant differences were found for CD45+ 、CD34+ cells in bone marrow cells. In contrast to two control groups (HFCL and HFCL/F), HFCL/EF (the Egr1 regulatory element-drived expression of FL gene therapy) resulted in a proportionally obvious increase in the number of the white blood cell at early stage after radiation, without significant differences being found for CD45+、CD34+ 、CFU-GM and marrow nucleared cells in bone marrow cells. Conclusions:The results suggested both in vivo and in vitro use of the gene therapy of FL gene regulated by Egr-1 promoter could protect hematopoiesis from irradiation-induced damage.     

增强型生物活性玻璃-胶原复合支架材料的成骨效能研究

Objective To study the in vivo and vitro biocompatibility and osteogenetic capacity of enhanced bioactive glass/collagen composite scaffold. Methods Bone marrow stromal cells(BMSCs)were collected and induced to osteoblast-like cells.The growth rate of BMSCs was detected and compared progressively through Alamar Blue.The RNAs of the cells were collected and detected for bone morphogenetic protein-2(BMP-2),alkaline phosphatase(ALP),collagen Ⅰ(Col-Ⅰ)through qRT-PCR on the fourth and seventh days.Scaffolds with induced osteoblasts were embedded into 3 nude mice subcutaneously in vivo and detected after 6 weeks.X-ray,qRT-PCR and tissue staining were used to detect the mRNA expressions of BMP-2,Col Ⅰ,osteocalcin(OCN)and ostcopontin(OPN)and bone formation. Results SEM(scanning electronic microscopy)showed BMSCs attached to the scaffold tightly and viably and proliferated actively on the scaffold.The growth rate in the experimental group was significantly higher after 7 days(P<0.05)than in the control group.qRT-PCR showed that the mRNA expressions of BMP-2,ALP and Col-Ⅰ in the experimental group were significantly higher than in the control group on the seventh day(P<0.05).X-ray showed that the dense images of embedded scaffolds were locally similar to those of normal bone after 6 weeks.qRT-PCR showed that the mRNA expressions of BMP-2,Col Ⅰ,OCN and OPN in the experimental group were significantly higher than those of normal bone(P<0.05).HE and Massort staining of the paraffin sections showed the scaffolds degraded generally and osteoblasts and chondrocytes proliferated abundantly and distributed irregularly.Bone formation could be observed obviously. Conclusion Enhanced bioactive glass/collagen composite scaffolds have good biocompatibility and osteogenetic capacity in vitro and vivo.     

弓形虫可溶性抗原混合液延长小鼠移植心脏存活时间

目的 探讨弓形虫可溶性抗原混合液(STAgs)延长小鼠移植心脏存活时间的作用及其作用机制.方法 通过在冰浴中超声粉碎弓形虫速殖子制备弓形虫STAgs.实验分为3组,每组受者9只.STAgs组和急性排斥反应(AR)组:供者为Balb/c小鼠,受者为C57BL/6小鼠,移植前4 d两组受者分别皮下注射STAgs 5μg和磷酸盐缓冲液100μl,同系对照组供、受者均为C57BL/6小鼠,术前未进行任何处理.分组后建立小鼠颈部异位心脏移植模型.术后观察移植心脏存活时间,术后第7天每组处死3只受者,获取移植心脏行病理学检查观察排斥反应,采用免疫组织化学检测移植心中CD4+和CD8+T淋巴细胞.结果 同系移植组在观察终点100 d时均存活,AR组和STAgs组移植心脏存活时间分别为(6.7±0.5)和(70.8±3.5)d,3组间两两比较,差异均有统计学意义(P＜0.05).术后第7天,同系移植组、AR组和STAgs组移植心排斥反应分级分别为0级、Ⅲ～Ⅳ级和0～Ⅰ级;免疫组织化学检测显示STAgs组CD4+和CD8+T淋巴细胞比例明显少于AR组,差异有统计学意义(P＜0.05).结论 弓形虫STAgs能显著延长小鼠移植心脏的存活时间,减轻移植心脏的排斥反应,县体机制可能与弓形虫STAgs可影响TH1/TH2比例相关,也可能通过刺激机体产生脂氧素A4抑制树突状细胞活化发挥作用.

Abstract：

Objective To investigate the effects of T. gondii soluble tachyzoite antigen (STAgs) on the survival time of mouse heart allograft and the possible mechanism. Methods The STAgs were prepared by pulverizing T. gondii tachyzoite with ultrasound on ice. Cervical heterotopic heart transplantations were done by using Balb/c mice as donors, and C57BL/6 mice as recipients.The recipients were classified randomly into three groups: syngeneic group, acute rejection group and STAgs-treated group. The recipients in acute rejection group and STAgs-treated group were injected subcutaneously with 0. 1 ml PBS and 0. 1 ml (5 μg) STAgs at the 4th day before transplantation respectively, and those in syngeneic group were not subjected to any treatment. The grafts were observed daily by cervical palpation, and the total cessation of cardiac contraction was defined as the endpoint. The heart allografts were harvested at the 7th day after transplantation for pathological examination and immunohistochemical staining for CD4+ T, CD8+ T. Results The recipients in syngeneic group were all alive at the 100th day after transplantation. The average survival time in acute rejection group and STAgs-treated group was (6.7± 0.5) days and (70.8± 3.5) days,respectively (P＜0.05). HE staining showed that the rejection on the 7th day after transplantation in syngeneic group, acute rejection group and STAgs-treated group was fallen into 0 degree, Ⅲ-Ⅳ degree and 0- Ⅰ degree, respectively. Immunohistochemical staining revealed that the CD4+ T and CD8+T were markedly down-regulated in STAgs-treated group as compared with those in acute rejection group. Conclusion T. gondii STAgs can significantly prolong the survival time of mouse heart allograft and inhibit the rejection probably by changing the ratio of TH1/TH2, or inhibiting the effect of dendritic cells by inducing the lipoxin A4.     

组织特异性胞嘧啶脱氨酶和5-氟胞嘧啶热化疗对裸鼠结肠癌肝脏转移的影响

Objective To investigate the effects of tissue specific cytosine deaminase/5-fluorocytosine (CD/5-FC) thermotherapy on hepatic metastasis of colonic carcinoma in nude mice. Methods Forty-five nude mice were randomly divided into control group, 5-FC group and 5-FC thermotherapy group according to the random number table (15 mice in each group). Mice models of hepatic metastasis of colonic carcinoma were established by portal vein injection of LoVo/CEACD cells. The hepatic metastasis rate and number of metastatic nodules of the 3 groups were compared by ehi-square test and one-way ANOVA. The pathological changes in tumor tissues and apoptotic index of tumor cells were observed. The expression of the CD gene in tumor tissues was detected by fluorescent quantitative RT-PCR and Western blot. Results The number of metastatic nodules and liver metas-tasis rate were 4.6±1.3 and 100.0% in control group, 2.2±1.0 and 60.0% in 5-FC group, 0.5±0.8 and 13.3% in 5-FC thermotherapy group, with statistical difference among the 3 groups (F=25.898, χ2=5.208, 19.548, 5.168, P<0.05). The mean apoptotic indexes of tumor cells of the 3 groups were 4.6%, 9.9% and 17.4%, respectively. Vacuolar degeneration, cell necrosis, cytolysis and apoptotic bodies were mostly observed in the 5-FC thermotherapy group. The expression of CD gene in tumor tissue was detected in all the groups. Conclusion Tissue specific CD/5-FC thermotherapy has inhibitory effects on the hepatic metastasis of LoVo cells transfected with CD gene.     

抗原特异性CD4+CD25+T细胞对同种异体胰岛移植影响及作用机制研究

目的:探讨抗原特异性CD4+CD25+Treg细胞免疫对同种异体胰岛急性移植排斥反应的影响和机制。方法:用MACS分选供体抗原特异性CD4+CD25+Treg细胞免疫糖尿病BALB/cByJ受体小鼠，以ICR小鼠胰岛为供体行同种异体胰岛移植。观察移植后小鼠的存活时间、移植前后外周血CD4+和CD8+T细胞亚群的变化和移植物中Th1/Th2细胞因子mRNA表达水平的变化。结果:抗原特异性Treg细胞联合胰岛移植组(C组)胰岛移植物平均生存期为(34.57±17.15)d，显著长于单纯胰岛移植组(B组)的(10.6±1.82) d (P<0.01)；移植后第3天，C组外周血CD4+/CD8+的值显著低于B组(P<0.01)；C组移植物中IL-10,TGF-β mRNA表达比B组显著增强。B组移植物中IL-1β，IL-2及IFN-γ mRNA表达明显强于C组。结论:抗原特异性CD4+CD25+ Treg细胞可通过调节Th2/Th1之间的反应平衡而延长同种异体胰岛移植物的存活时间。     

自体骨髓间质干细胞对异基因小鼠皮肤移植免疫排斥的影响

目的 观察自体骨髓间质干细胞(bone marrow stromal cells,BMSCs)对异基因小鼠皮肤移植的影响.方法 建立C57BL/6至BALB/c小鼠皮肤移植急性排斥模型,密度梯度离心法分离与贴壁法富集BALB/c小鼠BMSCs,自尾静脉输注.实验分3组,每组10只小鼠.A组:正常BALB/c小鼠+自体BMSCs,B组:仅行皮肤移植,C组:皮肤移植后BALB/c小鼠+自体BMSCs.检测各时间段皮肤组织病理表现以及相关细胞因子浓度变化.结果 由密度梯度离心法与贴壁法分离的细胞具有BMSCs的一般特征,C组较B组移植后皮肤免疫排斥反应明显减轻,IL-2、IFN-γ等细胞因子浓度在术后第7天(F=248 954.6,P＜0.05;F=148 311.7,P＜0.05)、第14天(F=117 372.3,P＜0.05:F=126 743.3,P＜0.05)均有降低.结论 异基因小鼠皮肤移植术后输注自体BMSCs可以抑制免疫排斥反应,其机制可能与减少IL-2、IFN-γ等相关细胞因子分泌有关.     

基因工程Treg细胞对小鼠异基因骨髓移植后GVHD和GVL效应的影响

目的 探讨输注慢病毒载体介导的小鼠基因工程调节性T淋巴细胞(Treg细胞)对小鼠异基因骨髓移植后移植物抗宿主病(GVHD)及移植物抗白血病(GVL)效应的影响.方法 利用慢病毒载体介导,将小鼠叉状头螺旋转录因子(Foxp3)基因转导入Balb/c小鼠的CD4+CD25-T淋巴细胞,即为基因工程Treg细胞.以Balb/c小鼠为供者.C57BL/6小鼠为受者,进行异基因骨髓移植,移植当天受者接受X线直线加速器全身照射.用随机数字表法将受者分为5组,每组10只.(1)单纯照射组:经受者尾静脉输注RPMI 1640培养液0.2 ml;(2)白血病对照组:经受者尾静脉输注供者骨髓细胞5×106个+C57BL/6小鼠T淋巴细胞白血病/淋巴瘤细胞株(EL4细胞)500个;(3)移植对照组:经受者尾静脉输注供者骨髓细胞5×106个+脾细胞5×106个+EL4细胞500个;(4)工程Treg组:经受者尾静脉输注供者骨髓细胞5×106个+脾细胞5×106个+EL4细胞500个+基因工程Treg细胞5×106个;(5)空载体对照组:经受者尾静脉输注供者骨髓细胞5×106个+脾细胞5×106个+EL4细胞500个+空载体转导的CD4+CD25-T淋巴细胞5×106个.每天观察受者存活情况;记录GVHD及白血病的发生情况;各组均于小鼠濒死前取其肝脏、小肠、皮肤、脾脏等组织,进行病理学观察;取长期存活(超过60 d)受者的骨髓细胞,检测嵌合情况.结果 单纯照射组、白血病对照组、移植对照组、工程Treg组和空载体对照组小鼠存活时间分别为(10.3±1.5)d、(20.7±1.9)d、(26.0±4.3)d、(49.0±17.7)d和(24.4±4.1)d,工程Treg组小鼠存活时间明显长于其他各组,差异有统计学意义(P＜0.05).白血病对照组小鼠肝、脾组织病理切片均存在白血病细胞浸润表现,移植对照组及空载体对照组小鼠肝脏、皮肤和小肠病理切片存在GVHD病理改变,而工程Treg组长期存活小鼠各组织病理切片结构基本正常,未见GVHD及白血病细胞浸润病理表现,该组GVHD评分明显低于移植对照组及空载体对照组.结论 小鼠异基因骨髓移植时联合输注基因工程Treg细胞可有效减少GVHD的发生并保留GVL效应.     

输注鼠基因工程Treg细胞抑制小鼠异基因骨髓移植后移植物抗宿主病

目的 探讨输注慢病毒载体介导的鼠基因工程调节性T淋巴细胞(Treg细胞)对小鼠异基因骨髓移植后移植物抗宿主病(GVHD)的影响.方法 利用慢病毒载体介导,将鼠又状头螺旋转录因子(Foxp3)基因转导入Balb/c小鼠的CD4~+ CD25~-T淋巴细胞,即为基因工程Treg细胞.以Balb/c小鼠为供者,C57BL/6小鼠为受者,进行异基因骨髓移植,实验分4组进行:(1)工程Treg组经受鼠尾静脉输注供鼠骨髓细胞5×10~6个+脾细胞5×10~6个+基因工程Treg细胞5×10~6个;(2)移植对照组经受鼠尾静脉输注供鼠骨髓细胞5×10~6个+脾细胞5x10~6;(3)单纯照射组经受鼠尾静脉输注RPMI 1640培养液0.2ml;(4)空载体对照组经受鼠尾静脉输注供鼠骨髓细胞5×10~6个+脾细胞5×10~6个十空载体转导的CD4~+ CD25~-T 淋巴细胞5×10~6个.每天观察受鼠存活情况;记录GVHD的发生情况;各组均于小鼠濒死前取其肝脏、小肠、皮肤等组织,进行病理学观察;取长期存活(超过60d)的受鼠骨髓细胞,检测嵌合情况.结果 单纯照射组、移植对照组、工程Treg组和空载体对照组小鼠存活时间分别为(8.8±0.6)d、(36.7±2.5)d、(51.6±4.0)d和(34.1±2.3)d,工程Treg组小鼠存活时间明显长于其他各组,差异有统计学意义(P<0.05).移植对照组及空载体对照组小鼠肝脏、皮肤和小肠病理切片均存在GVHD病理改变,工程Treg组长期存活小鼠的肝脏、皮肤和小肠常规病理切片结构基本正常,未见GVHD病理表现,该组GVHD评分明显低于移植对照组及空载体对照组.结论 小鼠异基因骨髓移植时联合输注基因工程Treg细胞可有效减少GVHD的发生,减轻其严重程度.     

体外光化学疗法对皮肤移植小鼠产生调节性T细胞的影响

目的  探讨输注体外光化学法处理的脾淋巴细胞对皮肤移植受体小鼠产生调节性T细胞(Treg)及移植物存活时间的影响。方法  以C57BL/6小鼠为供体,BALB/c小鼠为受体,建立小鼠皮肤移植模型。分离C57BL/6和BALB/c小鼠脾淋巴细胞(CSP、BSP),制备经8-甲氧基补骨脂素联合长波紫外线(PUVA)处理的小鼠脾淋巴细胞(PUVA-SP)。根据受者静脉输注的成分将实验动物随机分为5组(每组12只)：PUVA-BSP组、PUVA-CSP组、BSP组、CSP组及磷酸盐缓冲液(PBS)对照组,每组受体分别于术前7 d、手术当日和术后7 d按组别从尾静脉注入PUVA-BSP、PUVA-CSP、BSP、CSP或PBS。观察受体移植物的存活时间,检测受体外周血中CD4⁺CD25⁺Foxp3⁺Treg表达情况。结果  皮肤移植术后,PUVA-BSP组和PUVA-CSP组的受体小鼠外周细胞CD4⁺CD25⁺Foxp3⁺Treg的比例明显高于输注BSP组、CSP组和PBS对照组;PUVA-CSP组高于PUVA-BSP,BSP组和CSP组低于PBS对照组。PUVA-BSP组和PUVA-CSP组的受者小鼠移植皮片存活时间明显长于BSP组、CSP组和PBS对照组(均为P＜0.05)。结论  输注足够数量的PUVA-SP可诱导受体体内产生较多的CD4⁺CD25⁺Foxp3⁺Treg,可以显著延长移植物的存活时间。     

门静脉输注供者脾细胞诱导的供者特异性免疫低反应性

目的 探讨经门静脉输注供者脾细胞能否诱导皮肤移植小鼠产生供者特异性的免疫低反应性及其可能机制.方法 取Balb/c小鼠,随机分为空白对照组(经小鼠门静脉输注RPMI 1640培养液)、受者脾细胞组(经小鼠门静脉输注Balb/c小鼠脾细胞)、供者脾细胞组(经小鼠门静脉输注C57BL/6小鼠脾细胞)、空白移植对照组(经小鼠门静脉输注RPMI 1640培养液,7 d后移植C57BL/6小鼠的皮肤)、实验对照组(经小鼠门静脉输注Balb/c小鼠脾细胞,7 d后移植C57BL/6小鼠的皮肤)、实验组(经小鼠门静脉输注C57BL/6小鼠脾细胞,7 d后移植C57BL/6小鼠的皮肤)以及第三方移植组(经小鼠门静脉输注C57BL/6小鼠脾细胞,7 d后移植C3H小鼠的皮肤).记录空白移植对照组、实验对照组、实验组和第三方移植组移植皮肤的存活时间,并观察移植皮肤的病理学变化;脾细胞输注后7 d,分别获取空白对照组、受者脾细胞组和供者脾细胞组小鼠的外周血、脾脏和肝脏,用流式细胞仪测定样本中CD4+CD25+Foxp3+调节性T淋巴细胞(CD4+CD25+Foxp3+Treg细胞)的比例.结果 实验组移植皮肤的存活时间为(19.8±4.6)d,明显长于空白移植对照组、实验对照组和第三方移植组,但仍未达到长期存活.皮肤移植后7 d,空白移植对照组和实验对照组的移植皮肤呈现重度急性排斥反应的病理学改变,而实验组移植皮肤呈现中度急性排斥反应的病理学改变.供者脾细胞组外周血、肝脏和脾脏中CD4+CD25+Foxp3+Treg细胞比例明显高于空白对照组和受者脾细胞组.结论 门静脉输注供者脾细胞可特异性地延长供者皮肤移植物的存活时间,减轻移植物的排斥反应,该效应可能与受者体内的CD4+CD25+Foxp3+Treg细胞增加有关.     

体内注射白细胞介素10和转化生长因子质粒延长小鼠移植皮肤存活时间

目的 探讨体内注射白细胞介素10(IL-10)和转化生长因子β1(TGF-β1)质粒对小鼠移植皮肤存活时间的影响.方法 构建含IL-10和TGF-β1基因的质粒,以Balb/c小鼠为受者、Balb/c小鼠与C57BL/6小鼠杂交的F1代小鼠为供者,行皮片移植.移植当天,经尾静脉分别给受者快速注射不含基因的空白质粒(空白组)、含IL-10基因质粒(IL-10组)、含TGF-β1基因质粒(TGF-β1组)以及含IL-10和TGF-β1双基因的质粒(联合组),以后每2天注射1次,20 μg/次,共注射6次,观察移植皮肤存活时间.另取Balb/c小鼠,在输注C57BL/6小鼠脾细胞后,按前述分组及方法接受质粒快速注射,注射5次后,分离其脾细胞,以流式细胞仪检测脾细胞中CD4+ CD25+ T淋巴细胞含量.结果 移植皮片存活时间,空白组为(13.50±1.04)d,IL-10组为(13.83±1.16)d,TGF-β1组为(15.33±1.50)d,联合组为(21.33±3.20)d,联合组移植皮片存活时间明显长于其他3组(P<0.01).脾细胞中CD4+ CD25+ T淋巴细胞的含量,空白组为(6.58±1.86)%,IL-10组为(10.52±1.13)%,TGF-β1组为(14.44±0.42)%,联合组为(14.25±1.24)%,TGF-β1组和联合组的CD4+ CD25+ T淋巴细胞含量明显高于空白组和IL-10组(P<0.01).结论 体内注射IL-10和TGF-β1质粒可延长小鼠移植皮肤存活时间,并能提高CD4+ CD25+ T淋巴细胞含量.     

A differential requirement for CD8+ donor cells in the augmentation of allograft survival by posttransplantation administration of donor spleen cells and donor bone marrow cells

BACKGROUND: Peritransplant treatment with antithymocyte serum (ATS) and posttransplantation administration of donor bone marrow or donor splenocytes results in extended skin allograft survival. In this study, we examined the molecular basis of the tolerance promoting effect of donor bone marrow (BMC) cells and splenocytes with emphasis on the role of CD8 expression on the donor cells. METHODS: (C57BL/6J x A/J)F1 mice were treated on days -1 and +2 with ATS relative to transplantation with C3H/HeJ skin. On day +7, they were infused with CD8+ BMC, CD8- BMC, CD8+ splenocytes, or CD8- splenocyte donor subpopulations isolated by magnetic or fluorescence-based sorting. In additional experiments, B10.D2(R107) mice were treated in the same manner with C57BL/6 skin and BMC or splenocytes from C57BL/6 mice in which the CD8alpha gene had been inactivated. RESULTS: CD8+ donor bone marrow cells induced operational tolerance (defined as graft acceptance in the absence of chronic immunosuppression) in skin graft recipients at a dose that was reduced by 250-fold relative to unfractionated bone marrow cells (1.0x10(5) cells per recipient, median survival time (MST)=41 days vs. 2.5x10(7) cells per recipient, MST=49 days, P=0.40). Similarly, donor bone marrow cells from CD8 knockout mice did not promote graft acceptance (MST=98 days vs. animals not treated with bone marrow cells, MST=70 days, P=0.16). In contrast, the extension of graft survival by donor splenocytes did not require the presence of CD8+ donor cells because splenocytes depleted of CD8+ cells extended graft survival (MST=55 days) as well as unsorted splenocytes (44 days, P=0.2), and splenocytes from CD8 knockout animals (MST=145 days) extended graft survival at least as well as unsorted splenocytes (MST=74 days, P=0.4) CONCLUSIONS: These results suggest that the prolongation of graft survival by donor bone marrow is dependent on the presence of the CD8 molecule, whereas prolongation by donor splenocytes is not. Therefore, we suggest that the prolongation of graft survival by these cell types occurs via distinct molecular mechanisms probably mediated by different cell types.     

RNA干扰树突细胞主要组织相容性复合物1表达后诱导抑制性T细胞对小肠移植耐受的影响

目的探讨RNA干扰树突细胞（Dc）组织相容性复合物1（MHC-1）表达后获得的CD8＋CD28-抑制性T细胞（Ts）对小肠移植免疫耐受的的影响。方法通过siRNA干扰DC MHC—I表达后,诱导获得CD8^＋CD28^-Ts。建立由Wistar大鼠移植到SD大鼠的小肠移植模型36例,随机分为A组（转染实验组）、B组（未转染组,注射普通T细胞）和C组（移植对照组,注射生理盐水）。术后14d,每组各随机挑选6例,取移植大鼠小肠和血液标本行移植小肠组织病理学检查．并检测移植大鼠血清TGF-β、IFN-γ水平及回肠黏膜Na^＋-K^＋-ATP酶活性,观察移植大鼠的存活时间。结果术后第14天,A组移植大鼠血清中TGF—β和IFN-γ表达水平高于B、C组（P〈0．05）。A组大鼠肠黏膜Na^＋-K^＋-ATP酶活性为（6．3±1．0）kU／g,明显高于B组的（3．6±0．9）kU／g和C组的（2．9±1．3）kU／g（P〈0．05）。A组移植小肠病理Parks评分分级明显低于B组和C组（P〈0．05）。A、B和C组移植后大鼠中位生存时间分别为32．0、17．5和21．0d,A组存活时间明显优于B组和C组（P〈0．05）。结论将RNA干扰DCMHC—I表达所获得的CD8^＋CD28^-Ts过继小肠移植大鼠．可以减轻移植大鼠的损伤程度．抑制免疫排斥反应．