骨髓间充质干细胞在小鼠肝脏局部分化为肝细胞的实验研究

目的 探讨小鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）在肝内分化为肝细胞的可行性。方法腹腔注射丙烯醇致雌性C57BL／6小鼠全肝损伤后局部接受雄性同品系绿色荧光蛋白（green fluorescence protein，GFP）转基因小鼠骨髓间充质干细胞移植。移植后3周取骨髓嵌合小鼠肝脏做冰冻切片，荧光显微镜下观察GFP阳性细胞在肝内分布，石蜡切片行GFP抗体和白蛋白（albumin，Alb）抗体免疫组化双标法检测GFP阳性细胞在受体小鼠肝内的分化情况。结果移植后3周小鼠肝内可见GFP阳性细胞，并可见GFP和Alb双阳性细胞。结论异体骨髓间充质干细胞可在小鼠肝内分化为表达Alb的肝细胞，为肝组织的再生和修复提供了新思路，显示了其在肝病治疗中的应用潜力。     

甲基-β-环糊精对Ⅱ型肺泡上皮细胞增殖与转分化的影响

目的 观察甲基-β-环糊精(MβCD)破坏脂质微区结构对Ⅱ型肺泡上皮细胞(AEC Ⅱ)增殖、转分化及细胞周期的影响.方法 采用MβCD破坏体外培养的AEC Ⅱ细胞膜脂质微区(MβCD干扰组),以必需基本培养基(DMEM)作为对照.用血细胞计数器计数培养细胞,四甲基偶氮唑盐(MTT)比色法检测细胞增殖能力,流式细胞仪检测细胞周期,免疫荧光双标和蛋白质免疫印迹法(Western blotting)检测AEC Ⅱ特异性肺泡表面活性蛋白C(SP-C)及Ⅰ型肺泡上皮细胞(AEC Ⅰ)特异性水通道蛋白5(AQP5)的表达.结果 与对照组比较,MβCD组干扰后24、48、72 h细胞数及增殖能力显著降低[细胞数(×106/rml):2.74±0.56比8.05±0.92,4.45±0.68比10.52±0.81,7.82±0.59比11.39±0.81;MTT结果(A值):0.25±0.20比0.45±0.02,0.35±0.03比0.54±0.28,0.48±0.04比0.59±0.05,均P＜0.01＝;MβCD组干扰后24h G0/G1期细胞比例显著增多,S期细胞比例显著减少[G0/G1期:(60.06±1.65)%比(43.43±3.59)%;S期:(16.20±2.17)%比(34.07±2.63)%,均P＜0.05＝;MβCD组干扰后48 h、72 h SP-C蛋白表达明显增多(0.54±0.04比0.47±0.03,0.19±0.03比0.06±0.02),AQP5蛋白表达明显减少(0.30±0.04比0.43±0.06,0.39±0.04比0.59±0.04,P＜0.05或P＜0.01＝.结论 MβCD破坏体外培养AEC Ⅱ脂质微区结构后,细胞发生G1期阻滞,细胞增殖和转分化受到抑制.

Abstract：

Objective To study the destructive effects of the membrane lipid microdomain with methyl-β-cyclodextrin (MβCD) on the proliferation, transdifferentiation and cell cycle of type Ⅱ alveolar epithelial cell (AEC Ⅱ ). Methods The membrane lipid microdomain of AEC Ⅱ was destroyed by MβCD (MβCD interference group) in vitro, and then cultured with DMEM as control. Cell number was counted with hernacytometer; the proliferation rate was measured by methyl thiazolyl tetrazolium (MTT); flow cytometry was used to assay the cell cycle. The expressions of AEC Ⅱ -specific surfactant protein-C (SP-C)and AEC Ⅰ-specific aquaporin-5 (AQP5) were detected by immunofluorescence and Western blotting analyses. Results Compared with control group, cell number and the cell proliferation was decreased in MβCD interference group at 24, 48 and 72 hours after interaction [cell numbers (× 106/ml) : 2. 74±0. 56 vs.8. 05±0. 92, 4. 45±0. 68 vs. 10. 52±0. 81, 7.82±0. 59 vs. 11.39±0. 81; MTT results (A value) : 0. 25±0. 20 vs. 0. 45±0. 02, 0. 35±0.03 vs. 0. 54±0. 28, 0. 48±0. 04 vs. 0. 59±0. 05, all P＜0. 01]. MβCD could increase the percentage of cells in G0/G1 phases and decreased the percentage in S phases at 24 hours [G0/G1phases: (60. 06±1.65)% vs. (43.43±3. 59)% ; S phases: (16. 20±2.17)% vs. (34. 07±2. 63)%, both P＜0. 05]. Incubation of AEC Ⅱ with MβCD resulted in up-regulation of the expression of SP-C (0. 54±0. 04vs. 0. 47±0. 03, 0. 19±0. 03 vs. 0.06±0. 02) and down-regulation of AQP5 (0. 30±0. 04 vs. 0. 43±0. 06,0. 39±0. 04 vs. 0. 59±0. 04) at 48 hours and 72 hours after interaction (P＜0. 05 or P＜0. 01). Conclusion The destruction of membrane lipid microdomain by the MβCD can inhibi± proliferation and transdifferentiation of AEC Ⅱ , and induce cell cycle arrest in G1 phase.     

In vitro generation of insulin-producing beta cells from adult exocrine pancreatic cells

Aims/hypothesis Transplantation of insulin-producing beta cells from donors can cure diabetes, but they are available in insufficient quantities. In this study, we investigated the possibility of generating insulin-producing cells from adult rat exocrine cells cultured in the presence of growth factors.Methods Rat exocrine pancreatic cells were isolated and treated in vitro with epidermal growth factor (EGF) and leukaemia inhibitory factor (LIF). Analysis was performed by immunocytochemistry, DNA measurement and radioimmunoassay. Cells were transplanted to alloxan-treated (70 mg/kg) nude mice and glycaemia was monitored for 21 days. Nephrectomy was performed on day 15.Results In a 3-day culture period, addition of LIF plus EGF to the medium resulted in an 11-fold increase of the beta cell mass. This could not be attributed to the very low mitotic activity of contaminating beta cells. Furthermore, when contaminating beta cells were initially destroyed with alloxan, this effect was even more pronounced. The newly formed cells secreted insulin in response to glucose and were immunoreactive for C-peptide-I, Pdx-1 and GLUT-2, which are characteristics of mature beta cells. Electron microscopy showed that they also contained insulin-immunoreactive secretory granules. Some insulin-positive cells were immunoreactive for amylase and cytokeratin-20, or were binucleated, which are characteristics of exocrine cells. The cells were able to restore normoglycaemia when transplanted to alloxan-diabetic mice, and hyperglycaemia recurred upon removal of the graft.Conclusions/interpretation Our study shows that functional beta cells can be generated from exocrine tissue by transdifferentiation and thereby may offer a new perspective for beta cell therapy.     

支气管肺发育不良新生鼠模型肺泡上皮细胞标志物的表达变化及意义

目的 研究高氧致支气管肺发育不良(bronchopulmonary dysplasia,BPD)新生鼠模型中Ⅰ型肺泡上皮细胞(typeⅠalveolar epithelial cells,AECⅠ)和Ⅱ型肺泡上皮细胞(typeⅡalveolar epithelial cells,AECⅡ)特异性标志物表达变化及其意义.方法 80只新生Wistar大鼠,于生后12 h内随机分为模型组(吸入氧浓度为85%)和对照组(吸入空气),每组40只.于暴露第7、14、21天,分别进行肺组织HE染色观察病理学改变,免疫荧光双标染色观察AECⅠ标志物水通道蛋白5(aquaporin 5,AQP5)及AECⅡ标志物表面活性蛋白C(surfactant protein C,SP-C)表达及定位,Western blot方法检测AQP5和SP-C的蛋白表达水平,real-time PCR方法检测AQP5和SP-C的mRNA表达水平.结果 模型组大鼠肺组织表现出肺泡数目减少,体积增大,结构简单化,肺泡间隔增厚,次级分隔减少等肺泡化障碍表现.免疫荧光双标染色可见,模型组AQP5及SP-C表达明显增多,表达位置紊乱,且双染细胞/SP-C阳性细胞的比例明显增高(P<0.001).与对照组比较,模型组中AQP5及SP-C蛋白表达从高氧暴露7 d开始增加,增高的趋势持续至21 d.模型组中mRNA表达水平,AQP5从暴露7 d开始,SP-C从14 d开始较对照组明显升高(P<0.05),且两组间差异随高氧暴露时间延长更加明显(P<0.05).结论 暴露高氧中的新生鼠BPD模型肺组织中,AECⅠ标志物AQP5及AECⅡ标志物SP-C均表达上调,发生转分化的AECⅡ明显增多,表明在BPD肺损伤后的修复中存在AECⅡ过度转分化现象.     

雌激素诱导皮肤成纤维细胞向肌成纤维细胞表型转分化的机制

目的探讨雌激素诱导皮肤成纤维细胞向肌成纤维细胞转分化的可能途径和调控机制。方法将人皮肤成纤维细胞分为6组：对照组（A组）、雌激素组（B组）、雌激素＋ICI-182780组（C组）、雌激素＋SB203580组（D组）、雌激素＋PD98059组（E组）和雌激素＋SP600125组（F组）。各组细胞经同步化处理后,直接以雌激素刺激或经上述各激酶抑制剂预处理后再以雌激素刺激。收集细胞,一部分以单细胞逆转录聚合酶链反应（RT-PCR）检测α平滑肌肌动蛋白（alpha smooth muscleactin,（2-SMA）阳性表达百分比,另一部分细胞抽提总RNA后采用实时荧光定量RT—PCR检测α—SMA的表达水平变化。结果B组的α—SMA表达水平和阳性百分比与A组比较显著升高（P〈0．01）,而C组和F组与B组比较α—SMA表达水平和阳性百分比升高的作用则被显著抑制（P〈0．05）。结论在雌激素诱导成纤维细胞向肌成纤维细胞的转分化过程中,雌激素G受体及JNK—MAPK信号转导途径起到重要作用。     

周细胞在肾间质纤维化中的研究进展

周细胞是与内皮细胞接触并部分包埋于血管基底膜的间充质细胞.周细胞具有分化能力,其表面标志物根据不同组织、不同发育阶段、不同病理生理环境而发生变化.研究表明,周细胞是肾脏肌成纤维细胞的主要来源,多种信号通路参与了周细胞/成纤维细胞转分化为肌成纤维细胞的过程.本文综述了周细胞在肾间质纤维化发病机制中的研究进展,提出了周细胞...     

乙醛对大鼠肝星状细胞株HSC—T6激活、增殖及转分化的影响

目的：观察乙醛对大鼠肝星状细胞株（HSC-T6）激活、增殖并转分化为肌成纤维细胞的影响。方法：HSC—T6细胞常规培养及乙醛处理;VG染色法形态学及MTT法检测细胞增殖;ELISA法检测I型胶原蛋白（TIMPI）、纤维粘连素（FN）的表达;Westernblottin譬检测细咆内效应蛋白0L—SMA的表达。结果：VG染色乙醛束Ij激明显促进HSC贴壁及重叠、明显促进HSC增殖和显著增加胶原纤维蛋白的表达;MTT检测结果发现乙醛刺激明显增强HSC活性（P〈0．05）;ELISA结果表明乙醛刺激组较对照组TIMPI和FN的表达明显上调（P〈0．05）;Western blotting检测结果显示,乙醛刺激明显促进仅一SMA表达（P〈0．05）。结论：乙醛刺激可明显激活HSC～T6细胞株,并促进其增殖及转分化为肌成纤维细胞。     

PAX6基因过表达促进大鼠骨髓内皮祖细胞向角膜内皮细胞分化

目的 通过研究大鼠骨髓内皮祖细胞转染PAX6基因后角膜内皮细胞相关特征性基因的表达,探讨其作为角膜内皮细胞移植的种子细胞的可能性.方法 采用密度梯度离心法分离出大鼠骨髓单核细胞,接种到纤连蛋白铺层的细胞皿,以EGM-2培养液选择培养,获得大鼠骨髓内皮祖细胞.通过免疫荧光染色检测CD31、CD34、CD133、LDL、U...     

肝细胞生长因子对转化生长因子诱导肺泡Ⅱ型上皮细胞转分化的影响

目的:研究肝细胞生长因子(HGF)对转化生长因子β1(TGFβ-1)诱导肺泡上皮细胞转分化的影响。方法:应用TGF-β1诱导肺泡Ⅱ型上皮细胞转分化。将人肺腺癌A549细胞株培养液分为4组:①对照组;②HGF组;③TGFβ-1组;④HGF+TGFβ-1组,应用倒置相差显微镜观察细胞形态变化;免疫细胞化学方法检测肌成纤维细胞特异性标志物α-平滑肌肌动蛋白(α-SMA)、肺泡上皮细胞特异性标志物肺泡表面活性蛋白A(SP-A)的表达。结果:①TGF-β1组肺泡上皮细胞从原来典型的上皮细胞形态转变为长梭形肌成纤维细胞样形态。②TGF-β1组的细胞α-SMA的表达明显增加,SP-A的表达明显减低。③HGF组的细胞形态学、α-SMA、SP-A的表达与对照组无明显差异。④HGF+TGFβ-1组,多数细胞保持原来肺泡上皮细胞的形态,α-SMA表达明显低于TGFβ-1组、SP-A表达明显高于TGFβ-1组。结论:HGF能够在一定程度上负性调控TGFβ-1诱导的肺泡上皮细胞的转分化。