Objective To investigate the effects of syncytial formation induced by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spike protein(SARS-2-S)on the proliferation and migration of human lung adenocarcinoma A549 cells and mouse melanoma B16 cells in vitro.Methods Plasmids expressing human angiotensin-converting enzyme 2(hACE2)and SARS-2-S were constructed and respectively co-transfected with lentiviral packaging plasmids into HEK-293FT cells before the lentiviral supernatant was collected and infected with A549 cells which were screened by puromycin to obtain the A549 cells respectively that were stably transfected with hACE2(A549-A)and SARS-2-S(A549-S).The protein expression of A549-A and A549-S cells was verified by Western blotting.A549-A and A549-S cells were co-cultured before their syncytia were observed by fluorescence microscopy.Conditioned media(syncytial supernatant and non-syncytial supernatant)was collected to culture A549 cells,ovalbumin(OVA)-gene-modified B16 cells(B16-OVA),and B16-F10 cells in vitro.The CCK-8 assay and colony formation assay were used to assess the proliferation capacity of tumor cells,while the wound healing assay was employed to evaluate the migration capacity of tumor cells.Results Stable A549 cell lines expressing hACE2 and SARS-2-S were constructed.The SARS-2-S-induced syncytial formation system was established after co-culture of A549-A and A549-S cells.Both syncytial and non-syncytial supernatants significantly promoted the proliferation and migration of A549,B16-OVA,and B16-F10 cells in vitro,especially the syncytial supernatant.Conclusion SARS-2-S-induced syncytial formation promotes the proliferation and migration properties of A549 and B16 cells in vitro.

Objective:To investigate the clinical application and surgical effect of minimal invasive liposuction system (MILS) in lumbar abdominal liposuction.Methods:From July 2019 to March 2021, 985 patients were selected for lumbar and abdominal liposuction. Local anesthesia and intravenous anesthesia were used in all operations. The preoperative design and intraoperative procedures were carried out according to the requirements of MILS method. An average of 2 425 ml fat was aspirated, and the average operation time was 142.4 min.Results:After 3-18 (average 3.85 months) months of follow-up, 857 patients (87% of the patients) were discharged on the day of operation. The waist line curve of the patients was clear and the bulge of the upper and lower abdomen was significantly reduced. The satisfactory rate was 93.5%, and the incidence of complications was 2.8%. No severe complication occurred.Conclusions:MILS is a safe and effective method to perform the waist and abdominal liposuction scientifically and finely. The MILS method makes the lumbar abdominal liposuction standardized, reduces unnecessary procedure, shortens the liposuction learning curve, reduces the occurrence of complications, and achieves the best aesthetic effect.

Objective:To investigate the clinical application and surgical effect of minimal invasive liposuction system (MILS) method in the thighs liposuction.Methods:From July 2019 to March 2021, 868 patients were selected for thigh liposuction. Local anesthesia and intravenous anesthesia were used in all operations. The preoperative design and intraoperative procedures were carried out according to the requirements of MILS ? method. There was an average of 2 154.4 ml fat aspirated, and the average operation time was 156.8 min. Results:After 2-18 months of follow-up, 83.3% of the patients were discharged on the day of operation. The thigh line curve of the patients was fluent and the perimeter of upper thigh was significantly reduced. The satisfactory rate was 92.3%, and the incidence of complications was 2.8%. No severe complication occurred.Conclusions:MILS method is a safe and effective procedure to partition thighs liposuction area scientifically and finely. The MILS method makes the lumbar abdominal liposuction operation standardized, reduces unnecessary operation, shortens the liposuction learning curve, reduces the occurrence of complications, and achieves the best aesthetic effect.

Objective To investigate the effects ofpravastatin on the expression ofmicroRNA-155 (miR-155) and the functions of lipopolysaccharide (LPS)-treated extravillous trophoblast cells.Methods In vitro cultured HTR-8/SVneo cells were divided into the following groups:control group,enhanced plasmid with green fluoscent protein (pEGFP)-miR-155 group (transfected with green fluorescent protein-tagged miR-155),LPS group (treated with 100 ng/mL of LPS),miR-155 inhibitor+LPS group,pravastatin+LPS group (treated with 100 ng/mL of LPS following pretreatment with 12.50,25.00,50.00 and 100.00 μ g/ml of pravastatin),and pravastatin+pEGFP-miR-155 group (transfected with pEGFP-miR-155 following pretreatment with 50 μ g/ml of pravastatin).Levels of miR-155 in HTR-8/SVneo cells treated with different strategies were measured by real-time polymerase chain reaction.Expression of phosphorylated JunB (p-JunB) and p-FosB proteins was analyzed by Western blotting.Migration,invasion and apoptosis of HTR-8/SVneo cells were also analyzed.All data were analyzed with t test.Results (1) Compared with the control group,HTR-8/SVneo cells in the pEGFP-miR-155 group were characterized by shorter migration distance [(274.70± 18.82) vs (181.00±8.62) μ m],less transmembrane cells [(123.00±4.36) vs (63.00±6.08)] and enhanced apoptosis [(5.40± 0.68)％ vs (9.27±0.68)％] (all P＜0.05).(2) Compared with the LPS group,the miR-155 inhibitor+LPS group showed longer migration distance of HTR-8/SVneo cells [(166.30±5.07) vs (242.00±18.07) μm,P＜0.05],more transmembrane cells [(71.67±6.12) vs (109.00±7.81),P＜0.05] and decreased cell apoptosis [(14.40±1.69)％ vs (6.23± 0.44)％,P＜0.05].(3) Expression of miR-155 at mRNA level in the LPS group was increased as compared with that of the control group (1.65 0.07 vs 0.79±0.12,P＜0.05).Compared with the LPS group,pretreatment with 12.50,25.00,50.00 and 100.00 μ g/ml of pravastatin decreased the expression of miR-155 at mRNA level [(1.14±0.10),(1.02±0.10),(0.74±0.15) and (1.140.02)],especially at the concentration of 50 μμ g/ml (all P＜0.05).(4) Expression ofp-JunB and p-FosB proteins in the control,LPS and pravastatin (50 μ g/ml)+LPS groups were (0.33 ±0.06) vs (0.37±0.07),(1.22±0.20) vs (0.80±-0.13),and (0.31 ±0.02) vs (0.21 ±0.05),respectively,showing higher expression level in both p-JunB and p-FosB proteins in the LPS group compared with that of the other two groups (all P＜0.05).(5) Compared with the LPS group,the pravastatin (50 μμ g/ml)+LPS group showed increased migration distance [(166.30±5.07) vs (246.80± 13.42) μ m,P＜0.05],increased numbers of transmembrane cells [(71.67 ± 6.12) vs (95.33 ± 2.73),P＜0.05] and decreased cell apoptosis [(14.40± 1.69) vs (6.05 ± 0.35)％,P＜0.05].(6) Compared with the pEGFP-miR-155 group,the pravastatin (50.00.00 μμ g/mL)+pEGFP-miR-155 group showed longer migration distance [(197.50± 13.86) vs (275.80± 13.63) μ m,P＜0.05],more transmembrane cells [(52.67±5.49) vs (125.00±6.66),P＜0.05] and lower rate of cell apoptosis [(8.90± 1.00) vs (5.05±0.35)％,P＜0.05].Conclusions Pretreatment of extravillous trophoblast cells with pravastatin can protect them from apoptosis and loss of migratory and invasive abilities through inhibiting the activation of AP-1 and down-regulating the expression of miR-155,which may be a mechanism that inhibits the development of preeclampsia.

Objective:To evaluate the immunomodulating effect of oyster peptide on immunosup-pressed mice.Methods:ICR mice injected with cyclophosphamide (CTX)were adopted as the module group,with mice without treatment as the control group,and different dosages of oyster peptide (0.5 g/kg,1 .0 g/kg,and 2.0 g/kg)were given to the low,middle,and high groups for 1 5 days.The body weight,spleen,and thymus weight of the mice,structures under the microscope of the immune organs, numbers of white blood cells,ratios of T lymphocyte subsets,immune cytokines and numbers of nuclear cells,and DNA content in bone marrow were all assessed.Results:Compared with the control group, the structures of thymus and spleen of the mice in the CTX group appeared obscure and shrunk when ob-served under microscope,the number of their white blood cells declined (P =0.04),the proportion of their CD3 +T cells in peripheral blood declined (P =0.003),the proportion of their CD8 +T cells in pe-ripheral blood declined (P =0.002),the concentration of their IL-5 in peripheral blood significantly in-creased (P <0.01 ),the concentration of their nucleated cells and DNA density in bone marrow de-creased (P =0.04,P <0.01 ).Oyster could improve the structures of thymus and spleen of the immuno-suppressed mice.Compared with the CTX group,the number of white blood cells in 2.0 g/kg group in-creased (P =0.003),the proportion of CD3 +T cells in peripheral blood in 1 .0 g/kg group (P =0.04) and 2.0 g/kg group (P =0.02)increased,the proportion of CD8 +T cells in peripheral blood in 2.0 g/kg group increased (P =0.002),the concentration of IL-5 in peripheral blood in all the oyster treated groups increased (P <0.01 in 0.5 g/kg,1 .0 g/kg,and 2.0 g/kg groups),the concentration of IL-1 7 in peripheral blood in 2.0 g/kg group decreased (P =0.03),the concentration of nucleated cells in bone marrow of all the oyster treated groups increased (0.5 g/kg vs.CTX,P =0.04;1 .0 g/kg vs. CTX,P =0.02;2.0 g/kg vs.CTX P =0.01 ),the DNA content in bone marrow of all the oyster treated groups increased (P <0.01 in the 0.5 g/kg,1 .0 g/kg,and 2.0 g/kg groups).Conclusion:Oyster peptide could improve the structures of immune organs of the CTX-induced immunosuppressed mice,re-cover the imbalances of T lymphocyte subsets,improve the immune cytokines and increase numbers of nucleated cells and DNA content in bone marrow,thus improving the immunologic function.

ObjectiveTo investigate the relationship between levels of inflammatory cytokines, Ureaplasma infection in midtrimester amniotic fluid and spontaneous preterm delivery.MethodsFrom April 2009 to March 2012, a total of 1 865 pregnant women who underwent amniocentesis in midtrimester with known pregnant outcomes in Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School, among whom 43 cases who delivered spontaneously before 37 weeks, were classified as preterm group, and the control group consisted of 373 normal term delivery women selected from the other 1 785 cases who term delivered during the same period. Cytokines, including interleukin (IL)-10, IL-1β, IL-6, monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) in amniotic fluid, were measured by MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel and analysis system for liquid phase chips.Ureaplasma DNA was detected by polymerase chain reaction. Sum-rank test was used to compare the difference of cytokines between the two groups and receiver-operating characteristic curve and Logistic analysis were used to analyze the value of cytokines to predict spontaneous preterm delivery.ResultsThe median maternal age in preterm group was higher than in the control group [34(24-49) vs 29(20-47) years] (Z=-3.107,P=0.002), but there was no significant difference in levels of the five cytokines among women with different age in control group(20-24, 25-29, 30-34 and≥35 years old) (allP>0.05). The levels of IL-1β and IL-6 were not significantly different between two groups (allP>0.05), but levels of IL-10, MCP-1 and TNF-α in the preterm group were much higher than in the control group [11.22(4.79-468.73) vs 7.87(0.93-26.62), 1 358.86(553.16-7 635.81) vs 1 137.98 (311.80-5 196.33), 9.78(5.72-106.51) vs 8.37(2.56-30.20) pg/ml, respectively;Z were-4.333,-2.820 and-3.390, allP<0.01]. The areas under receiver-operating characteristic curve of IL-10, MCP-1 and TNF-αwere 0.70, 0.63 and 0.66. The appropriate cut-off level of IL-10 in amniotic fluid for prediction of preterm birth was 9.06 ng/ml, with a sensitivity of 74.4% and a specificity of 57.6%, and the latter increased to 100.0% when combined with IL-10, MCP-1 and TNF-α, but the sensitivity declined to 16.3%.Ureaplasma was identified in only two preterm cases and none in the controls. However, the levels of the five cytokines in the two infected cases in preterm group were 2.12–43.00 times of the rest cases in the same group, and 2.26–60.00 times of the controls.ConclusionsAlthough IL-10, MCP-1 and TNF-α levels in midtrimester amniotic fluid are increased, it is not able to predict spontaneous preterm birth neither independently nor combined together. Ureaplasma infection rate in amnion cavity is low, but may related to parts of spontaneous preterm delivery.

OBJECTIVETo explore the expressions of transforming growth factor-beta1 (TGF-beta1) and its signaling transduction molecule Smad 2 and their significance in the development of glomerulosclerosis.

METHODSUsing in situ hybridization and immunohistochemistry to detect Smad 2 mRNA expression and TGF-beta1, collagen IV, fibronectin expression in renal biopsies from 61 cases with a spectrum of glomerulonephritis including IgA nephropathy (40 cases), membranous glomerulonephritis (10 cases) and sclerosing glomerulonephritis (11 cases), compared with 11 cases of glomerular mild lesion with image analysis system.

RESULTSWith the exception of Smad 2 mRNA expression in mild type IgA nephropathy, all other types of diseased glomeruli showed increased expression of both TGF-beta1 and Smad 2 mRNA when compared with the 11 cases of mild glomerular lesions. The expressions of glomerular TGF-beta1 and Smad 2 mRNA positively correlated with collagen IV and fibronectin deposition in the glomeruli.

CONCLUSIONSTGF-beta1 and Smad 2 may be involved in the excessive deposition of glomerular extracellular matrix and play an important role in the development of glomerulosclerosis.

Collagen Type IV ; analysis ; DNA-Binding Proteins ; genetics ; Fibronectins ; analysis ; Glomerulonephritis ; metabolism ; Humans ; Immunohistochemistry ; Kidney Glomerulus ; chemistry ; RNA, Messenger ; analysis ; Smad2 Protein ; Trans-Activators ; genetics ; Transforming Growth Factor beta ; analysis ; Transforming Growth Factor beta1

OBJECTIVETo observe the localization of adrenomedullin (AM) in rat kidney tissue and its inhibitory effect on the growth of cultured rat mesangial cells (MsC).

METHODSA monoclonal antibody against AM developed by our laboratory was used to detect the localization of AM protein in rat kidney tissue by avidin-biotin complex immunohistochemistry. The expressions of AM and its receptor CRLR mRNA on cultured glomerular epithelial cells (GEC) and MsC were investigated by Northern blot assay, and the possible effect of AM secreted by GEC on MsC proliferation was observed using [3H]thymidine incorporation as an index.

RESULTSA specific monoclonal antibody against AM was succesfully developed. AM was immunohistochemically localized mainly in glomeruli (GEC and endothelial cells), some cortical proximal tubules, medullary collecting duct cells, interstitial cells, vascular smooth muscle cells and endothelial cells. Northern blot assay showed that AM mRNA was expressed only on cultured GEC, but not on MsC, however, AM receptor CRLR mRNA was only expressed on MsC. GEC conditioned medium containing AM can inhibit MsC growth and AM receptor blocker CGRP8-37 may partially decreased this inhibitory effect.

CONCLUSIONAM produced by GEC inhibits the proliferation of MsC, which suggests that AM as an important regulator is involved in glomerular normal physiological functions and pathologic processes.

Adrenomedullin ; Animals ; Antibodies, Monoclonal ; analysis ; Calcitonin Receptor-Like Protein ; Cell Division ; Cells, Cultured ; Epithelial Cells ; cytology ; metabolism ; Female ; Glomerular Mesangium ; cytology ; metabolism ; Kidney Glomerulus ; cytology ; Mice ; Mice, Inbred BALB C ; Peptides ; immunology ; metabolism ; physiology ; RNA, Messenger ; biosynthesis ; Receptors, Calcitonin ; biosynthesis ; genetics

Purpose　To investigate the significance of u-PA and PAI-1 expression on the glomeruli,and the effect of heparin on their expressions in rat anti-thy1 glomerulonephritis.　Methods　We analyzed the cell proliferation and the expression of u-PA/PAI-1 on the glomeruli by immunohistochemistry and quantitative analysis of immunostaining.　Results　The cell proliferation of the glomeruli decreased significantly at 7 th,14 th,21 st day after heparin treatment in comparison to the glomerulonephritic group(P<0.05 or 0.01).The expression of u-PA and PAI-1 on the glomeruli in glomerulonephritic and heparin-treated groups was higher than that in the control group.At 3 rd,7 th,14 th,21 st day,the glomerular hypercellularity in the glomerulonephritic group was closely related to the increased expression of u-PA and PAI-1(P<0.05 or 0.01).At 3 rd,7 th day,the decreased cell proliferation of the glomeruli in heparin-treated group had close relationship with the decreased expression of PAI-1(P<0.05).　Conclusions　In rat anti-thy 1 glomerulonephritis model,the expression of u-PA and PAI-1 increased with glomerular hypercellularity;heparin treatment can decrease the extent of glomerular hypercellularity in rat anti-thy 1 glomerulonephritis.The treatment function of heparin might be related with the inhibitory effect of PAI-1 expression on the glomeruli.