脓毒症大鼠肺基质金属蛋白酶-2/9表达与血必净干预

目的 观察创伤弧菌脓毒症大鼠肺基质金属蛋白酶(MMP)-2/9和组织型金属蛋白酶抑制剂(TIMP)-1/2的动态表达及血必净干预.方法 温州医学院生命科学院实验室,清洁级SD大鼠110只,随机(随机数字法)分为正常对照组(A组,n=10)、创伤弧菌脓毒症组(B组,n=50,采用大鼠左下肢皮下注射创伤弧菌悬液制作创伤弧菌脓毒症大鼠模型)及血必净干预组(C组,n=50,感染后0.5 h腹腔注射血必净4 mL/kg).B、C组大鼠于染菌后1,6,12,24,48 h麻醉后活杀,留取右肺标本.观察大鼠行为学变化,采用考马斯亮蓝法测肺通透性,采用逆转录-聚合酶链式反应(RT-PCR)法测肺MMP-2/9,TIMP-1/2 mRNA的表达,免疫组化法和双抗体夹心酶联免疫吸附法(ELISA)测肺MMP-2/9和TIMP-1/2的表达.数据采用单因素方差分析,并用LSD-t法进行组间两两比较,以P＜0.05为差异有统计学意义.结果 B组和C组肺通透性显著高于A组,C组显著低于B组.B组和C组MMP-2/9,TIMP-1/2mRNA显著升高,B组分别于6 h(0.344±0.108),6 h(1.230±0.377),12 h(0.523±0.098),12 h(0.280±0.070)达高峰(P＜0.05),C组分别于12 h(0.256±0.074),6 h(0.968±0.225),12 h(0.746±0.316),12 h(0.356±0.035)达高峰(P＜0.05),C组MMP-2/9mRNA升高趋势显著低于B组(P＜0.05),TIMP-1/2mRNA显著高于B组(P＜0.05).B组和C组MMP-2/9,TIMP-1/2蛋白也升高,B组分别于12 h(0.692±0.191),12 h(0.061±0.017),24 h(1384.42±91),24 h(41.04±3.60)达高峰(P＜0.05);C组分别于24 h(0.217±0.065),12 h(0.045±0.013),24 h(1617.22±103),24 h(47.66±3.58)达高峰(P＜0.05);C组MMP-2/9蛋白升高趋势低于B组(P＜0.05),TIMP-1/2蛋白早期与B组差别不大,后期显著高于B组(P＜0.05).结论 MMP/TIMP比例失衡是创伤弧菌脓毒症大鼠肺损伤机制之一,血必净可促进MMP/TIMP比例恢复平衡,对创伤弧菌脓毒症大鼠肺损伤具有保护作用.

Abstract：

Objective To detect the expression of MMP-2/9 and TIMP-1/2 in the lung of Vibrio vulnificus sepsis rats and observe the intervention of Xuebijing injection. Method One hundred and ten SD rats of clean grade were randomly(random number) divided into normal control group (group A, n = 10),Vibrio vulnificus sepsis group (group B, n = 50. Sepsis was reproduced in rats with subcutaneous injection in left lower limb with Vibrio vulnificus) and Xuebijin intervention group ( group C, n = 50. Rats were intraperitoneal(ip) with the dose of Xuebijing 4mL/kg at the time of 30 min later after infection). The rats in group B and C were sacrificed at 1 h, 6 h, 12 h, 24 h, 48 h after infection, the expression of MMP-2/9 and TIMP-1/2 were examined by PCR, Immunohistochemistry or ELISA methods, the lung permeability were measured by Coomassie Brilliant Blue method. Experimental data used single factor analysis of variance, and between groups by LSD method for pairwise comparison,P ＜0.05 statistically significant difference. Results The lung permeability increased both in group B and C compared with group A,and in group B were relatively higher. The lung MMP-2/9, TIMP-1/2mRNA expression in groups B and C compared with in group A was markedly higher, and reached the peak at 6 h(0. 344 ± 0. 108 ),6 h ( 1. 230 ± 0.377 ), 12 h (0.523 ±0.098),12 h(0.280±0.070) (P＜0.05) in group B while at 12 h(0.256 ±0.074),6 h(0.968±0.225) ,12 h(0.746 ±0. 316) ,12 h(0.356 ±0.035) (P ＜0. 05) in group C; the MMP-2/9mRNA expression in group C decreased(P＜0. 05) compared with the group B while the TIMP-1/2mRNA expression increased(P＜0. 05). The lung MMP-2/9, TIMP-1/2 protein expression in groups B and C compared with the group A(0.345±0.109) also increased, and the peak was at 12 h (0. 692 ± 0. 191 ), 12 h (0. 061 ±0.017) ,24 h(1384.42 ±91) ,24 h(41.04 ±3.60)in group B while at 24 h(0. 217 ±0.065) ,12 h(0. 045± 0. 013 ) ,24 h ( 1617.22 ± 103 ) ,24 h (47.66 ± 3.58 )in group C, the MMP-2/9 protein expression in group C was lower than in group B(P＜0.05), the TIMP-1/2 protein expression in group C was similar to in group B early while marked increased(P＜0.05)later. Conclusions MMP/TIMP imbalance was one of the mechanisms of the lung injury in the rats with Vibrio vulnificus sepsis, Xuebijing could restore the balance of MMP/TIMP ratio.     

MMP-9和TIMP-1在不明原因不孕症患者子宫内膜的表达

Objective To study the expression situation of matrix metalloproteinase-9(MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the endometrium of women with unexplained infertility and normal endometrium, and to explore the relationship between MMP-9/TIMP-1 expres-sion and unexplained infertility. Methods Immunohistochemieal assay(SP method) was employed to deteet the expression of MMP-9/TIMP-1 in 20 cases of impaired endometrium(unexplained infertility group,endometrial plantation window phase)and 20 cases of normal endometrium(heahhy control group). Results There were different levels of MMP-9/TIMP-1 expression in kytoplasms of glandular epicytes and stromal cells of all endometrial samples. The expression of MMP-9/TIMP-1 was signifi-cantly weaker in unexplained infertility group than that in healthy control group(P＜0.05). Conclusion Low expression of MMP-9/TIMP-1 in the endometrial plantation window phase may be one of impor-tant factors for unexplained infertility.     

MMP-2／MMP-9在乳腺癌组织中的表达及其与肿瘤浸润转移的关系

BACKGROUND: Matrix melloproteinases play a pivotal role in tumor invasion and metastasis, but little is known about the correlation between their expression and the prognosis of breast cancer.OBJECTIVE: To investigate the expression of matrix melloproteinases-2 and matrix melloproteinases-9 (MMP-2/MMP-9) and the distributive form of CollV (type IV collagen)in the tissues of breast carcinoma, and find out their relationship with tumor invasion and metastasis so as to disclose the mechanism of the tumor invasion and metastasis of breast carcinoma.SETTING and PARTICIPANTS: Eighty-two filed wax masses of samples from the removed breast carcinoma and 30 filed wax masses of samples from the control group were collected at random from the First Affiliated Hospital of Fujiatt Medical University.MAIN OUTCOME MEASURE: The expression of MMP-2, MMP-9 and the distributive form of CollV in the tissues of breast carcinoma and in the tissues without breast carcinoma, and the correlation of the results with clinieopathological tumor parameters.METHODS: Tissues were obtained from 82 patients with breast carcinoma and 30 without breast carcinoma. The expression of MMP-2, MMP-9 and the distributive form of ColIV were examined, and their correlation with clinicopathological tumor parameters was explored.RESULTS: ① The positive incidences of MMP-2 and MMP-9 expression in 82 cases of breast carcinoma were 52 % (43/82) and 54% (44/82) respectively, The distributive form of encapsulated CollV was 15% (12/82),and the distributive form of decomposed CollVwas 85% (70/82), which were different apparently from those of the control group, ② The expressions of MMP-2/MMP-9 were related to the distribution form of ColIV( P &;lt; 0.05).③The expression of MMP-9 was related with the metastasis of the axillary lymph node (X^2 =8, 1899, P &;lt; 0, 05), and the expressions of MMP-2 was not related with the metastasis of the axillary lymph node(X^2 = 3. 3590, P&;gt;0.05),CONCLUSION: MMP-2, MMP-9 play a pivotal role in tumor invasion and metastasis. The result is suggested that detection of MMP-2/MMP-9 helps to judge the malignant severity of breast carcinoma and its biological behavior.     

多器官功能障碍综合征大鼠肺组织血栓调节蛋白和基质金属蛋白酶-9的表达

Objective To determine the expressions of thrombomodulin (TM) and matrix metalloproteinase-9(MMP-9) in the lung of rats with multiple organ dysfunction syndrome (MODS) and to investigate the mechanism of lung injury in MODS. Method Forty adult mule Sprague-Dawley (SD) rats were randomly divided into two groups,namely the normal control group and the MODS model group. The rats of model group were further divided into four subgroups as per different intervals (6 h, 12 h, 24 h and 48 h) ,and there were 8 rats in each groups. The animal models of MODS were estabhshed by two hits,the left eyeball of each model rat was removed to bleed to 2 mL/100g,and four hours later, lipopolysaccharide (LPS 5 mg/kg) was injected into intraperitoneal cavity of model rats. The same volume of saline was injected intraperitoneally into rats of control group instead of LPS. All rats were sacrificed at various intervals. The histological changes in lung tissue were observed by naked eye and light microscope. The expressions of TM and MMP-9 proteins were deteceted by using immunohistechemistry. One-way ANOVA was used for comparison among multiple group. Results (1)There were no histopathological changes in lung of rats of control group, and the lung injury was serious in MODS rots. (2) Compared with the rots of con-trol group, the expression of TM in lung tissue of MODS rats increased 6 hours after LPS, reached peak 12 hours later(P <0.01),and then decreased during 24～48 period.There was no significant difference in expression of TM between two groups 48 hours later. Compared with control group, the expressions of MMP-9 in lung tissue of MODS rats didn't significantly increase 6 ～ 48 hours after LPS (P < 0.01). Conclusions There are endothelium damage and extracellular matrix damage found in the lung tissue of rats at the early phase of MODS. TM and MMP-9 are good biomarkers of endothelium and extracellular matrix damage, and they can be used for diagnosing and es-timating the severity of injury lungs at the early phase of MODS.     

脓毒症大鼠肺高迁移率族蛋白B1的表达及意义

目的 观察创伤弧菌脓毒症大鼠肺组织HMGB1表达和肺损伤的动态变化,并探讨HMGB1在创伤弧菌脓毒症肺损伤中作用.方法 温州医学院生命科学院实验室,清洁级SD大鼠60只,随机分为正常组(A组,n=10)和创伤弧菌脓毒症组(B组,n=50),采用大鼠左下肢皮下注射创伤弧菌悬液(浓度为6×108cfu/mL,剂量为0.1 mL/100 g)制作大鼠创伤弧菌脓毒症模型),B组于染菌后1、6、12、24、48 h后活杀(各时间点n=10),采用逆转录聚合酶链式反应(RT-PCR)和蛋白免疫印迹(Western blot)分别检测大鼠肺组织HMGB1基因与蛋白的表达,检测肺含水分数和光镜观察肺组织病理变化,数据采用单因素方差分析,并用LSD法进行组间两两比较,P＜0.05为差异有统计学意义.结果 B组染菌后12 h(1.161±0.358,P=0.013)、24 h(1.679±0.235,P=0.000)及48 h(1.258±0.274,P=0.004)大鼠肺组织HMGB1 mRNA表达量较A组(0.652±0.177)明显增高(P＜0.05),并于24 h达到高峰;与A组(0.594±0.190)比较,B组HMGB1蛋白表达量于感染后6 h(1.408±0.567,P=0.026)(P＜0.05)逐渐增加,24 h达到高峰(2.415±1.064,P=0.000);与A组(0.699±0.054)比较,B组大鼠肺含水分数于感染后6 h(0.759±0.030,P=0.001)、12 h(0.767±0.023,P=0.000)、24 h(0.771±0.043,P=0.000)和48 h(0.789±0.137,P=0.000)明显增大(P＜0.05),呈逐渐递增趋势;感染后12 h,大鼠肺内血管明显充血,间质水肿并伴炎性浸润,且逐渐加重,到48 h肺泡腔塌陷明显,肺泡间隔分界不清.结论 大鼠创伤弧菌脓毒症可导致肺脏损伤,HMGB1的表达增加可能是创伤弧菌脓毒症大鼠肺组织损伤的机制之一.

Abstract：

Objective To observe the dynamic changes of high mobility group protein B1 ( HMGB1 )expression in the lung of rats with Vibrio vulnificus sepsis so as to unravel the role of HMGB1 in lung injury.Methods Sixty rats of clean grade were randomly divided into normal control group ( A group, n = 10) and Vibrio vulnificus sepsis group (B group, n =50). Sepsis model was made in rats with subcutaneous injection of Vibrio vulnificus with concentration of 6 × 108 cfu/ml in dose of 0. 1 ml/100 g into left lower limb.The rats of group B were sacrificed 1 h, 6 h, 12 h, 24 h and 48 h after infection for taking lung tissues to detect the water content of lung and to observe the histopathological changes in lung under light microscope.The expression of HMGB1 mRNA and the level of HMGB1 protein in the lungs were detected by RT-PCR and Western blot, respectively. Data were analysed with ANOVA and LSD method for comparison between groups, and P ＜0.05 was considered statistically significant. Results Compared with the group A (0.652±0. 177), the expressions of HMGB1 mRNA in lung of rats of group B were significantly higher in 12 hours (1. 161 ±0.358, P=0.013), 24 hours (1.679 ±0.235, P =0.000) and 48 hours (1.258 ±0.274, P=0.004) and reached the peak in 24 h. Compared with group A (0.594 ±0. 190), the level of HMGB1 protein in rats of group B 6 h after infection ( 1. 408 ± 0. 567, P = 0. 026) was significantly increased (P＜0.05), and it reached peak in 24 h (2.415 ± 1.064, P =0.000) after infection. Compared with group A (0.699 ± 0.054), the lung water contents in rats of group B were significantly increased in 6 h (0.759±0.030, P=0.001), in 12 h (0.767 ±0.023, P =0.000), in 24 h (0.771 ±0.043, P=0.000) and in 48 h (0.789 ±0.137, P=0.000) after infection. Compared with group A, the pathological changes in the lung of rats in group B showed clearly marked pulmonary vascular congestion, interstitial edema and inflammatory cell infiltration, and those changes became more and more serious until alveolar sacs entirely collapsed and the boundaries of the alveolar septa could not be clearly identified in 48 h. Conclusions Vibrio vulnificus sepsis leads to the lung injury of infected rats, and the increase in the expression of HMGB1 mRNA in lung might be one of the mechanisms of lung injury in rats with Vibrio vulnificus sepsis.     

基质金属蛋白酶-2在胰腺癌组织中的表达及其临床意义

Objective To investigate the expression of matrix metalloproteinase-2 (MMP-2)in pancreatic carcinoma and the relationship between MMP-2 with tumour clinicopatholngical features. Methods The expression of MMP-2 was detected by S-P immunohistochemistry in 36 cases with pancreatic carcinoma and 14 normal pancreat-ic tissues. Results The positive expression rate of MMP-2 was 66.7% (24/36) in pancreatic carcinoma tissue and 14.3% (2/14) in normal pancreatic tissues (χ2 = 3. 587, P < 0.01 ) ;The expression rate of MMP-2 in pancreatic carcinoma tissue with positive-node was 86.7% ( 13/15 ) ,which was higher than that with negative-node,which was 52.4% ( 11/21 ) ( P < 0.05 ) ; As to TNM staging in pancreatic carcinoma, the expression rate of MMP-2 was 41.2% (7/17) with Ⅰ,Ⅱ staging and 89.5% (17/19) with Ⅲ,Ⅳ staging(χ2=9.418,P <0. 01 ) ;The expression rate of MMP-2 was 50.0% (5/10) ,66.7% (10/15) and 72.8% (8/11) in highly,moderately and poorly differentiated pancreatic carcinoma(P > 0.05 ). Conclusions The expression of MMP-2 is strengthened significantly in pancreatic carcinoma tissue and involved in turnout invasion and metastasis features; MMP-2 might be regarded as one more marker for the invasive properties of pancreatic carcinoma.     

Effects of rosiglitazone on the expression of nuclear factor-κB P65 and metalloproteinase-9 mRNA in peripheral blood monocyte-derived macrophages in patients with coronary artery disease

Objective To investigate the effects and mechanisms of rosiglitazone on the expressions of nuclear factor-κB and matrix metalloprotease (MMP-9) in peripheral blood monocyte-derived macrophages (MDMs) in patients with coronary heart disease. Method This was a clinical case-control study. Forty-eight actue coronary symdrome (ACS) patients (ACS group), and 20 patients with stable angina (SA) (control group) were collected. They were performed coronary arteriography in the Department of Cardiology of the Second Xiangya Hospital from March to April in 2007. Exclusion criteria included acute infection, trauma or surgery patients within four weeks, cerebral vascular accident, liver and kidney dysfunction, cancer, and so on. The peripheral blood mononuclear cells were isolated and transformed into MDMs with macrophage colony-stimulating factor treatment. The transformed MDMs were randomly assigned into subgrougs and incubated with 0 /μmol/L, 1 μmol/L, 10 μmol/L, 20 μmol/L of rosiglitazone respectively. The expressions of PPAR-γ mRNA, MMP-9 mRNA were determined by RT-PCR and nuclear factor-κB P65 (NF-KB P65) expression by immunohistochemistry. Multiple comparisons were examined for significant differences using analysis of variance (ANOVA). Results The basal expression of PPAR-y mRNA was lower, in contrast, the levels of NF-KB P65 and MMP-9 mRNA were higher in ACS group than control group. PPAR-γ mRNA expression were significantly upregulated in both ACS and control groups with rosiglitazone treatment. PPAR-γ mRNA expression was positive correlation, while the expressions of MMP-9 mRNA were negative correlation with the rosiglitazone concentration in the ACS group. Rosiglitazone inhibited the expression of NF-KB in a concentration-independent manner in ACS and control groups. Conclusions The expression of PPAR-y mRNA is inhibited, while the activity of NF-KB and expression of MMP-9 mRNA are enhanced in MDMs of ACS cases. Rosiglitazone intervention may inhibit NF-KB activity and MMP-9 expression by upregulation of PPAR-y expression in MDMS of patiens with ACS.     

Effects of rosiglitazone on the expression of nuclear factor-κB P65 and metalloproteinase-9 mRNA in peripheral blood monocyte-derived macrophages in patients with coronary artery disease

Objective To investigate the effects and mechanisms of rosiglitazone on the expressions of nuclear factor-κB and matrix metalloprotease (MMP-9) in peripheral blood monocyte-derived macrophages (MDMs) in patients with coronary heart disease. Method This was a clinical case-control study. Forty-eight actue coronary symdrome (ACS) patients (ACS group), and 20 patients with stable angina (SA) (control group) were collected. They were performed coronary arteriography in the Department of Cardiology of the Second Xiangya Hospital from March to April in 2007. Exclusion criteria included acute infection, trauma or surgery patients within four weeks, cerebral vascular accident, liver and kidney dysfunction, cancer, and so on. The peripheral blood mononuclear cells were isolated and transformed into MDMs with macrophage colony-stimulating factor treatment. The transformed MDMs were randomly assigned into subgrougs and incubated with 0 /μmol/L, 1 μmol/L, 10 μmol/L, 20 μmol/L of rosiglitazone respectively. The expressions of PPAR-γ mRNA, MMP-9 mRNA were determined by RT-PCR and nuclear factor-κB P65 (NF-KB P65) expression by immunohistochemistry. Multiple comparisons were examined for significant differences using analysis of variance (ANOVA). Results The basal expression of PPAR-y mRNA was lower, in contrast, the levels of NF-KB P65 and MMP-9 mRNA were higher in ACS group than control group. PPAR-γ mRNA expression were significantly upregulated in both ACS and control groups with rosiglitazone treatment. PPAR-γ mRNA expression was positive correlation, while the expressions of MMP-9 mRNA were negative correlation with the rosiglitazone concentration in the ACS group. Rosiglitazone inhibited the expression of NF-KB in a concentration-independent manner in ACS and control groups. Conclusions The expression of PPAR-y mRNA is inhibited, while the activity of NF-KB and expression of MMP-9 mRNA are enhanced in MDMs of ACS cases. Rosiglitazone intervention may inhibit NF-KB activity and MMP-9 expression by upregulation of PPAR-y expression in MDMS of patiens with ACS.     

针刺联合微创血肿抽吸术对家兔急性期脑出血灶周围脑组织损伤的影响

目的 观察针刺联合微创血肿抽吸术对家兔急性期脑出血灶周围脑组织损伤的影响.方法 共选取健康新西兰大白兔65只,将其随机分为假手术组、脑出血组、血肿抽吸组及血肿抽吸+针刺组.将脑出血组、血肿抽吸组及血肿抽吸+针刺组制成脑出血模型,制模后血肿抽吸组给予血肿抽吸治疗,血肿抽吸+针刺组则给予针刺及血肿抽吸联合治疗.分别于制模后6 h、24 h、3 d及7 d时称量不同组别脑组织干湿重并计算脑含水量,采用免疫组化法检测各组家兔血肿周围脑组织基质金属蛋白酶-9(MMP-9)表达,采用明胶酶谱技术检测各组家兔血肿周围脑组织MMP-9活性.结果 脑出血组、血肿抽吸组及针刺+血肿抽吸组脑含水量均较假手术组显著增高,随着时间进展,血肿抽吸组及针刺+血肿抽吸组脑含水量较脑出血组均有明显降低(P＜0.05或0.01),其中以制模后第3天时针刺+血肿抽吸组的下降幅度尤为显著(P＜0.01);免疫组化及明胶酶谱检测结果显示,各组家兔MMP-9阳性细胞表达及活性均随时间进展呈下降趋势,其中以制模后第3天时针刺+血肿抽吸组的下降幅度最为显著(P＜0.01).结论 针刺联合血肿抽吸术可显著减轻家兔急性期脑出血灶周围脑组织损伤,其治疗机制可能与抑制MMP-9表达有关.

Abstract：

Objective To investigate the effect of acupuncture combined with minimally invasive hematoma aspiration on a rabbit model of brain injury in the acute stage of intracerebral hemorrhage (ICH). Methods A total of 65 healthy New Zealand white rabbits were randomly divided into a sham operation (SO) group, an ICH group,a hematoma aspiration (HA) group, and an acupuncture and hematoma aspiration (AHA) group. Models of ICH were established in the latter three groups. The HA group was treated with minimally invasive hematoma aspiration and the AHA group was treated with both acupuncture and minimally invasive hematoma aspiration. At 6 hours and 1, 3 and 7 days after the ICH models were established, brain water content (BWC) was measured, and the expression of matrix metalloproteinase-9 ( MMP-9 ) in the rabbits' brains was detected by immunohistochemistry. MMP-9activity was detected by gelatin zymography. Results The BWCs of the ICH group, HA group and AHA group rabbits were significantly higher than those of the SO group. The BWCs of the HA group and AHA group animals descended significantly more than those in the ICH group as time went on, especially on the 3rd day in the AHA group. Immunohistochemistry and gelatin zymography showed that the expression and activity of MMP-9 in these test groups decreased with time, especially on the 3rd day in the AHA group. Conclusions Acupuncture combined with hematoma aspiration can reduce injury in the acute stage of ICH, and inhibition of the expression of MMP-9 may be the mechanism.     

基质金属蛋白酶家族的表达对急性单核细胞白血病细胞体外侵袭力的影响

目的 研究基质金属蛋白酶家族(TIMP-2、MT1-MMP、MMP-2)的表达对急性单核细胞白血病细胞体外侵袭力的影响.方法 以急性单核细胞白血病细胞株SHI-1细胞为研究对象,用定量PCR、Western blot法分别检测TIMP-2、MT1-MMP、MMP-2 mRNA和蛋白表达,并以NB4、K562、THP-1等人类其他白血病细胞株细胞为对照,进行比较.构建TIMP-2逆转录病毒载体,转染SHI-1细胞,G418筛选,有限稀释法挑选出单克隆S1、S2、S3细胞.RNA干扰(RNAi)法干扰SHI-1细胞TIMP-2、MT1-MMP、MMP-2表达.明胶酶谱法测定不同细胞和骨髓基质细胞(BMSC)共培养24 h后上清中MMP-2的表达,细胞侵袭实验测定SHI-1细胞侵袭人工基质膜的能力.结果 SHI-1细胞的TIMP-2、MT1-MMP、MMP-2 mRNA表达和蛋白表达均显著高于其他细胞(P＜0.05).SHI-1细胞和BMSC共培养上清中的MMP-2酶原和活化的MMP-2含量及细胞体外侵袭率均高于其他细胞(P＜0.05).单克隆S1、S2、S3细胞TIMP-2 mRNA表达水平分别是SHI-1细胞的3.0倍、2.0倍和1.5倍(P＜0.05),蛋白表达水平分别上调2.6倍、1.5倍和1.3倍(P＜0.01),体外侵袭率增加1.5～2.5倍(P＜0.05),活化的MMP-2含量增加1.5～3.0倍(P＜0.01).RNA干扰基因沉默效率为85%～98%.SHI-1细胞TIMP-2、MMP-2、MT1-MMP表达沉默后,细胞侵袭率分别下降60%～70%、50%～60%、40%～50%(P＜0.05).RNA干扰后的细胞培养上清中检测不到活化的MMP-2.结论 SHI-1细胞在mRNA水平和蛋白水平均高表达TIMP-2、MT1-MMP、MMP-2 mRNA,SHI-1细胞和BMSC共培养后这些分子的高表达促进MMP-2的活化,增强细胞的体外侵袭力.TIMP-2表达增加1.5～2.5倍对SHI-1细胞MMP-2的活化和细胞的体外侵袭力发挥的是增强作用,而不是抑制作用.

Abstract：

Objective To study the effect of matrix metalloproteinase 2 ( MMP-2), membrane type 1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase 2 (TIMP-2) expressions on the in vitro invasive capacity of acute monocytic leukemia SHI-1 cells. Methods SHI-1, NB4, K562, M937 and THP-1 human leukemia cell lines were cultured in vitro. The mRNA and protein expressions of TIMP-2, MMP-2 and MT1-MMP in different cells were detected by quantitative RT-PCR and western blot. A retroviral vector carrying human TIMP-2 cDNA was constructed and transfected into SHI-1 cells. Three subclone cells (S1, S2 and S3) were screened by G418 and selected by limiting dilution. RNA interference (RNAi) was used to knock down the expression of MMP-2, MT1-MMP and TIMP-2. Cell invasion capacity was performed through a reconstituted human basement membrane assays. Zymography was used to analyze the expression of MMP-2 in the supernatant of co-culture. Results The expressions of MMP-2, MT1-MMP and TIMP-2 in SHI-1 cells were higher than that in other leukemic cells at both mRNA and protein levels (P ＜ 0. 05 ). The amount of proMMP-2 and activated MMP-2 in the conditioned media from SHI-1 cells co-cultured with bone marrow stromal cells (BMSCs) was more than that from other cells (P ＜ 0. 05 ). The in vitro invasive capacity of SHI-1 cells were higher than that of other cells( P ＜ 0.05 ). The mRNA levels of TIMP-2 were increased by about 3 fold, 2 fold and 1.5 fold in S1, S2 and S3 cells, respectively( P ＜ 0.05 ), while the protein levels were by about 2.6 fold, 1.5 fold and 1.3 fold than that of SHI-1 cells, respectively(P ＜ 0.01 ). The invasion rates of subclone cells demonstrated a 1.5 - 2.5 fold' elevation ( P ＜ 0.05 ) and activated MMP-2 from their supernatants increased by 1.5 -2.0 fold(P＜0.01 ). The knock-down efficiency of siRNA was 85% to 98%. The down-regulation of TIMP-2, MMP-2 and MT1-MMP decreased the invasion rates of SHI-1 cells by 60% -70%, 50% - 60% and 40% - 50%, respectively ( P ＜ 0. 05 ). No activated MMP-2 in the supernatants from any knock-down cells could be found. Conclusions SHI-1 cells constitutively overexpress MMP-2,MT1-MMP and TIMP-2 at both mRNA and protein levels. After co-cultured with BMSCs the SHI-1 cells increased MMP-2 activation and cell invasion. An increase of TIMP-2 expression in SHI-1 cells reflects an activating effect on cells invasion and MMP-2 activation.     

MMP-9、TIMP-1及VEGF在大肠癌中的表达与临床病理和预后的相关性研究

目的研究MMP-9、TIMP-1及VEGF在大肠癌组织中的表达与临床病理和预后关系。方法对62例大肠癌患者的手术切除之癌组织和癌周正常组织,应用免疫组织化学方法检测其VEGF、基质金属蛋白酶(MMP-9)和组织金属蛋白酶抑制剂(TIMP-1)的表达情况,探讨这些因子表达与临床病理和预后关系。结果在62例大肠癌肿瘤组织:有29例VEGF表达阳性,阳性率46.8%;41例MMP-9表达阳性,阳性率为66.1%;39例TIMP-1表达阳性,阳性率为62.9%。这三种蛋白的表达水平与组织类型、组织分化程度、淋巴结转移和临床分期有统计学相关性(P<0.001)。MMP-9蛋白表达与五年生存率有影响(P<0.01),VEGF和TIMP-1蛋白表达对预后无影响(P>0.05)。结论大肠癌中MMP-9、TIMP-1及VEGF的表达,可能是癌肿发生、生长和浸润转移的重要因素,可作为判断肿瘤恶性程度和预后的重要指标。     

Effect of hemodialysis on the plasma level of type IV collagenases and their inhibitors

OBJECTIVES: Increased cell expression of matrix metalloproteinase-9 (MMP-9) was associated with the development of atherosclerosis and osseoarticular tissue destruction in hemodialysis patients. In this study, the pre- and post-HD plasma concentrations of type IV collagenases and their inhibitors in HD patients were examined. DESIGN AND METHODS: Commercial ELISA kits and Zymography techniques were used to assay the parameters in 40 patients pre- and post-HD session. RESULTS: After the hemodialysis process, MMP-9, MMP-2 and TIMP-2 levels were 124 +/- 72, 706 +/- 242 and 248 +/- 90 ng/mL, significantly different from the pre-HD values (187 +/- 148, 759 +/- 304 and 43 +/- 14 ng/mL). TIMP-1 were not affected by HD. Female subjects and patients with chronic glomerulonephritis had higher TIMP-2 than their counterparts (p < 0.05). The effect of gender on MMP-2 levels was interacted with that of membrane types (p < 0.01). CONCLUSION: These results indicated that the hemodialysis process tends to decrease the overall activity of the peripheral plasma MMP system in HD patients.     

子宫内膜异位症中金属蛋白酶9及其特异性组织抑制因子的表达及临床意义

目的:探讨基质金属蛋白酶9(MMP-9)、金属蛋白酶特异性组织抑制因子1(TIMP-1)在子宫内膜异位症患者内膜标本中表达水平的变化及其临床意义。方法:收集子宫内膜异位症患者的异位内膜56例(观察组)和非内异症患者的子宫内膜标本18例(对照组),采用RT-PCR半定量方法检测内膜标本中MMP-9、TIMP-1 mRNA表达率及表达强度。结果:观察组和对照组所有内膜标本均有TIMP-1 mRNA表达;观察组和对照组标本中MMP-9 mRNA的表达率分别为58.9%和50%;观察组异位内膜中MMP-9 mRNA的表达强度高于对照组(P<0.05);TIMP-1 mRNA的表达强度低于对照组(P<0.05);对于MMP-9/TIMP-1比值,观察组明显高于对照组(P<0.01),早期(I-II期)明显高于III、IV期(P<0.01)。结论:子宫内膜异位症患者内膜中TIMP-1 mRNA的表达减弱,降低了对MMP-9的抑制作用;同时子宫内膜异位症患者内膜MMP-9 mRNA的表达增强,加强了其侵袭能力,易于发生种植转移,这些因素可能在子宫内膜异位症的发生发展过程中发挥重要作用。     

大肠癌侵袭、转移CT征象与MMP-9及TIMP-1表达

目的探讨大肠癌侵袭和转移CT征象与癌组织中MMP-9及TIMP-1表达间的关系.方法对45例经手术病理证实且行螺旋CT平扫、动静脉期双期增强的大肠癌患者,观察每个病灶的浆膜侵犯和淋巴结转移情况.用免疫组化方法检测癌组织中MMP-9及TIMP-1表达情况,并分析其与CT征象的关系.结果CT征象上肿瘤的浆膜侵犯、淋巴结转移与MMP-9及TIMP-1蛋白表达有关(P＜0.05).结论大肠癌侵袭和转移CT征象可以在一定程度上反映MMP-9及TIMP-1的表达.     

非ST抬高性急性冠脉综合征患者血清MMP-9及TIMP-1与AngⅡ浓度改变意义

目的:观察不同类型冠心病患者血清MMP-9,TIMP-1和AngⅡ的血清浓度及相关关系,探讨非ST抬高性急性冠脉综合征发病机制。方法:冠心病患者分为非ST抬高性急性冠脉综合征和稳定心绞痛组,每组患者30例,另设对照组30例,比较各组间血清MMP-9,TIMP-1,MMP-9/TIMP-1和AngⅡ水平。结果:非ST抬高性急性冠脉综合征组血清MMP-9,MMP-9/TIMP-1和AngⅡ水平高于对照组,差异有统计学意义(P<0.01),但稳定心绞痛组血清MMP-9,TIMP-1,MMP-9/TIMP-1和AngⅡ水平与对照组比较差异无统计学意义(P>0.05),急性冠脉综合征组患者血清MMP-9,MMP-9/TIMP-1与AngⅡ水平呈正相关(P<0.01)。结论:血清MMP-9,MMP-9/TIMP-1和AngⅡ水平的增高与急性冠脉综合征相关,可作为评价冠状动脉粥样硬化斑块稳定性与病变严重程度的一个参考指标。     

复方藤梨根制剂抑制胃癌转移与MMP-9、TIMP-1和E-cad表达的关系研究

目的观察复方藤梨根制剂对胃癌细胞MMP-9、TIMP-1和E—cad蛋白表达的影响,探讨其抗胃癌细胞转移的作用机制。方法以SGC-7901胃癌细胞株建立胃癌裸鼠原位移植瘤模型,将裸鼠随机分为0．9％氯化钠溶液组（对照组）、复方藤梨根制剂低剂量、中剂量及高剂量组,接受连续21d灌服治疗。待裸鼠衰竭或死亡后解剖观察肿瘤转移等情况;用免疫组化方法检测肿瘤组织中MMP-9、TIMP-1和和E—cad蛋白表达的变化。结果根据胃癌裸鼠动物模型的转移部位进行赋值评分,氯化钠溶液与中、高剂量组比较差异有统计学意义（F=5．15,P〈0．05）,与低剂量组之间比较差异无统计学意义（F=1．27,P〉0．05）。经免疫组化法检测．对照组的胃癌细胞的MMP-9明显高于中、高剂量复方藤梨根制剂组（B3．86,P〈0．05）;对照组的胃癌细胞的,TIMP-1蛋白表达明显低于中、高剂量复方藤梨根制剂组（止4．30．P〈0．05）：而E—cad表达各组之间差异无统计学意义（F=0．41。P〉0．05）。结论复方藤梨根制剂可能是通过下调MMP-9和上调TIMP-1的途径发挥抗胃癌转移作用：而对E—cad的异常表达没有明显调节作用。     

MMP-9和TIMP-1在高危骨肉瘤中的表达及其诊断意义

目的研究高危骨肉瘤中MMP-9和TIMP-1的表达情况,探讨MMP-9／TIMP-1失衡在高危骨肉瘤中的诊断意义。方法对14例高危骨肉瘤、41例恶性程度低的骨肉瘤和15例正常骨组织行MMP-9和TIMP-1免疫组织化学染色,并对染色情况进行分析。结果高危骨肉瘤组的MMP-9的表达率为100％,呈普遍过表达,明显高于低恶性骨肉瘤组的73．2％的阳性率（P〈0．05）,而高危骨肉瘤组的TIMP-1的阳性率明显低于低恶性骨肉瘤组（P〈0．05）。高危骨肉瘤组的MMP-9／TIMP-1表达失衡率为42．8％,明显高于低恶性骨肉瘤组的12．2％（P〈0．05）。结论MMP-9与TIMP-1可作为诊断骨肉瘤的辅助指标。MMP-9与TIMP-1的表达失衡可以作为高危骨肉瘤的诊断指标之一.     

组织微阵列检测结直肠癌组织中MMP-2、MMP-9和TIMP-2蛋白的表达

目的 探讨MMP-2、MMP-9 和TIMP-2蛋白表达与结直肠癌侵袭转移的关系,寻找用于预测结直肠癌侵袭转移的分子标记物.方法 组织微阵列结合免疫组化技术检测结直肠癌及其癌旁正常肠黏膜上皮中的MMP-2、MMP-9 和TIMP-2蛋白表达.结果 结直肠癌组织中,MMP-9蛋白阳性表达率显著高于癌旁肠黏膜组织(P<0.001),TIMP-2蛋白阳性表达率显著低于癌旁黏膜组织(P<0.05).浸润至肠壁浆膜层的结直肠癌和Dukes'C、D期癌组织的MMP-2蛋白阳性表达率显著高于浸润肠壁浅、深平滑肌层和Dukes'A、 B期结直肠癌(P<0.05);肠壁局部淋巴结转移的结直肠癌中MMP-2、MMP-9 蛋白阳性表达率显著高于无淋巴结转移癌(P<0.05);Dukes'A、B期结直肠癌TIMP2蛋白阳性表达率显著高于Dukes'C、D期(P<0.05).TIMP2蛋白阳性的结直肠癌生存时间显著长于TIMP2阴性的结直肠癌(P<0.05).结直肠癌中MMP-2蛋白阳性表达与MMP-9表达呈正相关(P＜0.001),但TIMP-2蛋白阳性表达与MMP-2表达呈显著负相关(P<0.05).二变量多因素回归分析显示,MMP-2蛋白表达、癌组织的浸润深度和Dukes分期可作为预测结直肠癌淋巴结转移的独立指标.结论 结直肠癌MMP-9和TIMP-2蛋白共同高表达及MMP-2与TIMP-2蛋白的表达失衡在结直肠癌侵袭、转移中起重要作用.MMP-2蛋白表达可作为结直肠癌淋巴结转移的独立预测指标.     

结核性胸膜炎患者胸水中MMP-9和TIMP-1水平的临床意义

目的：探讨基质金属蛋白酶-9（MMP-9）、基质金属蛋白酶组织抑制物-1（TIMP-1）及MMP-9/TIMP-1比值在结核性胸膜炎致胸腔积液的形成过程中的作用及鉴别诊断中的价值。方法：采用ELISA法测定36例结核性胸膜炎、30例非结核性胸膜炎患者胸水中的MMP-9和TIMP-1浓度。结果：（1）结核性胸腔积液组胸水中MMP-9浓度、TIMP-1浓度和MMP-9/TIMP-1比值均显著高于非结核性胸腔积液组（83.76±47.36ng.mL^-1比38.83±16.53ng.mL^-1,600.17±121.46ng.mL^-1比481.67±96.36ng.mL^-1,0.147±0.097比0.081±0.018,P〈0.001）。（2）胸水中MMP-9、TIMP-1、MMP-9/TIMP-1比值在结核性胸腔积液诊断中的敏感性分别为88.9%、72.2%和80.6%,特异性分别为65.4%、63.5%和75.0%。采用胸水MMP-9和TIMP-1串联联合检测的敏感性和特异性分别为33.3%和94.2%,并联联合检测的敏感性和特异性分别为83.3%和53.8%。结论：MMP-9、TIMP-1及MMP-9/TIMP-1比例的失衡与结核性胸膜炎胸腔积液的形成密切相关,它们在结核性胸膜炎所致胸腔积液的诊断中有一定参考价值。