细胞内游离Ca2+在发育中大鼠皮层神经元无镁诱导惊厥性损伤中的作用

目的: 观察细胞内游离Ca2+([Ca2+]i)在培养的不同发育阶段皮层神经元无镁诱导惊厥性损伤中的作用,探讨惊厥性脑损伤年龄依赖性的可能机制.方法:体外培养6 d、17 d的胚胎大鼠皮层神经元用无镁细胞外液处理3 h,或于无镁处理前用NMDA(N-甲基-D-门冬氨酸)受体拮抗剂或Ca2+通道阻滞剂预处理,用MTT代谢率测定的方法检测神经元损伤,以Fluo-3作标记用激光共聚焦显微镜扫描的方法检测[Ca2+]i.结果:体外培养6 d、17 d的神经元单纯无镁组MTT代谢率较同期对照组降低.应用MK-801 10 μmol*L-1、AP-5 50 μmol*L-1、尼莫地平10 μmol*L-1预处理后再给无镁处理,培养6 d、17 d的神经元MTT代谢率均不同程度高于同期单纯无镁组.培养6 d、17 d的神经元相对荧光强度之间差异有显著性,两者与基线荧光强度比较差异亦有显著性.应用上述各种拮抗剂后,[Ca2+]i改变的峰值均明显低于同期单纯无镁组.结论: 在体外不同发育阶段的神经元,短暂无镁处理诱导惊厥样放电所引起的神经元线粒体功能损伤以及[Ca2+]i改变程度不同.这种[Ca2+]i改变的年龄依赖性可能是惊厥导致神经元损伤的年龄依赖性的机制之一.NMDA受体-Ca2+通道激活是导致这种[Ca2+]i改变及神经元损伤的关键环节.     

Adipose tissues differentiated by adipose-derived stem cells harvested from transgenic mice

Objective: To induce adipocyte differentiation in vitro by adipose-derived stromal cells (ASCs) harvested from transgenic mice with green fluorescent protein (GFP) and assess the possibility of constructing adipose tissues via attachment of ASCs to typeⅡcollagen scaffolds. Methods: Inguinal fat pads from GFP transgenic mice were digested by enzymes for isolation of ASCs (primary culture). After expansion to three passages of ASCs, the cells were incubated in an adipogenic medium for two weeks, and the adipocyte differentiation by ASCs in vitro was assessed by morphological observation and Oil Red O staining. Then they were attached to collagen scaffolds and co-cultured for 12 hours, followed by hypodermic implantation to the dorsal skin of nude mice for 2 months. The newly-formed tissues were detected by HE staining. Results: The cultured primary stem cells were fibroblast-like and showed active proliferation. After being incubated in an adipocyte differentiation medium, the lipid droplets in the cytoplasm accumulated gradually and finally developed into mature adipocytes, which showed positive in Oil Red O staining. A 0. 5-cm3 new tissue clot was found under the dorsal skin of the nude mice and it was confirmed as mature adipose tissues by fluorescent observation and HE staining. Conclusions: ASCs can successfully differentiate adipose tissues into mature adipocytes, which exhibit an adipocyte-like morphology and express as intracytoplasmic lipid droplets. It is an efficient model of adipose tissues engineered with ASCs and type I collagen scaffolds.     

大鼠脂肪干细胞复合胶原-壳聚糖-硫酸软骨素三维支架构建组织工程软骨

Objective To evaluate the character of the collagen-chitosan-chondroitin sulfate scaffold seeded with rat adipose tissue-derived stromal cells. Methods A dipose tissue were harvested from 6 weeks old Wistar rats and the stromal cells were harvested by type Ⅰ collagenase and then cultured in vitro. Type Ⅰ collagen was fully mixed with chitosan, freeze-dried and cross-linked with chondroitin sulfate, then freeze-dried again and sterilized by ethylene oxide. The pore diameter, water content, porosity of the scaffold were tested. The adipose tissue-derived stromal cells were digested, seeded into the plates, scaffold, and cen-trifuged into pellet, and then induced into cartilage. MTT detection for cell proliferation was done. After 3 weeks, the cell morphology, and cell proliferation and adhesion were observed, and chondrngenic differenti-ation was also analyzed. Results The pore diameter, water content, porosity tested for the scaffold showed an appropriate form. Cell proliferation showed faster in the scaffold and pellet culture system after 5 day, there was still cell proliferation in the scaffold system after 14 days but no obvious changes in the pellet cul-ture system; ceils on the scaffold proliferated densely showed by histological staining, but there was a scaf-fold structure residues in the inner layer. The finding of type Ⅱ immunohistochemistry stain showed that cells express strong positive for type Ⅱ collagen in the scaffold and pellet culture system whereas it was weakly positive in the plate culture system; the specific mRNA for cartilage, type Ⅱ collagen, aggrecan and SOX-9 were expressed in all three systems showed by RT-PCR, but type X collagen was expressed continu-ously in the plate culture system and expressed after 21 days in the pellet culture system, whereas it was not detected in the collagen-chitosan-chondroitin sulfate scaffold system. Conclusion The parameters of the collagen-chitosan-chondroitin sulfate scaffold were suitable in our study. The results suggested that it can promote the adipose tissue-derived stromal cells proliferation and chondrogenic differentiation better than the plate and pellet culture systems and maintain the phenotype of chondrocytes well; it is the optimal choice for cartilage tissue engineering in the future.     

脂肪干细胞免疫学性状的初步实验观察

目的初步研究脂肪干细胞(Adiposederivedstemcells,ADSC)表面免疫分子的表达以及体外免疫调节功能,以期为组织工程提供同种异体种子细胞来源。方法体外培养人脂肪抽吸术中获取的脂肪干细胞,体外培养至第二代,流式细胞仪检测免疫分子HLA、HLA、B7-1、B7-2、CD40的表达。1×105个/孔ADSC细胞分别刺激单一异体淋巴细胞或混合双向淋巴细胞反应,观察淋巴细胞增殖情况。同时观察ADSC经IFN-γ作用后,免疫分子表达与淋巴细胞增殖的调节情况。结果ADSC表达HLA类分子,但未检测到HLA类分子阳性表达。B7-1(CD80)、B7-2(CD86)、CD28、CD40未见明显阳性表达。人IFN-γ刺激48h后,HLA类分子表达明显增高,HLAI表达未见明显增高。异体或经IFN-γ作用的ADSC均未能刺激异体淋巴细胞增殖。同样数量的ADSC可明显抑制双相混合淋巴细胞增殖,经IFN-γ作用后抑制作用未见明显减弱。结论ADSC具有一定的体外调节淋巴细胞反应的能力,有可能成为组织工程同种异体细胞来源。     

EGF加强大鼠胃壁细胞的泌酸活动

以氨基比林聚集为间接的泌酸指标，本实验观察了急性分离的大鼠胃粘膜壁细胞对ＥＧＦ的反应。结果表明：１．急性分离后３０分钟左右，壁细胞对较大剂量的ＥＧＦ（１００ｎＭ）的反应为胃酸分泌的减少；２．急性分离后６０分钟左右，壁细胞对ＥＧＦ（０．１－１００ｎＭ）的反应为泌酸活动的加强。结果提示壁细胞被分离后经历一功能恢复的过程，ＥＧＦ对壁细胞的生理作用可能是加强其泌酸活动。     

Phenotypes and spontaneous cell cytotoxicity of mononuclear cells from patients with seronegative spondyloarthropathies: Ankylosing spondylitis,psoriatic arthropathy and pauciarticular juvenile chronic arthritis — Analysis of mononuclear cells from peripheral blood,synovial fluid and synovial membranes

Summary T-cell subpopulations and natural killer (NK) cells from peripheral blood, synovial fluid and synovial membranes from patients with seronegative spondyloarthropathies were investigated. Thirty-four patients with ankylosing spondylitis, sixteen patients with psoriatic arthropathy and six patients with pauciarticular juvenile chronic arthritis were studied. All the patient groups had normal proportions of T4⁺ and T8⁺ cells as well as normal T4/T8 ratios in peripheral blood. In the synovial fluids the T4/T8 ratios were reduced in ankylosing spondylitis and psoriatic arthropathy (p<0.05). Although both the T4 and T8 subpopulations were reduced, the T4/T8 ratios in the synovial membranes of patients with these two disorders tended to be within the normal range of that of peripheral blood. Increased numbers of T-cells in the synovial fluid from patients with ankylosing spondylitis expressed class II MHC antigens. The natural killer cell activity was normal in peripheral blood and synovial fluids of patients with ankylosing spondylitis and psoriatic arthropathy while it tended to be reduced, although not significantly, in pauciarticular juvenile chronic arthritis. Synovial membranes were almost devoid of NK cell activity. The number of Leu 7⁺ cells were reduced in synovial fluid of patients with psoriatic arthropathy (p<0.04), but not as significantly as in the two other patient groups.     

人骨形态发生蛋白-2基因转染脂肪源性间充质干细胞的成骨研究

目的 探讨人骨形态发生蛋白-2（hBMP-2）基因修饰的人脂肪源性间充质干细胞（ADSCs）的成骨能力及基因转染对ADSCs成骨分化的生物学调控。方法 流式细胞技术鉴定从人脂肪组织中提取的细胞为间充质来源的干细胞。将ADSCs转染hBMP＋2基因，免疫沉淀＋Western blotting法和酶联免疫吸附试验（ELISA）检测hBMP-2表达，确定hBMP-2表达量及表达稳定性，碱性磷酸酶（ALP）染色、ALP定量测定、钙结节染色及RT—PCR分析hBMP-2基因转染对ADSCs向成骨细胞转化的调控，并将转基因细胞注入裸鼠股部肌内，通过X线及组织学检查观察体内成骨情况。结果 流式细胞术证实从脂肪中提取的细胞为间充质来源的干细胞。转基因后免疫沉淀＋Western blotting法和ELISA法显示转染hBMP-2的ADSCs具有较高且稳定的hBMP-2的表达，转染21d后表达量无明显降低。ALP染色、ALP定量测定、钙结节染色及RT—PCR发现转基因ADSCs向成骨细胞方向发生分化。裸鼠肌内注射转基因细胞后2周即显示有异位骨形成，4周明显增多，对照组无骨组织形成。结论 转染hBMP-2基因的ADSCs在持续稳定高表达目的蛋白的同时，自身向成骨细胞发生分化，并在裸鼠体内形成异位骨。因此，ADSCs有望作为新的基因工程种子细胞。     

细胞移植治疗心肌梗死的研究与展望

细胞移植是治疗心肌梗死的一种新策略,本文对目前应用于临床研究的骨骼肌卫星细胞移植及骨髓干细胞移植治疗心肌梗死的进展进行综述。上述两种细胞移植均具有来源丰富、无排斥反应的特点,而且通过研究观察表明,上述两种细胞移植后,心脏功能均能得到改善,故具有美好发展前景。但二者亦均具有不足之处和需要解决的问题,还需进一步研究与探索。