ABSTRACT
Aim To investigate whether human umbili-cal cord mesenchymal stem cells(hUC-MSCs)exposed to inflammatory conditions could release large amounts of exosomes to induce regulatory T cells(Treg).Meth-ods hUC-MSCs were isolated by enzyme digestion method.(In vitro)interferonγ(IFN-γ)was added in-to hUC-MSCs to mimic inflammatory microenviron-ments,then exosomes were extracted from the superna-tant of normal conditional medium or IFN-γpretreated hUC-MSCs.Both sources of exosomes,Nor-hUC-exo and IFN-γ-stimulated hUC-exo, were identified by Nanoparticle Trafficking Analysis (NTA )and Western blot for the exosome-enriched protein CD63 .Next,hu-man peripheral blood mononuclear cells (PBMCs ) stimulated with PHA were respectively co-cultured with hUC-MSCs,IFN-γ-pretreated-hUC-MSCs,hUC-MSCs exosomes or IFN-γ-stimulated-hUC-MSCs exosomes for 5 days to assess the exosomes-T cells communication. The proliferation rate of PBMCs and frequency of CD4 +/CD25 +/Foxp3 + Treg were measured by flow cytometry.Results The isolated cells from human um-bilical cord tissue,which were positive for CD73, CD44,CD29,CD90 and HLA-ABC,but were nega-tive for CD31 and CD34,were mesenchymal stem cells indeed.After IFN-γtreatment,hUC-MSCs secreted nu-merous exosomes(P<0.05 ).Morerover,there was a significantly higher level of CD63 ,but no difference in diameter between Nor-hUC-exo and IFN-γ-stimulated hUC-exo.IFN-γ-stimulated hUC-exo had a superior a-bility compared with Nor-hUC-exo to suppress the pro-liferation of PHA stimulated PBMCs due to their upreg-ulation of the percentage of Treg (1 1.53 ±0.88% vs 6.60 ±0.56%,P <0.01 ).Conclusion hUC-MSCs could promote the expression of Treg to modulate im-munosuppression through exosomes,especially for IFN-γ-licenced exosomes,which might carry much immu-notherapeutic potential.
ABSTRACT
Aim To measure the methylation rate of bone marrow mesenchymal stem cells (BMMSCs) using ultra-performance liquid chromatography-tandem mass spectrometry, and determine the methylation rate in the process of senescence.Methods The DNA extracted from BMMSCs would be digested into individual deoxynucleosides using enzymatic hydrolysis.To quantify the global genomic DNA methylation rate,we developed a method using ultra-performance liquid chromatography-tandem mass spectrometry with multiple reaction monitoring to simultaneously measure the levels of 5-methyl deoxycytidine and deoxyguanosine in digested genomic DNA.Results The DNA methylation rate could be analyzed within two minutes after hydrolyzing 1μg DNA for an hour.The detection limit of DNA methylation rate was 5.00×10-4.The coefficient of variance of the intra-day and inter-day precision was within 7.12×10-2 and 0.119, respectively.With the subculture of BMMSCs, the methylation rate gradually decreased.The methylation rate of stem cells was the lowest in 4~6 generation, and gradually increased with the subculture.Conclusions Using this method, the preliminary data on DNA methylation of BMMSCs in the process of senescence are obtained.The method is simple and convenient for sample preprocessing, and the method is fast and accurate with good sensitivity and repeatability.
ABSTRACT
<p><b>OBJECTIVE</b>To evaluate the safety and short-term outcomes of laparoscopic-assisted surgery for rectal cancer by comparing the efficacy of laparoscopy and open surgery.</p><p><b>METHODS</b>Clinical data of patients with rectal cancer treated by laparoscopy or open surgery in Zhongshan Hospital from April 2011 to June 2012 were analyzed retrospectively, and the clinical outcomes between the two groups were compared.</p><p><b>RESULTS</b>Ninety-six rectal cancer patients undergoing laparoscopic surgery(LS) were enrolled. A total of 216 rectal cancer patients underwent open surgery(OS). There was no operative death in both groups. In LS and OS group, the overall completion rates of TME were 86.4%(83/96) vs. 89.3%(193/216)(P>0.05) respectively, and the overall anal reservation rates were 78.1%(75/96) vs. 75.0%(162/216)(P>0.05) respectively. The mean distance to proximal resection margin and distal resection margin respectively were (10.3±4.1) cm vs.(10.0±4.3) cm(P>0.05) and (3.4±0.9) cm vs. (3.6±1.4) cm(P>0.05) respectively. The mean number of harvested lymph nodes respectively were (12.8±5.2) vs.(13.7±6.4)(P>0.05). Compared to OS, LS presented less blood loss [(98.0±28.7) ml vs. (175.0±41.0) ml, P<0.05], shorter postoperative hospital stay [(9.4±4.9) d vs.(11.6±6.2) d, P<0.05], quicker postoperative recovery of bowel function[(2.7±0.9) d vs. (3.4±0.9) d, P<0.05], shorter postoperative time to intake semi-solid[(3.7±1.2) d vs. (4.4±1.5) d, P<0.05], less postoperative complications(15.6% vs. 25.9%, P<0.05), but longer operative time[(155.7±48.4) min vs. (120.0±26.7) min, P<0.05]. Postoperative follow-up was 6 to 24 months, and the local recurrence of LS and OS was 2.1% and 2.3%(P>0.05).</p><p><b>CONCLUSION</b>Laparoscopic surgery can obtain the same radical efficacy for rectal cancer as compared to open surgery.</p>