谷氨酰胺诱导大鼠热休克蛋白70mRNA的表达

热休克蛋白(heat shock protein,HSP)是感染或非感染(如创伤、休克)等致病因素作用于机体后,诱发机体细胞合成的一组高度保守的蛋白质,谷氨酰胺(Glu-tam ine,Gln)作为体液中最丰富的氨基酸之一,对机体多个脏器有保护作用。一些研究表明,Gln在体外可促进果蝇Kc细胞HSP的表达[1];Gln诱导的HSP表达对肠道上皮细胞有保护[2]。但Gln是否能在动物的各个脏器引起HSP的表达及时间和量效关系如何,尚未见明确报道。本研究对此进行了实验观察,拟为临床应用提供理论依据。1材料和方法1.1主要试剂力太(批号J20020081,Fresennius Kabi公司,德国),…     

缺氧诱导成骨细胞VEGF mRNA的RT-PCR表达

目的 观察在体外缺氧条件下，SD大鼠成骨细胞VEGFmRNA表达的变化。方法 酶消化法收集培养SD大鼠颅骨成骨细胞，用高纯氮气造成缺氧环境，应用RT-PCR技术观察不同时间段VEGF mRNA表达的改变。结果 缺氧6h后，VEGF mRNA明显增加，缺氧12h后，增至顶峰，缺氧24h后开始下降，缺氧48h接近正常水平。结论 缺氧在转录水平调节成骨细胞VEGF的表达。     

模拟失重对大鼠肝脏Hsp70及其基因表达的影响

目的 探讨模拟失重环境中大鼠肝脏组织Hsp70及Hsp70mRNA表达变化.方法 成年Wistar大鼠48只,体重(300±20)g).随机分为6组,按模拟失重时相分别为6、12、24、48、96、0 h组(地面对照组).采用尾悬吊法建立模拟失重动物模型.应用RT-PCR和Western blot技术分别检测各组大鼠肝组织Hsp70蛋白和mRNA的表达.结果 不同时相模拟失重环境下大鼠肝脏Hsp70mRNA水平均显著高于对照组(P<0.05),模拟失重6 h组肝脏Hsp70mRNA表达显著升高,12 h组达高峰,24 h和48 h略有下降,但仍然显著高于对照组(P<0.05).Hsp70蛋白表达水平在6 h组形成高峰,随模拟失重时间的延长,则呈明显下降趋势.96 h组降低到对照组水平.结论 模拟失重使大鼠肝脏组织中Hsp70蛋白和基因表达发生明显变化,提示肝脏Hsp70表达变化与失重应激反应和失重耐受有密切关系.     

Down-regulation of connective tissue growth factor and type I collagen mRNA expression by connective tissue growth factor antisense oligonucleotide during experimental liver fibrosis.

Transforming growth factor (TGF)-beta 1 is a major mediator of liver fibrosis. Connective tissue growth factor (CTGF) mediates TGF-beta 1 pro-fibrogenic effects in vitro, but its in vivo role is unknown. Both TGF-beta 1 and CTGF are overexpressed in hepatic stellate cells during liver fibrosis. We have used antisense oligonucleotides to examine the role of CTGF in carbon tetrachloride-induced liver fibrosis in mice. Mice received carbon tetrachloride together with CTGF or TGF-beta 1 antisense oligonucleotides for 2 weeks (preventive model), or carbon tetrachloride for 2 weeks followed by carbon tetrachloride and oligonucleotides for 2 more weeks (curative model). In both models, CTGF and TGF-beta 1 oligonucleotides decreased by more than 50 percent the mRNA expression of their targets. Type I collagen mRNA was also decreased by about 40 percent in the preventive experiment. Tissue inhibitor of matrix metalloproteinase-1 mRNA expression and fibrotic deposition evaluated by Sirius red staining were not modified in any group. In summary, our results suggest that hepatic stellate cells can be targeted in vivo with oligonucleotides, and that reducing CTGF levels can lead to a decrease in fibrogenesis as shown by the reduction in type I collagen expression. The lack of effect on fibrosis may be due to the persistence of high tissue inhibitor of matrix metalloproteinase-1 expression.     

Expression of connective tissue growth factor mRNA in human IgA nephropathy

Background. Connective tissue growth factor (CTGF) is a cysteine-rich member of a new family of growth regulators. Its upregulation is an important factor in the pathogenesis of mesangial matrix accumulation and progressive glomerulosclerosis. Methods. We evaluated the expression and localization of CTGF mRNA in renal tissues of 20 patients with IgA nephropathy (IgA-N) and 5 normal human kidneys (NHK), using high-resolution in situ hybridization with digoxigenin-labeled oligonucleotide. The expression level of CTGF mRNA was quantitated by counting all nuclei, as well as nuclei surrounded by CTGF mRNA-positive cytoplasm in at least ten randomly selected cross-sections of nonsclerotic glomeruli, and expressing the results as percentage of positive cells. Results. In both IgA-N and NHK, CTGF mRNA was mainly expressed in glomerular intrinsic cells, including mainly glomerular mesangial and epithelial cells, and some endothelial cells and cells of Bowman's capsule. CTGF mRNA-positive cells were abundant in tubulointerstitial fibrotic areas, especially in IgA-N with severe tissue damage. CTGF mRNA expression was also increased in vascular cells in IgA-N. The percentage of cells positive for CTGF mRNA was significantly higher in IgA-N than in NHK. Furthermore, the percentage of cells positive for CTGF mRNA was significantly greater in IgA-N with moderate mesangial proliferative lesions than in IgA-N with mild mesangial proliferative lesions and/or sclerotic lesions. Conclusions. Our study indicates that CTGF may play an important role in the development and progression of glomerulosclerosis and tubulointerstitial fibrosis in IgA-N. Received: July 17, 2000 / Accepted: October 21, 2000     

肝硬化大鼠肝组织生长抑素受体亚型mRNA表达的研究

目的 研究在大鼠肝硬化门静脉高压症形成过程中，肝组织生长抑素５种受体亚型ｍＲＮＡ的表达变化。方法 将大鼠３０只随机分为对照组，急性坏死组和肝硬化组。提取样本肝组织总ＲＮＡ，进行逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）后，琼脂糖凝胶电泳条带经过计算机图像分析处理，检测大鼠生长抑素受体亚型ｍＲＮＡ的表达。结果 正常肝组织中ｒＳＳＴＲ４ ｒＳＳＴＲ１，ｒＳＳＴＲ５的ｍＲＮＡ表达量均明显高于ｒＳＳＴＲ３，ｒＳ     

Liver metastasis and ICAM-1 mRNA expression in the liver after carbon dioxide pneumoperitoneum in a murine model

Background Liver metastasis of colorectal malignancies is an important prognostic factor. Several studies have demonstrated that carbon dioxide (CO₂) pneumoperitoneum enhances liver metastasis in animal models. Little is known about intercellular adhesion molecule-1 (ICAM-1) and tumor necrosis factor-alpha (TNF-(α) mRNA expression in the liver after CO₂ pneumoperitoneum. Methods Forty-five male BALB/c mice were randomly divided into three groups after intra-splenic tumor cell (colon 26) inoculation and the following procedures were performed: CO₂ pneumoperitoneum (n = 15), open laparotomy (n = 15), and anesthesia alone (n = 15). On day 7 after each procedure, the livers were excised and the number and diameter of the tumor nodules and the cancer index score were determined. Another 90 male BALB/c mice were randomly divided into three groups as described above, and they underwent each procedure (n = 30 each). After each procedure, the livers were excised on days 0, 1, 3, and ICAM-1 and TNF-α mRNA expression were examined by real-time RT-PCR using SYBR Green I. Results The number of tumor nodules and the cancer index score were larger in the CO₂ pneumoperitoneum group than in the control group (p < 0.05). The mean diameter of the tumor nodules was not different among the three groups. The expression of ICAM-1 in the CO₂ pneumoperitoneum group was higher than that in the other groups on day 1 (p < 0.05), and the TNF-α mRNA was higher than that in the control group on day 1 (p < 0.05). Conclusions CO₂ pneumoperitoneum enhances liver metastasis compared with anesthesia alone, and ICAM-1 expression in the liver after the pneumoperitoneum plays an important role in establishing liver metastasis in a murine model.     

脊髓损伤后热休克蛋白27、表皮脂肪酸结合蛋白和金属蛋白酶组织抑制因子-1的基因表达及甲基强的松龙对其表达的影响

目的研究脊髓损伤后热休克蛋白27(HSP27)、表皮脂肪酸结合蛋白(FABPs)和金属蛋白酶组织抑制因子-1(TIMP-1)的基因表达及甲基强的松龙(MP)对其表达的影响.方法SD大鼠30只.随机分为假手术组、单纯脊髓损伤组(损伤组)及脊髓损伤+大剂量MP治疗组(MP组),每组10只.应用改良的Allen's打击法致T8脊髓损伤.MP组大鼠伤后即刻从尾静脉内注射大剂量MP(30mg/kg).损伤后24h切取损伤平面上下0.5cm的脊髓组织,进行RT-PCR反应,检测HSP27、FABPs和TIMP-1的基因表达.结果术后24h假手术组HSP27、FABPs和TIMP-1的基因表达相对丰度分别为0.0643±0.0152、0.6413±0.1005和0.7091±0.0577;损伤组上述三个因子的表达升高,分别为1.0013±0.3861、1.2187±0.2851和0.8971±0.1092,与假手术组比较差异有显著性(P＜0.01、P＜0.05、P＜0.05);MP组上述三个因子的表达继续升高,分别为1.2858±0.1384、1.7122±0.1766和1.2081±0.1093,与损伤组比较差异有显著性(P＜0.05、P＜0.01和P＜0.01).结论脊髓损伤后,邻近损伤处的脊髓组织中HSP27、FABPs及TIMP-1的基因表达显著增高,大剂量MP能进一步促进三个因子表达,发挥组织保护作用.     

Glomerular expression of connective tissue growth factor mRNA in various renal diseases

SUMMARY:   Connective tissue growth factor (CTGF) is a cysteine-rich member of a new family of growth regulators. It is an important factor in the pathogenesis of mesangial matrix accumulation and progressive glomerulosclerosis. The present study was designed to elucidate the role of CTGF in diabetic nephropathy (DN), immunoglobulin A nephropathy (IgA-N), membranous nephropathy (MN), and minimal change nephrotic syndrome (MCNS). We evaluated the expression and localization of CTGF mRNA in surgically excised renal tissue samples from 10 patients with DN, 10 with IgA-N, 10 with MN, 10 with MCNS, and 10 normal human kidney (NHK) tissue samples, by using high-resolution in situ hybridization with digoxigenin-labelled oligonucleotide. To quantify CTGF mRNA expression, we counted all nuclei, and nuclei surrounded by CTGF-positive cytoplasm, in at least 10 randomly selected cross-sections of non-sclerotic glomeruli, and expressed the results as a percentage of total glomerular cells. In all glomeruli, CTGF mRNA was expressed mainly in glomerular intrinsic cells, including glomerular mesangial and epithelial cells and some cells of Bowman's capsule. The percentage of cells positive for CTGF mRNA was significantly higher in DN and IgA-N than in MN, MCNS and NHK. However, there was no significant difference in the percentage of CTGF mRNA-positive cells between DN and IgA-N. Our study indicates that CTGF may play an important role in the development and progression of glomerulosclerosis in DN and IgA-N, which are both accompanied by mesangial matrix expansion and comprise two major causes of end-stage renal failure.     

In situ hybridization of connective tissue growth factor mRNA in IgA nephropathy

Connective tissue growth factor (CTGF) is a member of the new family of growth regulators. It plays an important role of the pathogenesis of mesangial matrix accumulation and progressive glomerulosclerosis in IgA nephropathy (IgAN).
We investigated the expression and localization of CTGF mRNA in renal tissues of patients with IgAN and normal human kidneys (NHK), using in situ hybridization with digoxigenin-labelled oligonucleotide. Open renal biopsy tissues were obtained from 16 patients with IgAN. The renal pathology was categorized into four grades by light microscopic findings. The expression level of CTGF mRNA was quantified by counting all nuclei, as well as nuclei surrounded by CTGF mRNA-positive cytoplasm in randomly selected non-sclerotic glomeruli and expressing the results as the percentage of total cells.
Connective tissue growth factor mRNA was mainly expressed in glomerular mesangial and epithelial cells in both IgAN and NHK, and cells of Bowman's capsule. In IgAN, CTGF mRNA-positive cells were increased in tubulointerstitial fibrotic areas. The percentage of positive cells for CTGF mRNA was significantly higher in IgAN than in NHK. The percentage of positive cells for CTGF mRNA in each IgAN grade was significantly higher than that in NHK. Furthermore, the percentage of positive cells for CTGF mRNA was significantly greater in IgAN with moderate mesangial proliferative lesions (grade 2, grade 3) than in IgAN with mild mesangial proliferative lesions.
Our study suggests that CTGF may play an important role in the development and progression of glomerulosclerosis and tubulointerstitial fibrosis in IgAN.     

FHITmRNA及蛋白在膀胱移行细胞癌中的表达和意义

目的：研究膀胱移行细胞癌组织中脆性组氨酸三联体（fragilehistidinetriad,FHIT）mRNA及蛋白在膀胱移行细胞癌发生、发展中的作用。方法：采用RT—PCR和免疫组化SP法检测FHITmRNA及蛋白在62例膀胱移行细胞癌组织及35例正常组织中的表达情况。结果：FHITmRNA在癌组织及正常对照组中的缺失率分别为54．84％和2．86％。随着临床分期,FHITmRNA和蛋白异常表达呈增高趋势;并且随着膀胱移行细胞癌分化程度呈下降趋势。结论：FHIT基因与膀胱移行细胞癌的发生、发展过程密切相关,FHIT基因的检测可能成为膀胱移行细胞癌的预测指标。     

组织因子途径抑制物-2在肾癌组织中的表达及甲基化

目的 探讨组织因子途径抑制物-2(TFPI-2)对肾癌侵袭性及细胞凋亡的影响,TFPI-2在肾癌组织中的表达与基因启动子甲基化关系.方法 免疫组织化学、蛋白质印迹、荧光定量RT-PCR检测37例肾癌及11例正常肾脏组织TFPI-2基因表达;TUNEL法检测细胞凋亡;荧光定量甲基化特异性PCR检测TFPI-2基因启动子甲基化.结果肾癌与正常肾脏组TFPI-2蛋白质印迹条带吸光度(A)值分别为0.92±0.36、1.61±0.13;2组TFPI-2 mRNA相对表达量为0.0019±0.0011、0.0065±0.0008.随肾癌分期增加,TFPI-2表达下降.肾癌TFPI-2表达1～4级细胞凋亡指数分别为2.41%、3.90%、6.78%、9.57%.随TFPI-2表达增加,肾癌细胞凋亡指数上升.肾癌TFPI-2基因启动子甲基化阳性组与阴性组TFPI-2 mRNA相对表达量分别为0.0015±0.0011、0.0024±0.0009,2组蛋白质印迹条带A值为0.82±0.35、1.04±0.34.基因启动子甲基化阳性组TFPI-2表达低于阴性组.结论 肾癌TFPI-2基因表达与侵袭性呈负相关,促进细胞凋亡;启动子高甲基化是肾癌TFPI-2基因低表达机制之一.     

Injury induces increased monocyte expression of tissue factor: factors associated with head injury attenuate the injury-related monocyte expression of tissue factor

BACKGROUND: Activated monocytes are able to express tissue factor (TF), a potent procoagulant. The effect of injury on monocyte TF expression is not known. We have found that patients with head injury (HI) have increased antithrombin activity and decreased platelet function compared with non-head-injured trauma patients. Our objective was to determine whether injury increases TF expression by monocytes and whether this increased TF expression is attenuated in patients with HI. METHODS: We prospectively enrolled 37 trauma patients (meeting the entry criterion of an Injury Severity Score [ISS] > or = 9) and 11 healthy control subjects. We sampled blood on arrival and then at 24, 48, and 72 hours. We performed flow cytometry with antibody markers for monocytes (CD14), platelets (CD42a), and TF. We compared results of patients with HI (Glasgow Coma Scale score < or = 9 and Abbreviated Injury Scale Head/Neck score > or = 3) with patients without HI and with controls. RESULTS: Patients had a mean ISS of 23.9 +/- 2.3 (+/- SEM), mean age of 45 +/- 3 years, and mean length of stay of 17.9 +/- 3.2 days. Seventy-six percent were men, and 97% had blunt trauma. The overall mortality rate was 11%. Trauma patients had greater monocyte TF expression than controls for all time periods (p < 0.05). Trauma patients with HI had elevated monocyte TF expression compared with controls for the initial and 24-hour time periods, but they subsequently had more rapid return of monocyte TF expression to baseline (despite a higher ISS) than trauma patients without HI. Trauma patients both with and without HI had increased platelet-monocyte binding at each time versus controls. CONCLUSION: Trauma induces TF expression on monocytes. Patients with HI have attenuation of this expression by 24 hours after injury. The attenuation of TF expression by monocytes in HI parallels the increase in AT and the decrease in platelet function seen after HI. The correlation of TF expression with platelet-monocyte binding suggests that platelet binding may lead to monocyte activation.     

腹膜透析液诱导结缔组织生长因子在大鼠腹膜间皮细胞表达

目的 研究应用不同腹膜透析液对大鼠腹膜间皮细胞(RPMCs)结缔组织生长因子(CTGF)合成的影响。方法 分离培养的RPMC分为6组，分别以不同腹膜透析液[1.5% Dextrose(低糖组)、2.5% Dextrose(中糖组)、4.25% Dextrose(高糖组)、7.5% Icodextrin(糊精组) ]进行刺激培养，而无血清DMEM为阴性对照(对照组)， TGF-β1(2.5 ng/ml)为阳性对照(阳性对照组)。刺激培养24 h后，RT-PCR法检测RPMCs的CTGF mRNA、胶原Ⅰ mRNA、α-SMA mRNA表达。Western印迹法检测RPMCs的CTGF、胶原Ⅰ、α-SMA蛋白表达以及培养上清中的CTGF蛋白表达。结果 各组均见CTGF mRNA表达，高糖组、阳性对照组CTGF mRNA 表达水平显著高于中糖组、低糖组及对照组(P < 0.05)；中糖组与糊精组CTGF mRNA 表达亦显著上调(P < 0.05)。各组细胞均检测到CTGF蛋白质表达，为相对分子质量38 000及 25 000的2种亚型，与RT-PCR结果一致。高糖组、阳性对照组CTGF 蛋白表达水平显著高于糊精组、中糖组、低糖组及对照组(P < 0.05)。RPMCs培养上清液中检测出CTGF 38 000亚型的表达，其表达强弱趋势与CTGF在细胞中表达一致。高糖组、阳性对照组胶原I mRNA、蛋白质的表达水平显著高于对照组(P < 0.05)，其余各组间无显著性差异。各组α-SMA mRNA、蛋白质表达未见显著性差异(P > 0.05)。结论 正常培养的RPMCs表达低水平的CTGF。腹膜透析液、尤其是高浓度葡萄糖透析液，能明显上调CTGF表达的水平，这可能是导致长期腹膜透析过程中腹膜结构改变的机制之一。糊精腹膜透析液生物相容性可能优于高浓度葡萄糖透析液。     

以肿瘤-睾丸抗原mRNA为特异性标记评价肝癌患者肝移植的预后

目的探讨肝细胞癌 (hepatocellularcarcinoma ,HCC)患者原位肝移植 (orthotopiclivertrans plantation ,OLT)手术前后外周血中MAGE 1、SSX1及CTp11基因mRNA表达与预后的关系。方法用巢式RT PCR方法检测OLT手术前后外周血单个核细胞 (PBMC)中上述 3种基因的表达 ,并对 2 6例患者进行随访。结果HCC患者术前PBMC中MAGE 1、SSX1和CTp11基因mRNA的表达率分别为4 0 0 % (14 / 35 )、37 1% (13/ 35 )和 31 4 % (11/ 35 ) ,6 0 0 % (2 1/ 35 )患者的PBMC中至少可以检测到其中一种基因表达。 16例术前PBMC中检测到上述基因表达者 ,术后肿瘤复发 /转移率 (75 0 % )显著高于10例不表达这 3种基因者 (30 0 % ) (P =0 0 4 3) ;在术前及术后这 3种基因mRNA持续呈阳性或者由阴性转为阳性的 2 0例患者中 ,15例 (75 0 % )术后发生肿瘤复发 /转移 ,而 3种基因mRNA持续阴性或者由阳性转为阴性的 6例患者无 1例复发 /转移 (P =0 0 0 2 )。结论MAGE 1、SSX1及CTp11基因mR NA可以联合作为肿瘤特异性标志物用于检测HCC患者血循环中的肿瘤细胞 ,并有助于选择OLT受体及判断预后。     

生长抑素类似物奥曲肽对肝星状细胞结缔组织生长因子基因表达的影响

目的 研究生长抑素类似物奥曲肽(OCT)对体外培养的大鼠肝星状细胞(HSC)中结缔组织生长因子(CTGF)基因表达的影响。方法 从Sprague Dawley大鼠的肝脏中分离培养HSC,待其自发激活增殖及传代后,将其分为5组,其中4组加入不同浓度的OCT,余1组为不加OCT的对照组,培养72h,RT-PCR法检测各组细胞CTGF及转化生长因子β1(TGF-β1)mRNA表达的情况。结果 OCT能够明显抑制CTGF及FGF-β1mRNA的表达。在一定浓度范围内呈剂量依赖性。结论 OCT能够抑制激活的HSC中TGF和TGF-β1基因的表达,由此发挥其抗纤维化作用,为临床抗纤维化治疗提供新的思路。     

定量PCR技术检测增生性瘢痕组织纤维连接蛋白的基因表达

目的探讨纤维连接蛋白的表达水平与创伤后增生性瘢痕形成的关系。方法采用定量PCR技术,对纤维连接蛋白(Fibronectin,FN)在正常皮肤和增生性瘢痕中mRNA表达水平的变化进行比较。结果在增生性瘢痕中,FN的mRNA表达水平较正常皮肤增高。结论实验结果表明细胞外基质成分的变化在创伤修复失控形成中起重要作用。     

动脉粥样硬化闭塞症粥样斑块组织中血小板衍生生长因子表达的研究

目的：探讨血小板衍生生长因子（PDGF）同动脉粥样硬化闭塞症（ASO）之间的相互关系。方法：利用组织原位杂交技术对20例60处ASO血管进行PDGF基因表达检测。结果：ASO血管壁可见PDGF－A阳性表达，并主要分布于粥样斑块边缘和新生斑块增生的平滑肌细胞内。结论：PDGF主要分布于动脉粥样硬化早期病变血管组织内，可能在ASO的早期发挥作用，参于ASO的形成和发展。     

Cyclosporine induces myocardial connective tissue growth factor in spontaneously hypertensive rats on high-sodium diet

BACKGROUND: The introduction of cyclosporine (CsA) has led to an improvement in the prognosis of solid organ transplantation. However, drug-induced hypertension and nephrotoxicity, associated with the development of atherosclerosis and coronary heart disease, still worsen the long-term outcome of CsA-treated patients. Whether the CsA-induced myocardial changes are associated with the induction of connective tissue growth factor (CTGF), a recently found polypeptide implicated in extracellular matrix synthesis, is not known. METHODS: Spontaneously hypertensive rats (8-9 weeks old) were treated with CsA (5 mg x kg(-1) x d(-1) subcutaneously) for 6 weeks. The influence of angiotensin-converting enzyme inhibition (enalapril 30 mg x kg(-1) x d(-1) orally) and angiotensin-1 receptor blockade (valsartan 3 and 30 mg x kg(-1) x d(-1) orally) on CsA toxicity was also investigated. Myocardial morphology was examined, and vascular lesions were scored. Localization and the quantitative expression of CTGF, as well as collagen I and collagen III, mRNA were evaluated by in situ hybridization and Northern blot. RESULTS: CsA-induced hypertension and nephrotoxicity were associated with myocardial infarcts and vasculopathy of the coronary arteries. CsA increased myocardial CTGF, collagen I, and collagen III mRNA expressions by 91%, 198%, and 151%, respectively. CTGF mRNA expression colocalized with the myocardial lesions. Blockade of the renin-angiotensin system prevented vascular damage and the CsA-induced CTGF, collagen I, and collagen III mRNA overexpressions in the heart. CONCLUSIONS: CsA increases CTGF, collagen I, and collagen III mRNA expressions in the heart. The induction of CTGF gene is mediated, at least in part, by angiotensin II.