ABSTRACT
OBJECTIVE@#To construct short hairpin RNA interfering expression vector of TDRG1,and detect the specific interfering effect of TDRG1-shRNA expression vector on NTERA-2 cells.@*METHODS@#Oligos for short hairpin RNA targefing for TDRG1 were designed and connected to the expression vector pGPU6/GFP/Neo to construct the TDRG1 shRNA expression vector. The recombinant plasmid TDRG1-shRNA486, TDRG1-shRNA738, TDRG1-shRNA921 and lipofectamine ™2000 were used to generate and transfect shRNA into NTERA-2 cells. Expression of TDRG1 mRNA was assayed by RT-PCR.@*RESULTS@#TDRG1-shRNA expression vector was successfully constructed. TDRG1-shRNA486 was more effective in the suppression of TDRG1 with significant reduction of TDRG1 mRNA.@*CONCLUSION@#TDRG1-shRNA can interfere the expression of TDRG1 in NTERA-2 cells.
Subject(s)
Humans , Cell Line, Tumor , Genetic Vectors , RNA Interference , RNA, Messenger , RNA, Small Interfering , TransfectionABSTRACT
Objective To construct a prokaryotic plasmid to express the testis development related gene 1 (TDRG1) recombinant protein and obtain anti-TDRG1 mAb by immunizing mice, and to identify the biological properties of the mAb. Methods The coding sequence of TDRG1 was amplified by RT-PCR from normal human testis tissue and cloned into the vector pET21, and then was expressed in the E. coli BL 21(DE3) to get TDRG1 recombinant protein. The purified TDRG1 recombinant protein was injected to immunize the BALB/C mice to develop anti-TDRG1 mAb. Splenocytes of the immunized mice were collected and fused with the mouse myeloma cell line Sp2/0 cells. The hybridoma cells that secreted anti-TDRG1 mAb were subcloned with limited dilution. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate titers and subtypes of mAb. Western blot and immunohistochemistry were used to detect specificity of mAb.Results The prokaryotic plasmid expressing the recombinant protein was constructed, and the TDRG1 recombinant protein was expressed and purified. Two hybridoma cell lines that secreted anti-TDRG1 mAb were obtained. The titer of the mAb in ascites was 1∶1.6×10~6, and the subtype of the mAb was IgG_1. Westem blot and immunohistochemistry analysis indicated the mAb showed specific combination with TDRG1 protein in human testes.Conclusion The TDRG1 recombinant protein is highly purified and has strong antigenicity. The anti-TDRG1 mAb is produced successfully.
ABSTRACT
OBJECTIVE@#To determine the diagnostic value of real-time Rigiscan monitoring and Doppler ultrasonography following intracavernous injection of prostaglandin E1in erectile dysfunction(ED).@*METHODS@#From January 1998 to December 2007, 1 392 ED patients completed the test. PGE(1) was injected into corpus cavernosum penis near the radix penis with injection guns. After 1 or 2 minutes, the Rigiscan Plus device was used for real-time evaluation of penile rigidity. Optimal injected dosage of each patient with positive reaction was determined. Injected dosage of patients with negative reaction was gradually increased from to 60 microg. As the dosage was established, color Doppler ultrasonography was used to assess the morphodynamic features of cavernosal arteries.@*RESULTS@#Altogether 1 039 patients were positive, while 353 patients were negative by Rigiscan. In the 1 039 positive patients,663 were injected with 10 microg PGE(1), 265 patients 20 microg, 97 patients 30 microg,and 14 patients 60 microg while 89(13.4%),39(14.7%),41(42.3%),and 10(71.4%) patients were diagnosed vascular ED. Of the 353 negative patients, 267(75.64%) patients were diagnosed vascular ED. During intracavernous pharmacological testing,the more PGE(1) was used, the more vascular ED patients would be found(P<0.001).@*CONCLUSION@#PGE(1) is safe and effective for the erectile responses. The dosage of PGE(1) following intracavernous injection is related to vascular ED. Real-time Rigiscan monitoring combined with color Doppler ultrasonograph can be helpful to diagnose penile vascular diseases.