BDNF对H_2O_2诱导人肺癌细胞YTMLC-90凋亡的抑制效应

背景与目的:已证实在神经系统内外多种细胞中均可合成神经营养因子,如神经生长因子(nervegrowthfactor,NGF)、脑源性神经营养因子(brainderivedneurotrophicfactor,BDNF)和神经营养素3/4(NT-3/4)。这些神经营养因子不仅能促进神经细胞的存活、增殖和凋亡,也与癌细胞侵犯神经组织的活性有关;但BDNF与肺癌细胞的生物学行为是否相关尚未见报道。本文旨在研究BDNF对过氧化氢(H2O2)诱导人肺癌细胞YTMLC-90凋亡的作用。方法:用含人I型胶原a1链(COLIA1)基因启动子和增强子元件及在其3'端接BDNFcDNA的微基因pSVCEPBFCAT,转染人肺癌细胞YTMLC-90并驱动BDNF异位表达,采用四甲基偶氮唑蓝(MTT)显色法检测细胞增殖,吖啶橙荧光染色显微镜和电镜观察细胞形态及其超微结构的改变,琼脂糖凝胶电泳检测染色质DNA断裂。结果:经200μmol/LH2O2作用24h,转染pSVCEPBFCAT的YTMLC-90细胞生长抑制率为30.0%,而未转染YTMLC-90和转染空载体pSVCEPCAT的YTMLC-90细胞生长抑制率分别为84.60%和80.00%,实验组与两个对照组之间的差异均有显著性(P<0.01);对照组YTMLC-90细胞可见凋亡特有的形态学及生物化学改变,如胞浆和核固缩、染色质断裂、DNA电泳呈梯状条带,而转染pSVCEPBFCAT的YTMLC-90细胞未发现上述凋亡特征。结论:BDNF促进YTMLC-90?

苯丁酸钠体外对YTMLC-90细胞生长的影响

目的:观察苯丁酸钠(PB)体外对人个旧肺鳞状细胞癌YTMLC-90细胞生长的影响。方法:YTMLC-90细胞分为6组,对照组不作任何处理,余5组分别给予250、500、1000、2000和4000mg/LPB,分别作用48、72和96h。采用MTT法观察各组细胞生长情况,计算细胞生长抑制率。另取YTMLC-90细胞分为3组:对照组、500和1000mg/LPB组,分别作用48和72h,采用流式细胞术检测3组细胞的细胞周期及凋亡情况。结果:不同质量浓度的PB作用48、72和96h,各组细胞的生长抑制率相比,差异均有统计学意义(F=672.413、1633.321和1196.548,P均<0.05)。PB作用48和72h后,各组细胞G0/G1期、S期与G2/M期细胞百分率及凋亡率比较,差异均有统计学意义(F48h=508.347、224.522、19.890和111.849,F72h=311.979、102.923、247.432和177.018,P均<0.05)。与对照组相比,PB作用后YTMLC-90细胞生长缓慢,细胞阻滞于G0/G1期,凋亡增加(P<0.05)。结论:PB可抑制YTMLC-90细胞的生长,使其阻滞于G0/G1期,凋亡增加。

KISS-1基因对人肺鳞状细胞癌细胞YTMLC-90增殖及侵袭能力的影响

目的探讨肿瘤转移抑制基因KISS-1对人肺鳞状细胞癌细胞YTMLC-90增殖及侵袭能力的影响。方法通过基因转染技术,将携带KISS-1基因的pEGFP-N2-KISS-1质粒转染入人YTMLC-90细胞,同时设不携带KISS-1基因的空质粒转染组和空白对照组作为对照,用蛋白质印迹法检测转染后YTMLC-90细胞中KISS-1蛋白的表达以确定转染是否成功;噻唑蓝(MTT)法测定3组YTMLC-90细胞增殖情况;Millicell小室法检测3组YTMLC-90细胞的侵袭能力。结果成功将KISS-1基因转染入YTMLC-90细胞。pEGFP-N2-KISS-1质粒转染组YTMLC-90细胞的增殖活性与空质粒转染组和空白对照组差异无统计学意义(P>0.05),但YTMLC-90细胞穿透Millicell聚碳酸酯膜的细胞数较空质粒转染组和空白对照组明显减少[(35±5)×10~5个比(82±6)×10~5个、(84±5)×10~5个],差异有统计学意义(P<0.05)。结论 KISS-1基因对YTMLC-90细胞的增殖能力没有影响,但对其侵袭能力有抑制作用,提示KISS-1基因能够抑制肺癌的侵袭。

Epigenetic Enabled Normal Human Cells, Lead to <i>First Cell</i>’s Unique Division System, Driving Tumorigenesis Evolution

Normal cells must become cancer-enabling before anything else occurs, according to latest literature. The goal in this mini-review is to demonstrate special tetraploidy in the enabling process. This we have shown from genomic damage, DDR (DNA Damage Response) activity with skip of mitosis leading to diploid G2 cells at the G1 border in need of chromatin repair for continued cell cycling to the special tetraploid division system. In several studies specific methylation transferase genes were activated in normal human cells in tissue fields, containing different cell growth stages of the cancerous process. Histology studies, in addition to molecular chemistry for identification of oncogenic mutational change, were a welcome change (see below). In a study on melanoma origin, DDR also showed arrested diploid cells regaining cycling from methylation transferase activity with causation of 2n melanocytes transforming to 4n melanoblasts, giving rise to epigenetic tumorigenesis enabled First Cells. Such First Cells were from Barrett’s esophagus shown to have inherited the unique division system from 4n diplochromosomal cells, first described in mouse ascites cancer cells (below). We discovered that the large nucleus prior to chromosomal division turned 90° relative to the cytoskeleton axis, and divided genome reductive to diploid, First Cells, in a perpendicular orientation to the surrounding normal cells they had originated from. This unique division system was herein shown to occur at metastasis stage, implying activity throughout the cancerous evolution. Another study showed 4-chromatid tetraploidy in development to B-cell lymphoma, and that such cancer cells also proliferated with participation of this unusual division system. Such participation has long been known from Bloom’s inherited syndrome with repair chiasmas between the four chromatids, also an in vitro observation by us. Our cytogenetic approach also revealed that they believed mitotic division in cancer cells is wrong because such cell divisions were found to be from an adaptation between amitosis and mitosis, called amitotic-mitosis. Amitosis means division without centrosomes, which has long been known from oral cancer cells, in that MOTCs (microtubule organizing center) were lacking centrioles. This observation calls for re-introduction of karyotype and cell division studies in cancer cell proliferation. It has high probability of contributing novel approaches to cancer control from screening of drugs against the amitotic-mitotic division apparatus.

肺鳞癌细胞单克隆抗体的建立及鉴定

目的制备出高效价的抗人肺鳞癌单克隆抗体。方法以云南个旧肺鳞癌细胞YTMLC-90免疫的BALB/c小鼠脾细胞和小鼠骨髓瘤NS-1、SP2/0细胞为亲代细胞,应用鼠-鼠杂交瘤技术建立杂交瘤细胞系。ELISA法筛选,有限稀释法克隆化培养,ELISA法测定抗体效价,双抗体夹心法鉴定Ig亚类,制备染色体,G iemsa染色计数染色体数目,ABC免疫组化法行不同组织细胞的交叉检测。结果获得3株稳定分泌抗YTMLC-90细胞抗体的杂交瘤细胞株S2D1、N2D1及N3C5。培养上清效价分别为1∶512、1∶284及1∶1 024,腹水效价分别为1∶105、1∶104及1∶104;Ig亚类分别为IgG1、IgG2a及IgG3;染色体众数在90~110之间;3株抗人肺鳞癌单克隆抗体与其他组织细胞无交叉反应性。杂交瘤细胞体外传代培养6个月仍保持抗体稳定分泌。结论成功制备高效且特异的抗人肺鳞癌单克隆抗体。

三株抗人肺鳞癌单克隆抗体免疫组化分析

目的对三株抗云南个旧肺鳞癌YTMLC-90杂交瘤细胞S2D1、N2D1、N3C5所分泌的单克隆抗体进行不同组织细胞的免疫组化分析,探讨其特异性.方法用收集的原倍杂交瘤细胞上清常规ABC免疫组化法进行组织细胞学交叉检测,分析三个抗体与临床病理确诊的肺鳞癌组织、肺腺癌组织、肠癌组织、食道癌组织、肾癌组织、肺正常组织石腊切片和肺鳞癌YTMLC-90细胞系、肺腺癌GLC-82细胞系、舌鳞癌Tca-8113细胞系、鼻咽癌KB细胞系、胶质瘤U-251细胞系、膀胱癌T-24、B1u-87细胞系的交叉反应.结果杂交瘤细胞上清液与肺癌组织及肺鳞癌、肺腺癌细胞系YTMLC-90、GLC-82反应呈阳性,阳性率分别为82%、100%;与正常肺组织及肠癌组织、肾癌组织、食道癌组织及体外培养的其它各肿瘤细胞系均呈阴性反应.结论经免疫组化研究,三株单克隆抗体具有肺癌特异性,属于肺癌相关抗原的单克隆抗体.

肿瘤转移抑制基因KiSS-1对人肺鳞癌裸鼠移植瘤生长及转移能力的影响

目的探讨肿瘤转移抑制基因KiSS-1对人肺鳞癌裸鼠移植瘤生长及转移能力的影响。方法裸小鼠60只,随机分为三组,每组20只。实验组裸鼠皮下接种含有稳定转染KiSS-1基因的YTMLC-90-KiSS-1细胞,空转染组接种含有空载体pEGFP-N2转染的YTMLC-90细胞,对照组接种YTMLC-90细胞。建立肺癌皮下肿瘤动物模型,进行成瘤观察,成瘤后取材,RT-PCR检测三组皮下移植瘤KiSS-1mRNA的表达。结果三组裸鼠皮下接种细胞后,全部成瘤。三组皮下成瘤重量比较差异无统计学意义(P>0.05);接种YTMLC-90-KiSS-1细胞的实验组肺转移的发生率低于空转染组和对照组,差异有统计学意义(P<0.05);而实验组皮下移植瘤中KiSS-1 mRNA的表达量高于空转染组以及对照组,差异有统计学意义(P<0.05)。结论 KiSS-1基因对YTMLC-90细胞的成瘤能力没有影响,但对其转移能力有抑制作用,提示KiSS-1基因可能具有抑制肺癌侵袭和转移的能力。