三氧化二砷逆转乳腺癌细胞株MCF-7/ADM多药耐药的研究

目的探讨三氧化二砷（As2O3）体外逆转人乳腺癌细胞多药耐药性的作用及机制。方法采用MTT法检测As2O3的细胞毒作用和处理前后耐药细胞对化疗药物的敏感性,用流式细胞仪检测细胞内阿霉素浓度,通过RT-RCR检测MDR1基因的表达。结果 As2O3在0.25mg/L以下剂量时对MCF-7和MCF-7/ADM耐药细胞株的抑制率均小于15%,半数抑制率（IC50）分别为1.01m/L和1.28mg/L,无细胞毒剂量0.2mg/L的As2O3能部分逆转MCF-7/ADM细胞对阿霉素的耐药性。同时无细胞毒剂量0.2mg/L的As2O3能使MCF-7/ADM细胞内阿霉素浓度明显增加,MDR1表达下降。结论 As2O3具有体外部分逆转人乳腺癌细胞多药耐药性的作用,可能与增加细胞内药物积累、下调MDR1表达有关。     

卵巢癌细胞株COC1/DDP多药耐药逆转前后HLA、B7表达的实验研究

目的 :探讨卵巢癌多药耐药 (multidrugresistance ,MDR)细胞的免疫逃逸机制。方法 :利用三苯氧胺 (TAM)和维拉帕米 (VRP)为逆转剂 ,运用MTT法分析了TAM、ARP的逆转效应。采用流式细胞仪技术检测耐药亚株COC1/DDPMDR逆转前后及亲本株COC1细胞的HLA -Ⅰ、HLA -Ⅱ、B7-1、B7-2的表达。结果 :COC1/DDP和COC1均表达较强的HLA -Ⅰ类抗原 ,而HLA -Ⅱ、B7-1、B7-2的表达较较低。但COC1/DDP的B7-1、B7-2抗原的表达却高于COC1。经TAM、VRP作用后 ,COC1/DDP的耐药性发生逆转 ,但HLA、B7的表达无明显变化。结论 :TAM、VRP可以逆转卵巢癌的MDR细胞 ,敏感株细胞和多药耐药细胞有着不同的免疫逃逸机制 ,与耐药株相比 ,敏感株细胞更易逃避机体的免疫反应 ,过继免疫疗法可用于耐药肿瘤     

硫化砷诱导K562/ADM细胞凋亡

硫化砷（AS。S。）是中药雄黄的主要成份,研究表明砷剂对慢性粒细胞白血病及早幼粒细胞白血病均有促凋亡效应［’j。我们在对雄黄诱导细胞凋亡的基础上,对硫化砷诱导K562／ADM细胞凋亡进行初步研究,探讨其与化疗药间交叉耐药性的问题。l材料与方法l．l细胞K562RK562／ADM细胞由大连医科大学组胚教研室提供。常规培养干RPMll640完全培养液中,取对数生长期细胞进行实验。1．2药物l％AS。S。由本院制备,使用时以培养液稀释至所需药物浓度。1．3流式细胞仪（FCM）分析细胞周期不同药物浓度处理细胞36h后收集细胞。PBS洗两遍,4C…     

MGr1-Ag表达上调及下调的胃癌细胞株的建立及鉴定

为研究胃癌细胞耐药相关新基因MGr1 Ag对胃癌耐药细胞多药耐药性的调节作用及其机制。克隆MGr1 Ag的全长cDNA并构建其正反义真核表达载体 ,以脂质体介导法将正、反义真核表达载体分别转染入人胃癌细胞系MGC80 3与人胃癌多药耐药细胞系SGC790 1/VCR中。结果显示 ,经RT PCR扩增出大小约为 1.0kb的基因片段 ,成功克隆入pUCm T载体 ,经DNA测序证实为MGr1 Ag基因。将其克隆入pCDNA3 .1/V5 His后 ,经限制性核酸内切酶酶切鉴定 ,获得携有MGr1 Ag基因真核表达载体pCD NA3 1/V5 His MGr1 Ag及其反义表达载体pCDNA3 .1/V5 His anMGr1 Ag。以脂质体介导法将MGr1 Ag的正、反义真核表达载体分别转染入MGC80 3与SGC790 1/VCR中 ,经Westernblot证实 ,成功建立了MGr1 Ag表达上调及下调的胃癌细胞株 ,为研究MGr1 Ag参与耐药的分子机制奠定了基础     

肺癌组织MDR1和GST-πmRNA水平测定及其临床意义

用RT-PCR方法对51例手术切除肺癌组织和相应的癌周正常肺组织进行MDR1和GST-πmRNA检测.结果表明:MDR1 mRAN在正常肺组织和肺癌组织中表达阳性率分别13.7%(7/51)和43.1%(22/51);而GST-πmRNA阳性率则分别为19.6%(10/51)和51.0%(26/51).MDR1或GST-πmRNA表达与肿瘤病理类型、组织分化、TNM分期等均无明显的相关性(P＞0.05).19例(37.3%)发生MDR1和GST-πmRNA共表达.上述表明肺组织细胞在恶变过程中MDR1和GST-πmRNA表达均有所增加.提示,二者在肺癌先天性耐药机制中均占有重要的作用,而且二者在肺癌中的表达基本是一致的.     

辐射诱导人鼻咽癌细胞CNE1、CNE2多药耐药的研究

目的研究辐射诱导及逆转药物环孢霉素A（CsA）、干扰素a（IFNa）对鼻咽癌细胞CNE1、CNE2多药耐药表达的影响。方法通过体外辐射处理诱导鼻咽癌细胞CNEl、CNE2，流式细胞术、RT-PCR分析辐射诱导前后P-gp、MRP、TOPH、GST-π在蛋白、mRNA水平表达的改变。同时，研究CsA、IFN-α逆转耐药处理对放疗诱导的多药耐药的影响。结果辐射诱导后CNE1、CNE2细胞P-gp、MRP、GST-π在蛋白、mRNA水平表达上调，辐射后给予CsA、IFN-处理可以逆转辐射诱导的P-gp、MRP蛋白表达上调。结论辐射可以诱导鼻咽癌细胞CNE1、CNE2出现多药耐药，而CsA、IFN-可以逆转辐射诱导的多药耐药。     

体内多药耐药的显像研究

探讨了多药耐药产生的各种机制及体内P-糖蛋白(P-gp)和多药耐药相关蛋白(MRP)显像的情况。SPECT和PET都可以用来研究P-gp和MRP介导的转运情况。锝标记的甲氧异腈、p53和Q复合物都是P-gp泵的转运底物。显像剂如11C标记的秋水仙碱、维拉帕米、阿霉素等已经用于体内P-gp介导的转运水平的底量研究。白三烯是MRP的特异转运底物,因此N-11C-乙酰基白三烯E4可用于无创性评价MRP的转运功能。     

食管癌Mp53和mdr-1,mrp表达的相关性研究

目的　探讨食管癌组织中突变型 p5 3蛋白 (Mp5 3)与多药耐药基因 (mdr 1)和多药耐药相关蛋白基因 (mrp)的表达关系。方法　分别应用S P免疫组化法和逆转录 聚合酶链反应 (RT PCR)技术 ,对 5 4例食管癌组织的Mp5 3蛋白和mdr 1,mrp基因表达进行检测。 结果　食管标本的Mp5 3和mdr 1,mrp的阳性率均高于癌旁组织 (P <0 .0 1) ;癌组织Mp5 3阳性的mdr 1,mrp的阳性率高于Mp5 3阴性者 (P <0 .0 5 ) ;Mp5 3和mdr 1,mrp表达与食管肿瘤长度、分化程度和淋巴结转移无明显关系 (P >0 .0 5 )。随访发现 ,Mp5 3,mdr 1和 (或 )mrp阳性患者的 1,2 ,3年生存率 (73.5 %,39.1%,7.7%)低于Mp5 3和mdr 1,mrp阴性者 (82 .4 %,5 8.3%,37.5 %)。 结论　食管癌组织中Mp5 3蛋白表达阳性可能对肿瘤细胞的MDR起调控作用 ;Mp5 3和mdr 1,mrp的共表达还可能反映食管癌化疗效果差、易复发转移的生物学行为。     

耐多药肺结核病124例近期疗效观察

自1996年1月－1999年6月对住院的124例耐多药结核病治疗结果进行了分析。本组病例均系复治病例,平均病程5.84年,其中浸润型肺结核78例,慢性纤维空洞型肺结核46例。应用匡氏培养基培养出人型结核杆菌,并做药敏试验,其中耐2药占20.16％,耐3药占25.0％,耐4药占20.9％,耐5药及以上占33.7％。应用卡那霉素（K）、氧氟沙星（O）、对氨基水杨酸钠（P）为基础化疗方案,加用1或2种未曾应用过的抗痨药物,平均治疗3.3个月。结果显示,痰菌转阴率为66.9％,病灶吸收好转率为72.6％,空洞治疗有效率为50.0％,疗效较好,治疗过程中 未出现严重的毒副作用。笔者认为KOP组成的化疗方案对细胞内外的结核菌都有杀灭作用,3药联合协同作用好,药物副作用不多,是治疗多耐药结核病的较好化疗方案。     

99Tcm-MIBI显像对肿瘤多药耐药检测的应用

MDR(多药耐药)是目前肿瘤化疗失败的主要原因,对MDR的检测可以帮助化疗决策的制定,从而使肿瘤患者得到更有效的治疗。99Tcm-MIBI(99Tcm-甲氧基异丁基异腈)是mdr1基因编码的P-gp(P-糖蛋白)和MRP(多药耐药相关蛋白)的转运底物,肿瘤细胞内99Tcm-MIBI摄取减低表明其P-gp的高表达,并与MRP的表达相关。因此,99Tcm-MIBI显像可在治疗前预测对化疗的反应,并为选择更有效的化疗策略提供依据。99m     

CMH为KBv_(200)耐药相关中性鞘糖脂

为了研究耐药细胞株KBv2 0 0 中性鞘糖脂表达的规律 ,采用改良的Hakomori法提取、纯化了KB及其耐药细胞株KBv2 0 0 的中性鞘糖脂 ,行高效薄层层析 ,分析其含量的异同 ;并以糖脂合成抑制剂PPMP抑制KBv2 0 0 中性鞘糖脂的合成 ,观察了其对KBv2 0 0 耐药性的逆转作用。结果发现 ,KBv2 0 0 的CMH和CDH表达较KB强 ,尤以CMH为著 ,PPMP能抑制KBv2 0 0 细胞CMH的合成 ,并能逆转KBv2 0 0 对长春新碱 (VCR)的耐药性。说明CMH为KBv2 0 0 耐药相关中性鞘糖脂 ,抑制耐药细胞CMH的合成可能是逆转肿瘤多药耐药的一种新方法。     

CMH为KBv200耐药相关中性鞘糖脂

为了研究耐药细胞株KBv200中性鞘糖脂表达的规律，采用改良的Hakomori法提取、纯化了KB及其耐药细胞株KBv200的中性鞘糖脂，行高效薄层层析，分析其含量的异同；并以糖脂合成抑制剂PPMP抑制KBv200中性鞘糖脂的合成，观察了其对KBv200耐药性的逆转作用。结果发现：KBv200的CMH和CDH表达较KB强，尤以CMH为著，PPMP能抑制KBv200细胞CMH的合成，并能逆转KBv200对长春新碱（VCR）的耐药性。说明CMH为HBv200耐药相关中性鞘糖脂，抑制耐药细胞CMH的合成可能是逆转肿瘤多药耐药的一种新方法。     

In vitro and in vivo tracer characteristics of an established multidrug-resistant human colon cancer cell line.

99mTc-methoxyisobutylisonitrile (99mTc-MIBI) has been suggested as a tracer for the scintigraphic detection of multidrug resistance (MDR). The aim of this study was to compare MDR characteristics in vitro and in vivo by immunohistochemic and functional uptake assays in established tumor cell lines cultured and grown in severe combined immunodeficient (SCID) mice. METHODS: The presence of MDR was assessed in vitro in drug-resistant HT-29(mdr1) colon carcinoma cells and in nonresistant HT-29(par) cells by JSB-1 immunohistochemistry, uptake of the fluorescent dye Rhodamine 123, and quantitative measurement of 99mTc-MIBI accumulation. For in vivo imaging, SCID mice bearing subcutaneous xenografts of these cell lines were injected with 99mTc-MIBI and 18F-FDG for scintigraphic and PET examination. After imaging, tumors were analyzed by immunohistochemistry and electron microscopy. RESULTS: All HT-29(mdr1) cells cultured in vitro exhibited distinct JSB-1 immunoreactivity, although to a variable degree, whereas HT-29(par) cells were completely devoid of JSB-1 staining. Rhodamine 123 accumulated poorly in HT-29(mdr1) cells but strongly in HT-29(par) cells. Accumulation of 99mTc-MIBI was 0.05% +/- 0.01% of the activity of the external medium in HT-29(mdr1) cells, but about eight times higher in HT-29(par) cells (0.40% +/- 0.09%), a very low percentage compared with other tumor cell lines. No difference in 201TlCl accumulation was observed between both cell lines. In vivo, neither HT-29(par) nor HT-29(mdr1) tumors grown in SCID mice could be detected by 99mTc-MIBI scintigraphy. In FDG PET, both HT-29(mdr1) and HT-29(par) tumors were clearly visible. FDG uptake was, however, markedly higher in HT-29(par) than in HT-29(mdr1) tumors. Both tumor types were poorly vascularized, as shown histologically. JSB-1 immunoreactivity was absent in all HT-29(par) tumors examined, whereas the majority of HT-29(par) tumor cells were stained. Electron microscopy showed that HT-29(par) tumors contained significantly less mitochondria than hepatocytes of the SCID mouse liver, which displayed high 99mTc-MIBI uptake in our scintigraphy studies. CONCLUSION: Sufficient 99mTc-MIBI uptake is the major prerequisite for distinguishing successfully between drug-resistant and sensitive cells. Negative 99mTc-MIBI scintigrams are not necessarily associated with MDR expression. In some tumors, FDG may be an in vivo marker for MDR as suggested by PET.     

肝叶部分切除术后肝细胞DNA合成及细胞周期变化的实验研究

肝叶部分切除术后肝细胞ＤＮＡ合成及细胞周期变化的实验研究６３００３８重庆第三军医大学西南医院陈平，韩本立，陈娟，段恒春关键词肝再生；肝叶切除术；ＤＮＡ；细胞周期中国图书资料分类号Ｒ６５７．３肝脏再生是肝叶部分切除术后最基本的病理过程。但目前对再生肝细...     

肿瘤耐药相关鞘糖脂CMH对人树突状细胞B7表达的影响

为了探讨肿瘤耐药相关鞘糖脂CMH在耐药肿瘤细胞免疫逃避中的作用,用柱层析的方法分离纯化了KBv200细胞的耐药相关中性鞘糖脂CMH,采用流式细胞仪技术检测CMH作用前后人树突状细胞（DC）B7抗原的表达。结果显示,CMH明显地抑制DC B7抗原的表达,说明肿瘤耐药相关鞘糖脂可能是通过对DC的影响而抑制机体的免疫反应。     

Transcellular transport of radioiodinated 3-iodo-α-methyl-L-tyrosine across monolayers of kidney epithelial cell line LLC-PK<Subscript>1</Subscript>

OBJECTIVE: 3-[123I]iodo-alpha-methyl-L-tyrosine ([123I]IMT) is an imaging agent for amino acid transport. In order to obtain fundamental data related to tumor imaging with [123I]IMT and renal physiological accumulation of [123I]IMT, we investigated the transport characteristics of [125I]IMT in porcine kidney epithelial cell line LLC-PK1 using cell monolayers grown on microporous membrane filters. METHODS: LLC-PK1 monolayers were created on a collagen-coated microporous (3 microm) membrane (4.7 cm2). To examine transcellular transport (secretion and reabsorption) and accumulation, the monolayers were incubated for up to 90 min at 37 degrees C with 18.5 kBq [125I]IMT in Dulbecco's phosphate-buffered saline (pH 7.4) as an uptake solution. After incubation, transcellular transport was assessed by quantifying the radioactivity of the solutions on each side of the monolayer. For the accumulation experiment, the cells were solubilized in NaOH solution, and the radioactivity was quantified. For the inhibition experiment, the inhibitor was added at a final concentration of 1 mM. For the pH dependence experiment, the pH of the apical-side uptake solution was varied from pH 5 to pH 8. Transport of [14C]Tyr was examined for comparison. RESULTS: Bi-directional transcellular transport of [125I]IMT was observed, corresponding to secretion and reabsorption in proximal tubule. Accumulation of [125I]IMT from the basolateral side (1.62 +/- 0.15%) and the apical side (2.62 +/- 0.35%) was observed at 90 min. 2-Amino-bicyclo[2,2,1]heptane-2-carboxylic acid (a specific inhibitor of system L), L-Tyr (mother compound of [125I]IMT) and 2-aminoisobutyric acid (an inhibitor of system L and A) inhibited both directional transport (p < 0.01) and accumulation (p < 0.01). 2-(Methylamino)isobutyric acid (a specific inhibitor of system A) appeared to inhibit transport and accumulation, but the results were not significant. Decreasing apical pH significantly enhanced accumulation of [125I]IMT from both sides (p < 0.001), whereas accumulation of mother L-Tyr was significantly suppressed. CONCLUSIONS: The inhibition experiments suggest that the main contributor to [125I]IMT transport is system L, rather than Na+ -dependent transport, in both apical and basolateral membrane. [125I]IMT was transported by the system that transported L-Tyr, but the observed pH dependence of transport suggests that different mechanisms are involved in accumulation of [125I]IMT and [14C]Tyr.     

全反式维甲酸联合丁酸甘油酯诱导滤泡状甲状腺癌的实验研究

目的 探讨全反式维甲酸(ATRA)联合丁酸甘油酯(TB)诱导滤泡状甲状腺癌细胞株FTC-133钠/碘同向转运体(NIS)和甲状腺球蛋白(Tg)表达及其碘的摄取.方法 单独使用1 μmol/LATRA或0.1,0.25,0.5,1 mmol/L TB和联合使用该2种药物诱导FTC-133 48 h后,用实时定量PCR反应检测NIS mRNA和Tg mRNA的表达,蛋白印迹法检测NIS蛋白表达,放射免疫法检测Tg蛋白含量以及检测FTC-133诱导后摄碘变化.结果 TB可诱导FTC-133 NIS、Tg蛋白及mRNA的表达增高,并呈剂量依赖性.联合1 μmol/L ATRA及各浓度TB后较单独使用1 μmol/L ATRA及TB明显提高了FTC-133 NIS、Tg蛋白及mRNA的表达,并且增加了FTC.133对125I的摄取.结论 ATRA联合TB有效增强了FFC-133的摄碘能力,为低分化甲状腺癌的放射性碘治疗提供了一条新的研究途径.     

多药抗药基因mdrl探针的基因克隆

多药抗药基因（ｍｕｌｔｉｄｒｕｇｒｅｓｉｓｔａｎｔｇｅｎｅ，ｍｄｒｌ）的表达水平与细胞的耐药性直接相关，检测ＭＤＲ１的表达水平可预测化疗的效果以及预后。用分子原位杂交的方法可检测单个细胞中ＭＤＲ１的表达水平。采用ＰＣＫ扩增方法获得了一段特异的ＤＮＡ片段，并将其克隆到ｐＵＣ１８载体中，经ＤＮＡ序列分析证明与文献一致。此探针可用于临床标本的分子杂交检测。