骨髓间充质干细胞联合可降解支架体外构建组织工程带瓣静脉（英文）

背景:临床上治疗慢性静脉功能不全的主要方法是静脉瓣膜修复及带瓣静脉段移植,但这些方法创伤较大,且带瓣静脉来源有限。组织工程学和再生医学在修复病变血管方面取得的进步,而以自体来源的内皮细胞为种子细胞的组织工程带瓣静脉也见于了报道,但存在排出反应。目的:构建一个有可自我更新、修复、类似天然瓣膜结构并具有功能的带瓣静脉。方法:麻醉取Beagle犬的骨髓获取骨髓间充质干细胞,采用密度梯度离心和贴壁法获取骨髓间充质干细胞,并进行细胞的传代、冻存复苏、流式细胞仪检测和定向诱导分化。采用热致相分离技术,以聚(乳酸-乙醇酸)共聚物为基材,利用自制带瓣静脉模具制备三维组织工程带瓣静脉支架,制备组织工程带瓣静脉支架,并研究其形态结构。将骨髓间充质干细胞种植在支架上构建可降解的带瓣静脉,在体外培养2周。结果与结论:扫描电镜观察显示支架孔隙率高。培养的细胞符合骨髓间充质干细胞的形态特征,培养的细胞大部分表达CD29和CD44,不表达CD34和CD45。细胞毒性实验显示支架无毒性,有利于细胞增殖和迁移。将细胞种植在支架表面上培养后可形成单层细胞层。体外实验验证细胞支架复合物的瓣膜有一定的开闭功能。利用三维聚(乳酸-乙醇酸)共聚物支架和骨髓间充质干细胞可成功构建组织工程带瓣静脉,组织工程带瓣静脉将有可能作为静脉瓣膜的的替代物治疗静脉瓣膜疾病。     

心脏组织工程瓣脉动流培养系统的设计与构建

自行设计和制造平面和三维立体培养室及贮液室等构件,用医用硅胶管连接;转子泵作为动力源,贮液室通气口供给5%CO2 95%空气,恒温水浴箱保持构件37℃恒温,这样组成了种植细胞与生物瓣支架复合体的脉动培养系统,并进行生物力学和生物相容性测试,为心脏组织工程瓣的体外构建提供研究器材。结果显示,该装置密闭性能好,内环境能保持37±1℃、CO2浓度5%±1%、pH值6.8～7.5;流量在0.125～6.0L/min的范围内任意调节;同种瓣膜上的内皮细胞经2周培养后扩增约10倍;瓣膜支架的细菌和霉菌培养均为阴性,说明我们构建的脉动流培养系统能有效地模拟体内脉动流场实现种植细胞在体外的增殖、重塑,为心脏组织工程瓣的体外构建提供了一种新的实验方法。     

模拟微重力对许旺细胞三维培养影响的初步研究

目的初步探讨模拟微重力对许旺细胞三维培养的影响.方法分别把粘附在聚羟基乙酸支架上生长3、7、10及14天的许旺细胞和新鲜分离的原代许旺细胞悬液连同聚羟基乙酸支架送入转壁式生物反应器(RWVB)中旋转培养5天,观察细胞在支架上的生长情况及超微结构的改变.结果粘附在支架上的许旺细胞进入模拟微重力环境后细胞变圆变小,核染色质聚集,细胞器水肿扩张,核/浆比例明显增大,微丝微管松散紊乱,细胞在3天内快速凋亡或碎裂溶解;尚未贴壁的原代许旺细胞进入模拟微重力环境后在支架表面均匀贴壁生长、增殖,蛋白合成活跃,个别细胞内可见髓鞘样结构,5天内未见细胞死亡现象.结论贴壁生长3～14天的许旺细胞进入模拟微重力环境后表现为快速凋亡、溶解,而未贴壁的原代许旺细胞进入模拟微重力环境后则功能活跃表达及快速增殖,可用于构建比较理想的许旺细胞三维培养体系.     

低渗冻融联合生物酶法制备组织工程化静脉瓣脱细胞支架

目的:采用低渗冻融联合生物酶法制备组织工程化静脉瓣脱细胞支架,并与先前方法制备的静脉瓣脱细胞支架比较,以期获得一种性能较好的带瓣静脉脱细胞支架材料.方法:手术获得Beagle犬带瓣静脉分为4组,对照组、脱氧胆酸钠组、Triton组和冻融+生物酶组.处理后对各组标本行H-E染色,透射电镜观察,DAPI荧光染色,细胞相容性测试等观察.结果:3种脱细胞方法均能彻底去除细胞,DAPI荧光检测显示各组支架材料均无DNA成分残留;H-E染色及透射电镜显示,冻融+生物酶组胶原纤维排列整齐,未见明显的胶原纤维结构改变,其他2组可见胶原纤维断裂及结构紊乱现象;与其他2组脱细胞支架材料相比,冻融+生物酶组的组织细胞相容性好,可见内皮祖细胞在支架材料上生长良好.结论:结合渗透压改变的反复冻融加生物酶法,既可以较彻底除去带瓣膜静脉细胞成分,又保留了较完整的细胞外基质结构,具有良好的组织和细胞相容性,是较理想的带瓣膜静脉脱细胞支架制备方法.     

脱细胞绵羊带瓣静脉支架材料的制备及其免疫原性评价

目的:制备脱细胞绵羊带瓣静脉支架材料,通过探讨其对BALB/c小鼠体液免疫和细胞免疫的影响,以此评价该脱细胞基质的免疫原性,进而为脱细胞组织工程支架的安全性提供实验依据.方法:正常成年绵羊带瓣股静脉2cm长的片段,联合使用弱碱、非离子去垢剂及核酶进行脱细胞处理.H-E染色观察细胞组分残留及细胞外基质完整性.免疫组织化学...     

经液氮保存具有活性的同种生物瓣脱细胞支架的制备

目的为体外构建组织工程瓣提供脱细胞的同种生物瓣支架材料,并建立相应的技术方法。方法选取经液氮保存、具有活性的同种生物带瓣管道作为实验材料,用含十二烷基硫酸钠(SDS)的Tris低渗缓冲液脱去同种带瓣管道存在的细胞;固定剂固定后,分别行HE染色、胶原纤维和弹力纤维染色的光镜和扫描电镜观察、摄片。结果用质量浓度为0.03%SDS的Tris低渗缓冲液与同种瓣膜共同孵育48h,完全脱去了表面的内皮细胞(ECs),又较完整地保留了胶原纤维和弹力纤维等细胞外基质主要成分,其形态学结构在脱细胞前后无明显改变;瓣叶中心部位有部分成纤维细胞存留。而对于脱主动脉壁细胞,质量浓度为0.1%SDS更合适。结论质量浓度为0.03%～0.1%SDS的低渗缓冲液对制备脱细胞的同种生物瓣支架效果较佳,有利于同种瓣再内皮化时种植细胞的粘附和扩增,可进一步应用于组织工程瓣体外构建的研究。     

几种生物材料种植人成纤维细胞的比较

目的 通过人成纤维细胞种植，比较几种形式的聚酯材料，以及脱细胞生物组织基质的细胞亲和性，为组织工程血管及瓣膜研究选择支架材料。方法 ①聚氨酯(PU)、第3代聚烷酸酯[PHAs，分子表达式为p(3HB—co—3HH)]、PHAs／PU共混物分别涂在载波片上制成薄膜，将人成纤维细胞种植其上，培养3天。HE染色后于显微镜下每片取相同4点计数，得出平均值。②分别在PHAs／PU片、脱细胞牛心包片、脱细胞猪肺动脉瓣(简称猪瓣)种植人成纤维细胞，PHAs／PU片培养7天，后两者培养3天。用HE染色、扫描电镜分别观察细胞生长情况。结果 ①3组薄膜片上细胞平均数分别为(PU)196、(PHAs)124、(PHAs／PU)160。②扫描电镜显示PHAs／PU片表面约50％有细胞覆盖，细胞聚合成粗大束带状结构，牛心包表面紧密覆盖着厚实的鳞甲状细胞结合体，猪瓣瓣叶表面覆盖紧密联结的板状细胞结合体。③HE染色显示PHAs／PU片和牛心包片、猪瓣瓣叶单侧有联结较完整的细胞层覆盖，局部有3—4层重叠而成，牛心包片和猪瓣瓣叶上细胞与基质联结紧密。结论 ①PHAs混入PU后细胞亲和性显著提高，适宜作血管及补片支架材料；②脱细胞牛心包组织和猪瓣细胞亲和性明显好于合成材料，适合作血管、补片及带瓣通道组织工程支架。     

用于经导管二尖瓣置换术的支架有限元评估

目的：提出一种经导管二尖瓣置换术支架设计,评估支架与瓣叶植入后在体内相互作用产生的变形情况并分析其力学行为。方法：应用有限元方法,建立支架瓣叶组合模型,在瓣叶上施加压强载荷模拟其在人体内闭合的过程,分析其变形情况和应力应变结果以及受此影响的疲劳寿命。结果：本支架受瓣叶牵拉产生的最大顶端位移为0.24 mm,周期性应变的疲劳寿命约为2.05×10⁸次。结论：变形程度可接受但不可忽视,瓣叶的周期性牵拉对支架的影响可使其在人体内正常工作5～9年。     

Atelocollagen胶原支架在三维培养大鼠心肌中的生物相容性

目的探讨atelocollagen胶原支架在三维培养心肌细胞中的生物相容性,寻找三维培养心肌组织的条件和理想的支架材料。方法原代培养的大鼠心肌细胞经纯化后,将细胞悬液接种到胶原纤维支架上,动态观察第8d、16d、20d在atelocollagen胶原纤维支架上生长的细胞的变化,并进行光镜、扫描电镜和透射电镜观察。结果接种后第1d形成细胞-胶原支架搏动复合体,细胞与支架一起有节律的跳动;心肌细胞填充于支架网孔周围,并与支架融合;在透射电镜下,3个时间段均发现细胞与支架紧密相贴,融合在一起;在扫描电镜下发现,细胞在胶原纤维支架上贴附并充分伸展,相互融合成片。结论atelocollagen胶原支架的生物相容性较好,可作为三维培养细胞和组织的天然材料,适于心肌组织的立体培养。     

脱细胞猪主动脉瓣叶的生物力学特性和免疫反应性

目的：探讨脱细胞猪主动脉瓣叶构建组织工程心脏瓣膜支架的可行性。方法：经胰酶-EDTA、表面活性剂、核酸酶处理，去除瓣叶的细胞成分，测定脱细胞瓣叶的生物力学特性，同时行大鼠皮下包埋实验，观察其免疫反应性。结果：瓣叶中的细胞成分能完全去除，获得无细胞的纤维网状支架。脱细胞瓣叶与新鲜瓣叶有基本相同的应力-应变曲线及应力-松弛曲线，而弹性模量、面积比、松弛强度、断裂强度和断裂伸长率两者无显著差异。脱细胞瓣叶的免疫反应性明显降低。结论：猪主动脉瓣叶经脱细胞处理后可以作为组织工程心脏瓣膜的支架材料。     

Cell death and its suppression in human ovarian tissue culture

In women with premature ovarian failure, fertility may be preserved by ovarian tissue culture in vitro. However, techniques for tissue culture and follicle maturation have remained suboptimal. Our aim was to characterize ovarian tissue degeneration in cultures and to establish a model for cell death research in cultured ovarian tissue. Precise knowledge on the process resulting in cell death in cultured ovarian tissue will ultimately facilitate work aimed at improving long-term culture conditions. Ovarian tissue apoptosis was studied in a serum-free culture model in which nuclear DNA fragmentation was shown to occur within 24 h of the start of the culture. Activation of caspase-3 was detected in some stromal cells and a few oocytes. Since not all of the tissue exhibited signs of apoptosis and since DNA fragmentation increased over time, the tissue probably gradually dies by apoptosis. The antioxidant N-acetyl-L-cysteine (NAC; 25, 50 and 100 mmol/l) was found to inhibit this apoptosis. Thus, apoptosis appears to play a critical role in the degeneration of human ovarian cortical tissue cultures, and this cell death can be suppressed by NAC. The present tissue culture model can be used for identifying components capable of inhibiting cell death in vitro.     

Fibronectin binding properties of bacteriologic petri plates and tissue culture dishes

With the aid of a monoclonal antibody-based ELISA assay, the fibronectin binding properties of poly(styrene) bacteriologic and tissue culture petri plates were studied. After treatment of the plastics with serum, both the rate of fibronectin binding and the maximum amount of fibronectin bound were found to be lower for bacteriologic than tissue culture plates. In contrast, when treated with purified fibronectin rather than serum, bacteriologic and tissue culture plates bound fibronectin equally well. Thus, serum proteins are more effective in inhibiting fibronectin binding to bacteriologic petri plates than to tissue culture dishes. The fibronectin binding properties of plastic substrata could be enhanced by oxidation with H2SO4 or diminished by dissolution and recasting of tissue culture dishes. Thus, the fibronectin binding properties of bacteriologic and tissue culture dishes can be interconverted. Plastics with enhanced fibronectin binding properties (tissue culture plates) were found to be hydrophilic and good substrates for cell attachment and growth while plastics with decreased fibronectin binding characteristics were found to be hydrophobic and poor substrates for cell attachment and growth. The cell-adhesive properties of bacteriologic and tissue culture plastic substrata were found to vary during incubation with cells. While cells remained firmly attached and spread on tissue culture plastics over a period of 5 days or more, previously attached cells gradually detached from bacteriologic plastics at incubation times beyond 12 h. The gradual detachment of cells from bacteriologic plates probably explains the poor properties of bacteriologic plastics for the growth of anchorage-dependent cells, in particular.     

采用微载体技术大规模培养组织工程种子细胞

如何获得大规模、具有再生活力的种子细胞已经成为当前组织工程研究面临的最关键的限制性因素;针对这一限制性因素,研究人员对多个细胞培养系统进行了研究,其中包括微载体细胞培养系统,该系统最初主要用于细菌的大规模培养研究,但近年来的研究发现,微载体细胞培养系统也有助于种子细胞的体外大规模扩增;本对应用微载体技术大规模培养组织工程种子细胞进行了简要综述。     

Prenatal cytogenetic diagnosis of the fragile X chromosome: feasibility and speed of in situ clonal method in amniotic fluid cell tissue culture.

We have had experience with over 300 amniotic fluid specimens for prenatal diagnosis for the fragile X chromosome [fra(X)], and the flask method of tissue culture has been routinely utilized requiring extended tissue culture periods of 3-4 weeks. The use of the in situ clonal method of tissue culture for routine prenatal cytogenetic diagnosis of amniotic fluid cells has shortened tissue culture time and resulted in more rapid reporting; however, it has not been widely employed for fra(X) prenatal diagnosis. The simultaneous use of both methods of tissue culture has resulted in the detection of 2 cytogenetically fra(X) positive cases in amniotic fluid, with more rapid reporting and satisfactory expression of the fra(X) with the in situ clonal method. Thus, the use of the in situ clonal method of tissue culture for fra(X) prenatal diagnosis in amniotic fluid cells is feasible, faster and can serve as a more rapid cytogenetic adjunct to the newer DNA testing methods.     

Simplified organ cultures of human embryo trachea in the diagnosis of viral respiratory disease of children

Organ cultures of human embryonic trachea in test tubes were used as an adjunct to tissue cultures in the isolation of respiratory viruses from children in hospital. Fifty-one viruses were obtained from 127 specimens, giving an isolation rate of 40%. Fifteen viruses were isolated from the original tissue cultures and also after passage through organ culture. Thirty viruses were isolated from the original tissue culture only, and six viruses only from organ culture (three para-influenza, one influenza A, and one rhinovirus). An increase of 5% in virus isolation rate over that of standard tissue culture was obtained.     

Nonadherent,stationary organ culture of human respiratory mucosa

Summary Fragments of human adenoid tissue were grown in a tissue culture system where all artificial tissue handling, except for the explanation procedures and tissue culture condition in itself, was excluded. The fragments formed spheroids that were covered by a pseudostratified, ciliated epithelium. The epithelium rested on a basement membrane. The central parts of the spheroids consisted of fibroblasts and collagen fibers. This structure remained unchanged for the 40 days the fragments were observed in culture.     

Early detection of influenza virus by using a fluorometric assay of infected tissue culture.

A fluorometric substrate, 4-methylumbelliferyl-alpha-ketoside of N-acetylneuramide, was used directly on clinical specimens and infected tissue culture 24 h after inoculation for the detection of influenza viral neuraminidase. Viral neuraminidase was detected in infected tissue culture but not in clinical specimens. The sensitivity of the assay on tissue culture was 92%, and the specificity was 96%.     

Engineering three-dimensional bone tissue in vitro using biodegradable scaffolds: investigating initial cell-seeding density and culture period

New three-dimensional (3D) scaffolds for bone tissue engineering have been developed throughout which bone cells grow, differentiate, and produce mineralized matrix. In this study, the percentage of cells anchoring to our polymer scaffolds as a function of initial cell seeding density was established; we then investigated bone tissue formation throughout our scaffolds as a function of initial cell seeding density and time in culture. Initial cell seeding densities ranging from 0.5 to 10 x 10(6) cells/cm(3) were seeded onto 3D scaffolds. After 1 h in culture, we determined that 25% of initial seeded cells had adhered to the scaffolds in static culture conditions. The cell-seeded scaffolds remained in culture for 3 and 6 weeks, to investigate the effect of initial cell seeding density on bone tissue formation in vitro. Further cultures using 1 x 10(6) cells/cm(3) were maintained for 1 h and 1, 2, 4, and 6 weeks to study bone tissue formation as a function of culture period. After 3 and 6 weeks in culture, scaffolds seeded with 1 x 10(6) cells/cm(3) showed similar tissue formation as those seeded with higher initial cell seeding densities. When initial cell seeding densities of 1 x 10(6) cells/cm(3) were used, osteocalcin immunolabeling indicative of osteoblast differentiation was seen throughout the scaffolds after only 2 weeks of culture. Von Kossa and tetracycline labeling, indicative of mineralization, occurred after 3 weeks. These results demonstrated that differentiated bone tissue was formed throughout 3D scaffolds after 2 weeks in culture using an optimized initial cell density, whereas mineralization of the tissue only occurred after 3 weeks. Furthermore, after 6 weeks in culture, newly formed bone tissue had replaced degrading polymer.     

Study of the properties of a tissue smallpox vaccine manufactured by the Moscow Research Institute of Viral Preparations]

The tissue culture vaccine against smallpox has some important advantages over the dermal preparation: it is free from bacterial contamination, contains no serum proteins, and suitable for intradermal inoculation with jet injections. The virus for the tissue culture smallpox vaccine is grown in Japanese quail embryo cultures controlled for the absence of contaminating viruses. In trials of the tissue culture smallpox vaccine in 800 revaccinated volunteers no untoward reactions or complications were observed. The antigenic activity of the tissue culture smallpox vaccine was superior to that of dermal vaccine used in the same dose: the geometric mean neutralizing antibody titre after vaccination with the tissue and dermal preparations was 1 : 256 and 1 : 158, respectively, and the antibody rise was 4.5- and 2.5-fold.     

Sensitization of guinea-pigs to measles vaccines

Attenuated measles virus vaccine of chick embryo tissue culture origin and unadsorbed inactivated measles vaccines derived from monkey kidney tissue culture produced little or no sensitization of guinea-pigs, as monitored by the Schultz–Dale or passive cutaneous anaphylaxis techniques. Inactivated vaccines of both monkey kidney and chick embryo tissue culture origins were sensitizing when they contained a mineral adjuvant. The sensitizing activity was also present in the aqueous phase of Tween 80–ether-treated measles virus fluid and in uninfected tissue cultures of simian, avian and human origins.