Objective To observe the effects of different collagenase digestions on isolating human umbilical cord mesenchymal stromal cells (MSC) from Wharton’s jelly, to exam their differentiation ability and to investigate their passage effect on the immune phenotype. Methods Human umbilical cord samples were digested by collagenaseⅠorⅡorⅣfor 4-18 hours then were passed through sieves . Cells were collected by centrifugation then inoculated in DMEM/F12 medium at concentration within range of 4.8×103-1×104/cm2 to compare the effect of different digestions on MSC. Von kossa staining and tetracycline fluorescence was used to label the osteogenic differentiation capacity of MSC. Also RT-PCR was employed to identify the differentiate capacity of MSC into myocardial-like cells. The immunophenotype of MSCs were detected by flow cytometry after subculture. Results Using collagenaseⅠdigestion, the number of MSCs isolated from human umbilical cord in Wharton’s jelly and their vitality were much higher while the period to show cell extension and primary culture time were shorter than those using collagenaseⅡorⅣdigestions. The analysis of surface marker revealed that the expression of positive markers include CD29, CD44, CD73, CD90 and CD105 did not change with passages while the negative markers such as CD31, CD34 and HLA-DR increased significantly with passages;Differential experiments induced in vitro show that human umbilical cord MSC in wharton’s jelly had the ability to differentiate into osteoblasts and myocardial-like cells. Con?clusion The human umbilical cord MSC in Wharton’s jelly was successfully isolated by collagenaseⅠdigestion. This meth?od was simple with a high success rate while cell loss and damage were minimum. This makes large-scale cultivation possi? ble. Negative markers increased with cell passages. This phenomenon revealed that MSC showed directional differentiation.

BACKGROUND:The growth of mesenchymal stem cells in vitro in different conditioned media is different evidently. So it is necessary to choose a more suitable medium. OBJECTIVE:To contrast and observe the proliferation of human umbilical cord mesenchymal stem cells in three kinds of media and to check the immunophenotype and differentiation ability of mesenchymal stem cells. METHODS:Human umbilical cord mesenchymal stem cells were col ected by explant method in sterile conditions. After subculturing by T75 incubation bottles, the third generation of mesenchymal stem cells were cultured in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 medium containing 5%fetal bovine serum, Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 containing 10%fetal bovine serum and Mesen PRO RSTM medium. After 1, 3, 5, 7 days of culture, the cells were counted to draw a growth curve. Immunophenotype of the third generation of umbilical cord mesenchymal stem cells were determined by flow cytometry and the ability of osteogenic and adipogenic differentiation was also detected. RESULTS AND CONCLUSION:The third generation of cells cultured highly expressed CD44, CD73, CD90, CD105, but did not express CD29, CD31, CD34, HLA-DR. The oil red O staining showed a lot of little red lipid drops after adipogenic induction;alizarin red staining showed osteoblast-like cells after osteogenic induction, indicating umbilical cord mesenchymal stem cells in vitro have the potential of multi-directional differentiation. After observation and counting, the colony and shape of cells cultured in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 containing 10%fetal bovine serum were superior to those cultured in the other two media. Therefore, it is concluded that Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 containing 10%fetal bovine serum is preferred for cellsubculture.

BACKGROUND:Due to the difficulty in the control of delivery and col ection process, antibiotics are often added into the preservation fluid in order to avoid bacterial contamination but not affect cellgrowth and proliferation. OBJECTIVE:To observe the effects of meropenem on the proliferation and differentiation potential of umbilical cord-derived mesenchymal stem cells. METHODS:There were two groups in this experiment:control group, preservation fluid with penicil in-streptomycin (final concentration of 100 U/mL);experimental group, preservation fluid with meropenem (final mass concentration of 1.0 mg/L). 100 umbilical cord samples were col ected in each group, and the positive rate was calculated. After isolation and culture, the passage 3 cells were used to draw a growth curve, flow cytometry analysis was used for phenotype determination, and osteogenic and adipogenic differentiation of cells were detected. RESULTS AND CONCLUSION:The contamination rates were 3%(3/100) in the experimental group and 20%(20/100) in the control group, indicating that meropenem can obviously reduce the contamination rate. In the experimental group, the morphology, differentiation potential and cellphenotype of the passage 3 cells were al normal. The proliferation ability of cells showed no difference between the two groups. Therefore, meropenem can be added to the preservation fluid.

10.3969/j.issn.2095-4344.2013.23.004

BACKGROUND:High-quality and efficient umbilical cord-derived mesenchymal stem cells could be obtained by establishing and improving the method to avoid fungal contamination and by effectively decreasing pol ution rate of isolated culture during umbilical cord col ection. OBJECTIVE:To explore the effects of amphotericin B on growth and differentiation potential of umbilical cord-derived mesenchymal stem cells. METHODS:Umbilical cord-derived mesenchymal stem cells were separated from healthy ful-termed delivery fetus using col agenase digestion method and treated with amphotericin B. Subsequently, umbilical cord-derived mesenchymal stem cells were amplified and cultured in vitro with MesenPRO RSTM medium. The third passage of umbilical cord-derived mesenchymal stem cells in logarithmic phase was obtained to analyze their morphology, proliferation and immunophenotype, and induced to differentiate into osteoblasts and adipocytes in vitro. RESULTS AND CONCLUSION:After treatment with amphotericin B, umbilical cord-derived mesenchymal stem cells were successful y isolated and cultured in vitro. Flow cytometry results revealed that human umbilical cord-derived mesenchymal stem cells strongly expressed CD44, CD105 and CD73, CD90, and negatively expressed HLA-DR, CD29, CD31, and CD34. Amphotericin B-treated human umbilical cord-derived mesenchymal stem cells can stil differentiate into adipocytes and osteoblasts in vitro.

BACKGROUND: In vitro culture condition and culture efficiency are different in reported umbilical cord-derived mesenchymal stem cells, and lacked of unified standards. Different derived mesenchymal stem cells have different biological properties. Therefore, it is very necessary to establish a simple and high-performance culture system for umbilical cord-derived mesenchymal stem cells. OBJECTIVE: To observe the growth state of human umbilical cord-derived mesenchymal stem cells in different culture systems in vitro and adenovirus infection efficiency. METHODS: Mesenchymal stem cells were separated from healthy full-termed delivery fetus using collagenase digestion method and purified by adherent culture. These cells were cultured and amplified in DMEM (low glucose), MesenPRO RS~(TM) Medium and STEMPRO~(R) MSC SFM in vitro. The 3-5 passage mesenchymal stem cells were infected by the Ad5-EGFP, Ad5/11-EGFP, Ad5/35-EGFP as multiplicity of infection (MOI)=1, 10, 100. Viral infection and green fluorescence expression were observed at post-infection 24, 56 and 72 hours using inverted fluorescence microscope. RESULTS AND CONCLUSION: The cell morphology in STEMPR~(R) MSC SFM was different from other two culture system and these cells were not easy to adherent after trypsin digestion. Cell doubling time in the MesenPRO RS~(TM) Medium was shorter than other two groups. Mesenchymal stem cells were infected by Ad5/35-EGFP with higher efficiency than other two kinds of adenovirus, but part of cells appeared apoptosis. The infection efficiency of Ad5/11-EGFP was highest. The fluorescence intensity was gradually increased with increased MOI.

Objective To explore the effects of traditional Chinese medicinal herbs (TCMHs) on the expression of tyrosinase gene, melanogenesis and proliferation of melanocytes and elucidate the mechanism of TCMHs in promoting melanogenesis. Methods Seven TCMHs including Herba Ecliptae, Spica Prunellae, Caulis Spatholob, etc, which were known to be effective in activating tyrosinase in vivo, were selected. Brownish guinea pigs were selected as the experimental model. The mRNA in situ hybridization (ISH), Schmorl-staining and dopa-oxygenase staining were performed to observe the effects of TCMHs on gene expression of tyrosinase, melanogenesis and melanocyte proliferation. Results The mRNA ISH showed that these seven drugs, especially Herba Ecliptae,Spica Prunellae and Tribulus terrestris could significantly increase the number of positive cells and the intensity of hybridization signal in the treated group as compared with that in the control group (P 0.1). Conclusions These results suggested that these 7 TCMHs including Herba Ecliptae can upregulate the gene expression of tyrosinase, enhance the melanogenesis and promote the proliferation of melanocytes.

Objective To investigate sonographic characteristics of struma ovarii with conventional ultrasound and CEUS.Methods Ultrasound images of 65 patients with struma ovarii confirmed by pathology were retrospectively reviewed and analyzed,5 patients were examined with CEUS simultaneously.Results In all 65 patients,lesions in 14 (14/65,21.54 ％) were multocular,49 (49/65,75.34％) were cystic-solid component,2 (2/65,3.08％) were solid.Lesions in 39 (39/65,60.00％) contained dense latticed separation.The abundant blood flow was found in 18 cases (18/65,27.69％) with Doppler examination.Five cases were examined with CEUS,including multilocular lesions of 2 cases and cystic-solid lesions of 3 cases.Regular middle-degree intensity of cyst wall and septa were seen in all 5 cases.Among cystic-solid lesions of 3 cases,lesions of non-enhance pattern was seen in the solid areas of 1 case,irregular middle-high degree intensity pattern were seen in the solid areas of 2 cases,while non-enhance pattern could be seen in part of the solid areas of these 2 cases.All the cystic areas of these 5 cases showed non-enhance pattern.Conclusion The sonographic appearances of struma ovarii are usually multilocular or multilocular with solid component.Because of strum ovarii's special characteristic pathologic components,the imaging features of strum ovarii in conventional ultrasound and CEUS are atypical,thus preoperative diagnosis is quite difficult.

Objective: To effectively reduce the concentration of poisons in cleanroom, protect the health of workers, realize the optimization and automatic control of the new return air device. And the influence of initial concentration, air volume, temperature and relative humidity of formaldehyde on the purification effect of the new return air device was explored. Methods: The purification effect of the new return air device installed with the activated carbon and the photocatalyst purification net or ordinary activated carbon purification network was tested in a 60 m³ simulated cleanroom. The concentration of formaldehyde was determined by solution absorption-phenol reagent spectrophotometry. Based on the single factor experiment to determine the combination of two purification nets. The effects of air volume, initial formaldehyde concentration, temperature and relative humidity on the purification effect of the new return air device were investigated by orthogonal test. Then, the performance parameters of the return air device to purify formaldehyde were determined. Results: The formaldehyde purification efficiency of the two types of purification nets in the new return air device was higher than that of the ordinary activated carbon purification network (P<0.05) . The combination of activated carbon and photocatalyst purification net has no effect on the formaldehyde purification efficiency of the return air device (P>0.05) . According to the direct analysis and variance analysis, air volume was the most sensitive factor (F value is 18.894, P<0.05) , followed by initial concentration (F value is 16.128, P<0.05) , while temperature and relative humidity have little effect (F value is 0.041 and 0.599, respectively, P>0.05) . LSD analysis showed that there was no significant difference in the purification efficiency of formaldehyde between 475 m³/h and 626 m³/h (P>0.05) . From the perspective of formaldehyde purification efficiency and energy saving, when the air volume is set to 475 m³/h, the new return air device has higher purification efficiency for high concentration of formaldehyde. Conclusion The new return air device consisting of activated carbon and photocatalyst purification net can play a good purification role in cleanroom with different temperatures and different humidity. Its formaldehyde purification efficiency is affected by air volume and initial concentration.