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。     

力达霉素对神经胶质瘤细胞血管生成拟态的抑制及对细胞凋亡的影响

Objective To evaluate the in vitro effects of lidamycin upon vasculogenic mimicry and apoptosis induction in glioma cells. Methods Tube formation assay was performed to estimate the inhibitory effects of lidamycin upon vasculogenic mimicry in C6 and U87 glioma cells. The vasculogenic mimicry of glioma cells was photographed and enumerated. Annexin V-FITC/PI was used for determination of glioma cell apoptosis with flow cytometry. Results Vasculogenic mimicry assay indicated that 0. 1 nmol/L, 0. 5 nmol/L and 1 nmol/L lidamycin showed significant inhibition of vasculogenic mimicry in C6 and U87 cells. Comparing with C6 control group (14. 7 ± 1.2), 0. 1 nmol/L (12. 7 ± 0. 6), 0. 5 nmol/L (9. 0 ± 1.7) and 1 nmol/L (4. 7 ± 0. 6) lidamycin inhibited vasculogenic mimicry in C6 cells with statistical significances (P = 0. 013, P = 0. 005 and P = 0. 0002 respectively). Comparing with U87 control group (14.7±1.2), the vasculogenic mimicry of 0.1 nmol/L (10.0±2.0), 0.5 nmol/L (8.3±1.5) and 1 nmol/L lidamycin (4. 3±0. 6) treated U87 cells showed statistical significances (P =0. 025, P =0. 005 and P =0. 0009 respectively). The apoptotic ratios of same dosages lidamycin treated C6 cells and U87 cells showed a similar tendency as vaaculogenic mimicry inhibition (P < 0. 001). Lidamycin was more potent than neocarzinostatin in vasculogenic mimicry inhibition and apoptosis induction of C6 cells and U87 cells. Conclusion Lidamycin can inhibit vasculogenic mimicry and promote apotosis of glioma cells. Thus it is a promising drug in glioma treatment. Further researches on the therapeutic efficacy of enediyne antibiotics in glioma are needed.     

壳聚糖作为中枢神经系统支架材料的可行性研究

目的探索壳聚糖作为中枢神经系统组织工程支架材料的可行性。方法选择孕14～16dWistar大鼠剖宫取胎鼠脑进行分离、培养,经巢蛋白、胶原纤维酸性蛋白、特异性烯醇化酶和半乳糖脑苷酯免疫细胞化学染色鉴定获得神经干细胞,种植于壳聚糖膜和明胶膜表面培养,然后行巢蛋白免疫细胞化学染色,观察神经干细胞在壳聚糖膜和明胶膜表面的贴附、生长和分化状态,以及壳聚糖、明胶的降解速度。结果从胎鼠大脑分离获得的神经干细胞,巢蛋白染色呈阳性反应,可分化形成神经元和胶质细胞,分化细胞分别呈胶原纤维酸性蛋白、特异性烯醇化酶和半乳糖脑苷酯染色阳性反应。神经干细胞接种后12h即贴附于壳聚糖膜表面,24h壳聚糖膜和明胶膜均贴附牢固;48h明胶膜表面神经球周围首先出现带突起的分化细胞,壳聚糖膜迟至生长第3天才出现。神经干细胞接种生长后第3天,壳聚糖膜部分降解,明胶膜完全降解;第4天膜表面长满分化细胞;第5、6天壳聚糖膜大部分降解,分化细胞部分死亡。结论壳聚糖与神经干细胞生物相容,具有促细胞贴附和分化作用,由于机械性能差,不宜作为中枢神经组织工程的结构性生物支架材料,但可以单独或者联合促贴附物明胶作为促神经干细胞贴附和分化的支架表面修饰剂。     

胰岛素样生长因子1抑制小鼠耳蜗毛细胞凋亡

目的 利用耳蜗基底膜离体培养技术建立庆大霉素(gentamicin,GM)损伤模型,研究胰岛素样生长因子1(insulin-like growth factor-1,IGF-1)对小鼠耳蜗毛细胞(hair cell,HC)凋亡的影响及可能机制.方法 取新生小鼠耳蜗基底膜,在分别含有预先制备的培养液(A组),含有GM 0.5 mmol/L(B组),含有GM 0.5 mmol/L和IGF-1 1 ng/ml(C组),GM 0.5 mmol/L和IGF-1 10 ng/ml(D组),GM 0.5 mmol/L和IGF-1 50 ng/ml(E组),GM0.5 mmol/L和IGF-1 100 ng/ml(F组)的培养液中离体培养后制作耳蜗铺片.利用DNA末端转移酶介导的缺口末端标记法(terminal-deoxynucleotidyl transferase mediated nick end Iabeling,TUNEL)结合活性半胱氨酸蛋白酶-3抗体为标记物多重荧光免疫组织化学方法,激光共聚焦显微镜下观察各组耳蜗HC的凋亡情况.结果 耳蜗铺片观察显示A组各转及各组顶转未发现TUNEL染色阳性的凋亡HC.C、D、E、F各组耳蜗底转和中转HC的TUNEL染色阳性HC数较B组减少(P＜0.05),其中以D组减少最为明显.活性半胱氨酸蛋白酶-3抗体标记和TUNEL染色阳性的耳蜗HC凋亡的部位不完全重合.结论 GM可诱导离体培养的小鼠耳蜗HC凋亡的发生.应用IGF-1能够通过抑制其凋亡作用,在特定浓度达到最佳效果.半胱氨酸蛋白酶-3活化参与耳蜗HC凋亡过程.     

突触蛋白-Ⅰ在胚胎干细胞体外神经分化过程中的表达变化

目的探讨突触蛋白-I（synapsin-I）在胚胎干细胞（ESCs）体外神经分化过程中的表达变化及作用。方法采用“五步法”和维甲酸（RA）法两种途径体外诱导ESCs向神经细胞分化，并以另一种可向神经细胞分化的肿瘤细胞-PC12细胞的诱导过程作参照，从不同的途径、不同的细胞进行比较．通过免疫组织化学、RT-PCR、Western blot方法观察这一过程中synapsin-I的表达变化．找出synapsin-I在ESCs向神经细胞分化过程中表达变化的共同规律。结果结合形态学和其它神经特异性指标的变化，synapsin-I在ESCs和PC12细胞向神经细胞分化的过程中具有早期即有表达，后逐渐升高，至分化成熟阶段达最高，后期又逐渐下降的变化规律。结论在ESCs的分化过程中，synapsin-I的表达存在特定的时空规律，并与ESCs的形态学改变相关，提示synapsin-I可能对ESCs在神经分化过程中的形态分化起着重要的作用。