Objective To investigate the effect of bFGF on the grafting ability (grafing efficiency)of CD34 + cord blood cells seeded in 5 FU injured human bone marrow stromal cells.Methods Using combined methods of bone marrow stromal cell culture system,light microscopy and electron microscopy,flow cytometry,we investigated the toxic effects of 5 FU on human bone marrow stromal cells (hBMSC) and the benefit of bFGF on dynamics of hBMSC as well as the ability of the cord blood stem cell to bind hBMSC.Results After 3 hours incubation with 5 FU,significant changes in hBMSC cytosolic and nuclear morphology were observed.Nuclear splitting and abnormal shapes of the pseudopodia were found under the electron microscope.Flow cytometry revealed that apoptosis appeared before G 0/G 1,and that cells in G 0/G 1and G 2/M phases were remarkably lower,while the cells in S phase were remarkably higher than the controls.Two hours after adding the cord blood hematopoietic progenitor cells,the group treated with bFGF(injury with intervention of bFGF group,IBG)showed higher proprtion of the adherence layer compared to the group without adding bFGF(injury group,IG)(29% versus 12%) Conclusion 5 FU causes DNA damage in hBMSC.It also impairs the binding ability of cord blood stem cell to hBMSC.bFGF is able to repair hBMSC damage and improve the binding of CD34 + cord blood hematopoietic progenitor cells onto hBMSC.

Objective To elucidate the role of human bone marrow stromal cells (hBMSC) in combination with exogenous cytokines such as stem cell factor(SCF) and FLT 3 ligand (FL) in supporting the in vitro expansion of CD 34 + cells purified from cord blood.Methods CD 34 + cells were isolated from umbilical cord blood by using a high gradient magnetic cell sorting system(MACS),expanded in combination with SCF,IL3 (interleukin 3),IL 6 (interleukin 6),FL,EPO (erythropoietin),and were planted onto the pre established irradiated (20Gy) stroma layer(hBMSC plus combinations of cytokines) respectively.On day 10,total cells were counted, and hematopoietic progenitor cells were assessed by semisolid culture assay,CD 34 + cells were quantitated by FACS.Results After separation by MACS,the frequency of CD 34 + cells was 92%?0.04%.On day 2,almost all the inoculated cells adhered to the stroma layer,with only a small number in the supernatant.Then,cells in the supernatant increased gradually, but the percentage of CD 34 + cells decreased.Compared with control, expansion fold of CD 34 + cells, CFU GM, BFU E were significantly higher in hBMSC group (P 0.5).Conclusion ① CD 34 + cells from cord blood formed foci adherent to the monolayer and colony forming cells remained in the culture of 10days,indicating that hBMSC can support hematopoiesis in vitro;②using human bone marrow stromal cells in cooperation with exogenous cytokines may be a feasible way to expand hematopoietic stem/progenitor cells.

Objective:To investigate the expression and significance of Toll-like receptor 4 (TLR4) in renal tissue and peripheral blood of children with idiopathic nephrotic syndrome(INS).Methods:The renal biopsy tissues of 78 children with INS diagnosed in the First Affiliated Hospital of Xinxiang Medical University from October 2015 to June 2018 and normal renal tissues of 21 children (control group 1) were collected, and the expressions of TLR4 in the renal tissue was detected by using immunohistochemical method.The expression of TLR4 in different renal pathological types and clinical types of INS was compared, and the correlation of TLR4 with 24-hour urinary protein and serum albumin was analyzed.The expression levels of TLR4 in peripheral blood of children with INS before and after treatment (active stage and remission stage) and 23 healthy children (control group 2) were detected by enzyme linked immunosorbent assay(ELISA). The serum expression levels of TLR4 in different renal pathological types and clinical types of INS were compared, and the correlation of TLR4 with 24-hour urinary protein and serum albumin was analyzed; The correlation between TLR4 expression in renal tubules and in the serum of children with INS was also analyzed.Results:(1)Compared with the expression of TLR4 in normal renal tissues[(0.93±0.26)%], the expression of TLR4 in glomeruli and interstitium of all pathological types of INS [mesangial proliferative glomerulonephritis (MsPGN): (0.93 ± 0.21)%, focal segmental glomerulosclerosis (FSGS): (1.02±0.25)%, membranous glomerulonephritis(MN): (1.03±0.09)%, minimal change disease(MCD): (1.02±0.27)%]was not significantly different ( F=0.741, P=0.562), but the expression of TLR4 in renal tubules[MCD: (82.94±4.62)%, MN: (63.54±1.98)%, MsPGN(42.32±2.97)%, FSGS: (22.60±2.07)%] was significantly increased ( F=1 929.842, P<0.01), Especially, the expression of TLR4 in renal tubules of MCD type INS was significantly higher than that of MN, MsPG N and FSGS [MCD: (82.94±4.62)%, MN: (63.54±1.98)%, MsPGN: (42.32±2.97)%, FSGS: (22.60±2.07)%], and the differences were statistically significant(all P<0.01). TLR4 expression in renal tubules was the highest in steroid-sensitive nephrotic syndrome (SSNS) type and the lowest in INS patients with steroid-resistant nephrotic syndrome (SRNS) type, and the differences were statistically significant( F=220.951, P<0.01). (2)The expression of serum TLR4 in INS children at the active stage [MsPNG: (143.36±12.99) ng/L, FSGS(75.94±7.29) ng/L, MN(210.22±14.66) ng/L, MCD(283.93±21.58) ng/L]was significantly higher than that in INS children at remission stage [MsPNG: (29.51±4.93) ng/L, FSGS(15.66±3.78) ng/L, MN(45.40±5.73) ng/L, MCD(62.29±7.90) ng/L]and control group 2[(0.69 ± 0.33) ng/L], and the differences were statistically significant(all P<0.01); the expression of serum TLR4 in INS children at remission stage was significantly higher than that in the control group 2 ( F=286.287, P<0.01). TLR4 had the highest expression level in serum of MCD type INS children at active and remission stages, followed by MN and FSGS successively.The expression of serum TLR4 was highest in SSNS and lowest in SRNS, and the differences were statistically significant ( F=147.438, P<0.01). (3)The expression of TLR4 in renal tubules of children with INS[(62.82 ±20.94)%]was positively correlated with the expression of TLR4 in serum[(213.26±73.33) ng/L] ( r=0.852, P< 0.05). The expression levels of TLR4 in renal tubules and serum of INS patients at active stage were positively correlated with 24-hour urinary protein level[(123.05±33.55) mg/kg] ( r=0.401, 0.427, all P<0.05), and negatively correlated with serum albumin level[(19.54±3.55)g/L] ( r=-0.602, -0.617, all P<0.05). Conclusions:The expression of TLR4 in renal tubules and serum of children with INS increases, and may be related to different renal pathological types and clinical types of children with INS, as well as disease activity.