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.

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.

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 pylori 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 H 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 H 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 H pylori. CagA and VacA are not related to the ability of invasion and adhesion of H pylori in different cell lines in vitro.

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.

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。

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。

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。

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

细胞毒素的淋巴细胞是在有缺点的房间的有免疫力的反应和消除的组织的关键播放器。我们以前报导了生来的杀手(NK ) 房间进入目标肿瘤房间，导致在肿瘤房间以内的任何一个目标房间死亡或自我毁灭。然而，它留下了至于在成为主观以后的 NK 房间的命运逃犯并且 heterotypic cell-in-cell 过程最近是否与同型的 cell-in-cell 事件的不同，说出 entosis。这里，我们证明 NK 房间在肿瘤房间以内与 apoptosis 的最终的命运经历一个 cell-in-cell 过程并且表明成为主观过程要求肌动朊细胞骨架管理者， ezrin。设想 NK 房间怎么进入肿瘤房间，我们执行了 NK 房间成为主观的即时双颜色成像分析进肿瘤房间。令人惊讶地，大多数 NK 房间在他们的入口以后承诺规划房间死亡进肿瘤房间，它与在同型的 cell-in-cell 过程观察的 entosis 区别地不同。使内在化的 NK 房间的 apoptotic 房间死亡由 caspase 的激活是明显的 3 并且 DNA 破碎。而且，在成为主观以后的 NK 房间死亡被 caspase 禁止者稀释， Z-VAD-FMK，在肿瘤房间以内作为 NK 房间死亡的模式证实 apoptosis。决定为 NK 房间的入口必要的蛋白质因素进肿瘤房间，我们执行了基于 siRNA 的击倒的分析并且在 NK 房间成为主观发现了 ezrin 的一个关键角色。重要地， ezrin 的调停 PKA 的 phosphorylation 支持 NK 房间成为主观过程。我们的调查结果建议 ezrin 由管理 NK 房间成为主观进肿瘤房间的新奇规章的机制。

CD133^+ gallbladder carcinoma cells exhibit self-renewal ability and tumorigenicity

AIM:To identify cancer stem cells(CSCs) in human gallbladder carcinomas(GBCs).METHODS:Primary GBC cells were cultured under serum-free conditions to produce floating spheres.The stem-cell properties of the sphere-forming cells,including self-renewal,differentiation potential,chemoresistance and tumorigenicity,were determined in vitro or in vivo.Cell surface expression of CD133 was investigated in primary tumors and in spheroid cells using flow cytometry.The sphere-colony-formation ability and tumorigenicity of CD133+ cells were assayed.RESULTS:In vitro culture experiments revealed thatfloating spheroids were generated from primary GBC cells,and these sphere-forming cells could generate new progeny spheroids in serum-free media.Spheroid cells were differentiated under serum-containing conditions with downregulation of the stem cell markers Oct-4,Nanog,and nestin(P < 0.05).The differentiated cells showed lower spheroid-colony-formation ability than the original spheroid cells(P < 0.05).Spheroid cells were more resistant to chemotherapeutic reagents than the congenetic adherent cells(P < 0.05).Flow cytometry showed enriched CD133+ population in sphereforming cells(P < 0.05).CD133+ cells possessed high colony-formation ability than the CD133-population(P < 0.01).CD133+ cells injected into nude mice revealed higher tumorigenicity than their antigen-negative counterparts(P < 0.05).CONCLUSION:CD133 may be a cell surface marker for CSCs in GBC.

猪视网膜上皮细胞的分离培养与鉴定

旨在建立猪视网膜上皮细胞(PRECs)的分离纯化及传代培养方法。采用组织块贴壁法和消化法分离猪视网膜细胞(PRCs),经差速消化法纯化细胞,筛选确定最优培养条件并鉴定细胞生物学特性。结果:消化法分离的细胞约1周长成单层,组织块贴壁法约2周长满单层,但活力更高;PRCs主要形态为长梭形和大理石样,呈现为2种形态细胞混合生长;差速消化法纯化细胞后,经间接免疫荧光鉴定细胞角蛋白(CK18和CK19),确定该细胞为上皮细胞;细胞接种猪圆环病毒2型(PCV2)可产生明显病变。综上,本试验成功分离获得PRECs,含胎牛血清浓度为12%的α-MEM为最优培养基,该细胞支持PCV2增殖并产生明显的细胞病变,为进一步建立稳定传代的猪视网膜上皮细胞系及研究PCV2感染及致病机理提供基础。

大鼠螺旋神经节干细胞体外优化培养方法的研究

目的探索贴壁法进行螺旋神经节(Spiral Ganglion,SG)干细胞培养,同时与传统悬浮培养法对比培养的SG干细胞形态、增殖效率及分化表型。方法取新生1天SD大鼠耳蜗的螺旋神经节位置,以悬浮和单层贴壁的方式进行培养,观察SG干细胞的形态学特点。通过活/死细胞染色和Annexin-V/PI双染法比较其生长状态。通过Nestin、Sox2、Ki67等免疫荧光染色及生长曲线测定等方法对其增殖能力进行比较分析。体外胎牛血清诱导SG干细胞分化,通过Tuj1免疫荧光染色鉴定分化表型并比较分化效率。结果两种培养方式均可获得状态良好的高纯度SG干细胞,免疫荧光染色和生长曲线测定结果提示贴壁培养的干细胞的增殖活性较悬浮培养的干细胞更强。诱导分化后,两种培养方式获得的SG干细胞均可分化为Tuj1染色阳性的双极神经元样细胞。结论本研究结果表明单层贴壁可培养出状态良好的SG干细胞,并且其增殖能力较悬浮培养的干细胞更强。贴壁培养的干细胞可被可成功诱导分化为神经元。提示单层贴壁方式可作为一种稳定的SG干细胞培养方法,从而为SG干细胞的相关研究提供重要技术支持。

小鼠骨髓间充质干细胞分离方法的比较与探讨

目的探讨可高效分离小鼠骨髓间充质干细胞(mBMSCs)的技术方法。方法分别采用骨片消化爬片法、全骨髓贴壁法、骨片消化液上清法和骨片消化研钵法等四种方法分离7~9周龄雄性C57BL/6小鼠的mBMSCs;于倒置显微镜下观察原代mBMSCs形态;运用流式细胞术检测mBMSCs特征性免疫表型;采用多向分化诱导检测mBMSCs成骨分化能力与成脂分化能力。结果分离获得的原代细胞首次换液后于倒置显微镜下观察:骨片消化爬片法分离的细胞,仅见大量骨碎片和杂细胞,该法所分离细胞不再进行后续检测评估;全骨髓贴壁法分离的细胞,仅可见少量多角形贴壁细胞,夹杂大量杂细胞;骨片消化液上清法分离的细胞也可见较多长梭形、三角形贴壁细胞,但杂细胞较多;骨片消化研钵法分离的细胞,可见较多长梭形、多角形贴壁细胞,杂细胞少。分离细胞经培养传代到第3代(P3代)时,一方面采用流式细胞术分析mBMSCs特征性免疫表型,结果表明骨片消化液上清法和骨片消化研钵法分离的mBMSCs纯度均较高,全骨髓贴壁法不理想;另一方面,进行成骨分化诱导与成脂分化诱导,结果表明与全骨髓贴壁法分离的细胞相比,骨片消化液上清法和骨片消化研钵法所分离出的细胞具有较强的多向分化潜能。结论骨片消化液上清法和骨片消化研钵法分离获得的mBMSCs纯度较高、质量较好。

改良全骨髓贴壁法分离及培养大鼠骨髓间充质干细胞

目的建立简单、优化的分离及培养大鼠骨髓间充质干细胞(BMSCs)的方法,为组织工程支架研究提供细胞基础。方法分别采用常规和改良的全骨髓贴壁法分离和培养BMSCs。常规法按照传统全骨髓贴壁法,对骨髓冲洗液进行过筛和离心处理后加入完全培养基进行培养。在改良全骨髓贴壁法中,将骨髓冲出骨髓腔后直接加入完全培养基进行培养。倒置相差显微镜下观察两组细胞形态,采用细胞计数试剂盒(CCK-8)法比较两组BMSCs的增殖活性,流式细胞术检测表面标志物CD90、CD29、CD11b/c、CD45。对改良法培养的BMSCs进行定向诱导成骨及成脂分化,并通过茜素红染色检测其成骨分化潜能,通过油红O染色检测其成脂分化潜能。结果改良全骨髓贴壁法培养的BMSCs形态均一,细胞集簇排列呈“鱼群状”“漩涡状”。改良法比常规法培养的BMSCs增殖活性强(P<0.05)。改良法与常规法培养的BMSCs均高表达CD90和CD29,低表达CD11b/c和CD45。改良法培养的BMSCs成骨、成脂诱导分化后茜素红染色和油红O染色均为阳性。结论改良全骨髓贴壁法可成功分离并培养BMSCs,培养的BMSCs具有多向分化潜能,为组织工程支架的研究奠定了细胞基础。

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。