人血小板的冷冻干燥保存:细胞内外保护剂的作用

通过液相内吞途径将13mmol／L海藻糖载入人血小板内,采用牛血清白蛋白和海藻糖作细胞外保护剂,研究细胞内海藻糖和细胞外保护剂在血小板冻干保存中分别所起的作用。实验结果表明冻干血小板的活性主要依靠细胞外保护剂提供的保护作用而得以保存,13mmol／L的细胞内海藻糖只起到有益的补充作用。在13mmol／L细胞内海藻糖和1％牛血清白蛋白＋1％海藻糖细胞外保护剂同时存在的情况下,冻干保存40天的血小板复水后的数值恢复率约85％,显微镜观察细胞形态完整,对1U／ml凝血酶的聚集百分比为97．3％。     

胞内海藻糖对红细胞冷冻干燥保存效果的影响

冷冻干燥是长期保存人体红细胞最理想的方法之一。利用孵育的方法将非渗透性保护剂海藻糖载人人体红细胞中，以含海藻糖的保护剂对红细胞进行冻干保存实验，研究胞内海藻糖浓度对红细胞冻干保存的影响。在37℃下，以不同浓度海藻糖溶液（400mM，600mM，800mM，1000mM）对红细胞孵育7h。结果表明，随胞外海藻糖浓度的增加，胞内海藻糖的浓度相应升高，试验中海藻糖浓度最高达到35mmol／L。对新鲜与孵育后红细胞在相同条件下进行冻干实验，结果表明，孵育后的红细胞冻干回收率随胞内海藻糖浓度增加而增加，表明胞内海藻糖在冻干过程中对红细胞有保护作用。     

LEA蛋白特征片段及海藻糖对热敏性蛋白药物胰岛素冻干的协同保护研究

热敏性蛋白药物胰岛素的热稳定性较差,极易受制备方式和保存条件等影响而失活。故本研究分别利用高效液相色谱（HPLC）测定冻干样品中胰岛素的效价和差示扫描量热仪（DSC）测定冻干样品的玻璃化转变温度等重要参数,来研究胚胎发育晚期富集蛋白特征片段（LEA-motif, LEAM）和海藻糖冻干保护剂对胰岛素冻干保护效果的影响。冻干实验表明,两种保护剂均对胰岛素的活性具有保护效果,且LEAM的活性保护效果优于海藻糖。在效价和热稳定性方面,LEAM保护的胰岛素冻干品均高于海藻糖保护的胰岛素冻干样品。更为重要的是,两种保护剂协同保护时,少量海藻糖对于LEAM的保护特性具有增效作用。在冻干过程中,可有效提升药物玻璃态的稳定性,防止热致变性失活。由此可见,这种LEAM和海藻糖保护剂复合保护方法有望也可以应用在热敏性蛋白药物冻干粉针制备过程以保护药物活性。     

LEA蛋白特征片段及海藻糖对热敏性蛋白药物胰岛素冻干的协同保护

热敏性蛋白药物胰岛素的热稳定性较差,极易受制备方式和保存条件等影响而失活。故本研究分别利用高效液相色谱(HPLC)测定冻干样品中胰岛素的效价和差示扫描量热仪(DSC)测定冻干样品的玻璃化转变温度等重要参数,来研究胚胎发育晚期富集蛋白特征片段(LEA-motif,LEAM)和海藻糖冻干保护剂对胰岛素冻干保护效果的影响。冻干实验表明,两种保护剂均对胰岛素的活性具有保护效果,且LEAM的活性保护效果优于海藻糖。在效价和热稳定性方面,LEAM保护的胰岛素冻干品均高于海藻糖保护的胰岛素冻干样品。更为重要的是,两种保护剂协同保护时,少量海藻糖对于LEAM的保护特性具有增效作用。在冻干过程中,可有效提升药物玻璃态的稳定性,防止热致变性失活。由此可见,这种LEAM和海藻糖保护剂复合保护方法有望也可以应用在热敏性蛋白药物冻干粉针制备过程以保护药物活性。     

人肝癌细胞Hep-G2的冷冻干燥保存初探

冷冻干燥是长期有效保存细胞的手段之一。本文以肝癌细胞Hep-G2为研究对象,选取了不同体积分数的Me2SO、丙三醇及不同质量浓度的PVP、复方保护剂进行冷冻干燥保存,筛选出最佳保护剂配方。此外,将细胞在不同摩尔浓度的海藻糖溶液中孵育,通过检测细胞回收率、存活率和24 h贴壁率,探究胞内海藻糖对细胞冷冻干燥的影响。结果表明:添加40%PVP(w/v)+10%甘油(v/v)+15%FBS(v/v)+20%海藻糖(w/v)保护剂的细胞,在复水后回收率、存活率和24 h贴壁率分别为29.58%、42.18%和18.71%,与对照组相比三项指标均得到有效提高,该种保护剂的效果最佳;当胞外海藻糖摩尔浓度为800 mmol/L时,冻干效果最好,肝癌细胞Hep-G2在复水后回收率、存活率和24 h贴壁率分别为27.81%,66.65%和33.68%,存活率和贴壁率显著高于其他组。     

生物材料冻干保护剂的热分析

生物材料冻干保护剂的玻璃化转变温度的测量对生物材料冻干保护剂的选择、冻干工艺的设计和贮存条件的选择有重要的指导意义.以海藻糖、蔗糖、乳糖、麦芽糖、葡萄糖等五种糖以及牛血清白蛋白作为生物材料的冻干保护剂,利用差式扫描量热法测量了保护剂溶液和保护剂冻干样品的玻璃化转变温度,研究了牛血清白蛋白的加入、糖的浓度、糖的种类等三种因素对保护剂溶液和保护剂冻干样品玻璃化转变温度的影响.     

微滴喷射玻璃化的过程损伤分析及保护剂优化

微滴喷射玻璃化保存系统产生的微滴尺寸较小,在较低浓度的低温保护剂条件下即可实现玻璃化。本文采用微滴喷射玻璃化保存系统对HepG2细胞进行玻璃化保存,研究保护剂加载过程、喷射过程、接收过程及玻璃化/复温过程对细胞造成的损伤程度,并通过降低保护剂中Me2SO浓度、添加适量海藻糖来优化保护剂配方。结果表明,微滴喷射玻璃化保存各过程对细胞均有损伤,保护剂加载过程、喷射过程、玻璃化及复温过程对细胞造成的损伤较大,薄片接收过程对细胞造成的损伤小。另外,随着保护剂中Me2SO浓度的降低,低温保存后的细胞活性明显降低;保护剂浓度相同时,玻璃化保存效果较慢速冷冻效果好;适量的海藻糖能够起到增强低温保存效果的作用,过量则起到降低作用;以5%Me2SO+0.3 mol/L海藻糖作为低温保护剂玻璃化保存细胞时,细胞存活率达(92.42±0.95)%,24 h贴壁率达到(95.64±1.03)%,微滴喷射玻璃化效果最好。     

冷冻干燥保存人脐血实验研究

尝试用冷冻干燥法来长期保存脐血和有核细胞,并对冻干效果进行比较分析.选用聚乙烯吡咯烷酮(PVP)、蔗糖和甘露醇等作为冻干保护剂,先将样品冻结到-38℃,随后在-30℃搁板温度下进行升华干燥,最后在15℃搁板温度下进行解吸干燥.冻干后的样品用磷酸盐缓冲液复水,然后用血细胞计数板、流式细胞仪和扫描电镜进行检测.实验结果表明:在400 g/L PVP 200 g/L蔗糖 100 g/L甘露醇作为保护剂时细胞数目恢复率最高,为(75.0±4.1)%(P＜0.01),PI染色鉴定测得单个核细胞拒染率在400 g/L PVP 200 g/L蔗糖 10%胎牛血清作为保护剂时最高,为(98.57±0.68)%(P＜0.01),CD34 细胞回收率最高为35.59%.最终确定有核细胞的冻干效果比全血的要好,初步判定冷冻干燥法保存脐血是可行的.     

甘油预处理对红细胞冷冻干燥保存作用的实验研究

探索使用甘油作保护剂提高人体红细胞冷冻干燥保存效果的可能性。甘油预处理红细胞的目的是增加细胞质浓度,减小细胞的冷冻损伤。实验分为3组:新鲜对照组、20%甘油组与40%甘油组,冻干保存前后分别检查红细胞相关指标。结果表明,20%与对照组无显著差别;40%甘油组的冻干细胞回收率与血红蛋白回收率分别维持在冷冻干燥前的55.37±4.26%和53.49±3.85%,非常显著高于对照组(P<0.01);渗透脆性与超氧化物歧化酶(SOD)活性均显著高于对照组(P<0.05),但与新鲜红细胞无显著差异。研究结果为使用甘油作红细胞的冻干保护剂提供了理论基础。     

两种低温保护剂对皮肤组织β1整合素表达影响的比较

比较两种不同低温保护剂对皮肤组织β1integrin低温保存后表达的影响,为寻求皮肤组织低温保护剂的最佳配方提供试验依据。获取新鲜成人皮肤组织分为3组,新鲜对照组、海藻糖/二甲基亚砜(T/D)、二甲基亚砜/丙二醇(D/P)作为低温保护剂保存,-196℃液氮冻存7天、14天复温,免疫组织化学染色对各组间皮肤进行比较。并在此基础上,进一步采用RT-PCR方法对不同低温保护剂保存后皮肤的β1integrin基因水平进行了深入的研究。通过光镜图象观察和基因水平分析结果说明,0.5M海藻糖/二甲基亚砜能够很好保护皮肤组织,β1integrin的基因表达量与新鲜皮肤组相似。海藻糖与二甲基亚砜联合应用对皮肤组织β1integrin的保护作用优于传统组。     

Coupling of gelatin to inner surfaces of pore walls in spongy alginate-based scaffolds facilitates the adhesion,growth and differentiation of human bone marrow mesenchymal stromal cells

We have developed a novel wide-pore scaffold for cell 3D culturing, based on the technology of freeze-drying of Ca-alginate and gelatin. Two different preparation methodologies were compared: (i) freeze-drying of Na-alginate + gelatin mixed solution followed by the incubation of dried polymer in saturated ethanolic solution of CaCl₂; (ii) freeze-drying of the Na-alginate solution followed by the chemical “activation” of polysaccharide core with divinylsulfone with subsequent gelatin covalent attachment to the inner surfaces of pore walls. The scaffolds produced using the first approach did not provide adhesion and proliferation of human bone marrow mesenchymal stromal cells (MSCs). Conversely, the second approach allowed to obtain scaffolds with a high adherence ability for the cells. When cultured within the latter type of scaffold, MSCs proliferated and were able to differentiate into adipogenic, osteogenic and chondrogenic cell lineages, in response to specific induction stimuli. The results indicate that Ca-alginate wide-pore scaffolds with covalently attached gelatin could be useful for stem cell-based bone, cartilage and adipose tissue engineering.     

溶胶-凝胶生物活性玻璃与胶原复合多孔支架的生物相容性研究

实验前期用冷冻干燥法合成一种溶胶?凝胶生物活性玻璃(BG/COL)与粗胶原纤维复合的组织工程支架. 本实验将支架与鼠骨髓间充质干细胞(rMSCs)共同培养, 评价支架材料的细胞相容性. 并将复合了成骨细胞的支架材料植入裸鼠皮下, 探讨其异位成骨的性能. 研究结果显示rMSCs可以在BG/COL多孔支架材料表面成功粘附、铺展、并向多孔支架内部迁移, 随着培养时间的延长, 双链DNA(ds DNA)数量增多, 细胞增殖情况与对照组差异明显. 将种在复合材料上的骨髓间充质干细胞诱导培养14d后切片染色, 其碱性磷酸酶(ALP)和钙素表达均呈强阳性. 体内植入实验的裸鼠健康状况良好, 伤口完全愈合, 6w后BG/COL周边及内部有骨组织和血管生成. 由此证明, 这种新型的复合多孔支架材料具有良好的生物相容性, 其复合了成骨细胞的组织工程骨具有良好的诱导成骨的性能, 因此这种材料是理想的应用于骨组织修复和再生的组织工程支架材料.     

Influence of viscosity on chondrogenic differentiation of mesenchymal stem cells during 3D culture in viscous gelatin solution-embedded hydrogels

Differentiation of human bone marrow-derived mesenchymal stem cells(hMSCs)is regulated by a variety of cues of their surrounding microenvironments.In particular,mechanical properties of cell culture matrices have been recently disclosed to play a pivotal role in stem cell differentiation.However,it remains elusive how viscosity affects the chondrogenic differentiation of hMSCs during three-dimensional(3 D)culture.In this study,a 3 D culture system that was established by embedding viscous gelatin solution in chemically cross-linked gelatin hydrogels was used for 3 D culture of hMSCs in gelatin solutions with different viscosities.The influence of solution viscosity on chondrogenic differentiation of hMSCs was investigated.Viscous gelatin solutions promoted cell proliferation in the order of low,middle and high viscosity while elastic hydrogels restricted cell proliferation.High viscosity gelatin solution led to increased production of the cartilaginous matrix.Under the synergistic stimulation of chondrogenic induction factors,high viscosity was beneficial for the chondrogenic differentiation of hMSCs.The results suggested the role of viscosity should be considered as one of the dominant mechanical cues affecting stem cell differentiation.     

Calcium-phosphate ceramics and polysaccharide-based hydrogel scaffolds combined with mesenchymal stem cell differently support bone repair in rats

Research in bone tissue engineering is focused on the development of alternatives to autologous bone grafts for bone reconstruction. Although multiple stem cell-based products and biomaterials are currently being investigated, comparative studies are rarely achieved to evaluate the most appropriate approach in this context. Here, we aimed to compare different clinically relevant bone tissue engineering methods and evaluated the kinetic repair and the bone healing efficiency supported by mesenchymal stem cells and two different biomaterials, a new hydrogel scaffold and a commercial hydroxyapatite/tricalcium phosphate ceramic, alone or in combination.Syngeneic mesenchymal stem cells (5?×?10⁵) and macroporous biphasic calcium phosphate ceramic granules (Calciresorb C35^®, Ceraver) or porous pullulan/dextran-based hydrogel scaffold were implanted alone or combined in a drilled-hole bone defect in rats. Using quantitative microtomography measurements and qualitative histological examinations, their osteogenic properties were evaluated 7, 30, and 90 days after implantation. Three months after surgery, only minimal repair was evidenced in control rats while newly mineralized bone was massively observed in animals treated with either hydrogels (bone volume/tissue volume?=?20%) or ceramics (bone volume/tissue volume?=?26%). Repair mechanism and resorption kinetics were strikingly different: rapidly-resorbed hydrogels induced a dense bone mineralization from the edges of the defect while ceramics triggered newly woven bone formation in close contact with the ceramic surface that remained unresorbed. Delivery of mesenchymal stem cells in combination with these biomaterials enhanced both bone healing (>20%) and neovascularization after 1 month, mainly in hydrogel.Osteogenic and angiogenic properties combined with rapid resorption make hydrogels a promising alternative to ceramics for bone repair by cell therapy.     

Application of human amniotic mesenchymal cells as an allogeneic transplantation cell source in bone regenerative therapy

Autogenous mesenchymal stem cells (MSCs) have therapeutic applications in bone regenerative therapy due to their pluripotency. However, the ability of MSCs to proliferate and differentiate varies between donors. Furthermore, alternative sources of MSCs are required for patients with contraindications to autogenous cell therapy. The aim of this study was to evaluate the potential of mesenchymal cells from the human amniotic membrane (HAM) as a source of cells for allogeneic transplantation in bone regenerative therapy. Cells that retained a proliferative capacity of more than 50 population doubling level were distinguished from other HAM cells as HAMα cells and induced to osteogenic status—their in vivo osteogenesis was subsequently investigated in rats. It was found that HAMα cells were spindle shaped and were positive for MSC markers and negative for hematopoietic stem cell markers. Alkaline phosphatase activity and calcium deposition increased with osteogenic status of HAMα cells. The expression of osteocalcin mRNA was increased in HAMα cells cultured on calcium phosphate scaffolds. Moreover, xenografted HAMα cells remained viable and produced extracellular matrix for several weeks. Thus, this study suggests that human amniotic mesenchymal cells possess osteogenic differentiation potential and could be applied to allogeneic transplantation in bone regenerative therapy.     

连用DCS与显微方法的冷冻干燥系统及实验研究

研制了一种真空冷冻干燥装置,并在其中引入了DSC热分析和显微观察的功能,利用此连用DSC与显微方法的冷冻干燥系统,对低温保护剂海藻糖(trehalose)和聚乙烯醇(PVA)的水溶液进行了冷冻干燥实验,主要考察冷冻速率和溶液用量等不同冷冻条件对溶液冻结特性和升华过程的影响,并通过冻干溶液多孔结构的显微照片估计溶液冻结后形成冰晶的情况.实验结果表明,加快冷冻速率会导致不完全冻结并形成大量的细小冰晶,增加样品用量同样会引起过冷度增大,导致冰晶尺寸减小,使升华阻力增大,升华速率减慢.     

The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder

A key tenet of bone tissue engineering is the development of scaffold materials that can stimulate stem cell differentiation in the absence of chemical treatment to become osteoblasts without compromising material properties. At present, conventional implant materials fail owing to encapsulation by soft tissue, rather than direct bone bonding. Here, we demonstrate the use of nanoscale disorder to stimulate human mesenchymal stem cells (MSCs) to produce bone mineral in vitro, in the absence of osteogenic supplements. This approach has similar efficiency to that of cells cultured with osteogenic media. In addition, the current studies show that topographically treated MSCs have a distinct differentiation profile compared with those treated with osteogenic media, which has implications for cell therapies.     

Nanotopographical Cues Augment Mesenchymal Differentiation of Human Embryonic Stem Cells

The production of bone‐forming osteogenic cells for research purposes or transplantation therapies remains a significant challenge. Using planar polycarbonate substrates lacking in topographical cues and substrates displaying a nanotopographical pattern, mesenchymal differentiation of human embryonic stem cells is directed in the absence of chemical factors and without induction of differentiation by embryoid body formation. Cells incubated on nanotopographical substrates show enhanced expression of mesenchymal or stromal markers and expression of early osteogenic progenitors at levels above those detected in cells on planar substrates in the same basal media. Evidence of epithelial‐to‐mesenchymal transition during substrate differentiation and DNA methylation changes akin to chemical induction are also observed. These studies provide a suitable approach to overcome regenerative medical challenges and describe a defined, reproducible platform for human embryonic stem cell differentiation.     

羟基磷灰石球形颗粒表面微形貌构建及其对干细胞生物学行为的调控

通过调节溶胶-凝胶体系中羟基磷灰石(HA)粉末和甲壳素(Chitin)的质量比, 制备具有不同表面微形貌的HA球形颗粒。扫描电子显微镜(SEM)表征结果显示: 随着HA/Chitin质量比从4/1增加到35/1, 球形颗粒的表面微形貌发生了明显变化, 由粗糙渐趋平滑, 微米级皱褶逐渐减少至消失, 微孔隙率从(35%±0.8%)减少到(10.4%±0.7%)。体外细胞培养的结果表明具有微米级皱褶, 微孔隙率较高的粗糙表面具有引导干细胞铺展和增殖的作用, 微孔隙率低的平滑表面则具有引导干细胞轴向延伸及骨向分化的趋势。同时, HA球形颗粒表面微形貌对干细胞表面特征性抗原标志物的表达具有调控作用。