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Objective:To investigate the effect of the new nanomaterials,polyamidoamine dendrimers PAMAM-D,mediated foreign gene human decay accelerating factor(hDAF) for xenotransplantation transfected pig sperm cells.Methods:PAMAM-D/hDAF cDNA compounds were made.The compounds were divided into 0.2 μg,0.4μg,0.6μg,0.8 μg and 1.0μg groups (each group adding corresponding dose of PAMAM-D in accordance with the N/P ratio 10:1,20:1,40:1),then were digested by restriction enzymes.The compounds were incubated with washed pig sperm cells.Then the transfection efficiency was detected by in situ hybridization in the different groups.Results:The PAMAM-D molecule can prevent electrophoretic migration of DNA in the compound.After digested the compounds by restriction enzymes,DNA can not be degraded.The transfection efficiency was different in different groups.Among the total,the efficiency was higher in both groups of 0.4 μg and 0.6μg than that of others.The top was the group of 0.4 μg linear plasmid plus PAMAM-D when the N/P ratio was 20:1(47.5%±O.2%,167% vs control group).Conclusion:PAMAM-D can improve the efficiency of exogenous gene transfeeted pig sperm cells,which can reinforce the stable binding of exogenous DNA to sperm cells.
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Objective To investigate the expression of IFN-α of the peripheral blood monocytes (PBMC) stimulated by bacille Calmeue-Guérin (BCG) expressing recombinant human B7-2, and the antitumor effect of BCG activated killer cells(BAK). Methods Expression of human B7-2 was detected by SDS-PAGE and ELISA. Recombinant BCG and wild-type BCG were used to stimulate PBMC in different concentrations in vitro. Supernatant was collected at various time points and IFN-α was detected by an enzyme-linked immunosorbent assay (ELISA). MTT assay was used to observe the effects of recombinant BCG on proliferation of T cell, and LDH assay was used to study antitumor cytotoxicity of BAK cells. Results The concentration of human B7-2 in culture was 3.8 U/ml by ELISA. Compared with wild-type BCG, recombinant BCG can induce more IFN-α. The results of the LDH release assay showed that the anti-tumor activity of BAK cells stimulated by recombinant BCG was 2.14 fold higher than that of wild-type BCG. Conclusion The expression of IFN-α in PBMC stimulated by recombinant BCG is higher than that stimulated by wild-type BCG, suggesting that enhanced antitumor activity of BAK when bladder cancer cells could be enchanced by using recombinant BCG.
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Objective To construct a new secreting recombinant hIFN-?-2B-BCG to provide a new tool for the treatment of bladder tumor. Methods BCG was genetically engineered to secrete recombinant human interferon-alpha 2B by transforming of shuttle plasmid phIFN-?-2B. Expression of hIFN-? was readily detectable by ELISA. Results The phIFN-?-2B was transformed in BCG correctly,and the value of hIFN-?-2B in supernatant of recombinant BCG culture was calculated to approximately 997.2 pg/ml. Conclusions This study demonstrates that the recombinant phIFN-?-2B can be expressed in BCG secretively. As the construction of the plasmid and its transformation and expression in BCG were accomplished successfully, a foundation of reformed BCG and new vaccine will be established.
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Objective To construct transgenic mice tissue-specifically expressing human decay accelerating factor (DAF) on the vascular endothelium for xenotransplantation. Methods Transgenic mice expressing hDAF were generated by microinjection of a hDAF-minigene under the control of the human intercellular adhesion molecule-2 (ICAM-2) promoter. PCR analysis and Southern blot analysis of genomic DNA were used to examine the presence of the transgene in the genome of the offspring, and the expression was detected by RT-PCR and flow cytometry respectively. Immunohistochemical assay was performed to detect the distribution of hDAF on the tissues from these transgenic mice. An ex vivo perfusion model was used to compare hearts from these hDAF mice with wild-type hearts.Results After microinjection of gene, 21 of 133 mice born were shown to be transgenic. Human DAF gene was expressed on the surface of leucocytes in 8 of the 21 hDAF-integrated mice. Expression levels of 8 founders ranged from 70 % to 95 % of that on human leucocytes. Human DAF were strongly expressed on the vascular endothelium of heart, kidney and liver, with little or no positive staining observed on non-endothelial cells. These endothelial-specific hDAF hearts displayed prolonged survival compared to wild-type hearts during perfusion with 20 % human plasma and maintained approximately 20 % maximum work. Conclusions The introduced hDAF gene has been specifically expressed on the vascular endothelium of transgenic mice. The hDAF tissue-specifically expressed on vascular endothelium could enhance xenograft survival.
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Objective To investigate the causes,diagnosis and management of Wunderlich syndrome. Methods A total of 19 cases (15 men and 4 women) of Wunderlich syndrome were reported.Their average age was 47 years (range,11-75 years).Imaging examinations (B-ultrasound,IVU,CT,arteriography,MRI) together with laboratory testing and history supported the etiologic diagnosis of tumorous disease (n=10),non-tumorous disease (n=7) and unknown causes (n=2).Surgical management was performed in 9 cases of kidney tumor and 1 of ureter stone with obstructive hydronephrosis.Conservative treatment was adopted in the rest 9 cases.In 2 of the conservatively treated cases,the bleeding artery was embolized because of persistent bleeding. Results In the 9 tumor cases undergoing surgery,pathology confirmed renal cell carcinoma in 7,malignant fibrous histocytoma in 1 and transitional cell carcinoma of the renal pelvis in 1.These 9 cases all recovered after operation and experienced follow-up for 6 months to 3 years.Of them,5 cases died of tumor recurrence and metastasis;3 survived disease-free for 9 months to 2 years;1 had liver metastasis 1 year after operation and survived with tumor for 11 months.One case of ureter stone got significant improvement.The 9 cases undergoing conservative treatment had satisfactory results. Conclusions The main cause of Wunderlich syndrome is renal tumor.The conservative approach appears to be the most acceptable option for the patients with Wunderlich syndrome who have no malignant tumorous cause.
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Objective To study the regulatory effect of TGF ? 1 on the expression of multiple genes in prostatic stromal cells in vitro. Methods The primary culture of prostatic stromal cells (including fibroblasts and smooth muscle cells ) have been established and cultured to 4～6 passages. Then the cells were cultured in the medium with various concentration of TGF ? 1(0.01, 0.10, 1.00 and 10.0 ng/ml)for 48h. By semi quantitative RT PCR method, the androgen receptor(AR), TGF ? 1, bFGF and smoothlin mRNA were measured. Results The expression of AR could be stimulated by low concentration of TGF ? 1 ( P