目的:探讨虎杖苷联合生长因子诱导人脐带间充质干细胞(hUCMSC)向神经元样细胞分化的过程及Notch信号分子的表达。方法:体外分离培养hUCMSC。取第4代细胞,在不同剂量的虎杖苷和生长因子诱导下向神经样细胞分化。诱导后显微镜下观察细胞...目的:探讨虎杖苷联合生长因子诱导人脐带间充质干细胞(hUCMSC)向神经元样细胞分化的过程及Notch信号分子的表达。方法:体外分离培养hUCMSC。取第4代细胞,在不同剂量的虎杖苷和生长因子诱导下向神经样细胞分化。诱导后显微镜下观察细胞形态变化,应用细胞免疫荧光技术检测神经元特异性标志物3型β微管蛋白(β-tubulin-Ⅲ)的蛋白表达,荧光定量PCR法检测β-tubulin-Ⅲ、微管相关蛋白2(MAP2)、孤束核受体相关因子1(Nurr1)及Notch通路中Notch1受体蛋白和的靶基因发状分裂相关增强子1(hairy and enhancer of split 1,HES1)表达。结果:诱导后部分细胞胞体收缩变圆,折光性增强,突起变细增多,末端出现分支,呈典型的神经元样细胞。各诱导组的β-tubulin-Ⅲ、MAP2基因表达均高于对照组。β-tubulin-ⅢmRNA在低剂量和高剂量虎杖苷联合生长因子组高于单纯生长因子诱导组,MAP2 mRNA的表达在高剂量虎杖苷联合生长因子组最高。Nurr1 mRNA仅在高剂量虎杖苷联合生长因子组显著上调。细胞免疫荧光结果显示,低剂量和高剂量虎杖苷联合生长因子组部分细胞β-tubulin-Ⅲ表达阳性,单纯生长因子诱导组仅少量细胞表达β-tubulin-Ⅲ,对照组几乎不表达,与mRNA变化一致。Notch1和HES1的mRNA在高剂量虎杖苷联合生长因子组表达下调。结论:虎杖苷能够诱导hUCMSCs分化为神经元样细胞,并抑制Notch信号分子表达,且存在剂量依赖性。低水平的Notch信号激活可能有利于神经细胞的分化。展开更多
目的探讨微小RNA-34b(mi R-34b)促进关节软骨细胞基质退变的分子机制。方法提取10例骨关节炎(OA)患者和7例创伤性截肢者的膝关节软骨组织,实时定量反转录聚合酶链反应(RT-q PCR)法检测组织标本中mi R-34b表达水平。利用Ta rg e ts c a ...目的探讨微小RNA-34b(mi R-34b)促进关节软骨细胞基质退变的分子机制。方法提取10例骨关节炎(OA)患者和7例创伤性截肢者的膝关节软骨组织,实时定量反转录聚合酶链反应(RT-q PCR)法检测组织标本中mi R-34b表达水平。利用Ta rg e ts c a n基因信息软件预测得到mi R-34b靶基因为Sma d 3,构建Sma d 3野生型及突变型3′非编码区(3′-UTR)-荧光素酶报告载体,利用荧光素酶报告基因实验检测mi R-34b对Smad3基因野生型及突变型3′-UTR荧光素酶活性的影响。体外培养SW1353细胞,分为A、B、C和D组,分别加入等量脂质体、无义序列、mi R-34b模拟物(mi R-34b mimic)和mi R-34b抑制物(mi R-34b inhibitor)。RT-q PCR法检测mi R-34b、Smad3、Ⅱ型胶原和基质金属蛋白酶-13(MMP-13)的表达水平。结果 OA软骨组织中mi R-34b表达水平较正常膝关节软骨组织明显上调(P<0.05)。SW1353细胞转染后,与A组比较,C组mi R-34b表达水平明显升高(P<0.05),D组明显降低(P<0.05);A组与B组比较,差异无统计学意义(P>0.05)。Targets Can软件预测mi R-34b的潜在靶基因为Smad3,荧光素酶报告基因实验证实mi R-34b直接靶向抑制靶基因Smad3;转染mi R-34b mimic和mi R-34inhib itor后,与A组比较,C组Sma d 3 m RNA表达水平明显降低(P<0.05),D组明显升高(P<0.05);A组与B组比较,差异无统计学意义(P>0.05)。转染后,与A组比较,C组Ⅱ型胶原m RNA表达水平明显降低(P<0.05),D组明显升高(P<0.05);C组MMP-13m RNA明显升高(P<0.05),D组明显降低(P<0.05)。结论 mi R-34b可能通过抑制其靶基因Sma d 3的表达进而诱导人关节软骨细胞基质退变,从而促进OA的发生、发展。展开更多
Objective To investigate the expression and regulation of programmed cell death protein 1(PD1),B lymphocyte and T lymphocyte attenuator(BTLA)in peripheral blood of patients with non-small cell lung cancer(NSCLC);to ex...Objective To investigate the expression and regulation of programmed cell death protein 1(PD1),B lymphocyte and T lymphocyte attenuator(BTLA)in peripheral blood of patients with non-small cell lung cancer(NSCLC);to examine the correlation of the mRNA levels between PD and BTLA in NSCLC.Methods Flow cytometry was used to detect the expression of PD1 and BTLA on the surfaces of CD8^+T cells andγδ+T cells in the peripheral blood samples collected from 32 in-patients with stage IV NSCLC and 30 healthy individuals.We compared the expression of PD1 and BTLA on the surfaces ofγδ+T cells in the NSCLC patients with bone metastasis before and after the treatment of zoledronic acid.The correlations of PD1 and BTLA,as well as their ligands were analyzed using Pearson correlation analysis with the cBioPortal data platform.Results The frequency of PD1 on the surfaces of CD8^+T cells was significantly higher than that of theγδT cells in both healthy controls(t=2.324,P=0.024)and NSCLC patients(t=2.498,P=0.015).The frequency of PD1 on CD8^+T cells,rather than onγδ+T cells,was significantly upregulated in advanced NSCLC patients compared with that in healthy controls(t=4.829,P<0.001).The PD1+BTLA+γδT cells of the healthy controls were significantly lower than that of the NSCLC patients(t=2.422,P=0.0185).No differences in percentage of PD1+γδ+and BTLA+γδ+T cells were observed in 7 NSCLC patients with bone metastasis before and after zoledronic acid treatment.PD1 was positively correlated with BTLA in both lung adenocarcinoma(r=0.54;P<0.05)and lung squamous cell carcinoma(r=0.78;P<0.05).Conclusions The upregulation of co-inhibitory molecules occurs on the surfaces of both CD8^+T cells andγδT cells in advanced NSCLC,suggesting that these molecules were involved in regulating the inactivation of CD8^+T cells andγδ+T cells,immune escape and tumor invasion.展开更多
文摘目的:探讨虎杖苷联合生长因子诱导人脐带间充质干细胞(hUCMSC)向神经元样细胞分化的过程及Notch信号分子的表达。方法:体外分离培养hUCMSC。取第4代细胞,在不同剂量的虎杖苷和生长因子诱导下向神经样细胞分化。诱导后显微镜下观察细胞形态变化,应用细胞免疫荧光技术检测神经元特异性标志物3型β微管蛋白(β-tubulin-Ⅲ)的蛋白表达,荧光定量PCR法检测β-tubulin-Ⅲ、微管相关蛋白2(MAP2)、孤束核受体相关因子1(Nurr1)及Notch通路中Notch1受体蛋白和的靶基因发状分裂相关增强子1(hairy and enhancer of split 1,HES1)表达。结果:诱导后部分细胞胞体收缩变圆,折光性增强,突起变细增多,末端出现分支,呈典型的神经元样细胞。各诱导组的β-tubulin-Ⅲ、MAP2基因表达均高于对照组。β-tubulin-ⅢmRNA在低剂量和高剂量虎杖苷联合生长因子组高于单纯生长因子诱导组,MAP2 mRNA的表达在高剂量虎杖苷联合生长因子组最高。Nurr1 mRNA仅在高剂量虎杖苷联合生长因子组显著上调。细胞免疫荧光结果显示,低剂量和高剂量虎杖苷联合生长因子组部分细胞β-tubulin-Ⅲ表达阳性,单纯生长因子诱导组仅少量细胞表达β-tubulin-Ⅲ,对照组几乎不表达,与mRNA变化一致。Notch1和HES1的mRNA在高剂量虎杖苷联合生长因子组表达下调。结论:虎杖苷能够诱导hUCMSCs分化为神经元样细胞,并抑制Notch信号分子表达,且存在剂量依赖性。低水平的Notch信号激活可能有利于神经细胞的分化。
文摘目的探讨微小RNA-34b(mi R-34b)促进关节软骨细胞基质退变的分子机制。方法提取10例骨关节炎(OA)患者和7例创伤性截肢者的膝关节软骨组织,实时定量反转录聚合酶链反应(RT-q PCR)法检测组织标本中mi R-34b表达水平。利用Ta rg e ts c a n基因信息软件预测得到mi R-34b靶基因为Sma d 3,构建Sma d 3野生型及突变型3′非编码区(3′-UTR)-荧光素酶报告载体,利用荧光素酶报告基因实验检测mi R-34b对Smad3基因野生型及突变型3′-UTR荧光素酶活性的影响。体外培养SW1353细胞,分为A、B、C和D组,分别加入等量脂质体、无义序列、mi R-34b模拟物(mi R-34b mimic)和mi R-34b抑制物(mi R-34b inhibitor)。RT-q PCR法检测mi R-34b、Smad3、Ⅱ型胶原和基质金属蛋白酶-13(MMP-13)的表达水平。结果 OA软骨组织中mi R-34b表达水平较正常膝关节软骨组织明显上调(P<0.05)。SW1353细胞转染后,与A组比较,C组mi R-34b表达水平明显升高(P<0.05),D组明显降低(P<0.05);A组与B组比较,差异无统计学意义(P>0.05)。Targets Can软件预测mi R-34b的潜在靶基因为Smad3,荧光素酶报告基因实验证实mi R-34b直接靶向抑制靶基因Smad3;转染mi R-34b mimic和mi R-34inhib itor后,与A组比较,C组Sma d 3 m RNA表达水平明显降低(P<0.05),D组明显升高(P<0.05);A组与B组比较,差异无统计学意义(P>0.05)。转染后,与A组比较,C组Ⅱ型胶原m RNA表达水平明显降低(P<0.05),D组明显升高(P<0.05);C组MMP-13m RNA明显升高(P<0.05),D组明显降低(P<0.05)。结论 mi R-34b可能通过抑制其靶基因Sma d 3的表达进而诱导人关节软骨细胞基质退变,从而促进OA的发生、发展。
基金Fund supported by the Healthcare Technology Plan of Zhejiang Provincial Health Bureau(No.2016KYB292)the Technology Plan of Science and Technology Bureau of Jiaxing,Zhejiang province(No.2016AY23054)~~
文摘Objective To investigate the expression and regulation of programmed cell death protein 1(PD1),B lymphocyte and T lymphocyte attenuator(BTLA)in peripheral blood of patients with non-small cell lung cancer(NSCLC);to examine the correlation of the mRNA levels between PD and BTLA in NSCLC.Methods Flow cytometry was used to detect the expression of PD1 and BTLA on the surfaces of CD8^+T cells andγδ+T cells in the peripheral blood samples collected from 32 in-patients with stage IV NSCLC and 30 healthy individuals.We compared the expression of PD1 and BTLA on the surfaces ofγδ+T cells in the NSCLC patients with bone metastasis before and after the treatment of zoledronic acid.The correlations of PD1 and BTLA,as well as their ligands were analyzed using Pearson correlation analysis with the cBioPortal data platform.Results The frequency of PD1 on the surfaces of CD8^+T cells was significantly higher than that of theγδT cells in both healthy controls(t=2.324,P=0.024)and NSCLC patients(t=2.498,P=0.015).The frequency of PD1 on CD8^+T cells,rather than onγδ+T cells,was significantly upregulated in advanced NSCLC patients compared with that in healthy controls(t=4.829,P<0.001).The PD1+BTLA+γδT cells of the healthy controls were significantly lower than that of the NSCLC patients(t=2.422,P=0.0185).No differences in percentage of PD1+γδ+and BTLA+γδ+T cells were observed in 7 NSCLC patients with bone metastasis before and after zoledronic acid treatment.PD1 was positively correlated with BTLA in both lung adenocarcinoma(r=0.54;P<0.05)and lung squamous cell carcinoma(r=0.78;P<0.05).Conclusions The upregulation of co-inhibitory molecules occurs on the surfaces of both CD8^+T cells andγδT cells in advanced NSCLC,suggesting that these molecules were involved in regulating the inactivation of CD8^+T cells andγδ+T cells,immune escape and tumor invasion.
