ABSTRACT
Objective To investigate the effects and its mechanisms of bradykinin B2 receptor (B2R) on the growth and function of human extravillous trophoblast cells (HTR-8/SVneo cells).Methods B2R expression plasmid (pcDNA3.1-B2R) was constructed and B2R-specific small interfering RNA (siRNA) was synthesized.HTR-8/SVneo cells were divided into four groups and transfected with pcDNA-3.1 (blank plasmid group),pcDNA3.1-B2R (B2R expression plasmid group),siRNA negative control and B2R-specific siRNA,respectively.Quantitative real-time reverse transcription-polymerase chain reaction and Western blot were used to detect the changes in the expression of B2R,matrix metalloproteinase-2,matrix metalloproteinase-9,cyclin D1 and vascular endothelial growth factor-A at both mRNA and protein levels in HTR-8/SVneo cells.Cell counting kit-8 and flow cytometry were used to detect cell activity and cell cycle,respectively.Cell migration assay and cell invasion assay were used to detect cell migration and invasion,respectively.Tube formation assay was used to evaluate the tube formation abilities of HTR-8/SVneo cells.All data were analyzed with t test.Results (1) Compared with the blank plasmid group,expression of B2R in HTR-8/SVneo cells in the B2R expression plasmid group were significantly increased at both mRNA (5.06±0.49 vs 1.00±0.28,t=7.226,P=0.002) and protein levels (1.34 ± 0.07 vs 1.00± 0.05,t=3.727,P=0.006).And the expression of B2R in HTR 8/SVneo cells transfected with B2R-specific siRNA were significantly reduced at both mRNA (0.34±0.05 vs 1.00±0.17,t=3.667,P=0.021) and protein levels (0.74±0.03 vs 1.00±0.05,t=4.097,P=0.006) comparing with the siRNA negative control group.(2) Compared with the blank plasmid group,HTR-8/SVneo cells being transfected with B2R expression plasmid showed a higher proliferation activity (1.50 ±0.03 vs 1.34± 0.04) promoting G0/G1 to S phase transition;compared with the siRNA negative control group,B2R-specific siRNA inhibited the proliferation of HTR-8/SVneo cells (1.06 ± 0.04 vs 1.20± 0.02) and arrested the cell cycle at G0/G 1 phase (all P＜0.05).(3) Compared with the blank plasmid group,B2R expression plasmid significantly increased the HTR-8/SVneo cell migration distance [(80.67±0.33) vs (41.33±5.24) μm],the number of cells penetrating matrigel gel (360.70 ±12.33 vs 268.70 ±14.45) and the number of cells having tube-like structures (28.20 ± 2.47 vs 14.00± 1.67),while significantly decrease was shown in these three parameters in B2R-specific siRNA group comparing with the siRNA negative control group [HTR-8/SVneo cell migration distance:(56.00±3.51) vs (87.00±1.53) μ m,number of cells penetrating matrigel gel:143.30± 12.91 vs 252.30± 17.07;number of tube-like structures:6.25±1.49 vs 15.75 ±2.02;all P＜0.05].(4) Expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 at mRNA level,and expression of cyclin D1 and vascular endothelial growth factor-A increased in the B2R expression plasmid group than in the blank plasmid group,and decreased in the B2R-specific siRNA group than in the siRNA negative control group at both mRNA and protein levels (all P＜0.05).Conclusions B2R might enhance the activity,migration,invasion and tube formation ability of human extravillous trophoblast cells through promoting the expression of matrix metalloproteinase-2,matrix metalloproteinase-9,cyclin D1 and vascular endothelial growth factor-A.
ABSTRACT
Objective To investigate the interference and associated mechanism of hnman tissue kallikrein (HK) gene on renal interstitial fibrosis in rats with 5/6 nephrectomy. Methods Human kallikrein cDNA was packed in a recombinant adeno-associated virus(rAAV)-based plasmid vector. The rAAV-HK was produced by transfection in 293 cells. Twenty-four male Wistsr rats were divided into sham operation and operation groups. The rats with 5/6 nephrectomy were randomly divided into simple operation, control and experiment groups. The rats in experiment group received single dose rAAV-HK via the tail vein with 1×1011 pfu. Before nephrectomy and every month after surgery until the rats were sacrificed, the caudal arterial pressure was measured using tail cuff blood pressure determinator. Three months after HK gene delivery, the rats were sacrificed. The expression of HK in rats was assessed by RT-PCR, Western blot and enzyme-linked immunosorbent assay (ELISA). The pathological changes of renal interstitium were evaluated by Masson stainning, and the distribution of bradykinin B2 receptor (BKB2R) and angiotensin Ⅱ typel receptor (ATIR) was examined by immunohistochemistry. The expressions of BKB2R, AT1R, p-MAPK protein in renal tissue were detected by Western blot. Results Three months after HK gene delivery, the systolic blood pressure of experiment group was significantly decreased compared with the control group [(163±13) nun Hg vs (217±16) mm Hg, P<0.01](1 mm Hg=0.133 kPa). Compared with sham rats, the rats in simple operation group and control group had much more renal interstitial collagen deposition and more serious fibrosis performance, but renal interstitial collagen deposition and fibrosis were significantly ameliorated in the rats of experiment group. In addition, the tubulointerstitial injury index of HK transgenic rats was significantly lower than that of the rats in control group (1.33±0.73 vs 3.01±0.62, P<0.01). Up-regnlating expression of bradykinn B2 receptor protein and down-regulating expression of AT1 receptor and p-MAPK protein were found in renal tissues of experimental group after three months (P<0.05). Conclusion HK gene delivery significantly alleviates renal interstitial fibrosis in rats with 5/6 nephrectomy through regulating the expression of bradykinin B2 receptor, AT1 receptor and p-MAPK in renal tissue.