创伤性脑损伤后白细胞介素1β在星形胶质细胞中的表达

目的:探究创伤性脑损伤后白细胞介素1β(IL-1β)在星形胶质细胞中的表达。方法:采用免疫印迹半定量检测创伤性脑损伤后IL-1β、星形胶质细胞标志物-胶质纤维酸性蛋白(GFAP)的表达,免疫荧光双重染色检测IL-1β在星形胶质细胞中的定位表达。结果:IL-1β表达在损伤后8 h就有增高,1 d时最高,4 d时有所降低,且1 d和4 d时可定位表达在星形胶质细胞中。结论:损伤后增高的IL-1β表达可能对促进星形胶质细胞的活化具有重要的调节作用。     

靶向IL-6慢病毒介导RNA干扰实验性基因治疗类风湿关节炎

目的:探讨靶向小鼠IL-6基因RNA干扰慢病毒载体颗粒(LV-IL-6-RNAi)对小鼠巨噬细胞株J774A.1细胞IL-6表达的影响,及其对胶原诱导性关节炎(CIA)的治疗作用。方法:设计合成3对特异性针对小鼠IL-6基因的小干扰RNA(siRNA)序列,筛选最高效siRNA序,构建高效LV-IL-6-RNAi。LV-IL-6-RNAi感染小鼠巨噬细胞株J774A.1细胞,并设慢病毒阴性对照(LV-NC-RNAi)、培养基空白对照,RT-qPCR检测J774A.1细胞的IL-6、肿瘤坏死因子-α(TNF-α)和IL-1β mRNA相对表达水平,以检测LV-IL-6-RNAi体外干扰效率。取DBA/小鼠构建CIA模型并随机分为4组:LV-IL-6-RNAi组、LV-NC-RNAi组、甲氨蝶呤(MTX)阳性对照组、PBS空白对照组,分别关节腔注射LV-IL-6-RNAi、LV-NC-RNAi、腹腔注射MTX、PBS溶液,记录CIA小鼠关节炎评分,ELISA检测小鼠IL-6、IL-1β和TNF-α血清水平,组织病理检测炎症关节的炎症细胞浸润数。结果:成功构建了高效LV-IL-6-RNAi。...     

siRNA沉默波形蛋白表达抑制大鼠急性脊髓损伤胶质瘢痕的形成

背景:脊髓损伤后胶质瘢痕形成物理屏障抑制轴突跨越损伤部位,被认为是神经修复过程中的不利因素.目的:研究siRNA干扰波形蛋白表达对反应性星形胶质细胞的影响,探讨其在脊髓损伤后胶质瘢痕形成中的意义.方法:①选取生长良好的第3代星形胶质细胞,分为未转染组、siRNA-NC组、siRNA-Vimentin组,利用siRNA干...     

慢病毒载体介导的RNA干扰抑制PC12细胞MyD88基因的表达

目的研究慢病毒介导的RNA干扰对PC12细胞的转染效率和对MyD88基因的抑制效率。方法构建针对大鼠MyD88基因3个靶点的ShRNA慢病毒载体,PC12细胞按照不同的转染条件分为正常组(DMEM完全培养液)、DMEM加入polybrene组、EN.iS(Enhance infection solution)组、EN.iS加入polybrene组。荧光显微镜下观察不同组的转染效率,采用Real-timePCR和Western blot检测不同的靶点对MyD88的不同沉默效率。结果病8毒滴度为1×10TU/ml,MOI为100时,PC12细胞在EN.iS组的转染效率最高,转染试剂polybrene对转染效率无明显影响,沉默效率最高的靶点是5'-CATAC GCAACCAGCAGAAA-3'。结论慢病毒介导的RNA干扰是一种高效的基因沉默手段,可以对PC12细胞中MyD88的功能进行长期的研究。     

不同年龄大鼠大脑皮质损伤后的星形胶质细胞反应

对幼年（３月龄）、成年（１２月龄）与老年（２７月龄）三组雄性Ｗｉｓｔａｒ大鼠的躯感皮质进行针刺后，在１～２１ｄ时间内用光镜与电镜观察损伤后的星形胶质细胞反应，探讨年龄因素对星形胶质细胞反应的影响。结果表明：反应性星形细胞在水肿、细胞分裂、吞噬、肥大与胶质界膜形成等方面均表现出程度上和发生时间上的年龄差异。幼年大鼠的星形细胞不仅水肿更为显著且出现更早，并且表现出向变性与最后死亡以及向代偿性合成增强两个方向发展、三组动物的星形细胞分裂活动以术后３ｄ最明显，尤以幼年组更多见，并出现活跃的吞噬现象．     

慢病毒介导的RNA干扰技术应用于抑制黑色素瘤细胞转移的特异性研究

目的 应用慢病毒介导的RNA干扰技术,对该技术能特异性的抑制黑色素瘤细胞转移进行研究.方法 以人巢蛋白(Human nestin)的信使RNA编码序列作为干扰靶点,把慢病毒基因PLL3.7作为质粒载体,人巢蛋白的“短发夹”RNA(shRNA)表达质粒和阴性对照shRNA表达质粒(不包含所有已知信使RNA),分别感染黑色素瘤细胞株UACC903细胞并流式分选获得高纯度的巢蛋白干扰或对照组细胞.用划痕法检测评估各组黑色素瘤细胞迁移能力.结果 巢蛋白下调的UACC903细胞侵袭能力和迁移能力均下降(P＜0.05).结论 慢病毒介导的RNA能有效地沉默巢蛋白基因的表达,从而能特异性的抑制黑色素瘤细胞迁移.     

慢病毒介导的RNA干扰下调IL-6抑制脊髓顿挫损伤后Caspase-3表达

目的通过慢病毒介导的RNA技术干扰IL-6的表达,探究脊髓顿挫损伤后Caspase-3的表达变化。方法Allen′s打击法制备脊髓顿挫伤(SCC)模型,BBB评分确认造模成功;免疫组织化学和Q-PCR技术定位和定量测定IL-6和Caspase-3的表达变化;Gene MANIA网站预测IL-6与Caspase-3的联系;慢病毒介导的RNA干扰技术构建IL-6干扰模型后,Q-PCR技术定量测定Caspase-3的表达变化。结果 SCC后SD大鼠BBB评分显著低于假手术组(P0.05);IHC结果显示:IL-6和Caspase-3均在在脊髓白质胶质细胞表达;Q-PCR结果显示:IL-6在损伤后12h表达显著增加,3d、28d表达持续下降;Caspase-3在损伤后12h、3d、28d表达持续下降。生物信息学分析结果:IL-6和Caspase-3在大鼠体内存在共定位关系。干扰组Caspase-3表达比空载组显著降低(P0.05)。结论在SCC后,抑制IL-6表达可降低Caspase-3表达水平。     

中枢神经系损伤后不同时期星形胶质细胞的变化

将神经毒６－羟多巴胺注入大鼠一侧中脑腹侧被盖区，用胶质原纤维酸性蛋白抗体对损伤鼠中脑切片进行免疫组织化学ＡＢＣ法检测，观察星形胶质细胞在受损伤１ｄ至３０ｄ的不同时期的变化，术后１￣３ｄ，针道所经处见到ＧＦＡＰ阳性反应的细胞纤维，其胞体增大，纤维短粗。术后１周，针道周围反应性胶质细胞增多，有些反应性胶质细胞发出长的突起横向延伸垂直于针道，损害区附近血管增生，紧贴血管外周的胶质细胞也出现反应性变化。术     

大鼠脊髓损伤后星形胶质细胞特异性表达Gc的研究

用免疫组织化学方法观察了脊髓的星形胶质细胞在损伤后出现的抗原性改变并对其改变的意义进行了探讨。实验选用Wistar大鼠 2 0只。实验组 10只 ,对脊髓 T1 0 节段进行完全横断 ;对照组 10只 ,只进行 T1 0 椎板切除术 ,不损伤脊髓。在术后第 1、3、5、7、14 d分别对 2 0只大鼠灌流固定 ,并取出 3 cm长手术段脊髓。用 anti-Galactocerebrosides( anti-Gc)和 anti-glial fibrillaryacidic protein( anti-GFAP)抗体对脊髓进行标记。结果表明 :脊髓损伤后第 7d,增生肥大的星形胶质细胞可以同时被 anti-GF AP和 anti-Gc标记 (荧光双标 )。此抗原表型改变至术后 14 d依然显现。被双标的星形胶质细胞在形态上与成熟的正常胶质细胞基本相同 ,而少突胶质细胞只为 anti-Gc单独标记。对照组脊髓星形胶质细胞和少突胶质细胞只为 anti-GFAP、anti-Gc分别标记。本实验结果提示 :大鼠脊髓受损后 ,星形胶质细胞出现 GFAP和 Gc二种抗原表型。此结果首次表明成熟哺乳动物脊髓损伤后星形胶质细胞也可出现类似少突胶质细胞特异性抗原抗体改变。这可能是星形胶质细胞对脊髓创伤的一种特异性反应。这种变化可为探索脊髓损伤区域微环境的变化对脊髓损伤修复的影响提供新的线索     

白细胞介素-6受体在大鼠小脑间位核中的表达

我们以往的研究表明小脑皮层颗粒细胞表达白细胞介素-6受体(interleukin-6 receptor,IL-6R),该受体介导了白细胞介素-6(interleukin-6,IL-6)的神经保护作用。然而,大鼠小脑核团是否也表达IL-6R目前尚不清楚。为此,本研究应用RT-PCR、Western blot和免疫组织化学方法分别在基因、蛋白和细胞水平检测IL-6R在大鼠小脑间位核的表达。结果显示:(1)在小脑间位核PCR扩增产物的凝胶电泳图上,在365 bp位置可见一清晰的条带,与IL-6R mRNA预计扩增片断的长度一致;(2)在West-ern blot实验体系中,IL-6R免疫反应阳性条带出现在分子量为80 kD处;(3)免疫组织化学结果显示,小脑间位核中出现大量IL-6R免疫反应阳性细胞。以上结果表明:小脑间位核细胞在mRNA和蛋白水平均表达IL-6R,提示IL-6在小脑间位核内可能具有重要作用。     

Increased interleukin-6 messenger RNA expression in macrophage-cocultured endometrial stromal cells in adenomyosis

PROBLEM: To determine the effects of macrophage on endometrial stromal cells (ESCs) in women with adenomyosis. METHOD OF STUDY: Eutopic endometrium was obtained and separated into single ESC in 10 women with adenomyosis (study group) and 11 without adenomyosis (control group). ESCs were then cultured alone or with macrophage for 24 hr. RESULTS: Immunohistochemistry identified the presence of interleukin-6 (IL-6), IL-8, and IL-10 in ESCs. Real-time quantitative PCR revealed that the IL-6 mRNA was significantly expressed in macrophage-cocultured ESCs in adenomyosis than that in the controls, but was not different in ESCs cultured alone between the two groups. The levels of IL-8 and IL-10 mRNA were similar in ESCs either cultured alone or with macrophage between women with and without adenomyosis. CONCLUSION: IL-6 mRNA was significantly expressed in ESCs after in vitro coculture with macrophage in adenomyosis. This aberrant behavior of ESCs might play a role in the formation of ectopic endometrial implants in adenomyosis.     

两型星形胶质细胞损伤后胰甘油三酯脂酶的表达

目的:通过建立两型星形胶质细胞体外机械损伤模型,于损伤后不同时间点检测两型星形胶质细胞中胰甘油三酯脂酶(PTL)的表达变化。方法:采用震荡培养和差速贴壁法分离纯化新生SD大鼠的1型星形胶质细胞(T1As)和2型星形胶质细胞(T2As),以real-time PCR和Western Blot分别检测损伤后的两型星形胶质细胞中PTL mRNA和蛋白表达变化。结果:损伤后两型星形胶质细胞中PTL表达均出现先升高后降低的趋势,而在T2As中的表达远高于在T1As中的表达水平(P<0.01)。结论:PTL在T2As中高表达提示,中枢神经系统损伤后再生与修复过程中,T2As对脂质代谢及髓鞘形成发挥作用。     

Osteoblasts induce prostate cancer proliferation and PSA expression through interleukin-6-mediated activation of the androgen receptor

Prostate cancer (CaP) metastases selectively develop in bone as opposed to other sites through unknown mechanisms. Interleukin-6 (IL-6) is considered to contribute to CaP progression and is produced at high levels in osteoblasts. We hypothesized that osteoblast-derived IL-6 in the bone microenvironment contributes to the fertile soil for CaP growth. Accordingly, human CaP cells, LNCaP, C4-2B and VCaP, were treated with conditioned medium (CM) collected from human osteoblast-like HOBIT cells grown in androgen-depleted medium. We found that CM induced proliferation, prostate-specific antigen (PSA) protein and mRNA expression in a dose-dependent manner in these cell lines as determined by ELISA and real-time PCR, respectively. CM also activated the PSA promoter in these cells. Both HOBIT and primary osteoblast (POB) cells produced high levels of IL-6 measured by bioassay. LNCaP, C4-2B and VCaP cells expressed IL-6, but at much lower levels then the HOBIT and POB and they also expressed the IL-6 receptor mRNA, indicating they can respond to IL-6. Anti-IL-6 antibody added to HOBIT or POB CM dose-dependently inhibited the CM-induced cell proliferation and PSA expression in these CaP cell lines. HOBIT CM induced nuclear translocation of the AR and this was inhibited by anti-IL-6 antibody. Additionally, the antiandrogen bicalutamide inhibited HOBIT CM-induced cell proliferation. These results demonstrate that osteoblasts promote CaP growth through IL-6-mediated activation of the AR. Furthermore, these data underscore the importance of cross-talk between tumor and the bone microenvironment in the development of CaP bone metastases.     

Alternative splicing of interleukin-6 mRNA in mice

Expression of mRNA for interleukin-6, interleukin-6Δ3, and interleukin-6Δ5 was detected in placental tissue (second and third trimesters of pregnancy) and spleen of mice immunized with sheep erythrocytes in high dose. We hypothesize that translation of mRNA yields proteins capable of binding to individual subunits of the interleukin-6 receptor and possessing effector functions. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 138, No. 7, pp. 87–90, July, 2004     

Effects of interleukin-1 and dexamethasone on interleukin-6 production and growth in human meningiomas

Interleukin-6 (IL-6) has been shown to be released by cultured human meningioma cells and may be a positive or negative regulator of tumour growth. IL-6 protein and mRNA levels have been examined in a series of meningiomas. In 14 cases, the results are compared with the effects of IL-6 and dexamethasone on growth and IL-6 secretion in vitro. Tumours with the highest in vivo IL-6 mRNA expression also showed maximum induction of IL-6 and increased cellular proliferation on IL-1 stimulation in vitro. Dexamethasone decreased the IL-1-stimulated IL-6 release in all cases. Meningiomas which had little or no IL-6 message were refractory to IL-1 control of IL-6. Remarkably, these formed the group of meningiomas that increased their growth rate in response to dexamethasone. © 1997 John Wiley & Sons, Ltd.     

Sirtuin SIRT6 suppresses cell proliferation through inhibition of Twist1 expression in non-small cell lung cancer

SIRT6 is a member of the NAD⁺-dependent class III deacetylase sirtuin family. Current studies have revealed that SIRT6 plays important roles in the epigenetic regulation of genes expression and contribute to the proliferation, differentiation and apoptosis of cancer cells. However, the biological function of SIRT6 in lung cancer has not been elucidated. The present study showed that the mRNA and protein levels of SIRT6 were decreased in human non-small cell lung cancer (NSCLC) tissues and cell lines. MTT assay showed that overexpression of SIRT6 could inhibit the proliferation in NSCLC cells. In contrast, SIRT6 knockdown using small interfering RNA promoted NSCLC cells proliferation. On the molecular level, we found that SIRT6 inhibited the expression of Twist1 both at the mRNA and protein levels in NSCLC cells. Taken together, these results demonstrated for the first time that SIRT6 suppressed NSCLC cells proliferation via down-regulation of Twist1 expression and might provide novel therapeutic targets in the treatment of lung cancer.     

应用RNAi抑制细胞中绿色荧光蛋白的表达

目的 在293T和Mel细胞中研究RNA干扰（RNA interference，RNAi）对绿色荧光蛋白（enhanced green fluorescent protein，eGFP）表达的沉默作用。方法 以巢式PCR方法从人胚肾293T细胞中克隆出依赖于RNA聚合酶Ⅲ的H1启动子，并用于驱动RNAi片段的合成；构建能抑制eGFP特异性表达的RNAi载体（TRl）。将eGFP载体和RNAi干扰载体共转染293T和Mel两类细胞中，应用荧光显微镜观察、逆转录-PCR、荧光辅助细胞分选技术和定量逆转录-PCR方法分析上述细胞中RNAi对eGFP表达的抑制情况。结果 RNAi载体能有效地使293T和Mel细胞中红系特异的eGFP表达量均降低约50％以上。结论 本文构建的RNAi载体能有效的抑制目的基因eGFP在细胞中的表达。