ICAP-1对脐静脉内皮细胞PI3K表达及细胞增殖的影响

目的 研究整合素胞浆结构域关联蛋白-1(ICAP-1)对人脐静脉内皮细胞(HUVEC)增殖及PI3K信号通路表达的影响,以进一步研究其生物学作用及其在血管形成中的作用机制.方法 通过构建PCDNA3.1-ICAP-1质粒并转染HUVEC,采用MTT法测定HUVEC增殖化,Western Blot检测ICAP-1及PI3K表达.结果 PCDNA3.1-ICAP-1转染组ICAP-1和PI3K表达明显增加,PCDNA3.1-ICAP-1转染组与正常对照组、PCDNA3.1-GFP转染组比较表达差异有统计学意义(P<0.05).PCDNA3.1-ICAP-1转染组较PCDNA3.1-GFP转染组和正常对照组细胞增殖能力增强,差异有统计学意义(P<0.05).结论 ICAP-1具有促进内皮细胞增殖的作用,ICAP-1可能通过上调PI3K表达,促进内皮细胞增殖达到促进血管新生的作用.     

老年白内障晶状体上皮细胞凋亡及凋亡基因的表达

目的探讨老年白内障与晶状体上皮细胞凋亡的关系。方法TUNEL法检测凋亡细胞百分率；免疫组化法检测P53、bax在老年白内障晶状体上皮细胞中的蛋白表达；RT-PCR检测p53、bcl-2在老年白内障晶状体上皮细胞中的mRNA的表达水平。结果凋亡细胞百分率为5%～47.4%，P53蛋白在老年白内障晶状体上皮细胞中的表达率为15%～28%，bax蛋白的表达率为5%～16%，p53mRNA的表达率为0.48～0.78，baxmRNA的表达率为0.34～0.67。结论老年白内障的发生与晶状体上皮细胞凋亡存在一定的相关性。     

辛伐他汀抑制血管平滑肌细胞增殖及对PTEN、p27蛋白表达的影响

目的：观察辛伐他汀（simvastatin）对血管平滑肌细胞（VSMC）增殖的抑制作用及其对细胞周期和周期蛋白依赖性激酶抑制物p21、p27，细胞G1－S期转换重要调节基因PTEN、c-myc蛋白表达的影响，并进一步观察simvastatin是否通过抑制脂质代谢的中间产物－甲羟戊酸的合成而上调PTEN、p27蛋白的表达及PTEN和p27是否存在上下游关系。方法：[^3H]－胸腺嘧啶核苷酸（[^3H]-TdR）掺入测定VSMC DNA合成，流式细胞仪检测细胞周期情况，Western印迹杂交法检测p21、p27及PTEN、c-myc蛋白表达，人工合成PTEN反义寡核苷酸，并以正义寡核苷酸为对照，以脂质体介导转染细胞，以Western印迹杂交流检测转染效率并观测其对PTEN、p27蛋白表达的影响。结果：simvastatin以剂量依赖关系抑制血清诱导的VSMC[^3H]-胸腺嘧啶核苷酸掺入，使G0／G1期细胞比例明显增多，S期细胞比例显减少，并上调p27及信号通路中常位于p27上游的PTEN的蛋白表达，但不影响p21、c-myc蛋白表达，PTEN反义寡核苷酸下调PTEN蛋白的表达后并不影响p27蛋白表达，200μmol/L甲羟戊酸能显抑simvastatin诱导的PTEN、p27蛋白表达升高。结论：simvastatin能抑制VSMC增殖，使细胞周期停滞于G0／G1期，这一过程可能通过不同的信号途径分别上调PTEN、p27这两个调控G1－S周期转换的基因而实现，而非通过PTEN－p27这一经典通路，simvastatin对PTEN、p27的调节与其抑制甲羟戊酸的合成有关。     

莲心碱对血管平滑肌细胞增殖及热应激蛋白70和P53表达的影响

采用培养的内皮素所致血管平滑肌细胞增殖模型。用氚-胸腺嘧啶核苷掺入法、流式细胞术、Westernblot及Northernblot分析方法，观察了莲心减对血管平滑肌细胞增殖的作用及对热应激蛋白70及其mRNA和抑癌基因P53表达的影响。结果发现，遂心碱能逆转内皮素所致的~3H－TdR掺入量增多，阻止血管平滑肌细胞由Go／G1期进入DNA合成期（S期）和有丝分裂期（G2／M期）．并能使效应激蛋白70及mRNA表达减弱，P53抑癌基因及mRNA表达增强。以上结果提示，莲心碱能抑制血管平滑肌细胞增殖，这可能与热应激蛋白70及P53的调控有关。     

阿托伐他汀对人脐静脉内皮细胞增殖活性的影响

目的观察阿托伐他汀对人脐静脉内皮细胞增殖(humanumbilicalveinendothelialcells,HUVEC)及超微结构的影响。方法将HUVEC分为3组进行培养,包括正常对照组、重组人白细胞介素6(rhIL6)刺激组和阿托伐他汀组。通过MTT法测定各组细胞的增殖活力,并用透射电镜观察各组细胞超微结构的改变。结果①正常对照组、rhIL6组及阿托伐他汀组的吸光度(A)分别为0.172±0.014、0.257±0.058及0.184±0.021;②rhIL6作用下,HUVEC粗面内质网上核糖体较对照组明显增多,而阿托伐他汀可抑制rhIL6对HUVEC的这种超微结构改变。结论阿托伐他汀能有效抑制rhIL6,进而阻止HUVEC增殖。     

马兜铃酸对肾小管上皮细胞UⅡ、GPR14 mRNA表达的影响

目的观察马兜铃酸对肾小管上皮细胞(NRK-52E细胞)中尾加压素Ⅱ(UⅡ)及其受体GPR14 mRNA表达的影响。方法在常规培养的NRK-52E细胞中分别加入5、10、20 ng/mL马兜铃酸和单纯培养液(对照组)。培养48 h后用Trizol法提取总RNA,采用RT-PCR法检测UⅡ、GPR14 mRNA。结果用5、10、20 ng/mL马兜铃酸处理48 h后,NRK-52E细胞中UⅡmRNA表达量(相对值)分别为0.53±0.07、0.55±0.05、0.62±0.04,均明显高于对照组的0.41±0.08,P均<0.05;GPR14 mRNA表达量(相对值)分别为0.23±0.06、0.38±0.07、0.51±0.07,20 ng/mL马兜铃酸处理组明显高于对照组的0.22±0.03,P<0.05。结论马兜铃酸以剂量依赖方式促进肾小管上皮细胞UⅡ及GPR14 mRNA表达。     

差异性应激对青、老年大鼠海马HSP70 mRNA表达的影响

目的 观察重复低强度强迫游泳应激后青、老年大鼠海马HSP70 mRNA表达及习惯化表达;增强应激强度对青、老年大鼠海马HSP70 mRNA表达的影响.方法 采用反转录-聚合酶链反应(RT-PCR)方法 ,观察重复强迫温水游泳应激导致青年组(2个月龄)、老年组(16个月龄)大鼠海马HSP70 mRNA表达的变化和增强应激强度对HSP70 mRNA表达的影响 .结果 重复强迫温水游泳可导致两组大鼠HSP70 mRNA表达习惯化,当再给予4℃冰水游泳应激时,可引起HSP70 mRNA表达增加;老年组大鼠HSP70 mRNA表达在多时段均低于青年组大鼠(P＜0.05).结论 重复应激可以导致HSP70 mRNA表达习惯化,高强度应激可以逆转低强度应激引起的习惯化效应;老年组大鼠应激后HSP70 mRNA表达在多时段低于青年组大鼠.     

高糖对体外培养肾小管上皮细胞增殖的影响

利用体外培养胎儿肾小管上皮细胞在不同糖浓度刺激下采用四甲基偶氮多胍(MTT)渗入法观察肾小管上皮细胞的增殖情况.结果生长在糖浓度25mmol／L中的上皮细胞24小时为0.196±0.023(吸光度),48小时为0.194±0.027;在糖浓度5.15mmol／L中,24小时为0.240±0.028,48小时为0.236±0.027;两者差异(P＜0.05).结论高糖对体外培养肾小管上皮细胞的增殖起到一定的抑制作用.     

Influencing factors of rat small intestinal epithelial cell cultivation and effects of radiation on cell proliferation

INTRODUCTIONCrypt epithelial cells in normal small intestineproliferate at a high speed. But they are verydifficult to culture in vitro and passage stably. A lotof studies have been done[1-16]. Some domestic labsisolated and cultured crypt cells from embryonalintestines and aseptic animal intestine, but failed.We introduced normal rat epithelial cell line-IEC-6from the USA and its living condition for stablepassage was successfully established after trials. Thecell line was testified to be the small intestinalepithelial cell by electron microscopy,immunihistochemistry and enzymatic histoch-emistry. It has been applied to some relatedresearch work[17-21]. It was found that manyfactors were involved in the culture system. Ourpresent study focuses on the culture method and theinfluencing factors on IEC-6.     

Influence of acid and bile acid on ERK activity, PPAR_Y expression and cell proliferation in normal human esophageal epithelial cells

AIM: To observe the effects of acid and bile acid exposure on cell proliferation and the expression of extracellular signal-regulated protein kinase (ERK) and peroxisome proliferator-activated receptor Y (PPAR_Y) in normal human esophageal epithelial cells in vitro. METHODS: In vitro cultured normal human esophageal epithelial cells were exposed to acidic media (pH 4.0-6.5), media containing different bile acid (250μmol/ L), media containing acid and bile acid, respectively. Cell proliferation was assessed using MTT and flow cytometry. The expressions of phosphorylated ERK1/2 and PPAR_Y protein were determined by the immunoblotting technique. RESULTS: Acid-exposed (3 min) esophageal cells exhibited a significant increase in proliferation ratio, S phase of the cell cycle (P<0.05) and the level of phosphorylated ERK1/2 protein. When the acid-exposure period exceeded 6 min, we observed a decrease in proliferation ratio and S phase of the cell cycle, with an increased apoptosis ratio (P<0.05). Bile acid exposure (3-12 min) also produced an increase in proliferation ratio, S phase of the cell cycle (P<0.05) and phosphorylated ERK1/2 expression. On the contrary, deoxycholic acid (DCA) exposure (> 20 min) decreased proliferation ratio. Compared with bile acid exposure (pH 7.4), bile acid exposure (pH 6.5, 4) significantly decreased proliferation ratio (P<0.05). There was no expression of PPAR_Y in normal human esophageal epithelial cells. CONCLUSION: The rapid stimuli of acid or bile acid increase proliferation in normal human esophageal epithelial cells by activating the ERK pathway.     

Influence of acid and bile acid on ERK activity, PPARγ expression and cell proliferation in normal human esophageal epithelial cells

AIM: To observe the effects of acid and bile acid exposure on cell proliferation and the expression of extracellular signal-regulated protein kinase (ERK) and peroxisome proliferator-activated receptor gamma (PPARgamma) in normal human esophageal epithelial cells in vitro. METHODS: In vitro cultured normal human esophageal epithelial cells were exposed to acidic media (pH 4.0-6.5), media containing different bile acid (250 mumol/L), media containing acid and bile acid, respectively. Cell proliferation was assessed using MTT and flow cytometry. The expressions of phosphorylated ERK(1/2) and PPARgamma protein were determined by the immunoblotting technique. RESULTS: Acid-exposed (3 min) esophageal cells exhibited a significant increase in proliferation ratio, S phase of the cell cycle (P<0.05) and the level of phosphorylated ERK(1/2) protein. When the acid-exposure period exceeded 6 min, we observed a decrease in proliferation ratio and S phase of the cell cycle, with an increased apoptosis ratio (P<0.05). Bile acid exposure (3-12 min) also produced an increase in proliferation ratio, S phase of the cell cycle (P<0.05) and phosphorylated ERK(1/2) expression. On the contrary, deoxycholic acid (DCA) exposure (>20 min) decreased proliferation ratio. Compared with bile acid exposure (pH 7.4), bile acid exposure (pH 6.5, 4) significantly decreased proliferation ratio (P<0.05). There was no expression of PPARgamma in normal human esophageal epithelial cells. CONCLUSION: The rapid stimuli of acid or bile acid increase proliferation in normal human esophageal epithelial cells by activating the ERK pathway.     

PI3K抑制剂LY294002对人晶状体上皮细胞E2F-1、cyclinE蛋白表达的影响

目的探讨PI3K抑制剂LY294002对人晶状体上皮细胞（LECs）增殖及E2F-1、细胞周期素E（cyclinE）蛋白表达的影响。方法人LECs常规培养,分为实验组和对照组,实验组加入PI3K抑制剂LY294002（25μmol/L）,对照组加入同体积DMSO,两组分别培养36 h后,MTT比色法测定细胞增殖状况,Western blot法检测细胞中E2F-1及cyclinE蛋白表达。结果实验组细胞增殖受到明显抑制,与对照组比较差异有统计学意义（P〈0.05）,人LECs中E2F-1及cyclinE蛋白表达降低（P〈0.05）。结论 PI3K抑制剂LY294002可抑制人LECs的增殖,可能与下调E2F-1及cyclinE蛋白的表达水平相关。     

Helicobacter pylori and mitogen-activated protein kinases regulate the cell cycle, proliferation and apoptosis in gastric epithelial cells

Background and Aims:  Helicobacter pylori infection activates mitogen-activated protein kinases (MAPK) and modulates cell proliferation and apoptosis. However, the relationship between H. pylori infection and MAPK signaling in controlling cell proliferation and apoptosis is not clear, nor has the role of MAPK on the gastric epithelial cell cycle and proliferation been established. Therefore, we investigated the effects of H. pylori infection and MAPK inhibition on these processes.
Methods:  Gastric epithelial cell lines (AGS and MKN45) were infected with H. pylori and/or treated with MAPK inhibitors. Cell cycle and apoptosis were measured by flow cytometry. Cell cycle proteins and proliferation were monitored by western blot and cell count, respectively.
Results:  Infection with H. pylori resulted in dose-dependent MAPK activation, cell cycle arrest, reduced proliferation and increased apoptosis. The effect of H. pylori and MAPK at various cell cycle checkpoints was noted: MEK1/2 and p38 inhibition increased H. pylori -induced cell cycle G₁ arrest, while JNK inhibition reduced G₁ arrest. MEK1/2 inhibition increased p21, p27 and cyclin E and JNK inhibition additionally increased cyclin D1 expression. Both inhibitors decreased cell proliferation. All inhibitors enhanced apoptosis after H. pylori infection. We also detected MAPK cross-talk in AGS cells: p38 and JNK inhibitors increased ERK activation. The p38 inhibitor increased JNK and the MEK1/2 inhibitor decreased JNK activation only during H. pylori infection.
Conclusions:  These results suggest H. pylori and MAPK differentially regulate the cell cycle, proliferation and apoptosis in gastric epithelial cells. The imbalance between H. pylori infection and MAPK activation likely contributes to the H. pylori -induced pathogenesis.     

人肝癌细胞膜上HSP70的表达

用流式细胞仪和Cell-ELISA检测了 4株人肝癌细胞膜上HSP70的表达。结果表明正常情况下人肝癌细胞膜上HSP70的表达较低 ,经热处理后表达增加 (P <0 0 1)并显示出细胞之间的差异 (P <0 0 1)。推测其低表达可能与肝癌细胞逃逸机体免疫监视有关 ,对制定肝癌的防治对策有重要的指导意义。     

左旋肉碱对氧化应激损伤肾小管上皮细胞的保护作用

目的观察左旋肉碱对过氧化氢（H2O2）应激损伤人肾小管上皮细胞的保护作用,并探讨其可能机制。方法用H2O2作用于人肾小管上皮细胞系HK-2,建立肾小管上皮细胞氧化应激损伤模型;MTT法检测左旋肉碱预处理后HK-2细胞的活力;用酶化学法测定HK-2细胞超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH—Px）、过氧化氢酶（CAT）活性及总抗氧化能力（T—AOC）、丙二醛（MDA）;荧光显微镜观察及流式细胞仪测定细胞内活性氧（ROS）及HK-2细胞凋亡率。结果左旋肉碱预处理12h能抑制H2O2损伤所导致的HK-2细胞活力降低,增加细胞中SOD、GSH—Px和CAT含量,提高细胞T-AOC,降低细胞中MDA和ROS水平,抑制HK-2细胞凋亡。结论左旋肉碱对氧化应激所致的肾小管上皮细胞损伤具有保护作用,其机制可能与增强细胞抗氧化能力、减少自由基生成、抑制脂质过氧化反应及细胞凋亡有关。     

结核分枝杆菌热休克蛋白Hsp70在原核表达载体中的克隆表达与鉴定

目的对结核分枝杆菌的一种热休克蛋白Hsp70基因进行克隆,鉴定,并在原核系统中表达。方法以结核杆菌H37Rv菌株基因组DNA为模板,用PCR法对基因Hsp70进行扩增,产物与载体质粒pET-22b(+)构建表达Hsp70的重组质粒,将此重组质粒先转化到E.coliDH5α内提取质粒,酶切鉴定,再转化入表达宿主E.coliBL21(DE3)PlysS菌株内,对转化菌株以IPTG进行诱导后,破菌,离心,上清进行SDS-PAGE,通过电转移将胶中蛋白转到硝酸纤维素薄膜上后进行免疫印迹分析。结果电泳发现转化了重组质粒的菌株有表达蛋白,所表达的蛋白质相对分子质量为70 000,抗体检测有特异带大小为70kDa。结论成功进行了结核杆菌分泌热休克蛋白Hsp70基因的克隆表达。     

肝癌相关抗原kineetin基因重组蛋白致敏的树突状细胞对T细胞增殖活化的影响

目的观察肝癌相关抗原kineetin基因重组蛋白（kinectln—MBP）致敏的树突状细胞（DC）对T细胞增殖活化能力的影响。方法从正常人外周血中分离单个核细胞,用rhGM—CSF、rhIL-4联合诱导扩增DC;用kinectin-MBP（kinectin—MBP组）、麦芽糖结合蛋白（MBP组）分别致敏DC,对照组不处理。采用ELISA法检测细胞上清液中的IL-12、IFN-γ,MTT法检测各组DC刺激自体T淋巴细胞增殖的能力。结果kinectin—MBP组上清液中IL-12、IFN-γ含量分别为（615．32±7．93）、（544．28±7．17）pg／ml,MBP组分别为（382．13±5．21）、（308．46±6．44）pg／ml对照组组分别为（299．40±10．22）、（228．03±6．70）pg/ml,三组相比,P均〈0．05。kinectin—MBP组T淋巴细胞增殖指数为53．14±0．62,MBP组为22．14±0．31,对照组为10．86±0．65,三组相比,P均〈0．001。结论体外诱导扩增的DC经kinectin-MBP致敏后具有很强的刺激自体T细胞增殖的能力。