Reversal of Multidrug Resistance and Inhibition of Phosphorylation of AKT in Human Ovarian Cancer Cell Line by Wild-type PTEN Gene

The reversing effect of wild-type PTEN gene on resistance of C13K cells to cisplatin and its inhibitory effect on the phosphorylation of protein kinase B (AKT) were studied. The expression of PTEN mRNA and protein in OV2008 cells and C13K cells were semi-quantitatively detected by using RT-PCR and Western blotting. Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine2000. The expression of PTEN mRNA was monitored by RT-PCR and the expression of PTEN, Akt, p-Akt protein were ana- lyzed by Western blotting in PTEN-transfected and non-transfected C13K cells. Proliferation and chemosensitivity of cells to DDP were measured by MTT, and cell apoptosis was detected by flow cytometry after treatment with cisplatin. The expression of PTEN mRNA and protein in OV2008 cells were significantly higher than those in C13K cells. After transfection with PTEN gene for 48 h, the expression of PTEN mRNA and protein in C13K cells were 2.04±0.10, 0.94±0.04 respectively and the expression of p-Akt protein ( 0.94±0.07) was lower than those in control groups (1.68±0.14, 1.66±0.10) (P< 0.05). The IC50 of DDP to C13K cells transfected with PTEN (7.2±0.3 μmol/L) was obviously lower than those of empty-vector transfected cells and non-transfected cells (12.7±0.4 μmol/l, 13.0±0.3 μmol/L) (P<0.05). The apopototis ratio of wild-type PTEN-transfected, empty vector transfected and non-transfected C13K cells were (41.65±0.87)%, (18.61±0.70)% and (15.28 ±0.80)% respectively, and the difference was statistically significant (P<0.05). PTEN gene plays an important role in ovarian cancer multidrug resistance. Transfection of PTEN could increase the ex- pression of PTEN and restore drug sensitivity to cisplatin in human ovarian cancer cell line C13K with multidrug-resistance by decreasing the expression of p-Akt.     

Reversal of multidrug resistance and inhibition of phosphorylation of AKT in human ovarian cancer cell line by wild-type PTEN gene

Summary The reversing effect of wild-type PTEN gene on resistance of C13K cells to cisplatin and its inhibitory effect on the phosphorylation of protein kinase B (AKT) were studied. The expression of PTEN mRNA and protein in OV2008 cells and C13K cells were semi-quantitatively detected by using RT-PCR and Western blotting. Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine2000. The expression of PTEN mRNA was monitored by RT-PCR and the expression of PTEN, Akt, p-Akt protein were analyzed by Western blotting in PTEN-transfected and non-transfected C13K cells. Proliferation and chemosensitivity of cells to DDP were measured by MTT, and cell apoptosis was detected by flow cytometry after treatment with cisplatin. The expression of PTEN mRNA and protein in OV2008 cells were significantly higher than those in C13K cells. After transfection with PTEN gene for 48 h, the expression of PTEN mRNA and protein in C13K cells were 2.04 ± 0.10, 0.94 ± 0.04 respectively and the expression of p-Akt protein (0.94 ± 0.07) was lower than those in control groups (1.68 ± 0.14, 1.66 ± 0.10) (P < 0.05). The IC₅₀ of DDP to C13K cells transfected with PTEN (7.2 ± 0.3 μmol/L) was obviously lower than those of empty-vector transfected cells and non-transfected cells (12.7 ± 0.4 μmol/l, 13.0 ± 0.3 μmol/L) (P<0.05). The apopototis ratio of wild-type PTEN-transfected, empty vector transfected and non-transfected C13K cells were (41.65 ± 0.87)%, (18.61 ± 0.70)% and (15.28 ± 0.80)% respectively, and the difference was statistically significant (P<0.05). PTEN gene plays an important role in ovarian cancer multidrug resistance. Transfection of PTEN could increase the expression of PTEN and restore drug sensitivity to cisplatin in human ovarian cancer cell line C13K with multidrug-resistance by decreasing the expression of p-Akt. This project was supported by a grant from the National Natural Sciences Foundation of China (No. 30571950) and National Key Basic Research Program Foundation (NO.2002CB513107).     

Effect of cytokines on the efficiency of gene transfer into murinehematopoietic progenitors

Ｅｆｆｅｃｔｏｆｃｙｔｏｋｉｎｅｓｏｎｔｈｅｅｆｆｉｃｉｅｎｃｙｏｆｇｅｎｅｔｒａｎｓｆｅｒｉｎｔｏｍｕｒｉｎｅｈｅｍａｔｏｐｏｉｅｔｉｃｐｒｏｇｅｎｉｔｏｒｓ￥（杨建民）（宋献民）（卢大儒）（闵碧荷）（李川晟）（孟沛霖）ＹａｎｇＪｉａｎｍｉｎ；Ｓｏ...     

应用抑制消减杂交（SSH）克隆肺腺癌多药耐药细胞特异表达基因

目的 克隆和筛选肺腺癌多药耐药细胞特异性表达基因。方法 将肺腺癌多药耐药细胞（SPC-A-1/CDDP）作为实验组。肺腺癌细胞（SPC-A-1）作为对照组,应用抑制消减杂交技术,构建实验组特异表达cDNA消减文件;用斑点杂交初步筛选cDNA消减文库后,将获得的阳性克隆进行测序序和同源性分析（Genebank）。结果 建立了一个肺腺癌多药耐药细胞（SPC-A-1/CDPP）特异表达cDNA消减文库,斑点杂交初步筛选显示23个克隆中有SPC-A-1/CDPP特异表达cDNA片断,测序和同源性分析表明2个cDNA片断为新序列,其余cDNA片断与已知基因有96%～100%的同源性。结论 2个新的cDNA序列可能为未知肺腺癌多药耐药相关基因序列;抑制消减杂交是克隆特异表达基因的有效方法。     

人肺腺癌多药耐药细胞系的建立及其生物学特征

目的 建立人肺腺癌多药耐药细胞系。方法 以顺铂为诱导药物,人肺腺癌细胞系SPC-A-1为诱导对象,采用大剂量冲击与逐步增加剂量相结合的方法,诱导建立人肺腺癌多药耐药细胞系SPC-A-1/CDDP;MTT法测定药物敏感性,透射和扫描电镜观察形态变化,常规染色体检测分析染色体变化。结果 用192d建成人肺腺癌多药耐药细胞系SPC-A-1/CDDP,它对顺铂的耐药指数为11.2,对顺铂、5-氟尿嘧啶、丝裂霉素、长春新碱和足叶乙甙等药物有不同程度的耐药性,但对羧基喜树碱无耐药性,电镜观察可见SPC0-A-1/CDDP细胞胞核不规则,较大,有丰富的微绒毛。结论 本研究建立了稳定的人肺腺癌多药耐药细胞系（SPC-A-1/CDDP）,可应用于下游实验。     

人前列腺癌多药耐药细胞系的建立

目的:建立人前列腺癌多药耐药细胞系。方法：以顺铂为诱导药物,人前列腺癌细胞PC3M为诱导对象,采用大剂量冲击与逐步增加剂量相结合的方法,诱导建立人前列腺癌多药耐药细胞系PC3M/CDDP;MTT法检测药物敏感性,免疫细胞化学法检测P-糖蛋白（P-gP）、多药耐药相关蛋白(MRP)的表达。结果：经顺铂诱导建立的人前列腺癌细胞系PC3M/CDDP对顺铂、丝裂霉素、阿霉素、5-氟尿嘧啶、长春新碱等药物的50%抑制浓度(IC₅₀)分别为（0.603±0.102）、（0.201±0.056）、（0.267±0.067）、（1.676±0.321）和（0.657±0.227）mg•L-1,与PC3M细胞系比较差异有显著性（P<0.05）;免疫细胞化学染色结果显示,PC3M/CDDP细胞系MRP、P-gP表达强度明显大于PC3M细胞系（P<0.05）;PC3M/CDDP细胞系的细胞倍增时间为40.5 h,与PC3M细胞系（48.6 h）比较差异无显著性（P>0.05）。结论：成功建立稳定的人前列腺癌 PC3M/CDDP细胞系,该细胞系具有对多种抗癌药耐药及细胞增殖未受影响的特点,可应用于下游实验。     

高增殖潜能内皮祖细胞促进脐血造血祖细胞体外扩增

目的：研究脐血来源高增殖潜能内皮祖细胞支持脐血造血干／祖细胞体外扩增的能力。方法：利用第3代的HPP—EPC联合细胞因子培养脐血造血干／祖细胞,分因子组、非接触培养组和接触培养组。体外培养脐血CD34＋细胞10d后,从细胞总数,CD34＋细胞数,各系造血集落形成细胞数等比较各组的扩增效率。结果：接触组细胞总数扩增倍数（44．6±8．7）倍和非接触组（39．5±7．3）倍均高于因子组（24．8±5．6）倍,P〈0．01。接触组CD34＋细胞数扩增倍数（8．8±2．1）倍高于因子组（2．9±0．6）倍和非接触培养组（3．3±0．6）倍,P〈0．01。接触组集落扩增倍数和非接触组均分别高于因子组,P〈0．05,且接触组各造血集落扩增数均分别高于非接触组,各P〈0．05。接触组扩增后CD34＋CD38＋细胞（10．8％±2．7％）明显高于因子组（4．1％±0．9％）和非接触培养组（3．8％±0．8％）,P〈0．01。结论：脐血来源高增殖潜能内皮祖细胞能支持脐血CD34＋细胞体外扩增。     

转基因法建立膀胱癌多药耐药细胞株

目的:建立高效、稳定的人膀胱癌多药耐药性细胞模型。方法:采用基因重组技术构建真核表达载体pcDNA3.1( )/bcl-2,通过脂质体将人bcl-2基因转染膀胱癌细胞BIU-87,RT-PCR检测基因转录水平,应用MTT比色法进行耐药性检验。结果:酶切鉴定和基因测序证明真核表达载体pcDNA3.1( )/bcl-2构建成功;RT-PCR分析表明,转染bcl-2基因的BIU-87/bcl-2细胞的bcl-2基因表达水平较BIU-87细胞和转染空载体的BIU-87/neo细胞显著提高;与BIU-87细胞和BIU-87/neo细胞相比,BIU-87/bcl-2细胞具有较高的耐药性。结论:基因转染法是建立人膀胱癌多药耐药细胞模型的理想方法。     

bcl-xL基因介导白血病细胞多药耐药的形成

观察ｂｃｌ－ｘＬ基因可否介导白血病细胞的多药耐药,并探讨其机制。方法采用脂质体法将ｂｃｌ－ｘＬｃＤＮＡ转导入ＨＬ－６０细胞,免疫印迹法检测Ｂｃｌ－ｘＬ及抗凋亡蛋白Ｂａｘ的表达;ＭＴＴ法测定呈足乙甙、柔红霉素、阿霉素对转染细胞的细胞毒性;流式细胞仪定量检测凋亡细胞及细胞内药物浓度。     

Multidrug resistance pglycoprotein function of bone marrow hematopoietic cells and the reversal agent effect

Intheprocessofchemotherapyfortu-mors,multidrugresistance(MDR)P-gly-coprotein(P-gp)expressionoftenoccurstoinduceMDR.PreviousstudiesconfirmedthatmanynormaltissuecellsalsoexpressedP-gp['J.So,whenthedrugsthatdisturbP-gpfunctionwereusedtoreverseMDRofthetumorceIls,thenormalP-gpfunctionofthesecellswouldbeaffectedtocausedamage.Recently,new-typeMDRreversalagentswerebeingdeveIopedandtheirphar-macologicalactionwasagainsttheP-gponthetumorcellmembrane[2].However,theeffectsoftheMDRreversalagentson…     

阿霉素诱导大肠癌LoVo细胞产生多药耐药中端粒酶逆转录酶的变化

目的:观察在阿霉素诱导大肠癌LoVo细胞产生多药耐药过程中,端粒酶逆转录酶基因表达,明确端粒酶是否参与了多药耐药机制. 方法:采用阿霉素浓度递增法,建立人大肠癌细胞多药耐药模型LoVo/Adr;用MTT法鉴定耐药LoVo/Adr细胞的耐药性;以流式细胞术检测其周期分布;用RT-PCR方法检测hTERT mRNA水平在诱导耐药过程中的变化. 结果:历经8 mo 、传代67次连续培养,建立了人大肠癌耐药模型LoVo/Adr细胞株;LoVo/Adr对阿霉素、长春新碱、丝裂霉素、环磷酰胺和5-氟尿嘧啶耐药倍数分别是61倍、14倍、3倍、9倍和1倍;LoVo/Adr细胞与LoVo细胞相比,S期细胞减少,而G1, G2期增多;亲本LoVo细胞的hTERT mRNA呈低水平表达,在阿霉素诱导初期即显著增高,随后基本保持高表达状态,并不随着耐药性增加而增强. 结论:耐药株LoVo/Adr是一个典型的具有多药耐药性表型的耐药模型,端粒酶参与了其耐药机制.     

胃癌多药耐药基因1表达及其与生物学行为的关系

目的了解胃癌多药耐药基因1(mdr1)的表达及其与生物学行为的关系。方法应用现代信息技术,收集有关文献进行综合分析。结果正常和病理胃均可表达mdr1,胃癌的mdr1表达水平最高。胃癌的mdr1表达和胃癌对抗肿瘤药物敏感性及病人的生存期、生存率呈负相关,和组织分化、肿瘤浸润、分期及转移的关系不肯定,和肿瘤发生部位、大小、组织类型、患者年龄、性别之间无相关性。结论胃癌mdr1基因表达和生物学行为相关。     

人VEGF 165基因转染大鼠内皮祖细胞及对其增殖的影响

目的探讨人血管内皮细胞生长因子165（hVEGF165）基因转染骨髓源性内皮祖细胞（EPCs）及对EPCs增殖的影响。方法体外分离、培养、鉴定EPCs,脂质体介导pcDNA3-hVEGF165质粒转染组、pcDNA3空质粒转染组、空白对照组。ELISA检测EPCs表达VEGF蛋白,MTT法检测hVEGF165基因修饰对EPCs增殖的影响。结果FITC-UEA-Ⅰ和DiI-ac-LDL荧光双染证实分化的EPCs,脂质体介导pcDNA3-hVEGF165质粒转染组EPCs培养基上清中表达VEGF,hVEGF165基因转染促进EPCs增殖。结论EPCs可以成功转染hVEGF165基因,并且可促进EPCs的增殖,为进一步研究hVEGF165基因修饰EPCs治疗血管内膜损伤性疾病提供实验依据。     

bcl-2基因的表达在卵巢恶性肿瘤多药耐药中的意义

目的探讨bcl-2基因与卵巢恶性肿瘤细胞多药耐药的关系。方法采用体外药敏实验(MTT法)检测药物敏感性,免疫组织化学方法检测45例卵巢恶性肿瘤组织bcl-2蛋白的表达,并对相关的临床病理因素进行分析。结果卵巢恶性肿瘤组织中bcl-2蛋白表达阳性率为55.6%,且不同病理类型、临床分期和术后残留病灶大小与卵巢恶性肿瘤组织中bcl-2蛋白的表达无关(P>0.05),bcl-2的表达与卵巢恶性肿瘤的耐药性呈显著的正相关(P<0.05)。结论bcl-2基因可能参与了卵巢恶性肿瘤组织的化疗耐药性,bcl-2蛋白表达的检测可以预测卵巢恶性肿瘤化疗敏感性。     

川芎嗪对转基因多药耐药细胞K562/MDR耐药性的逆转作用

目的：研究川芎嗪（TMP）与环孢菌素A（CsA）单独及联合逆转人红白血病多药耐药细胞K562/MDR的多药耐药性（MDR）,并进一步探讨其耐药逆转机制。方法：分别以倍比稀释后不同浓度的TMP（50～6　400 mg?L^-1 ）和CsA（0.5～256 mg·L^-1 ）作用于体外培养的K562/MDR细胞72 h,采用MTT法检测细胞生长率,确定TMP和CsA的非细胞毒性剂量;采用非细胞毒性剂量,实验分为5组：G1组（TMP+ADM+K562/MDR）、G2组（CsA+ADM+K562/MDR）、G3组（TMP+CsA+ADM+K562/MDR）、阴性对照组（以K562/S代替K562/MDR）、空白对照组（以1640培养基代替药物）,检测各组细胞生长抑制50%的ADM浓度,即IC₅₀,计算耐药倍数和逆转倍数;利用荧光分光光度法检测各组细胞内ADM浓度;流式细胞术检测跨膜糖蛋白P-gp的表达情况。结果：TMP及CsA的非细胞毒性剂量分别为320和2.0 mg·L^-1,K562/MDR细胞的耐药倍数为19.2倍;TMP及CsA均能降低K562/MDR细胞的耐药性,逆转倍数分别为5.2及9.6倍,并且两者联合应用,逆转倍数为15.6倍;TMP和CsA单独及联合应用均能明显增加K562/MDR细胞内ADM浓度;TMP及CsA单独应用均能降低K562/MDR细胞P-gp的表达,并且两者联合应用使K562/MDR细胞P-gp的表达率明显降低,与阴性对照组比较差异有显著性（P<0.01)。结论：TMP和CsA单独及联合应用可逆转K562/MDR细胞对化疗药物ADM的MDR,且两者具有协同作用。其逆转机制可能为降低P-gp水平,升高细胞内ADM浓度,增强对ADM的敏感性,从而发挥治疗肿瘤的作用。     

重组人源MPG基因真核表达载体构建及转染非小细胞肺癌多药耐药细胞的研究

目的构建人N甲基化嘌呤DNA糖基化酶(NmethylpurineDNAglycosylase,MPG)基因真核表达载体,并研究其在稳定转染的人非小细胞肺癌多药耐药细胞中的表达情况。方法应用分子克隆技术构建pCMVScript/MPG重组真核表达载体;应用脂质体介导的基因转染技术将其导入人非小细胞肺癌多药耐药细胞株A549/CDDP内;应用G418筛选稳定表达的转染细胞;应用PCR检测pCMVScript载体上的NEOr基因,应用RTPCR及Westernblot检测MPG基因的表达。结果构建产物经限制性酶切分析及基因序列测定证实为pCMVScript/MPG重组表达载体;转染pCMVScript/MPG载体组(MP组)及转染pCMVScript载体组(P组)细胞内检测到NEOr基因,未转染组(C组)则未检测到;MP组细胞内MPGmRNA水平较P组及C组细胞显著增高(P<0.01);MP组细胞内MPG蛋白质水平较P组及C组细胞显著增高(P<0.01)。结论成功地构建了人MPG基因表达载体,并在人非小细胞肺癌多药耐药细胞A549/CDDP获得了稳定、高效的表达。     

mdr1基因在多药耐药相关的几种人胃癌细胞系的表达

目的探讨mdr1在多药耐药相关的几种人胃癌细胞系中的转录、翻译、P-gp功能变动趋势.方法培养SGC7901/VCR(人胃癌多药耐药细胞亚系)、SGC7901和BGC823(人胃腺癌细胞系)于RPMI1640,用RT-PCR和原位杂交检测mdr1mRNA的表达,用免疫印迹和免疫组化检测P-gp的表达,用流式细胞仪检测阿霉素在细胞内的蓄积.结果半定量RT-PCR显示,SGC7901/VCR mdr1和β-actin吸收峰面积之比为5.63,SGC7901为0.61,BGC823为0.85.杂交信号呈紫蓝色颗粒,分布于胞质.免疫印迹显示SGC7901/VCRP-gp的表达最强,SGC7901最弱.P-gp阳性呈棕黄色颗粒,位于细胞膜上.SGC7901中,少数细胞P-gp表达极强.SGC7901/VCR平均光密度为(1.8310±0.8401),SGC7901为(0.3590±0.2512),BGC823为(0.6260±0.4996)(P＜0.05).SGC7901/VCR阿霉素特异荧光强度为(6.59±50.30),SGC7901为(35.88±14.55),BGC823为(27.44±7.06)(P＜0.001).结论在多药耐药相关的人胃癌细胞系SGC7901/VCR、SGC7901和BGC823中,mdr1基因均表达,P-gp具有药物外排功能,SGC7901/VCR的mdr1表达最强,SGC7901最弱,BGC823居中.     

Liposome-mediated functional expression of multiple drug resistance gene in human bone marrow CD34<Superscript>+</Superscript> cells

Summary The expression and functional activity of multiple drug resistance (MDR1) gene in human normal bone marrow CD34⁺ cells was observed. Human normal bone marrow CD34⁺ cells were enriched with magnetic cell sorting (MACS) system, and then liposome-mediated MDR1 gene was transferred into bone marrow CD34⁺ cells. Fluorescence-activated cell sorter was used to evaluate the expression and functional activity of P-glycoprotein (P-gp) encoded by MDR1 gene. It was found that the purity of bone marrow CD34⁺ cells was approximately (91±4.56) % and recovery rate was (72.3±2.36) % by MACS. The expression of P-gp in the transfected CD34⁺ cells was obviously higher than that in non-transfected CD34⁺ cells. The amount of P-gp in non-transfected CD34⁺ cells was (11.2±2.2) %, but increased to (23.6±2.34) % 48 h after gene transfection (P<0.01). The amount of P-gp was gradually decreased to the basic level one week later. The accumulation and extrusion assays showed that the overexpression of P-gp could efflux Rh-123 out of cells and there was low fluorescence within the transfected cells. The functional activity of P-gp could be inhibited by 10 μg/ml verapamil. It was suggested that the transient and highly effective expression and functional activity of P-gp could be obtained by liposome-mediated MRD1 transferring into human normal bone marrow CD34⁺ cells. CAO Wenjing, female, born in 1968, Doctor in Charge     

氨磷汀对化疗中外周血造血干/祖细胞的保护作用

目的　评估氨磷汀 (amifostine ,AMF)在保护造血干 /祖细胞免受化疗药物依托泊甙 (etoposide ,VP 16)损伤方面的作用。方法　提取新鲜和冻存的外周血造血干细胞 (PBSC)和HL 60细胞 ,分为AMF VP 16组、VP 16组和空白对照组 ,用台盼蓝拒染法检测细胞活性 ,CFU GM集落培养计数 ,流式细胞仪测定细胞的凋亡率。结果　新鲜和冻存的PB SC样本中 ,AMF VP 16组的细胞存活率和集落形成能力较VP 16组显著提高 (均P <0 .0 5 ) ;HL 60细胞样本中 ,AMF VP 16组与VP 16组凋亡率的差异无统计学意义 (P >0 .0 5 )。结论　AMF能保护造血干 /祖细胞免受化疗药物VP 16的损伤 ,并且不影响VP 16对HL 60细胞的杀伤效果。     

进展期胃癌多药耐药基因表达的临床意义

目的 探讨多药耐药基因（MDR1基因）在人胃癌组织中的表达及其与临床病理的关系。方法 采用逆转录－多聚酶链反应（RT－PCR）法检测了215例手术切除的进展期胃癌组织中的MDR1基因的表达。实验数据采用SAS软件中的χ^2检验和Fisher’s exact P做统计学处理。结果 MDRI基因的阳性率为31．63％（68／215）,与年龄、性别、组织学类型、分化程度、淋巴结转移、Borromann分型及TNM分期等无关,但在分化差的肿瘤中有增高趋势,如黏液腺癌及印戒细胞癌中达41．67％及50．00％。结论 化疗前胃癌组织中MDRI基因即存在较高的表达率,这为选用化疗药物和MDR逆转剂提供了参考指标,但不能作为制定化疗方案的唯一指标。