Minimally manipulated autologous adherent bone marrow cells (ABMCs):a promising cell therapy of spinal cord injury

Spinal cord injury（SCI）is a devastating ailment that results in drastic life style alterations for the patients and their family members（Mc Donald and Sadowsky,2002）.Damage post injury causes necrosis,edema,hemorrhage and vasospasm.Post injury,secondary damage is caused by ischemia,

Neuronal-like cell differentiation of non-adherent bone marrow cell-derived mesenchymal stem cells

Non-adherent bone marrow cell-derived mesenchymal stem cells from C57BL/6J mice were sepa- rated and cultured using the ＂pour-off＂ method. Non-adherent bone marrow cell-derived mesen- chymal stem ceils developed colony-forming unit-fibroblasts, and could be expanded by supple- mentation with epidermal growth factor. Immunocytochemistry showed that the non-adherent bone marrow cell-derived mesenchymal stem cells exposed to basic fibroblast growth factor/epidermal growth factor/nerve growth factor expressed the neuron specific markers, neurofilament-200 and NeuN, in vitro. Non-adherent bone marrow cell-derived mesenchymal stem cells from 13-galactosidase transgenic mice were also transplanted into focal ischemic brain （right corpus striatum） of C57BL/6J mice. At 8 weeks, cells positive for LacZ and 13-galactosidase staining were observed in the ischemic tissues, and cells co-labeled with both 13-galactosidase and NeuN were seen by double immunohistochemical staining. These findings suggest that the non-adherent bone marrow cell-derived mesenchymal stem cells could differentiate into neuronal-like cells in vitro and in vivo.

THE EFFECT OF PHENYLACETATE ON THE EXPANSION AND CYTOTOXIC ACTIVITY OF ADHERENT LAK CELLS FROM PATIENTS WITH HEPATOCELLULAR CARCINOMA

Objective: To improve the preparation of adherent lymphokine-activated killer (A-LAK) cells and study the synergistic anti-tumor effect of phenylacetate (PA) and A-LAK cells. Methods: A-LAK cells were obtained from peripheral blood mononuclear cells (PBMC) of patients with hepatocellular carcinoma (HCC) by using L-phenylalanine methyl ester (PME) to deplete immunosuppressive monocytes. The proliferation of SMMC7721 cell line treated with PA was studied. A-LAK cells were treated with the supernatant of SMMC7721 cells which had been pretreated with PA and the changes of the proliferation and anti-tumor activity of A-LAK cells were investigated. Results: The expansion of A-LAK cells was significantly higher than that of non-adherent LAK (NA-LAK) cells as well as regular LAK cells. The growth of SMMC7721 cells was significantly suppressed by PA. The supernatant of cultured tumor cells intensively suppressed the proliferation and cytotoxicity of A-LAK cells, but the suppressive effect of supernatant treated with PA previously was decreased. Conclusion: A-LAK cells could be simply prepared by using PME, and showed a synergistic anti-tumor effect with the combination of PA.

Self-assembled IKVAV Peptide Nanofibers Promote Adherence of PC12 Cells

Lack of biocompatibility and bioactivity is a big problem for the synthetic materials that have been generated for neural tissue engineering. To get around the problem and generate better scaffold for neural tissue repair, we intended to generate nano-fibers by self-assembly of polypeptide IKVAV. Bioactive IKVAV Peptide-Amphiphile （IKVAV-PA） was first synthesized and purified, the property of which was analyzed and determined by high-performance liquid chromatography （HPLC） and mass spectrometry （MS）. Then, by addition of hydrogen chloride （HC1）, self-assembly of IKVAV-PA was induced in vitro and nano-fibers formed as shown by transmission electron microscopy （TEM）. The effect of IKVAV nanofibers on adherence of PCI2 cells was assayed in cell culture and the results showed that the rates of adherence of PC12 increased significantly when the density of IKVAV was within a certain range （0.58 μg/cm^2 to 15.6 μg/cm^2）. However, its effect on the rates of adherence did not significantly alter with time, whether after 1 hour or 3 hours of culture. In general, we showed that IKVAV-PA can successfully self-assemble to form nanofiber, and promote rapid and stable adherence of PC12 cells, and the effect of the self-assembled IKVAV to promote PCI2 cells adherence is dosage-dependent within a certain range of densities.

Internalization of NK cells into tumor cells requires ezrin and leads to programmed cell-in-cell death

Cytotoxic lymphocytes are key players in the orchestration of immune response and elimination of defective cells. We have previously reported that natural killer （NK） cells enter target tumor ceils, leading to either target cell death or self-destruction within tumor cells. However, it has remained elusive as to the fate of NK cells after internalization and whether the heterotypic cell-in-cell process is different from that of the homotypic cell-in-cell event recently named entosis. Here, we show that NK cells undergo a cell-in-cell process with the ultimate fate of apoptosis within tumor cells and reveal that the internalization process requires the actin cytoskeletal regulator, ezrin. To visualize how NK cells enter into tumor cells, we carried out real-time dual color imaging analyses of NK cell internalization into tumor cells. Surprisingly, most NK cells commit to programmed cell death after their entry into tumor cells, which is distinctively different from entosis observed in the homotypic cell-in-cell process. The apoptotic cell death of the internalized NK cells was evident by activation of caspase 3 and DNA fragmentation. Furthermore, NK cell death after internalization is attenuated by the caspase inhibitor, Z-VAD-FMK, confirming apoptosis as the mode of NK cell death within tumor cells. To determine protein factors essential for the entry of NK cells into tumor cells, we car- ried out siRNA-based knockdown analysis and discovered a critical role of ezrin in NK cell internalization. Impor- tantly, PKA-mediated phosphorylation of ezrin promotes the NK cell internalization process. Our findings suggest a novel regulatory mechanism by which ezrin governs NK cell internalization into tumor cells.

Adherence and invasion of mouse-adapted H pylori in different epithelial cell lines

AIM： To assess the adhesion and invasion abilities of different mouse adapted H py/or/strains in different cell lines in vitro and investigate their effects on the virulence factors cagA and vacA. METHODS： The adherence and invasion abilities of different N pylori strains in different epithelial cell lines were examined by the gentamycin protection assay. The null mutants of cagA and vacA were processed by direct PCR mutation method. The morphologic changes of different cell lines after N pylori attachment were examined by microscopy. RESULTS： The densities of adherence to and invasion into cells in vitro were different from those in the mouse infection experiments. 88-3887 strain could invade and adhere to cells stronger than SS1 and X47. All tested strains had better adhering and invasive abilities in SCG-7901 cell. CagA and vacA minus mutants had the same invasion and adherent abilities as their wild types. In all strains and cell lines tested, only AGS cell had the significant hummingbird phenotype after inoculation with the 88-3887 wild-type. CONCLUSION： Both the host cells and the bacteria play important parts in the invasion and adhesion abilities of Hpylori. CagA and VacA are not related to the ability of invasion and adhesion of Hpylori in different cell lines in vitro.

Factors Associated with Non-Adherence to Treatment in Sickle Cell Patients Monitored at the National Reference Center for Sickle Cell Disease in Niger

Introduction: Sickle cell disease is a real public health problem in the world and particularly in Niger where the prevalence of the S gene is estimated at 25% and that of the homozygous forms at between 1% and 2%. Treatment combines quarterly follow-up of patients and management of complications. The objective of this study was to identify the potential explanatory factors of non-adherence to treatment in sickle cell patients followed at the national reference center for sickle cell disease in Niger. Methods: This is a cross-sectional study of sickle cell cases followed at the CNRD in Niger. The population consisted of all sickle cell patients followed in this center in 2021. The data collection techniques were individual interviews and documentary reviews. Non-adherence was assessed with the Girerd test. Descriptive statistical tests and simple and multiple logistic regression models were performed. Results: A total of 368 patients were enrolled. The median age is 7 years (4;10) and the sex ratio is 1.04. Ninety-eight (98) or 26.6% were compliant and 270 (73.4%) were non-compliant. In multivariate analysis, the factors independently and negatively associated with non-adherence to treatment were schooling (adjusted OR [95% CI], p-value), 0.17 [0.10 - 0.30];p Conclusion: The factors influencing treatment compliance identified in this study are all modifiable. To prevent the complications of sickle cell disease, we must fight against ignorance, make care services accessible and make care free.

T-cadherin基因在HepG2中的功能研究

目的证实T-cadherin的重新表达是否能抑制肝癌细胞的增殖、侵袭及转移等恶性生物学行为。方法收集2014年5月—2015年10月长沙医学院外科30份肝癌患者的新鲜癌组织和癌旁2cm以上肝组织标本,按随机原则分成实验组和空白对照组各15份。运用RT-PCR技术检测其表达含量;向肝癌细胞株Hep G2转染目的 T-cadherin基因mimics;通过MTT实验、划痕实验与Transwell侵袭实验分别检测转染mimics前后细胞增殖、迁移、侵袭能力的改变。结果 (1)T-cadherin基因在肝癌细胞中表达明显下调,在癌旁组织表达上调。(2)与空白对照组对比,转染T-cadherin mimics的Hep G2在48 h^72 h细胞增殖有统计学意义(P<0.01);同空白对照组对比,24 h内细胞侵袭有明显的统计学意义(P<0.002);同空白对照组相比,24 h^48 h细胞迁移有显著统计学意义(P<0.05),48 h^72 h细胞迁移有非常显著统计学意义(P<0.01)。结论 T-cadherin基因参与了Hep G2细胞增殖、生长、分化、侵袭等环节。

Sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent cells

Objective To study the sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent cells.Methods Sensitivity and specificity of several common methods for apoptosis determination were evaluated (Apo2.7-expression, Annexin V-binding, TUNEL-reaction, poly-(ADP-ribose)-polymerase-(PARP) cleavage and single-stranded-DNA (ssDNA) staining). Apoptosis was induced by oxidative stress generated by hydrogen peroxide in 3 cultured cells types growing as adherent monolayer (MiaPaCa-2, Hep-G2 and human skin fibroblasts), necrosis was induced by depletion of cellular ATP using sodium azide. Cells positively stained by the respective apoptosis assay were quantified and alterations of cell morphology were monitored by fluorescence microscopy. The date was analyzed by one-way analysis of variance and significance test of correlation coefficient.Results One hour after apoptosis induction significant cell fractions were positively stained for ssDNA (33% with MiaPaCa-2 cells, 35% with Hep-G2 cells, 56% with human skin fibroblasts). PARP-cleavage was less sensitive compared to the ssDNA-staining. Apo2.7-expression, Annexin V-binding and TUNEL-reaction were not applicable to detect early apoptosis induced by oxidative stress (below 2 hours), but were efficiently monitoring late apoptosis. Specificity of ssDNA-staining was complete with each cell type even 4 hs after induction of necrosis by the highest sodium azide concentration. In contrast, the same experimental conditions resulted in 50%-90% positively stained necrotic cells by using Apo2.7-expression, TUNEL-reaction or AnnexinV-binding. Surprisingly, specificity of PARP-cleavage was highly depending on the respective cell type.Conclusions Our study prove that among the five methods investigated only ssDNA-staining allowed to completely differentiate apoptosis from necrosis, and is thus suitable to reliably detect early as well as late apoptosis. Therefore, the ssDNA-staining may be used as reference method to clearly identify apoptosis induced by oxidative stress in adherent cells. The TUNEL-reaction, annexin-V-binding and Apo-2.7-expression may be used to quantify the number of apoptotic and necrotic cells especially at later stages but without discrimination of apoptosis and primary or secondary necrosis.

Histone deacetylase inhibitor promotes differentiation of embryonic stem cells into neural cells in adherent monoculture

Background Embryonic stem （ES） cells poss unlimited self-renewal capacity and the ability to differentiate into cell of all three germ layers in vitro. Induced differentiation of ES cells to neural lineage cells has great potential in basic study of neurogenesis and regeneration therapy of neurodegenerative diseases. Histone deacetylase （HDAC） inhibitors enhance histone acetylation so that globularly activate gene expression and may initiate multilineage differentiation. In this study,we aimed to develop a method to induce the differentiation of ES cells to neural cells combining HDAC inhibition and neural cellselection.Methods In this study, we used HDAC inhibitor sodium butyrate （NAB） to induce the differentiation of mouse embryonic stem cells to neural cells through monolayer culture. After differentiation initiation by histone deacetylase inhibitor sodium butyrate, neural cells were induced and selected with a serum free culture system. Results Homogeneous neurons without glial cells demonstrated by molecular marker expression were differentiated with the method. The resultant neurons were excitable. Conclusion The method combined differentiation induction effect of HDAC inhibitors and selective culture system to derive neural cells from ES cells, and implied the involvement of epigenetic regulation in neural differentiation.

奶牛小肠上皮细胞系细胞染色体核型与G-带分析

为了鉴定奶牛小肠上皮细胞系(BIECs-21)的生物学遗传稳定性,体外贴壁培养BIECs-21细胞,制备染色体标本和G-带标本,分析BIECs-21细胞的染色体核型和G-带。结果表明:BIECs-21细胞染色体数为2n=60,包括常染色体29对和性染色体(X和Y染色体)1对。其中,29对常染色体均为端部着丝点染色体;性染色体中,X染色体是较大的中着丝粒染色体,Y染色体是较小的中着丝粒染色体。除X、Y性染色体外,BIECs-21细胞常染色体的G-带带纹的数量和位置无显著差异。BIECs-21细胞与荷斯坦牛的正常染色体核型相比,染色体数目及形态结构均没有明显变化,证明该细胞系遗传特性稳定,可以用于牛肠道相关疾病分子机制的研究。

原代大鼠主动脉血管干细胞的体外培养扩增及鉴定

目的采用“切段外膜贴壁培养法”体外培养扩增出原代大鼠主动脉血管干细胞,为开展血管重构及相关疾病研究提供实用的工具细胞。方法无菌分离2~3周龄Sprague-Dawley大鼠胸腹主动脉,切成长约2.0 mm的血管节段,均匀种瓶,经外膜贴壁固化后进行原代培养,待细胞生长融合度达80%~90%时传代。镜下观察细胞形态及其生长特性,采用流式细胞仪分析目的细胞表面标志物CD分子表达情况,成脂、成骨诱导分化实验检测其多向分化潜能。结果接种3 d后,少量梭形、星形或多角形的细胞从血管节段周围爬出;5~6 d后形成岛屿状细胞集落,细胞增殖迅速,呈放射状向外扩大,并出现细胞克隆现象;7~8 d后细胞融合成片,呈涡旋状分布;传至第3代后,细胞匀质性较高,表现为典型的“成纤维样”排列生长。流式细胞仪分析细胞主要表达CD44、CD73、CD90,阳性率分别为80.3%、62.2%、46.8%;低表达CD34、CD45、CD11b/c,阳性率分别为1.1%、0.2%、0.2%。体外诱导分化实验表明目的细胞具有成脂、成骨分化潜能。结论“切段外膜贴壁培养法”可成功分离培养出具有间充质干细胞特性的大鼠主动脉血管干细胞。

L929细胞无血清悬浮驯化及其在流产衣原体灭活疫苗中的应用研究

为通过悬浮细胞培养方法规模化生产流产衣原体抗原,采用逐步降血清悬浮驯化法使贴壁L929细胞逐步适应悬浮培养环境,最终获得了一株可无血清悬浮培养的L929细胞株。将流产衣原体接种于L929悬浮细胞上,研究不同的促感染试剂以提高收菌量,并选择出最佳的培养方法制备流产衣原体灭活疫苗。以25μL、50μL、100μL的剂量免疫Balb/c小鼠,攻毒后检测其免疫保护效果。结果显示,当该L929悬浮细胞初始接种密度为5×10^(5)/mL时,培养72 h后细胞浓度可达到约7×10^(6)/mL,细胞活力在95%以上;在细胞密度为6×10^(6)/mL,接菌量为5.5×10^(4) IFU/mL,加入脂质体2000的量为1.7μL/mL、放线菌酮浓度为0.4μg/mL时,菌量增殖倍数最大,48 h内约扩增了52倍。经流产衣原体悬浮培养细胞灭活疫苗免疫后,小鼠产生的抗体水平随免疫剂量的增加而提升;接种50μL、100μL疫苗组小鼠的脾淋巴细胞增殖水平、IFN-γ、IL-2的含量远高于接种25μL疫苗和注射PBS组的小鼠;对免疫后的小鼠进行攻毒,接种疫苗组小鼠的脾脏、十二指肠、子宫中的流产衣原体基因组含量远低于未接种疫苗组小鼠。注射PBS组及接种25μL疫苗组的小鼠子宫出现了不同程度的坏死及炎症,而其余两组小鼠的子宫无异常变化。结果表明,本试验成功驯化出1株L929悬浮培养细胞株,并且通过悬浮培养工艺制备的流产衣原体灭活疫苗免疫效果良好,接种50μL、100μL该灭活疫苗可以有效抑制流产衣原体的感染,该L929细胞全悬浮培养株的成功驯化为疫苗规模化生产提供了技术支持。

LMH贴壁细胞悬浮驯化及病毒增殖研究

研究旨在实现LMH贴壁细胞悬浮培养、稳定传代、高密度生长,并应用于血清4型禽腺病毒(FAdV-4)悬浮培养。研究进行LMH贴壁细胞低含量血清适应驯化,并进行无血清悬浮培养筛选及FAdV-4 RP-4毒株接毒盲传试验。结果显示:LMH悬浮细胞可在无血清培养基中传代第3代后,最终细胞密度达4.00×10^(6)cells/mL以上;FAdV-4连续培养至第4代,毒价可达10^(8.13)TCID_(50)/0.1 mL。研究表明,用特定培养基可使LMH贴壁细胞实现悬浮驯化,并可应用于FAdV-4稳定增殖。

益生乳酸菌对奶牛阴道上皮细胞粘附作用的研究

益生菌对奶牛阴道上皮细胞粘附能力的强弱是其发挥益生作用的前提条件,因此粘附能力是益生菌筛选的重要标准之一。为筛选出可以制备微生态制剂的益生菌菌株,本实验首先通过碳氢化合物粘着法测试了魏斯氏菌(1#菌)、海氏肠球菌(3#菌)、乳酸明串珠菌(5#菌)和营养泥杆菌(9#菌)的疏水性能,随后将细菌作用于奶牛阴道上皮细胞并且利用革兰氏染色法对细菌的粘附能力进行研究。结果表明,疏水率由强到弱依次为:9#菌、5#菌、3#菌和1#菌,其中9#菌对3种溶剂的疏水率均显著高于其它3株细菌(p<0.05);单一菌株中,9#菌株的粘附性显著高于1#、3#和5#菌株(p<0.05),混合菌株1+3#的粘附能力显著高于其它混合菌株和单一菌株(p<0.05)。研究结果提示,1+3#混合菌株更适合用于微生态制剂的研发。

植物乳杆菌NCU116的模拟人体肠道上皮细胞黏附性能

益生菌对人体肠道上皮细胞表面的黏附性能是益生菌筛选的重要指标之一。采用体外细胞模型(人体结肠癌细胞系HT-29)测定植物乳杆菌NCU116的黏附性能,并研究不同因素对NCU116黏附HT-29的影响。结果表明:植物乳杆菌NCU116的黏附能力较强,为(4.78±0.21)CFU/cell。黏附菌数随着菌液浓度增大而显著增大,当菌液浓度达到1.0×108CFU/mL时趋于饱和;稳定期的菌体黏附效果较好;黏附菌数随着作用时间的延长而增加,到2.0h时逐渐趋于饱和;外部环境的pH值对黏附性能影响很大,偏酸性环境更有利于黏附;Ca2+、Mg2+对黏附作用没有显著影响。植物乳杆菌NCU116对体外细胞模型HT-29的黏附性能较好,在食品行业和保健业中具有良好的发展前景。

细胞外基质促日本血吸虫培养细胞贴壁作用的研究

目的 研究肝、肺生物基质及鼠尾胶三种细胞外基质 (ECM )对日本血吸虫培养细胞的促贴壁作用。方法 灌注法获取 2 1d虫龄的日本血吸虫虫体 ,冷消化法制备细胞悬液 ,将密度为 2× 10 6/ml的细胞悬液分别接种于均匀铺敷三种基质及未铺敷基质 (对照 )的各组培养瓶中进行培养 ,定时计数贴壁细胞数 ,计算贴壁率。结果 随着培养时间从 8h— 4 8h ,各组日本血吸虫细胞的贴壁率逐渐增高 ,4 8h后下降。同一培养时间 ,基质不同 ,细胞的贴壁率不同。培养 4 8h时细胞的贴壁率 ,鼠尾胶组、肝与肺基质组分别为 6 3 2 7%、4 8 95 %、4 5 36 % ;统计学处理发现 ,它们之间差异显著 ;鼠尾胶组与肝、肺基质组比较 ,p <0 0 1;肝与肺基质组之间比较 ,p <0 0 5 ;对照组与各基质组比较 ,均具显著差异 (p <0 0 1)。 结论 ECM对日本血吸虫培养细胞具有明显的促贴壁作用。其中 ,鼠尾胶对日本血吸虫细胞的促贴壁作用最强 ,其次是肝生物基质 ;肺生物基质的促贴壁作用相对较弱 ,但也强于对照组 (p <0 0 1)。

贴壁培养细胞台盼蓝拒染试验的方法学探讨

本工作旨在探讨贴壁培养细胞台盼蓝拒染试验的方法,为其合理应用提出建议。用正常培养及NaN_3作致死处理的人视网膜色素上皮细胞株-19,进行原位贴壁细胞和培养体系上悬液中细胞的台盼蓝拒染实验,并比较其原位法与计数板法所测活细胞率。与计数板法相似,随染液浓度增高或染色时间延长,原位台盼蓝拒染实验的活细胞率检测值逐渐增加。培养体系上悬液中脱落细胞的存活率显著低于贴壁的细胞,NaN_3处理使脱落细胞增加。因此,对于贴壁牢固的培养细胞,原位台盼蓝染色是检测活细胞率的实用方法。根据细胞从培养表面上消化下来的难易程度和脱落到培养上悬液中的数目多少,可选用原位染色法或/和计数板法台盼蓝拒染试验,从而检测贴壁培养细胞的总存活率。