Expansion of human umbilical cord derived mesenchymal stem cells in regenerative medicine

BACKGROUND Stem cells are undifferentiated cells that possess the potential for self-renewal with the capacity to differentiate into multiple lineages.In humans,their limited numbers pose a challenge in fulfilling the necessary demands for the regeneration and repair of damaged tissues or organs.Studies suggested that mesenchymal stem cells(MSCs),necessary for repair and regeneration via transplantation,require doses ranging from 10 to 400 million cells.Furthermore,the limited expansion of MSCs restricts their therapeutic application.AIM To optimize a novel protocol to achieve qualitative and quantitative expansion of MSCs to reach the targeted number of cells for cellular transplantation and minimize the limitations in stem cell therapy protocols.METHODS Human umbilical cord(hUC)tissue derived MSCs were obtained and re-cultured.These cultured cells were subjected to the following evaluation pro-cedures:Immunophenotyping,immunocytochemical staining,trilineage differentiation,population doubling time and number,gene expression markers for proliferation,cell cycle progression,senescence-associatedβ-galactosidase assay,human telomerase reverse transcriptase(hTERT)expression,mycoplasma,cytomegalovirus and endotoxin detection.RESULTS Analysis of pluripotent gene markers Oct4,Sox2,and Nanog in recultured hUC-MSC revealed no significant differences.The immunophenotypic markers CD90,CD73,CD105,CD44,vimentin,CD29,Stro-1,and Lin28 were positively expressed by these recultured expanded MSCs,and were found negative for CD34,CD11b,CD19,CD45,and HLA-DR.The recultured hUC-MSC population continued to expand through passage 15.Proliferative gene expression of Pax6,BMP2,and TGFb1 showed no significant variation between recultured hUC-MSC groups.Nevertheless,a significant increase(P<0.001)in the mitotic phase of the cell cycle was observed in recultured hUC-MSCs.Cellular senescence markers(hTERT expression andβ-galactosidase activity)did not show any negative effect on recultured hUC-MSCs.Additionally,quality control assessments consistently confirmed the absence of mycoplasma,cytomegalovirus,and endotoxin contamination.CONCLUSION This study proposes the development of a novel protocol for efficiently expanding stem cell population.This would address the growing demand for larger stem cell doses needed for cellular transplantation and will significantly improve the feasibility of stem cell based therapies.

Power generation expansion planning approach considering carbon emission constraints

Decarbonization of the power sector in China is an essential aspect of the energy transition process to achieve carbon neutrality.The power sector accounts for approximately 40%of China’s total CO_(2) emissions.Accordingly,collaborative optimization in power generation expansion planning(GEP)simultaneously considering economic,environmental,and technological concerns as carbon emissions is necessary.This paper proposes a collaborative mixedinteger linear programming optimization approach for GEP.This minimizes the power system’s operating cost to resolve emission concerns considering energy development strategies,flexible generation,and resource limitations constraints.This research further analyzes the advantages and disadvantages of current GEP techniques.Results show that the main determinants of new investment decisions are carbon emissions,reserve margins,resource availability,fuel consumption,and fuel price.The proposed optimization method is simulated and validated based on China’s power system data.Finally,this study provides policy recommendations on the flexible management of traditional power sources,the market-oriented mechanism of new energy sources,and the integration of new technology to support the attainment of carbon-neutral targets in the current energy transition process.

Long-term Performance of Moderate Heat Portland Cement with Double-expansive Sources

The long-term performance of moderate heat Portland cement with double-expansive sources （DE cement） in the system of high MgO clinker and gypsum was studied by XRD, SEM/EDAX and test methods for strength and expansion of cement. Results indicate that the periclase particle, whose size was 5-7.5μm in DE cement clinker containing 4.8 % MgO, existed individually. The periclase hydration in hardened DE cement paste started at about 60 days and completed up to 2 000 days, and ettringite in the paste was stable from 3 days to 2 000 days. Under the conditions of 4.5%-5.0 % MgO in clinker and 2.8%-3.4 %SO3 in cement, ettringite expansion and brucite expansion in DE cement paste had a continuity, entirety and stability. At the ages of 90, 365,730 and 2 000 days the expansion of the paste reached 0.07%-0.11%, 0.16%-0.21%, 0.21%-0.27 %, and 0.29%-0.38%, respectively. The results suggest that by using this cement in mass concrete it may compensate its temperature shrinkage and autogenous shrinkage to some extent.

Efficient Technique for Long-Term <i>in Vitro</i>Storage of Transgenic Aspen Genotypes

In vitro culture of isolated cells from tissues and organs is sometimes used to preserve and reproduce unique genotypes of woody plants. The technique, however, requires regular subculturing which raises storage costs and creates risks for contamination and accumulation of somaclonal variations. We examined the effects of sugar composition of culture medium, the length of photoperiod, light intensity, and ambient temperature on the survival of plant material in vitro. The study was performed on 49 genotypes of Populus tremula (46 transgenic genotypes carrying GFP-, Xeg- and Gus-genes, and 3 control (wild-type) genotypes). It was shown that effective storage of plants was achieved through optimization of the combined effects of all storage parameters under study. Based on the experimental data, we developed a protocol for long-term in vitro storage of desirable genotypes without subculture and with a survival rate of up to 98%. The best results were obtained when the plant material was pre-cultured on a WPM medium containing 15 g/L sucrose, 7.5 g/L sorbitol and 7.5 g/L mannitol, and then stored at +4°C under a 24-hour light day cycle with only 8 hours of light per day and maximum light intensity of 2000 lux. Post-storage recovery was done by culturing on a medium containing 1 mg/L gibberellic acid. The developed method can be used for effective in vitro storage of the studied genotypes for up to 24 months without subculture.

Experimental Study and Design of Balloon- expandable Endovascular Stent Expansion

The application background and experimental research overview of medical endovascular stent are presented. Based on the analytical comparison of the current research achievements, the life cycle of medical endovascular stent is pointed out and the characteristics of stent expansion in the life cycle are emphasized on. The experimental scheme of in vitro stent expansion based on the machine vision technology in LabVIEW is presented. The selection and usage of the chosen component devices and design of measurement program for experiment are expatiated. A special drug-loading stent was expanded on the assembled platform of selected equipments and experimental results were analyzed. The experimental scheme presented in the paper provides powerful experimental support for the optimization of stent design and computer simulation of stent expansion by the finite element analysis.

黑枸杞花青素联合人脂肪源性血管外膜细胞支持脐血造血干/祖细胞的增殖

背景:黑枸杞花青素(Anthocyanins in Lycium ruthenicum Murr,ALRM)是黑枸杞中重要的活性成分之一,具有抗氧化、免疫调节等功效。人脂肪源性血管外膜细胞(CD146^(+)hAD-PCs)是骨髓间充质干细胞的前体细胞,在体外具有促进造血干/祖细胞增殖与分化的功能。ALRM联合CD146^(+)hAD-PCs对脐血造血干/组细胞的体外支持作用有待于研究。目的:探讨ALRM联合CD146^(+)hAD-PCs对脐血CD34^(+)造血干/祖细胞体外扩增的支持作用。方法:CCK-8法检测不同质量浓度ALRM(0,200,400,600,800,1000 mg/L)对CD146^(+)hAD-PCs增殖的影响;流式细胞术检测ALRM对CD146^(+)hAD-PCs细胞周期的影响。共培养实验分为空白组、ALRM组、CD146^(+)hAD-PCs组、ALRM+CD146^(+)hAD-PCs组,分析ALRM联合CD146^(+)hAD-PCs对脐血CD34^(+)造血干/祖细胞的体外支持作用。共培养1,2,4周,比较扩增后细胞数量、集落形成单位数量,流式细胞仪检测细胞免疫表型,ELISA检测细胞因子水平。结果与结论:(1)ALRM质量浓度为200 mg/L时,CD146^(+)hAD-PCs活力最高,CD146^(+)hAD-PCs的G_(0)/G_(1)期细胞比例下降,S期、G_(2)/M期细胞比例上升(P<0.01)。(2)脐血CD34^(+)造血干/祖细胞数量变化:在共培养1,2,4周时ALRM+CD146^(+)hAD-PCs组高于ALRM组(P均<0.05),在共培养2,4周时ALRM+CD146^(+)hAD-PCs组高于CD146^(+)hAD-PCs组(P均<0.05),ALRM组与空白组随着共培养时间延长细胞数量逐渐减少。(3)集落形成能力及免疫表型分析:在共培养1,2周时ALRM+CD146^(+)hAD-PCs组的集落形成单位数量高于CD146^(+)hAD-PCs组和ALRM组(P均<0.05);在共培养1,2,4周时ALRM+CD146^(+)hAD-PCs组CD45^(+)、CD34^(+)CD33^(-)细胞比例高于CD146^(+)hAD-PCs组(P均<0.01)。(4)细胞因子变化:在共培养4周时ALRM+CD146^(+)hAD-PCs组的白细胞介素2水平高于ALRM组、CD146^(+)hAD-PCs组(P<0.05);在共培养2,4周时ALRM+CD146^(+)hAD-PCs组白细胞介素3水平高于CD146^(+)hAD-PCs组(P<0.05);在共培养1周时ALRM+CD146^(+)h AD-PCs组的粒细胞集落刺激因子水平高于CD146^(+)hAD-PCs组,在共培养2周时高于ALRM组、CD146^(+)hAD-PCs组(P<0.01);在共培养1,2,4周时ALRM组、ALRM+CD146^(+)hAD-PCs组的干扰素γ水平低于CD146^(+)hAD-PCs组(P<0.05)。(5)由于空白组无基质细胞,脐血CD34^(+)造血干/祖细胞在共培养1周之后就无法计数,未进行免疫表型、集落分析和细胞因子检测。(6)结果表明:ALRM可以通过促进CD146^(+)hAD-PCs增殖和细胞周期转化进而促进脐血CD34^(+)造血干/祖细胞的体外扩增,在造血干细胞移植研究方面具有重要价值。

挤压膨化结合酶解制备低GI代餐粉及其体外消化率测定

以藜麦、燕麦、青稞、苦荞为主要原料,辅以薏仁、葛根、桑叶、菊粉、低聚果糖等,利用酶解植物性原料及挤压膨化工艺制备代餐粉。以分散性指数和感官评分为指标,通过单因素试验和Box-Behnken试验结合优化原料挤压膨化工艺,并对原料代餐粉、挤压膨化代餐粉和酶解挤压膨化代餐粉的品质、体外消化率和血糖生成指数进行测定。结果表明:酶解挤压膨化代餐粉的最佳挤压膨化工艺为挤压温度150℃,螺杆转速810 r/min,喂料速度340 r/min,该工艺下代餐粉的分散性指数98.81%,感官评分89分。三种粉品质差异显著,酶解挤压膨化代餐粉的分散性指数和感官品质最高,酶解挤压膨化代餐粉eGI值为51.56,属于低GI食物,可为低GI代餐粉的开发提供一定的参考。

以开设体外诊断产业特色方向培养医学检验创业人才的探索

体外诊断(in vitro diagnosis,IVD)产业发展迅猛,我国IVD每年复合增长率保持在15%~20%,增速明显高于全球平均水平,IVD企业急需大量医学检验体外诊断复合型人才。然而,近40多年来,我国高校医学检验专业办学模式、培养目标及课程体系设置等变化不大,培养目标单一,课程设置同质化严重,医学检验专业人才培养与岗位需求严重脱节。为培养体外诊断产业复合型医学检验人才,以行业发展和岗位需求为导向,我院设置了“体外诊断产业”特色拓展方向,与体外诊断企业共同成立“体外诊断现代产业学院”,修订了人才培养方案,优化了课程体系,主编了相应课程创新特色教材,组织牵头全国高校医学检验专业毕业生体外诊断企业在线招聘会,效果显著。

Zinc enhances the cell adhesion,migration,and self-renewal potential of human umbilical cord derived mesenchymal stem cells

BACKGROUND Zinc(Zn)is the second most abundant trace element after Fe,present in the human body.It is frequently reported in association with cell growth and proliferation,and its deficiency is considered to be a major disease contributing factor.AIM To determine the effect of Zn on in vitro growth and proliferation of human umbilical cord(hUC)-derived mesenchymal stem cells(MSCs).METHODS hUC-MSCs were isolated from human umbilical cord tissue and characterized based on immunocytochemistry,immunophenotyping,and tri-lineage differentiation.The impact of Zn on cytotoxicity and proliferation was determined by MTT and Alamar blue assay.To determine the effect of Zn on population doubling time(PDT),hUC-MSCs were cultured in media with and without Zn for several passages.An in vitro scratch assay was performed to analyze the effect of Zn on the wound healing and migration capability of hUC-MSCs.A cell adhesion assay was used to test the surface adhesiveness of hUC-MSCs.Transcriptional analysis of genes involved in the cell cycle,proliferation,migration,and selfrenewal of hUC-MSCs was performed by quantitative real-time polymerase chain reaction.The protein expression of Lin28,a pluripotency marker,was analyzed by immunocytochemistry.RESULTS Zn at lower concentrations enhanced the rate of proliferation but at higher concentrations(>100μM),showed concentration dependent cytotoxicity in hUC-MSCs.hUC-MSCs treated with Zn exhibited a significantly greater healing and migration rate compared to untreated cells.Zn also increased the cell adhesion rate,and colony forming efficiency(CFE).In addition,Zn upregulated the expression of genes involved in the cell cycle(CDC20,CDK1,CCNA2,CDCA2),proliferation(transforming growth factorβ1,GDF5,hypoxia-inducible factor 1α),migration(CXCR4,VCAM1,VEGF-A),and self-renewal(OCT4,SOX2,NANOG)of hUC-MSCs.Expression of Lin28 protein was significantly increased in cells treated with Zn.CONCLUSION Our findings suggest that zinc enhances the proliferation rate of hUC-MSCs decreasing the PDT,and maintaining the CFE.Zn also enhances the cell adhesion,migration,and self-renewal of hUC-MSCs.These results highlight the essential role of Zn in cell growth and development.

空气膨化关键工艺对青稞藜麦姜米茶品质的影响

本试验以青稞、藜麦为主原料,研制出了青稞藜麦姜米茶。通过单因素试验,研究了空气膨化温度和时间对青稞藜麦姜米茶总酚含量、体外降血糖活性和感官品质的影响。结果表明:青稞藜麦姜米茶制备的最佳工艺条件为空气膨化温度180℃,空气膨化时间10 min。在最佳工艺条件下制备的青稞藜麦姜米茶,汤色清澈透亮,茶味清香,鲜醇甘爽,此时青稞藜麦姜米茶的品质最好。

基于RNA测序的SHED体外连续扩增后差异表达基因的研究

目的:利用RNA测序(RNA sequencing,RNA-seq)技术研究人脱落乳牙干细胞(stem cells derived from human exfoliated deciduous teeth,SHED)体外长时期扩增至20代(P20)后基因表达的差异,初步探讨其体外扩增衰老相关的信号通路。方法:从健康儿童脱落的乳牙中分离牙髓干细胞,在常规条件下将其扩增培养至20代,利用RNA-seq筛选出差异表达的基因,并对其进行相关生物学信息分析,寻找SHED体外连续扩增衰老相关的信号通路。结果:RNA-seq结果显示,SHED第4代(P4)和P20代差异表达的基因共有1884个,其中上调表达的基因有575个,下调表达基因1309个,这些差异表达的基因分布在生物过程(biological progress,BP)、细胞组成(cellular component,CC)和分子功能(molecular function,MF)等生物学过程中。早、晚期代次的SHED差异基因及相关蛋白之间的作用主要富集在剪接体、核糖体、细胞周期、p53通路相关的信号通路上。结论:本研究揭示了SHED体外连续扩增培养至第20代后基因表达谱的改变,为后续进一步研究其细胞体外衰老机制指明了方向。

不同磷酸化合物扩增外周血γδ T细胞的效率及条件优化

目的研究异戊烯焦磷酸(IPP)与帕米磷酸钠(PAM)体外扩增成人外周血γδT细胞的效率及条件优化。方法梯度离心法分离人外周血单个核细胞(PBMC),分别加入(1.0、5.0、10.0、15.0)μg/mL IPP或(2.0、5.0、8.0、12.0)μg/mL PAM及(100.0、200.0、500.0)IU/mL IL-2,置于100 mL/L小牛血清的RPMI1640培养体系培养,观察并收集细胞,采用流式细胞仪检测刺激后CD3+TCRδ2+的γδT细胞数量以及比例,并计算其扩增效率。结果经过14 d扩增培养后,IPP组的γδT细胞数量在淋巴细胞中比例可上升为81.3%,PAM组的γδT细胞数量在淋巴细胞中比例上升为78.5%,表明IPP和PAM均能有效刺激扩增γδT细胞,2组的扩增效率无统计学差异(P>0.05)。结论 PAM具有与IPP相似的体外扩增γδT细胞的能力,PAM扩增法可能成为扩增γδT细胞更为经济实用的选择。

蛋白质合成抑制剂亚胺环己酮(CHX)对猪卵母细胞体外成熟的影响

研究了蛋白质合成抑制剂亚胺环己酮 (CHX)对猪卵母细胞体外成熟过程中的GVBD、染色质凝集、MⅡ期成熟及卵丘细胞扩展的作用。结果表明 :( 1)培养液中添加CHX ,可抑制卵母细胞GVBD的发生 ,而且此作用是浓度依赖性的 ,但CHX的抑制效果是完全可逆的 ;( 2 )在含 10 μg/mlCHX液中分别培养 0、 6、 12和 2 4h后转入正常培养液再继续培养至 4 8h ,卵母细胞成熟率分别为 84 1%、 77 1%、 4 8 9%和 2 7 8% ;( 3 )正常培养液中培养 0、 6、 12、 2 4、 3 6和 4 8h后 ,再转入浓度为 10 μg/mlCHX液中继续培养至 4 8h ,卵母细胞成熟率分别为 0、 0、 0、 3 1 3 %、 65 4 %和 79 5 % ;( 4 )CHX对卵丘细胞扩展的影响随培养时间延长而增强 ,在CHX中处理时间为 16h或更长 。

人脐血CD34^+细胞体外DEXTER基质细胞培养的动态观察

目的 动态观察人脐血CD3 4+ 细胞DEXTER基质细胞培养的情况。方法 应用MACS磁珠分离系统分离脐带血CD3 4+ 细胞 ,按照DEXTER法行脐血基质细胞培养 ,观察不同时间、不同细胞因子组合基质细胞的生长状态。结果 SCF及SCF +BFGF组合可促进基质细胞集落的生成 ,脐血基质细胞在生长情况、组成成分、细胞形态等方面与骨髓基质细胞有不同的生物学特性。结论 脐血CD3 4+ 细胞在生长因子的辅助下能促进基质细胞集落的形成 ,有望成为新的基质细胞来源。

恶性肿瘤患者外周血淋巴细胞体外扩增后亚群的变化

目的:观察23例恶性肿瘤患者外周血淋巴细胞在体外经扩增培养后总CD3+、CD3+CD4+、CD3+CD8+淋巴细胞及CD3-CD16+56+(NK细胞)、CD3+CD16+56+(NKT细胞)的变化,初步观察淋巴细胞体外扩增培养后回输患者的生物安全性。方法:体外大规模诱导和扩增恶性肿瘤患者外周血淋巴细胞及单个核细胞,然后应用流式细胞术测定扩增前后CD3+、CD3+CD4+、CD3+CD8+、CD3-CD16+56+、CD3+CD16+56+细胞在淋巴细胞中所占百分比的变化。结果:CD3+、CD3+CD8+、CD3+CD16+56+细胞比例较培养前明显增加(P<0.01),而CD3+CD4+和CD3-CD16+56+细胞比例则明显的降低(P<0.01)。所有患者细胞回输治疗后无明显的毒副反应。结论:在本研究体系下体外诱导扩增培养肿瘤患者自体外周血NK细胞及致敏淋巴细胞效率较高,细胞毒性细胞比例增高较大,具有良好的生物安全性。

GM-CSF对脐血CD34^+巨核祖细胞体外扩增及分化的影响

本实验旨在研究GMCSF对脐血CD34+细胞诱导分化为巨核细胞的影响。采用免疫磁珠法分选CD34+细胞,在含有TPO+IL3+SCF并添加了不同浓度(5、20、100ng/ml)的GMCSF的无血清培养基中进行培养。培养6、10、14天后计数单个核细胞(MNC),检测CD41+细胞比例和CFUMK。结果表明,培养14天后3种不同浓度GMCSF对MNC均有明显的扩增作用,其中以20和100ng/mlGMCSF的扩增效果较好。3种不同浓度的GMCSF均使CD41+细胞比例增加,20和100ng/ml与5ng/mlGMCSF相比更能提高CD41+细胞的比例。5和20ng/ml的GMCSF能促进CFUMK的形成,但100ng/ml的GMCSF却抑制CFUMK的形成。结论:在TPO+IL3+SCF细胞因子组合中添加GMCSF有利于促进脐血CD34+细胞诱导分化为巨核细胞。

脐血造血干/祖细胞体外扩增及移植动物模型分析

目的 探讨脐血造血干 /祖细胞体外扩增和体内重建造血的潜能。方法 利用造血干 /祖细胞培养、体外扩增、移植动物模型等技术 ,研究不同比密ficoll分离液分离的脐血造血干 /祖细胞的造血活性。结果 比密 1.0 6 4 g/ml分离液分离的 10 5个脐血MNC中 ,CFU GM集落数为 373± 2 89,BFU E为 12 1± 70。在G3(GM CSF和IL 3融合蛋白 )、IL 6、TPO(thrombopoietin)作用下 ,1.0 6 4 g/ml分离液分离的脐血造血干 /祖细胞在体外扩增 14d ,CFU GM扩增 5 2 .2倍。给经 8.5Gy致死剂量照射的小鼠输注 1.0 6 4 g/ml分离液分离的脐血造血干 /祖细胞 ,体内产生的脾结节 (CFU S)是输注 1.0 77g/ml分离液分离细胞的 2 .2倍。结论 比密 1.0 6 4 g/ml分离液分离的脐血造血干 /祖细胞 ,在体外具有较高的增殖。