Diagnostic Significance of Detecting Peripheral Blood Granulocyte CD55/CD59 and FLAER in Anemia and PNH / 现代检验医学杂志

Objective To investigate the clinical significance of CD55,CD59 and Aeromonas hydrophila toxin variant (FLAER) in the diagnosis of anemia and paroxysmal nocturnal hemoglobinuria (PNH).Methods Collected 30 healthy controls,22 cases of PNH,33 cases of aplastic anemia (AA),37 cases of iron deficiency anemia (IDA),45 cases of megaloblastic anemia (MA),30 cases of hemolytic anemia (HA) and 31 cases of myelodysplastic syndrome (MDS) from January 2009 to March 2017,CD55,CD59 and FLAER negative cell ratio of peripheral blood neutrophil of them were detected by multipa rameter flow cytometry.Results The detection rates of FLAER in PNH,AA and MDS groups were higher than those of CD55 and CD59,but there was significant difference in AA (x2 =7.759,5.518,P=0.005,0.019＜0.05).The average CD55,CD59 and FLAER deletion rate in PNH and AA group were significantly higher than those in normal control group and other groups (t=2.163～17.890,P=0.000～0.038＜0.05).The number of FLAER in PNH group was higher than CD59 and CD59 was higher than CD55 with the statistically significant difference (t=2.503 ～ 6.308,P=0.000 ～0.016＜ 0.05).Conclusion CD55,CD59 and FLAER have important value in the diagnosis of PNH and differential diagnosis with other anemia diseases,and can also be used to detect the presence of MDS and AA in patients with PNH.FLAER outperforms CD59,CD59 outperforms CD55.

The study of isolation and culture in vitro of human umbilical cord mesenchymal stem cells and their biological properties / 重庆医学

Objective To identify a detailed biological characterization of mesenchymal stem cells (MSCs) isolated from hu‐man umbilical cord(UC) tissue regarding their morphology ,immunophenotype ,purity and proliferative capacity and establish a rea‐sonably cultured and amplified system .Methods After stripping off arteries and veins ,the remaining parts of umbilical cord were cut into 1 mm3 small sections and cultured with DMEM/F12 containing 10% fetal bovine serum .Adhere cells were obtained and the morphology of the cells was observed under inverted phase contrast microscope .The growth curves of them were drawn by CCK‐8 and the cell cycle and surface antigens (CD29 ,CD73 ,CD90 ,CD105 ,CD31 ,CD14 ,CD34 ,CD45 ,CD11b ,HLA‐DR) were detected by flow cytometry .Results Seven to ten days after primary culture ,adhere cells came out of fragments .The MSCs harvested were a high purity and mainly presented as a fibroblast‐like morphology .UC‐MSCs had a strong ability of proliferation through the cell growth curve .The special surface antigens CD29 ,CD73 ,CD90 ,CD105 were positive expression ,while CD31 ,CD14 ,CD34 ,CD45 , CD11b ,HLA‐DR were negative .More than 80% cells of MSCs were found at G0/G1 phase .Conclusion Human UC‐MSCs could be cultured and proliferated in vitro .

Cytotoxity of pomalidomide combined CAR-T cell for multiple myeloma cell RPMI8226 and U266 / 中华血液学杂志

<p><b>OBJECTIVE</b>To observe the cytotoxity of CD138-CAR-T cells on human multiple myeloma cell RPMI8226 and U266 cells and explore the impact of pomalidomide on the cytotoxity of CD138-CAR-T on RPMI8226 and U266 cells.</p><p><b>METHODS</b>The cytotoxity of CD138-CAR-T and CD138-CAR-T combined pomalidomide on RPMI8226 and U266 was detected by CFSE/7AAD. The effctor cells were co-cultured with target cells at 5:1 for 18 h, and then the supernatant were collected and used for ELISA assays.</p><p><b>RESULTS</b>After 18 h co-culture, the cytotoxity of CD138-CAR-T on RPMI8226 and U266 was significantiy higher than control (P<0.01). There was no significant change on the cytotoxity of pomalidoide combined with CD138-CAR-T on RPMI8226 and U266. The results showed that co-cultured system contribted to a markedly increased production of IFN-γ, after adding pomalidomide to the co-cultured system. It can significantly enhance the production of IFN-γ, compared with CD138-CAR-T alone.</p><p><b>CONCLUSION</b>CD138-CAR-T had significantly cytotoxity on U266 and RPMI8226. Pomalidomide could promote CD138-CAR-T cells IFN-γ production.</p>

Effect of cobalt chloride-induced hypoxia on proliferation of human umbilical cord-derived mesenchymal stem cells and related gene and protein expressions / 中国组织工程研究

BACKGROUND:Cobalt chloride (CoCl2) may promote the proliferation of human umbilical cord-derived mesenchymal stem cels (hUC-MSCs) in a time- and concentration-dependent manner, and meanwhile, CoCl2 can regulate the expression of genes and proteins in hUC-MSCs. OBJECTIVE:To explore the effects of CoCl2 induced-hypoxia on the proliferation of hUC-MSCs and gene and protein expressions in hUC-MSCs, thereby establishing an effective method for MSCs culture and amplificationin vitro. METHODS: hUC-MSCs were extracted using tissue explant method. Under hypoxia conditions induced by CoCl2 (0, 100, 150, 200, 250 μmol/L) for different periods (0, 1, 2, 3, 4 days), flow cytometry was used to identify cel surface-associated antigens; cel counting kit-8 was used to detect cel proliferation; RT-PCR was used to determine levels of hypoxia inducible factor-1α, inducible nitric oxide synthase, stromal cel-derived factor-1, interleukin-6, transforming growth factor-β mRNA; western blot assay was used to detect protein expression of hypoxia inducible factor-1α. RESULTS AND CONCLUSION:The cels were positive for CD29, CD73, CD90, CD105, while negative for CD31, CD14, CD34, CD45, CD11b, HLA-DR. Moreover, the antigen expression was not affected by CoCl2 induced-hypoxia. CoCl2 induced-chemical hypoxia could promote the proliferation of hUC-MSCs in a time- and concentration-dependent manner. RT-PCR results showed thatunder hypoxia, hypoxia inducible factor 1α, inducible nitric oxide synthase and stromal cel-derived factor-1 mRNA expressions were significantly up-regulated, but interleukin-6 and transforming growth factor-β mRNA expressions were down-regulated significantly (P < 0.05). Additionaly, the protein expression of hypoxia inducible factor 1α was increased under hypoxia conditions. These findings indicate that CoCl2 induced-hypoxia environment may promote the proliferation of hUC-MSCs and the optimal concentration of CoCl2 is 200μmol/L. However, a higher concentration of CoCl2 (≥ 250μmol/L) inhibits the proliferation of hUC-MSCs, and the mechanism may be related to the increase of hypoxia inducible factor-1α at protein and mRNA levels.

Effects of paeoniflorin on the proliferation of bone marrow mesenchymal stem cells / 中国组织工程研究

BACKGROUND:Studies have shown that paeoniflorin functions as replenishing blood and treatment of autoimmune diseases, and bone marrow mesenchymal stem cels also play an important role in the body’s blood and immune function. However, paeoniflorin effects on bone marrow mesenchymal stem cel proliferation and cytokine secretion and expression are rarely reported. OBJECTIVE:To investigate the effect of paeoniflorin on proliferation of bone marrow mesenchymal stem cels and the expression of interleukin-6. METHODS:Human bone marrow mesenchymal stem cels were separated and culturedin vitro by density gradient centrifugation combined with attachment method. The biological characteristics of bone marrow mesenchymal stem cels were identified by flow cytometry and osteogenic/adipogenic induction. The proliferation of bone marrow mesenchymal stem cels under different concentrations of paeoniflorin was detected by MTT method. The mRNA expression and secretion of interleukin-6 in the supernatant of bone marrow mesenchymal stem cels were detected by RT-PCR and ELISA, respectively. RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cels were isolated successfuly and had osteogenic and adipogenic differentiation potential. Compared with the controlgroup, 2 μmol/L and 10 μmol/L paeoniflorin significantly promoted the proliferation of bone marrow mesenchymal stem cels. 10 μmol/L paeoniflorin could significantly decrease the proportion of bone marrow mesenchymal stem cels in G0/G1 phase and increase this proportion in S phase. Compared with the control group, the experimental group could significantly increase the secretion and mRNA expression of interleukin-6 (P < 0.01). It is concluded that paeoniflorin at certain concentrations can obviously promote the proliferation of bone marrow mesenchymal stem cels, and increase the expression and secretion of interleukin-6.

Role of miR-124 in breast cancer and its underlying mechanism / 中国肿瘤临床

Objective:To evaluate the role of miR-124 in breast cancer and its underlying mechanism. Methods:Quantitative re-verse transcription-polymerase chain reaction (qRT-PCR) was employed to quantify the expression level of miR-124 in the breast can-cer cell lines and matched tissues of 52 patients. Cell proliferation, invasion, and migration of MDA-MB-231 and T-47D were deter-mined by miR-124 overexpression in vitro. Luciferase vectors (pMIR-SP1 3'UTR) were also constructed. The predicted target gene of miR-124 was identified via luciferase activation assay. The mRNA and protein expression of SP1 was detected via qRT-PCR and West-ern blot, respectively. Results:MiR-124 was decreased in breast cancer tissues and cell lines. This result is correlated with metastatic capacity, TMN stages, and prognosis in breast cancer tissues. In breast cancer cell lines, ectopic overexpression of miR-124 inhibited cell proliferation, invasion, and migration in vitro. MiR-124 mimics significantly inhibited luciferase activation (P<0.05) in HEK293 cells and could significantly decrease the mRNA (P<0.05) and protein expression levels of SP1 in MDA-MB-231 and T-47D cells. Con-clusion:MiR-124 could be inhibited in breast cancer. The low miR-124 expression is associated with poor prognosis. In addition, miR-124 could inhibit cell proliferation, invasion, and migration by targeting SP1. These findings confirm that miR-124 downregulation may be a key mechanism for breast cancer carcinogenesis.

Effects of low dose X-ray irradiation on proliferation and differentiation of mesenchymal stem cells derived from human umbilical cord into adipocytes and osteoblasts / 中华放射医学与防护杂志

Objective To observe the effects of low dose irradiation (LDR) on proliferation,adipogenesis and osteogenic potential of human umbilical cord mesenchymal stem cells (hucMSCs).Methods hucMSCs were isolated from Wharton's jelly tissue of human umbilical cord by modified tissuepiece inoculation,and flow cytometry was used to detect the expression of specific marker in the hucMSCs.The hucMSCs were randomly divided into two groups:irradiation group undergoing irradiation with the doses 50,100,or 200 mGy respectively,and control group without irradiation.MTT method was applied to evaluate the proliferation of the hucMSCs at different time points with various doses irradiation.The third passage hucMSCs were randomly divided into two groups:irradiation group undergoing low dose irradiation of 200 mGy,and control group without irradiation,and then underwent induction by adipocytic and oesteocytic differentiation induction fluids respectively so as to differentiate into adipocytes and osteoblasts.Oil red O staining was used to detect the activity of alkaline phophatase (ALP),and RT-PCR was used to detect the mRNA expression of core binding factor alpha 1 in human osteoblast.Results After 9-12days,fibroblasts began to swim out of the tissue piece with a confluence rate of 80% 2 weeks later.Within 7 days the absorption values of the hucMSCs undergoing different irradiation doses 2,3,4,5,and 6 days later were all significantly higher than those of the control group(F = 159.17,448.81,265.15,183.93,and 181.83 ,all P <0.01),with the proliferation rates of the 100 mGy subgroup being the highest.After being induced liquid,vacuoles were observed in the irradiated group 2 days later.21 days later,the adipogenic rates of irradiated group was significantly higher than that of the control group (t = 28.25,P <0.01).The ALP activity increased in the irradiated group compared with control group (t=16.87,P <0.01) .The expression level of Cbf-α1 mRNA was up-regulated obviously (t = 14.16,P＜0.01).Conclusions LDR promotes the proliferation of hucMSCs,and accelerates the hucMSCs' differentiation into adipocytes and osteoblasts.

Autologous bone marrow-derived mesenchymal stem cells and peripheral blood stem cells cotransplantation in treatment of hematological malignant diseases / 中华内科杂志

Objective To explore the feasibility and safety of cotransplantation of autologous bone marrow-derived mesenehymal stem cells (MSCs) and peripheral blood stem cells in hematological malignant diseases and to observe its effect on hematopoietic reconstruction after cotransplantation. Methods Adult human MSCs were isolated from the healthy bone marrow of the patient himself with Percoll (1. 073 g/ml) and cultured in Dulbecco's modified Eagle's medium with low glucose containing 10% AB type human serum. After conditioning regimen of high-dose chemotherapy and radiotherapy, cotransplantation of autologous bone marrow-derived MSCs and peripheral blood stem cells was done in five patients with hematological malignant diseases. Results The process of the infusion was safe and there were no adverse reactions or other toxicities related to the infustion of MSCs. The median time to achieve neutrophil counts greater than 0. 5 × 109/L was 9.4 days ( ranging from 8 to 11 days) after cotransplantation and platelet counts greater than 20 × 109/L 12. 2 days (ranging from 10 to 14 days). Conclusion Cotransplantation of autologous bone marrow-derived MSCs and peripheral blood stem cells in hematological malignant diseases is feasible and safe. The rapid hematopoietic reconstruction after cotransplantation shows that MSCs have an effect on hematopeiesis, but the mechanism is still to be investigated.

Negatively regulatory effects of bone marrow-derived mesenchymal stem cells supernatant on allogeneic lymphocytes proliferation / 中国组织工程研究

BACKGROUND: Preliminary study has proven that adult human bone marrow-derived mesenchymal stem cells (MSCs) suppress peripheral blood lymphocytes proliferation.But the mechanism was still to be investigated.OBJECTIVE: To study the negatively regulatory effect of adult human MSCs on allogeneic lymphocyte proliferation by cell-free condition.DESIGN,TIME AND SETTING: Cytological observation in vitro,which was performed in the Lanzhou General Hospital,Lanzhou Military Area Command of Chinese PLA between October 2005 and December 2007.MATERIALS: The bone marrow sample was provided by the allo-transplantation donor.The peripheral blood lymphocytes were provided by the healthy volunteer.METHODS: Adult human MSCs were separated with Percoll + adherence method.Allogeneic peripheral blood lymphocytes were obtained from healthy donors with Ficoll solution and the cell concentration was adjusted as 2×109/L for use.100μ L MSCs culture supernatant was taken out in 96-well plates.The groups were following: A superuatant + 3-day MSCs culture media (100 μ L/well); B superuatant + phytohemagglutini (PHA; 1 g/L,5 μ L); C medium + LG-DMEM culture media containing 10% fetal bovine serum (100 μ L); D medium + PHA (1 g/L,5 μ L).The cells were incubated at 37 ℃ with 5% CO2 in a fully humidified atmosphere for three days.MAIN OUTCOME MEASURES: Effect of MSCs supematant on proliferation and transformation of variant lymphocytes.RESULTS: Peripheral blood lymphocytes proliferation was suppressed as compared with the blank control group and PHA group after MSCs culture,and the inhibition ratio was 9.00% (P ＜ 0.05).When lymphocytes were stimulated by PHA,the suppression effects were even stronger and the inhibition ratio was 20.91% (P ＜ 0.01).CONCLUSION: Adult human MSCs supernatant can suppress peripheral blood lymphocytes proliferation and transformation; furthermore,PHA can enhance the inhibitory effect,suggesting the negative regulation is at least in part due to indirectly inhibiting lymphocytes via soluble cytokines.

Immunocompetent cells and immune regulator improve apopotosis of mesenchymal stem cells by down-regulating heme oxygenase-1 / 第三军医大学学报

Objective To investigate the effect of immunocompetent cells and immune regulator on the apoptosis of human mesenchymal stem cells ( MSCs) and on mRNA expression of heme oxygenase-1. Methods MSCs were cultured by density gradient centrifugation and then identified by flow cytometry. RT-PCR was used to detect HO-1 mRNA expression and flow cytometry was used to analyze cell apoptosis after the stimulation of IFN-? and PHA-activated T cells. Results The mRNA expression of Heme oxygenase-1 was observed in MSCs and decreased after the stimulation of IFN-? and activated T cells. IFN-?,znpp-Ⅸ and combined these two caused obvious cell apoptosis in MSCs,with an apoptotic rate of ( 56. 50 ? 0. 16) % ,( 56. 85 ? 2. 27) % ,and ( 82. 53 ? 2. 65) % respectively. All of them had a significant difference compared with the normal MSCs [( 7. 56 ? 1. 43) % ,P

Dynamic changes of CD34~+ cell and T lymphocyte subset from different populations after G-CSF treatment / 第三军医大学学报

Objective To explore the dynamic changes of CD34 + cells and T lymphocyte subsets and best time of harvesting peripheral blood stem cell when G-CSF was used for peripheral blood stem cell mobilization in donors and patients. Methods A total of 12 donors and 16 patients who received chemotherapy for 7 d were injected G-CSF of 300 ?g/d to mobilize peripheral blood stem cells for 5 d and flow cytometry were used to detect the changes of CD34 + cells and T lymphocyte subsets everyday for 5 d. Results ① Before G-CSF treatment, there were obvious differences in bone marrow CD34 + cells between patients and donors (P

Observation on Dynamic Changes of Flavonoids Contents of Citrus grandis(L.)Osbeck var.tomentosa Hort / 广州中医药大学学报

【Objective】To observe the dynamic changes of flavonoids contents of Citrus grandis(L.)Osbeck var.tomentosa Hort..【Methods】The total flavonoid content was determined by spectral photometric analysis and naringin content by HPLC,and the fingerprints of Citrus grandis(L.)Osbeck var.tomentosa Hort.were studied by HPLC.【Results】With the increase of the fruit age,total flavonoid contents in the peel and the leaves of Citrus grandis(L.)Osbeck var.tomentosa Hort.decreased obviously.Fingerprints results showed that at the early fruit age the rhoifolin content in the peel increased with the fruit age and began to decrease 62 days later,while the rhoifolin content in the leaves showed no changes.【Conclusion】In terms of active components contents and economic value,the optimal collecting time for Citrus grandis(L.)Osbeck var.tomentosa Hort.is:young fruit,34 days,and immature fruit,55 days.

Random Amplified Polymorphic DNA Analysis of Germplasm Resources of Exocarpium Citri Grandis / 广州中医药大学学报

Objective To investigate the influence of producing areas and sexual reproduction on the genetic features of Citrus grandis‘Tomentosa’. Methods The genetic distance in different species was counted,and the DNA fingerprint of germplasm resources of Citrus grandis ‘Tomentosa’ was established after random amplified polymorphic DNA analysis of Citrus grandis ‘Tomentosa’ and Citrus grandis (L.) Osbeck of various species from different producing areas. Results The genetic distance index D of Citrus grandis ‘Tomentosa’ and Citrus grandis (L.) Osbeck of various species from different producing areas was in the range of 0.01~0.64. Conclusion Sexual reproduction and the changes of producing areas can result in the genetic diversity of Citrus grandis ‘Tomentosa’.

Studies on Genuineness of Exocarpium Citri Grandis / 广州中医药大学学报

Objective To explore the genuineness of Exocarpium Citri Grandis(ECG) from Huazhou city of Guangdong province.Methods We used the method of high performance liquid chromatography to detect the naringin content in ECG from different producing areas of Huazhou city.Random amplified polymorphic DNA analysis was used for the examination of genetic distance,and plasma-atomic emission spectrometry for the detection of soil elemental abundance of 8 elements such as aluminium(Al),kalium(K),calcium(Ca),ferrum(Fe),titanium(Ti),boron(B),magnesium(Mg),and manganese(Ma).The correlation of the above three parameters was analyzed by statistical software SPSS 11.5.Results Ca abundance in the surface soil layer had an obvious effect on the content of naringin,and the difference of Al and K abundance in subsoil layer was correlated with the genetic distance of ECG.Conclusion The genuineness of ECG is probably related with the abundance of phlopopitum in the soil of producing areas of Huazhou city.

Ways for the supervision of department directors / 中华医院管理杂志

Objective To explore ways for the supervision of department directors. Methods Job responsibility agreements with department directors were signed and a series of assessment standards were established. Results The methods adopted were standardized, individualized and easy to use. Conclusion Job responsibility agreements with department directors and a standard assessment system are currently effective measures for the supervision of department directors.

Influence of Different Processing Technique on the Content of Naringin in Exocarpium Citri Grandis / 中药新药与临床药理

Objective To assess the influence of different processing technique on the quality of Exocarpium citri grandis.Methods The RP-HPLC method was adopted to determine the naringin content in Exocarpium citri grandis.The chromatographic conditions were: Kromasil ODS column(250 mm?4.6 mm),mobile phase being methanol-water-acetic acid(35∶61∶4),flow rate 1.0 mL/min,detection wavelength at 283nm and column temperature at 25 ℃.Results There were obvious differences in the naringin content of Exocarpium citri grandis which was processed by different methods: the naringin content processed by electro static drying method was much higher than that by bake drying method, natural drying method and vacuum drying method.Conclusion The electro static drying method is the best way for drying Exocarpium citri grandis.