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Objective To establish a stable and efficient method of culturing imDCs in vitro,and to explore the effect of GW5074, which blocks ERK1/2 signal pathway in the process of imnature dentritic cells (imDCs) on inducing differentiation of the na(i)ve allogeneic CD4+ T cells into Treg cells in vitro. Methods The imDCs and mature DCs (mDCs) were isolated and cultured from the peripheral blood mononuclear cells (PBMC) derived from a healthy adult male volunteer, and they were identified by cell morphology, cell surface marker and cell functions respectively. Na(i)ve CD4+ T cells were isolated from newborn umbilical vein blood and were divided into 5 groups to be cultured: (1) Blank control group: Na(i)ve CD4+ T cells were cultured alone;(2) Positive control group: The irrDCs were Middle-concentration GW5074 group;(5) High-concentration GW5074 group. In the last three groups, imDCs and na(i)ve CD4+ T cells were co-cultured, the same as the positive control group, but these groups were added by GW5074 dilution at the concentrations of 8, 24, and 40μmol/Lrespectively. After co-culture for 5 days, the transformation ratio from naive CD4+T cells to Treg T cells was detected by flow cytometry. Results On the surface of imDCs, there was stronger pression of CD1a, but weaker expression of CD80 and CD83. On the contrary, on the surface of mDCs, there was weaker expression of CD1a, but stronger expression of CD80 and CD83. The stimulation index in imDCs group and mDCs group was 1.12±0.03 and 2.85±0. 07 respectively. The transformation ratio of Treg T cells in blank control group, positive control group, low-concentration GW5074 group, middle-concentration GW5074 group and high-concentration GW5074 group was (5. 81±1.36)%, (35.73±2.07)%, (22.53±2.11)%, (11.55±1.73)%, and (4.97±1.83)%respectively. One-way ANOVA analysis revealed that there was no significant difference between high-concentration GW5074 group and blank control group, P＞0. 05, but significant difference between the remaining groups, P＜0.01. Conclusion High purity of imDCs can be obtained from PBMC by induction with rhGM-CSF and rhIL-4. ERK1/2 signal pathway plays a role in inducing the immune tolerance. GW5074 can inhibit differentiation of na(i)ve CD4+ T cells into Treg T cells.

8.Effects of Acute Sacral Neuromodulation at Different Frequencies on Bladder Overactivity in Pigs.

Xing LI ; Limin LIAO ; Guoqing CHEN ; Zhaoxia WANG ; Han DENG

International Neurourology Journal 2017;21(2):102-108

PURPOSE: We investigated the effects of different stimulation frequencies on the inhibition of bladder overactivity by sacral neuromodulation (SNM) in pigs. METHODS: Implant-driven stimulators were used to stimulate the S3 spinal nerve in 13 pigs. Cystometry was performed by infusing normal saline (NS) or acetic acid (AA). SNM (pulse width, 210 µsec) at frequencies ranging from 5 to 50 Hz was conducted at the intensity threshold at which observable perianal and/or tail movement was induced. Multiple cystometrograms were performed to determine the effects of different frequencies on the micturition reflex. RESULTS: AA-induced bladder overactivity significantly reduced the bladder capacity (BC) to 34.4%±4.7% of the NS control level (354.4±35.9 mL) (P<0.05). During AA infusion, SNM at 5 Hz did not significantly change the BC (48.1%±6.9% of the NS control level) (P>0.05), but SNM at 15, 30, and 50 Hz significantly increased the BC to 54.5%±7.1%, 55.2%±6.5%, and 57.2%±6.1% of the NS control level (P<0.05), respectively. No significant differences were found among the results obtained using frequencies of 15, 30, and 50 Hz (P>0.05). CONCLUSIONS: This study demonstrated that 15 Hz was an appropriate frequency for SNM and that frequencies higher than 15 Hz did not lead to better surgical outcomes.
Acetic Acid ; Reflex ; Spinal Nerves ; Swine* ; Tail ; Urinary Bladder* ; Urinary Bladder, Overactive ; Urination