1.Role of a disintegrin and metalloprotease with thrombospondin type 1 motifs in aged rat myocardium after ischemic preconditioning
Yong WANG ; Congxin HUANG ; Jinsong CHENG ; Yifeng ZHOU ; Hui WANG ; Wenjing WU ; Wenqiang LIAO ; Jianyan WEN ; Yuannan KE ; Jingang ZHENG
Chinese Journal of Geriatrics 2011;30(1):54-58
Objective To investigate the effect of ischemic preconditioning (IPC) on the expression of a disintegrin and metalloprotease with thrombospondin type 1 motifs (ADAMTS-1), and to study whether the application of small interfering (si)RNA specifically targeting ADAMTS-1 would help to recover IPC protection in the aged heart. Methods The 32 young (4 months) and 32 aged(24 months) male Sprague-Dawley (SD) rats were assigned randomly to IPC group (n=20) and sham operated group (n= 12) respectively. Myocardial samples from the ischemic-reperfused region were harvested for detecting the ADAMTS-1 expression. In addition, the 110 aged SD rats were assignedrandomly to ADAMTS-1 siRNA group and control group (n=55, each). The effects of ADAMTS-1siRNA transfcction on the expression of ADAMTS-1 protein, myocardial infarction survival rate,heart function and myocardial infarction size after IPC were observed.Results Twenty-four hours after IPC, the ADAMTS-1 protein expression increased significantly in iscbemic-reperfused region both in young and aged rats (P<0. 05), and the protein expression was higher in aged rats than in young rats (P<0.05). In young-IPC group, the absorbency showed ADAMTS-1 protein expression at 0 hrs and 24 hrs after IPC were 0. 05±0.01 and 0.12±0.03 by immunohistochemical staining, and were 0.68±0. 16 and 1. 17±0.21 by Western blots respectively. In aged-IPC group, the absorbency showed ADAMTS-1 protein expression at 0 hrs and 24 hrs after IPC were 0.07±0. 03 and 0.21 ±0.04 by immunohistochemical staining, and were 0. 76±0. 21 and 1. 48±0. 17 by Western blots. In the aged rats, ADAMTS-1 siRNA transfection inhibited ADAMTS-1 protein expression (0. 66±0. 19and 0.78±0.21, by Western blots at 0 hrs and 24 hrs after IPC, P>0.05), but didn't improve myocardial infarction survival rates [ADAMTS-1 siRNA group and sham operated group: 14.3% (5/35) vs. 17.1 %(6/35), P>0.05], left ventricular fractional shortening [(14.0±3.2)% vs. (13.0±2.9)%, P>0.05] and myocardial infarction size[(39.0±4.1)% vs. (38.0±5.3)%, P>0.05].Conclusions ADAMTS-1 expression induced by IPC increases significantly in aged versus in young rats. ADAMTS-1 knockdown by siRNA inhibits ADAMTS-1 protein expression but cannot recover the age-associated loss of IPC protection.
2.Effects of SUMOylation on the subcellular localization and function of DAXX.
Ling LI ; Juan WEN ; Qin-Hui TUO ; Duan-Fang LIAO
Acta Physiologica Sinica 2013;65(1):89-95
Death domain-associated protein (DAXX) as a multifunctional nuclear protein widely resides in nucleolus, nucleoplasm, chromatin, promyelocytic leukaemia nuclear bodies (PML-NBs) and cytoplasm. It plays significant roles in transcriptional regulation, apoptosis, cell cycle and other biological activities. Small ubiquitin-like modifier (SUMO) is required for SUMOylation which is a highly conserved post-translational modification in a wide variety of cellular processes. Numerous studies demonstrated that SUMOylation has a great effect on the subcellular localization and functional regulation of DAXX. This review will provide a summary for SUMOylation of DAXX.
Adaptor Proteins, Signal Transducing
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physiology
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Gene Expression Regulation
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Humans
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Nuclear Proteins
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physiology
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Sumoylation
3.Regulation of naotai recipe on the expression of HIF-lα/VEGF signaling pathway in cerebral ischemia/reperfusion rats.
Yi CHEN ; Hui-bin ZHU ; Jun LIAO ; Ya-qiao YI ; Guo-zuo WANG ; Le TONG ; Jin-wen GE
Chinese Journal of Integrated Traditional and Western Medicine 2014;34(10):1225-1230
OBJECTIVETo observe the therapeutic angiogenesis effect of naotai recipe (NR) on local ischemia/reperfusion (I/R) injury of rats by observing signaling pathway of hypoxia-inducible factor-lα (HIF-1α) and vascular endothelial growth factor (VEGF).
METHODSTotally 120 Sprague-Dawley (SD) rats were randomly divided into 4 groups, namely, the normal control group (n =12), the sham-operation group (n =12), the I/R model group (n =48), and the NR group (n =48). Cerebral I/R injury models were established using thread suture method. Rats in the I/R model group and the NR group were sub-divided into 4 sub-groups according to the 1st, 3rd, 5th, and 7th I/R day (n =12). The phenomenon of neovasculization was observed by immunofluorescence staining. The protein and mRNA expression levels of HIF-la, VEGF-A, and VEGFR II receptor were detected by RT-PCR.
RESULTSThere were a large amount of labels for neovasculization in the ischemic area of the NR group. Double-immunofluorescence labeling [vWF (red) and BrdU (green)] was observed in the NR group. Compared with the model group, the HIF-1α protein expression was obviously enhanced on the 1 st day of I/R (P <0.01), and the VEGF protein expression started to enhance on the 3rd day in the NR group (P <0.01). The VEGFR protein expression level was the highest in the NR group on the 5th day of I/R (P <0.01). The protein expression of VEGF and HIF-1α started to decrease on the 7th day of I/R.
CONCLUSIONNR could strengthen angiogenesis after I/R by elevating the expression of HIF-lα and activating HIF-lα/VEGF signaling pathway.
Animals ; Brain Ischemia ; metabolism ; Cerebral Infarction ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Hypoxia-Ischemia, Brain ; metabolism ; Ischemia ; Neovascularization, Pathologic ; Rats, Sprague-Dawley ; Reperfusion Injury ; Signal Transduction ; Vascular Endothelial Growth Factor A ; biosynthesis
4.The Relationship between nm23 and p16 Gene Synergy Expression and Gastric Cancer Biological Characteristic and Prognosis
Guifang LI ; Yizhou LUO ; Chijun DUAN ; Qun ZHAI ; Hai SUN ; Hui LIAO ; Hongsheng WEN ; Xuezhi WANG ; Ge GAN
Chinese Journal of Cancer Biotherapy 1996;0(04):-
Objective: To examine and analyze the nm23, p16 gene expression in gastric cancer tissure,and follow up patients 5 years to discuss the relationship between nm23 and p16 gene synergy expression and gastric cancer biological characteristic and prognosis.Methods: nm23 and p16 protein in gastric cancer tissue and control were detected by immunohistochemistry,and the patients had been followed up for 5 years. Results: In 84 samples of gastric cancer, nm23 positive rate was 46.43%, p16 was 44.05%, the positive rate of gastric cancer tissue and metastasitic lymph node was lower than that of normal control, normal tissue near cancer or benign polyp,and these two genes were related to the depth of tumor invasion and clinical stage.The mortality and recurrence-metastasis rate was higher in these low expression group, and had a shorter median survive period. Conclusion: Abnormal expression of nm23 and p16 gene plays a important role in gastric cancer recurrence and devolepment and may be one of markers for evaluating tumor biological behavior and prognosis.
5.Inhibitory effect of ginkgolide B on angiogenesis in chronic inflammation.
Xue-yu OU-YANG ; Wen-jie WANG ; Wen-hui LIAO ; Xiao-hong CHEN
Acta Pharmaceutica Sinica 2005;40(4):311-315
AIMTo investigate the inhibitory effect of ginkgolide B on angiogenesis in chronic inflammation and the possible mechanisms.
METHODSThe murine chronic granulomatous air pouch model was used to observe the anti-angiogenesis effect of ginkgolide B. The vascular index was determined by colorimetry of carminic acid, and angiogenesis was observed by histology method. The interleukin-1beta (IL-1beta) levels in mice serum and in supernatants of U937 cell culture stimulated by phorbol 12-myristate 13-acetate (PMA) were detected by radioimmunoassay (RIA). The tumor necrosis factor-alpha (TNF-alpha) levels in mice serum and in supernatant of U937 cell culture were measured by cytotoxicity bioassay. The mRNA expression of IL-1beta and TNF-alpha of U937 cell culture was investigated by RT-PCR.
RESULTSOral administration of ginkgolide B 25 and 100 mg x kg(-1) was shown to significantly inhibit the vascular index of murine chronic granulomatous air pouch model with the inhibitory rate of 22.52% and 25.29%, respectively. This result was supported by histological observation. Concomitantly, the IL-1beta levels in mice serums were also significantly decreased with the inhibitory rate of 50.61% and 58.66%; so were the TNF-alpha levels with the inhibitory rate of 28.91% and 52.41%. Ginkgolide B at concentration of 1 x 10(-5) to 1 x 10(-8) mol x L(-1) could also reduce both the IL-1beta and TNF-alpha contents in the supernatants of U937 cell culture stimulated by PMA, but the scopes of changes were much different. For IL-1beta the IC50 was 1.93 x 10(-8) mol x L(-1), while ginkgolide B at concentration of 1 x 10(-5) mol x L(-1) only decreased the release of TNF-alpha by 25.99%. Furthermore, ginkgolide B at concentrations of 1 x 10(-5) to 1 x 10(-7) mol x L(-1) was shown to significantly inhibit TNF-alpha mRNA expression of U937 cells; and at concentrations of 1 x 10(-5) and 1 x 10(-6) mol x L(-1) could inhibit IL-1beta mRNA expression.
CONCLUSIONGinkgolide B was shown to significantly inhibit angiogenesis of the murine chronic granulomatous air pouch model, reduce the IL-1beta and TNF-alpha levels in mice serums, and significantly inhibit IL-1beta and TNF-alpha mRNA expression and protein secretion in supernatants of U937 cell culture. It was suggested that reduction of proangiogenic cytokines IL-1beta and TNF-alpha secretion may contribute to the anti-angiogenesis effect of ginkgolide B in the murine chronic granulomatous air pouch model.
Animals ; Cell Line ; Diterpenes ; pharmacology ; Female ; Fibrinolytic Agents ; pharmacology ; Fibroblasts ; cytology ; metabolism ; Ginkgolides ; Granuloma ; metabolism ; pathology ; Humans ; Inflammation ; metabolism ; pathology ; Interleukin-1 ; biosynthesis ; genetics ; Lactones ; pharmacology ; Mice ; Mice, Inbred BALB C ; Neovascularization, Pathologic ; pathology ; Platelet Activating Factor ; antagonists & inhibitors ; RNA, Messenger ; biosynthesis ; genetics ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics ; U937 Cells ; metabolism
6.Identification and transcriptional activity analysis of core regulatory region of human guanylate binding protein 5 gene promoter
YE Ting ; YANG Kang ; WANG Tian-tian ; LIAO Yu-jiao ; DU Wen-qian ; HUANG Min ; JIANG Pei-wen ; LI Min-hui ; YANG Ping
Chinese Journal of Biologicals 2023;36(2):138-144
Objective To construct luciferase reporter plasmids of truncated fragments of different lengths of human guanylate binding protein 5(GBP5)gene promoter and analyze the transcriptional activity of each fragment to determine the core regulatory region.Methods GBP5promoter sequence was amplified by PCR,truncated into five fragments of different lengths and connected to pGL3-basic plasmid.The constructed recombinant plasmids pGL3-GBP5-11/21/31/41/51were transfected into 293FT cells and detected for luciferase activity.The binding sites of transcription factors in GBP5promoter region were predicted by JASPAR software,and Yin-Yang transcription factor 1(YY1)targeting the core regulatory region was selected and verified for the transcriptional regulatory activity.The CDS sequence of YY1 was amplified by PCR to construct the overexpression plasmid pIRES2-EGFP-YY1,which was then co-transfected to 293FT cells with plasmids pGL3-GBP5-21(-1 623 ~ +47 bp)and internal reference plasmid pRL-CMV,and detected for luciferase activity to analyze the regulation of transcription factor YY1 on GBP5 promoter activity.Results Colony PCR and double enzyme digestion identification proved that the plasmid of human GBP5 promoter reporter gene was correctly constructed;JASPAR software predicted that there were multiple transcription factor binding sites such as STAT1,YY1 and Foxp3 in GBP5promoter region.Double luciferase activity assay showed that pGL3-GBP5-21(-1 623 ~ +47 bp)showed the highest promoter activity,while the promoter activity of pGL3-GBP5-41(-520 ~ +47 bp)decreased significantly,suggesting that the core region of GBP5 promoter was located at upstream-1 623 ~-520 bp of 5 'UTR;Overexpression of YY1 significantly activated the GBP5 promoter activity and regulated the expression of GBP5.Conclusion The core regulatory region of human GBP5 promoter was located in upstream-1 623 ~-520 bp of the 5 'UTR,with a binding site of transcription factor YY1 existing in this region.Meanwhile,overexpression of YY1 significantly effected the activity of GBP5 promoter.
7.Application of serum protein profiling in diagnosis, prognosis and evaluation of curative effect of pancreatic adenocarcinoma.
Jing-hui GUO ; Wen-jing WANG ; Ping LIAO ; Chun-yan ZHANG ; Da-yong JIN ; Wen-hui LOU ; Shun-cai ZHANG
Chinese Journal of Oncology 2010;32(1):33-36
OBJECTIVETo establish decision tree and logistic regression classification models for diagnosing pancreatic adenocarcinoma (PaCa) and for screening serum biomarkers related to evaluation of different stages and curative effects.
METHODSSerum samples obtained from subjects with pancreatic adenocarcinoma (n = 58) and normal pancreas (n = 51) were applied to strong anion exchange chromatography (SAX2) chips for protein profiling by SELDI-TOF-MS to screen multiple serum biomarkers. Biomarker Wizard software and several statistical methods including algorithm of decision tree, logistic regression and ROC curves were used to construct the decision tree or logistic regression classification models.
RESULTSAverage of 61 mass peaks were detected at the molecular range of 2000-30,000, ten decision trees with the highest cross validation rate were chosen to construct the classification models, which can differentiate PaCa from normal pancreas with a sensitivity of 83.3% and a specificity of 100%. Logistic regression was used to achieve the AUC (0.976 +/- 0.011, P < 0.001) with a sensitivity of 77.6% - 91.4% and a specificity of 92.2% - 100%. Six mass peaks were combined by logistic regression to achieve the AUC 0.897 +/- 0.054, 0.978 +/- 0.021 and 0.792 +/- 0.107 (P < 0.05) in the three groups (patients at stage I and II, stage II and III, stage III and IV). One mass peak (M/Z 4,016) was screened (P < 0.05) significantly between the preoperative and postoperative PaCa samples and the intensity decreased weeks after operation.
CONCLUSIONDecision tree and logistic regression classification models of the mass peaks screened by SELDI-TOF-MS serum profiling can be used to differentiate pancreatic adenocarcinoma from normal pancreas, and is superior to CA 199. The detected mass peaks are helpful for the evaluation of curative effect and prognosis of pancreatic adenocarcinoma.
Adenocarcinoma ; blood ; diagnosis ; pathology ; surgery ; Adult ; Aged ; Aged, 80 and over ; Area Under Curve ; Biomarkers, Tumor ; blood ; Blood Proteins ; analysis ; Chromatography, Ion Exchange ; methods ; Decision Trees ; Female ; Humans ; Logistic Models ; Male ; Middle Aged ; Neoplasm Staging ; Pancreatic Neoplasms ; blood ; diagnosis ; pathology ; surgery ; Prognosis ; Protein Array Analysis ; Proteomics ; ROC Curve ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
8.tMfn2 gene transfer promotes vascular smooth-muscle cells apoptosis via activation of the mitochondrial cell death pathway.
Li ZHAO ; Wei ZHOU ; Si-kun WANG ; Hua LIAO ; Wen-juan ZHANG ; Guang-hui CHEN ; Xiao-mei GUO
Chinese Journal of Cardiology 2009;37(7):639-643
OBJECTIVETo investigate the effect of tMfn2 gene transfer on promoting the apoptosis of vascular smooth-muscle cells (VSMCs).
METHODSVSMCs were infected by adenovirus-mediated tMfn2 (Adv-tMfn2) or adenovirus-mediated Mfn2 (Adv-Mfn2). FACS analysis, cell death ELISA and TUNEL staining were used to investigate the role of tMfn2 and Adv-Mfn2 gene transfer on VSMCs apoptosis. Western blot was used to analyze the protein expression of p-Akt, Bcl-2, Bax and cleaved caspase-9.
RESULTSFACS and ELISA results showed that tMfn2 was superior to Mfn2 in promoting VSMCs apoptosis and tMfn2 gene transfer induced VSMCs apoptosis in a time-dependent manner (P < 0.01). TUNEL staining evidenced that there were more positive-apoptotic VSMCs in tMfn2 group than that in Mfn2 group (P < 0.01). The protein expressions of phosphorylated Akt and Bcl-2 were significantly decreased, whereas Bax and cleaved caspase-9 protein expressions were significantly upregulated in tMfn2-transfected VSMCs.
CONCLUSIONStMfn2 transfer promoted apoptosis of VSMCs in a time dependent manner via the PI3K-Akt signaling pathway and mitochondrial apoptotic pathway.
Adenoviridae ; Animals ; Apoptosis ; Caspase 9 ; metabolism ; Cells, Cultured ; Membrane Proteins ; genetics ; Mitochondria ; metabolism ; Mitochondrial Membranes ; metabolism ; Mitochondrial Proteins ; genetics ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Myocytes, Smooth Muscle ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Transfection ; bcl-2-Associated X Protein ; metabolism
9.Tissue-engineered conduit using bladder acellular matrix and bladder epithelial cells for urinary diversion in rabbits.
Wen-Biao LIAO ; Chao SONG ; Yong-Wei LI ; Si-Xing YANG ; Lin-Chao MENG ; Xin-Hui LI
Chinese Medical Journal 2013;126(2):335-339
BACKGROUNDFor muscle invasive bladder cancer, radical cystectomy is the most effective treatment now and urinary diversion is often necessary. The use of intestinal tissue for urinary diversion is frequently associated with complications. In this study, we aimed to make a tissue-engineered conduit (TEC) using bladder epithelial cells and bladder acellular matrix (BAM) for urinary diversion in rabbits.
METHODSBladder epithelial cells of rabbit were cultivated and expanded in vitro, then seeded on BAM, and cultured for 7 days. Then cell-seeded graft was used to make TEC. In the experimental group, most of bladder of the rabbit was removed while bladder trigone was retained. The proximal end of TEC was anastomosed with bladder trigone and the distal end was anastomosed with the abdominal stoma. In the control group, TEC was made using unseeded BAM. Haematoxylin and eosin staining was conducted, respectively, at 1, 2, 4, and 8 weeks postoperatively. Immunohistochemistry was performed 8 weeks postoperatively. Intravenous urography, retrograde pyelography, and cystoscopy of TEC were made at 12 weeks postoperatively.
RESULTSAll animals were alive in the experimental group. Haematoxylin and eosin staining showed epithelial coverage in TEC. Immunohistochemistry showed anti-cytokeratin AE(1)/AE(3) antibody and anti-ZO1 antibody positive, confirming there were mature and functional epithelial cells on the lumen of TEC. Retrograde pyelography and intravenous urography showed that TEC developed well and that there was no obstruction. In the control group, four rabbits were dead within 2 weeks and scar formation, atresia, and severe hydronephrosis were found.
CONCLUSIONSWe successfully made TEC using BAM and bladder epithelial cells for urinary diversion in rabbits. The lumen of this new TEC covered mature epithelial cells and could prevent urinary extravasation.
Animals ; Epithelial Cells ; cytology ; Male ; Rabbits ; Tissue Engineering ; methods ; Urinary Bladder ; cytology ; Urinary Diversion ; methods
10.Level VI and II-V cervical lymph node metastasis in differentiated thyroid carcinoma.
Zhen-dong LI ; Hui-lei DONG ; Shu-chun LI ; Wen-chao FU
Chinese Journal of Otorhinolaryngology Head and Neck Surgery 2007;42(12):915-918
OBJECTIVETo explore the characters of the cervical lymph node metastasis in differentiated thyroid carcinoma, and to provide evidence for proper surgery of differentiated thyroid carcinoma.
METHODSFrom 1984 to 2000, 99 cases with differentiated thyroid carcinoma were performed thyroidectomy and neck dissection. The patients were followed up. A retrospective analysis was performed. Results In 99 cases with differentiated thyroid carcinoma, there were 61 papillary carcinoma, 13 papillary and follicular mixed carcinoma, 25 follicular carcinoma. According to 2002 UICC TNM classification, 60 cases were staged I, 1 case staged II, 5 cases staged III, 33 cases staged IV. Lobectomy and isthmectomy was performed in 80 cases, lobectomy was resected and opposite subtotal lobectomy in 15 cases, total thyroidectomy in 4 cases. One hundred and four neck dissection were performed in 99 cases (5 cases were bilateral neck dissection ). Among them, 66 (68 sides) were radical neck dissection, 33 (36 sides) were modified neck dissection. Pathological results showed that lymph nodes were positive in 86 sides of 83 cases. The rate of cervical lymph node metastasis was 83.8% (83/99). The positive rates of lymph node were 37.5% (39/104) in level VI and 76.9% (80/104) in II-V, which was statistically different (chi2 = 33.01, P < 0.01). The cervical lymph node metastasis in lateral area (level II-V) and that in VI had not relationship (chi2 = 2.08, P > 0.05). Ten and 15 year survival rates of all 99 cases were 88.3% and 84.5% respectively.
CONCLUSIONSThe occurrence of lymph node metastasis in level VI and level II was different and no relationship .One can not judge whether lateral neck metastasis by the lymph node statue in level VI only . Although they all had good prognosis, patients with positive nodes in level VI were not worse than that in lateral neck (II-V).
Adolescent ; Adult ; Aged ; Carcinoma, Papillary ; pathology ; Cell Differentiation ; Child ; Female ; Humans ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; pathology ; Male ; Middle Aged ; Neck ; Neck Dissection ; Neoplasm Staging ; Prognosis ; Retrospective Studies ; Thyroid Neoplasms ; pathology ; Young Adult