表皮生长因子受体反义基因转染对人恶性胶质瘤细胞体外生长的影响

目的探讨表皮生长因子受体（ＥＧＦＲ）反义基因转染对人恶性胶质瘤细胞Ｕ８７ＭＧ体外生长的影响。方法构建ＥＧＦＲ反义基因表达载体，应用脂质体转染法将其导入Ｕ８７ＭＧ细胞；ＰＣＲ方法鉴定转染克隆；Ｗｅｓｔｅｒｎ印迹杂交检测ＥＧＦＲ蛋白表达水平；通过绘制生长曲线和软琼脂集落形成实验测定反义ＥＧＦＲ基因对恶性胶质瘤细胞体外生长的抑制作用。结果转染反义ＥＧＦＲ基因的细胞克隆株ＡＳ１ＡＳ６的ＥＧＦＲ蛋白表达水平均有不同程度的降低；ＡＳ１ＡＳ６细胞体外生长明显减慢，ＡＳ１ＡＳ６细胞克隆形成率明显降低。表明Ｕ８７ＭＧ细胞体外生长速度和恶性转化能力均与ＥＧＦＲ表达呈正相关。结论反义ＥＧＦＲ基因转染可以明显抑制Ｕ８７ＭＧ细胞生长，ＥＧＦＲ对Ｕ８７ＭＧ细胞生长具有重要的调控作用     

阻断TGF α-EGFR自泌环对胰腺癌细胞体外生长的影响

目的研究阻断ＴＧＦα－ＥＧＦＲ自泌环对胰腺癌细胞生长的影响。方法构建了反义ＥＧＦＲ的表达载体ｐＣＭＶ－ＡＳ－ＥＧＦＲ,转染已经反义ＴＧＦα转化的胰腺癌ＰＣ－７细胞,经Ｇ４１８筛选,获得稳定的双重转化细胞系ＰＣ－７／ＡＳ－ＴＧＦα／ＡＳ－ＥＧＦＲ。经Ｓｏｕｔｈｅｒｎｂｌｏｔ、Ｎｏｒｔｈｅｒｎｂｌｏｔ、１２５Ｉ－ＥＧＦ结合试验分析双重转染细胞系基因的整合及表达。ＤＮＡ凝胶电泳、流式细胞术、原位凋亡检测细胞凋亡。结果双重转染细胞系有外源ＴＧＦα基因的整合及表达,内源性ＥＧＦＲ及ｃｙｃｌｉｎＤ１ｍＲＮＡ表达下调、细胞表面ＥＧＦＲ受体表达下降,与反义ＴＧＦα单独作用比较,反义ＥＧＦＲ与反义ＴＧＦα的联合作用更强。３Ｈ－ＴｄＲ掺入率由２５％降至１４．５％。生长抑制率由７８．８％提高到８６．０％、软琼脂集落形成能力完全丧失。双重抑制后,细胞更容易发生凋亡。结论对胰腺癌中异常信号传导途径的阻断,能明显地抑制肿瘤恶性生物学行为,使肿瘤细胞恶性表型部分逆转。     

阻断TGF α—EGFR自泌环对胰腺癌细胞体外生长的影响

目的 研究阻断ＴＧＦα－ＥＧＦＲ自泌环对胰腺癌细胞生长的影响。方法 构建了反义ＥＧＦＲ 表达载体ｐＣＭＶ－ＡＳ－ＥＧＦＲ。转染反应反义ＴＧＦα转化的胰腺癌ＰＣ－７细胞,经Ｇ４１８筛选,获得稳定的双重转化细胞系ＰＣ－７／ＡＳ－ＴＧＦα／ＡＳ－ＥＧＦＲ。经Ｓｏｕｔｈｅｒｎｂｌｏｔ,Ｎｏｒｔｈｅｒｎｂｌｏｔ,＾１２５Ｉ－ＥＧＦ结合试验分析双重转染细胞系基因的整合及表达。ＤＮＡ凝胶电泳,流式细胞术,。原位     

A COMPARISON OF THE EFFECTS OF THE PROPOFOL VERSUS MIDAZOLAM DURING TOTAL INTRAVENOUS ANESTHESIA FOR GYNECOLOGICAL SURGERY PR

ＡＣＯＭＰＡＲＩＳＯＮＯＦＴＨＥＥＦＦＥＣＴＳＯＦＴＨＥＰＲＯＰＯＦＯＬＶＥＲＳＵＳＭＩＤＡＺＯＬＡＭＤＵＲＩＮＧＴＯＴＡＬＩＮＴＲＡＶＥＮＯＵＳＡＮＥＳＴＨＥＳＩＡＦＯＲＧＹＮＥＣＯＬＯＧＩＣＡＬＳＵＲＧＥＲＹＰＲＯＣＥＤＵＲＥＳＹｅＴｉｅｈｕ（叶...     

反义N—ras1DNA片段部分抑制人膀胱癌BIU—87细胞VEGF基因的表达

本文报道将载有反义人Ｎ－ｒａｓｌ（ｅｘｏｎｌ）ＤＮＡ片段的重组表达质粒ｆＰＧＶ１－ＭＴ－Ｎｒａｓｌ（Ａ）导入人膀胱移行细胞癌ＢＩＵ－８７细胞系，可以部分抑制ＢＩＵ－８７细胞中ＶＥＧＦｍＲ－ＮＡ的表达。提示ＶＥＧＦ基因的表达可受到反义Ｎ－ｒａｓ１ＤＮＡ片段的调控。本研究不仅增加了从分子水平对ＢＩＵ－８７细胞的认识，有助于进一步确定该细胞的生物学特征，还为以后具有Ｎ－ｒａｓ癌基因及ＶＥＧＦ基因表达的膀胱癌进行反义癌基因治疗提供一定的理论和实验依据。     

pLTKcSN／VPC及GCV系统的旁观者效应观察

目的：探讨单纯疱疹病毒Ｉ型胸苷激酶基因（ＴＫ）逆转录病毒载体生产细菌（ｐＬＴＫｃＳＮ／ＶＰＣ）和更昔洛韦（ＧＣＶ）系统杀伤恶性胶质瘤细胞过程中的旁观者效应。方法：采用大鼠胶质瘤细胞Ｃ６、人恶性胶质瘤Ｕ８７ＭＧ和它们的转染ＴＫ阳性细胞Ｃ６ＴＫ，Ｕ８７ＴＫ，将Ｃ６与Ｃ６ＴＫ，Ｕ８７ＭＧ和Ｕ８７ＴＫ按比例（ＴＫ阳性细胞占细胞总体的０－１００％，１０％梯度）分别混合培养于９６孔板中，每种混合比例设６孔。     

EFFECTS OF ALVEOLAR MACROPHAGE CONDITIONED MEDIA FROM INTERSTITIAL LUNG DISEASE PATIENTS ON THE PROCOLLAGEN mRNA EXPRESSION I

ＥＦＦＥＣＴＳＯＦＡＬＶＥＯＬＡＲＭＡＣＲＯＰＨＡＧＥＣＯＮＤＩＴＩＯＮＥＤＭＥＤＩＡＦＲＯＭＩＮＴＥＲＳＴＩＴＩＡＬＬＵＮＧＤＩＳＥＡＳＥＰＡＴＩＥＮＴＳ　ＯＮ　ＴＨＥＰＲＯＣＯＬＬＡＧＥＮｍＲＮＡＥＸＰＲＥＳＳＩＯＮＩＮＨＵＭＡＮＬＵＮＧＦＩＢＲ...     

The future pattern of surgical department and training of surgeons in community hospitals.

ＴＨＥＦＵＴＵＲＥＰＡＴＴＥＲＮＯＦＳＵＲＧＩＣＡＬＤＥＰＡＲＴＭＥＮＴＡＮＤＴＲＡＩＮＩＮＧＯＦＳＵＲＧＥＯＮＳＩＮＣＯＭＭＵＮＩＴＹＨＯＳＰＩＴＡＬＳＩｎｔｈｅｗａｋｅｏｆｔｈｅｒｅｆｏｒｍａｎｄｏｐｅｎｉｎｇｐｏｌｉｃｙ，ａｒａｐｉｄｅｃｏｎｏ...     

转染TNFα基因对胶质瘤细胞致瘤性的影响

研究转染肿瘤坏死因子α（ｔｕｍｏｒｎｅｃｒｏｓｉｓｆａｃｔｏｒα,ＴＮＦα）基因的胶质瘤细胞的致瘤性。方法运用ＭｏＭＬＶ逆转录病毒载体,将ＴＮＦα基因转导至人胶质瘤细胞ＳＨＧ－４４,用Ｇ４１８筛选出阳性细胞克隆,以细胞培养检测细胞生长抑制,以裸鼠接种研究其致瘤性。结果转染细胞培养上清液中ＴＮＦα含量分别为（５１９８．７±３７５７．４）和（３２１７．４±１１８０．６）ｐｇｍｌ（Ｐ＜０．０５）,其生物活性达３２０．０ｕｍｌ。运用ＲＴ－ＰＣＲ检测ＴＮＦαｍＲＮＡ特异性表达。细胞培养发现：转染ＴＮＦα基因的细胞生长受抑制,Ｇ２－Ｍ期缩短而Ｇ０－Ｇ１期延长。裸鼠接种试验发现转染ＴＮＦα基因的胶质瘤细胞致瘤性下降,早期出现明显坏死。未转染ＴＮＦα基因的胶质瘤细胞混有１０％的转染ＴＮＦα基因的细胞,其生长也受到抑制。结论转染ＴＮＦα基因的胶质瘤细胞的致瘤性下降。     

EFFECTS OF β－ENDORPHIN ON PHYTOHEMAGGLUTININ－INDUCED LYMPHOCYTE PROLIFERATION AND MOUSE PLAQUE－FORMING CELL RESPONSE VIA AN O

ＥＦＦＥＣＴＳＯＦβ－ＥＮＤＯＲＰＨＩＮＯＮＰＨＹＴＯＨＥＭＡＧＧＬＵＴＩＮＩＮ－ＩＮＤＵＣＥＤＬＹＭＰＨＯＣＹＴＥＰＲＯＬＩＦＥＲＡＴＩＯＮＡＮＤＭＯＵＳＥＰＬＡＱＵＥ－ＦＯＲＭＩＮＧＣＥＬＬＲＥＳＰＯＮＳＥＶＩＡＡＮＯＰＩＯＩＤＲＥＣＥＰＴＯＲＭ...     

靶向表皮生长因子受体的小分子干扰RNA抑制TJ905人脑胶质瘤细胞增殖和侵袭的实验研究

目的　比较靶向表皮生长因子受体 (EGFR)的小分子干扰RNA与反义RNA构建体对TJ90 5人脑胶质瘤细胞增殖和侵袭的抑制作用。方法　选择人EGFR的二个siRNA靶序列构建靶向EGFR的siRNA表达质粒 ,并以反义EGFRRNA为对照进行脂质体介导的TJ90 5人脑恶性胶质瘤细胞系表达。应用免疫荧光和蛋白印迹检查EGFR的表达水平 ;应用TUNEL法分析细胞凋亡 ,流式细胞术分析细胞周期变化 ,MTT法分析细胞增殖 ;蛋白印迹检测基质金属蛋白酶 9的表达 ,明胶酶谱分析MMP9酶活性 ,Transwell法分析侵袭能力。结果　与TJ90 5细胞和转染空载体的TJ90 5细胞比较 ,转染反义EGFRRNA使EGFR表达下调 82 % ,转染siRNA表达质粒组EGFR表达下调 90 %和 92 %。TJ90 5组和空载转染组几乎没有凋亡细胞 ,反义EGFR转染组与siRNA表达质粒组的凋亡率明显增加 ( χ2 =31 5 4 9,P <0 0 0 1) ;siRNA表达载体转染后S期指数较对照和反义RNA转染组细胞明显减少 ,与TJ90 5组和空载转染组比较 ,转染组细胞自第 1天起存活率均明显下降 (P <0 0 0 1) ,且反义组与siRNA表达载体转染组之间存活率差异有显著意义 (P <0 0 1) ;同时蛋白印迹发现转染siRNA表达载体后MMP 9的表达明显下调 ,明胶酶谱分析发现在反义组与siRNA表达载体转染组MMP 9酶活性明显下降 ,以siRNA     

反义CDK4基因抑制人结肠癌细胞HT29生长

目的 将反义CDK4导入人结肠癌细胞株HT29细胞,观察其对肿瘤细胞生长的影响。方法 采用lipofectamine转染方法转染HT29细胞,Northern印迹,Western印迹,形态学和流式细胞术等方法用于转染效果的鉴定。结果 转化细胞有反义CDK4的表达,而内源性CDK4mRNA表达和蛋白合成下调,并且转化细胞的恶性行为及表型部分逆转,细胞生长受到抑制、软琼脂集落形成能力明显降低,同时揭示G1期阻滞。HT29-asCDK4细胞有较高的凋亡率。结论 反义CDK4基因可抑制HT29细胞的生长和增殖、诱导其凋亡。为结直肠癌的基因治疗提供了一种新的可能的途径。     

Induction of apoptosis and inhibition of proliferation in Hep-2 by antisense survivin RNA in vitro

Objective.. To study induction of apoptosis and inhibition of proliferation in Hep-2 by antisense survivin RNA. Methods： Antisense survivin RNA expression vector was constructed and then was transfected to human laryngeal carcinoma cell line Hep-2 by lipofectamine. HpEGFP/survivin cells （transfected with the combinant of antisense survivin RNA） were obstained by using G418. The levels of survivin protein before and after transfection were determined by Western-blot. Proliferation activity was measured by MTT assay. The experiment of colony formation in soft agar was carried out for assessing ability of proliferation of Hep-2 cell. Apoptosis was assessed by flow cytometry and acrdine orange（AO）. Results： After antisense survivin RNA plasmids were transfected, the level of survivin protein was inhibited in Hep-2. Compared with control, proliferation of HpEGFP/survivin cells were suppressed significantly. The experiment of colony formation in soft agar showed the ability of colony formation decreased in HpEGFP/survivin cells compared to control （P〈0.05）. Apoptosis rate increased about 1.81-folds compared with control. Conclusion.. The antisense survivin RNA can partly inhibit the level of surviivin protein expression in Hep-2 and can induce apoptosis and inhibit the proliferation of Hep-2 by down-regulating the expression of endogenous survivin in vitro.     

EGFR反义RNA的转染对人类鼻咽癌CNE－2细胞EGFR的表达下调及恶性表型的抑制

目的 利用反义RNA抑制靶基因表达的策略，下调EGFR的表达而探讨对人类低分化鼻咽癌CNE-2细胞的恶性表型的抑制性效应。方法 将人EGFR的N-端1．35kb片段反向构建人逆转录病毒表达载体pLXSN中，并用脂质体介导转染人鼻咽癌CNE-2细胞，G418筛选并分离阳性克隆，将两个EGFR反义cDNA转染的克隆细胞命名为CNE-2／AS4和CNE-2／AS8，而空载体转染的细胞命名为CNE-2／pLXSN。125I-EGF配体结合分析细胞膜上EGFR的表达，台盼蓝染色法测定细胞生长的改变，并用软琼脂集落形成实验检测细胞转化，最后，将筛选的各组阳性克隆细胞分别注射人裸鼠皮下，不同时间观察肿瘤生长的抑制改变及肿瘤的转移状况。结果配体结合实验结果显示。两个选择的克隆CNE-2／AS4和CNE-2／AS8细胞表面EGFR的数量分别较未转染细胞CNE-2下降18％、45％，表明EGFR反义RNA的表达下词了细胞膜上EGFR的表达；同时，EGFR反义RNA表达的CNE-2细胞生长速率和软琼脂生长能力也较对照组细胞明显降低。注射入裸鼠皮下后，EGFR反义RNA表达的阳性克隆细胞表现出肿瘤生长减慢。淋巴结和肺转移能力也明显降低。结论这些实验结果提示EGFR反义cDNA转染的CNE-2细胞能下调：EGFR的过量表达并部分抑制鼻咽癌的恶性表型，这为进一步阐明：EGFR在鼻咽癌的发生、演化中的功能作用提供了有用的工具。     

The effects of antisense PTEN gene transfection on the growth and invasion of glioma cells

Objective:To study the effects of antisense PTEN gene on the growth and invasion of glioma cells. Methods: A pcDNA3. 1/Hygro(-) recombinant plasmid containing antisense PTEN gene fragment was constructed. Glioma cells of primary culture were transfected with antisense PTEN gene vector and stably transfected clones were selected. Then, the different growth and invasion abilities and the different MMP9 mRNA expressions of three kinds of cells were observed, including the transfected cells, untrans-fected cells and the cells transfected with empty vector. Results:The abilities of growth and invasion of the transfected cells and the expressions of MMP9 mRNA were obviously enhanced. Conclusion: Antisense PTEN gene could have a negative impact on the growth and invasion of primary culture glioma cells.     

Role of cyclinD1 and CDK4 in the carcinogenesis induced by silica

To study the role of cyclinD 1 and CDK4 in malignant transformation of human fetal lung diploid fibroblast cell line （2BS） induced by silica. Methods Recombination vectors with sense and antisense pXJ41-cyclinD1 and pXJ41-CDK4 were constructed, and then transfected into the malignant transformed cells induced by silica, respectively. At the same time, pXJ41-neo was used as the control. Results During the progress of the malignant transformation of 2BS cells induced by silica, cyclinD 1 and CDK4 were overexpressed. Antisense RNA suppressed cyclinD 1 and CDK4 gene expression in the antisense pXJ41-cyclinD1 and pXJ41-CDK4 transfected cells. Antisense RNA led to cell cycle arrest, resulting in lengthened G1 phase （the percentages of cells in the G1 phase changed from 45.1% to 52.7% and 58.0% for cyclinD1 and CDK4 transfected cells, respectively）, and eventually attenuated the increase of the proliferation of malignant transformed cells induced by silica. Compared with malignant transformed cells induced by silica, cells transfected with antisense pXJ41-cyclinD1 and pXJ41-CDK4 showed obviously reduced growth rates. On the 8th day, the suppression rates were 58.69 and 77.43% （the growth rate of malignant transformed cells induced by silica was 100%）, doubling time changed from 21.0 h to 31.4 h and 21.0 h to 42.7 h, respectively, the growth capacities on soft agar of cells transfected by antisense pXJ41-cyclinD1 and pXJ41-CDK4 decreased obviously. Conclusion CyclinD 1 and CDK4 play an important role in maintaining transformed phenotype of the cancer cells.     

EGFR反义RNA的转染对人类鼻咽癌CNE-2细胞EGFR的表达下调及恶性表型的抑制

目的利用反义RNA 抑制靶基因表达的策略,下调EGFR的表达而探讨对人类低分化鼻咽癌CNE-2细胞的恶性表型的抑制性效应。方法将人EGFR的N-端1.35kb片段反向构建入逆转录病毒表达载体pLXSN中,并用脂质体介导转染人鼻咽癌CNE-2细胞,G418筛选并分离阳性克隆,将两个EGFR 反义cDNA转染的克隆细胞命名为CNE-2/AS4 和CNE-2/AS8,而空载体转染的细胞命名为CNE-2/pLXSN。125I-EGF配体结合分析细胞膜上EGFR的表达,台盼蓝染色法测定细胞生长的改变,并用软琼脂集落形成实验检测细胞转化,最后,将筛选的各组阳性克隆细胞分别注射入裸鼠皮下,不同时间观察肿瘤生长的抑制改变及肿瘤的转移状况。结果配体结合实验结果显示,两个选择的克隆CNE-2/AS4 和CNE-2/AS8细胞表面EGFR的数量分别较未转染细胞CNE-2下降18%、45%,表明EGFR反义RNA的表达下调了细胞膜上EGFR的表达;同时,EGFR反义RNA表达的CNE-2细胞生长速率和软琼脂生长能力也较对照组细胞明显降低。注射入裸鼠皮下后,EGFR反义RNA表达的阳性克隆细胞表现出肿瘤生长减慢,淋巴结和肺转移能力也明显降低。结论这些实验结果提示EGFR 反义cDNA转染的CNE-2细胞能下调EGFR的过量表达并部分抑制鼻咽癌的恶性表型,这为进一步阐明EGFR在鼻咽癌的发生、演化中的功能作用提供了有用的工具     

靶向survivin基因RNAi沉默表达对恶性胶质瘤细胞侵袭的影响

目的 探讨存活素(survivin)基因对胶质瘤细胞侵袭的影响和可能机制.方法 应用survivin基因小干扰RNA(siRNA)转染处理胶质瘤SNB19细胞后,分别采用荧光实时定量RT-PCR和Western blot检测survivin基因mRNA和蛋白水平,分别采用软琼脂集落培养试验和Boyden小室模型试验检测癌细胞集落生长数和侵袭力.采用Western blot 方法检测癌细胞尿激酶纤溶酶原激活物(uPA)蛋白水平.结果 survivin siRNA转染组细胞的survivin基因mRNA和蛋白水平明显下调.转染组胶质瘤细胞集落生长数和癌细胞穿膜细胞数明显下调,且与浓度相关.转染组癌细胞uPA蛋白水平明显下降.结论 Survivin基因在胶质瘤细胞侵袭中起着重要作用,采用靶向survivin siRNA转染可抑制其侵袭能力.其机制可能与下凋uPA基因表达有关.