急性肝衰竭大鼠载肝细胞生长因子纳米粒子肝细胞移植后有丝分裂指数和Ki-67表达变化及意义

目的 探讨急性肝衰竭(ALF)大鼠载肝细胞生长因子(HGF)的聚乳酸-氧-羧甲基壳聚糖(PLA-O-CMC)纳米粒子肝细胞移植后有丝分裂指数和Ki-67抗原的表达变化及意义.方法 D-氨基半乳糖腹腔内注射制作大鼠ALF模型,48 h后分别将Ⅰ型胶原(Ⅰ组)、PLA-O-CMC纳米粒子(Ⅱ、Ⅳ组)、载HGF的PLA-O-CMC纳米粒子(Ⅲ组)培养的大鼠肝细胞(均为5×107个,5 ml)移植到ALF大鼠腹腔内.以每日静脉注射HGF 10μg/kg×7 d(Ⅳ组)、5 Ml RPMI 1640腹腔内注射(Ⅴ组)作为对照.观察其14 d存活率、血清及肝组织HGF浓度、肝组织病理及有丝分裂指数(MI)、Ki-67变化.结果移植后14 d Ⅰ～Ⅴ组ALF大鼠的存活率分别为50.00％、68.75％、81.25％、75.00％、18.75％.各纳米移植组高于V组.Ⅲ组3 d时肝组织HGF浓度最高,平均为5882.91 μG／L.Ⅲ组肝组织结构恢复更快,5 d时平均MI 10.20％0、7 d时平均MI 10.20‰、7 d时Ki-67平均标记指数16.8‰,均高于Ⅴ组.结论 应用载HGF的PLA-O-CMC纳米粒子培养的大鼠肝细胞腹腔内移植治疗ALF大鼠能促进肝再生相关抗原表达.

Abstract：

Objective To evaluate the change and significance of Ki-67 antigen expression follow ing hepatocyte transplantation using hepatocyte growth factor (HGF) loaded polylactic acid-O-carboxymethylchitosan (PLA-O-CMC) nanoparticles in rats with acute liver failure (ALF). Methods ALF models of rats were established by D-galactosamine intraperitoneal injection. Rat hepatocytes type Ⅰ collagen suspension (group Ⅰ ),rat hepatocytes co-cultured with PLA-O-CMC nanoparticles ( groups Ⅱ ,Ⅳ ) and rat hepatocytes co-cultured with HGF loaded PLA-O-CMC nanoparticles ( group Ⅱ ) (5 × 107 cells each group,5 ml) were transplanted into the abdominal cavity of SD rats at 48 h after D-Gal injection. Intravenous injection of HGF ( 10 μg/kg for 7 days) ( group Ⅳ ) and RPMI 1640 injection (group Ⅴ ) were used as the negative groups. The 14th-day survival rate of rats,pathological change and mitotic index of liver,Ki-67 antigen labeling index of ALF rat livers were observed. Results The 14th-day survival rate in groups Ⅰ -Ⅴ was 50.00％,68. 75％,81.25％,75.00％,18.75％,and that in nano-transplanted groups was higher than in group Ⅴ. At the 3rd day,the concentration of HGF in liver tissue of group Ⅲ,average 5882. 91 μg/L was the highest. The hepatic lobules structure in group Ⅲ recovered faster. The average mitotic index in group Ⅱ at the 5th day was 10. 20‰ and the average Ki-67 labeling index at the 7th day was 16. 8‰,which were all higher than in group Ⅴ. Conclusion Intraperitoneal transplantation of HGF loaded PLA-O-CMC nanoparticle-attached hepatocytes for treatment of ALF can promote the liver regenerationassociated antigen expression.     

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.     

不同全麻对口腔恶性肿瘤患者免疫功能的影响

目的 评价不同全麻对口腔恶性肿瘤患者免疫功能的影响.方法 择期拟行口腔恶性肿瘤根治术患者60例,年龄49～64岁,体重50～71kg,ASA分级Ⅰ或Ⅱ级,采用随机数字表法,将患者随机分为3组(n=20):Ⅰ组采用全凭静脉麻醉,静脉注射眯达唑仑、瑞芬太尼、顺阿曲库铵和异丙酚麻醉诱导,静脉输注异丙酚和瑞芬太尼维持麻醉;Ⅱ组采用静吸复合麻醉,麻醉诱导同Ⅰ组,吸入七氟醚或静脉输注异丙酚复合瑞芬太尼维持麻醉;Ⅲ组采用吸入全麻,吸入七氟醚,静脉注射瑞芬太尼和顺阿曲库铵麻醉诱导,吸入七氟醚,静脉输注瑞芬太尼维持麻醉.于麻醉诱导前30 min(T0)、麻醉后1、3、5 h、术毕、术后24、48、72 h(T2～7)时采集外周静脉血,采用流式细胞仪测定T淋巴细胞亚群及NK细胞、B淋巴细胞百分比.结果 与T0时比较,Ⅰ组～Ⅲ组T1-5时CD3+、CD4+、CD4+/CD8+比值、NK细胞百分比和B淋巴细胞百分比降低,Ⅱ组和Ⅲ组T6时CD3+、CD4+、CD4+/CD8+比值及NK细胞百分比降低(P＜0.05或0.01);与Ⅰ组比较,Ⅱ组T2,3,6时CD4+、CD4+/CD8+比值和Ⅲ组T2～6时CD3+、CD4+、CD4+/CD8+比值及NK细胞百分比降低(P＜0.05或0.01);与Ⅱ组比较,Ⅲ组T4.5时CD4+、CD4+/CD8+比值及NK细胞百分比降低(P＜0.05).结论 与吸人麻醉和静吸复合麻醉比较,咪达唑仑、瑞芬太尼和异丙酚全凭静脉麻醉对口腔恶性肿瘤患者免疫功能的抑制程度较低.

Abstract：

Objective To investigate the effects of different general anesthesia protocols on immune function in patients with oral malignant tumor. Methods Sixty ASA Ⅰ or Ⅱ patients undergoing elective radical operation for oral malignant tumor were randomly divided into 3 groups ( n = 20 each): group Ⅰ total intravenous anesthesia (TIVA); group Ⅱ combined intravenous-inhalational anesthesia (IV-INH) and group Ⅱ inhalational anesthesia (INN). Anesthesia was induced and maintained with propofol and remifentanll in group Ⅰ; with sevoflurane,propofol and remifentanil in group Ⅱ and with sevoflurane and remifentanil in group Ⅲ. Peripheral venous blood samples were taken at 30 min before (To) and 1 h (T1), 3 h (T2 ) and 5 h (T3) after induction of anesthesia, the end of operation (T4 ) and at 24 h (T5 ), 48 h (T6 ) and 72 h (T7) after operation for determination of the percentages of T lymphocyte subsets (CD3+ , CD4+ , CD8+ , CD4+/CD8+ ratio). Natural killer (NK) cells (CD16+ ,CD56+ ) and B lymphocyte (CD19+ ) with flow cytometer. Results The percentages of CD3+ , CD4+ , NK cells, B lymphocyte and CD4+/CD8+ ratio were significantly decreased during and after operation at T1-5 in all groups and the percentges of CD3+ ,CD4+ ,NK cells and CD4+/CD3+ ratio were decreased at T6 in groups Ⅱ and Ⅲ as compared with the baseline values before anesthesia at To. The percentage of CD4+ cells and CD4+/CD8+ratio were significantly lower during anesthesia at T2,3,6 in group Ⅱ than in group Ⅰ . The percentages of CD4 +and NK cells and CD4+/CD8+ ratio were significantly higher after operation at T4,5 in group Ⅱ than in group Ⅲ.The percentages of CD3 + , CD4 + , NK cells and CD4 +/CD8 + ratio were significantly lower at T2-6 in group Ⅲthan in group Ⅰ . Conclusion TIVA with midazolam, propofol and remifentanil has less impact on immune function than inhalational and combined intravenous-inhalational anesthesia in patients with oral malignant tumor under-going elective radical operation.     

联合内皮祖细胞移植对小鼠骨髓移植预处理中肝脏内皮损伤的修复作用

目的 研究联合内皮祖细胞(EPC)移植对小鼠骨髓移植预处理中肝脏内皮损伤的修复作用.方法 将C57BL/6小鼠分为4组,每组10只.(1)正常对照组:小鼠不做任何处理,仅作为正常对照;(2)单纯照射组:单次给予全身照射(TBI)预处理,不进行骨髓移植;(3)单纯移植组:给予单纯照射组相同的TBI预处理,TBI后4 h内经小鼠尾静脉输注C57BL/6小鼠骨髓单个核细胞5×106/只;(4)联合移植组,小鼠的处理方式与单纯移植组相同,仅在骨髓移植的同时经尾静脉输注C57BL/6小鼠EPC 5×105/只.TBI后第2、4、7、14、21天,检测各组小鼠肝脏重量的变化情况,并于TBI后第4、7、14、21天对各组小鼠肝脏进行组织病理学检查.结果 单纯照射组、单纯移植组和联合移植组小鼠肝脏重量均于TBI后第2天开始明显增加,于第14天达到高峰,峰值分别为正常对照组的(1.65±0.15)倍(P＜0.05)、(1.61±0.06)倍(P＜0.05)和(1.11±0.4(0)倍(P＜0.05);以后均呈下降趋势,第21天时单纯照射组和单纯移植组肝脏重量仍明显高于正常对照组(P＜0.05),但联合移植组小鼠肝脏重量已完全恢复正常.组织病理学检查显示,单纯照射组小鼠肝窦内皮损伤明显,肝细胞水肿及严重的炎症细胞浸润,第7天时肝细胞水肿、坏死较前明显加重,几乎无存活的肝血窦内皮细胞;第14天时单纯移植组小鼠肝窦内皮损伤较前有所减轻,但到第21天时仍未恢复正常;联合移植组小鼠第7天时肝窦内皮及肝细胞水肿、坏死程度均较轻,到第14天时已基本恢复正常.结论 造血干细胞移植前的预处理会造成受者肝脏内皮损伤,且此损伤持续存在;移植时联合输注EPC能修复肝窦内皮的损伤.

Abstract：

Objective To study the repair function of united endothelial progenitor cells (EPC)transplantation on injured liver endothelium by bone marrow transplantation (BMT) conditioning.Methods C57BL/6 mice were divided into four groups randomly: normal control group, without any treatment; irradiation alone group, administered a total body irradiation(TBI) pretreatment, without BMT; (3) BMT alone group: C57BL/6 mice were infused with bone marrow mononuclearcells (MNC) 5 × 106/only through caudal vein not more than 4 h after the same TBI pretreatment as the irradiation alone group; united transplantation group: receiving the same way as the BMT alone group, but C57BL/6 mice were infused with EPC 5 × 105/only at the same time. Two, 4, 7, 14, and 21 days after the TBI, the changes of the liver weight were observed regularly. The histopathological examination of liver was done at the 4th, 7th, 14th, and 21st day after the TBI. Results In irradiation alone group, BMT alone group and united transplantation group the liver weight began to increase significantly on the day 2 and peaked at 14th day after the TBI, and the peaks were respectively (1.65±0. 15) times (P＜0. 05), (1.61 ±0.06) times (P＜0.05), and (1.11 ±0.40)times (P＜0. 05) of those in normal control group. At the day 14, the liver weight in irradiation alone group, BMT alone group and united transplantation group began to decrease, and on the day 21 the liver weight in united transplantation group had been completely restored to normal level, however the liver weight in irradiation alone group and BMT alone group were still significantly heavier than that in normal control group (P＜0. 05). Liver histopathological examination revealed that there were obvious sinusoidal endothelial cells (SEC) injury, hepatocyte edema and severe inflammatory cell infiltration in irradiation alone group, and on the day 7 the hepatocyte edema and necrosis were significantly worse than before, and almost no alive SEC were found. On the day 14 the injury of SEC in BMT alone group was lighter than before, but on the day 21 the injury had not returned to normal. On the day 7 the injury of SEC, hepatocyte edema and necrosis were alleviated in united transplantation group as compared with irradiation alone group and BMT alone group, and on the day 14 the injury had returned to normal basically. Conclusion The transplantation conditioning could damage recipient liver endothelium and the injury would persist, and united EPC infusion could repair the injured SEC following BMT.     

鞘内注射DREAM-shRNA对神经病理性痛大鼠脊髓背角p-CREB表达的影响

Objective To investigate the effects of intrathecal (IT) DREAM-short hairpin RNA (DREAM-shRNA) on expression of phosphorylated cyclic AMP response element binding protein (p-CREB) in the spinal dorsal horn in a rat model of neuropathic pain. Methods Adult male SD rats weighing 280-320 g were anesthetized with intraperitoneal 10% chloral hydrate. Neuropathic pain was induced by chronic constrictive injury (CCI) to sciatic nerve. IT catheters were placed according to the method described by Yaksh on 3rd day after CCI. Twenty-four rots in which IT catheter was successfully implanted were randomly divided into 4 groups (n = 6 each) : group Ⅰ sham operation (group S) ; group Ⅱ neuropathic pain (group NP) ; group Ⅲ RNA interference (group RNAi) and group Ⅳ blank vector (group BV). Lentivius with DREAM-shRNA 5 μl was injected IT in group RNAi, and blank vector 5 μl in group BV, and once a day for 7 days, starting from the day 8 after CCI. The mechanical pain threshold was measured at day 1 before CCI (T0 ,baseline) and day 7-14 after CCI (T1-8). The animals were killed on 15th day after CCI. The L4-6 lumbar segment of the spinal cord was removed for determination of the expression of green fluorescent protein (GFP) and p-CREB by immuno-fluorescent method.Results The mechanical pain threshold was significantly decreased as compared with the baseline at T0 in all 4 groups and returned to the baseline levels at T5-8 in group S and RNAi, but remained low in group NP and BY. The mechanical pain threshold was significantly lower after CCI/sham operation and significanty higher at T8 in group RNAi than in the other 3 groups. The expression of p-CREB in the spinal dorsal horn was up-regulated in group NP, RNAi and BV as compared with group S, and in group NP and BV as compared with group RNAi. The green fluorescence was observed in group RNAi but not in the other 3 groups. Conclusion IT DREAM-shRNA can ameliorate neuropathic pain in rats through inhibiting the expression of p-CREB in the spinal dorsal horn.     

Role of spinal cord TNF-α in the development of bone cancer pain in mice

Objective To investigate the role of spinal cord TNF-a in the development of bone cancer pain in mice. Methods Seventy-two 4-6 week old C3H/He mice weighing 18-25 g were randomly divided into 3 groups (n = 24 each) : group I sham operation (group S) ; group II bone cancer pain (group BCP) and group Ⅲ etanercept (group E). Bone cancer pain was induced by implantation of osteosarcoma NCTC 2472 cells into the intramedullary space of right femur in group II and Ⅲ . Group Ⅲ received intraperitoneal etanercept 100 μg at 3 days before and immediately before and day 3 and 6 after tumor cell inoculation. In group S culture medium α-MEM containing no cancer cell was injected instead. The paw withdrawal threshold to mechanical stimuli (PWMT) and paw withdrawal latency to thermal stimuli ( PWTL) were measured before inoculation (baseline) and at day 3, 5,7, 10, 14 after inoculation respectively. Eight animals were killed on the 7th, 10th, and 14th day after inoculation in each group. The spinal cords were removed and TNF-α mRNA expression in the spinal cord was determined by RT-PCR. Results Cancer pain was significantly attenuated by pretreatment with etanercept. The TNF-α mRNA expression in the spinal cord was significantly increased after inoculation and was significantly attenuated by pretreatment with etanercept in group Ⅲ . Conclusion Spinal cord TNF-a is involved in the development of bone cancer pain in mice.     

骨髓间充质干细胞体外对1型糖尿病大鼠淋巴细胞的免疫调节作用

目的 观察骨髓间充质干细胞(MSCs)体外对1型糖尿病(T1DM)大鼠淋巴细胞表型及增殖能力的影响,探讨其抑制淋巴细胞增殖的机制.方法 分离、培养和鉴定大鼠MSCs,噻唑蓝(MIT)比色法观察该细胞对淋巴细胞增殖能力的影响,应用流式细胞术分析MSCs对植物血凝素(PHA)作用下淋巴细胞凋亡,周期水平和CD4+CD25+调节性T细胞亚群(CD4+CD25+Tregs)比例的影响.结果 大鼠MSCs表型为CD29+、CD90+、CD106+、CD34-、CD45-,对PHA刺激的淋巴细胞增殖有抑制作用,以淋巴细胞:MSCs为1∶1时(C组)抑制作用最强;共培养体系中,大部分淋巴细胞处于G0/G1期;C组淋巴细胞凋亡水平(58.05±0.89)%显著高于对照组(43.35±0.86)%(P＜0.05);CD4+CD25+Tregs的比例C组(22.76±1.15)%显著高于对照组(5.80±0.68)%(P＜0.05).结论 MSCs体外可显著抑制PHA刺激的T1DM大鼠淋巴细胞的增殖,其机制与CD4+CD25+Tregs比例增高密切相关.

Abstract：

Objective To observe the effects of bone marrow mesenchymal stem cells (MSCs) on the lymphocytes of rats with type 1 diabetes mellitus (T1DM) in vitro, and investigate the inhibitory effect of MSCs on lymphocytes proliferation and the underlying mechanism. Methods MSCs were isolated from SD rats, cultured in vitro, purified and then identified by testing the phenotypes with flow cytometry (FCM). The third-generation MSCs were planted in 24-well plates. After treated with mitomycin C, MSCs were co-cultured for 72 h with the T1 DM rat's lymphocytes activated by phytohemagglutinin (PHA). The proliferation of lymphocyte was measured by methyl thiazol tetrazolium (MTT) method. FCM analysis was done to investigate the apoptosis, cell cycle and the proportion of CD4+ CD25+ regulatory T cells of the T1 DM rat's lymphocytes after co-cultivation. Results The phenotypes of MSCs from normal SD rats were CD29 + , CD90 +, CD106 + , CD34-, CD45 -. MSCs obviously inhibited the lymphocyte proliferation stimco-culture system, most of the lymphocytes were arrested at G0/G1 phase. The apoptosis rate of lymphocytes (58.05 ± 0. 89)% in group C was increased significantly as compared with the control group (43.35± 0.86 ) % ( P ＜ 0. 05 ) as well as the proportion of CD4 + CD25 + regulatory T cells (22.76 ± 1.15 ) % vs (5.80 ± 0. 68) %. Conclusion In vitro, MSCs can obviously inhibit the T1 DM rat' s lymphocytes proliferation stimulated with PHA via increasing the proportion of CD4 + CD25 + regulatory T cells.     

Role of spinal cord TNF-α in the development of bone cancer pain in mice

Objective To investigate the role of spinal cord TNF-a in the development of bone cancer pain in mice. Methods Seventy-two 4-6 week old C3H/He mice weighing 18-25 g were randomly divided into 3 groups (n = 24 each) : group I sham operation (group S) ; group II bone cancer pain (group BCP) and group Ⅲ etanercept (group E). Bone cancer pain was induced by implantation of osteosarcoma NCTC 2472 cells into the intramedullary space of right femur in group II and Ⅲ . Group Ⅲ received intraperitoneal etanercept 100 μg at 3 days before and immediately before and day 3 and 6 after tumor cell inoculation. In group S culture medium α-MEM containing no cancer cell was injected instead. The paw withdrawal threshold to mechanical stimuli (PWMT) and paw withdrawal latency to thermal stimuli ( PWTL) were measured before inoculation (baseline) and at day 3, 5,7, 10, 14 after inoculation respectively. Eight animals were killed on the 7th, 10th, and 14th day after inoculation in each group. The spinal cords were removed and TNF-α mRNA expression in the spinal cord was determined by RT-PCR. Results Cancer pain was significantly attenuated by pretreatment with etanercept. The TNF-α mRNA expression in the spinal cord was significantly increased after inoculation and was significantly attenuated by pretreatment with etanercept in group Ⅲ . Conclusion Spinal cord TNF-a is involved in the development of bone cancer pain in mice.     

肾移植术后早期严重肺部感染患者外周血CD4+T淋巴细胞计数的临床意义

Objective To explore the clinical implication of peripheral blood CD4+ T-cell counts in renal allograft recipients with severe pulmonary infection in the early stage after kidney transplantation. Methods From February 2007 to June 2008, we investigated the variation of peripheral blood CD4+ T-cell counts using flow cytometry in 28 cases of severe pulmonary infection 1 ～6 months after kidney transplantation (infection group), and 30 cases (control group) randomly selected that had stable situation and normal kidney function in the same period. Results CD4+ T-cell counts on the day of admission in infection group were significantly lower than in control group (184.1 ±117.5/μl vs. 518.6±232.7/μl, P<0.01 ). In infection group, 5 patients died and 4 of them had obviously declining trends of CD4+ T-cell counts during hospitalization course. Comparing to the day of admission, CD4+ T-cell counts of those survivors in infection group were significantly increased (184.1±117.5/μl vs. 406.5±163.9/μl, P<0.01) when infections were controlled. ROC analysis showed that CD4+ T-cell counts on the day of admission were accurate enough to identify who were susceptible to infection. In detail, the area under the curve (AUC) was 94.9% (P<0.01). CD4+ T-cell counts of 220/μl displayed the minimal misdiagnosis rate. Conclusions The variations of CD4+ T-cell counts are correlated to onset and progression of severe pulmonary infection in the early stage after kidney transplantation. Those who had CD4+ T-cell counts lower than 220/μl were at high risk of pulmonary infection. Direct measure and dynamic analysis of CD4+ T-cell subset have an important role in optimizing treatment and predicting prognosis of severe pulmonary infection in the early stage after kidney transplantation.     

肾移植术后早期严重肺部感染患者外周血CD4+T淋巴细胞计数的临床意义

Objective To explore the clinical implication of peripheral blood CD4+ T-cell counts in renal allograft recipients with severe pulmonary infection in the early stage after kidney transplantation. Methods From February 2007 to June 2008, we investigated the variation of peripheral blood CD4+ T-cell counts using flow cytometry in 28 cases of severe pulmonary infection 1 ～6 months after kidney transplantation (infection group), and 30 cases (control group) randomly selected that had stable situation and normal kidney function in the same period. Results CD4+ T-cell counts on the day of admission in infection group were significantly lower than in control group (184.1 ±117.5/μl vs. 518.6±232.7/μl, P<0.01 ). In infection group, 5 patients died and 4 of them had obviously declining trends of CD4+ T-cell counts during hospitalization course. Comparing to the day of admission, CD4+ T-cell counts of those survivors in infection group were significantly increased (184.1±117.5/μl vs. 406.5±163.9/μl, P<0.01) when infections were controlled. ROC analysis showed that CD4+ T-cell counts on the day of admission were accurate enough to identify who were susceptible to infection. In detail, the area under the curve (AUC) was 94.9% (P<0.01). CD4+ T-cell counts of 220/μl displayed the minimal misdiagnosis rate. Conclusions The variations of CD4+ T-cell counts are correlated to onset and progression of severe pulmonary infection in the early stage after kidney transplantation. Those who had CD4+ T-cell counts lower than 220/μl were at high risk of pulmonary infection. Direct measure and dynamic analysis of CD4+ T-cell subset have an important role in optimizing treatment and predicting prognosis of severe pulmonary infection in the early stage after kidney transplantation.     

CTLA4-Ig基因对大鼠肝移植后免疫细胞浸润及细胞凋亡的影响

目的研究腺病毒介导的细胞毒性T淋巴细胞相关抗原4-Ig(cytolyticT-lymphocyteassociatedantigen4-Ig,CTLA4-Ig)基因对大鼠肝移植后移植物中免疫细胞浸润和细胞凋亡的影响。方法将大鼠原位肝移植模型分为排斥对照组、环孢素A(CsA)组和CTLA4-Ig组。分别于术后1,3,5,7,12d,用免疫组织化学法和缺口末端标记技术(TUNEL法)分别测定移植物中CTLA4-Ig基因的表达和巨噬细胞、CD8 T细胞浸润及细胞凋亡,并以病理形态学变化作参照。结果静脉注射重组CTLA4-Ig基因腺病毒7d后,大鼠肝脏CTLA4-Ig稳定表达,在肝移植60d后仍呈阳性;CTLA4-Ig组汇管区巨噬细胞、CD8 T细胞浸润明显较排斥对照组少;细胞凋亡指数在术后3、5和7d明显低于排斥对照组(P<0·01),汇管区巨噬细胞、CD8 T细胞浸润数和凋亡指数与排斥反应分级均显著相关。结论重组CTLA4-Ig基因腺病毒经静脉一次给药后能在大鼠肝脏稳定表达,并通过抑制移植物中免疫细胞浸润及移植物细胞凋亡,抑制移植后急性排斥反应。     

细胞毒性T淋巴细胞相关抗原4诱导新鲜异体骨移植免疫耐受的实验研究

目的　利用外源性细胞毒性T淋巴细胞相关抗原4 ( cytotoxic T lymphocyte- associated antigen 4immuno globlin,CTL A4 - Ig)阻断T细胞反应的第2信号,诱导新鲜同种异体骨移植免疫耐受。　方法　采用近交系小鼠BAL B/C 6 6只作为骨移植的受体,进行异位肌袋一次及第2次骨移植。一次移植分为3组,每组18只,一组移植C5 7BL/6小鼠骨骼,注射L6 (对照品) ,为AL组;一组移植C5 7BL/6小鼠骨骼,注射CTL A4 - Ig,为AC组;一组移植同系BAL B/C小鼠骨骼,注射PBS缓冲液,为AB组。在第2、4及6周,进行血淋巴细胞亚群分析,血清抗体测定,供体细胞及抗原二次刺激实验及组织学观察。另取12只BAL B/C小鼠,进行第2次移植实验,供体为C5 7BL/6小鼠的骨骼,注射CTL A4 - Ig后2周,分别移植C5 7BL/6 ( BC组)和C3H( BH组)小鼠的骨骼,检测产生免疫耐受的特异性。　结果　AL组与AB组比较,产生了强烈的免疫排斥反应,移植后2、4及6周,CD4 T细胞的增殖明显增加( P<0 .0 5 ) ,血清抗体明显增多( P<0 .0 5 ) ,供体细胞及骨抗原二次刺激的细胞增殖也明显增加( P<0 .0 5 ) ;AC组免疫排斥反应较轻,在CD4 T细胞的增殖、血清抗体及二次刺激的细胞增殖方面均与AB组相似( P>0 .0 5 ) ;组织学观察也显示,异体骨周围的淋巴细胞浸润消失,软骨诱导及新骨形     

阻断第二信号诱导骨移植后对淋巴细胞亚群的抑制作用

目的　研究细胞毒性T淋巴细胞相关抗原(CTLA4Ig)对新鲜同种异体骨移植后血淋巴细胞亚群的影响。方法　在组织相容性抗原完全不同的两组小鼠间进行异位骨移植,于移植后第0、1天,腹腔注射CTLA4Ig100μg,分别于术后2、4、6周取受体外周血,利用流式细胞仪技术测定血淋巴细胞亚群CD     

CTLA4-Ig基因修饰骨髓间充质干细胞抑制大鼠肝移植排斥反应

目的探讨细胞毒性T淋巴细胞相关抗原4免疫球蛋白(CTLA4-Ig)基因转染骨髓间充质干细胞(MSC)在抑制大鼠原位肝移植排斥反应的作用及机制。方法采用重组腺病毒(Ad)5-CTLA4-Ig转染MSC。转染72 h后,提取细胞总蛋白,采用蛋白质印迹法检测转染后MSC中CTLA4-Ig的蛋白表达。采用细胞计数试剂盒(CCK)-8方法检测未转染和转染后的MSC对外周血淋巴细胞增殖的抑制作用。以雄性Lewis大鼠为供体(40只);以雄性Brown Norway(BN)大鼠为受体(40只)。采用改良的Kamada两袖套法进行原位肝移植,建立大鼠原位肝移植急性排斥反应模型。40只受体大鼠随机分为4组,每组10只。其中对照组(A组),于肝移植时门静脉输注生理盐水;MSC治疗组(B组),于肝移植时门静脉输注MSC;转基因MSC治疗组(C组),于肝移植时门静脉输注转基因MSC;免疫抑制剂治疗组(D组),于肝移植时门静脉输注生理盐水,术后即给予环孢素(CsA)1.5 mg/(kg·d)肌内注射,连续8 d。每组大鼠取5只观察生存情况。每组其余5只于术后第9日处死,检测外周血细胞因子白细胞介素(IL)-2、IL-4、干扰素(IFN)-γ水平,光学显微镜下观察肝组织病理学变化和排斥反应程度。结果重组Ad5-CTLA4-Ig转染MSC 72 h后,蛋白质印迹法可检测到转染后的MSC中有CTLA4-Ig的蛋白表达。当未转染的MSC∶外周血单核细胞比例为1∶10、1∶20时,MSC抑制淋巴细胞增殖的作用分别为85.60%、76.69%。重组Ad5-CTLA4-Ig转染MSC 72 h后,在相同的数量比下,其抑制淋巴细胞增殖的作用分别为90.50%、84.20%;与未转染的MSC比较,转染后抑制淋巴细胞增殖的作用增强(P0.05)。A、B、C、D组大鼠肝移植术后存活时间分别为(13±3),(41±6),(90±15),(102±18)d。A、B、C组的大鼠术后存活时间比较差异有统计学意义(P0.05),C组和D组的大鼠术后存活时间比较差异无统计学意义(P0.05)。与A组比较,B组和C组的IL-4水平明显升高;与B组比较,C组的IL-4水平明显升高,差异均有统计学意义(均为P0.05);C组和D组的IL-4水平比较,差异无统计学意义(P0.05)。与A组比较,B组和C组的IL-2、IFN-γ水平明显降低,C组的IL-2、IFN-γ水平亦低于B组,差异均有统计学意义(均为P0.05),C组和D组的IL-2、IFN-γ水平比较,差异无统计学意义(P0.05)。大鼠肝组织病理检查结果显示,A组移植肝发生重度排斥反应,B组移植肝亦发生排斥反应,但与A组比较程度较轻。C组与D组移植肝有轻度排斥反应。结论重组Ad-CTLA4-Ig转染MSC可抑制肝移植排斥反应,其效果优于MSC单独应用。     

肝细胞移植治疗急性肝功能衰竭的免疫排斥研究

目的研究肝细胞腹腔移植治疗急性肝功能衰竭的免疫排斥反应。方法用胶原酶灌注法分离幼猪及BALB/c和C57BL/6小鼠肝细胞;用腹腔注射CCl4的方法建立C57BL/6急性肝功能衰竭模型;将实验动物分为同基因移植组、同种异基因移植组和异种移植组。各组动物肝细胞移植入急性肝功能衰竭小鼠腹腔内,观察受体小鼠存活情况,比较各组动物T细胞亚群、免疫球蛋白水平和细胞因子的变化。结果①受体小鼠存活情况:同基因移植组为8/10,同种异基因移植组为6/10,异种移植组为3/10,同基因移植组和同种异基因移植组受体的生存情况明显好于异种移植组(P<0.05)。②T细胞亚群变化:移植后7 d内,同基因移植组外周血中CD4+和CD8+T细胞均无明显变化,同种异基因移植组CD4+T细胞于移植后3 d时达高峰(P<0.05),而CD8+T细胞7 d内无明显变化,异种移植组CD4+和CD8+T细胞移植后7 d内均明显升高,且于移植后3 d时达高峰(P<0.05)。③免疫球蛋白水平:同基因移植组血清免疫球蛋白IgM和IgG水平在移植后0.5、1和3 d时未见明显变化,同种异基因移植组和异种移植组的IgM在移植后1 d时达高峰(P<0.05),而IgG在移植后3 d时达高峰(P<0.05)。④血清细胞因子水平:移植后7 d,同种异基因移植组和异种移植组的IFN-γ、TNF-α及IL-2血清浓度明显高于同基因移植组(P<0.05);异种移植组的IL-6血清浓度明显高于同基因移植组和同种异基因移植组(P<0.05)。结论无论同种还是异种肝细胞移植,初期均发生了强烈的CD4+及CD8+T细胞参与的细胞免疫和较强体液免疫反应。     

骨髓输注联合阻断共刺激通路延长大鼠皮肤移植物的存活时间

目的 探讨骨髓输注联合阻断共刺激通路对大鼠移植皮肤存活的影响及可能机制.方法 以Lewis大鼠为受者,皮肤移植前经尾静脉输注供者(BN大鼠)骨髓细胞2×108 个,并于骨髓输注当天、输注后第2、4、6及8天腹腔注射抗CD25单克隆抗体,同时按照分组要求腹腔注射细胞毒性T淋巴细胞相关抗原4融合蛋白(CTLA4Ig组)、抗CD154单克隆抗体(抗CD154单抗组)以及CTLA4Ig和抗CD154单克隆抗体(联合处理组),于骨髓输注后第8天移植BN大鼠的皮肤.另以仅行皮肤移植者为对照(对照组).观察各组移植物抗宿主病(GVHD)的发生情况、外周血中供者细胞嵌合率、T淋巴细胞凋亡率及移植皮肤的存活时间.结果 各组均未观察到GVHD的发生.骨髓输注后第7天即可在CTLA4Ig组、抗CD154单抗组及联合处理组观察到嵌合现象,至第21天时,嵌合率仍维持于一定水平,联合处理组明显高于其它三组(P＜0.01).骨髓输注后第7及21天,CTLA4Ig组、抗CD154单抗组及联合处理组间两两比较,T淋巴细胞凋亡率的差异无统计学意义,但均显著高于对照组(P＜0.05,P＜0.01).CTLA4Ig组、抗CD154单抗组及联合处理组移植皮肤的存活时间显著长于对照组(P＜0.01),联合处理组移植皮肤存活时间为(16.7±3.1)d,明显长于CTLA4Ig组和抗CD154单抗组(P＜0.05).结论 骨髓输注联合阻断共刺激通路能够延长移植皮肤存活时间,其机理可能与诱导嵌合和T淋巴细胞凋亡有关.     

腺病毒介导的hCTLA4-Ig和FasL基因转移诱导大鼠同种异体肾移植长期存活的作用

目的 探讨腺病毒介导hCTLA4-Ig和FasL基因转移延长异基因大鼠肾移植物存活时间及其相关机制.方法 供、受者分别为SD系大鼠和Wistar系大鼠,建立大鼠肾移植模型.实验分为4组:对照组、空载病毒Ad-EGFP处理组、Ad-CTLA4-Ig处理组、Ad-CTLA4-Ig+Ad-FasL处理组.对照组不予处理,其他各组在供肾冷保存时,经供肾动脉灌注(1×10~9-5 ×10~9)PFU/ml Ad-EGFP、Ad-CTLA4-Ig、Ad-CTLA4-Ig+Ad-FasL.术后比较各组的存活时间、移植肾组织学、肾功能和免疫组织化学等的改变.结果 肾移植受者平均存活时间Ad-CTLA4-Ig+Ad-FasL处理组(64.67±6.41)d、Ad-CTLA4-Ig处理组(31.33 ±6.77)d与对照组(8.17±1.17)d,空载病毒Ad-EGFP处理组(8.00±1.55)d比较明显延长(P均＜0.01),并且Ad-CTLA4-Ig+Ad-FasL处理组优于Ad-CTLA4-Ig处理组(P＜0.01);对照组和空载病毒Ad-EGFP处理组术后血清肌酐浓度迅速上升,其余两组移植肾功能保持稳定.结论 Ad-CTLA4一Ig和Ad-FasL介导的基因转移能够诱导异基因肾移植物长期存活;该效应为供体特异性,与调节性T细胞和同种反应性T细胞的删除有关.     

基因转移CTLA4-Ig和抗CD154抗体在异种胰岛移植排斥反应中的作用

目的 探讨基因转移细胞毒性T细胞相关抗原4免疫球蛋白(CTLA4-Ig)和抗T细胞分化群154(CD154)抗体在异种胰岛移植排斥反应中的作用及机理.方法 建立人-大鼠异种胰岛移植模型,用携带CTLA4-Ig基因的重组腺病毒感染移植胰岛细胞,并用抗CD154抗体进行治疗,观察糖尿病大鼠胰岛移植后血糖变化、生存情况及移植物病理形态学改变,检测移植物CTLA4-Ig、胰岛素的表达和移植大鼠白细胞介素2(IL-2)、肿瘤坏死因子(TNF)-α的水平变化.结果 (1)糖尿病大鼠移植后2 d血糖降至正常,对照组血糖平均在移植后8 d升高,抗体治疗组、转染组和联合治疗组血糖分别在18、25和36 d升高.(2)对照组、抗体治疗组、转染组和联合治疗组的移植物存活时间分别为(10.0±2.1)d、(22.0±8.2)d、(28.0±6.5)d和(37.0±9.3)d,各组间比较差异有统计学意义(P＜0.05);移植大鼠生存时间分别为(21.0±5.7)d、(35.0±6.5)d、(48.0±8.5)d和(65.0 ±12.5)d,各组间比较差异有统计学意义(P＜0.05).(3)对照组在移植后1周内,IL-2、TNF-α的水平均急剧上升,较移植前显著升高(P＜0.01).(4)各治疗组移植物见成片的胰岛细胞团,未见淋巴细胞浸润,转染组和联合治疗组移植物可见CTLA4-Ig和胰岛素的表达.结论 基因转移CTLA4-Ig和抗CD154抗体均可抑制异种胰岛移植排斥反应,二者联合效果优于单独使用.     

Notch2基因在大鼠肝移植术后胆管损伤中的表达及其意义

目的 探讨大鼠肝移植术后损伤胆管中Notch 2基因的表达及其意义.方法 将96只健康雄性SD大鼠随机分为3组,A组:未手术组;B组:假手术组;C组:采用改良"二袖套"法建立大鼠肝移植.于术后1、7、14、28 d检测总胆红素(TBIL)、γ-GT、谷丙转氨酶(ALT),同时处死大鼠取肝组织,苏木素-伊红(HE)染色观察病理改变,免疫组织化学、Western blot检测Notch 2基因的表达.结果 大鼠肝移植术后TBIL(32.05±3.28)μmol/L,γ-GT(118.30±17.40)μmol/L明显升高(P＜0.05),HE染色显示损伤胆管增生(14 d),部分胆管破坏、消失(28 d).免疫组织化学显示,A和B组胆管细胞Notch 2染色阴性,C组胆管细胞Notch 2染色强阳性,Western blot检测显示Notch 2蛋白在C组肝组织中呈逐渐升高的强表达(P＜0.05).结论 大鼠肝移植术后随胆管损伤加重,Notch 2基因表达显著升高,该基因可能参与了胆管损伤.

Abstract：

Objective To investigate the expression and clinical significance of Notch 2 expression in postoperative injured bile duct of rat liver transplantation. Methods Ninety-six healthy male SD rats were divided into three groups randomly: un-operation group (A), Sham operation group ( B), and group of liver transplantation model by modified "two-muff" techniques (C). Total bilirubin (TBIL), γ-GT and alanine aminotransferase (ALT) were determined at 1st, 7th, 14th, 28th day after transplantation and the expression of Notch 2 in the liver was detected by immunohistochemisty and Western blotting at the same time. Results TBIL (32. 05 ±3. 28) μmol/L, and γ-GT ( 118.30 ± 17.40) μmol/L were increased obviously after liver transplantation ( P ＜ 0. 05 ). Hematoxylin-eosin ( HE ) staining showed the injured bile ducts obviously proliferated after 14 days, and partly posttransplanted bile ducts were destructed and vanished after 28 days. Immunohistochemisty revealed that Notch 2 staining in bile ducts was negative in groups A and B, but Notch 2 staining in hyperplasitic bile ducts was strong positive in group C. Western blotting confirmed that Notch 2 protein was upregulated gradually in the damaged bile ducts after rat liver transplantation (P ＜ 0. 05 ). Conclusion Bile duct injury is aggravated after rat liver transplantation, and Notch 2 gene expression is up-regulated remarkably.     

CTLA4Ig基因对大鼠胰岛移植后排斥反应的治疗作用

目的　研究CTLA4Ig基因在糖尿病大鼠体内表达及其产物对胰岛移植物存活的作用。方法　利用Lipofectin载体包裹CTLA4IgcDNA质粒后转染鼠胰岛和肌肉细胞 ,检测移植后CT LA4Ig表达和T淋巴细胞转化率。结果　胰岛移植术后 7dT淋巴细胞转化试验 ,实验组 (A组 )和对照组 (B组 )每分钟脉冲数 (cpm)分别为175 .7± 98.2 ,2 5 4.4± 116 .3 ,两组比较差异显著 (P <0 .0 5 )。A组胰岛移植第 7d ,2只大鼠血清CTLA4Ig呈阳性 (阳性率 2 0 % )。A、B两组胰岛移植后血糖维持正常时间分别为 (14.8± 12 .3)d和 (3 .6± 5 .1)d ,两组比较差异显著 (P <0 .0 5 )。A、B两组大鼠平均存活时间分别为 (2 4.0± 10 .8)d和 (10 .8± 4.8)d ,两组比较 ,差异有极显著性 (P <0 .0 1)。结论　脂质体包裹的CTLA4IgcDNA转染肌细胞和胰岛细胞 ,可以在受体大鼠胰岛细胞或肌肉组织中表达 ,其表达产物可使胰岛移植物和受体鼠存活时间明显延长 ,抑制细胞免疫活性 ,发挥其治疗排斥反应的作用