Bone marrow stromal cell versus neural stem cell transplantation in a C6 glioma rat model

BACKGROUND： Embryonic neural stem cells （NSCs） have provided positive effects for the treatment of glioma. However, the source for embryonic NSCs remains limited and high amplification conditions are required. Bone marrow stromal cells （BMSCs） have been proposed for the treatment of glioma. OBJECTIVE： To investigate biological changes in NSCs and BMSCs following transplantation into rat models of glioma. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Embryonic Stem Cell Research Laboratory of Yunyang Medical College from February 2006 to August 2008. MATERIALS： The rat C6 glioma cell line was purchased from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; mouse anti-bromodeoxyuridine （BrdU） monoclonal antibody and Cy3-1abeled goat anti-mouse IgG antibody was purchased from Upstate, USA. METHODS： A total of 95 Sprag6ue Dawley rats were randomly assigned to three groups： NSC （n = 35）, transplanted with 〉 6 × 10^6 NSCs via left medial hind limb; BMSC （n = 35）, transplanted with 〉 1 × 10^6 BMSCs via left medial hind limb; model group （n = 25）, injected with the same volume of 0.1 mmol/L phosphate buffered saline. MAIN OUTCOME MEASURES： Gliomal growth and size were assessed by nuclear magnetic resonance, and glioma morphological features were observed following hematoxylin-eosin staining and BrdU immunohistochemistry 3 and 4 weeks following transplantation. RESULTS： The average survival of rats in the BMSC, NSC, and model groups was 4.03, 4.28, and 3.88 weeks. At 3 weeks, there was no significant difference in the average glioma diameter between the BMSC and model groups （P 〉 0.05）. However, gliomal diameter was significantly decreased in the NSC group compared with the model group （P 〈 0.05）. At 4 weeks, there was no statistical difference between the groups （P 〉 0.05）. BrdU immunohistochemistry revealed that BMSCs and NSCs appeared to migrate to the gliomas. CONCLUSION： NSCs inhibited glioma cell growth and prolonged rat survival. BMSCs did not significantly suppress glioma cell growth.

Cell viability and dopamine secretion of 6-hydroxydopamine-treated PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells

In the present study, PC12 cells induced by 6-hydroxydopamine as a model of Parkinson＇s Disease, were used to investigate the protective effects of bone marrow-derived mesenchymal stem cells bone marrow-derived mesenchymal stem cells against 6-hydroxydopamine-induced neurotoxicity and to verify whether the mechanism of action relates to abnormal a-synuclein accumulation in cells Results showed that co-culture with bone marrow-derived mesenchymal stem cells enhanced PC12 cell viability and dopamine secretion in a cell dose-dependent manner. MitoLight staining was used to confirm that PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells demonstrate reduced levels of cell apoptosis. Immunocytochemistry and western blot analysis found the quantity of α-synuclein accumulation was significantly reduced in PC12 cell and bone marrow-derived mesenchymal stem cell co-cultures. These results indicate that bone marrow-derived mesenchymal stem cells can attenuate 6-hydroxydopamine-induced cytotoxicity by reducing abnormal α-synuclein accumulation in PC12 cells.

Reduced glutathione alleviates the toxic effect of 6-hydroxydopamine on bone marrow stromal cells

We studied the effect of reduced glutathione on bone marrow stromal cells （BMSCs） treated with 6-hydroxydopamine （6-OHDA）, which shows a toxic effect on dopaminergic neurons. The proliferation of BMSCs treated with 6-OHDA decreased, while that of BMSCs treated with reduced glutathione increased. The proliferation of BMSCs treated with both 6-OHDA and reduced glutathione was significantly higher compared with that treated with 6-OHDA alone. These findings indicate that reduced glutathione alleviates the toxic effect of 6-OHDA on BMSCs.

肾癌患者中外周血淋巴细胞亚群、T-reg、MDSC的表达及临床意义

目的 采用流式细胞术分析肾癌患者MDSC、T-reg、CD_(3)^(+)CD4^(+)T细胞、CD_(3)^(+)CD_(8)^(+)T细胞和CD3-CD_(1)^(+)6CD_(5)^(+)6细胞的表达情况,以及各项指标与肾癌进展的相关性,探讨各项指标在肾癌治疗预后中的相关价值。方法 选取2021年9月至2022年11月住院的肾癌患者44例为肾癌组,选取29例健康人作为对照组。清晨,空腹采集3 mL静脉血。所有肾癌患者均已通过组织病理学诊断。采用全血9色荧光11参数流式细胞术检测肾癌患者和健康体检者MDSC、Treg、CD_(3)^(+)CD_(4)^(+)T细胞、CD_(3)^(+)CD_(8)^(+)T细胞、CD_(3)^(+)CD_(4)^(+)T细胞/CD_(3)^(+)CD_(8)^(+)T细胞比值和CD3-CD_(1)^(+)6CD_(5)^(+)6细胞表达水平。收集患者的临床数据:包括年龄、性别、BMI、临床分期、肿瘤大小、病理类型。结果 肾癌组与对照组之间性别、年龄、BMI差异均无统计学意义(P>0.05)。肾癌组与对照组之间外周血CD_(3)^(+)CD_(4)^(+)、CD_(3)^(+)CD_(8)^(+)、CD_(3)^(+)CD_(4)^(+)/CD_(3)^(+)CD_(8)^(+)、NK、T-reg、PMN-MDSC、M-MDSC水平差异有统计学意义(P<0.05)。肾癌患者CD_(3)^(+)CD_(4)^(+)、CD_(3)^(+)CD_(8)^(+)的表达水平低于对照组(P<0.05),T-reg、PMN-MDSC、M-MDSC的表达水平高于对照组(P<0.05)。肾癌周血中患者外的CD_(3)^(+)CD_(4)^(+)、CD_(3)^(+)CD_(8)^(+)、CD_(3)^(+)CD_(4)^(+)/CD_(3)^(+)CD_(8)^(+)、NK、T-reg、PMN-MDSC、M-MDSC的表达水平与性别、年龄、BMI、病理学类型水平无显著相关(P>0.05)。CD_(3)^(+)CD_(8)^(+)T、CD_(3)^(+)CD_(4)^(+)/CD_(3)^(+)CD_(8)^(+)、NK与肿瘤大小、临床分期之间无明显相关性(P>0.05),CD_(3)^(+)CD_(4)^(+)、M-MDSC、PMN-MDSC、Treg与肿瘤大小、临床分期有相关性(P<0.05)。经Logistic回归分析结果显示:外周血CD_(3)^(+)CD_(4)^(+)淋巴细胞低表达,T-reg、PMN-MDSC、M-MDSC的高表达可能与肾癌患者分期相关(P<0.05)。通过构建ROC曲线结果显示:按照临床分期进行分组,“0”为Ⅰ期、Ⅱ期组,“1”为,Ⅲ期、Ⅳ期组,T-reg、PMN-MDSC、M-MDSC血清指标水平检测肾细胞Ⅲ期、Ⅳ期患者的AUC均>0.70,均有一定的评估价值。T-reg、PMN-MDSC、M-MDSC、M-MDSC+Treg、PMN-MDSC+Treg与参考线比较差异有统计学意义(P<0.05),其中PMN-MDSC+Treg联合检测的差异性最为显著(P<0.05)。结论 肾癌患者与对照组相比CD_(3)^(+)CD_(4)^(+)T细胞、CD_(3)^(+)CD_(8)^(+)T、CD3-CD_(1)^(+)6CD_(5)^(+)6细胞有明显降低,CD4/CD8比值升高,PMN-MDSC、M-MDSC和Treg细胞明显升高,提示机体的免疫功能受损。CD_(3)^(+)CD_(4)^(+)、PMN-MDSC、M-MDSC、T-reg在肿瘤大小、临床分期中表达水平不同,肿瘤直径越大,临床分期越晚,PMN-MDSC、M-MDSC、T-reg表达水平越高。外周血MDSC、T-reg与肾癌临床分期相关,外周血MDSC、T-reg对于肾癌的发生、发展和预后可能有一定的提示意义。

微小RNA-22-3p调控Kruppel样因子6表达对骨髓间充质干细胞心肌样分化的影响

目的探究微小RNA-22-3p(miR-22-3p)调控Kruppel样因子6(KLF6)表达对骨髓间充质干细胞(BMSC)心肌样分化的影响。方法分离并培养大鼠BMSC,将第3代BMSC分为对照组、5-氮杂胞苷(5-AZA)组、模拟物阴性对照组、miR-22-3p模拟物组、miR-22-3p模拟物+空载质粒组、miR-22-3p模拟物+KLF6过表达质粒组;实时荧光定量PCR(qRT-PCR)检测细胞中miR-22-3p及KLF6表达;免疫荧光化学染色法检测细胞结蛋白(Desmin)、肌钙蛋白T(cTnT)、间隙连接蛋白43(Cx43)表达;Western blot检测Desmin、cTnT、Cx43、KLF6蛋白表达;流式细胞术检测BMSC凋亡情况;采用双荧光素酶报告基因实验分析miR-22-3p和KLF6的靶向关系。结果与对照组比较,5-AZA组BMSC中miR-22-3p(q=7.971,P<0.001)、Desmin(q=7.876,P<0.001)、cTnT(q=10.272,P<0.001)、Cx43表达(q=6.256,P<0.001)以及细胞凋亡率(q=12.708,P<0.001)显著增加,而KLF6 mRNA(q=20.850,P<0.001)及蛋白表达(q=11.080,P<0.001)显著降低;与5-AZA组和模拟物阴性对照组比较,miR-22-3p模拟物组BMSC中miR-22-3p(q=3.591,P<0.001;q=11.650,P<0.001)、Desmin(q=5.975,P<0.001;q=13.579,P<0.001)、cTnT(q=7.133,P<0.001;q=17.548,P<0.001)、Cx43表达(q=4.571,P=0.037;q=11.068,P<0.001)显著增加,而KLF6 mRNA(q=7.384,P<0.001;q=28.234,P<0.001)及蛋白表达(q=4.594,P=0.036;q=15.945,P<0.001)显著降低,miR-22-3p模拟物组细胞凋亡率显著低于5-AZA组(q=8.216,P<0.001);与miR-22-3p模拟物+空载质粒组比较,miR-22-3p模拟物+KLF6过表达质粒组KLF6 mRNA(q=23.891,P<0.001)及蛋白表达(q=13.378,P<0.001)显著增加,Desmin(q=9.505,P<0.001)、cTnT(q=10.985,P<0.001)、Cx43表达(q=8.301,P<0.001)显著降低,细胞凋亡率增加(q=4.713,P=0.029)。双荧光素酶报告基因实验发现KLF6是miR-22-3p的潜在靶基因。结论miR-22-3p通过抑制KLF6表达来促进BMSC心肌样分化。

APE1/Ref-1 siRNA抑制多发性骨髓瘤骨髓基质细胞IL-6及IL-8分泌的体外研究

目的体外通过APE1/Ref-1 siRNA敲低多发性骨髓瘤骨髓基质细胞(bone marrow stromal cells,BMSCs)APE1/Ref-1的表达,观察BMSCs的增殖及分泌细胞因子IL-6、IL-8的变化,初步探讨BMSCs APE1/Ref-1表达的功能特点。方法通过免疫细胞化学染色法定量检测35例初治、11例复发/难治多发性骨髓瘤患者及10例正常人BMSCs APE1/Ref-1的表达特点及其差异,经Adv5-APE1/Ref-1 siRNA感染BMSCs后,流式细胞仪检测BMSCs细胞周期的变化;ELISA法检测BMSCs分泌IL-6、IL-8的水平变化情况。结果多发性骨髓瘤BMSCs的APE1/Ref-1蛋白阳性表达率显著高于正常BMSCs APE1/Ref-1蛋白阳性表达率(P<0.05),且多发性骨髓瘤BMSCs的APE1/Ref-1呈细胞核及核浆共同表达方式。Adv5-APE1/Ref-1 siRNA感染敲低多发性骨髓瘤及正常BMSCs APE1/Ref-1的表达量呈进行性减少(P<0.01),同时发现APE1/Ref-1 siRNA对多发性骨髓瘤BMSCs抑制作用更明显。Adv5-APE1/Ref-1 siRNA感染BMSCs后对正常人及骨髓瘤患者BMSCs分泌细胞因子IL-6、IL-8的量有显著的抑制作用,特别是感染72h后,骨髓瘤患者及正常人的BMSCs分泌IL-6[初治患者(246.29±46.51)pg/ml,复发/难治患者(365.09±75.25)pg/ml]、IL-8[初治患者(118.77±18.08)pg/ml,复发/难治患者(188.71±33.76)pg/ml]的量最低,与其他时段比较差异有统计学意义(P<0.01)。结论多发性骨髓瘤BMSCs APE1/Ref-1的表达特点不同于正常BMSCs,可能导致其功能差异;APE1/Ref-1 siRNA敲低了MMBMSCs APE1/Ref-1的表达,同时明显抑制了其IL-6、IL-8的分泌,减少了对骨髓瘤细胞的促增殖和凋亡作用。

胰岛素对高糖环境下大鼠颌骨骨髓基质细胞成骨分化过程中peroxiredoxin-6表达的影响

目的:探讨胰岛素对高糖环境下大鼠颌骨骨髓基质细胞(rat bone marrow stromal cells,rBMSCs)成骨分化过程中peroxiredoxin-6表达的影响。方法:从Wistar大鼠下颌骨中分离培养骨髓基质细胞,分别于不同糖浓度(5.5、25、45 mmol/L)及胰岛素(0、10-5mol/L)的干预下成骨诱导(5.5 mmol/L组、5.5 mmol/L+Ins组、25 mmol/L组、25 mmol/L+Ins组、45 mmol/L组、45 mmol/L+Ins组)。成骨诱导21 d后,进行茜素红染色,矿化结节定量分析。成骨诱导1、3、7、14、21 d后,用ELISA试剂盒检测peroxiredoxin-6蛋白的表达。采用SPSS15.0软件包对数据进行统计学分析。结果:45 mmol/L高糖环境显著抑制rBMSCss的矿化,胰岛素可改善高糖对rBMSCs矿化的抑制。45 mmol/L组peroxiredoxin-6的表达较5.5 mmol/L组和25 mmol/L组均显著下降。矿化诱导第21天,各组peroxiredoxin-6蛋白的表达均达到最高峰。在25 mmol/L和45 mmol/L的糖浓度下,10-5mol/L的胰岛素可显著上调rBMSCs peroxiredoxin-6蛋白的表达。结论:高糖环境抑制peroxiredoxin-6蛋白的表达,胰岛素可改善高糖环境对peroxiredoxin-6蛋白在大鼠颌骨骨髓基质细胞成骨分化过程中表达的影响。Peroxiredoxin-6蛋白在大鼠颌骨骨髓基质细胞成骨分化后期表达显著。

Hepal-6细胞热休克后裂解蛋白致敏的树突状细胞瘤苗对肝细胞癌瘤内CD25^+Foxp3^+Treg细胞的影响

目的:观察注射Hepal-6细胞热休克后裂解蛋白致敏的骨髓来源树突状细胞(bone marrowderived dendritic cells,BMDCs)瘤苗对小鼠肝细胞癌(hepatocellular carcinoma,HCC)瘤内CD25+叉头盒转录因子P3(forkhead box p3,Foxp3)+调节T淋巴细胞(regulatory T cells,Tregs)浸润的影响.方法:在粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulatingfactor,GM-CSF)和白介素-4(interleukin-4,IL-4)诱导下体外扩增BMDCs,使用Hepal-6细胞热休克后裂解蛋白体外致敏BMDCs制备瘤苗,荧光免疫化学染色和FACS检测致敏前后BMDCs CD11c、CCR7、CD80和CD86的表达变化.使用Hepal-6细胞皮下注射的方法制备小鼠(C57BL/6J)HCC模型,成瘤小鼠分组注射Hepal-6细胞热休克后裂解蛋白致敏的BMDCs瘤苗(足垫部和瘤内,每7 d注射1次,共2次),并另设对照(空白对照组、BMDCs组和Hepal-6细胞裂解蛋白组).在治疗结束后9 d获取组织标本,免疫荧光组织化学染色和FACS检测瘤苗注射后肿瘤内CD8+T细胞和CD25+Foxp3+Tregs细胞的浸润情况.结果:光镜和扫描电镜显示:GM-CSF和IL-4在体外诱导扩增的BMDCs具有树突状细胞特征性的形态特征,且免疫细胞化学染色显示:该细胞表达CD11c,CCR7,CD80和CD86.使用Hepal-6细胞热休克后裂解蛋白致敏的BMDCs组,与对照组(BMDCs组和Hepal-6细胞裂解蛋白组)相比该组细胞CD11c(67.2±4.49 vs 52.4±5.20,58.4±4.43,P<0.01),CCR7(65.4±5.34 vs 45.9±5.04,57.0±3.46,P<0.01),CD80(62.9±4.69 vs 46.9±4.75,54.4±3.47,P<0.01)和CD86(73.3±3.58 vs 60.1±2.98,63.7±3.10,P<0.01)的表达均明显增高.使用Hepal-6细胞热休克后裂解蛋白致敏的BMDCs瘤苗为HCC荷瘤小鼠进行注射治疗,治疗后的检测结果显示:该组小鼠瘤内CD8+T细胞的浸润明显高于对照组(空白对照组、BMDCs组和Hepal-6细胞裂解蛋白组)(55.0±4.11 vs 38.2±3.34,44.6±4.29,45.6±4.92,P<0.01),而同时瘤内CD25+Foxp3+Tregs细胞的浸润则明显低于相应对照组(0.37±0.028 vs 1.31±0.020,0.77±0.057,0.57±0.062,P<0.05).结论:使用Hepal-6细胞热休克后裂解蛋白致敏的BMDCs瘤苗进行治疗,可增强HCC小鼠瘤内CD8+T细胞的浸润,并同时减少CD25+Foxp3+Tregs细胞的浸润,该瘤苗具有抗肿瘤免疫效果.

构建高效载体OPEI沉默TRAF6促进骨关节炎软骨再生的研究

目的·构建低毒性的关节滑膜高效率siRNA转染载体OPEI,抑制肿瘤坏死因子受体相关因子6 (tumor necrosis factor receptor-associated factor 6,TRAF6),观察其对骨关节炎(osteoarthritis,OA)模型骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成软骨能力的影响。方法·通过内侧半月板切除术建立SD大鼠OA模型(OA组,n=20);另设假手术组(n=10),半月板保持完好。造模后3个月后采集手术部位软骨和滑膜,免疫组织化学法检测TRAF6的表达,Western blotting方法检测白介素-1β (interleukin-1β,IL-1β)诱导的大鼠原代滑膜细胞中MMP13、TRAF6、p-p65的表达。在无水厌氧环境中合成小分子聚乙烯亚胺(polyethylenimine,PEI)衍生物OPEI,琼脂糖凝胶电泳检测OPEI包裹siRNA的能力,动态光散射测量形成的OPEI/siRNA复合物的粒径和Zeta电位,MTT法检测形成的复合物对大鼠原代滑膜细胞的细胞毒性,流式细胞术分析复合物对滑膜细胞凋亡的影响,并利用激光共聚焦技术及荧光显微镜观测体内外OPEI在滑膜细胞中转染siRNA的效率,阿尔新蓝染色检测软骨细胞基质蛋白聚糖含量。结果·与假手术组相比,TRAF6在大鼠OA模型的滑膜及软骨中的表达显著升高;抑制TRAF6表达可显著降低IL-1β刺激的原代滑膜细胞中MMP13和p-p65的表达。构建的OPEI载体在大鼠原代滑膜细胞中的siRNA转染效率高达99.33%,在大鼠膝关节腔内注射OPEI/Cy3-siRNA后第3日、第7日均显示大量滑膜细胞摄取siRNA。OPEI/siTRAF6复合物转染大鼠原代滑膜细胞2 d,Western blotting结果显示OPEI/siTRAF6组在质量比值为3∶1、4∶1和5∶1时,TRAF6基因敲除效率分别为49.05%、74.61%和83.18%。OPEI/siTRAF6沉默TRAF6基因后,与对照组(OPEI/siNC)相比,IL-1β刺激条件下软骨细胞阿尔新蓝染色显著增强。结论·OPEI是低毒性高效率siRNA转染载体,在滑膜细胞中沉默TRAF6可促进骨关节炎软骨再生。

毕西巴尼、Leustroducsin及Z-100对人骨髓基质细胞株白介素6产生的作用

我们使用Ｌｅｕｓｔｒｏｄｕｃｓｉｎ、毕西巴尼（ＯＫ－４３２）及Ｚ－１００三种细菌制剂作为诱导剂，通过培养人骨髓基质细胞株ＢＭ０１，观察此三种物质对ＢＭ０１白细胞介素６（ＩＬ６）产生的影响，用ＥＬＩＳＡ法检测培养液中ＩＬ６的含量。结果显示，三种诱导剂均可能ＢＭ０１分泌ＩＬ６增多，以Ｌｅｕｓｔｒｏｄｃｓｉｎ的诱导作用最显著。

Study on the dualistic mechanisms for dysregulated expression of interleukin 6 in multiple myeloma

Objective: To explore the source cells of interleukin-6 (IL-6) among bone marrow cells in multiplemyeloma (MM). Methods: Expressions of IL-6 in bone marrow mononuclear cells (BMMNCs) were detected using double labeled immunofluorometry, and the IL-6 activities in the supernatants of co-culture system of myelomacell line KM3 and bone marrow stromal cells (BMSCs) were measured using B9 cell proliferation test. ResultS: IL6 molecules were expressed not only in cytoplasndc γ-globin positive cell but also in type I collagen or von Willebrand Factor(vwr)positive cells. After fixation, both myeloma cells and BMSCs hardly secreted IL-6. IncreasedIL-6 activities were found in the supernatants after co-culture of unfixed my8loma cell line KM3 and fixed BMSCsor of fixed KM3 cells and unfixed BMSCs. Furthermore, BMSCs from MM patients induced KM3 cells to secretsignificantly more IL-6 in the supernatants than in BMSCs did controls (P<0. 01). Couclusiou: Both the BMSCsand myeloma cells secret IL-6 simultaneously in MM. Their interaction with each other promotes IL-6 over production. Both autocrine and paracrine mechanisms of IL-6 dysregulated expression are involved in the pathogenesisof MM.

Effect of growth and differentiation factor 6 on the tenogenic differentiation of bone marrow-derived mesenchymal stem cells

Background Recent studies showed that bone marrow-derived mesenchymal stem ceils （BMSCs） had risk of ectopic bone formation. In this study, we aimed to investigate the effect of growth and differentiation factor 6 （GDF-6） on the tenogenic differentiation of BMSCs in vitro, and then combined with small intestine submucous （SIS） to promote tendon regeneration in vivo. Methods The BMSCs were isolated from the green fluorescent protein （GFP） rats, and were characterized by multi-differentiation assays following our previous study protocol. BMSCs cultured with different concentrations of GDF-6, without growth factors served as control. After 2 weeks, mRNA expression and protein expression of tendon specific markers were examined by qRT-PCR and Western blotting to define an optimal concentration of GDF-6. Mann-Whitney U-test was used to compare the difference in relative mRNA expression among all groups; P 〈0.05 was regarded as statistically significant. The GDF-6 treated BMSCs combined with SIS were implanted in nude mice and SD rat acute patellar tendon injury model, the BMSCs combined with SIS served as control. After 12 and 4 weeks in nude mice and tendon injury model, the samples were collected for histology. Results After the BMSCs were treated with different concentration of GDF-6 for 2 weeks, the fold changes of the specific markers （Tenomodulin and Scleraxis） mRNA expression were significantly higher in GDF-6 （20 ng/ml） group （P 〈_0.05）, which was also confirmed by Western blotting result. The BMSCs became parallel in orientation after GDF-6 （20 ng/ml） treatment, but the BMSCs in control group were randomly oriented. The GDF-6 （20 ng/ml） treated BMSCs were combined with SIS, and were implanted in nude mice for 12 weeks, the histology showed neo-tendon formation. In the SD rat patellar tendon window injury model, the histology also indicated the GDF-6 （20 ng/ml） treated BMSCs combined with SIS could promote tendon regeneration. Conclusions GDF-6 has tenogenic effect on the tenogenic differentiation of BMSCs, and GDF-6 （20 ng/ml） has better tenogenic effect compared to other concentrations. The GDF-6 （20 ng/ml） treated BMSCs combined with SIS can form neo-tendons and promote tendon regeneration.

JAK2／STAT3途径在IL-6抑制LPS诱导的树突状细胞分化成熟中的作用

目的观察JAK2/STAT3信号途径在IL-6抑制LPS诱导的小鼠髓源性树突状细胞(bone marrow-derived dendritic cells,BMDCs)分化成熟中的作用。方法分离小鼠骨髓细胞,采用黏附法结合GM-CSF和IL-4刺激法在体外培养BMDCs;用相差倒置显微镜观察BMDCs的形态特点;用LPS诱导DCs成熟;采用流式细胞术观察IL-6处理与否,LPS诱导的BMDCs在表型上的变化;Western blot检测BMDCs细胞质p-JAK2、总STAT3和p-STAT3的表达水平。结果形态观察和FCM分析的结果表明:体外成功培养了纯度较高的高表达CD11c的BMDCs,LPS能明显诱导BMDCs表面MHC-Ⅱ类分子和共刺激分子CD80、CD86、CD40的表达上调;而经过IL-6处理后,LPS诱导BMDCs表面MHC-Ⅱ类分子、CD80、CD86、CD40表达上调的能力被显著削弱(P<0.01)。Western blot结果显示,与单独用LPS处理的BMDCs相比,经IL-6处理后的BMDC中p-JAK2、p-STAT3表达随作用时间增加而明显上调,提示IL-6促进JAK2/STAT3信号途径活化。结论JAK2/STAT3信号途径参与了IL-6抑制LPS诱导的BMDCs成熟过程。

银屑病骨髓间充质干细胞分泌白介素-6和血管内皮生长因子的检测

目的:研究银屑病患者骨髓间充质干细胞(MSCs)细胞因子分泌水平及其与病情活动的相关性,进一步探讨银屑病的发病机制。方法:采用密度梯度离心分离和贴壁培养法对24例银屑病患者和20例正常对照骨髓MSCs进行分离培养,采用ELISA方法测定MSCs培养上清中白介素(IL)-6和血管内皮生长因子(VEGF)的表达。结果:银屑病患者骨髓MSCs培养上清液IL-6浓度较正常人明显增高(P<0.01),VEGF浓度较正常人明显降低(P<0.01)。患者IL-6及VEGF水平与银屑病皮损面积及严重度指数(PASI)评分无相关性(P>0.05)。结论:银屑病患者骨髓MSCs细胞因子分泌异常,银屑病患者骨髓造血微环境有异常,进而对银屑病造血干细胞发育可能产生影响。

前列腺素E2调控骨髓间充质干细胞成骨分化最佳浓度及其效应基因

背景:前列腺素E2不仅是参与机体炎症反应的炎性递质,而且在骨组织形成与改建过程中起着非常重要的调节作用,且前列腺素E2在其他干细胞如神经干细胞、胚胎干细胞的增殖分化中,也表现出类似的调节作用。目的:研究前列腺素E2对骨髓间充质干细胞增殖与骨向分化的影响,检测其促增殖和成骨分化的最佳剂量,筛选出前列腺素E2的效应基因,评价前列腺素E2诱导成骨的作用机制。方法:提取SD大鼠骨髓间充质干细胞并鉴定,配制10-2,10-3,10-4,10-5,10-6 g/L不同质量浓度的前列腺素E2溶液,分别与第3代骨髓间充质干细胞共培养,空白组为含体积分数10%胎牛血清的L-DMEM。MTT比色法及碱性磷酸酶定量检测,筛选出前列腺素E2促骨髓间充质干细胞增殖及成骨分化的最佳剂量。利用基因芯片筛选出最佳成骨诱导浓度前列腺素E2诱导后14 d的骨髓间充质干细胞与未诱导组的差异表达基因。结果与结论:不同质量浓度前列腺素E2诱导实验数据进行统计学分析后发现前列腺素E2最佳促骨髓间充质干细胞增殖浓度为1×10-4 g/L,前列腺素E2最佳促骨髓间充质干细胞成骨分化浓度为1×10-5 g/L。基因芯片筛选发现Cxcl13、Cd55基因及PPAR、MAPK信号通路可能与前列腺素E2促骨髓间充质干细胞向成骨分化关系密切。

骨髓间充质干细胞复合多孔纳米羟磷灰石/聚酰胺6整复大鼠颅骨缺损的实验研究

目的初步探讨纳米羟磷灰石/聚酰胺6(n-HA/PA6)多孔材料对体外培养的大鼠骨髓间充质干细胞(BM-SCs)增殖及分化的影响,以及BMSCs作为种子细胞、多孔n-HA/PA6作为支架材料构建组织工程化骨修复大鼠颅骨极限骨缺损的可行性及整复效果。方法矿化诱导的第3代BMSCs与n-HA/PA6多孔材料复合培养,MTT检测细胞增殖,ALP染色检测骨向分化。将BMSCs与n-HA/PA6复合物植入大鼠颅骨8mm骨缺损处,4、8、16周时,应用组织学、扫描电镜观察植入物与骨组织交界处的成骨修复情况,并与单纯n-HA/PA6植入组修复效果进行比较。结果与n-HA/PA6复合培养的BMSCs生长良好,细胞增殖未受影响,ALP染色阳性。BMSCs复合n-HA/PA6植入4周时,有较多新骨长入支架孔穴;8周时,材料和宿主骨融为一体,接近正常骨;16周时,材料和天然骨形成骨性结合。单纯n-HA/PA6组植入4周时,新骨形成较少;8周时,新骨明显增加,但骨钙化程度较低;16周时,2组无明显差异。结论多孔n-HA/PA6支架材料对种子细胞BMSCs的增殖和骨向分化无影响;BMSCs作为种子细胞、n-HA/PA6多孔复合体作为支架材料构建组织工程化骨能够加速界面骨愈合,有效修复颅骨缺损,具有潜在的骨组织工程应用前景。

IL-6体外诱导成年大鼠骨髓破骨细胞形成的实验观察

目的 :探讨IL - 6对成年大鼠骨髓破骨细胞生成诱导的影响。方法 :采用 12周龄SD大鼠无菌条件下取出股骨 ,剪去两骺端 ,以α -MEM将骨髓细胞冲出 ,然后将细胞悬液接种于预置盖玻片或牙本质片的 2 4孔培养板内 ,次日实验组换含IL - 6 (10U/ml)的α -MEM培养液继续培养 2周 ,对照组培养液不含IL - 6。结果 :培养一周左右光镜下观察实验组可见明显单核细胞融合成多核细胞现象 ,多核细胞数量增多。经TRAP染色呈阳性反应 ,电镜下可观察到典型的破骨细胞骨吸收现象。结论 :IL - 6 (10U/ml)

多发性骨髓瘤骨髓基质细胞IL-6和TNF-α的表达及其临床意义

目的研究骨髓瘤骨髓基质细胞(BMSCs)分泌的白介素-6(IL-6)和肿瘤坏死因子α(TNF-α)在多发性骨髓瘤(MM)进展中的变化和意义以及人脐血源基质细胞(hUCBDSCs)分泌IL-6和TNF-α的水平。方法用ELISA法检测单独培养的hUCBDSCs和骨髓瘤BMSCs以及它们与KM3细胞共培养后IL-6、TNF-α在培养上清液中的浓度。结果(1)单独培养和共培养时骨髓瘤BMSCs分泌IL-6和TNF-α的水平高于hUCBDSCs;(2)共培养后骨髓瘤BMSCs和hUCBDSCs分泌IL-6和TNF-α的水平明显高于共培养前;(3)Ⅲ期患者BMSCs分泌IL-6的水平高于Ⅰ期和Ⅱ期患者,但是TNF-α水平无变化。结论IL-6与MM的进展有关;hUCBDSCs表达IL-6和TNF-α的水平较骨髓瘤BMSCs低。

前列腺素E1干预大鼠骨髓间充质干细胞的旁分泌和迁移

背景:目前还缺乏前列腺素E1对骨髓间充质干细胞旁分泌和迁移影响的研究。目的:观察前列腺素E1与骨髓间充质干细胞共培养对细胞旁分泌和迁移的影响。方法:取第3代骨髓间充质干细胞与10μg/L前列腺素E1共培养,在3,6,9,12,24,48,72 h各时间点收集培养孔内上清液100μL,利用酶联免疫吸附法检测不同时间骨髓间充质干细胞分泌血管内皮生长因子水平。采用细胞划痕实验及Transwell迁移实验观察前列腺素E1干预后骨髓间充质干细胞的迁移能力。结果与结论:(1)前列腺素E1作用3 h后骨髓间充质干细胞分泌血管内皮生长因子水平开始升高,作用24 h时血管内皮生长因子分泌达到峰值,随后逐渐下降;(2)细胞划痕实验和Transwell迁移实验显示,前列腺素E1作用后迁移的骨髓间充质干细胞显著增多(P<0.05);(3)结果可见,前列腺素E1可促进骨髓间充质干细胞分泌血管内皮生长因子,并能提高它的迁移能力。

let-7f对骨髓间充质干细胞增殖的影响

背景:micro RNA let-7f和炎性因子白细胞介素6对骨髓间充质干细胞的具体作用及两者之间是否存在联系尚不可知。目的:分析let-7f及白细胞介素6表达水平对骨髓间充质干细胞增殖的影响及两者的关系。方法:1构建let-7f过表达及抑制慢病毒载体,分别转染SD大鼠骨髓间充质干细胞,实验分为4个组:转染上调组(转染LV-rno-let-7f-up)、转染下调组(转染LV-rno-let-7f-down)、阴性转染组(转染空病毒)、未转染组(不进行病毒转染)。q RT-PCR检测各组细胞let-7f表达水平。MTT法、流式细胞术和ELISA等方法检测不同let-7f表达水平下细胞增殖情况及白细胞介素6表达水平,q RT-PCR及Western blot检测各组Cyclin D1 m RNA和蛋白表达水平。2预测let-7f潜在的靶基因,构建野生型/突变型白细胞介素6 3’UTR报告基因载体,分别与let-7f/let-7f inhibitor共转染293T细胞系,测定荧光素酶活性。结果与结论:let-7f转染上调组较未转染组及阴性转染组细胞增殖能力及克隆能力明显增强,G1期细胞数明显减少,S期明显增多,凋亡减少(P<0.05);转染下调组结果与其相反。let-7f转染上调组细胞培养液中的白细胞介素6表达水平低于未转染组及阴性转染组(P<0.05);转染下调组细胞培养液中的白细胞介素6则明显升高(P<0.05);Western blot、定量PCR显示let-7f转染上调组Cyclin D1蛋白及m RNA表达上调(P<0.05);转染下调组表达均下调(P<0.05);未转染组与阴性转染组所有结果无明显差异(P>0.05)。野生型白细胞介素6 3’UTR报告基因载体与let-7f共转染细胞的荧光素酶活性显著减低(P<0.05)。结果表明上调let-7f表达水平可显著促进骨髓间充质干细胞增殖活性及克隆形成能力,减少凋亡,而下调let-7f表达水平则出现抑制作用。白细胞介素6过表达可抑制骨髓间充质干细胞增殖,考虑白细胞介素6是let-7f的靶基因,let-7f可能通过抑制白细胞介素6表达而促进细胞增殖。