Comparative Study of the Two High-Efficient Strategies for in vitro Generation of Human Umbilical Cord Blood-derived Natural Killer Cells / 中国实验血液学杂志

OBJECTIVE@#To explore the similarities and variations of biological phenotype and cytotoxicity of human umbilical cord blood natural killer cells (hUC- NK) after human umbilical cord blood-derived mononuclear cells (hUC-MNC) activated and expanded by two in vitro high-efficient strategies.@*METHODS@#Umbilical cord blood mononuclear cells (MNC) from healthy donor were enriched by Ficoll-based density gradient centrifugation. Then, the phenotype, subpopulations, cell viability and cytotoxicity of NK cells derived from Miltenyi medium (denoted as M-NK) and X-VIVO 15 (denoted as X-NK) were compared using a "3IL" strategy.@*RESULTS@#After a 14-day's culture, the contents of CD3-CD56+ NK cells were elevated from 4.25%±0.04% (d 0) to 71%±0.18% (M-NK) and 75.2%±1.1% (X-NK) respectively. Compared with X-NK group, the proportion of CD3+CD4+ T cells and CD3+CD56+ NKT cells in M-NK group decreased significantly. The percentages of CD16+, NKG2D+, NKp44+, CD25+ NK cells in X-NK group was higher than those in the M-NK group, while the total number of expanded NK cells in X-NK group was half of that in M-NK group. There were no significant differences between X-NK and M-NK groups in cell proliferation and cell cycle, except for the lower percentage of Annexin V+ apoptotic cells in M-NK group. Compared with X-NK group, the proportion of CD107a+ NK cells in M-NK group were higher under the same effector-target ratio (E∶T) (P<0.05).@*CONCLUSION@#The two strategies were adequate for high-efficient generation of NK cells with high level of activation in vitro, however, there are differences in biological phenotypes and tumor cytotoxicity.

Developing process and term analysis of WFAS standard: General Requirements for the Risk Control in the Safe Use of Acupuncture / 中国针灸

In order to promote the application of WFAS standard, General Requirements for the Risk Control in the Safe Use of Acupuncture and the safe practice of acupuncture technology worldwide, the paper introduces the developing process and main contents of this standard, explains the developing purpose, scope, ideas, methods and basis, and analyzes the definition of the relevant terms. Through strictly complied with the development procedure of standard, the terms related to acupuncture risk in this standard are defined. The connotations of 5 special terms are clarified, i.e. "acupuncture risks" "adverse events of acupuncture" "adverse reactions of acupuncture" "acupuncture accidents" and "acupuncture negligence". The range, rank, control flow and source of risk, as well as the control measures are determined. The standard extracts the underlying common problems and basic requirement of the safe practice of acupuncture so as to lay a framework for the development of the relevant technical standards of acupuncture.

Comparison of the Biological Functions between Human Bone Marrow Derived CD106 Mesenchymal Stem Cells and CD106 Subgroup / 中国医学科学院学报

Objective To analyze the differences in biological functions between bone marrow(BM)-derived CD106 mesenchymal stem cells(MSCs)and the CD106 subgroup. Methods The MSCs from normal BM were isolated and expanded.The subgroups of CD106 and CD106 MSCs were sorted.The cell proliferation and adhesion functions,chemotactic activities,adipogenic and osteogenic potentials,senescence,and senescence protein 21(p21)were detected.The capacity of translocation into nucleus of nuclear factor-kappa B(NF-κB)when stimulated by tumor necrosis factor(TNF-α)was measured. Results The proliferative ability was higher in CD106 MSCs than that in CD106 MSCs.In 48 hours,the value of optical density(OD)was significantly higher in CD106 MSCs than that in CD106 subgroup(1.004±0.028 0.659±0.023,=3.946,=0.0225).In 72 hours,this phenomenon was even more pronounced(2.574±0.089 1.590±0.074,=11.240,=0.0000).The adhesive capacity of CD106 MSCs was significantly stronger than that of CD106 subgroup(0.648±0.018 0.418±0.023,=7.869,=0.0002).Besides,the metastasis ability of CD106 MSCs were significantly stronger than that of CD106 subgroup(114.500±4.481 71.000±4.435,=6.900,=0.0005).The CD106 MSCs had signifcnatly lower proportions of senescent cells.The expression of aging protein p21 in CD106 MSCs was significantly lower than that in CD106 MSCs [(17.560±1.421)% (45.800±2.569)%,=9.618,=0.0000].Furthermore,there were no visible pigmenting cells after β-galactosidase staining in CD106 MSCs subgroup.However,in CD106 MSCs,some colored green cells were detected.The rate of NF-κB translocation into nucleus after stimulated by TNF-α was significantly higher in CD106 MSCs than CD106 MSCs [(37.780±3.268)% (7.30±1.25)%,=8.713,=0.0001]. Conclusion Bone marrow-derived CD106 MSCs possess more powerful biological functions than CD106 MSCs.

Effect of CD106 Mesenchymal Stem Cell on Bone Marrow Vascular Failure in Patients with Aplastic Anemia / 中国医学科学院学报

Objective To investigate the vascularization ability of mesenchymal stem cells(MSCs)and explore its influencing factors in aplastic anemia(AA) patients. Methods MSCs were isolated from the bone marrow of AA patients(AA MSCs) and normal controls(N MSCs) were cultured and then evaluated by flow cytometry and immunofluorescene staining technique.The expression level of vascular cell adhesion molecule-1(CD106) was detected by gene sequencing,and the content and fluorescene intensity of CD106MSCs was determined by fluorescence-activated cell sorting.The content of CD105CD106MSCs in fresh AA bone marrow was measured,followed by the determination of the capability of endothelial differentiation from AA MSCs and N MSCs with immunofluorescene analysis;finally,the capability of CD31cell differentiation from CD106-blocking N MSCs and its tubular structures formation in matrigel were tested.Results The expression of CD106 in AA patients was defective(decreased by 12.13 times when compared with N MSCs) and the concentration and fluorescene degree of CD106MSCs was also decreased in AA patients [(28.03±17.71)% vs.(59.61±12.26)%,P=0.000].The content of CD105CD106MSCs decreased significantly in the fresh bone marrow [(0.33±0.10)% vs.(2.98±0.46)%,P=0.0005].Besides, the capability of CD31cell differentiation from AA MSCs was significantly delayed [(13.67±1.50)% vs.(43.24±0.96)%,P=0.0004].Also,the capability of CD31cell differentiation and tubular structures formation of CD106-blocking N MSCs was also obviously decreased [(26.00±2.65)% vs.(91.78±2.44)%,P=0.000;(13.81±1.98)mm vs.(68.12±6.78)mm,P=0.0015].Conclusion The deficient or decreased expression of CD106MSCs accelerate the bone marrow vascularization failure in AA patients.

Use of a disposable circumcision suture device versus conventional circumcision: A systematic review and meta-analysis / 亚洲男科学杂志(英文版)

This systematic review assessed the safety and efficacy of the disposable circumcision suture device (DCSD) and conventional circumcision (CC) in the treatment of redundant prepuce and phimosis. Two independent reviewers conducted a literature search for randomized controlled trials (RCTs) using the DCSD and CC for the treatment of redundant prepuce or phimosis in China and abroad. Nine RCTs (1898 cases) were included. Compared with the CC group, the DCSD group had a shorter operative time (standardized mean difference [SMD] = -21.44; 95% confidence intervals [95% CIs] [-25.08, -17.79]; P < 0.00001), shorter wound healing time (SMD = -3.66; 95% CI [-5.46, -1.85]; P < 0.0001), less intraoperative blood loss (SMD = -9.64; 95% CI [-11.37, -7.90]; P < 0.00001), better cosmetic penile appearance (odds ratio [OR] =8.77; 95% CI [5.90, 13.02]; P < 0.00001), lower intraoperative pain score, lower 24-h postoperative pain score, lower incidence of infection, less incision edema, and fewer adverse events. There were no differences between the CC and DCSD groups in the incidences of dehiscence, or hematoma. The results of this meta-analysis indicate that the DCSD appears to be safer and more effective than CC. However, additional high-quality RCTs with larger study populations are needed.

Human Umbilical Cord-derived Mesenchymal Stem Cells Secrete Interleukin-6 to Influence Differentiation of Leukemic Cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the effect of human umbilical cord-derived mesenchymal stem cells (UC-MSC) on the differentiation of leukemic cells.</p><p><b>METHODS</b>The co-culture system of UC-MSC with acute promyelocytic leukemic cell line NB4 cells was constructed in vitro,and the differentiation status of the leukemic cells was assessed by cell morphology,nitroblue tetrazolium reduction test,and cell surface differentiation marker CD11b.</p><p><b>RESULTS</b>UC-MSC induced the granulocytic differentiation of NB4 cells. When UC-MSC and a small dose of all-trans retinoic acid were applied together,the differentiation-inducing effect was enhanced in an additive manner. Interleukin (IL)-6Ra neutralization attenuated differentiation and exogenous IL-6-induced differentiation of leukemic cells.</p><p><b>CONCLUSION</b>UC-MSC can promotd granulocytic differentiation of acute promyelocytic leukemia cells by way of IL-6 and presented additive effect when combined with a small dose of all-trans retinoic acid.</p>

Clinical effect of circumcision stapler in the treatment of phimosis and redundant prepuce / 中华男科学杂志

<p><b>OBJECTIVE</b>To observe the clinical effect and safety of circumcision stapler in the treatment of phimosis and redundant prepuce.</p><p><b>METHODS</b>We treated 120 patients with redundant prepuce or phimosis using circumcision stapler and another 60 by conventional dorsal-incision circumcision. We observed intraoperative blood loss, operation time, postoperative pain, wound healing time, cosmetic appearance of the penis, and postoperative complications and compared them between the two groups of patients.</p><p><b>RESULTS</b>Stapler circumcision showed obvious advantages over the conventional method in intraoperative blood loss ([2. 3 ± 1. 3] vs [15.6 ± 2.9] ml), operation time ([7.1 ± 1.4] vs [22.6 ± 4.6] min), wound healing time ([12.0 ± 2.9] as [16.3 ± 3. 1] d), postoperative pain score (1. 9 ± 1. 3 vs 5. 2 ± 1. 7), incision edema, and cosmetic appearance of the penis (all P <0. 05). Besides, stapler circumcision exempted the patients from stitch-removal pain. However, the incidence rate of postoperative local ecchymosis was significantly higher in the circumcision stapler group than in the conventional circumcision group (20. 8% vs 8. 3% , P <0. 05).</p><p><b>CONCLUSION</b>Circumcision stapler, with its advantages of easier manipulation, shorter operation time, better cosmetic penile appearance, less pain, and fewer complications, is superior to conventional circumcision in the treatment of phimosis and redundant prepuce.</p>

Adipogenic potentials of mesenchymal stem cells from human bone marrow, umbilical cord and adipose tissue are different / 中国实验血液学杂志

Mesenchymal stem cells (MSCs) could be obtained from many sources, and there are differences between them. This study was purposed to compare and analyze the basic biological characteristics of umbilical cord, adipose tissue-and bone marrow-derived MSC (UC-MSCs, AD-MSCs and BM-MSCs). The MSCs were isolated from umbilical cord, adipose tissue and bone marrow were cultured; the morphology of UC-MSCs, AD-MSCs and BM-MSCs was observed by using microscopy; the immunophenotype, differentiation potential and expression of peroxisome proliferation-activated receptor-γ (PPAR-γ) mRNA were detected by using flow cytometry, differentiation test (von kossais and 0:1 red O staining) and quantitative fluorescent PCR, respectively. The results showed that the UC-MSCs, AD-MSCs and BM-MSCs displayed similar morphology under confocal microscope after being stained with rhodamine phalloidin and DAPL. The immunophenotypes of these three originated cells conform to coincide with identification criterion for MSCs, and showed similar expression level. During adipogenic induction the adipogenic potential of these MSCs was different, AD-MSCs exhibited the highest adipogenic potential, UC-MSCs displayed the lowest, while potential of BM-MSCs get between; however, the osteogenic differentiation potential of UC-MSCs, AD-MSCs and BM-MSCs was similar. The PCR detection showed that the expression level of PPAR-γ mRNA was the highest in AD-MSCs and the lowest in UC-MSCs, while expression level in BM-MSCs get between, these results were identical with the adipogenic potential, suggest that the difference of adipogenic potential in 3 kinds of MSCs was associated with basic expression level of PPAR-γ mRNA. It is concluded that UC-MSCs, AD-MSCs and BM-MSCs exhibit similar morphology, the immunophenotypes of these MSCs coincide with identification criterion for MSCs, the osteogenic potential of these MSCs is similar, while the adipogenic potential and the expression level of PPAR-γ mRNA are different. The difference-associated mechanisms need to further study.

Biological characteristics of exosomes secreted by human bone marrow mesenchymal stem cells / 中国实验血液学杂志

This study was aimed to explore the immunoregulatory function and capability supporting the angiogenesis of exosomes secreted by bone marrow mesenchymal stem cells (BMMSC) from healthy persons. Supernatant of BMMSC (P4-P6) was collected for exosome purification. Transmission electron microscopy (TEM) and Western blot were used to identify the quality of isolated exosomes. The amount of exosomes was quantified through bicinchoninic acid (BCA) protein assay. Human peripheral blood mononuclear cells (PBMNC) were isolated from healthy donor and added with isolating exosomes. After co-cultured for 72 h, IFN-γ from the co-culture system was detected by ELISA. The expression of miRNA-associated with immunity were detected by real-time reverse transcription polymerase chain reaction (Real-time RT-PCR). The interactions between exosomes and human umbilical vein endothelial cells (HUVEC) were observed with confocal microscopy. Subconfluent HUVEC were harvested and treated with the indicated concentration of exosomes. Nude mice were injected subcutaneously with exosomes or PBS as control to verify the ability of angiogenesis. The results showed that diameter range of exosomes was range from 40 to 160 nm. The isolated exosomes expressed the CD9. There was approximately linear relation between the secretion of exosomes and cell density. The exosomes suppressed the production of IFN-γ from PBMNC, and contained miRNA associated with immune regulation such as miR301, miR22 and miR-let-7a. Exosomes induced vascular tube formation in vitro and vascularization of Matrigel plugs in vivo. It is concluded that the BMMSC-derived exosomes can regulate immunity and support vascularization.

Comparison of the therapeutic effect on skeletal fluorosis and impact on urine fluoride value among fire needle therapy, electroacupuncture and calcium carbonate D3 / 中国针灸

<p><b>OBJECTIVE</b>To observe the impacts on skeletal fluorosis pain, joint motor dysfunction and urine fluoride excretion in the treatment with fire needle therapy, electroacupuncture and calcium carbonate D3.</p><p><b>METHODS</b>The randomized controlled trial was adopted. Ninety-five patients were randomized into a fire needle group (31 cases), an electroacupuncture group (33 cases) and a calcium carbonate D3 group (31 cases). In the fire needle group and the electroacupuncture group, Ashi points, Dazhui (GV 14), Geshu (BL 17), Quchi (LI 11), Hegu (LI 4), Xuehai (SP 10) points were selected and stimulated with fire needle and electroacupuncture separately, three times a week. In the calcium carbonate D3 group, calcium carbonate D3 tablets was prescribed for oral administration, 600 mg each time, twice a day. The duration of treatment was 2 months in the electroacupuncture group and calcium carbonate D3 group and 1 month in the fire needle group. VAS score, the range of motion (ROM) and urine fluoride value were compared before and after treatment in the patients of the three groups.</p><p><b>RESULTS</b>After treatment, VAS value and ROM were improved significantly in the patients of the three groups (all P < 0.05), the difference was not significant in comparison of the three groups (all P > 0.05). After treatment, the urine fluoride value was increased significantly in the fire needle group [(7.89 +/- 3.61) mg/L vs (9.81 +/- 4.17) mg/L, P < 0.01] and was increased in the electroacupuncture group [(7.53 +/- 3.46) mg/L vs (8.97 +/- 4.21) mg/L, P < 0.05]. The difference was not significant in comparison before and after treatment in the calcium carbonate D3 group (P > 0.05).</p><p><b>CONCLUSION</b>The fire needle therapy, electroacupuncture and calcium carbonate D3 all have the clinical value in the prevention and treatment of skeletal fluorosis and the difference in the therapeutic effect has not been discovered among them yet at present. But it has been found that the fire needle therapy and electroacupuncture display the active significance in the promotion of urine fluoride excretion.</p>

Effects of rapamycin on biological characteristics of bone marrow mesenchymal stem cells from patients with aplastic anemia / 中国实验血液学杂志

This study was aimed to investigate the effects of rapamycin on biological function and autophagy of bone marrow mesenchymal stem cells (BM-MSC) from patients with aplastic anemia so as to provide experimental basis for the clinical treatment of aplastic anemia (AA) with rapamycin. BM-MSC were treated with different concentrations of rapamycin (0, 10, 50, 100 nmol/L) for 48 h, the expression of LC3B protein was detected by Western blot to observe the effect of rapamycin on cell autophagy; cell apoptosis and cell cycles were detected by flow cytometry; the proliferation of BM-MSC of AA patients was measured by cell counting kit-8; the adipogenic differentiation of BM-MSC were tested by oil red O staining after adipogenic induction for 2 weeks; the adipogenic related genes (LPL, CFD, PPARγ) were detected by real-time PCR. The results showed that the proliferation and adipogenesis of BM-MSC of AA patients were inhibited by rapamycin. Moreover, the autophagy and apoptosis of BM-MSC were increased by rapamycin in a dose-dependent way.Rapamycin arrested the BM-MSC in G0/G1 phase and prevented them into S phase (P < 0.05). It is concluded that rapamycin plays an critical role in inhibiting cell proliferation, cell cycles, and adipogenesis, these effects may be related with the autophagy activation and mTOR inhibition resulting from rapamycin.

Efficacy comparison between electroacupuncture and dyne-35 in treatment of polycystic ovary syndrome / 中国针灸

<p><b>OBJECTIVE</b>To compare the difference in clinical efficacy on polycystic ovary syndrome (PCOS) between electroacupuncture (EA) and dyne-35 and to explore the effect mechanism.</p><p><b>METHODS</b>Sixty-five patients were randomized into an EA group (33 cases) and a western medication group (32 cases). In the EA group, the selected acupoints were Danzhong (CV 17), Qimen (LR 14), Zhongwan (CV 12), Tianshu (ST 25), Guanyuan (CV 4), Zigong (EX-CA 1), Sanyinjiao (SP 6), Zusanli (ST 36) and Taichong (LR 3), etc. After the arrival of qi, electric stimulation was attached to the acupoints for 30 min. The treatment was given 3 times a week. In the western medication group, dyne-35 was prescribed on the 5th day of natural menstruation or withdrawal bleeding, one tablet a day, continuously for 21 days. The treatment cycle was 3 months in the two groups. The menstrual condition, body mass, body mass index (BMI), serum testosterone (T), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and LH/FSH were compared before and after treatment in the two groups. The clinical efficacy was assessed in the two groups.</p><p><b>RESULTS</b>The total effective rate was 90.6% (29/32) in the EA group and was 93.3% (28/30) in the western medication group. The efficacy was similar in the two groups (P > 0.05). After treatment, the levels of LH and LH/FSH were all reduced significantly in the two groups (all P < 0.01). After treatment, T level in serum was reduced apparently in the western medication group (P < 0.05). Before and after treatment, the differences in body mass and BMI in the EA group were more significant than those in the western medication group (P < 0.01, P < 0.05).</p><p><b>CONCLUSION</b>EA is the effective method for PCOS, similar to that of dyne-35. The effect of it for weight loss is superior to dyne-35 and no apparent adverse reactions happen. The effect mechanism of EA is related to the regulation of serum sexual hormone levels and their ratio, as well as to the regulation of body lipid metabolism.</p>

Optimization of phenoxazinone synthase production by response surface methodology and its application in Congo red decolourization

Background: Enzymatic decolourization has been recently proposed as a promising and eco-friendly method for treatment of synthetic dye-contaminated wastewaters. However, the processes require large quantities of enzymes, attracting significant attention in developing efficient methods for mass production of multifunctional enzymes. Several methods such as response surface methodology (RSM) and orthogonal experiment have been applied to optimize the parameters in bioprocesses for enzyme production. Results: In the present study, a laccase-like enzyme, phenoxazinone synthase (PHS) originated from Streptomyces antibioticus was recombinantly expressed in Escherichia coli BL21 (DE3). The production of PHS in E. coli BL21 was optimized by response surface methodology based on Box-Behnken design. A full third-order polynomial model was generated by data analysis with Statistica 8.0 in which the optimal conditions for PHS production were calculated to be 1.525 mM CuSO4 and 16.096 hrs induction at temperature of 29.88ºC. The highest PHS production under optimal conditions was calculated to be 4098.51 U/l using the established model. Average PHS production obtained from actual production processes carried out under the calculated optimal conditions was 4052.00 U/l, very close to the value predicted by the model. Crude PHS was subsequently tested in Congo red decolourization which exhibited a low decolourization rate of 27% without mediator. Several mediators were found to improve PHS-catalyzed Congo red decolourization, with the highest rate of 73.89% obtained with 2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) as mediator under optimized conditions of 4000 U/l PHS activity, 10 μM ABTS, 100 μM Congo red, and 8 hrs reaction time. Conclusion: Our results indicated that PHS recombinantly produced in E. coli BL21 was a prospective enzyme for decolorizing reactive dye Congo red.

Isolation and biological characteristics of mesenchymal stem cells derived from human placenta decidua basalis / 中国实验血液学杂志

Comparing to bone marrow mesenchymal stem cells (MSCs), placenta-derived MSCs have the advantages of adequate sources, low immunogenicity, little risk of viral contamination, and no ethical controversy, and thus possess a better prospect for clinical application. Placental tissue not only includes chorionic and amniotic, but also contains decidua basalis which locate in the maternal placenta surface. The biological characteristics of MSCs isolated from decidua basalis have not been well studied. This study was aimed to investigate the biologic characteristics of placenta decidua basalis-derived MSC from placenta decidua basalis (DB) by enzymatic digestion. Short tandem repeats (STR) test was used to identify the cells derived from the maternal placenta surface. Growth rate of decidua basalis mesenchymal stem cells (DB-MSC) was measured by MTT. Cell cycle and cell phenotype were detected by flow cytometry. Inducing differentiation was used to evaluate multipotency of DB-MSC. For testing the immunosuppression of DB-MSC, they were co-cultured with peripheral blood mononuclear cells (PBMNC) stimulated by phytohemagglutinin (PHA) and then IFN-γ in the co-cultured media was quantified by ELISA. The results showed that the cells were derived from the maternal placenta by STR analysis. DB-MSC showed typical fibroblast morphology in the culture and were positive for the MSC surface markers: CD90, CD73, CD105, CD44 and negative for CD45, CD11b, and CD34. DB-MSC underwent osteogenic, adipogenic and chondrogenic differentiation in inducing medium. DB-MSC could inhibit the secretion of IFN-γ by PBMNC. It is concluded that the cells are isolated from placenta decidua basalis and possess the basic characteristics of MSC. DB-MSC can be an important maternal autologous MSC and may be a safe and effective treatment for immune system diseases, which makes the DB-MSC as an important source of autologous MSC from mother. DB-MSC can be safely for the treatment of the mother's immune system diseases.

Human umbilical cord mesenchymal stem cells reduce the sensitivity of HL-60 cells to cytarabine / 中国实验血液学杂志

This study was purposed to investigate the impact of human umbilical cord-derived mesenchymal stem cells (hUC-MSC) on the sensitivity of HL-60 cells to therapeutic drugs so as to provide more information for exploring the regulatory effect of hUC-MSC on leukemia cells. Transwell and direct co-culture systems of HL-60 and hUC-MSC were established. The apoptosis and cell cycle of HL-60 cells were detected by flow cytometry. RT-PCR and Western blot were used to detect the mRNA and protein levels of Caspase 3, respectively. The results showed that the apoptosis of HL-60 induced by cytarabine (Ara-C) decreased significantly after direct co-cultured with hUC-MSC cycle mRNA (P < 0.05). The similar phenomenon was observed in transwell co-culture system. Cell cycle of HL-60 cells were arrested at G0/G1 phase and did not enter into S phase (P < 0.05) and the expression of Caspase-3 mRNA and protein in HL-60 cells were reduced (P < 0.05). It is concluded that hUC-MSC protected HL-60 from Arc-C induced apoptosis through regulating the cell cycle and down-regulating expression of Caspase 3 in HL-60 cells. In addition, this effect is caused by the soluble factors from hUC-MSC.

Culture of pancreatic progenitor cells in hanging drop and on floating filter / 中国医学科学院学报

<p><b>OBJECTIVE</b>To construct a method to culture pancreatic progenitor cells in hanging drop and on floating filter,and to examine if pancreatic progenitor cells can differentiate into mature endocrine cells with this method.</p><p><b>METHODS</b>Murine embryos at day 12.5 were isolated and digested into single cells,which were then cultured in hanging drop for 24h and formed spheres.Spheres were cultured on the filter for 6 days,which floated in the dish containing medium.During culture,the expressions of pancreas duodenum homeobox-1(PDX-1)and neurogenin3(Ngn3)were determined.The expressions of endocrine and exocrine markers,insulin,glucagon,and carboxypeptidase(CPA)were determined on day 7 by immunohistochemistry.Insulin secretion of spheres stimulated by glucose was detected by ELISA.The changes of pancreatic marker expressions during culture were monitored by real-time polymerase chain reaction(PCR).</p><p><b>RESULTS</b>One day after the culture,there were still a large amount of PDX-1 positive cells in pancreatic spheres,and these cells proliferated.On day 3,high expression of Ngn3 was detected,and the Ngn3-positive cells did not proliferate.On day 7,The expressions of endocrine and exocrine markers in the differentiated pancreatic progenitor cells were detected,which were consistent with that in vivo.Insulin was secreted by spheres upon the stimulation of glucose.</p><p><b>CONCLUSION</b>In hanging drop and on floating filter,pancreatic progenitor cells can differentiate into mature endocrine cells.</p>

Discussion on the complementation of different effects of acupuncture and Chinese drugs and clinical application / 中国针灸

Complementation of different effects of acupuncture and Chinese drugs generalizes not only the objective interrelations between acupuncture and Chinese drugs with varied actions when the two therapies are applied simultaneously but also the clinical therapeutic efficacy produced either by acupuncture or Chinese drugs singly. This view of complementation has been promoted on the basis of the understanding of the mechanism and efficacy of acupuncture and Chinese drugs as well as their interrelations concerning the different actions complementary most appropriately to one another in treatment of diseases. The presented view will be of theoretical value in working out the efficacy and mechanism of acupuncture and Chinese drugs applied at the same time and help the clinical doctors understand further the properties of actions of acupuncture and Chinese drugs so as to strengthen more specifically the integration of acupuncture with Chinese drugs and improve the clinical therapeutic effect.

Long term in-vitro expansion reduces immune modulation function of placental chorionic villi mesenchymal stem cells / 中国实验血液学杂志

The main aim of this study was to investigate the biological activities and immune modulation changes of chorionic villi mesenchymal stem cells (CV-MSC) after long term culture. The morphology of the CV-MSC of passage 3 and passage 9 were observed by microscopy, and their phenotypes were detected by flow cytometry. CV-MSC of passage 3 and 9 were co-cultured with PHA-stimulated PBMNC, and IFN-γ concentration in culture medium was detected by ELISA. The mRNA expression of COX-2, HGF and HLA-G in CV-MSC were detected by real-time PCR. The results showed that after long term culture, the CV-MSC kept the MSC morphology and most of the phenotypes including CD31, CD34, CD44, CD45, CD62L, CD73, CD90, CD105, CD117, CD151, CD235a, CD271 and HLA-DR, while the CD49d was significantly up-regulated. Immune modulation ability of CV-MSC was reduced and the mRNA expression of COX-2 and HGF was down regulated after long term culture, but the expression of HLA-G did not found to be obvious change. It is concluded that the long term in vitro expansion changes the expression of CD49d and reduces immune modulation of CV-MSC.

Effect of 15-deoxy-Δ(12), 14-prostaglandin J2 on the cytokines in the culture supernatant of bone marrow mesenchymal stem cells / 中国实验血液学杂志

15-Deoxy-Δ(12), 14-prostaglandin J2 (15d-PGJ2), a well known peroxisome proliferator activated receptor (PPAR) γ ligand, has been shown to inhibit cellular proliferation and induce apoptosis and differentiation. However, whether 15d-PGJ2 influences the cytokines in the culture supernatant of bone marrow mesenchymal stem cells (BM-MSC) is unknown. This study was purposed to investigate the influence of 15d-PGJ2 on cytokines in the culture supernatant of BM-MSC. The fibroblast-like cells attached to the culture dish from bone marrow of healthy donors were isolated. The immunophenotype and differentiation potential of the obtained cells were detected by flow cytometry and oil red O and von kassa staining respectively to confirm that these cells were BM-MSC. Thereafter, the BM-MSC were cultured with complete medium supplemented with 10, 20, 40 and 60 µmol/L 15d-PGJ2 for 24 hours respectively. The real-time PCR was used to assay the PPARγ mRNA level, the confocal immuno fluorescence technique was used to detect the expression level of PPARγ. The results showed that the BM-MSC underwent apoptosis and got detached from the culture dish when the concentration of 15d-PGJ2 was no less than 20 µmol/L. The PPARγ mRNA level of BM-MSCs cultured with medium containing 10 µmol/L 15d-PGJ2 was higher than that cultured without 15d-PGJ2, and the difference was statistically significant (P < 0.05). The enhancement of PPARγ expression was observed after stimulated by 15d-PGJ2. The protein chip detecting the culture supernatants of BM-MSC cultured with 10 µmol/L 15d-PGJ2 or without 15d-PGJ2 for 24 hours demonstrated that expression levels of some of the cytokines varied. It is concluded that the down-regulation of TIMP-2 exists after treatment of 15d-PGJ2, which is statistical significant.

IL-1β promotes the hematopoietic support of human umbilical cord mesenchymal stem cells / 中国实验血液学杂志

This study was aimed to investigate the effect of IL-1β on hematopoietic support of human umbilical cord mesenchymal stem cells (hUC-MSC). 2×10(6) hUC-MSC were seeded in 75 cm(2) flasks, after adherence to wall for 2 h, 10 ng/ml IL-1β was added in hUC-MSC supernatant and cultured for 36 h, then the culture supernatants and cells were harvested. The effect of conditioned medium with/without IL-1β on CD34(+) cell hematopoietic support was observed, mRNA expression changes of hUC-MSC cultured in medium with/without IL-1β were monitored by real time PCR, the differences in hematopoiesis-related factors were detected by ELISA. The results showed that the conditioned culture medium of hUC-MSC with IL-1β enhanced the ability to form colony of CD34(+) cells, especially CFU-G and CFU-GM in vitro; IL-1β promoted the mRNA expression of GM-CSF, G-CSF, IL-6 on MSC; IL-1β also promoted the secretion of GM-CSF, G-CSF, and IL-6 protein from hUC-MSC. It is concluded that IL-1β enhances hematopoietic support capacity especially, capability of MSC to myeloid differentiation.