Antiarrhythmic Efficacy of Neferine Assessed by Programmed Electrical Stimulation in a Canine Model of Electropharmacology

An ishemic ventricular tachyarrhythmias canine model was established in open-chest dogs subjected to programmed electrical stimulation (PES)for 5-8 days after acute myocardial infarction. The electrophysiologic effects of neferine (Nef) and procainamide (PA) were observed in this model. With routine methods of PES,ventricular tachycardia (VT)and ventricular fibrillation (VF) could be reproducibly initiated. Both drugs lengthened the QTc interval (P【0.01) and effective refractory period(ERP)of normal and ischemic ventricular myocardia (NERP and IERP) respectively (P【0.01), decreased the dispersion of ERP in ischemic myocardium and the dispersion of ERP in left ventricle (P【0.01), and increased the diastolic excitability threshold of normal and ischemic ventricular myocardia (P【0.01). The two compounds prevented the PES-induced VT or VF (Nef group P【0.01, PA group P【0.05) and ischemia-induced VF (P【0.05). The results indicated that neferine and procainamide may be effective in preventing the onset of reentrant ventricular tachyarrhythmias after myocardial ischemic damage in dogs.

Reversal of Multidrug Resistance by Neferine in Adriamycin Resistant Human Breast Cancer Cell Line MCF-7/ADM

Objective: To study the reversal effect of neferine on adriamycin (ADM) resistant human breast cancer cell line MCF-7/ADM. Methods: The cytotoxic effect of Nef or ADM was determined by 3-[4, 5-dimethylthiazol-2.-yl], 5-diphenyl tetraxolium bromid (MTT) assay. Apoptosis and the expression of P-glycoprotein (P-gp) were detected by flow cytometry (FCM). The intracellular ADM concentration was measured by HPLC. Results: Nef at 1, 5, 10 mol/L decreased the IC50 of ADM to MCF-7/ADM from 11.63 g/mL to 4.59, 2.44, 0.27 g/mL respectively. MCF-7/ADM could resist the apoptosis induced by ADM while Nef (1-10 mol/L) could augment ADR-mediated apoptosis. Nef (10 mol/L) increased the accumulation of ADM up to 2.88 fold in MCF-7/ADM but not in sensitive cells MCF-7/S and reduced the expression of P-gp in MCF-7/ADM cells. Conclusion: Nef can circumvent multidrug resistance (MDR) of MCF-7/ADM cells and the mechanism was associated with the increase of intracellular accumulation of ADM and the reduced expression of P-gp in MCF-7/ADM cells.

Synergistic Effect of Hyperthermia and Neferine on Reverse Multidrug Resistance in Adriamycin-resistant SGC7901/ADM Gastric Cancer Cells

Multidrug resistance（MDR） plays a major obstacle to successful gastric cancer chemotherapy.The purpose of this study was to investigate the MDR reversal effect and mechanisms of hyperthermia in combination with neferine（Nef） in adriamycin（ADM） resistant human SGC7901/ADM gastric cancer cells.The MDR cells were heated at 42℃ and 45℃ for 30 min alone or combined with 10 μg/mL Nef.The cytotoxic effect of ADM was evaluated by MTT assay.Cellular plasma membrane lipid fluidity was detected by fluorescence polarization technique.Intracellular accumulation of ADM was monitored with high performance liquid chromatography.Mdr-1 mRNA,P-glycoprotein（P-gp）,γH2AX expression and γH2AX foci formation were determined by real-time PCR,Western blot and immunocytochemical staining respectively.It was found that different heating methods induced different cytotoxic effects.Water submerged hyperthermia had the strongest cytotoxicity of ADM and Nef combined with hyperthermia had a synergistic cytotoxicity of ADM in the MDR cells.The water submerged hyperthermia increased the cell membrane fluidity.Both water submerged hyperthermia and Nef increased the intracellular accumulation of ADM.The water submerged hyperthermia and Nef down-regulated the expression of mdr-1 mRNA and P-gp.The water submerged hyperthermia could damage DNA and increase the γH2AX expression of SGC7901/ADM cells.The higher temperature was,the worse effect was.Our results show that combined treatment of hyperthermia with Nef can synergistically reverse MDR in human SGC7901/ADM gastric cancer cells.

Effects of plant extract neferine on cyclic adenosine monophosphate and cyclic guanosine monophosphate levels in rabbit corpus cavernosum in vitro

Aim： To further investigate the relaxation mechanism of neferine （NED, a bis-benzylisoquinoline alkaloid extracted （isolated） from the green seed embryo of Nelumbo nucifera Gaertn in China, on rabbit corpus cavernosum tissue in vitro. Methods： The effects of Nef on the concentrations of cyclic adenosine monophosphate （cAMP） and cyclic guanosine monophosphate （cGMP） in isolated and incubated rabbit corpus cavernosum tissue were recorded using ^125I radioimmunoassay. Results： The basal concentration of cAMP in corpus cavernosum tissue was 5.67 ± 0.97 pmol/mg. Nef increased the cAMP concentration in a dose-dependent manner （P 〈 0.05）, but this effect was not inhibited by an adenylate cyclase inhibitor （cis-N-[2-phenylcyclopentyl]azacyclotridec-1-en-2-amine, MDL-12, 330A） （P 〉 0.05）. The accumulation of cAMP induced by prostaglandin Et （PGEt, a stimulator of cAMP production） was also augmented by Nef in a dose-dependent manner （P 〈 0.05）. The basal concentration of cGMP in corpus cavernosum tissue is 0.44 ± 0.09 pmol/mg. Nef did not affect this concentration of cGMP, either in the presence or in the absence of a guanyl cyclase inhibitor （1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, ODQ） （P 〉 0.05）. Also, sodium nitroprusside （SNP, a stimulator of cGMP production）-induced cGMP production was not enhanced by Nef （P 〉 0.05）. Conclusion： Nef, with its relaxation mechanism, can enhance the concentration of cAMP in rabbit corpus cavernosum tissue, probably by inhibiting phosphodiesterase activity. （Asian JAndro12008 Mar; 10： 307-312）

Inhibitory Effects of Neferine on Na_v1.5 Channels Expressed in HEK293 Cells

Neferine, a bisbenzylisoquinoline alkaloid in Lotus Plumule, was proved to have a wide range of biological activities. In the present study, using whole-cell patch-clamp technique, we investigated the effects of neferine on Nav1.5 channels that are stably expressed in HEK 293 cells. We found that neferine potently and reversibly inhibited Nav1.5 currents in a concentration dependent manner with a half-maximal inhibition（IC50） being 26.15 μmol/L. The inhibitory effects of neferine on Nav1.5 currents were weaker than those of quinidine at the same concentration. The steady-state inactivation curve was significantly shifted towards hyperpolarizing direction in the presence of 30 μmol/L neferine, while the voltage-dependent activation was unaltered. Neferine prolonged the time to peak of activation, increased the inactivation time constants of Nav1.5 currents and markedly slowed the recovery from inactivation. The inhibitory effect of neferine could be potentiated in a frequency-dependent manner. These results suggested that neferine can block Nav1.5 channels under the open state and inactivating state and it is an open channel blocker of Nav1.5 channels.

The Effect of Neferine on Foam Cell Formation by Anti-low Density Liporotein Oxidation

Oxidatively modified low density lipoprtein (LDL) plays an important role in atheroslerosis (AS) development. To investigate the role of neferine (Nef) in anti-LDL oxidation and foam cell formation, the lipoprotein was derived and subjected to three different treatments: N-LDL (normal LDL), Cu(2+) +LDL and Cu(2+)+Nef+LDL. The LDLs were put at 25℃ for 24 h and the thiobarbituric acid reactive substance (TBARS) values were determined. They were 0. 57 ±0. 02, 6.01±0. 22 and 2. 26±0. 13 nmol/mg protein, respectively. The difference was very significant (P<0.01) for each two groups by t test. Mouse peritoneal macrophage (MΦ) were exposed to 50 μg protein/ml of Cu(2+) + LDL and Cu(2+)+Nef+LDL at 37℃ for 60 h. The tryglyceride (TG) and total cholesterol (TC) content in Mad were assayed. The results showed that Cu(2+) + LDL was more efficient than Cu(2+)+Nef+LDL in stimulating lipid accumulation in MΦ(P <0. 001). The study demonstrated that Nef could inhibit Cu(2+)-mediated LDL oxidation and thereby inhibiting macrophage-derived foam cell formation.

Relaxation mechanisms of cavernosum tissue in vitro neferine on the rabbit corpus

Aim： To investigate the relaxation mechanisms of neferine （Nef） on the rabbit corpus cavemosum tissue in vitro. Methods： Strips of rabbit corpus cavemosum were mounted in organ chambers. The effects of Nef were examined on isolated muscle strips precontracted with phenylephrine （PE） alone, in the presence of NW-nitro-L-arginine （LNNA, a nitric oxide synthase inhibitor）, 1-H-[ 1,2,4]oxadiazolo[4,3-tx]quinoxalin- 1-one （ODQ, a guanylyl cyclase inhibitor）, indomethacin （cyclooxygenase inhibitor）, tetraethylammonium （Ca^2＋ -activated K^＋ channel blocker）, 4-aminopiridine （4-AP ,voltage dependent K^＋ channel blocker） and glibenclamide （ATP sensitive K^＋channel blocker）. The effects of Nef on KCl-induced contraction of isolated muscle strips were also investigated. The procedure of calcium absencecalcium addition was designed to observe the effect of Nef on two components of the contractile responses to PE based on the source of Ca^2＋ （extracellular vs. intracellular）. Results： Corpus cavemosum strips relaxed in response to Nef （10-9-10-4 mol/L） in a concentration-dependent manner with an IC50 of 4.60 × 10^-6 mol/L. However, they were not affected by LNNA, ODQ, indomethacin or K^＋-channel blockers. Nef （10^-6 mol/L, 10^-5 mol/L） concentration dependently reduced the maximal contraction response of isolated strips induced by KC1 to 79.3% ± 5.5% and 61.5% ±3.2%, respectively （P 〈 0.01）. In the calcium absence-calcium addition procedure, Nef 10.5 mol/L inhibited both intracellular calcium-dependent and extracellular calcium-dependent contraction induced by PE （2 × 10^5 mol/L） （P 〈 0.05）. The inhibition ratios were 26.2% ± 5.4% and 48.3% ±7.6%, respectively. Conclusion： The results of the present study suggest that Nef possesses a relaxant effect on rabbit corpus cavemosum tissues, which is attributable to the inhibition of extracellular Ca^2＋ influx and the inhibition of release of intracellular stored Ca^2＋, but not mediated by the release of nitric oxide, prostaglandins or by the activation of potassium channels.

SENSITIVITY OF LEUKEMIC CELL LINE HL-60 TO COMBINATION OF NEFERINE AND ARSENIC

Objective： To determine whether neferine （Nef） enhances the sensitivity of human myeloid leukemia HL-60 cells to arsenic trioxide （ATO）. Methods： Apoptosis was detected by DNA electrophoresis. Giemsa staining was used to observe the apoptotic cells under microscope. The apoptotic rates of cells were analyzed using flow cytometry. The inhibitory rates of cell growth were assayed by MTT, and the expression of P-gp was determined by flow cytometry. Results： Low doses of ATO （1.0 μmol/L） only partially inhibitory percentage of cell growth at 72 h was （9.92±3.03） % （P〉0.05, vs control）. The combination of 1.0 μmol/L ATO with 2.0 μmol/L Nef inhibited （45.27±4.93） % of cell growth, and induced apoptosis in leukemic cells more significantly than wither ATO or Nef on their own （P〈0.01）. Moreover, ATO-induced expression of P-gp in leukemic cells was inhibited significantly by Nef. Conclusion： These results indicate that Nef significantly increases sensitivity of leukemic cells to ATO, which might be associated with inhibitory expression of P-gp gene. Combined use of the two agents could be a novel and attractive strategy in leukemia treatment.

HIV-1Nef调节内皮细胞黏附分子ICAM-1的表达

目的研究HIV-1的调节基因Nef对ECV304细胞ICAM-1表达的影响,从而为分析HIV-1感染引起内皮细胞生物学活性的变化,以及为阐明Nef参与HIV-1致病的分子机制奠定基础。方法应用本实验室已经建立保存的HIV-1Nef基因在内皮细胞的稳定表达细胞株ECV304-Nef和其阴性对照细胞株ECV304 pcDNA 3.1(+),通过RT-PCR、实时定量PCR(real-time PCR)、Western blot、FCM和细胞黏附试验分析ECV304-Nef细胞ICAM-1的表达水平。结果 RT-PCR、real-time PCR结果显示ECV304-Nef细胞ICAM-1 mRNA表达水平明显升高,为对照组的(4.3±0.2)倍;Western blot结果示ECV304-Nef细胞I-CAM-1蛋白的表达水平高于对照组;FCM分析显示ECV304-Nef细胞和对照组细胞ICAM-1阳性细胞百分率分别为(35.3±2.2)%和(12.5±0.8)%(P<0.01),两组间ICAM-1表达有显著差异。细胞黏附实验观察到ECV304-Nef细胞黏附的Jurkat细胞数明显多于对照组,荧光仪定量分析结果显示ECV304-Nef细胞黏附的Jurkat细胞的荧光强度值显著高与对照组(P<0.05)。结论本实验证实了HIV-1Nef基因可以上调血管内皮细胞细胞黏附分子ICAM-1的表达。

HIV-1 Nef通过Erk/Mapk途径调节内皮细胞黏附分子ICAM-1的表达

目的:研究HIV-1Nef基因对内皮细胞ECV304细胞ICAM-1表达的影响及其信号途径。方法:选用Nef基因稳定表达细胞株ECV304-Nef和对照细胞株ECV304pcDNA3.1(+),应用Westernblot分析ECV304-Nef细胞ERK的磷酸化水平,利用ERK磷酸化抑制剂通过Westernblot、流式细胞术分析ECV304-Nef细胞ICAM-1的表达水平与信号分子ERK磷酸化的相关性。结果:Westernblot显示ECV304-Nef细胞ICAM-1和p-ERK蛋白的表达水平均高于对照组;流式细胞术检测结果表明ECV304-Nef细胞和对照组ICAM-1阳性细胞百分率分别为(35.3±2.2)%和(12.5±0.8)%(P<0.01)。加入p-ERK抑制剂PD98059后,ECV304-Nef细胞p-ERK水平被显著抑制,ICAM-1降至对照组水平,ECV304-Nef细胞和对照组细胞ICAM-1阳性细胞百分率分别为(11.4±1.1)%和(10.4±1.5)%(P>0.05)。结论:HIV-1Nef基因上调血管内皮细胞细胞黏附分子ICAM-1的表达与ERK信号分子磷酸化有关,为HIV-1感染的致病机制及临床治疗提供实验基础。

HIV-1病毒Nef基因克隆、表达、纯化及其抗体的制备

目的:运用基因工程方法制备HIV-1 Nef重组蛋白及其抗体。方法:用PCR技术从HIV-1H×B2质粒中扩增HIV-1 Nef基因,将测序鉴定过的HIV-1 Nef基因克隆到原核表达载体pET28a(+)上,应用酶切鉴定、测序、SDS-PAGE及Western blot等方法鉴定基因片段的正确性及表达蛋白的特异性。纯化的重组蛋白免疫兔3次后,ELISA法测定兔多克隆抗体滴度。用其制备的多克隆抗体通过免疫组织化学方法测定表达Nef基因的内皮细胞。结果:成功地构建了Nef基因的原核表达质粒,测序证明HIV-1Nef基因全长为621bp,编码206个氨基酸。在大肠杆菌BL21(DE3)中高效表达的重组蛋白为包涵体的形式。HIV-1 Nef蛋白N端融合6×His标签便于纯化及鉴定。获得的高纯度HIV-1 Nef融合蛋白,免疫动物后,可产生特异的高滴度抗体(ELISA滴度达1∶10000)。将转染Nef基因的内皮细胞,应用免疫组化的方法证明其抗体有高度的特异性。结论:构建了高表达HIV-1 Nef蛋白的原核表达系统,制备的Nef重组蛋白免疫动物,获得了特异、高效价的抗体,为研究Nef基因的生物学活性提供了实验材料和依据。

非复制重组痘苗病毒表达人免疫缺陷病毒Bostwana C亚型Nef蛋白及免疫效果的观察

从含人类免疫缺陷病毒Ⅰ型(HIV 1)BostwanaC亚型全基因的质粒pJM4 HIV中克隆了nef基因,并利用非复制型痘苗病毒载体构建表达Nef蛋白的重组病毒NTVJ1175nef,经PCR和Southernblot鉴定,nef基因正确整合到痘苗病毒基因组的J片段上;感染人源细胞后,经Westernblot和免疫荧光检测表明,重组病毒能很好地表达Nef蛋白,并定位于细胞质中。NTVJ1175nef免疫BALB/c小鼠后,经Pep IFN ′γ Assay法检测,可诱导产生针对表位肽P1特异的可分泌IFN ′γ的CD+8T细胞(占脾细胞总数0 20%);经乳酸脱氢酶(LDH)法检测证实,诱导的细胞毒性T细胞(CTL)可特异性地杀伤表位肽P1特异P815靶细胞。这些结果表明,NTVJ1175nef具有良好的细胞免疫原性,为下一步构建表达包含HIV早期抗原的多组分重组痘苗病毒候选疫苗奠定了免疫学基础。

HIV-1C亚型调控蛋白Nef在大肠杆菌中的表达

目的表达人免疫缺陷病毒1型C亚型调控蛋白Nef,为研制HIV疫苗寻找新的途径。方法通过PCR扩增,获得HIV-1C亚型Nef基因片段,并将其克隆到原核表达载体pGEX-5X-1中,转化大肠杆菌BL21(DE3),IPTG诱导表达后,以SDS-PAGE和Westernblot分析表达产物。结果表达产物是相对分子质量为50000的GST融合蛋白,且能与p27单克隆抗体发生特异性反应。结论重组Nef蛋白在大肠杆菌中得到了有效表达。

重组慢病毒载体介导HIV-1 Nef蛋白对原发性渗出性淋巴瘤细胞系和血管内皮细胞增殖作用影响的初探

目的:构建含有HIV-1负调控因子(Nef)基因的重组慢病毒表达载体,并检测Nef蛋白在人胚肾293T细胞、体腔渗出性淋巴瘤细胞BCBL-1和人血管内皮细胞EA.hy926中的表达情况及其对BCBL-1和EA.hy926细胞增殖的影响。方法:从本实验室已构建好的含Nef基因的重组真核表达质粒pCI-neo-Nef中扩增出Nef基因,插入到慢病毒载体pHAGE-CMV-MCS-Izs-Green中构建成pHAGE-Nef,利用脂质体将其与包装质粒psPAX2和包膜质粒pMD2.G共转染293T细胞。荧光显微镜下观察293T细胞中绿色荧光蛋白(GFP)的表达并收集培养上清,此上清经0.45μm滤器过滤后即获得慢病毒悬液。梯度稀释法测定慢病毒滴度。将重组慢病毒分别感染293T、BCBL-1和EA.hy926细胞,用Western blot检测Nef蛋白的表达。通过细胞增殖实验检测Nef蛋白对BCBL-1和EA.hy926细胞增殖的影响。结果:限制性内切酶鉴定和核酸序列测序证实成功构建了含HIV-1 Nef基因的重组慢病毒表达载体,病毒滴度为1×107 TU/ml。以重组慢病毒分别感染293T、BCBL-1和EA.hy926细胞,均能检测到Nef蛋白的表达,且Nef蛋白能够抑制BCBL-1和EA.hy926细胞的增殖作用。结论:成功构建了含HIV-1 Nef基因的慢病毒表达载体,获得的慢病毒不仅能够有效感染293T、BCBL-1和EA.hy926细胞,而且能够介导Nef蛋白在这些细胞中表达。重组慢病毒载体介导的Nef蛋白能够抑制BCBL-1和EA.hy926细胞的增殖。

早期半定量检测HIV-1 nef RNA的水平

目的 早期半定量检测HIV - 1nefRNA。方法 以nef基因引物nef6 ,nef7和β-actin基因引物BA1,BA4 在同一反应体系中对标本进行逆转录聚合酶链反应 ,用BIO - 1D软件对条带密度进行分析 ,半定量测定HIV - 1nefRNA水平。结果 所试 7例实验标本均扩增出HIV - 1nef和 β -actin的基因产物。且通过BIO - 1D软件分析条带密度 ,判断艾滋病病毒感染情况。结论 该方法灵敏 ,简便 ,经济 。

莲子心及Nef对实验性糖尿病及肥胖大鼠模型的影响

目的 观察莲子心及甲基莲心碱 (neferine ,Nef)对链脲佐菌素 (streptozotocin ,STZ)和高糖高脂制作的大鼠糖尿病及肥胖模型的影响 ,并与二甲双胍 (metformin ,Met)作对照。方法 STZ(30mg/kg)一次性ip后给予高糖高脂饮食 8周造模 ,莲子心乙醇提取物 5 0 0mg(生药 ) /kg、Nef 10mg/kg灌胃给药 1个月。结果 Nef用药后空腹血糖 (FBG)显著低于给药前及模型组 (P <0 .0 1) ,与Met组相当 ,莲子心空腹血糖 (FBG)低于模型组 (P <0 .0 5 ) ;莲子心的总胆固醇 (TCHO)、甘油三脂(TG)和低密度脂蛋白 (LDL -C)显著低于模型组 (P <0 .0 5 ,P <0 .0 1) ,而Nef的TCHO、TG显著低于模型组 (P <0 .0 5 ,P <0 .0 1)。结论 提示莲子心及Nef对实验性糖尿病及肥胖大鼠有一定的降低血糖及调节血脂作用。

利培酮氯丙嗪对首发精神分裂症患者眼动分析NEF的影响

目的探讨眼动分析中凝视点数和反应性探索分析能否作为首发精神分裂症患者的诊断和治疗效果的客观依据以及这两种指标是否受不同抗精神病药物治疗的影响。方法将符合入组标准的53例精神分裂症患者随机分为氯丙嗪组24例、利培酮组29例进行治疗,疗程6w。随机抽取30例正常健康者为对照组,于患者组治疗前及治疗6w末测评三组眼动分析中凝视点数来分析其认知功能的改善情况。结果氯丙嗪组、利培酮组眼动分析中凝视点数评分均显著低于对照组(P<0.01),治疗6w末凝视点数评分虽有显著升高(P<0.01),但仍均显著低于对照组,差异均有极显著性(P<0.01)。结论凝视点数测评可作为首发精神分裂症患者诊断和判定疗效的客观依据,并且可能受药物的影响。

抗Nef蛋白单抗的制备与鉴定及应用——Ⅰ:应用合成Nef蛋白肽分析抗Nef蛋白单抗的特异性

本文报告了应用杂交瘤技术制备出97个鼠抗HIV重组Nef蛋白的单抗,并应用24个按Nef蛋白氨基酸顺序合成的部分重叠的肽(Over lapping peptides)鉴定其肽特异性以及采用“肽扫描”(Pepscan;Pin ELISA)法进一步研究单抗的抗原表位特异性。文中对合成肽在抗原性分析以及抗蛋白单抗的肽特异性和表位特异性鉴定上的应用与优点进行了讨论。

应用人工合成的HIV nef蛋白肽制备抗nef单抗

本文用人工合成的HIV nef蛋白肽免疫Belb/c小鼠,应用活体融合法制备了11个抗nef单克隆抗体。ELISA测定表明,11个单抗均能与nef肽和nef蛋白反应,其中3、7、9、10和11号抗体与nef肽和nef蛋白的反应性相近似;4、5、6和7号抗体与nef蛋白的反应性明显比肽强。所有单抗均不与HIV的其它蛋白反应。应用免疫荧光检测抗nef单抗与HIV感染细胞的反应性表明,大部分抗体均与细胞浆着色,而无一般膜荧光的典型征象。

Nef对乳腺癌细胞survivin与RASSF2基因的调控及细胞凋亡

目的探讨甲基莲心碱(Nef)对乳腺癌细胞survivin、RASSF2基因的调控及细胞凋亡情况。方法将在ATCC细胞库购买的乳腺癌细胞株zr-75-30细胞分为癌细胞组(CC组):单纯zr-75-30细胞不做其他处理,正常培养;低浓度组(LC组):将zr-75-30细胞与浓度为5 mg/kg甲基莲心碱混合培养;中浓度组(MC组):将zr-75-30细胞与浓度为10 mg/kg甲基莲心碱混合培养;高浓度组(HC组):将zr-75-30细胞与浓度为20 mg/kg甲基莲心碱混合培养。通过流式细胞术、Hoechst 33258荧光染色法、Western blot法、免疫组化法、qRT-PCR法检测zr-75-30细胞凋亡情况、survivin的蛋白表达、RASSF2的表达情况、survivin、RASSF2 mRNA的表达含量来探讨Nef对乳腺癌细胞survivin、RASSF2基因的调控及细胞凋亡的情况。结果CC组zr-75-30细胞荧光强度较弱,凋亡数量较少,LC组、MC组及HC组细胞的细胞核出现核固缩的概率显著高于CC组(均P<0.05),其中,HC组与LC组、MC组相比,HC组细胞凋亡细胞数量最多(均P<0.05)。通过Western blot法,对4组zr-75-30细胞内的survivin蛋白进行免疫印迹,灰度显示,HC组zr-75-30细胞中survivin蛋白表达最低,CC组zr-75-30细胞survivin蛋白表达高于LC组、MC组及HC组(均P<0.05);LC组细胞survivin蛋白表达高于MC组、HC组(均P<0.05)。在zr-75-30细胞中RASSF2阳性染色,呈现紫色颗粒。HC组zr-75-30细胞中RASSF2阳性数较多,而在CC组zr-75-30细胞中RASSF2阳性数较少;HC组与LC组、MC组相比,HC组zr-75-30细胞中RASSF2阳性数明显较高(均P<0.05)。HC组zr-75-30细胞中的survivin mRNA表达量最低,CC组zr-75-30细胞中表达量最高;HC组与LC组、MC组相比,survivin mRNA含量明显较低(均P<0.05),RASSF2 mRNA表达含量与survivin相反。结论甲基连心碱对乳腺癌(zr-75-30)细胞的凋亡具有促进作用,且与浓度呈正相关,在20 mg/kg浓度下促进效果最佳,其作用原理可能与甲基连心碱抑制细胞中survivin表达及上调RASSF2表达有关。