脂联素受体在正常SD大鼠乳鼠心肌细胞的分布和表达

目的通过体外培养SD大鼠乳鼠心肌细胞,研究脂联素受体1(AdipoR1)、脂联素受体2(AdipoR2)及T-钙黏蛋白(T-cadherin)三种脂联素受体在大鼠乳鼠心肌细胞上的分布和表达。方法采用酶消化法原代培养乳鼠心肌细胞,通过α-肌动蛋白免疫荧光法对原代培养的心肌细胞进行鉴定,并在倒置相差显微镜下观察细胞生长状态。选用原代培养96h的单层心肌细胞进行实验,使用RT-PCR和细胞免疫组织化学方法检测AdipoR1、AdipoR2、T-cadherin的mRNA和蛋白在心肌细胞中的表达情况。结果在SD大鼠乳鼠心肌细胞上均检测到了AdipoR1、AdipoR2、T-cadherin的mRNA和蛋白表达,其中以AdipoR1和T-cad-herin的mRNA和蛋白表达量高,与AdipoR2的表达量相比差异有统计学意义(P<0.01)。结论脂联素受体1、脂联素受体2和T-钙黏蛋白,三种脂联素受体在SD大鼠乳鼠心肌细胞上均有表达,并以脂联素受体1和T-钙黏蛋白的表达为主,脂联素受体2表达量较少。     

Methylation status of the T-cadherin gene promotor in peripheral blood mononuclear cells is associated with HBV-related hepatocellular carcinoma progression

DNA methylation is one of the epigenetic mechanisms to regulate gene expression and frequently occurs in human cancer cells. T-cadherin (CDH13) is a new member of the cadherin superfamily and possesses multiple functions. Our study included 26 normal controls (NCs), 65 chronic hepatitis B patients (CHB), 14 liver cirrhosis patients (LC) and 157 hepatocellular carcinoma patients (HCC). We mainly focused on the mRNA expression and methylation status of CDH13 in peripheral blood mononuclear cells (PBMCs), which were detected by semi-quantitative real-time polymerase chain reaction (RT-qPCR) and methylation-specific polymerase chain reaction (MSP) respectively. The CDH13 mRNA level was lower in HCC, especially in early-stage of HCC than in NCs and CHB groups (p < 0.05). Methylation frequency of the CDH13 promoter was significantly higher in HCC patients than in the NCs and CHB groups (67.52 % vs 0.00 %, p < 0.001, 67.52 % vs 52.31 %, p < 0.05, respectively). CDH13 mRNA level was significantly and relatively lower in methylated groups than in unmethylated groups among the whole participants. The methylation level of CDH13 promoter in HCC might be influenced or partly influenced by some critical factors such as TBil, ALB and AFP (p < 0.05). As an important factor in signaling pathway regulating by CDH13 to promote carcinogenesis, JNK level was significantly higher in HCC which had a higher methylation frequency than in NCs, CHB and LC (p < 0.05). Furthermore, the combination of the methylated CDH13 level and AFP level showed a better score: AUC = 0.796 (SE = 0.031, 95 %CI 0.735–0.857; p < 0.001) in male and AUC = 0.832 (SE = 0.057, 95 %CI 0.721–0.944; p < 0.001) in female compared to AFP alone for diagnosing HCC from NCs, CHB and LC. The methylation of CDH13 promoter was an independent predictor for assessing the prognosis of HCC patients (r=-1.378 p < 0.05). In conclusion, hypermethylation of CDH13 in PBMCs was associated with the underexpression of mRNA and the high risk of HCC. The methylation status of the CDH13 promoter in PBMCs was a potential noninvasive biomarker to predict the prognosis of HCC patients.     

耐力运动对老年小鼠心肌T-钙粘蛋白的影响及其效应研究

目的 观察耐力运动对老年小鼠心肌组织T-钙粘蛋白及其配体脂联素和下游相关分子表达的影响,探讨T-钙粘蛋白的作用。 方法 选取30只17月龄C57雄性小鼠,采用随机数字表法将其分为老年对照组及老年运动组,每组15只;另取15只2月龄C57雄性小鼠纳入为青年对照组。老年运动组小鼠进行12周耐力运动,老年对照组及青年对照组小鼠均正常饲养、无特殊干预。于老年运动组小鼠最后一次运动结束24 h后处死3组小鼠。采用免疫印迹法检测各组小鼠心肌组织T-钙粘蛋白及其配体脂联素表达、腺苷酸活化蛋白激酶(AMPK）磷酸化水平、凋亡及自噬相关信号蛋白B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)、Beclin-1等表达;采用免疫组化法观察各组小鼠心肌T-钙粘蛋白、脂联素及p-mTOR细胞定位和阳性表达。 结果 T-钙粘蛋白定位于心肌细胞胞膜及血管内皮处,脂联素定位于心肌细胞胞膜、胞浆及血管内皮处,p-mTOR定位于心肌细胞胞浆处。与青年对照组比较,老年对照组心肌T-钙粘蛋白、脂联素、AMPK活化水平、抑凋亡蛋白bcl-2、促自噬蛋白Beclin-1表达显著下调（P<0.05）,促凋亡蛋白bax、半胱氨酸天冬氨酸蛋白水解酶-9(Caspase-9)表达及自噬相关蛋白mTOR活化水平均显著上调（P<0.05）;与老年对照组比较,老年运动组心肌组织T-钙粘蛋白及配体脂联素、AMPK活化水平、bcl-2、Beclin-1蛋白表达均显著上调（P<0.05),bax、Caspase-9蛋白表达、mTOR磷酸化水平均显著下调（P<0.05）。 结论 耐力运动可增强老年小鼠心肌T-钙粘蛋白表达,促进脂联素与心肌组织结合,刺激AMPK活化,从而发挥对老龄心肌组织的保护作用。     

结肠癌T-cadherin表达水平分析与5-Aza-CdR对其表达和结肠癌细胞增殖、侵袭及凋亡的影响

背景与目的：结肠癌是临床常见恶性肿瘤,探讨抑癌蛋白T-cadherin在结肠癌中的表达情况及其与患者临床病理学特征的关系,并分析5-Aza-CdR对T-cadherin表达和结肠癌细胞增殖、侵袭及凋亡的影响。方法：收集福建医科大学附属第二医院2015—2016年40例手术切除的结肠癌组织及癌旁组织新鲜样本,并经过病理学检查验证,分别提取总RNA和总蛋白质。采用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR）分析T-cadherin mRNA的表达,采用蛋白质印迹法（Western blot）分析T-cadherin蛋白水平,并分析T-cadherin的mRNA表达变化与患者临床病理学特征的关系。进一步以人结肠癌细胞系HT-29为研究对象,采用甲基化抑制剂5-Aza-CdR处理HT-29细胞,分别采用RTFQ-PCR和Western blot分析T-cadherin表达变化,采用细胞计数试剂盒（cell counting kit-8,CCK-8）分析细胞增殖,采用Transwell实验验证细胞侵袭能力,采用流式细胞术分析细胞凋亡。结果：T-cadherin在结肠癌组织的蛋白水平和mRNA表达均明显低于癌旁组织,其mRNA表达与淋巴结转移（F=5.316,P=0.009 3）和分化程度（F=5.807,P=0.006 4）明显相关,而与其他病理变量（包括性别、年龄、肿瘤大小和肿瘤浸润深度）无明显相关。药物5-Aza-CdR可以显著上调HT-29细胞中T-cadherin的表达水平,抑制HT-29细胞增殖、侵袭并促进细胞凋亡。结论：T-cadherin表达可能与结肠癌恶性程度密切相关,药物5-Aza-CdR处理可上调结肠癌细胞T-cadherin的表达,并抑制结肠癌细胞的增殖、迁移,促进结肠癌细胞凋亡,提示T-cadherin可能是结肠癌患者使用甲基化抑制剂5-Aza-CdR治疗的靶点。     

Reciprocal altered expression of T-cadherin and P-cadherin in psoriasis vulgaris

BACKGROUND: The most characteristic change in psoriasis vulgaris is markedly increased, persistent keratinocyte proliferation. The underlying mechanism of excessive epidermal growth is controversial. We previously found and reported that T-cadherin was expressed in keratinocytes and confined to the basal layer of mouse and human skin. Invasive cutaneous squamous cell carcinoma showed a loss of T-cadherin expression. Another study showed that T-cadherin was a negative growth regulator of epidermal growth factor in T-cadherin transfectant neuroblastoma cells. OBJECTIVES: To obtain insight into the role of T-cadherin in keratinocyte proliferation and to investigate further the pathogenesis of psoriasis vulgaris, we examined the expression of T-cadherin, as well as E- and P-cadherin, in psoriasis vulgaris. METHODS: Four untreated active psoriatic skin samples from psoriasis vulgaris patients and four normal human skin samples from plastic surgery were collected, cryosectioned and immunohistochemically stained by antihuman T-, P- and E-cadherin antibodies. Further, the immunofluorescence intensities of T- and P-cadherin on the basal layer of the epidermis were quantitatively measured by the histogram function of LSM 510 software installed in a Zeiss laser scanning confocal microscope. The data were statistically analysed by Student's t-test. RESULTS: It was observed that T-cadherin was weakly and discontinuously expressed on the basal layer of psoriatic skin, while it was intensively expressed on all basal keratinocytes in normal human skin. In contrast, P-cadherin was strongly expressed throughout the entire epidermal layer in psoriatic skin samples, although its expression is restricted to the basal cell layer in normal human skin. There were no obvious differences in E-cadherin expression between normal human skin and psoriatic skin. Statistical analyses showed that the immunofluorescence intensity of T-cadherin in the basal cell layer of psoriatic skin (35 +/- 9.08) was significantly decreased compared with that in normal human skin (131.75 +/- 3.49, P = 2.46 x 10(-6)). There was a significant increase (P = 0.00139) in the immunofluorescence intensity of P-cadherin in the basal layer of psoriatic skin (68.25 +/- 12.13) compared with normal human skin (26 +/- 4.90). CONCLUSIONS: The present study demonstrates that there is downregulation of T-cadherin expression and upregulation of P-cadherin expression in psoriatic skin, which are considered to be involved in the hyperproliferation of keratinocytes in psoriasis vulgaris.     

黏附分子T-cadherin表达诱导C6胶质母细胞瘤细胞G2期阻滞

目的 研究T-cadherin分子表达抑制胶质母细胞瘤C6细胞增殖的机制。方法 利用脂质体转染pcDNA3和pcDNA3-T-cadherin质粒后获得的表达和不表达T-cadherin的C6细胞克隆，以流式细胞仪检测两种克隆细胞的细胞周期的变化，同时分析其细胞周期与细胞分裂指数的关系。结果 转染后表达T-cadherin的C6细胞在去血清培养48h使细胞周期同步后，分别用血清刺激培养12h后行流式细胞仪检测，与转染空载体的1、2克隆相比，转染T-cadherin基因后表达T-cadherin的3、4克隆于G2／M期的细胞比例显著增加（3、4克隆为52．60％和51．84％，1、2克隆为16．17％和13．47％），而G1期的细胞比例显著下降（3、4克隆为0．66％和0．39％，1、2克隆为50．6％和57．9％）。在血清刺激0、4、8、12、24和48h时，空载体克隆1在各时间点的分裂指数与其G2／M期的细胞比例一致，而表达T．cadherin的克隆3在各时间点的分裂指数显著低于其G2／M期的细胞比例数。结论 T．cadherin分子表达能诱导C6细胞G2期细胞阻滞，该机制可能是T-cadherin抑制C6细胞增殖的主要机制。     

黏附分子T-cadherin表达对C6细胞恶性特性的影响

目的 探讨T-cadherin分子表达对胶质母细胞瘤C6细胞的恶性生物学特征的影响。方法 C6细胞分别转染pcDNA3．T-cadherin和pcDNA3表达质粒后。筛选3个表达不同水平T．cadherin的C6克隆和2个不表达T．cadherin的C6克隆，研究T-cadherin分子表达水平对C6细胞的细胞黏附、迁移及聚集能力的影响。结果 与不表达T-cadherin的C6细胞相比．表达T-cadherin的C6细胞在附着后1h和4h时的细胞黏附能力均显著增强，分别增强61．2％和25．1％（P〈0．01），而其细胞迁移能力平均下降72．6％（P〈0．010）。同时，T-cadherin表达诱导显著的同源细胞聚集，聚集指数平均下降52．4％（P〈0．01）。结论 T-cadherin分子表达显著抑制胶质母细胞瘤C6细胞的恶性生物学特性。     

T-钙黏蛋白对黑素瘤侵袭力的影响

目的探讨T-钙黏蛋白(T-cadherin)对黑素瘤侵袭力的影响。方法体外培养黑素瘤细胞株B16F10,将pEGFP-N1空载体和构建的pEGFP-N1-T-cadherin转染B16F10细胞,采用RT-PCR和免疫组化SP法检测T-cadherin基因及蛋白水平的表达情况;应用transwell细胞侵袭实验测定转染T-cadherin后肿瘤细胞的侵袭力。结果 RT-PCR和免疫组化SP法检测结果表明,细胞可转录和表达T-cadherin;转染T-cadherin组穿过transwell小室膜的细胞数目(2.20±0.49)明显少于未转染组(16.80±1.39)和转染pEGFP-N1空载体组(15.00±1.26),差异有统计学意义(P<0.05),未转染组与转染pEGPF-N1空载体组差异无统计学意义(P>0.05);MTT法结果显示,转染T-cadherin组穿过小室膜细胞的吸光度(OD)值(0.124±0.003)明显低于未转染组(0.263±0.006)和转染pEGFP-N1空载体组(0.247±0.012),差异有统计学意义(P<0.05),未转染组与转染pEGFP-N1空载体组的差异无统计学意义(P>0.05)。结论 T-cadherin对黑素瘤细胞的体外侵袭能力有抑制作用。     

黏附分子T-cadherin表达对胶质母细胞瘤C6细胞的生抑制作用

目的 探讨T—cadherin分子表达对胶质母细胞瘤C6细胞增殖的影响。方法 利用脂质体将pcDNA3和pcDNA3．T—cadherin表达质粒转染C6细胞，以克隆形成试验和细胞增殖试验研究T-cadherin表达对C6细咆增殖的影响。结果 转pcDNA3．T-cadherin质粒的C6细胞形成的细胞克隆数显著少于转染pcDNA3载体质粒的C6细胞形成的克隆数，两者差异有统计学意义（P〈0．01）。转染后表达T—cadherin分子C6细胞的生长速度明显慢于转染空载体不表达T—cadherin的C6克隆（P〈0．01），且T—cadherin表达水平与C6细胞增殖速度呈负要关。结论 T—cadherin分子表达显著抑制胶质母细胞瘤C6细胞的增殖。