应用骨髓间充质干细胞复合关节软骨脱细胞基质修复兔软骨缺损的实验研究

目的研究骨髓间充质干细胞(BMSCs)复合关节软骨脱细胞基质修复兔关节软骨缺损的效果。方法选取36只3~4个月龄的健康新西兰兔,分离其骨髓间充质干细胞(BMSCs),并在体外进行培养扩增。将BMSCs与关节软骨脱细胞基质在体外培养瓶中进行复合培养约3 d。将实验对象随机分为3组,分别进行3种不同的处理,实验结果用关节软骨组织学评分标准进行评分。结果移植手术后12周,移植培养好的骨髓间充质干细胞和关节软骨脱细胞基质复合物的实验组修复组织和周围组织整合良好,且修复效果显著优于移植单独的关节软骨脱细胞基质组和空白对照组。结论应用骨髓间充质干细胞和关节软骨脱细胞基质的复合物能有效的修复兔关节软骨缺损。     

壳聚糖衍生物对异丙肾上腺素诱导的H9c2心肌损伤的保护作用

目的 探究5种壳聚糖衍生物对大鼠心肌细胞的保护作用。方法 在5种壳聚糖衍生物对大鼠心肌细胞H9c2的细胞形态学和生长增值研究基础上,通过建立H9c2细胞异丙肾上腺素损伤模型,研究5种壳聚糖衍生物对损伤H9c2细胞的保护作用。结果 N-乙酰氨基葡萄糖、磺酸化壳寡糖、低脱乙酰度壳寡糖、高脱乙酰度壳寡糖、羧甲基壳聚糖均能不同程度地促进H9c2细胞的生长,对损伤H9c2细胞均有保护和增殖促进作用,N-乙酰氨基葡萄糖、磺酸化壳寡糖、低脱乙酰度壳寡糖、高脱乙酰度壳寡糖的最佳作用浓度均为50 μg?mL-1,羧甲基壳聚糖的最佳作用浓度为100 μg?mL-1。     

人骨髓间充质干细胞对再生障碍性贫血患者T细胞活化的影响

目的探讨人骨髓间充质千细胞在体外对再生障碍性贫血患者T细胞活化的影响。方法从人骨髓分离培养间充质干细胞，用尼龙棉柱分离T淋巴细胞，分别以不同数量间充质干细胞加入到植物血凝素刺激的再生障碍性贫血患者T淋巴细胞活化体系中，通过流式细胞仪获取数据，计算CD3^＋CD25^＋和CD3^＋CD38^＋T细胞的表达率。结果当间充质干细胞为1×10^4／孔时，与单独培养的再生障碍性贫血患者T淋巴细胞（对照组）相比，间充质干细胞对再生障碍性贫血患者T淋巴细胞CD25及CD38的表达呈明显抑制作用，差异有统计学意义（P〈0．01）。当间充质干细胞为1×10^3／孔时，与对照组相比无统计学意义。结论间充质干细胞在体外抑制再生障碍性贫血患者T细胞的活化，且这种抑制作用具有数量依赖性。     

不同分子量高脱乙酰度壳聚糖的制备及表征

目的制备不同分子量高脱乙酰度壳聚糖。方法在强碱备件下通过不同方式的水解从原料壳聚糖制备一系列高脱乙酰度壳聚糖，用HAc／H2O2体系对制备的高脱乙酰度壳聚糖进行不同时间的降解得到中低分子量高脱乙酰壳聚糖，并用乌氏黏度计测定其黏均分子量。结果在实验条件下成功制备了一系列中低分子量高脱乙酰壳聚糖，并用红外和核磁光谱进行分析表征。结论采用10mol·L^-1 NaOH水溶液110℃间歇水解的方法可以制备90％～100％脱乙酰度的壳聚糖，用0．35mol·L^-1 HAc／0．45mol·L^-1 H2O2体系可以制备黏均分子量10，000～100，000Da的壳聚糖样品。     

壳聚糖及其衍生物体外抗幽门螺杆菌作用及影响因素

目的研究壳聚糖及其衍生物在体外对幽门螺杆菌(Hp)的抑菌作用及其影响因素。方法采用打孔法检测了不同浓度、pH值、脱乙酰度壳聚糖及羧甲基壳聚糖在体外对Hp的抑菌作用。结果①壳聚糖和羧甲基壳聚糖在体外对3株Hp标准菌株具有普遍的抑菌作用;②在pH 6~4范围内,随pH值降低,抗菌作用增强,差异有显著性(P<0.01),最佳pH值为4;③70%、88.5%脱乙酰度壳聚糖及羧甲基壳聚糖的抗Hp作用差异有显著性(P<0.01~0.05),抑菌强度依次为DD70壳聚糖、DD88.5壳聚糖和羧甲基壳聚糖;④在质量浓度为10 g.L-1~50 g.L-1范围内羧甲基壳聚糖抗Hp作用差异无显著性;在质量浓度为5 g.L-1~20 g.L-1范围内70%、88.5%脱乙酰度壳聚糖抗Hp作用差异也无显著性(P>0.05)。结论①壳聚糖和其衍生物对Hp有普遍的抑菌作用;②壳聚糖及其衍生物的抗Hp作用受多种因素影响,其中pH值对壳聚糖抗菌作用的影响最为明显,在pH值6~4范围内,壳聚糖的抗菌活性随着pH值的下降而增强;③壳聚糖的脱乙酰度、结构(化学修饰)及分子量也影响壳聚糖的抗菌活性,不同的细菌所要求的脱乙酰度、修饰基团和分子量有着明显的差异。     

体外中药干预骨髓间充质干细胞向心肌细胞分化的研究进展

骨髓间充质干细胞具有自我更新、增殖和多向分化的潜能,骨髓间充质干细胞在体外可通过化学药物及中药干预诱导分化为心肌细胞,将其应用于治疗缺血性心脏病,可减少梗死面积,改善心功能,为心脏病的治疗开辟了新途径。大量研究表明,中药在骨髓间充质干细胞增殖和定向分化过程中具有促进作用,不仅可在体内作用于骨髓间充质干细胞促进其增殖、分化,还可通过影响体外微环境促进其功能的建立与存活。     

组织工程心脏瓣膜体外构建的初步研究

目的:初步体外构建组织工程人工心脏瓣膜。方法:种子细胞选用胸骨骨髓,抽取后分离出人骨髓间充质干细胞,体外培养,扩增待用。脱细胞瓣膜支架的构建应用破膜剂曲拉通-脱氧胆酸钠对新鲜猪主动脉瓣膜36片(实验组)进行处理48h后,与对照组未经处理的猪主动脉瓣膜(36片)进行对比,在光镜和电镜下观察脱细胞效果。将人骨髓间充质干细胞接种于脱细胞瓣膜支架上静态培养7d,观察细胞-支架复合体生长状况。结果:曲拉通-脱氧胆酸钠法脱细胞彻底,保持原有纤维结构。种植的人骨髓间充质干细胞7d后可生长于瓣膜支架的表面。结论:曲拉通-脱氧胆酸钠法是一种较为理想的猪瓣膜脱细胞方法;人骨髓间充质干细胞能在脱细胞猪瓣膜支架上较好地黏附和生长,从而初步构建组织工程人工心脏瓣膜。     

骨髓间充质干细胞在肝纤维化中的研究进展

骨髓间充质干细胞为多能干细胞的一种类型,其日后可分化成为脂肪细胞、软骨细胞、骨细胞、神经元细胞、肝细胞等。经过多项实验资料表明,骨髓间充质干细胞可减轻或抑制肝纤维化,并且不诱发免疫排异,在治疗肝硬化、修复肝细胞损伤的前景非常突出。     

HSV-1体外感染骨髓间充质干细胞的实验研究

目的:在体外原代培养大鼠骨髓间充质干细胞,观察单纯疱疹病毒1型(HSV-1)感染骨髓间充质干细胞情况。方法:分离骨髓间充质干细胞,并对其作鉴定。用HSV-1感染骨髓间充质干细胞,提取总DNA,PCR法扩增骨髓间充质干细胞内的HSV-1特异性片段。结果:骨髓间充质干细胞经14d诱导后,碱性磷酸酶含量增高,形成钙结节,表现出成骨细胞特性。HSV-1感染骨髓间充质干细胞,细胞出现典型的病变,PCR法成功扩增出骨髓间充质干细胞内的HSV-1特异性片段。结论:大鼠骨髓间充质干细胞在体外可以向成骨细胞方向分化,可作为组织工程学的种子细胞。HSV-1可以在体外感染骨髓间充质干细胞。     

黄芪多糖对电离辐射骨髓间充质干细胞向神经分化潜能的影响

目的研究黄芪多糖对X射线照射人骨髓间充质干细胞向神经分化的干预作用。方法采用CCK-8法筛选不同浓度的黄芪多糖作用于2Gy X射线辐射人骨髓间充质干细胞的增殖能力;细胞形态计数X射线对骨髓间充质干细胞分化为神经细胞数量的影响;甲苯胺蓝染色检测X射线对骨髓间充质干细胞分化为神经细胞中尼氏体的表达量;Western blot检测神经巢蛋白(Nestin)及神经元特异性烯醇化酶(neuron-specific enolase,NSE)表达变化。结果与对照组相比,X射线照射后细胞增殖水平明显降低(P<0.05),与单纯照射组比较,加药照射组骨髓间充质干细胞增殖水平显著增加(P<0.05),其中黄芪多糖浓度为50 mg·L~(-1)时促增殖作用最强,设为最佳药物浓度;单纯辐射后骨髓间充质干细胞分化为神经细胞的数量明显降低(P<0.05),当给予黄芪多糖干预后分化为神经细胞的数量明显上升(P<0.05);辐射后骨髓间充质干细胞分化为神经细胞中尼氏体的表达量降低(P<0.05),当给予黄芪多糖干预后,尼氏体的表达量明显增多;单纯辐射后Nestin及NSE表达量降低(P<0.05),而当给予黄芪多糖干预后,Nestin和NSE表达量升高(P<0.05)。结论黄芪多糖对电离辐射人骨髓间充质干细胞向神经分化潜能的影响具有保护作用。     

人脐血间充质干/祖细胞诱导为神经样细胞的实验研究

目的探讨人脐血间充质干细胞用于神经细胞再生的可行性与人脐血间充质干/祖细胞最佳的诱导培养条件。寻找一种合适的细胞来源进行移植以代替受损的神经元、星形胶质细胞和少突胶质细胞来修复神经损伤。方法将脐血单个核细胞置于低血清(2%)达氏修正依氏培养基中培养,生成贴壁细胞层,依传代方法,用相同培养条件进行扩增,扩增后的贴壁细胞置入细胞培养液(加入新生大鼠神经细胞分离培养上清液)中,并添加表皮生长因子(EGF)和碱性成纤维细胞生长因子(bFGF)进行诱导分化。采用流式细胞术、免疫组织化学法分别检测被诱导的神经样细胞的表面标志及细胞特异性标志、结构,并进行分析。结果脐血间充质干/祖细胞克隆在脐血单个核细胞中出现频率为0.5×10-6,在传20代时,可有效扩增1.5×106倍,诱导后,70%的脐血间充质干/祖细胞分化为神经样细胞。结论采用上述扩增与诱导条件,脐血间充质干/祖细胞可得到有效扩增,并可高效向神经样细胞分化,从而证实脐血是一种细胞替代治疗前景光明的细胞来源。     

A new lipid emulsion formulation with high antimicrobial efficacy using chitosan.

The antimicrobial activity of chitosan in lipid emulsions as well as in aqueous solutions was investigated. Two types of long-chained chitosan were used differing in the molecular weights, degree of the deacetylation and their viscosity: type I, mol. weight 8.7 x 10(4) g/mol, 92% degree of deacetylation and a viscosity of 14 mPa s, type II, mol. weight of 5.32 x 10(5) g/mol, 73% degree of deacetylation and a viscosity of 461 mPa s. In order to assess the pH optimum of the antimicrobial activity of the biopolymer, suspensions of the microorganisms Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and Aspergillus niger were incubated at different pH-values in lactic acid solution (1% w/v) containing different concentrations of chitosan up to 1.5% (w/v). Emulsion formulations containing either 0.25%, 0.5% or no chitosan, respectively, were inoculated with the same microorganisms and were incubated at 25 degrees C. The aqueous solutions as well as the emulsions were examined for microbial counts on agar plates after different periods of incubation. After 24 h of incubation in aqueous solutions only the cfu numbers of the bacteria were reduced. Both types of chitosan revealed a pH optimum of their antibacterial activity at pH 5.0-5.1 for P. aeruginosa, and at pH 5.3 for S. aureus. In addition, chitosan with a mol. weight of 8.7 x 10(4) g/mol, high degree of deacetylation and low viscosity showed a higher antimicrobial activity than the other chitosan type of this study. It was found that lipid emulsions containing 0.5% chitosan (type I) conformed to the requirements of the preservation efficacy test for topical formulations according to the European Pharmacopoeia while the emulsion without chitosan and a lactic acid solution with and without the biopolymer did not conform. In hemolysis studies on human erythrocytes, the hemolytic activity of the lipid emulsions with chitosan was assessed. These emulsions showed a negligible hemolytic behavior. The results indicate a use of chitosan as antimicrobial preservative in emulsion formulations for mucosal as well for parenteral applications.     

Evaluation of process parameters involved in chitosan microsphere preparation by the o/w/o multiple emulsion method

Abstract

Chitosans are interesting biopolymers largely studied for applications in the medical and pharmaceutical fields. In this work, an o/w/o multiple emulsion technique was used for the preparation of hydrophobic drug loaded microspheres. Moreover, the influence of critical variables (concentration of acetic acid in the polymer solution and drug-polymer ratio) on microsphere morphology and drug content was evaluated. Two chitosans of different molecular weights and deacetylation degree were employed; ketoprofen, a non-steroidal anti-inflammatory drug, was chosen as the hydrophobic model drug. The multiple emulsion method produced well-formed microspheres with good yields. Acetic acid concentration in the polymeric solutions influenced particle size and drug content of the microspheres. The highest drug encapsulation efficiencies were obtained for the lowest theoretical drug/chitosan ratio.     

人脂肪间充质干细胞分离培养及其生物学特性的实验研究

目的探索人脂肪间充质干细胞（adipose tissue—derived mesenchymal stem cells，ADMSCs）分离培养的方法及体外扩增的条件，观察ADMSCs的生物学特性。方法以腹部手术患者皮下脂肪组织为材料，采用I型胶原酶消化法及贴壁法分离培养ADMSCs，在含10％胎牛血清的低糖DMEM培养基中贴壁培养，倒置显微镜观察，流式细胞仪检测细胞表面标记CD29、CD44、CD105、CD31、CD34、CD106的表达，透射电镜及扫描电镜下观察ADMSCs超微结构，流式细胞仪测定细胞周期。结果原代和传代细胞呈梭形外观，生长增殖能力良好。CD29、CD44、CD105均呈阳性表达，阳性率分别为95.3％、98．6％和86．5％；而CD31、CD34、CD106阳性率分别为3．5％、2．6％、1．3％。透射电镜观察显示ADMSCs表现出早期幼稚细胞形态的特点，流式细胞仪检测显示84．8％的细胞处于G0/G1期。结论酶消化法能有效地从人脂肪组织分离培养人ADSCs，细胞生长稳定，增殖能力活跃，为今后ADMSCs的分离培养提供了更简单有效的方法。     

Chitosans as Absorption Enhancers for Poorly Absorbable Drugs. 1: Influence of Molecular Weight and Degree of Acetylation on Drug Transport Across Human Intestinal Epithelial (Caco-2) Cells

Purpose. Chitosan has recently been demonstrated to effectively enhance the absorption of hydrophilic drugs such as peptides and proteins across nasal and intestinal epithelia (1–3). In this study, the effect of the chemical composition and molecular weight of chitosans on epithelial permeability and toxicity was investigated using monolayers of human intestinal epithelial Caco-2 cells as a model epithelium. Methods. Eight chitosans varying in degree of acetylation (DA) and molecular weight were studied. The incompletely absorbed hydrophilic marker molecule ¹⁴C-mannitol was used as a model drug to assess absorption enhancement. Changes in intracellular dehydrogenase activity and cellular morphology were used to assess toxicity. Results. Chitosans with a low DA (1 and 15%) were active as absorption enhancers at low and high molecular weights. However, these chitosans displayed a clear dose-dependent toxicity. Chitosans with DAs of 35 and 49% enhanced the transport of ¹⁴C-mannitol at high molecular weights only, with low toxicity. One chitosan (DA = 35%; MW = 170kD) was found to have especially advantageous properties such as an early onset of action, very low toxicity, and a flat dose-absorption enhancement response relationship. Conclusions. The structural features of chitosans determining absorption enhancement are not correlated with those determining toxicity, which makes it possible to select chitosans with maximal effect on absorption and minimal toxicity.     

子宫内膜腺上皮细胞和基质细胞的分离培养

目的　建立一种获得高产量子宫内膜腺上皮细胞和基质细胞的分离培养方法。方法　通过胶原酶消化法、系列过滤法及贴壁纯化等技术分离纯化培养人子宫内膜腺上皮细胞和基质细胞 ,并通过光镜及免疫细胞化学染色进行鉴定。结果　腺上皮细胞呈蝌蚪形 ,漩涡状生长。角蛋白免疫细胞化学染色阳性 ,纯度约 90 %。基质细胞呈梭形或多角形 ,平行状生长。波形蛋白染色反应阳性 ,纯度达 95 %。每例子宫肌瘤切除标本可获得 (10～2 5 )× 10 6 原代基质细胞和 (4～ 6 )× 10 6 原代腺上皮细胞 ,每例诊断性刮宫获得的原代细胞数约为前者的 1/ 2～2 / 3。结论　改良培养程序可获得高产量的纯化的子宫内膜腺上皮细胞和基质细胞。     

兔骨髓间充质干细胞的分离、培养、鉴定及Dil荧光标记

摘要 目的　探讨兔骨髓间充质干细胞(MSCs)体外分离培养、表型鉴定和标记的方法。方法　采用密度梯度离心法及贴壁分离筛选法分离培养MSCs;采用免疫细胞化学方法检测细胞表面标志抗原CD29,CD106的表达,进行表型鉴定;DiI标记第3代MSCs,观察标记效率。结果　体外培养的原代MSCs48h内可见少量贴壁细胞,7-8d达到90%汇合;免疫细胞化学方法检测细胞表面标志抗原CD29,CD106为阳性;DiI进行细胞标记后,荧光显微镜下见所有MSCs均被标记为红色荧光,提示DiI标记法敏感性好,标记效率高。结论　此培养方法能在短时间内获得大量MSCs,操作简单,成功率高,可以作为培养兔MSCs的常规方法,为构建组织工程尿道提供充足的种子细胞,并进一步用于治疗重度尿道下裂、尿道下裂残废和较长的后尿道狭窄等顽症。     

人脐带问充质干细胞体外培养、鉴定及其诱导分化的研究

目的通过体外分离、培养及鉴定人脐带间充质干细胞（humanumbilicalcordmesenchymalstemcells,hUCMSCs）,探讨其多向分化潜能。方法取正常足月新生儿脐带,采用组织贴壁培养法分离原代hUCMSCs,观察细胞生长形态。采用流式细胞仪技术检测hUCMSCs细胞表型及细胞周期。通过成神经细胞诱导和成脂肪细胞诱导,鉴定hUCMSCs的多向诱导分化能力。结果成功分离和培养hUCMSCs原代细胞,经流式细胞仪鉴定高表达间质细胞标志CD44和CDl05,阳性率为96．73％和96．10％,整合素受体CD29阳性率为99．53％;低表达造血系标志CD34和CD45阳性率为0．80％和1．91％,人白细胞抗原HLA-DR阳性率为1．41％。细胞周期检测hUCMSCs主要处于G0／G1期。hUCMSCs成神经细胞诱导,出现神经元样细胞;免疫组化检测神经巢蛋白（Nestin）呈阳性表达。hUCMSCs成脂肪细胞诱导,出现空泡样脂肪滴,油红O染色可见脂质沉积。结论hUCMSCs具有多向分化潜能,可跨胚层诱导分化为多种组织类型的细胞。     

大鼠胃平滑肌细胞收缩模型及其药物研究

目的 建立胃平滑肌收缩的实验模型并且对相应治疗药物的作用进行评价。方法 采用分离大鼠胃底平滑肌,用胶原酶和胰蛋白酶进行消化培养,得到离体平滑肌细胞悬液。用Trypan blue稀释法检查,通过带刻度显微镜观察和标测细胞的形态和长度,并观察在不同的时间范围内细胞形态和长度的变化情况,计算细胞存活率和存活时间。于细胞悬液加入各种神经介质乙酰胆碱(ACh)、5-羟色胺(5-HT、组织胺(His)引起刺激反应,同时加入其相应的拮抗剂阿托品(atropine)、多潘立酮(Domp)、苯海拉明(Diph)时,观察细胞形态和长度的前后变化,从而对模型和药物的作用进行评价。结果 每只大鼠胃底均能收获1×10~6以上个细胞,细胞存活率在95％以上,细胞至少可存活2.5 h。ACh能引起胃平滑肌细胞的收缩,具有明显的量效关系,1×10~(-8)mol·L~(-1)即可产生最大收缩反应(38.0％)。atropine对乙酰胆碱的拮抗作用有明显的量效关系。5-HT对大鼠胃底平滑肌细胞具有明显的收缩作用,井有明显的量效关系。Domp能够拮抗5-HT的细胞收缩作用,并具有一定的量效关系。5-HT的量效曲线比较平坦,可能至少有2种受体参与细胞的收缩作用,它们引起细胞收缩的最大效应分别为20.7％和10.5％。His引起的平滑肌细胞收缩,在1×10~(-13)～1×10~(-10) mol·L~(-1)内有?     

Mesenchymal stem cells: cell biology and potential use in therapy

Mesenchymal stem cells are clonogenic, non-haematopoietic stem cells present in the bone marrow and are able to differentiate into multiple mesoderm-type cell lineages e.g. osteoblasts, chondrocytes, endothelial-cells and also non-mesoderm-type lineages e.g. neuronal-like cells. Several methods are currently available for isolation of the mesenchymal stem cells based on their physical and immunological characteristics. Because of the ease of their isolation and their extensive differentiation potential, mesenchymal stem cells are among the first stem cell types to be introduced in the clinic. Recent studies have demonstrated that the life span of mesenchymal stem cells in vitro can be extended by increasing the levels of telomerase expression in the cells and thus allowing culture of large number of cells needed for therapy. In addition, it has been shown that it is possible to culture the cells in xeno-free environment without affecting their growth or differentiation potential. Finally, the mesenchymal stem cells seems to be hypoimmunogenic and thus allogenic mesenchymal stem cells transplantation is possible. It is envisaged that mesenchymal stem cells can be used in systemic transplantation for generalized diseases, local implantation for local tissue defects, as a vehicle for genes in gene therapy protocols or to generate transplantable tissues and organs in tissue engineering protocols. The results of these initial trials are very encouraging and several clinical trials are under way to study the efficacy and long-term safety of therapeutics based on mesenchymal stem cells.