铜绿假单胞菌耐药性分析及金属酶基因检测

目的分析中南大学湘雅医院临床分离铜绿假单胞菌抗菌药物敏感性及金属酶流行情况,以期指导临床用药。方法收集2010年11月—2011年4月临床分离非重复铜绿假单胞菌200株,所有菌株均经VITEK-2全自动微生物鉴定与药敏试验分析系统进行鉴定和药敏试验。采用双纸片协同试验作为金属酶表型初筛试验,初筛阳性菌株采用聚合酶链反应(PCR)检测IMP、VIM、SPM和NDM4种金属酶基因型以及Ⅰ类整合酶基因。结果 200株铜绿假单胞菌分离自ICU患者55株,占27.5%。药敏试验结果显示,该菌对美罗培南耐药率最低(8.5%),对庆大霉素耐药率最高(38.5%)。6株铜绿假单胞菌金属酶初筛试验阳性,检出率3.0%,经基因检测发现IMP型阳性菌株2株(2株均产IMP-1菌株),未检出VIM、SPM及NDM型金属酶,且这6株铜绿假单胞菌Ⅰ类整合酶基因均为阳性。结论该院产金属β内酰胺酶的铜绿假单胞菌检出率较低,主要金属酶基因型为IMP-1。铜绿假单胞菌耐药仍较严重,但对碳青霉烯类抗生素耐药率低。

多重耐药鲍曼不动杆菌携带金属酶与整合酶基因特征及同源性分析

目的了解吉林地区多重耐药鲍曼不动杆菌(Multidrug-resistant Acinetobacter baumanni,MDRAB)金属酶与整合酶基因的类型,并对菌株进行同源性分析.方法收集吉林地区2所三级甲等医院临床标本分离鉴定的鲍曼不动杆菌38株.多重耐药株携带金属酶的初筛实验-双纸片协同试验:金属酶与整合酶基因的检测-聚合酶链反应(PCR),并对其进行同源性分析.结果在所有MDRAB菌株亚胺培南-EDTA协同试验中,15株(39.4%)阳性,16株(42.1%)携带整合酶基因Ⅰ,2株(5.2%)携带整合酶基因Ⅱ,2株(5.2%)携带blaVIM型金属酶基因,1株(2.6%)携带blaNDM-1型金属酶基因,未检测出金属酶基因blaIMP,blaSIM-1和整合酶基因Ⅲ.结论该地区临床分离的MDRAB耐药率高,可能与其携带金属酶基因和整合酶基因有关,A型MDRAB为医院感染主要流行株.

临床分离铜绿假单胞菌耐药机制研究

目的探讨临床分离的铜绿假单胞菌的耐药性及10种金属酶基因的存在情况和生物被膜产生能力,为临床合理选用抗菌药物提供依据。方法对2016年5~11月临床分离的89株铜绿假单胞菌,采用VITEK2 COMPACT仪器鉴定菌株,并检测抗菌药物敏感性,采用聚合酶链反应(PCR)检测菌株是否存在NDM-1、IMP、VIM、SPM、GIM、SIM、DIM、TMB、KHM、AIM等金属酶基因,采用结晶紫半定量方法检测菌株的生物被膜形成能力。结果 89株铜绿假单胞菌对碳青酶烯类抗菌药物亚胺培南的敏感率为73.03%,耐药率和中介率分别为17.98%和8.99%。89株均未检出NDM-1、IMP、VIM、SPM、GIM、SIM、DIM、TMB、KHM、AIM金属酶基因。92.13%的铜绿假单胞菌具有产生生物被膜能力,大部分菌株产生中量和大量生物被膜,分别占34.83%和41.57%。结论该组铜绿假单胞菌的耐药与携带NDM-1、IMP、VIM、SPM、GIM、SIM、DIM、TMB、KHM、AIM等金属酶基因无关,产生生物被膜为其主要耐药机制之一。

Expressions of inducible nitric oxide synthase and matrix metalloproteinase-9 and their effects on angiogenesis and progression of hepatocellular carcinoma

AIM： To determine the expressions of inducible nitric oxide synthase （iNOS） and matrix metalloproteinase-9 （MMP-9） in hepatocellular carcinoma （HCC） and to investigate the relationship between iNOS and MMP-9 expression and their effects on angiogenesis and progression of HCC.METHODS： In this study, we examined iNOS, MMP-9, and CD34 expression in specimens surgically removed from 32 HCC patients and 7 normal liver tissues by immunohistochemical staining. Meanwhile, microvessel density （MVD） was determined as a marker of angiogenesis by counting CD34-positive cells. RESULTS： The positive rates of iNOS and MMP-9 expression were 71.88% （23/32） and 78.13% （25/32） in HCC. MMP-9 expression was significantly correlated with tumor size, capsule status, TNM stage, and risk of HCC recurrence （P = 0.032, P= 0.033, P= 0.007, and P= 0.001, respectively）. There was also a significant relationship between iNOS expression and capsule status and risk of HCC recurrence （P = 0.049 and P = 0.004, respectively）, but no correlation between iNOS expression and tumor size and TNM stage. There was a positive association between MVD and TNM stage and risk of HCC recurrence （P = 0.037 and P = 0.000, respectively）. The count of MVD was significantly different in different iNOS and MMP-9 immunoreactivity groups （F= 17.713 and 17.097, P= 0.000 and P = 0.000, respectively）. The examination of Spearman＇s rank correlation coefficient showed that there was a significant positive correlation between MVD and iNOS, MMP-9 immunoreactivity （r = 0.754 and 0.751, P= 0.000 and P=-0.000, respectively）. There was also a significant association between MMP-9 and iNOS expression in HCC （P = 0.010）. CONCLUSION： Nitric oxide （NO） produced by iNOS could modulate MMP-9 production and therefore contribute totumor cell angiogenesis and invasion and metastasis in HCC. The strong expression of iNOS and MMP-9 in HCC may be helpful in evaluating the recurrence of HCC, predicting poor prognosis. For patients with strong expression of MMP-9 and iNOS, the optimal treatment scheme needs to be selected.

Relationship between matrix metalloproteinase-2 mRNA expression and clinicopathological and urokinase-type plasminogen activator system parameters and prognosis in human gastric cancer

AIM: To investigate the relationship between matrix metalloproteinase-2 (MMP-2) mRNA expression and clinicopathologic and urokinase-type plasminogen activator (uPA) system parameter and prognosis in human gastric cancer. METHODS: Expression of MMP-2 mRNA, uPA, and uPA-R mRNA in tumor tissues and ≥5 cm adjacent normal tissues from 67 cases of gastric cancer was studied using RT-PCR and Northern blot respectively.Survival analyses were done using the Kaplan-Meier method. RESULTS: The expression rates of MMP-2 mRNA,uPA and uPA-R mRNA in tumor tissues (31%,41%,and 51%, respectively) were significantly higher than those in ≥5 cm adjacent tissues (19%, 11%, and 9%; X2=4.59,43.58, and 53.24 respectively, P<0.05,0.0001,and 0.0001, respectively). Expression of MMP-2 mRNA was significantly correlated with lymph node metastasis (metastasis: 61.9%, no metastasis: 39.1%, X2= 7.61, P<0.05),Lauren's classification of diffuse/mixed types:54.2%,intestinal type: 26.3%,X2 = 4.25, P<0.05, expression of uPA and uPA-R mRNA (uPA+: 55.1%, uPA-: 22.2% and uPA-R+: 54.9%, uPA-R-: 18.8%, X2=5.72 and 6.40 respectively, P<0.05).Kaplan-Meier survival analysis of MMP-2 mRNA expression did not show significant difference in all 67 cases, but revealed an association of the expression of MMP-2 mRNA, uPA, and uPA-R mRNA with worse prognosis (P= 0.0083, 0.0160, and 0.0094, respectively). CONCLUSION: MMP-2 may play an important role in the development of invasion and metastasis of gastric cancer.

No association of the matrix metalloproteinase 1 promoter polymorphism with susceptibility to esophageal squamous cell carcinoma and gastric cardiac adenocarcinoma in northern China

AIM: To investigate association of the 2Gor1Gsingle nudeotide polymorphism (SNP) in matrix metalloproteinase 1 (MMP1) promoter with susceptibility to esophageal squam-ous cell carcinoma (ESCC) and gastric cardiac adenocarcinoma (GCA) in a population of North China. METHODS: MMP1 promoter SNP was genotyped by polymerase-chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis in 417 cancer patients (234 ESCC and 183 GCA) and 350 healthy controls. RESULTS: The genotype frequencies of the MMP1 promoter SNP in healthy controls were 55.4% (2G/2G), 30% (1G/2G) and 14.6% (1G/1G), respectively. The genotype and allelotype distribution in ESCC and GCA patients was not significantly different from that in healthy controls (all lvalues were above 0.05). Compared with the 1G/1Ggenotype, neither the 2G/2Gnor in combination with the 1G/2G genotype significantly modified the risk of developing ESCC and GCA, the adjusted odds ratio was 1.28 (95%CI = 0.78-2.09), 1.23 (95%CI = 0.38-2.05) in ESCC and 1.39 (95%CI = 0.80-2.41), 1.34 (95%CI = 0.74-2.40) in GCA, respectively. When stratified by smoking status and family history of upper gastrointestinal cancer, the 2G/2Ggenotype alone or in combination with the 1G/2G genotype also did not show any significant influence on the risk of ESCC and GCA development. In addition, influence of the MMP1 SNP on lymphatic metastasis in ESCC and GCA was also not obs-erved. CONCLUSION: The 2Gor 1GSNP in the MMP1 promoter might not modify the risk of ESCC and GCA development and might not be used as a stratification marker to predict the potential of lymphatic metastasis in these two tumor types.

Matrix metalloproteinases and their expression in mammary gland

The matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that play a key role in both normal and pathological processes involving tissue remodeling events. The expression of these proteolytic enzymes is highly regulated by a balance between extracellular matrix (ECM) deposition and its degradation, and is controlled by growth factors, cytokines, hormones, as well as interactions with the ECM macromolecules. Furthermore, the activity of the MMPs is regulated by their natural endogenous inhibitors, which are members of the tissue inhibitor of metalloproteinases (TIMP) family. In the normal mammary gland, MMPs are expressed during ductal development, lobulo-alveolar development in pregnancy and involution after lactation. Under pathological conditions, such as tumorigenesis, the dysregulated expression of MMPs play a role in tumor initiation, progression and malignant conversion as well as facilitating invasion and motastasis of malignant cells through degradation of the ECM and basement membranes. Matrix metalloproteinases and their expression in mammary gland

Theoretical basis of a beneficial role for vitamin D in viral hepatitis

Abnormal bone metabolism and dysfunction of the calcium-parathyroid hormone-vitamin D axis have been reported in patients with viral hepatitis. Some studies suggested a relationship between vitamin D and viral hepatitis. Genetic studies have provided an opportunity to identify the proteins that link vitamin D to the pathology of viral hepatitis (i.e., the major histocompatibility complex class Ⅱ molecules, the vitamin D receptor, cytochrome P 450 , the renin-angiotensin system, apolipoprotein E, liver X receptor, toll-like receptor, and the proteins regulated by the Sp1 promoter gene). Vitamin D also exerts its effects on viral hepatitis via non-genomic factors, i.e., matrix metalloproteinase, endothelial vascular growth factor, prostaglandins, cyclooxygenase-2, and oxidative stress. In conclusion, vitamin D could have a beneficial role in viral hepatitis. Calcitriol is best used for viral hepatitis because it is the active form of the vitamin D 3 metabolite.

Expression of tissue inhibitor of matrix metalloproteinases-1 during aging in rat liver

AIM: To investigate the expression and role of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) during natural aging in rat liver and to detect the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9. METHODS: The rats were divided into 3-mo-old group (n=5), 10-mo-old group (n=5) and 24-mo-old group (n=5). Histopathologic changes of liver were observed with HE and Masson stain. The location and protein expressions of TIMP-1 were determined by immunohistochemistry and Western blot; message RNA (mRNA) levels were measured in livers from rats of various ages by semi-quantitative reverse transcriptional polymerase chain reaction (RT PCR). In addition, the expression of MMP-2 and MMP-9 was assessed by RT-PCR and Western blot. RESULTS: Histologic examination showed that the aging liver had excessive fatty degeneration and collagen deposition. Immunohistochemical staining showed that TIMP-1 related antigen in livers was located in cytoplasm. The protein expression of TTMP-1 was significantly higher in the oldest animals and the mRNA expression was increased significantly in the 24-mo-old rats (t= 4.61, P= 0.002<0.05, 24-vs 10-mo-old rats; t=4.31, P=0.003<0.05, 24- vs 3-mo-old rats). The expression of MMP-2 and MMP-9 had no change during aging; the ratios TIMP-1/MMP-2 and TIMP-1/MMP-9 in aging liver were significantly higher than those in maturation and young livers. CONCLUSION: TIMP-1 may play an important role in the process of liver aging.

Expression of MMP-13 and its correlation with prognosis in non-small cell lung cancer

Objective:To investigate the expression of MMP-13 in non-small cell lung cancer(NSCLC),so as to analyze its correlation with prognosis of NSCLC. Methods:MMP-13 expression was detected in 99 NSCLC tissues and 32 normal lung tissues by immunohistochemical method. Results:(1) Expression of MMP-13(51.5%,51/99) in cancer tissues was signifi-cantly higher than that in normal tissues 0%(P < 0.05). Expression level of MMP-13 was significantly related to lymph node metastasis and clinical stage(P < 0.01).(2) Kaplan-Meier analysis showed that expression level of MMP-13 was closely cor-relate with the prognosis of NSCLC. Multivariate Cox model analysis suggested that the survival time was significantly related to clinical stage and the expression of MMP-13. Conclusion:MMP-13 is an independent factor that affect prognosis.

Effect of quercetin on expression of matrix metallo - proteinases and tissue inhibi tor of matalloproteinase -1 in cultured rat hepatic stellate cells

Objective: To study the effects of quercetin (QU) on matrix metallo-proteinases (MMPs) , the tissue inhibitor of matalloproteinase-1 (TIMP-1) , procollagen I and 2 proteoglycans (decorin and biglycan) mRNA expression in cultured rat hepatic stellate cell line HSC-T6 cells. Methods: Cells were treated with different concentrations of QU (12. 5, 25, 50 μmol/L) or drug solvent (0. 1 % Me2SO) for 24 h. mRNA expression was determined by reverse transcription polymerase chain reaction (RT-PCR). Results: QU (12.5 - 50 μmol/L) enhanced collagenase (rat MMP-13) and membrane typel-MMP (MMP-14) mRNA expression, decreased procollagen I mRNA expression in a concentration-dependent manner, but did not affect gelatinase-A (MMP-2) , TIMP-1, decorin and biglycan expression. Conclusion: QU may decrease matrix deposition and increase matrix degradation, which might be beneficial to liver fibrosis.

Prognostic value of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 in bladder carcinoma

OBJECTIVES: To detect the level of matrix metalloproteinase-2 (MMP-2) mRNA and the tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA in bladder transitional cell carcinoma (BTCC), and to estimate the prognosis for bladder tumor based on the quality and quantity of MMP-2 and TIMP-2 mRNA. METHODS: Thirty-five samples of human BTCC and 15 normal fresh bladder tissues were studied by RT-PCR analysis followed by computer-assisted image analysis. RESULTS: The level of the MMP-2 mRNA in BTCC was significantly increased compared with that in normal bladder epithelium. The positive rates of MMP-2 and TIMP-2 mRNA were 71.4% and 65.7% in BTCC, and 66.7% and 60.0% in the normal bladder wall. The expression intensity of the MMP-2 mRNA by image analysis tended to increase with tumor grading and staging, which showed statistical significance. Similarly, the MMP-2 to TIMP-2 ratio also showed statistically significant difference between normal bladder tissue and bladder carcinoma (P

Improvement in post-partum uterine involution in rats treated with Apios americana

Objective: Apios americana,a plant used as a staple ingredient of native American diets,has various properties,including anti-cancer,anti-hyperglycemic,hypotensive,and anti-inflammatory activity.In Japan,Apios is used as a post-natal medication.After parturition,women undergo a period of recovery as they return to pre-pregnancy conditions.However,few health products that aid post-partum recovery are on the market.We explored whether Apios can accelerate the post-partum recovery process,in particular the involution of the uterus.Methods: Female rats kept in individual cages were mated with two male rats,with the exception of the control group(female rats without mating,on basal diet;n=6).After delivery,rats were divided into five groups based on their diet: basal diet(model;n=6);basal diet+oral intake at 5.4 g/kg of Chanfukang granules(a Chinese patent medicine preparation for post-partum lochia)(positive;n=6);basal diet containing 10% Apios powder(low;n=6);basal diet containing 20% Apios powder(medium;n=6);basal diet containing 40% Apios powder(high;n=6).Five days later,uteri and spleens were weighed.Uterus and spleen indices for each rat were calculated by dividing visceral weight by the total weight.Hormone and cytokine concentrations were measured using enzyme-linked immunosorbent assay(ELISA).Histological analysis of uteri was completed using hematoxylin and eosin(H&E) staining.Expression of matrix metalloproteinases and inhibitors in uteri was measured by western blotting.Results: Our results showed that Apios treatment reduced the post-partum uterus index and regulated the hormone concentrations.Moreover,we found that the process of uterine involution was accelerated,based on morphological changes in the uterus.In addition,our results indicated that Apios alleviated the inflammatory response induced by the involution process.Transforming growth factor β was also found to be regulated by Apios.There were significant downregulation of matrix metalloproteinases and upregulation of their inhibitors by Apios,which suggested that Apios increased the rate of the collagen clearance process.Conclusions: These results,based on experimental observations at the molecular and protein levels,verified our hypothesis that Apios can improve uterine involution,and demonstrated the potential application of Apios in post-partum care.

Variants of genes encoding collagens and matrix metalloproteinase system increased the risk of aortic dissection

Aortic dissection （AD） is a devastating, heterogeneous condition of aorta. The homeostasis between collagens and matrix metalloproteases （MMPs）/tissue inhibitors of MMPs （TIMPs） system in the extracellular matrix plays an important role for structure and functions of aorta. However, our knowledge on association between variants of genes in this system and pathogenesis of AD is very limited. We analyzed all yet known coding human genes of collagens （45 genes）, MMPs/TIMPs （27 genes） in 702 sporadic AD patients and in 163 matched healthy controls, by using massively targeted next-generation and Sanger sequencing. To define the pathogenesis of potential disease-causing candidate genes, we performed transcriptome sequencing and pedigree co-segregation analysis in some genes and generated Col5a2 knockout rats. We identified 257 pathogenic or likely pathogenic variants which involved 88.89% （64/72） genes in collagens-MMPs/TIMPs system and accounted for 31.05% （218/702） sporadic AD patients. In them, 84.86% patients （185/218） carried one variant, 12.84% two variants and 2.30% more than two variants. Importantly, we identified 52 novel probablY pathogenic loss-of-function （LOF） variants （20 nonsense, 16 frameshift, 14 splice sites, one stop-loss, one initiation codon） in 11.06% （50/452） AD patients, which were absent in 163 controls （P=2.5-10-5）. Transcriptome sequencing revealed that identified variants induced dyshomeostasis in expression of collagens-TIMPs/MMPs systems. The Col5a2-/- rats manifested growth retardation and aortic dysplasia. Our study provides a first comprehensive map of genetic alterations in collagens-MMPs/TIMPs system in sporadic AD patients and suggests that variants of these genes contribute largely to AD pathogenesis.

Effect of warming Yang and removing blood stasis method on matrix metalloproteinases/tissue inhibitor metalloproteinases levels secreted by cultured endometrial cells from patients with endometriosis

OBJECTIVE:To investigate the effect of Chinese medicines using the warming Yang and removing blood stasis method on levels of matrix metalloproteinases(MMPs)/tissue inhibitor metalloproteinases(TIMPs) secreted by cultured endometrial cells from patients with endometriosis.METHODS:Ectopic and eutopic endometrial cells obtaind from 15 endometriosis patients were cultured in vitro,and divided randomly into five groups:high dose;moderate dose;low dose;nemestran;blank control.The three dose groups were treated with a decoction prepared according to the principle of warming Yang and removing blood stasis;nemestran and 0.9%NaCI were administered to the nemestran group and balnk control group,respectively.Eutopic endometrial cells obtaind from 10 hysteromyoma patients were cultured in vitro,as the normal control group,0.9%NaCI were administered to the normal control group.Cell culture supernatants were collected and levels of matrix metalloproteinase-1(MMP-1),matrix metalloproteinase-2(MMP-2),matrix metalloproteinase-9(MMP-9),tissue inhibitor metalloproteinase-1(TIMP-1) and tissue inhibitor metalloproteinase-2(TIMP-2) detected by enzyme-linked immuno sorbent assay(ELISA).RESULTS:Compared with the normal control group,levels of MMP-1,MMP-2,and MMP-9 in eutopic and ectopic endometrium cell supernatants in the blank control group were increased,whereas levels of TIMP-1 and TIMP-2 were decreased(P <0.05).Compared with the blank control group,levels of MMP-1 and MMP-2 in ectopic and eutopic endometrium cell supernatants cultured in low-dose,middle-dose,and high-dose groups were decreased,whereas levels of TIMP-1 and TIMP-2 were increased significantly(P < 0.05).CONCLUSION:The warming Yang and removing blood stasis method affects expression of MMPs andTIMPs.

The effects of sodium hyaluronate on mRNA expressions of matrix metalloproteinase-1,-3 and tissue inhibitor of metalloproteinase-1 in cartilage and synovium of traumatic osteoarthritis model

Objective: To observe the influence of intra-articular injection of sodium hyaluronate (HA) on the mRNA expressions of matrix metalloproteinase-1,-3 (MMP-1,-3) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in cartilage and synovium of traumatic osteoarthritis (OA).Methods: Sixteen white rabbits underwent unilateral anterior cruciate ligament transection (ACLT) were divided into 2 groups randomly 5 weeks after transection. The experimental group rabbits received 0.3 ml of 1% HA by intra-articular injection once a week. Animals in the control group were treated under the same conditions using physiological saline. Ten weeks following surgery, cartilage and synovium were harvested. The mRNA expressions of MMP-1, MMP-3 and TIMP-1 were analyzed using reverse transcription-polymerase chain reaction (RT-PCR).Results: In synovium, the mRNA expression of MMP-3 was suppressed in the HA injection group. HA treatment had no effect on the MMP-3 expression in cartilage. No significant difference of MMP-1 and TIMP-1 expressions in cartilage and synovium was found between the HA injection group and the control group.Conclusions: One of the mechanisms of the therapeutic effect of HA may be the inhibition of expression of MMP-3 in synovium during early stage of traumatic OA.