The Apoptosis of Bovine Lens Epithelial Cells Induced by Proteasome Inhibitor MG132

To investigate the effect of proteasome inhibitor MG132 on the apoptosis of bovine lens epithelial cells （BLECs）, the cells were treated with MG132 at different concentrations for12, 24 and 36 h. The cell viability was analyzed by MTT assay and the effect of MG132 on the apoptosis of BLECs was assessed by flow cytometry （FCM）. The results showed that after treatment for the same period, the inhibitory effect of MG132 on BLECs proliferation was enhanced with the increment of the concentration of MG132 （0, 2, 5, 10, μmol/L） （P〈0.05）. The 50% inhibiting concentration （IC50） was 2.03 μmol/L when the BLECs were treated with MG132 for 36 h. MG132 also induced the apop- tosis of BLECs obviously. FCM showed that the apoptosis index of the cells treated by MG132 at 2 μmol/L for 12 h was （20.24±1.51）%, and that of the control was （0.98±0.20）% respectively （P〈0.01, n=3）. It was concluded that MG132 could lead to apoptosis of BLECs. The decrease of proteasome activity may play an important role in the formation and development of cataract.     

紫外线对人晶状体上皮细胞凋亡的诱导及Bcl-2,Bax基因的影响

目的:研究紫外线照射体外培养人晶状体上皮细胞(human lens epithelial cell,HLEC)对凋亡的诱导、凋亡调控基因(Bax,Bcl-2)表达的变化,探讨紫外线诱导HLEC凋亡的机制。方法:以实验室培养的HLEC细胞株为研究模型,采用同一紫外线光源对HLEC进行照射。按紫外线照射时间将HLEC分为0,5,10,15及30 min组。采用Annexin V+PI双染流式细胞计数对HLEC凋亡进行检测,用原位杂交的方法检测各组Bax,Bcl-2 mRNA的表达。结果:随紫外线照射时间的延长HLEC凋亡率增加,Bcl-2阳性细胞率逐渐降低;而Bax阳性细胞率逐渐增加。HLEC凋亡率与Bcl-2和Bax的比率呈负相关(r=-0.874,P<0.05)。结论:紫外线照射可诱导HLEC凋亡,Bax和Bcl-2可能参与了紫外线诱导的HLEC凋亡的基因调控过程。     

老年性白内障晶状体上皮细胞超微结构的研究

目的　观察老年性白内障晶状体上皮细胞超微结构的改变 ,探讨细胞凋亡在老年性白内障形成中的作用。方法　老年性白内障患者 2 4例 ,行囊外或超声乳化联合后房型人工晶状体植入术 ,术中取中央部晶状体前囊膜 ,分别行光镜、扫描电镜、透射电镜观察。结果　老年性白内障晶状体上皮细胞的超微结构改变有细胞形态大小不一 ,扁平细胞多无正常细胞结构 ,低柱状及柱状细胞结构大体正常 ,但细胞间隙增大 ,胞浆内可见空泡变性 ,部分细胞溶解坏死或发生皱缩 ,未见凋亡细胞。结论　老年性白内障的发生与晶状体上皮细胞变形坏死密切相关 ,与晶状体上皮细胞凋亡无关     

姜黄素抑制晶状体上皮细胞增殖的信号转导机制

目的:探讨姜黄素(curcumin,Cur)抑制重组人表皮生长因子(recom binanthuman epidermal growth factor,rhEGF)诱导的牛晶状体上皮细胞(lens epithelial cell,LEC)增殖的信号转导机制。方法:采用荧光分光光度法检测Cur作用后LEC内游离Ca2 浓度;应用放射免疫分析法检测Cur作用后LEC内cAMP和cGMP含量的变化。结果:经50μg/LrhEGF作用后,LEC内游离Ca2 浓度明显升高,Cur可使LEC内游离Ca2 浓度进一步升高。经50μg/LrhEGF作用后,LEC内cAMP浓度明显下降,cGMP浓度明显升高;而Cur则可使rhEGF作用后的LEC内cAMP浓度明显升高,cGMP浓度明显降低。结论:Cur可抑制rhEGF诱导的LEC增殖,其抑制细胞增殖的作用可能是通过多条信号转导途径来实现的。     

人类晶体上皮细胞的体外培养与观察

目的：建立人类晶体上皮细胞培养的简便方法并观察原代和传代细胞的生物学特征和组织学变化。方法：将人工晶体植入术中取下的前囊膜分割成小碎片，吸管转移至培养瓶底部进行原代培养，7—10d后常规方法进行传代培养，采用相差显微镜观察活体细胞的增殖活动和形态学变化。结果：人类晶体上皮细胞在体外培养时可以存活并传代，但是其生存能力与供体年龄有关。传代后期细胞发生纤维化改变。结论：本方法简便，有效。可用于实验研究。     

Caspase-3 and its inhibitor Ac-DEVD-CHO in rat lens epithelial cell apoptosis induced by hydrogen in vitro

Objective To investigate the role of caspase-3 and its inhibitor Ac-DEVD-CHO in rat lens epitheli alcell apoptosis induced by hydrogen peroxide (H2O2 ) in vitro.Methods Rat lenses were incubated in modified Eagle‘s medium containing 2 mmol/L H2O2 to induce apoptosis in vitro. Apoptosis in lens epithelial cells was assessed by transmission electron microscopy and annexin V-propidium iodide (PI) double staining flow cytometry after 12, 24 and 48 h of incubation. The activity of caspase-3 was analyzed by western blotting.Results Observations under transmission electron microscopy revealed that 2 mmol/L H2O2 could effectively induce lens epithelial cell apoptosis in vitro. Caspase-3 activity increased during cell apoptosis and the peak measurement occurred at 24 h after treatment with H2O2. Cell apoptosis was blocked by caspase-3 inhibitor Ac-DEVD-CHO.Conclusions The activation of caspase-3 plays an important role in executing apoptosis in H2O2-treated lens epithelial cells and in the formation of cataract. The caspase-3 inhibitor Ac-DEVD-CHO may effectively prevent lens epithelial cell apoptosis caused by oxidative injury.     

上皮性卵巢癌组织中P53和Survivin蛋白的表达

目的:探讨上皮性卵巢癌组织中P53蛋白和Survivin蛋白的表达情况.方法:应用免疫组织化学法检测61例上皮性卵巢癌组织(浆液性腺癌43例,黏液性腺癌18例;高分化30例,中分化21例,低分化10例;伴淋巴结转移30例,无淋巴结转移31例)中P53和Survivin蛋白的表达.结果:61例上皮性卵巢癌组织中P53蛋白阳性表达32例;Survivin蛋白阳性表达40例.P53和Survivin蛋白的表达与上皮性卵巢癌组织学分型无关(P＞0.05 );与分化程度、临床分期和淋巴结转移有关(P＜0.05).上皮性卵巢癌组织中Survivin蛋白的表达与P53蛋白表达呈正相关(r=0.558,P＜0.05).结论:P53和Survivin蛋白与上皮性卵巢癌组织的发生、发展及转移有关,可作为判断卵巢癌预后不良的指标.     

卵巢上皮性肿瘤组织中VEGF与P53的表达

目的:探讨卵巢上皮性肿瘤组织中血管内皮细胞生长因子(VEGF)及P53蛋白的表达。方法:应用免疫组织化学SP法和形态定量学方法,检测卵巢上皮性肿瘤组织(良性囊腺瘤17例,交界性肿瘤15例,恶性肿瘤29例)中VEGF和P53蛋白的表达,并测定其积分光密度(IOD)和平均光密度(MOD)。结果:良性、交界性及恶性肿瘤组织中VEGF的IOD和MOD值分别为213.6±94.8和0.19±0.07、419.4±71.6和0.26±0.08、667.2±32.9和0.41±0.03,P53蛋白表达的IOD和MOD值分别为164.2±44.8和0.17±0.04、521.3±57.1和0.24±0.07、872.2±37.4和0.41±0.04,良性肿瘤分别与交界性肿瘤和恶性肿瘤相比,IOD和MOD值差异均有统计学意义(P<0.01或0.05)。且VEGF与P53表达呈正相关(rs=6.24,P<0.05)。结论:卵巢上皮性肿瘤的发展中VEGF起重要作用,P53蛋白可能参与卵巢肿瘤的血管生成调节。     

过氧化氢诱导鼠晶状体上皮细胞凋亡中 Caspase-2的表达及意义

目的探讨过氧化氢诱导鼠白内障形成过程中晶体上皮凋亡细胞中半胱氨酸天冬氨酸酶-2(Cas-pase-2)的表达及意义。方法大鼠晶状体器官离体培养后，用免疫组织化学方法检测晶体上皮细胞Caspase-2的表达。结果随过氧化氢损伤时间的处长，晶体上皮细胞Caspase-2的表达逐渐增强，与晶体混浊程度正相关。结论过氧化氢可以诱导鼠晶体上皮细胞Caspase-2的表达，后者可能参与晶体上皮细胞凋亡和白内障的形成。     

野生型P53基因诱导人肝癌HepG2/5-Fu耐药细胞株凋亡的研究

目的 探讨野生型P53基因与人肝癌耐药的相关性，为肝癌的临床治疗提供新的依据。方法 以IONP—PLL纳米颗粒为基因转运载体，将wtp53基因转入HepG2／5-Fu耐药性细胞株，MTT法、台盼兰拒染实验和Hochest33258染色等分析5-Fu对转染有wtp53基因的HepG2／5-Fu细胞的杀伤效应。结果 转染wtp53基因的HepG2／5-Fu细胞对5-Fu呈剂量依赖性杀伤效应和时间依赖性杀伤效应，在5-Fu作用下，能发生明显的凋亡效应。结论 以wtp53基因为靶点的肝癌基因治疗有望成为治疗晚期肝癌的一种新的治疗策略。     

紫外线对晶状体上皮细胞凋亡、水含量及SOD活性的影响

目的 :探讨紫外线对体外培养的晶状体上皮细胞凋亡作用以及对晶状体水含量和超氧化物歧化酶 (superoxidedismutase,SOD)活性的影响 .方法 :紫外线诱导离体牛晶状体白内障形成 ,以TUNEL技术检测紫外线照射后培养 3,6 ,12 ,2 4h的晶状体上皮凋亡细胞 ,并测定晶状体水含量和SOD活性 .结果 :TUNEL法显示 ,照射组晶状体上皮细胞经紫外线照射后培养 6h出现凋亡细胞 ,对照组晶状体上皮细胞中无明显的凋亡细胞 ;照射组 12h后水含量明显升高 (P <0 .0 1)及SOD活性明显降低 (P <0 .0 1) .对照组水含量和SOD活性无明显变化 .结论 :紫外线诱导晶状体上皮细胞发生凋亡 ,并使晶状体水含量和SOD活性发生改变 ,紫外线诱导的晶状体上皮细胞凋亡是白内障形成的早期机制之一     

上皮性卵巢肿瘤P53的表达

为了解卵巢癌变过程中Ｐ５３的变化及其意义，应用免疫组化技术，研究了５例正常卵巢，２１例卵巢腺瘤，１０例交界性肿瘤，４０例上皮性卵巢癌及１９例大网膜转移灶组织中Ｐ５３蛋白的变化。结果发现：Ｐ５３表达仅见于卵巢癌组织中，浆液性癌的阳性表达率（１５／２２）显著高于粘液性癌（３／１３）和子宫内膜样癌（１／５）（Ｐ＜００５）；Ｐ５３在卵巢癌原发灶和其相应大网膜转移灶中的表达基本一致；Ｐ５３标记指数与卵巢癌组织学分级有关，Ⅱ、Ⅲ级癌的Ｐ５３标记指数显著高于Ⅰ级癌（Ｐ＜００５）。结果提示：Ｐ５３蛋白聚集变化主要发生在卵巢癌变的晚期，Ｐ５３表达有助于良恶性病变的鉴别；卵巢癌为单中心起源；Ｐ５３标记指数与卵巢癌的组织学分级呈正相关     

人晶状体上皮细胞体外培养方法的改良

目的改良现有人晶体上皮细胞的培养方法,建立稳定的体外培养模型。方法用改良培养法对人胚胎眼晶体前囊膜进行培养,并利用形态学检查方法进行检测和鉴定。结果组织块法在加入培养基24 h内即可见细胞生长,且保持上皮细胞形态,1wk左右细胞融合。在体外可传5代以后细胞衰老,存活者呈成纤维细胞状。消化法中2 d后见细胞壁生长,1周左右细胞融合。结论成功地改进了建立晶状体上皮细胞体外培养模型的方法,为研究后囊膜混浊发病机制提供了方法学基础。     

阿霉素诱导兔眼后囊晶状体上皮细胞凋亡及其凋亡基因的表达

目的:探讨阿霉素对兔晶状体摘除术后诱导后囊晶状体上皮细胞凋亡并观察凋亡相关基因(P53、Bcl-2)的表达.方法:在12只兔眼晶状体囊外摘除术中分别注入0.2ml阿霉素(0.4mg./L),在术后3周、8周分别行TUNEL法和免疫组化SABC法染色,计算P53、Bcl-2基因表达阳性百分率.结果:术后3、8周TUNEL法标记的阳性凋亡细胞比对照组明显增多(P＜0.05).P53蛋白表达阳性率在3、8周比对照组显著增多(P＜0.01).Bcl-2蛋白表达阳性率在3周时显著减少(P＜0.01),在8周时仍比对照组减少(P＜0.05).结论:阿霉素诱导兔后囊晶状体上皮细胞凋亡,用药组P53基因表达而Bcl-2基因低表达,提示P53、Bcl-2基因可能参与晶状体上皮细胞凋亡的调控.     

Effects of rapamycin on expression of Bcl-2 and Bax in human lens epithelial cells and cell cycle in rats

The effects of rapamycin on the expression of Bcl-2 and Bax protein in in vitro cultured human lens epithelial cells(LECs) and cell cycle were investigated in order to provide the theoretical basis for the development of new inhibitory drugs for clinical prevention and treatment of after-cataract.The cultured LECs of second and third passages were collected and treated with rapamycin.The LECs were transferred into 96-well culture plates and divided into 6 groups,and each group was set to have 8 duplicate wells.In the negative control group,the LECs were given culture medium only,and in the blank control group,only culture medium was given.In the four rapamycin-treated groups,different concentrations(20,40,60 and 80 ng/mL) of rapamycin were given.After treatment for 24,48 and 72 h,the absorbance(A) values in each well were determined by MTT assay.The cell cycles of all groups were detected by using flow cytometry.Real-time fluorescent quantitative polymerase chain reaction(RFQ-PCR) and Western blot were used to detect the mRNA and protein expression of Bcl-2 and Bax respectively.MTT assay showed that rapamycin could inhibit proliferation of LECs in a time-and dose-dependent manner.Flow cytometry revealed that rapamycin could block the conversion of LECs from G1 phase to S phase,resulting in the increase of cells in G1 phase and the decrease of the cells in S phase.RFQ-PCR indicated that rapamycin could down-regulate the expression of Bcl-2 mRNA,but up-regulate the expression of Bax mRNA,suggesting it could induce apoptosis of LECs.Western blot demonstrated that rapamycin could suppress the expression of Bcl-2 protein,but promote the expression of Bax protein.It is concluded that rapamycin could inhibit proliferation of LECs probably not only by blocking the progression of cell cycle,but also by promoting the induction of apoptosis.     

老年性白内障晶状体上皮细胞的形态学变化

目的 观察老年性白内障晶状体上皮细胞的形态学改变，探讨其与白内障形成的关系。方法 采集老年性白内障晶状体囊外除术中取出的前囊膜，采用铺片及ＨＥ染色、平板包埋及制备超薄切片等技术，分别在光镜和电镜下观察晶状体上皮细胞的密度、细胞形态和超微结构变化。结果 白内障 晶状体上皮细胞密度较正常晶状体明显减小（Ｐ〈０．０１），６０岁以上组细胞低于６０岁以下组（Ｐ〈０．０１）。光镜下，形态异常的细胞散在分布，     

P53蛋白在喉癌细胞中的表达

（1）目的：对喉癌发生，发展的分子生物学机制进行初步的探讨。（2）方法：喉癌手术新鲜标本30例，喉正常粘膜16例为对照组。采用流式细胞术检测喉癌组织及正常粘膜的P53蛋白的含量，基因蛋白以荧光指 数（FI）作为定量分析的指标，以FI大于正常粘膜的x 2s为阳性。（3）结果：a、癌组织中P53的阳性率高达96．7％，其FI均明显高于正常粘膜（P＜0．01）。b.临床Ⅲ、Ⅳ期癌组织P53的FI明显高于Ⅰ、Ⅱ期（P＜0．01），T3,T4病变的P53的FI明显高于T1、T2病变（P＜0．05）；淋巴结转移组的P53的FI明显高于无淋巴结转移组（P＜0．01）。（4）结论：P53蛋白在喉癌中的表达明显高于喉正常粘膜，而且与临床分期，T分级及淋巴结转移呈正相关，与年龄、肿瘤大小和病理分级无关。     

Mitochondrial proteomic analysis of isopsoralen protection against oxidative damage in human lens epithelial cells

Objective:To investigate the protective effects of the natural medicinal monomer isopsoralen（ISR） with estrogenic activity against oxidative damage in human lens epithelial cells B3（HLE-B3） caused by hydrogen peroxide（H₂O₂） and to pursue the possible mitochondrial proteomic regularity of the protective effects.Methods: HLE-B3 cells were treated with H₂O₂（300μmol/L）,β-estradiol(E₂:10^-8 mol/L) and H₂O₂,ISR(10^-5 mol/L) and H₂O₂,or left untreated.Altered expressions of all mitochondrial proteins were analyzed by protein array and surfaceenhanced laser desorption ionization time of flight mass spectrometry（SELDI-TOF-MS）.The mass/charge（m/z） ratios of each peak were tested by the Kruskal-Wallis rank sum test,and the protein peak value of the m/z ratio for each treatment by pair comparison was analyzed with the Nemenyi test.Results:H₂O₂ up-regulated the expressions of two protein spots（with m/z of 6532 and 6809）.E₂ mitigated the oxidative damage,and the expression of one protein spot（m/z 6532） was down-regulated.In contrast,ISR down-regulated both of protein spots（m/z 6532 and 6809）.Conclusions:ISR could effectively inhibit H₂O₂-induced oxidative damage in HLE-B3 cells.The protein spot at m/z of 6532 might be the target spot of ISR against oxidative damage induced by H₂O₂.     

反义c-myc寡核苷酸对半乳糖诱导的晶体上皮细胞生长的抑制作用

目的探讨反义c-myc寡核苷酸(ASODN)对半乳糖诱导的兔晶体上皮细胞(LEC)生长的影响.方法人工合成与c-myc基因第二外显子翻译起始区序列互补的寡核苷酸,用半乳糖和反义寡核苷酸处理培养的晶体上皮细胞,应用细胞计数法和MTT法观察反义c-myc寡核苷酸对半乳糖诱导的晶体上皮细胞增殖的影响.结果 2.5～10.0 μmol/L反义c-myc寡核苷酸能抑制半乳糖诱导的晶体上皮细胞的增殖,10.0 μmol/L时作用较显著.结论反义c-myc寡核苷酸对半乳糖诱导的白内障晶体上皮细胞的生长增殖有抑制作用.     

黄芪注射液对高糖诱导的肾小管上皮细胞凋亡的影响

[目的] 研究黄芪注射液对高糖环境下肾小管上皮细胞(HK-2)凋亡的影响。[方法] 传代培养人近曲肾小管上皮细胞,细胞用无血清培养基同步化24 h后,将细胞分为5组:低糖对照组、高糖组、高糖+不同浓度黄芪注射液组(2、20、200 μg/mL),每组设3个复孔。将细胞置于37 ℃恒温培养箱中培养至24、48、72 h,收集细胞及细胞上清液。用流式细胞仪测定细胞凋亡率。[结果] 24、48、72 h,低糖对照组细胞凋亡率明显低于高糖组(P<0.05)。细胞培养24 h,黄芪干预组200和20 μg/mL细胞凋亡率明显低于高糖组(P<0.05)。细胞培养48和72 h,黄芪注射液干预组细胞凋亡率明显低于高糖组(P<0.05)。黄芪注射液干预组各组之间凋亡率比较差异也都有显著性(P<0.05),其干预作用呈剂量依赖性。[结论] 黄芪注射液能抑制高糖诱导的肾小管上皮细胞的凋亡,有助于糖尿病肾病的治疗。