Objective To use an improved technique to construct the recombinant adeno-associated virus 2 (rAAV2) mediated gene which can transfer human tissue inhibitor of metalloproteinase-1 (TIMP1). Methods Human TIMP1 gene was amplified from pDNR-LIB plasmid by PCR and cloned into the rAAV2 vector pSNAV to recombinant pSNAV-TIMP1, then was transferred into BHK-21 cells by means of lipofectamine. Using G418 selection, a mixed cell named BHK-21/rAAV2-TIMP1 was isolated, which was capable to express TIMP1. The cell was subsequently infected with recombinant herpes simplex virus 1 (rHSV1-rc/△UL2) that was able to package the rAAV2-TIMP1. After purification, rAAV2-TIMP1 was obtained. Results The rAAV2 carrying human TIMP1 gene was constructed successfully. The viral titer of the rAAV2-TIMP1 was 1×1012 v.g./ml. Conclusion rAAV2-TIMP1 was constructed successfully, which would provide experimental basis for carrying the TIMP1 into hepatocellular carcinoma effectively and inhibiting the invasiveness and migratory capacity of hepatocellular carcinoma in vitro and in vivo models.
Abstract: Objective To generate a eukaryotic expression plasmid-pcDNA3.1/human tissue inhibitor of metalloproteinase-1(hTIMP-1)enhanced green fluorescent protein (EGFP), carrying hTIMP-1 and labeled with EGFP, and to examine the expression of hTIMP-1 in vascular smooth muscle cells (SMCs) transferred with hTIMP. Methods The recombinant plasmids of pcDNA3.1/hTIMP-1-EGFP were obtained bypolymerase chain reaction (PCR) amplification, splicing, and insertion of complementary deoxyribonucleic acid (cDNA) fragments of hTIMP-1 and EGFP. The target gene was transferred to the primarily cultured SMCs (pcDNA3.1/hTIMP-1-EGFP transferred group) by using cationic liposome mediated gene transfection technique. EGFP expression was detected by fluorescence microscopy, and the transfection rate was determined by flow cytometry. Reverse transcriptase polymerase chain reaction (RTPCR), Western blotting, and other techniques were used to detect the expression of hTIMP-1 gene. The biological activity of matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9(MMP-9) were studied by zymographic analysis of gelatinases. Blank plasmidpcDNA3.1 transferred SMCs (blank plasmid pcDNA3.1 transferred group) and untransferred SMCs (untransferred group) were used as control. Results In cDNA3.1/hTIMP-1-EGFP transferred group,the growth ability of SMCs was profoundly inhibited, bright green fluorescence was observed by fluorescence microscopy 24 hours after transfection in SMCs,the rate of transfection analyzed with flow cytometry was 15%,RT-PCR results showed that the genome of hTIMP-1 transferred SMCs contained a 646 bp specific fragment of hTIMP-1 gene, Western blotting results proved hTIMP-1 protein expression in SMCs transferred by hTIMP-1, zymographic analysis of elatinases showed decreased activity of MMP-2 and MMP-9, compared to those in blank plasmidpcDNA3.1 transferred group and untransferred group, significant differences were observed (Plt;0.05). Conclusion The generation of a eukaryotic expression plasmid carrying TIMP-1 gene and its expression in SMCs provide a sound basis for hTIMP-1 gene therapy.
Objective To find the difference between the collagenase activity and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression level in normal urethral tissue and in urethral scar tissue, and to study the effect of collagenase activity and TIMP-1 expression level on the degradation of urethral scar. Methods The urethral tissues were derived from 10 human surgical specimens of urethral stricture scar and 10 human normal urethral specimens from patients with brain death. The collagenase activity was detected by ELISA assay, and the TIMP-1 mRNA level by RT-PCR. Results The collagenase activity of urethral scar tissue was (15.32±2.29) U and lower than that of normal urethral tissue (24.67±6.78) U, there was significant difference between them (P lt; 0.01). The TIMP-1 expression level of urethral scar tissue was higher than that of normal urethral tissue, there was significant difference between them (P lt; 0.05). Conclusion The high level of TIMP-1 expression and the low collagenase activity in urethral scar tissue may inhibit the degradation of urethral scar, and may be one of important causes of the scar tissue hyperplasia.
Objective To observe the expression of matrix metalloproteinase-9 (MMP-9), its tissue inhibitor of matrix metalloproteinase (TIMP-1), inducible nitric oxide synthase (iNOS) and contents of nitric oxide (NO) in the ocular tissues of Sprague-Dawley (SD) rats with endotoxin induced uveitis（EIU）. Methods Ninety SD rats were randomly divided into experimental (81 rats) and control group (9 rats). The model of EIU was induced in rats in experimental group by injecting with lipoplysaccharide (LPS) 200 μl into the hind feet pads, while the rats in the control group were not injected. Nine rats were executed 0, 6, 12, 18, 24, 48, 72, 96 hours and 7 days, respectively, after injecting with LPS; the NO content and concentration of protein in the aqueous humor in blood plasma, aqueous humor, and uveal tissues were detected. The expressions of MMP-9, TIMP-1 and iNOS in the ocular tissues were detected by immunohistochemistry, and the average absorbance (A) value was evaluated by computer medical image analysis system. Results iNOS, MMP-9 and TIMP-1 expressed in the epithelial cells of iris and ciliary body and exudated inflammatory cells of rats. The concentration of protein in the aqueous humor, the contents of NO in blood plasma, aqueous humor, and uveal tissues, and A value of MMP-9 had obvious relativity with the inflammatory extent, while no positive correlation was found between the inflammatory extent and the A value of iNOS and TIMP-1. Expression of iNOS was found 6 hours after injection, reached the peak after 12 hours, and then dropped gradually. The expression of TIMP-1 could be seen 24 hours after injection, and reached its peak after 72 hours. Conclusion The content of NO and expressions of iNOS, MMP-9 and TIMP-1 changes from the beginning and during the development of EIU, which suggests that NO, iNOS, MMP-9 and TIMP-1 are involved in the pathologic process of EIU. (Chin J Ocul Fundus Dis, 2005, 21: 371-374)
Objective To explore the effects of overexpression of human tissue inhibitors of metalloproteinase-1 (hTIMP-1) on proliferation of human liver cancer cell line HepG2 in vitro. Methods A recombinant adenoviral vector containing full-length cDNA of hTIMP-1 was generated and transfected into HepG2. The viral titer was checked by measuring GFP, and the expression of hTIMP-1 in vitro was detected by the techniques of Western blot and semi-quantitative RT-PCR. The ultrastructure was observed by transmission electron microscope and the effects of overexpression of hTIMP-1 on proliferation of HepG2 in vitro was analyzed by MTT assay and growth curve. Results The resultant AdhTIMP-1 was successfully constructed and the expression of hTIMP-1 was detected by Western blot and RT-PCR. The growth and proliferation of HepG2, which had been transfected with AdhTIMP-1, was significantly inhibited. Conclusion The proliferation of HepG2 was markedly inhibited by recombinant adenovirus-mediated overexpression of hTIMP-1, which may pave the way for further application in liver gene therapy.
Objective To explore the protein expressions of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1)， and to investigate their relationships between their serum concentration before operation and the infiltration and metastasis of thyroid carcinoma. Methods The protein expressions of MMP-9 and TIMP-1 in 32 cases of thyroid carcinomas, 23 cases of adjacent tissues and 30 cases of benign hyperplastic lesions were measured by using immunohistochemistry. The preoperative serum concentrations of MMP-9 and TIMP-1 in 21 cases of thyroid carcinomas and 19 cases of benign hyperplastic lesions were determined by enzyme-linked immunosorbent assay. Results The positive expression rates of MMP-9 and TIMP-1 in tumor tissues were significantly higher （75.0%,56.3%）than those in adjacent tissues and benign hyperplastic lesions （30.4%, 21.7%; 26.7%, 23.3%) P＜0.05. There were correlations between the expressions of MMP-9 and TIMP-1 and the local infiltrative degrees, lymph node metastasis and TNM stage （P＜0.05）. There was a negative correlation between the expression of MMP-9 and the expression of TIMP-1 （r=-0.509， P=0.003）. The concentration of MMP-9 in serum of thyroid carcinoma patients was (122.60±36.20) ng/ml, whereas TIMP-1 was (59.44±38.65) ng/ml, both of which were significantly higher compared to those of benign group (P＜0.05). In addition, there was a positive correlation between the expressions of MMP-9/TIMP-1 in the carcinoma tissues and their concentrations in serum （P＜0.05）.Conclusion To detection the expressions of MMP-9 and TIMP-1 in the lesion and their concentrations in the serum may not only contribute to the differential diagnosis of thyroid tumors, but may also help to predict the prognosis of the carcinoma.