EHSH1蛋白在中枢神经系统中的功能研究进展

<正>唐氏综合征(Down syndrome,DS)又称为先天愚型或21三体综合征,是最常见的胎儿先天性缺陷疾病之一,其主要特征为严重的先天性智力障碍、特殊的面容,并常伴有各种先天性畸形,其出生率约占活产新生儿的1/800～1/700,是人类第21号染色体部分或全部出现三倍体所致。患者到了四五十岁大都     

Design of Dendrimer Modified Carbon Nanotubes for Gene Delivery

Objective: To investigate the efficiency of polyamidoamine dendrimer grafted carbon nanotube (dendrimer-CNT) mediated entrance of anti-survivin oligonucleotide into MCF-7 cells, and its effects on the growth of MCF-7 cells. Methods: Antisense survivin oligonucleotide was anchored onto polyamidoamine dendrimer grafted carbon nanotubes to form dendrimer-CNT-asODN complex and the complex was characterized by Zeta potential, AFM, TEM, and 1% agarose gel electrophoresis analysis. Dendrimer-CNT-asODN complexes were added into the medium and incubated with MCF-7 cells. MTT method was used to detect the effects of asODN and dendrimer-CNT-asODN on the growth of MCF-7 cells. TEM was used to observe the distribution of dendrimer-CNT-asODN complex within MCF-7 cells. Results: Successful synthesis of dendrimer-CNT-asODN complexes was proved by TEM, AFM and agarose gel electrophoresis. TEM showed that the complexes were located in the cytoplasm, endosome, and lysosome within MCF-7 cells. When dendrimer-CNT-asODN (1.0 μmol/L) and asODN (1.0 μmol/L) were used for 120 h incubation, the inhibitory rates of MCF-7 cells were (28.22±3.5)% for dendrimer-CNT-asODN complex group, (9.23±0.56)% for only asODN group, and (3.44±0.25)% for dendrimer-CNT group. Dendrimer-CNT-asODN complex at 3.0 μmol/L inhibited MCF-7 cells by (30.30±10.62)%, and the inhibitory effects were in a time- and concentration- dependent manner. Conclusion: Dendrimer-CNT nanoparticles may serve as a gene delivery vector with high efficiency, which can bring foreign gene into cancer cells, inhibiting cancer cell proliferation and markedly enhancing the cancer therapy effects.     

乳腺癌脑转移与Slit/Robo信号通路的关系

乳腺癌患者发生脑转移的几率逐年呈上升趋势,临床上乳腺癌脑转移的机率可高达30%,从诊断乳腺癌到发现脑转移的中位时间为34个月[1].由于脑转移瘤患者其原发乳腺癌多为晚期,且脑转移瘤大多为实质性,易复发,如果未得到及时的治疗,其自然病程是进展性神经恶化,平均生存期为1～2个月,因此乳腺癌脑转移成为促进乳腺癌患者死亡的一个重要因素.     

抑制信号转导和转录激活因子-3活化对恶性胶质瘤细胞LN229运动能力的影响

目的 观察抑制信号转导和转录激活因子-3( STAT3)的活化对调控人恶性胶质瘤LN229细胞运动能力的影响.方法 应用JAK/STAT3信号通路抑制剂AG490处理LN229细胞株,Westem blot检测STAT3、磷酸化STAT3( p-STAT3)的表达.噻唑蓝(MTr)比色法筛选能够阻断LN229细胞STAT3活化的最适AG490浓度;划痕实验和趋化实验检测AG490给药后,LN229细胞运动能力的变化;F-肌动蛋白( F-actin)实验检测AG490对LN229细胞肌动蛋白聚合能力的影响.结果 AG490可有效抑制LN229细胞STAT3的持续活化.50 μmol/L浓度的AG490在不影响LN229细胞存活时(t=1.862,P＞0.05),能够有效抑制STAT3的磷酸化,对照组和50μmol/L AG490处理组的p-STAT3/STAT3的密度比值分别为0.530±0.040和0.291±0.036(t =4.821,P＜0.01).伤口愈合实验可见50μmol/L AG490处理的LN229细胞非定向运动能力降低,24h时对照组运动的距离为(0.290±0.049) mm;实验组为(0.150±0.054) mm(P ＜0.05).体外趋化实验显示50 μmol/LAG490处理的LN229细胞定向运动能力降低(P＜0.01),表皮细胞生长因子(EGF)浓度为10 μg/L时对照组穿过膜的细胞数为93.92±9.52;实验组为24.52±3.04.50μmol/L浓度的AG490抑制LN229细胞肌动蛋白聚合能力(P＜0.05).结论 STAT3信号转导通路参与调控胶质母细胞瘤LN229的细胞运动过程,阻断STAT3的活化可以抑制恶性胶质瘤的运动能力.     

水通道蛋白4调控恶性胶质瘤细胞凋亡的研究

Objective To investigate the effects of apuaporin 4 (AQP4) on apoposis of LN229 glioblastoma cells by AQP4 small RNA interference (siRNA) technology and the molecular mechanism.Methods AQP4 expression was knocked down in LN229 glioblastoma cells by AQP4 siRNA (scr/LN229 and siAQP4/LN229 cells). The expression level of AQP4 protein in LN229 cells was detected by Western blotting. Stable clones were used to measure cells survival ratio (6 days) by methyl thiazol tetrazolium ( MTT) assay. Flow cytometry (FCM) was used to examine the content of cytochrome C in siAQP4/LN229 and scr/LN229. Western blotting was used to measure the levels of cytochrome C, bcl-2 and Bad. Nu/Nu mice were subcutaneously injected with siAQP4/LN229 and scr/LN229 cells to study the growth of tumor in the two groups (20 mice in each group). Results Western blotting showed that the expression of AQP4 was decreased significantly; MTT assay suggested that the survival ratio was decreased with deficiency of AQP4: the survival ratio of scr/LN229 was (587.00 ±4.68)%, and the ratio of siAQP4/N229 was (317. 00 ± 26. 30)% at sixth day (P＜0. 05). The mean fluorescence density of cytochrome C in scr/LN229 and siAQP4/LN229 by FCM was (57.2 ± 16.64) and (468.8 ±46.9) respectively (P＜0.05). The relative intensity of cytochrome C and Bad in siAQP4/N229 cells was ( 1. 62 ±0. 16) and (1. 30 ±0. 24) respectively, and that in scr/LN229 cells was (0. 83 ±0. 29) and (0. 56 ±0. 21) respectively (P＜0. 05). The intensity of bcl-2 in siAQP4/N229 cells (0.53 ±0.03) was decreased compared to scr/LN229 cells (0. 73 ± 0. 12) ( P ＜ 0. 05). The subcutaneous mouse xenograft model showed that the mean tumor volume of scr/LN229 group was (402. 67 ±34. 27) mm3, and that of siAQP4/N229 group was (65. 15 ±32. 12) mm3 at the 6th week. Conclusion AQP4 can regulate apoptosis of glioblastoma cell line LN229, which may be related with the release of cytocherome C, Bad and bcl-2.     

PTBP1在胶质瘤进展中的作用研究

目的:探讨多聚嘧啶区结合蛋白1（PTBP1）在胶质瘤进展中的作用。方法:利用多个数据库分析PTBP1在胶质瘤组织和正常脑组织中的表达水平及其与胶质瘤的组织学级别和患者预后的关系。构建稳定敲低PTBP1的LN229细胞系,通过Western印迹和RT-qPCR技术验证细胞中PTBP1的敲低效率。通过EdU实验检测细胞的增殖能力。利用Transwell实验检测细胞的迁移和侵袭能力。结果:PTBP1在胶质瘤组织中较正常脑组织表达更高（P＜0.000 1）,其表达水平随组织学级别的升高而升高（P＜0.000 1）;PTBP1高表达的胶质瘤患者总生存期（OS）明显缩短（P＜0.000 1）;稳定敲低PTBP1的LN229细胞系,细胞的增殖速度变慢（t=3.579,P=0.037 3）,迁移（t=13.16,P＜0.000 1）和侵袭能力（t=3.111,P=0.035 8）显著下降。结论:PTBP1在胶质瘤中高表达且与胶质瘤的组织学级别和患者不良预后呈正相关;PTBP1促进胶质瘤细胞的增殖、迁移、侵袭。     

二氢嘧啶脱氢酶基因T85C位点突变与乳腺癌细胞5-氟尿嘧啶敏感性的关系

目的:探讨二氢嘧啶脱氢酶基因（DPYD）T85C位点突变对乳腺癌细胞5-氟尿嘧啶（5-FU）敏感性的影响。方法:从肝脏组织中提取mRNA,扩增得到野生型DPYD编码序列。利用定点突变技术,获得T85C定点突变的DPYD编码序列。将GFP、野生型和突变型DPYD序列,连入PCDH-CMV-MCS-EF1-Puro载体。转染上述三种质粒到MDA-MB-231细胞中,获得稳定表达的细胞株。MTT法检测不同浓度5-FU（0、1、5、10、50μg/mL）处理24h后,细胞存活情况。结果:酶切验证三种重组质粒,观察到目的序列。测序验证定点突变质粒,突变位点发生T到C的改变。相比对照组,突变型细胞的存活率显著增高（P <0.01）,且突变型细胞存活率高于野生型（P <0.05）。结论:DPYD基因突变影响乳腺癌细胞对5-FU敏感性,T85C突变型DPYD细胞对5-FU的敏感性显著降低。

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细胞内磁热疗诱导人肺腺癌细胞SPC-A1凋亡的体外实验研究

对体外培养的人肺腺癌细胞株SPC-A1分别施以水浴热疗、细胞外磁热疗及细胞内磁热疗,收集细胞分别用透射电镜,DNA凝胶电泳及流式细胞仪等方法分析检测细胞的凋亡及凋亡率。结果表明:43.8℃处理2h,细胞外磁热疗及水浴热疗没有明显的细胞凋亡,其凋亡率分别为7.78%、5.66%;经细胞内磁热疗后用TEM观察到细胞的典型的凋亡形态;用DNA琼脂糖凝胶电泳观察到凋亡条带;其细胞凋亡率为36.59%。氨基硅烷纳米Fe3O4诱导的细胞内磁热疗优于细胞外磁热疗及水浴热疗。导致细胞凋亡的主要原因不是热,而是细胞质中的磁粒与细胞的相互作用。     

Slit2在乳腺组织的表达与乳腺癌脑转移的关系

目的 检测神经轴突导向蛋白2(Slit2)在不同乳腺肿瘤组织中的表达,探讨Slit2与乳腺癌脑转移的相关性.方法 采用免疫组织化学LSAB法检测对24例发生脑转移的乳腺浸润性导管癌(IDC)、71例未发生脑转移的乳腺浸润性导管癌、22例乳腺导管内癌和23例乳腺腺纤维瘤组织中Slit2的表达.结果 Slit2在浸润性导管癌中有脑转移患者的阳性表达率(13%)明显低于无脑转移患者(59%)(P＜0.05);在乳腺导管内癌和浸润性导管癌中的阳性表达率(59%和48%)均明显低于乳腺纤维瘤(87%,P＜0.05);但前两者间差异无统计学意义(P＞0.05);在50岁以上乳腺癌患者中的阳性表达率(62%)明显高于50岁及以下患者(34%,P＜0.05);在生存期＜5年的乳腺浸润性导管癌患者组织中Slit2的阳性表达率(18%)明显低于生存期＞5年的患者(59%)(P＜0.05).Slit2阴性的患者总生存时间明显短于Slit2阳性患者(P＜0.01).Slit2在乳腺癌中的表达与肿瘤大小、淋巴结转移状态、病理学分期、组织学分级均无明显相关(P＞0.05).结论 乳腺癌中Slit2的表达和乳腺癌脑转移呈负相关,和乳腺浸润性导管癌患者的发病年龄及预后呈正相关,可成为判断乳腺癌预后和脑转移的分子标志物.