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.     

AG1478对U87细胞增殖、细胞周期和凋亡的影响

目的 观察表皮生长因子受体抑制剂Tyrphostin AG1478对人脑胶质瘤U87细胞增殖、细胞周期和细胞凋亡的影响.方法 噻唑蓝(MTT)比色法检测不同浓度Tyrphostin AG1478作用于体外培养的人脑胶质瘤U87细胞24 h后的细胞生存率,流式细胞仪检测不同浓度Tyrphostin AG1478作用于体外培养的人脑胶质瘤U87细胞24 h后细胞周期分布和细胞凋亡.结果 5、10、15、20 μmol/L的Tyrphostin AG1478作用人脑胶质瘤U87细胞24 h后细胞生存率分别为(7 8.93±11.95)%、(46.42±4.12)%、(42.13±7.54)%和(37.48±4.69)%;0、10、20 μmol/L的Tyrphostin AG1478作用人脑胶质瘤U87细胞24 h后细胞凋亡率分别为(10.19±3.15)%、(32.02±1.60)%和(54.35±2.80)%;0、10、20 μmol/L的Tyrphostin AG1478作用人脑胶质瘤U87细胞24 h后细胞主要分布在G0～G1期,分别为(51.20±1.21)%、(78.61±1.57)%和(82.73±0.77)%,实验组与对照组比较差异均有统计学意义(P＜0.05).结论 Tyrphostin AG1478抑制体外人脑胶质瘤细胞增殖,阻滞细胞周期在G0～G1期,诱导细胞凋亡,均呈浓度依赖性.

Abstract：

Objective To investigate the effects of Tyrphostin AG1478 on glioma U87 cells proliferation, cells cycle and apoptosis. Methods U87 cells were cultured for 24 h in the medium which contained AG1478 with different concentrations (0, 5, 10, 15, 20 μmol/L). The methyl thiazolyl tetrazolium(MTT) assay was used to detect the survival rate, and the cells cycle and apoptosis of the cells were examined by using flow cytometry. Results The cells proliferation was obviously inhibited by AG1478 in a dose-dependent manner. The survival rate of the cells in 5, 10, 15, 20 μmol/L AG1478 groups was (78.93 ±11.95)%, (46.42 ±4. 12)%, (42. 13 ±7.54)% and (37.48 ±4.69)% respectively, which was all significantly higher than that in 0 μ mol/L AG1478 group (P ＜0. 05 ). The apoptotic rate in 10, 25μmol/L AG1478 groups was (32.02 ± 1.60)% and (54. 35 ± 2. 80)% respectively, significantly higher than that in 0 μmol/L AG1478 group ( P ＜ 0. 05 ). The cells cycle was obviously inhibited by AG1478 in a dose-dependent manner. The percentage of cells treated with 10 and 20 μmol/L AG1478 in Go-G1 phase was ( 78. 61 ± 1.57 ) % and ( 82. 73 ± 0. 77 ) % respectively, significantly higher than that in 0 μmol/L AG1478 group (P ＜ 0. 05 ). Conclusion The growth inhibition of glioma U87 cells caused by AG1478 may be associated with apoptotsis induction and the G0-G1 arrest. AG1478 was expected to become a new anti-tumor drug in human glioma.     

人内皮型一氧化氮合成酶基因转染抑制人血管平滑肌细胞增殖及其机制

目的 探讨人内皮型一氧化氮合成酶基因(heNOS)转染抑制人血管平滑肌细胞(HVSMCs)增殖的机制.方法 以AdCMV-heNOS病毒感染复数分别为50、150、250、300、450 MOI,转染HVSMCs,放射免疫法检测转染HVSMCs中的环一磷酸鸟苷(cGMP)的表达变化;Western blot检测血管平滑肌细胞中p21、p27蛋白的变化,流式细胞术分析对细胞周期分布及凋亡的影响.结果　(1)转染120 h,A570值分别为1.410±0.081、1.357±0.150、1.303±0.311、0.995±0.248、0.731±0.101,其中感染复数300 MOI明显稳定抑制血管平滑肌细胞的增殖.(2)转染72 h,未转染组、Ad-LacZ转染组、Ad-heNOS转染组cGMP的含量分别为(7.91±0.39)、(8.36±0.34)、(12.89±2.06)μnol/L,差异有统计学意义(P＜0.01).(3)转染48 h,Westem blot检测转染组p27、p21表达明显上调,而未转染组虽也有p21、p27的表达,但两组差异有统计学意义(P＜0.05).(4)无血清转染48 h后血清刺激24 h,未转染组、Ad-LacZ转染组、Ad-heNOS转染组G0/G1期分别为(64.23±1.58)%、(64.96±1.36)%、(76.03±2.27)%,差异有统计学意义(P＜0.01).(5)转染3 d,第1天,未转染组、Ad-LacZ转染组、Ad-heNOS转染组细胞凋亡率分别为(4.70±0.56)%、(5.53±0.74)%、(8.53±1.06)%,差异无统计学意义(P＞0.05).第3天,细胞凋亡率分别为(5.40±0.62)%、(8.30±0.80)%、(9.30±0.90)%,差异无统计学意义(P＞0.05).结论　heNOS基因转染HVSMCs抑制细胞增殖,通过p21、p27上调导致细胞周期的阻滞,无诱导细胞凋亡.

Abstract：

Objectiye To invesigate the effect of human endothelial nitric oxide synthase (heNOS ) gene transfer on the proliferation of in vitro cultured human vascular smooth muscle cells (HVSMCs)and the mechanism. Methods The HVSMCs were transfeced with multiplicity of infection (MOI) of 50,150,250, 300,450. cGMP was measured by radioimmunoassay in HVSMCs. The expression levels of p21 and p27 were detected by Western blotting. Cell cycle and apoptosis were assayed by flow cytometry. Results (1) At 120th h, the A570values were respectively 1.410±0.081, 1.357 ±-0. 150, 1.303±0.311,0. 995 ±0. 248 and 0. 731 ±0. 101 at MOI of 50, 150,250,300,450. The proliferation of HVSMCs was significantly and stably inhibited with MOI 300 of AdCMV-heNOS; (2) Af72nd h after the gene transfer,cGMP levels were increased in heNOS-transduced ( 12. 89 ±2. 06) compared to LacZ- (8.36 ±0. 34) and non-tranduced (7. 91 ± 0. 39) cells ( P ＜ 0. 01 ); (3) At 48th h after the gene transfer, the expression of heNOS in HVSMCs up-regulated p21 and p27 (P＜0. 05); (4) After 48 h tronsfected with sersuln-depriveol and for 24 h serum stimulation, the cell cycle was significantly arrested in G0/G1 phase in heNOStransduced group, and G0/G1 was respectively ( 64. 23 ± 1.58 ) %, ( 64. 96 ± 1.36 ) %, ( 76. 03 ±2. 27 ) % in non-, LacZ- and heNOS-transduced cells; ( 5 ) At first and third day afer the gene transfer,there was no increase in apoptosis at the first day in all transduced cells ( P ＞ 0. 05 ). Conclusion Adenovirus-mediated heNOS gene transfer to HVSMCs inhibits cell proliferation via up-regulation of p21 and p27 resulting in a delay in cell progression not apoptosis.     

重组人内皮抑素对结肠癌细胞SW620增殖和凋亡的影响

目的 观察重组人内皮抑素对结肠癌细胞SW620增殖和凋亡的影响.方法 在培养液中加入梯度浓度的重组人内皮抑素(0.1、0.5、1.0,5.0、10.0、20.0mg/L)分别作用24、48、72 h,采用噻唑蓝(MTT)比色法检测细胞生长,流式细胞术检测细胞周期和凋亡,扫描及透射电镜观察细胞超微结构的变化.结果 内皮抑素对SW620细胞的增殖有抑制作用.其中,作用48 h的抑制率分别为13.46%、15.38%、20.82%、24.71%、30.12%和37.44%,呈剂量依赖关系.给药后24 h和48 h,内皮抑素组细胞G0/G1期比例增高,而s期细胞比例降低(P＜0.05).72h两组差异无统计学意义(P＞0.05).24 h和48 h的凋亡率分别为(1.430±0.145)%和(1.760±0.054)%,均高于对照组(0.670±0.181)%和(1.260±0.138)%(P＜0.05).透射电子显微镜下,内皮抑素组细胞微绒毛减少,染色质厚薄不均,位于核膜下.结论 重组内皮抑素可以在体外直接抑制结肠癌细胞的增殖,诱导凋亡,影响细胞骨架等结构.

Abstract：

Objective To investigate the effect of endostatin on the proliferation of colon cancer SW620 cells. Methods The SW620 cells were treated by recombinant human endostatin(0. l ,0. 5,1.0,5.0,10.0,20.0 mg/L). The effect of endostatin on the proliferation, cell cycle, apoptosis and ultrastructure of SW620 cells were examined after 24,48 and 72 h. Results The proliferation of SW620 cells were inhibited by endostatin compared with the control group. The inhibiting rates were 13. 46% ,15. 38% ,20. 82% ,24. 71% ,30. 12% and 37.44% .respectively (P ＜0.05). Endostatin arrested SW620 cells at G0/G1 phase (P ＜ 0. 05); The apoptotic rates were (1.430 ± 0. 145) % and (1. 760 ± 0. 054) % in endostatin group after 24 and 48 h, respectively. This was a significant increase when compared with the control group after 24 and 48 h (0. 670 ±0. 181)% and (1.260 ±0. 138)% (P＜0. 05). Under TEM,both the number of protrusions and microfilaments of SW620 cells in the endostatin group decreased. Conclusion Recombination human endostatin inhibits the proliferation of SW620 cells,induce the apoptosis,and changes the skeleton of SW620 cells directly.     

冠心病与内皮型一氧化氮合酶基因多态性的关系

目的 探讨内皮型一氧化氮合酶(eNOS)基因-786T/C,4a4b,894G/T等3个多态性位点与冠心病(CAD)发病相关.方法 对146例中国汉族人群CAD患者和113例正常对照进行遗传学分析,应用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)和PCR技术分析2个SNP位点即-786T/C和894G/T,以及1个VNTR位点4a4b,检测各位点基因型和等位基因频率,采用HaploView 4.0及SPSS 13.0软件经x2检验比较两组间各位点基因型及等位基因频率的差异.结果 CAD组中eNOS基因-786T/C位点CC基因型频率为2.0%,4a4b位点4a/4a基因型频率为5.4%,对照组eNOS基因-786T/C位点CC基因型频率为0.0%,4a4b位点4a/4a基因型频率为0.9%,差异有统计学意义(P＜0.05).CAD组和对照组在eNOS基因的894G/T位点等位基因和基因型频率分布差异均无统计学意义(P＞0.05).结论 eNOS基因-786T/C和4a4b多态性与中国汉族人群CAD存在关联,C等位基因和4a等位基因可能是CAD发病的危险因素.eNOS基因894G/T位点与CAD发病无明显相关.

Abstract：

Objective To investigate the relationship between the 3 polymorphisms ( -786T/C,4a4b,894G/T) in endothelial nitric oxide synthase (eNOS) gene and coronary artery disease (CAD).Methods 146 patients with CAD and 113 healthy unrelated individuals in a Chinese Han nation were involved.The genotype and allele frequency of each polymorphism of the eNOS gene in these patients and normal controls were examined by using polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) or PCR methods.Genotypes and allele frequency were analyzed by HaploView 4.0 and SPSS13.0 software.Results The frequency of CC genotype of the -786T/C was 2.0%,and that of 4a/4a genotype of the 4a4b was 5.4% in CAD.The frequency of CC genotype of the - 786T/C was 0.0%,and that of 4a/4a genotype of the 4a4b was 0.9% in controls ( P＜0.05 ).There were significant differences in both allele and genotype frequency of -786T/C and 4a4b between CDA group and control group.Between patients with CAD and controls,there were no significant differences in the frequency of the genotypes and alleles of the 894G/T in eNOS gene.Conclusion The - 786T/C and 4a4b polymorphisms of eNOS gene may be associated with CAD.The individuals with C allele of - 786T/C and 4a allele of 4a4b are susceptible to CAD.There is no significant correlation between 894G/T polymorphism in eNOS gene and CAD.     

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.     

不同时点应用维拉帕米对心肌缺血再灌注损伤的保护作用

目的 观察不同时点应用维拉帕米(VP)对大鼠心肌缺血再灌注损伤的保护作用,并探讨其心肌保护的作用机制.方法 建立大鼠心肌缺血再灌注模型,将18只雄性SD大鼠随机分为3组,每组6只.Verapamil-1组于结扎前10 min开始泵入维拉帕米稀释液(0.25 mg/kg),Verapamil-2组于再灌前10 min开始泵入维拉帕米稀释液(0.25 mg/kg),IR组于结扎前10 min开始泵人生理盐水(2ml/kg).再灌注后60min处死大鼠.检测血清肌钙蛋白T(cTnT)含量、缺血区心肌组织Caspase-3表达水平;组织形态学分析心肌损伤程度.结果　Verapamil-1组血清cTnT含量、心肌组织Caspase-3表达量(4.60±1.12)ng/L,(39.51±5.01)%较IR组(7.70±1.31)ng/L,(51.10±5.30)%和Verapamil-2组(7.23±1.03)ng/L,(49.35±4.95)%明显降低,差异有统计学意义(P＜0.05);Verapamil-2组血清cTnT含量、心肌组织Caspase-3表达水平和IR组比较差异无统计学意义(P＞0.05);Verapamil-1组心肌组织形态学损伤程度较IR组和Verapamil-2明显降低,差异有统计学意义(P＜0.05);Verapamil-2组心肌组织形态学损伤程度和IR组比较差异无统计学意义(P＞0.05).结论　结扎前10 min开始给予维拉帕米对心肌缺血再灌注损伤有明显保护作用,再灌注前10 min开始给予维拉帕米对心肌缺血再灌注损伤无保护作用.

Abstract：

Objective To investigate the effect of verapamil administered at different time points on myocardial ischemia-reperfusion injury in rats, and explore the mechanism of myocardial protection.Methods The model of myocardial ischemia reperfusion in rats was established and 18 male SD rats were randomly divided into 3 groups,n =6 each. Verapamil dilution (0. 25 mg/kg) was pumped into verapamil1 group 10 min before ischemia, and verapamil dilution (0. 25 mg/kg) was pumped into verapamil-2 group 10 min before reperfusion. Normal saline (2 ml/kg) was pumped into IR group 10 min before ischemia.Rats were killed 60 min after reperfusion. The levels of serum cardiac Troponin T (cTnT) and the expression of myocardial Caspase-3 were evaluated. Histomorphological methods were used to analyze the extent of myocardial injury. Results The levels of serum cTnT and the expression of myocardial Caspase-3 in verapamil-1 group (4.60 ± 1.12) ng/L, (39.51 ±5.01)% were significantly lower than those in IR group (7. 70 ± 1.31 ) ng/L, (51.10 ±5. 30)% and verapamil-2 group (7. 23 ± 1.03) ng/L, (49. 35 ±4. 95 ) % ( P ＜ 0. 05 ). The levels of serum cTnT and the expression of myocardial Caspase-3 had no significant difference between verapamil-2 group and IR group (P ＞ 0. 05 ). The extent of myocardial injury in verapamil-1 group was significantly lower than that in IR group and verapamil-2 group (P ＜ 0. 05 ). The extent of myocardial injury had no significant difference between verapamil-2 group and IR group (P ＞0. 05). Conclusion Starting from 10 min before ischemia, verapamil has protective effects on myocardial ischemia/reperfusion injury. Starting from 10 min before reperfusion, verapamil does not provide protection on myocardial ischemia reperfusion injury.     

食管癌易感性与细胞毒性T淋巴细胞相关抗原4基因多态性的关系

目的 探讨细胞毒性T淋巴细胞相关抗原4(CTLA4)基因3个位点基因型及单倍型频率分布与食管癌易感性的关系.方法 应用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法,检测205例食管癌患者(男113例,女92例)和205例与病例组同性别、同年龄的正常对照者CTLA4基因第一外显子区+49A/G、启动子区-1661A/G和-1772A/G位点的基因型,采用条件Logistic回归模型分别进行基因多态性、单倍型与食管癌易感性的相关分析.结果 CTLA4+49位点基因型AG和AA均增加食管癌发病风险(P＜0.01,OR=2.280;P＜0.O1,OR=2.192).-1661位点AG基因型频率在病例组也高于对照组(P＜0.01,OR=1.848),而GG基因型在两组间分布差异无统计学意义(P＞0.05);-1772位点各基因型频率在病例组和对照组分布差异均无统计学意义(P＞0.05).单倍型分析显示AAG单倍型可增加食管癌的风险(P＜0.01,0R=5.035),而GAA单倍型则降低食管癌风险(P＜0.01,0R=0.413).结论 CTLA4基因+49A/G和-1661A/G位点基因多态性与食管癌易感性相关,单倍型分析进一步证实AAG单倍型为食管癌的危险因素,而GAA单倍型是其保护因素.     

重组生长激素促短肠大鼠结肠代偿机制的探讨

目的　探讨重组生长激素促短肠大鼠结肠代偿的作用机制。方法　将短肠大鼠分成肠内营养（EN）和肠内营养+重组生长激素（EG）两组。结果　自术后第15天起,EG组体重的减轻明显低于EN组(P＜0.05),术后第21天起体重已大于术前;EG组氮平衡的改善明显好于EN组;血浆蛋白水平也高于EN组(P＜0.05);EG组结肠壁胰岛素样生长因子1(IGF-1)mRNA含量比EN组明显升高〔(1291±43）vs.（1026±42）,P＜0.05〕,IGF-1受体mRNA含量明显降低〔(899±5）vs.（1113±7）,P＜0.05〕;EG组的血IGF-1水平明显高于EN组〔(455±107）ng/mlvs．（329±68）ng/ml,P＜0.05〕,GH水平也显著升高〔(9.7±3.3）ng/mlvs.（5.8±2.4）ng/ml,P＜0.05〕。结论　重组生长激素可促使短肠大鼠IGF-1和结肠壁IGF-1mRNA的合成及残留肠道对营养物质吸收。     

IL-10基因启动子区G-1082A及C-592A多态性与IgA肾病相关性研究

目的：探讨IL-10基因启动子区域G-1082A、C-592A多态性与汉族人群IgA肾病（IgAN）发病间关系。方法：用SSP-PCR方法对180例IgA肾病（IgAN）患者和163例健康对照组IL-10基因启动子区域-1082、-592位点单核苷酸多态性进行分析。结果：-1082位点IgA肾病患者AG/GG基因型频率显著高于正常对照组（为21.0% vs 11.7%,P〈0.05）;-1082位点G等位基因频率显著高于正常对照组（为11.0% vs 6.4%,P〈0.01）;携带有G等位基因者患IgA肾病危险性是携带有A等位基因者1.8倍,95%CI为1.12-3.20。-592位点IgA肾病患者AA、CA、CC基因型与正常对照组相比,无统计学差异（11.11% vs 16.56%;46.67% vs 51.53%;42.22% vs 31.90%,P〉0.05）;-592位点C等位基因频率与正常对照组相比,无统计学差异（32.21% vs 32.50%,P〉0.05）。结论：IL-10基因G-1082A是中国汉族人群IgA肾病患者的易感基因,携带G等位基因者患IgA肾病的危险性是携带A等位基因者的1.8倍。     

严重烧伤患者CD14基因多态性对高迁移率族蛋白B1表达的影响

目的 了解LPS受体CD14C-159T基因多态性对严重烧伤患者伤后高迁移率族蛋白B1(HMGB1)合成、释放的影响以及与脓毒症的关系.方法 采集35例烧伤总面积大于或等于30%TBSA患者伤后1、3、5、7、14、21、28 d静脉血.另设11名志愿者作为健康对照组.采用PCR-限制性片段长度多态性方法检测CD14-159C/T基因多态性,ELISA法检测血浆HMGB1水平,RT-PCR法检测HMGB1 tuRNA表达.对数据行χ~2检验、方差分析和t检验.结果 35例患者的CD14基因C-159T基因型中,CC纯合子型7例占20.0%、TC杂合子型16例占45.7%、TT等位基因纯合子型12例占34.3%.T等位基因和C等位基因分布的频率为57.2%和42.8%.验证表明,此研究群体达到了Hard-Weinberg平衡.在CD14C-159T基因型中,CC纯合子型患者发生脓毒症的概率较TC杂合子型、TT等位基因纯合子型低.3例CC纯合子型脓毒症患者中,仅1例死亡;9例TC杂合子型脓毒症患者中4例死亡;7例TT等位基因纯合子型脓毒症患者中4例死亡.与健康对照组比较伤后1 d患者血浆HMGB1水平即迅速升高,伤后14、21、28 d TC杂合子型、TT等位基因纯合子型患者血浆HMGB1水平均显著高于CC纯合子型(F值为3.5671、4.2035、3.8529,P<0.05或P<0.01).伤后14 d脓毒症组患者外周血白细胞HMGB1 mRNA表达量为1.5±0.5,显著高于非脓毒症组患者(1.2±0.4,t=-2.205,P<0.05).伤后7、21 d脓毒症组患者血浆HMGB1水平分别为(44±29)、(25±15)ng/mL,均高于非脓毒症组患者的(26±12)、(10±6)ng/mL(t值分别为-2.355、-3.872,P<0.05或P<0.01).结论 CD14C-159T基因多态性可显著影响严重烧伤后HMGB1的合成与释放,并与烧伤患者脓毒症易感性有关.     

线粒体ATP敏感性钾通道阻断剂对在体大鼠肺缺血后处理的作用及其机制

目的 探讨缺血后处理(IPO)对大鼠在体肺缺血-再灌注损伤(I/R)的保护作用及线粒体ATP敏感性钾通道(mitoKATP)在缺血后处理效应中的作用.方法 将Wistar大鼠35只随机分为5组:假手术组(Sham组)、缺血再灌注损伤组(I/R组)、缺血后处理组(IPO组)、缺血再灌注损伤+5-羟基葵酸盐组(I/R+5-HD组)、缺血后处理+5-羟基葵酸盐组(IPO+5-HD组).观察各组肺组织中丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性、湿/干比值(W/D)以及病理形态学改变.结果 I/R组与Sham组比较MDA含量增加[(5.07±1.60)nmol/mg prot比(1.43±0.41)nmol/mgprot,P＜0.01],SOD活性减低[(12.38±2.24)U/mg prot比(45.51±5.42)U/mg prot,P＜0.01],W/D比值增高(5.45±0.82比3.05±0.47,P＜0.01),肺组织形态及超微结构明显受损;IPO+5-HD组与IPO组比较MDA含量增加[(3.74±0.71)nmol/mg prot比(2.60±0.43)nmol/mg prot,P＜0.01],SOD活性减低[(22.91±2.71)U/mg prot比(28.74±2.03)U/mg prot,P＜0.01],W/D比值增高(4.64±0.79比3.89±0.60,P＜0.01),肺组织形态及超微结构明显受损;IPO组与I/R组比较,肺组织MDA含量减少[(2.60±0.43)nmol/mg prot比(5.07±1.60)nmol/mg prot,P＜0.01],SOD活性增高[(28.74±2.03)U/mg prot比(12.38±2.24)U/mg prot,P＜0.01],W/D比值减低(3.89±0.60比5.45±0.82,P＜0.01),肺组织病理形态学改变轻于I/R组;I/R+5-HD组与I/R组比较,肺组织MDA含量[(5.14±1.30)mol/mg prot比(5.07±1.60)mol/mg prot,P＞0.05)、SOD活性[(11.65±1.82)U/mg prot比(12.38±2.24)U/mg prot,P＞0.05]、W/D比变化(5.54±0.61比5.45±0.82),差异无统计学意义(P＞0.05),肺组织病理形态学改变无明显差异.IPO+5-HD组的各项指标介于IPO组和I/R组之间.结论 缺血后处理能减轻大鼠在体肺缺血再灌注损伤,mitoKATP参与了肺缺血后处理效应.

Abstract：

Objective To investigate the protective effect of ischemic postconditioning (IPO) on lung ischemic reperfusion (L/R) in rats in vivo and the mechanism of mitochondrial ATP-sensitive potassium channel (mitoKATP) blocker in the ischemic postconditioning. Methods Thirty five Wistar rats were randomly divided into 5 groups: sham group, I/R group, ischemic postconditioning (IPO) group, I/R +5-hydroxydecanoate (I/R + 5-HD) group, IPO + 5-HD group. The concentration of malondialdehyde (MDA) and activity of superoide dismutase (SOD) were determined in the lung homogenate, wet to dry weight ratio (W/D) was measured and pathological changes were also observed. Results The levels of MDA[(5.07±1.60) vs (1.43 ±0.41) nmol/mg prot,P＜0. 01]and W/D (5.45 ±0.82 vs 3.05 ±0. 47,P ＜0. 01 ) were increased significantly in I/R group as compared with sham group, while the activity of SOD[( 12. 38 ±2. 24) vs (45.51 ±5.42) U/mg prot,P ＜0. 01]was decreased, and the injury of lung tissues was significantly aggravated in IPO + 5-HD group as compared with IPO group[MDA: (3.74 ±0. 71 ) nmol/mg prot vs (2. 60 ± 0. 43 ) nmol/mg prot , P ＜ 0. 01]; W/D: 4. 64 ± 0. 79 vs 3. 89 ± 0. 60,P＜0.01; SOD:[(22.91 ±2.71) U/mg prot vs (28.74±2.03) U/mg prot,P＜0. 01]. The levels of MDA[(2.60±0.43) vs (5.07 ±1.60) nmol/mg prot,P＜0. 01]and W/D (3.89 ±0.60 vs 5.45 ±0. 82,P ＜0. 01 ) were decreased significantly in IPO group as compared with I/R group, the activity of SOD[(28.74±2.03) vs (12.38 ±2.24) U/mg prot,P＜0. 01]increased and lung tissue histological damage attenuated. The difference in MDA[(5.14 ± 1.30) vs (5.07 ± 1.60) nmol/mg prot, P ＞ 0. 05],W/D (5.54±0.61 vs5.45 ±0.82,P＞0.05) and SOD[(11.65 ±1.82) vs (12.38 ±2.24) U/mgprot,P ＞ 0. 05]levels had no statistical significance between I/R + 5-HD group and I/R group, and the injury of lung tissues had no significant difference too. Each index in IPO + 5-HD group was between IPO and I/R groups. Conclusion Ischemic postconditioning can attenuate the lung I/R injury, and mitoKATP plays a vital role in the protective procession of ischemic postconditioning on lung ischemic reperfusion.     

血管内皮钙黏蛋白的表达与CASP脓毒症大鼠肠系膜微循环的关系

目的 通过监测血管内皮钙黏蛋白的表达并测定CASP脓毒症大鼠肠系膜微循环的血流速度,分析相互之间的关系.方法 参照脓毒症CASP脓毒症大鼠造模标准造模,根据静脉留置针孔径分组,每组6只,脓毒症A组22 G(0.9 mm×25 mm,33 ml/min),脓毒症B组20G(1.1 mm×32 mm,54 ml/min),脓毒症C组18G (1.3 mm×32 mm,80 ml/min),脓毒症D组14 G(2.0 mm×45 mm,270 ml/min),另外设置对照组(无操作组,n=6),运用活体显微镜技术测定各组脓毒症大鼠6h的血流速度,免疫组化半定量分析各组大鼠肠系膜钙黏蛋白的表达.结果 对照组肠系膜微循环血流速度为(583.21 ±52.39) μm/s,较脓毒症D组(213.30 ±52.39) μm/s明显升高(P＜0.05),脓毒症A组(482.71 ±58.62) μm/s,较脓毒症D组明显升高(P＜0.05).对照组肠系膜钙黏蛋白免疫组化半定量评分(11.17±0.34)分,较脓毒症D组(5.43±0.43)分明显升高(P＜0.01),脓毒症A组(10.07±0.30)分,较脓毒症D组明显升高(P＜0.05).结论 CASP脓毒症大鼠肠系膜钙黏蛋白的表达量与膜微循环血流速度呈正相关,可间接反映脓毒症病变程度.     

酸性HEPES-KH液对离体未成熟心肌的作用

目的 观察不同pH值HEPES-KH复灌液对离体未成熟心肌的影响.方法 建立Langendorff离体灌注模型,分为2组:缺血/再灌(I/R,n=8),用pH 7.4 HEPES-KH液灌流20 min,缺血60min,恢复灌注30min;酸性灌注组(E,n=8),用pH7.4HEPES-KH液灌流20min,缺血60min后,应用pH 6.8、7.1和7.4 HEPES-KH液顺次灌注5、5、20 min.以血流动力学指标、生化指标作为观察指标.结果 E组与I/R组比较,左心室功能恢复、三磷酸腺苷含量(ATP)(0.93±0.12比0.56±0.04,P＜0.01)、超氧化物歧化酶活性(183.47±9.72比120.17±6.21,P＜0.01)、心肌线粒体Ca2+-ATP酶活性(16.74±1.42比6.78±0.64,P＜0.01)和心肌线粒体合成ATP的能力(105.37±9.51比50.83±4.75,P＜0.01)明显增强,在心肌含水量(74.56±1.68比86.20±2.33,P＜0.01)、丙二醛含量(1. 97±0.17比2.88±0.32,P＜0.01)、肌酸激酶(64.56±4.69比88.48±5.86,P＜0.01)和乳酸脱氢酶漏出率漏出率(96.41±6.57比128.42±9.80,P＜0.01)、心肌细胞内Ca2+含量(2.25±0.28比4.48±0.74,P＜0.01)和心肌线粒体Ca2+含量(36.10±4.05比68.29±6.90,P＜0.01)明显减少.结论 复灌初期应用梯度酸性复灌液对离体未成熟心肌具有明显保护作用.

Abstract：

Objective To study the protective effects of different pH HEPES-KH reperfusate solutions on immature myodium. Methods The isolated Langendorff perfused model from immature rabbit hearts was established. The rabbits in ischemia/reperfusion (I/R) group were perfused with pH7.4HEPES-KH solutions preischemia and postischemia. In experimental (E) group, pH 6. 8, pH 7. 1 and pH 7. 4 HEPES-KH solutions were perfused for 5, 5 and 20 min postischemia, respectively. The hemodynamics and biochemistry were tested. Results The left ventricular function was significantly improved, adenosine triphosphate (ATP) content (0. 93 ±0. 12 vs 0. 56 ±0. 04,P ＜0. 01 ), superoxide dismutase activity ( 183.47 ±9. 72 vs 120. 17 ± 6. 21, P ＜ 0. 01 ), Ca2+ -ATPase activity of mitothondia ( 16. 74 ± 1.42 vs 6. 78 ± 0. 64, P ＜ 0. 01 ), ATP activity of mitochondria ( 105.37 ± 9. 51 vs 50. 83 ± 4. 75, P ＜ 0. 01 ) were significantly increased in E group as compared with those in I/R group. Myocardial water content (74. 56 ± 1.68 vs 86. 20 ±2. 33 ,P ＜0. 01 ), malondialdehyde content ( 1.9710. 17 vs 2. 88 ±0. 32,P ＜0. 01 ), dehydrogenase (64. 56 ± 4. 69 vs 88. 48 ± 5. 86, P ＜ 0. 01 ) and creatine kinase leakage (96. 41 ±6.57 vs 128.42 ±9.80,P＜0.01), myocardial cell Ca2+ content (2.25 ±0.28 vs 4.48 ±0.74,P＜0.01) and mitochondrial Ca2+ content (36. 10 ±4.05 vs 68.29 ±6.90,P＜0.01) in E group were reduced as compared with those in I/R group. Conclusion pH paradox might be one of important mechnisms for immature myocardial I/R injury, and acidic perfusate, at the beginning of reperfusion, might attenuate pH paradox and ameliorate functional recovery on isolated immature rabbit hearts.     

LAMP2A和MEF2D在增龄过程中大鼠髓核组织的表达及其意义

目的 探讨在增龄过程中SD大鼠髓核组织中LAMP2A和MEF2D的表达及其意义.方法 3、12、24月龄SD大鼠各8只,建立青年大鼠组、中年大鼠组和老龄大鼠组模型,透射电镜检测髓核细胞中自噬体的表达;逆转录-聚合酶链反应(RT-PCR)和Western blot法测定髓核组织中LAMP2A和MEF2D的表达.结果 不同年龄组大鼠髓核细胞中均有自噬体的表达;RT-PCR和Western blot法显示LAMP2A在3、12、24月龄组中均有表达(0.91±0.07、1.12±0.11、1.32±0.28和0.45±0.06、0.55±0.08、0.63±0.11),且在老龄组表达较青年组表达明显增高(P＜0.01和P＜0.05),在3、12、24月龄组中,RT-PCR和Western blot法亦显示MEF2D均有表达(0.66±0.10、0.55±0.08、0.52±0.08和0.46±0.03、0.43±0.02、0.41±0.04),且24月龄组表达明显较青年组低(P均＜0.01).结论 自噬存在于椎间盘退变过程中;分子伴侣介导的自噬活性的增加可能与椎间盘退变的发展进程有关.LAMP2A介导的分子伴侣自噬性细胞死亡可能通过MEF2D进行调节.     

受体诱导一氧化碳经PI3 K/Akt信号途径抑制冷缺血再灌注诱导的移植心细胞凋亡

目的 观察受体诱导一氧化碳(CO)对移植心冷缺血再灌注(I/R)损伤中细胞凋亡的影响,并探讨其机制.方法 以BALB/C小鼠建立同系移植心冷I/R损伤模型.受体麻醉前3 h以二氯甲烷(MC)500 mg/kg灌胃诱导CO(MC组,n=12),或橄榄油灌胃(IR组,n=12);在MC组的基础上受体在移植心恢复血供前1 h腹腔注射PI3K抑制剂LY294002(40 mg/kg,LY组,n=10)或二甲亚砜(DMSO组,n=10);检测移植后3、24 h移植心细胞凋亡指数(AI)、磷酸化Akt(p-Akt)蛋白、bcl-2与bax蛋白表达比值;设正常对照组(N组,n=5).结果 受体以MC灌胃后血液中碳氧血红蛋白(COHb)浓度与心肌组织CO含量均在3 h达到峰值,分别为(9.82±0.84)%和(2.25±0.08)pmol/mg;与IR组比较,MC组明显降低移植心AI[3 h:(8.65±2.01)%比(19.28±4.94)%,P＜0.01;24 h:(5.82±2.36)%比(10.54±3.66)%,P＜0.05]、激活Akt蛋白(3 h:P＜0.01;24 h:P＜0.05)、上调bcl-2/bax比值(3 h:1.97±0.16比0.46±0.07,P＜0.01;24 h:1.89±0.10比0.51±0.04,P＜0.01);与MC组比较,LY组明显增加AI[3 h:(17.95±4.92)%,P＜0.01;24 h:(9.75±3.14)%;P＜0.01]、抑制Akt蛋白激活(P＜0.01)、下调bcl-2/bax比值(3 h:0.47±0.06,P＜0.01;24 h:0.52±0.03,P＜0.01);DMSO组与MC组的各个指标差异无统计学意义(P＞0.05).结论 受体诱导C0能明显抑制冷I/R诱导的移植心细胞凋亡,其机制可能与通过PI3K/Akt信号途径上调bcl-2/bax比值有关.