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Yang L.,Lanzhou University | Li T.,Lanzhou University | Lu Y.,University of Sichuan | Luo G.,Peoples Hospital of Guizhou | And 2 more authors.
Academic Journal of Second Military Medical University

Objective To observe C4d deposition in renal allografts of rats undergoing chronic allograft nephropathy (CAN), and to analyze the effects of immunosuppressants on deposition of C4d in peritubular capillaries. Methods The renal grafts of Fisher 344 rats were ortho topically transplanted into Lewis rats to create CAN models, and all the recipients were given cyclosporine A (CsA) 10 mg/(kg · d) X10d after operation. The models were then divided into 5 groups (each n=9): Group A was normal saline control group, only receiving vehicle orally; Group B, C, D, and E received CsA 6 mg/(kg · d), RAPA0. 8 mg/(kg · d), FK506 0. 15 mg/(kg · d), and MMF 20 mg/(kg · d), respectively. The renal allografts were harvested after three rats were sacrificed at the 4th, 8th and 12th weeks post-transplantation. The histological changes were assessed according to Banff 97 standard. The deposition of C4d was detected by immunofluorescence method. Results C4d deposition in peritubular capillary (PTC) was found in all the allografts at the 4th week after transplantation, while there were no obvious clinical pathological changes of CAN in all groups, and the Banff scores were not significantly different among different groups (P > 0. 05). CAN manifestations of different degrees were observed 8 weeks after operation, with increased C4d deposition in the PTC. Severest CAN was observed at the 12th week after operation, accompanied by the most C4d deposition in the PTC. C4d deposition was positively correlated with the severity of CAN (r=0. 894, P = 0. 000). Compared with the control group, CsA and FK506 showed no significant effect on C4d deposition (P>0. 05); however, MMF and RAPA significantly decreasedC4d deposition (P<0. 05). Conclusion Deposition of C4d in PTC may appear in allografts earlier than the pathological changes of CAN, and the deposition is associated with the progression of CAN. MMF and RAPA can inhibit the progression of CAN, while CsA and FK506 can not. Source

Hu X.,Chongqing Medical University | Wang J.-Y.,Peoples Hospital of Guizhou | Yuan J.,Central Laboratory | Wan X.,Peoples Hospital of Guizhou | And 2 more authors.
Academic Journal of Second Military Medical University

Objective: To construct a recombinant lentivirus carrying C57BL/6 mouse estrogen receptor α (Erα) and to infect mouse neurons, so as to pave a way for further studying the relationship of Erα with some nervous system diseases. Methods: Erα gene was inserted into the main virus vector LV-GFP-flag to construct recombinant lentiviral vector LV-Erα-flag, which was confirmed by agarose gel electrophoresis (AGE) and DNA sequencing. Recombinant lentivirus V-Erα-flag was produced by 293T cells following the co-transfection of LV-Erα-flag with the packaging plasmids pHelper 1.0 and pHelper 2.0, and the virus titer was examined. The neurons of C57BL/6 mouse were cultured using a serum-free culture medium, and then control lentivirus V-GFP-flag was used to infect the neurons. The infection efficiency and apoptosis rate were examined by flow cytometry to obtain optimal multiplicity of infection (MOI). GFP expression was detected daily under an inverted fluorescent microscope. After that, V-Erα-flag with the optimal MOI was used to infect neurons; the expression of Erα mRNA and protein in the neurons was detected by quantitative real-time PCR and Western blotting analysis. Results: AGE and DNA sequencing confirmed that LV-Erα-flag was successfully constructed, and packaged into 293T cells with a titer of 2×108 TU/ml. Control lentivirus V-GFP-flag could infect neurons, with the infection efficiency being (89.8±4.03)% and the cell apoptosis rate being only (3.6±0.29)% when MOI = 5. Neurons could survive in the culture for at least 8 weeks, during which the GFP was persistently expressed, indicating the lentivirus could efficiently and stably infect the neurons. The expression of Erα mRNA and protein was greatly increased in neurons infected with V-Erα-flag (MOI = 5). Conclusion: The recombinant lentivirus carrying Erα has been constructed successfully, which can infect neurons and lead to increase the expression of Erα mRNA and protein. Source

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