程控降温后液氮保存对自体外周血造血干细胞含量和活性的影响

目的：观察程控降温后液氮保存对自体外周血干细胞含量和活性的影响。方法：13例接受APBSCT患者分离的造血干细胞利用程控降温仪进行冷冻,然后置液氮液相内（－196℃）保存,检测患者冻存前后的有核细胞数,CFU－GM,CD34＋细胞。结果：冻存前后CFU－GM,有核细胞数,CD34＋细胞统计学之间无显著性差异。结论：自体外周血干细胞体外冷冻处理和保存是有效和适当的。     

夹层胶原"三明治"法冻存复苏肝细胞的实验研究

目的 研究胶原、表皮生长因子对新鲜分离及冻仔复苏后Sprague-Dawley（SD）大鼠肝细胞活率、形态和功能的影响,期望建立一种较为完善的肝细胞体外培养及冻存复苏体系,为肝细胞移植、生物人工肝及相关肝细胞研究获取充足有效的肝细胞来源提供必须的技术支持和理论依据。方法 采用胶原酶二步原位循环肝脏灌注法分离SD大鼠肝细胞,测定不同条件培养及冻存复苏后各组肝细胞的活性及功能。结果 双层胶原及双层胶味＋表皮生长因子组细胞活率、蛋白质含量及细胞功能均优于普通组（P〈0．05）。结论 双层胶原体外培养体系更接近于肝细胞体内生长环境,能有效的减少各种理化因素对肝细胞的损伤,有利于肝细胞在体外长期保存,是一种较为理想的培养及冻存复苏体系。     

大规模猪肝细胞低温保存方法的建立

目的　建立大规模猪肝细胞的低温保存方法以满足生物型人工肝治疗的需要。方法　用酶法从中国实验用小型猪肝脏分离出猪肝细胞 ;加入本实验室配制的含有 10 %二甲基亚砜 (dimethylsulfoxide,DMSO)的营养液中 ;分别采用两种降温方法使 (12 )× 10 10 猪肝细胞保存在 - 196℃液氮中 ;1个月后复温 ,观察在同一条件下培养不同时间后猪肝细胞形态、存活率和白蛋白、尿素、葡萄糖合成功能及对利多卡因的转化功能。结果　用两种降温方法保存的肝细胞 ,复温后细胞的存活率均较高 (梯度降温组和程控降温组存活率分别较冻存前降低了 4 7%和 8 6 %) ,其中前者肝细胞的尿素、葡萄糖合成功能较后者高。结论　所建立的冻存方法简单易行 ,可保存大量的猪肝细胞。     

中国仓鼠卵巢细胞集落的简易原位贴壁冻存方法

目的：目前通用的细胞冻存方法是将细胞在保护剂的作用下是悬浮冻者，这样细胞自然相互混合，复苏后不能保持原来的位置关系，本工作探索在需要时将中国仓鼠卵巢细胞（ＣＨＯ）在贴壁状态下原位冻存的可能性，方法：将转促红细胞生成素（ＥＰＯ）表达质粒形成的ＣＨＯ集落细胞在贴壁状态下加上１０％的二甲亚砜保护作用，然后分阶段降温并保存在－７０℃冰箱，两个月后复苏检测细胞的存活情况及其目的产物的表达情况，结果：集落至少     

低温冻存基质细胞特性的研究

目的：探讨骨髓细胞低温冻存后贴壁基质细胞培养生长的基本特性，为低温冻存基质细胞的应用提供实验依据。方法：取新鲜骨髓和经Dexter长期培养法培养14d的骨髓贴壁基质细胞（称“新鲜基质细胞”），用5％二甲基亚砜－6％羟乙基淀粉为保护剂，－80℃冰箱降温，－196℃液氮冻存（前者称“冰存骨髓”，后者称“冻存基质细胞”）2周，复温后，再用Dexter法培养这些细胞，检测细胞增殖、细胞形态、细胞化学染色、细胞表面及基质细胞支持另一骨髓造血细胞形成的鹅卵石造血区、长期培养起始细胞等为指标，比较它们的生长特性、成分及功能。结果：生长特性：冻存骨髓比新鲜骨髓出现贴壁细胞、细胞丛和细胞融合成片迟1－2d，冻存基质细胞融合成片比新鲜基质细胞迟12－18h；冻存骨髓与新鲜骨髓，冻存基质细胞与新鲜基质细胞增殖数比明显低。细胞成分；冻存骨髓比新鲜骨髓形成的成纤维细胞、内皮细胞比率下降，而巨噬细胞和脂肪细胞比率升高，冻存基质细胞上述现象更明显；内含凋亡小体的细胞冻存基质细胞多于新鲜基质细胞，冻存骨髓细胞多于新鲜骨髓；冻存骨髓和冻存基质细胞台盼蓝拒染率分别为95．2％、89．5％；冻存骨髓、冻存基质细胞CD14、人白细胞DR抗原表达百分率比新鲜骨髓、新鲜基质细胞高，CD45、CD33反之。细胞功能；新鲜骨髓、新鲜基质细胞、冻存骨髓和冻存基质细胞形成的鹅卵石造血区和长期培养起始细胞，生长良好，无显著差异。结论：不管是骨髓细胞还是经培养生成的贴壁基质细胞，经5％二甲基亚砜－6％羟乙基淀粉冷冻冻存、复温后培养，生物特性有一定损伤，但仍有较好的支持造血功能。     

冻存乳猪肝细胞的胶原凝胶培养研究

目的 探索乳猪肝细胞的冻存以及培养条件和方法。方法 采用两步法分离乳猪肝细胞．液氮冻存4个月后复苏．与鼠尾胶原液混合后接种培养,或将肝细胞接种于胶原被覆的培养瓶中．然后被覆上第2层胶原培养．观察培养细胞的形态、白蛋白mRNA的表达、尿素合成及天冬氨酸氮基转移酶(AST)的漏出量等。结果 冻存肝细胞的活性维持较好．并成功地被固定于混合凝胶或三明治形凝胶中,较好地保持了尿素合成以及白蛋白mRNA表达能力。结论 此冻存条件适合于猪肌细胞的长期保存,胶原凝胶可为肝细胞提供更接近体内的培养环境,较好地维持了肝细胞的功能和形态学特征。     

人细胞融合肝癌疫苗的制备及其融合细胞百分率的检测

手术病人新鲜肝癌组织及部分脾脏，分离纯化、激活B细胞，应用PEG融合肝癌细胞与激活B细胞，经培养灭活制得肿瘤疫苗。同时测定了融合肝癌疫苗的融合率、冻存后复苏活细胞百分率。结果显示，3例融合细胞的融合率分别为66.84%、74.43%、76.55%，融合细胞经DMSO和甘油冻存后复苏活细胞百分率分别为95%、97%。研究表明，人细胞融合肝癌疫苗具有很高的融合率和冻存后复苏活细胞百分率，便于制备及储存，适合临床应用。     

低温冻存脐血树突状细胞的生物特性研究

目的 考察经低温保存的脐血树突状细胞所表现的生物特性，为树突状细胞的应用提供保存方法。方法 取新鲜脐血细胞和脐血细胞培养产生的树突状细胞，加5％二甲基亚砜－6％羟乙基淀粉的保存剂，－80℃冰箱降温，－196℃保存，40℃复温（前者称“冻存脐血”，后者称“树突状细胞”），再用树状突细胞培养增殖法培养，检测；细胞增殖量，台盼蓝拒染回收率，不染色和染色观察，表面抗原，树状突细胞的混合淋巴细胞刺激指数、抗毒－杀灭效应处理率，比较其变化。结果 形成树突状细胞的时间冻存脐血培养比新鲜脐血迟，细胞增殖量低，冻存脐血台盼蓝拒染为95.8%。冻存树突状细胞形成贴壁树突样细胞比新鲜树突状细胞迟，细胞数增殖量低，冻存树突状细胞的台盼蓝拒染为88.7%。冻存脐血与新鲜脐血比，冻存树突状细胞与新鲜树突状细胞比，其树突样细胞量、CD1a、CD83、HLA-DR表达低；冻存脐血与新鲜脐血比，冻存树突状细胞与新鲜树突状细胞比，刺激指数、效应处理率均低。结论 用5％二甲基亚砜－6％羟乙基淀粉低温保存脐血细胞或经培养增殖的脐血树突状细胞，复温后培养，树突状细胞的生物特性受到了损伤，但仍完整，回收率＞85％。     

大鼠胚胎大脑组织的冷冻保存研究

用组织培养测定活性的方法研究了胎龄18d大鼠胚胎大脑组织的适宜冻存条件,较系统地观察了有关低温保护剂、冷冻和复温速率、保存时间等条件对胎鼠脑组织活性的影响。结果表明,以下条件能使冻存组织获得良好的培养结果:以1mol/L的二甲亚砜作为低温保护剂,以1℃/min的速率冷冻至-70℃,液氮保存,37℃水浴快速复温。在液氮中保存60d活性无明显变化。2mol/L甘油对胎脑组织也有一定的保护作用,但不如1mol/L二甲亚砜,而聚乙烯吡咯烷酮无明显保护作用。冷冻速率等于或超过5℃/min,则组织活性急剧下降,说明冷冻速率对胚胎大脑组织活性影响较大。     

大鼠肝细胞球形体低温保存条件的初步探索

目的探索有效的肝细胞球形体低温保存方法,促进生物人工肝及其他研究的应用。方法采用两步法分离大鼠肝细胞,经无血清培养基(SFM)摇摆培养48h形成肝细胞球形体。将球形体继续培养(对照组),或置于4℃SFM,SFM+1mmol/L去铁胺(Def),SFM+1μmol/L环孢霉素A(Cs A),SFM+1mmol/L Def+1μmol/L Cs A保存液中24h或48h,继续培养4d或5d后观察低温保存后肝细胞球形体的存活率、超微结构变化,以及白蛋白和尿素合成情况。结果Def和Cs A能很好地保护球形体肝细胞,在4℃SFM+Def+Cs A保存液中保存24h,肝细胞球形体功能、结构等变化及尿素合成水平与对照组比较差异无统计学意义,而单独放在SFM中进行低温保存后,细胞活性明显下降。在4℃保存48h后,球形体肝细胞超微结构发生明显变化,死亡细胞增多,但SFM+Def+Cs A组和SFM+Def组细胞结构优于SFM组和SFM+Cs A组,细胞存活率及尿素合成水平明显高于后两组(P<0.05),但合成白蛋白量均处于较低水平,各组间差异无统计学意义(P>0.05)。结论低温状态下肝细胞球形体可保持良好的存活率和功能24h,低温保存能为细胞治疗提供良好的细胞材料。     

体外培养人胎肝细胞与永生化L-02肝细胞的生物学性状比较

目的 对比体外培养的人胎肝细胞与L-02肝细胞形态学及增殖分化特征的异同，初步评价其用于移植的可行性，探讨介入性肝细胞移植术的理想供体来源。方法 分离培养14～24周的引产胎儿肝细胞，放射免疫法测定上清液中甲胎蛋白(AFP)与白蛋白(ALB)含量，免疫细胞化学法检测细胞角蛋白19(CK—19)的表达。同法检测传代培养L-02肝细胞的蛋白表达。结果人胎肝细胞分离活率达95％，在体外存活最长达3周，可同时检测到AFP、ALB及CK—19表达，其中ALB分泌峰值42．06μg／ml；L-02肝细胞增殖迅速，AFP与CK-19表达阴性，ALB表达在10μg／ml水平，部分细胞多次传代后发生形态变异，ALB表达缺失。结论人胎肝细胞具有潜在的双向分化能力，是肝细胞移植较为合适的供体来源；L-02肝细胞不适用于移植。     

Cell refractive index: Models,insights, applications and future perspectives

Cell refractive index (RI) is an intrinsic optical parameter that governs the propagation of light (i.e., scattering and absorption) in the cell matrix. The RI of cell is sensitively correlated with its mass distribution and thereby has the capability to provide important insights for diverse biological models. Herein, we review the cell refractive index and the fundamental models for measurement of cell RI, summarize the published RI data of cell and cell organelles and discuss the associated insights. Illustrative applications of cell RI in cell biology are also outlined. Finally, future research trends and applications of cell RI, including novel imaging techniques, reshaping flow cytometry and microfluidic platforms for single cell manipulation are discussed. The rapid technological advances in optical imaging integrated with microfluidic regime seems to enable deeper understanding of subcellular dynamics with high spatio-temporal resolution in real time.     

黄连素对乳腺癌细胞生长、迁移和放射敏感性的影响

目的 研究黄连素对乳腺癌细胞生长、迁移和放射敏感性的影响.方法 选取人乳腺癌细胞MDA-MB-231和MCF-7,用MTT法检测细胞的生长和增殖;划痕愈合法观察细胞迁移能力;细胞流式技术测定细胞周期改变;磷脂酰丝氨酸外翻分析法检测细胞凋亡;Western blot法测定蛋白表达水平;细胞克隆形成法观察黄连素与辐射的联合作用.结果 黄连素明显抑制人乳腺癌MCF-7和MDA-MB-231细胞的生长,并显现剂量-效应和时间-效应关系,MCF-7和MDA-MB-231细胞对黄连素表现出同样的敏感性.黄连素可以诱导剂量依赖性的G0/G1期细胞阻滞,40 μmol/L的黄连素处理后MDA-MB-231和MCF-7细胞早期凋亡分别高达86.63％和66.62％,与未处理的对照组相比,差异均有统计学意义(t=75.15和43.75,P＜0.01).黄连素明显降低了细胞周期调节相关蛋白Cyclin B1和抑凋亡蛋白Bcl-2的表达水平,而增加了促凋亡蛋白Bax和Caspase-3的表达水平,但不影响细胞周期调节蛋白Cyclin D1的表达水平.低剂量(≤5μmol/L)黄连素可以明显降低细胞的迁移能力.采用单靶多击模型拟合曲线发现,与单独照射组相比,5 μmol/L黄连素预处理4h后MDA-MB-231细胞和MCF-7细胞的辐射增敏比分别为1.12和1.22.结论 黄连素通过调节细胞凋亡和细胞周期阻滞抑制乳腺癌细胞生长和迁移,并可提高乳腺癌细胞的放射敏感性.     

骨形态发生蛋白和转化生长因子—β及碱性成纤维细胞生长因？…

观察骨形态发生蛋白（ＢＭＰ）、转化生长因子－β、碱性成纤维细胞生长因子在骨折修复中的表达及分布情况,进而探讨其作用机制。方法以成年雄性新西兰兔为研究对象,制作桡骨骨折愈合模型。伤后不同时期处死取材,分别进行这和ＢＭＰ、ＴＧＦ－β和ｂＦＧＦ免疫组化染色观察。结果（１）伤后３ｄ开始形成原始骨痂。１周时肉芽组织中的间质细胞开始分化为软骨细胞,软骨形成后再进行软骨内化骨。４周时形成连接骨折端的桥接骨痂。２     

[131I]FIAU labeling of genetically transduced, tumor-reactive lymphocytes: cell-level dosimetry and dose-dependent toxicity

Purpose Donor T cells have been shown to be reactive against and effective in adoptive immunotherapy of Epstein-Barr virus (EBV) lymphomas which develop in some leukemia patients post marrow transplantation. These T cells may be genetically modified by incorporation of a replication-incompetent viral vector (NIT) encoding both an inactive mutant nerve growth factor receptor (LNGFR), as an immunoselectable surface marker, and a herpes simplex virus thymidine kinase (HSV-TK), rendering the cells sensitive to ganciclovir. The current studies are based on the selective HSV-TK-catalyzed trapping (phosphorylation) of the thymidine analog [¹³¹I]-2′-fluoro-2′-deoxy-1-β-D-arabinofuransyl-5-iodo-uracil (FIAU) as a means of stably labeling such T cells for in vivo trafficking (including tumor targeting) studies. Because of the radiosensitivity of lymphocytes and the potentially high absorbed dose to the nucleus from intracellular ¹³¹I (even at tracer levels), the nucleus absorbed dose (D _n ) and dose-dependent immune functionality were evaluated for NIT⁺ T cells labeled ex vivo in [¹³¹I]FIAU-containing medium. Methods Based on in vitro kinetic studies of [¹³¹I]FIAU uptake by NIT⁺ T cells, D _n was calculated using an adaptation of the MIRD formalism and the recently published MIRD cellular S factors. Immune cytotoxicity of [¹³¹I]FIAU-labeled cells was assayed against ⁵¹Cr-labeled target cells [B-lymphoblastoid cells (BLCLs)] in a standard 4-h release assay. Results and conclusion At median nuclear absorbed doses up to 830 cGy, a ⁵¹Cr-release assay against BLCLs showed no loss of immune cytotoxicity, thus demonstrating the functional integrity of genetically transduced, tumor-reactive T cells labeled at this dose level for in vivo cell trafficking and tumor targeting studies.     

药物性重型肝炎1例肝细胞体内移植治疗的效果

目的探讨人肝细胞体内移植治疗药物性重型肝炎患者的有效性与安全性。方法分离健康志愿者捐献的肝脏,获得人原代肝细胞并冷冻保存,复苏后经股动脉插管移植到脾脏,观察治疗前后患者临床症状、血液生化指标及脾脏核磁共振信号的改变。结果人肝脏可获取2×1010肝细胞,复苏后肝细胞活率在70%以上,移植的肝细胞数为2×109个。移植后1个月,患者临床症状明显改善,血BIL、NH3、ALT、AST明显降低,PA水平明显升高。出院50天后随访各项血生化指标均恢复正常,脾脏内可见肝细胞信号。结论肝细胞体内移植是一项安全有效的治疗方法,移植的肝细胞能够在脾脏内增殖、分化,替代或部分恢复肝脏合成、解毒和代谢功能;将为终末期肝病治疗开辟一项新的治疗途径。     

Asialoglycoprotein-receptor-targeted hepatocyte imaging using 99mTc galactosylated chitosan

This study investigated the usefulness of 99mTc hydrazinonicotinamide-galactosylated chitosan (HGC) in hepatocyte imaging. HGC was obtained by coupling the galactose moiety of both lactobionic acid and succinimidyl 6-hydrazinonicotinate hydrochloride (succinimidyl HYNIC). The coupled product was then radiolabeled with 99mTc using stannous chloride and tricine as reducing agent and coligand, respectively. Labeling efficiency was >90% both in room temperature and in serum up to 24 h after injection. The hepatic uptake properties of 99mTc HGC were studied in Balb/C mice. 99mTc HGC and 99mTc hydrazinonicotinamide chitosan (HC) were intravenously injected into mice, with receptor binding identified by coinjection with 9 and 14 mg of free galactose. Images were acquired with a gamma-camera. After injection via the tail vein of the mice, 99mTc HGC showed high selectivity for the liver, while 99mTc HC without a galactose group showed low liver uptake. In addition, the hepatic uptake of 99mTc HGC was blocked by coinjection of free galactose. Tissue distribution was determined at three different times (10, 60 and 120 min). The liver accumulated 13.16+/-2.72%, 16.11+/-5.70% and 16.55+/-2.28% of the injected dose per gram at 10, 60 and 120 min after injection, respectively. 99mTc HGC showed specific and rapid targeting of hepatocytes. It is a promising receptor-specific radiopharmaceutical with potential applications in liver imaging for the evaluation of hepatocytic function.     

Radiation risk of tissue late effects,a net consequence of probabilities of various cellular responses

Late effects from the exposure to low doses of ionizing radiation are hardly or not at all observed in man probably due to the low values of risk coefficients that preclude statistical analyses of data from populations that are exposed to doses less than 0.2 Gy. In order to arrive at an assessment of potential risk from radiation exposure in the low dose range, the microdosimetry approach is essential. In the low dose range, ionizing radiation generates particle tracks, mainly electrons, which are distributed rather heterogenously within the exposed tissue. Taking the individual cell as the elemental unit of life, observations and calculations of cellular responses to being hit by energy deposition events from low LET type are analysed. It emerges that besides the probability of a hit cell to sustain a detrimental effect with the consequence of malignant transformation there are probabilities of various adaptive responses that equip the hit cell with a benefit. On the one hand, an improvement of cellular radical detoxification was observed in mouse bone marrow cells; another adaptive response pertaining to improved DNA repair, was reported for human lymphocytes. The improved radical detoxification in mouse bone marrow cells lasts for a period of 5–10 hours and improved DNA repair in human lymphocytes was seen for some 60 hours following acute irradiation. It is speculated that improved radical detoxification and improved DNA repair may reduce the probability of spontaneous carcinogenesis.Thus it is proposed to weigh the probability of detriment for a hit cell within a multicellular system against the probability of benefit through adaptive responses in other hit cells in the same system per radiation exposure. In doing this, the net effect of low doses of low LET radiation in tissue with individual cells being hit by energy deposition events could be zero or even beneficial. Since there was no simple additivity of equal effects from repeated exposures to equal doses and because of the potential effect of adaptive cell responses on the spontaneous evolution of malignancy in tissue, the extrapolation of risk with absorbed dose reaching down to zero, does not appear to be generally valid.