[Role of a latent soluble TNF receptor type I (hsTNFRI) fusion protein in targeted treatment of endometriosis].
OBJECTIVE
To construct a latent human soluble tumor necrosis factor receptor I (hsTNFRI) using the latency associated protein (LAP) of transforming growth factor-beta1 (TGF-beta1) fused via a matrix metalloproteinase (MMP) cleavage site to hsTNFRI so as to detect the latent biological activity of LAP-MMP-hsTNFRI fusion protein.


METHODS
A double-stranded deoxyoligonucleotide coding for MMP cleavage site was cloned into plasmid pcDNA3.1. LAP and hsTNFRI cDNA were then inserted into both two sides of MMP cleavage site. After being transferred by LAP-MMP-hsTNFRI fusion gene with liposome, the expression of fusion protein in COS-7 cells was detected by RT-PCR and Western blot. The inhibitory effect of fusion protein upon cytotoxicity of TNF-alpha was detected by methyl thiazolyl tetrazolium (MTT) assay before and after the fusion protein incubated in MMP or peritoneal fluid from endometriosis patients.


RESULTS
The recombinant plasmid LAP-MMP-hsTNFRI-pcDNA3.1 was constructed successfully and was expressed effectively in COS-7 cells. The MTT assay showed that there was no difference in the mortality rate of L929 cells between LAP-MMP-hsTNFRI-pcDNA3.1 and empty vector transfection groups (P > 0.05). The mortality rates of L929 cells with 800 ng/L TNF-alpha in LAP-MMP-hsTNFRI-pcDNA3.1 transfection group after incubation with MMP or peritoneal fluid from endometriosis patients were (44.5 +/- 2.4)% and (33.8 +/- 1.9)% respectively. And it was lower than the pre-incubation period (58.1 +/- 2.4)% (P < 0.05).


CONCLUSIONS
The biological activity of LAP-MMP-hsTNFRI fusion protein can be made latent by LAP and activated by peritoneal fluid from endometriosis. Thus a new method has been provided for a targeted therapy of endometriosis.
Xiong
Zhou-fang
ZF
Department of Obstetrics & Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, China.
Hu
Sha
S
Wang
Ze-hua
ZH
D001202:Ascitic Fluid / Q000166:cytology; D002460:Cell Line; D004715:Endometriosis / Q000628:therapy*; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics* / Q000627:therapeutic use*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000627:therapeutic use; D014162:Transfection
D047888:Receptors, Tumor Necrosis Factor, Type I; D011993:Recombinant Fusion Proteins