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.
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 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.