In order to explore further the regulatory factors to the potentiality in inducing osteogenesis by fibroblasts, the fibroblasts were isolated, and purified from human skin, and were grown in incubation in the media of EGF, IL-6, TNF-alpha and BMP2 at different concentrations for two weeks, then, the markers for osteogenic features were investigated by biochemistry, histochemistry and electron microscopic observations. It was found that the combined use of TNF-alpha and BMP2 could stimulate fibroblasts to secrete alkaline phosphatase, osteocalcin and collagen, and the morphological changes of the fibroblasts were also very striking. In the extracellular matrix, the collagen fibrils, with or without periodicity, were arranged regularly or randomly oriented, and numerous minute calcium granules were interspersed among them. The fibroblasts were interwoven one on top of another in the form of multilayer structure and on the surface, there were secreting granules and piling up of calcium crystals which coalessed steadily and increased in size in forming bony nodules. It was considered that TNF-alpha and BMP2 were capable of inducing the fibroblasts to form bone.
Objective To investigate the possibility of ectomesenchymal stem cell of human embryo facial process in differentiating into osteoblasts.Methods Ectomesenchymal stem cells of human embryo facial process were isolated and cultured in mineralized promoting solution containing 10 mmol/L β-glycerophosphate, 100 μg/ml ascorbic acid and 10 nmol/L dexamethasone supplemented with 15% FBS. The morphological change was observed by phase contrast microscopy. The characteristics of cells was identified by immunohistochemistry assay. Alkaline phosphatase activity was tested and the form of mineralized nodules was tested with Von Kossa staining. The expression of osteocalcin was identified by RT-PCR.Results There were significant changes in the shape of the cells after 3 days cultured in mineralized promoting solution. The cells became larger and the shape changed from fibroblast-like to multilateral. The result for anticollogen typeⅠstaining was positive. The alkaline phosphatase activity increased. Mineralized nodules were formed aftercultured 25 days by Von Kossa staining. RT-PCR assay showed induced cells expressed osteocalcin.Conclusion Ectomesenchymal stem cells of humanembryo facial process can be induced to differentiate into osteoblasts by mineralized promoting solution.
Objective To explore the in vitrodifferentiation of the rat mesenchymal stem cells (MSCs ) into the skeletal muscle cells induced by the myoblast differentiation factor (MyoD) and 5-azacytidine. Methods The MSCs were taken from the rat bone marrow and the suspension of MSCs was made and cultured in the homeothermia incubator which contained 5% CO2at 37℃. The cells were observed under the inverted phase contrast microscope daily. The cells spreading all the bottom of the culture bottle were defined as onepassage. The differentiation of the 3rd passage of MSCs was induced by the combination of 5-azacytidine, MyoD, transforming growth factor β1, and the insulin like growth factor 1. Nine days after the induction, the induced MSCs were collected, which were analyzed with the MTT chromatometry, theflow cytometry, and the immunohistochemistry. Results The primarily cultured MSCs grew as a colony on the walls of the culture bottle; after the culture for 5-7 days, the cells were shaped like the fibroblasts, the big flat polygonal cells, the medium sized polygonal cells, and the small triangle cells; after the culture for 12 days, the cells were found to be fused, spreadingall over the bottle bottom, but MSCs were unchanged too much in shape. After the induction by 5-azacytidine, some of the cells died, and the cells grew slowly. However, after the culture for 7 days, the cells grew remarkably, the cell volume increased gradually in a form of ellipse, fusiform or irregularity. After theculture for 14 days, the proliferated fusiform cells began to increase in a great amount. After the culture for 18-22 days, the myotubes increased in number and volume, with the nucleus increased in number, and the newly formed myotubes and the fusiform myoblst grew parallelly and separately. The immunohistochemistry for MSCs revealed that CD44 was positive in reaction, with the cytoplasm ina form of brown granules. And the nucleus had an obvious border，and CD34 was negative. The induced MSCs were found to be positive for desmin and specific myoglobulin of the skeletal muscle. The flow cytometry showed that most of the MSCs and the induced MSCs were in the stages of G0/G1，accounting for 79.4％ and 62.9％，respectively; however, the cells in the stages of G2/S accounted for 20.6％ and 36.1％. The growth curve was drawn based on MTT，which showed that MSCs weregreater in the growth speed than the induced MSCs. The two kinds of cells did not reach the platform stage，having a tendency to continuously proliferate.ConclusionIn vitro,the rat MSCs can be differentiated into the skeletal muscle cells with an induction by MyoD and 5-azacytidine, with a positive reaction for the desmin and the myoglobulin of the skeletal muscle. After the induction, the proliferation stage of MSCs can be increased, with a higher degree of the differentiation into the skeletal muscle.
Objective To investigate auto-cortex of crystalline lens-induced neovascular epiretinal membrane（NVERM）by micro-injuring posterior c apsule of crystalline lens. Methods twenty four C57BL/6 mouse between 4-6 weeks were selected, and divided into two groups randomly: auto-cortex of crystalline group and the control group. The auto-cortex of crystalline group was treated by penetrating the posterior capsule of lens and washing out the lens cortex into the mouse vitreous using PBS (phosphate buffered solution), while the control group were injected PBS into vitreous merely. Clinical change s were followed by slit-lamp examination and photograph. The eye balls were enu cleated at the day of 3, 7, 14 and 28 after operation. Both HE and immunohistoch emistry were used to detect the pathological changes. Results postoperative one to three days, 11 of 12 mouse in autocortex of crystalline g roup, lens appear to alba turbid at different levels one after another, and then develop into highdensity chinaware white. Postoperative (po) three days, HE s taining shows cortex of lens debris transmigrated in vitreous cavity, and some o f which approached to internal limiting membrane and lead it to rough and discon tinue; Po7-14 days, the capillary in retina expanded, migrated and broke though t internal limiting membrane which got to the pro retina and became the new ves sels. And typical NVERM were observed. Po28 days, some vascularslike structure formed in vitreous cavity. None of mouse in control group developed NVERM. Conclusion Auto-cortex of crystalline lens can induced neovascular epiretinal membrane in C57BL/6 mouse. (Chin J Ocul Fundus Dis,2008,24:118-121)
目的 探讨HIF-1α和BAK蛋白在胃癌中的表达情况，以及二者在胃癌中的相互关系及作用。方法 应用免疫组化SABC染色法检测80例胃癌组织和20例正常胃组织中的HIF-1α和BAK蛋白的表达情况。结果 胃癌中HIF-lα和BAK蛋白的表达阳性率分别为56.3%（45/80）和67.5%（54/80），而在胃正常组织中分别为5.0%（1/20）和20.0%（4/20），二者在胃癌中的表达显著高于胃正常组织，其差异有统计学意义（P＜0.05）。HIF-1α蛋白表达与胃癌组织的浸润范围、分化程度及淋巴结转移有关（P＜0.05），与临床分期、年龄及性别无关（P＞0.05）；BAK蛋白表达与胃癌浸润及分化程度相关（P＜0.05），与淋巴结转移、临床分期、年龄及性别无关（P＞0.05）。胃癌组织中HIF-1α与BAK蛋白的阳性表达之间呈正相关（列联系数r=0.056，P＜0.05）。结论 HIF-1α与BAK蛋白在胃癌的临床分期及浸润转移中存在关系，这对于研究胃癌的发生和发展，以及对于探索以二者为靶点的抗肿瘤治疗有重要意义。
目的 研究活动期多发性肌炎患者外周血白细胞细胞因子信号转导蛋白抑制因子（SOCS）1、SOCS2、SOCS3和细胞因子诱导的含SH2区域蛋白1（CIS）与正常人表达的差异，探讨SOCS在多发性肌炎发病中可能的作用。 方法 2011年6月－12月，采用实时荧光定量聚合酶链反应法检测了14例活动期多发性肌炎患者和14例正常人外周血白细胞中SOCS1、SOCS2、SOCS3和CIS1基因的相对表达量。 结果 与对照组相比，多发性肌炎症患者外周血白细胞基因SOCS 1～3表达明显降低（P值均＜0.05），CIS1基因的表达较对照组明显升高（P＜0.05），差异有统计学意义。 结论 SOCS基因家族可能参与了多发性肌炎的发病，该蛋白分子家族的成员可能会成为多发性肌炎治疗的一种新的候选基因。
Objective To investigate the method and conditions of isolation，proliferation of multipotent mesenchymal stem cells（MSCs）from human umbilical cord blood in vitro, and to induce osteogenic and adipogenic differentiation directly for identification. Methods Human umbilical cord blood was collected in asepsis condition, isolated by density gradient centrifugation,or sedimented red cell with methylcellulose, and then the same centrifugation was done, or obtained by negative immunodepletion of CD34+. These isolated mononuclear cells were used to carry on plastic adherent culture. To obtain single cellderived colonies, these cells were proliferated clonally in medium which consists of L-DMEM orMesencultTM medium and 10% fetal calf serum（FCS） respectively, then their differentiation potentiality to osteoblasts and lipoblasts was tested. Results The mononuclear cells isolated by sedimented and centrifugated way cultured in MesencultTM medium and 10%FCS were most available. These adhesive cells could become obviously short rodshape or shuttle-shape cells after 5-7 days.The colonies form well in 3rdpassage cells. The mononuclear cells obtained by onlycentrifugalized in density gradient were hard to form colony, isolated by immunomagnetic beads were hard to culture. The surface antigens of these colonies cells presented CD29, CD59, CD71 but not CD34,CD45 and HLADR etc. The colony cells differentiating into osteoblasts that produce mineralized matrices, stained by alizarin red, and differentiating into adipocytes that accumulate lipid vacuoles, stained by oil red. Conclusion MSCs can be isolated from human umbilical cord blood and proliferate it in vitro. The way that mononuclear cells are sedimented red cell by methylcellulose and cultured by MesencultTM medium and 10% FCS is the valid method of isolation. Proliferation colonies cells present matrix cell immunophenotypes, and candifferentiate into osteoblasts and adipocytes.
Objective To establ ish a new induction method from human embryonic stem cells (hESCs) differentiating into hepatocyte-l ike cells using an adherent culture system with single-step induction. Methods Undifferentiated hESCs were cultured on Matrigel-coated culture plates for 4 days, hepatic differentiation was initiated at 60%-70% confluence by adding Activin A for 5 days. Then the induction medium was replaced by hepatocyte induction medium (HIM) supplemented with fibroblast growth factor 1 (FGF-1) and bone morphogenetic protein 4 (BMP-4) for another 6 days. Finally, the cells were treated with HIM adding hepatocyte growth factor (HGF) and Oncostatin M (OSM) for 5-7 days. The characteristics of differentiated cells were determined by morphology, immunofluorescence staining, RT-PCR, and Periodicacid- Schiff (PAS) test. Results Differentiated cells treated with Activin A, FGF-1, BMP-4, HGF, and OSM sequentially were morphologically larger and became spherical, oval or polygon. Some cells had 2 or 3 nuclei, suggesting that the cells have a hepatocyte-l ike morphology. Differentiated cells at first induction stage could be stained positive by SOX17 and Forkhead (FOX)A2 after induction by Activin A. Then they turned to be α fetoprotein (AFP) and α1 antitripsin positive cells at second induction stage after induction by FGF-1 and BMP-4. Finally, the differentiated cells treated with HGF and OSM showed PAS possitive for glycogen detection. The differentiated cells at various stages also expressed at early (SOX17, FOXA2, and GATA-4),middle (AFP, albumin, and cytokeratin 18), and mature (alcohol dehydrogenase 1C and Cytochrome P4501B1) stage hepatic genes, respectively. Conclusion Using a simple-step induction method and by suppl ied with cytokines consequently, hESCs can be induced to differentiate into hepatocyte-l ike cells. The differentiation method can provide seed cells for hepatic tissue engineering or cell-therapy.