川芎嗪对凝血酶诱导的血管平滑肌细胞增殖的影响

目的观察川芎嗪（Tetramethylpyrazine,TMP）对凝血酶诱导的血管平滑肌细胞（vascular smooth muscle cell,VSMC）增殖的影响及可能机制。方法酶消化法培养兔胸主动脉VSMC。WST-8比色法、流式细胞仪及免疫细胞化学测定TMP对凝血酶诱导的VSMC增殖、细胞周期及c-fos表达的影响。结果TMP浓度依赖性地抑制凝血酶诱导的VSMC增殖,20mg/L TMP能够抑制凝血酶诱导的VSMC G0/G1期向S期的转变及c-fos表达。结论TMP浓度依赖性地抑制凝血酶诱导的VSMC增殖、机制与其抑制c-fos表达和抑制VSMC细胞周期G0/G1期向S期的转变有关。     

雌激素对PDGF诱导的血管平滑肌细胞增殖及细胞周期的影响

目的探讨17β-雌二醇（E2）对血管平滑肌细胞（VSMC）增殖及细胞周期的影响。方法分离培养VSMC,血小板源生长因子（PDGF）诱导其增殖,应用MTT法及流式细胞仪检测不同浓度E2（10和100 nmol/L）对传代VSMC增殖活性及细胞周期的影响。结果E2（10和100 nmol/L）作用下VSMC增殖活性下降,且与浓度有关;对VSMC细胞周期的分布影响主要表现为处于G0/G1期细胞数增多,G2/S期的细胞数减少（P<0.01）。结论E2具有抑制VSMC增殖的作用,其部分机制可能是通过阻滞VSMC G0/G1期向S的转化有关。     

泽泻汤对氧化型低密度脂蛋白诱导血管平滑肌细胞增殖的影响

目的观察泽泻汤对血管平滑肌细胞(VSMC)周期蛋白Cyclin D1、Cyclin E、增殖细胞核抗原(PCNA)和p27表达的影响,探讨泽泻汤在氧化型低密度脂蛋白(ox-LDL)诱导的VSMC增殖中的作用和机制。方法通过体外50 mg/L ox-LDL诱导VSMC,建立VSMC增殖的动脉粥样硬化细胞模型;应用空白血清及20%泽泻汤含药血清干预。MTS法检测泽泻汤含药血清对VSMC增殖的影响;Western blot检测增殖相关蛋白Cyclin D1、Cyclin E、PCNA和p27表达水平。结果 50 mg/L ox-LDL可明显诱导VSMC增殖。与ox-LDL组比较,泽泻汤含药血清可显著抑制ox-LDL诱导的VSMC增殖,下调细胞Cyclin D1、Cyclin E和PCNA的表达,同时促进p27蛋白表达。结论泽泻汤具有抗VSMC增殖的作用,机制可能与上调p27蛋白和抑制Cyclin D1、Cyclin E、PCNA表达有关。     

球囊扩张术对血管平滑肌细胞COX-2表达的影响及其增殖凋亡分子机制研究

目的研究球囊扩张术后血管平滑肌细胞(VSMC)中环氧化酶2(COX-2)mRNA表达及选择性COX-2抑制剂处理VSMC后,细胞周期蛋白D1(Cyclin D1)、凋亡蛋白Bcl-2的变化,明确COX-2表达与VSMC增殖凋亡分子机制的相关性。方法用RT-PCR检测20只兔腹主动脉球囊拉伤前后VSMC中COX-2的mRNA表达水平；体外实验将选择性COX-2抑制剂NS-398,作用于兔VSMCs,运用MTF法分别于0,24h,48h,72h检测细胞增殖状态；流式细胞仪观察NS-398对细胞凋亡的影响,进一步采用Western blot检测药物作用前后Cyclin D1、Bcl-2的表达。结果兔腹主动脉球囊拉伤后VSMC COX-2mRNA的表达水平明显高于正常VSMC(P＜0．01),为正常组2．42倍；对照组S及G2/M期DNA百分含量与处理组比值分别为1．31,1．62(P＜0．01),NS-398呈时间、剂量依赖性方式抑制VSMC增殖,促进其凋亡。同时,72h时空白组与NS-398(75μmol/L)处理组Cyclin D1、Bcl- 2表达水平比值分别为2．37和3．81(P＜0．01),故两者表达水平随作用时间延长而下降。结论COX-2在球囊扩张术后血管平滑肌细胞中高表达可能在VSMC过度增殖、凋亡受阻中起重要作用。选择性COX-2抑制剂NS-398可能通过Cyclin D1,Bcl-2影响VSMC的增殖与凋亡,提示COX-2抑制剂可作为预防血管成形术后再狭窄新的候选药物。     

β-淀粉样肽(25-35)对PC12细胞Cyclin D1、CDK4、pRb、E2F1基因表达的影响

目的 观察β-淀粉样肽(25-35)[β-amyloid peptide (25-35),Aβ25-35]对体外血清饥饿培养PC12细胞的Cyclin D1、CDK4、pRb、E2F1基因表达的影响.方法 用终浓度为25 μmol/L Aβ25-35处理PC12细胞,流式细胞仪检测分析细胞周期的改变,通过RT-PCR检测Cyclin D1、CDK4、E2F1基因mRNA表达变化,Western印迹检测Cyclin D1、CDK4、pRb蛋白表达的变化.结果 流式细胞仪分析表明血清饥饿培养24 h可使约90%PC12细胞停滞于G0/G1期,25 μmol/L Aβ25-35诱导组8、16、24 h与对照组比较,S期百分率明显增加(P＜0.01),16 h后细胞凋亡率明显增加(P＜0.01),可见明显的亚二倍体峰(Ap峰);Aβ25-35浓度诱导血清饥饿培养的PC12细胞0～20 h,Cyclin D1、CDK4、pRb 、E2F1 mRNA和蛋白表达增高.结论 Aβ25-35诱导同步化于G0/G1的PC12细胞重新进入细胞周期,并阻滞于S期,同时出现凋亡,可能与增加Cyclin D1、CDK4、pRb 、E2F1 mRNA和蛋白的表达有关.     

Flavopiridol通过调节CDK4/CDK6/cyclinD1复合物表达抑制肝癌细胞增殖

目的研究flavopiridol对肝癌细胞Huh7增殖、凋亡和细胞周期的影响。方法Flavopiridol处理Huh7细胞后,检测细胞增殖和凋亡,进行细胞周期分析。Westernblot检测flavopiridol对cDK4／cDK6／cyclinD1复合物表达的影响。结果Flavopiridol可显著抑制Huh7细胞增殖;Flavopiridol可剂量依赖性导致Huh7细胞发生凋亡,空白对照组凋亡细胞2．65％,550nmol／L剂量处理组凋亡细胞14．17％;Flavopiridol可剂量依赖性引起Huh7细胞G1期阻滞,空白对照组G1期细胞51．06％,550nmol／L剂量处理组G1期细胞57．53％;Flavopiridol可抑制Huh7细胞Gl期相关的蛋白细胞周期素依赖激酶（CDK）4,CDK6,细胞周期素D1（cyclinD1）的表达。结论Flavopiridol通过抑制肝癌细胞G1期cDK4／cDK6／cyclinD1蛋白复合物表达,引起细胞G1期阻滞并发生凋亡,从而抑制细胞增殖。     

同型半胱氨酸促血管平滑肌细胞增殖的机制

目的　研究同型半胱氨酸 (HCY)对血管平滑肌细胞 (VSMC)细胞周期、细胞周期素和 P2 7蛋白表达的影响。方法　用流式细胞技术测定细胞周期、细胞周期素和 P2 7蛋白表达量。结果　 HCY促进 VSMC增殖和细胞 S期合成 ,同时使细胞周期素 D、E、A表达明显增加 ,P2 7蛋白表达下降。结论　 HCY促进 VSMC增殖和细胞 S期合成可能是通过增加细胞周期素 D、E、A的表达 ,抑制 VSMC中 P2 7蛋白的表达来实现的。     

淫羊藿苷对人肝癌HepG2细胞的作用及其机制研究

目的:探讨淫羊藿苷(ICA)对HepG2肝癌细胞的作用及其可能机制。方法:给人肝癌HepG2细胞施加不同浓度的ICA(0～40μM)培养,用CCK-8法和集落形成试验测定细胞增殖能力,通过流式细胞术分析检测细胞周期进程和细胞凋亡情况,蛋白免疫印迹法分析细胞周期相关蛋白CyclinD1、CDK4、凋亡相关蛋白Bcl-2、Bax、以及芳烃受体(AHR)蛋白的表达水平。结果:ICA可抑制HepG2细胞的集落形成和细胞增殖,诱导细胞周期阻滞于G0～G1期,并诱导肝癌细胞凋亡。此外,ICA上调AHR蛋白表达,且其对HepG2细胞增殖的影响被AHR拮抗剂CH223191抑制。结论:ICA可能通过激活AHR信号抑制人肝癌HepG2细胞的增殖和诱导细胞周期阻滞于G0～G1期,并诱导细胞凋亡。     

下调ATAD2表达对胃癌细胞增殖的影响及其机制探讨

目的观察下调三磷酸腺苷酶家族蛋白2(ATAD2)表达对胃癌细胞增殖的影响,并探讨其作用机制。方法将过表达ATAD2胃癌细胞株MGC-803分为4组,A、B、C组分别给予siRNA1、siRNA2、Neg.siRNA转染48h,D组不转染。采用流式细胞仪检测MGC-803细胞周期,Western blot法检测ATAD2蛋白及细胞周期相关蛋白Cyclin D1、CDK4、Rb。结果与C、D组比较,A、B组G0/G1期细胞比例升高(P均<0.01),S期细胞比例降低(P均<0.05),细胞周期蛋白Cyclin D1和CDK4表达减少,但不引起Rb的表达变化。结论下调ATAD2表达能够抑制胃癌细胞增殖、细胞周期进展,其作用机制与降低Cyclin D1、CDK4等细胞周期相关调节蛋白表达有关。     

丹参酮ⅡA诱导PC-3细胞凋亡的细胞周期调控机制

目的研究丹参酮ⅡA诱导PC-3人前列腺癌细胞凋亡的细胞周期调控机制。方法 PC-3人前列腺癌细胞与不同浓度的丹参酮ⅡA共同培养48 h后,MTT法检测细胞增殖能力;流式细胞仪分析细胞周期;检测细胞周期调控因子CyclinD1和CDK4的表达。结果与对照组相比,0.25、0.5、1 mg/L丹参酮ⅡA组G0/G1期细胞数明显增多,S期细胞数明显减少(P<0.01);CyclinD1和CDK4的表达量均随丹参酮ⅡA剂量增加而减少(P<0.05,P<0.01)。结论丹参酮ⅡA拮抗PC-3人前列腺癌细胞增殖,其主要机制可能与CyclinD1及CDK4低表达所致细胞周期抑制有关。     

Cell Cycle Kinetic Analysis of Colorectal Neoplasms Using a New Automated Immunohistochemistry-Based Cell Cycle Detection Method

We have recently developed a new method called the immunohistochemistry-based cell cycle detection (iCCD), which allows the determination of cell cycle phases on a cell-by-cell basis. This automated procedure can be performed on tissue sections and involves triple immunostaining for geminin, cdt1, and γ H2A.X, which are nuclear proteins expressed sequentially, with a few overlaps, during the cell cycle. In the current study, we applied this technique to resected specimens of colorectal neoplasm to determine the usefulness of iCCD for the pathological examination of colorectal cancers.We examined 141 cases of colorectal cancers. Normal mucosa and adenomas were analyzed as controls.In nonneoplastic mucosa, we observed a pattern of distribution of the cells positive for these cell cycle markers. Adenomas showed a slight distortion in this pattern, the geminin-positive cells, indicative of S/G2/M phase, were localized in the upper one-third region of the crypts. In neoplastic mucosa, the marker expression pattern was disorganized. Compared with normal mucosa, colorectal neoplasms showed an increased proportion of geminin-positive cells and decreased percentages of cdt1-positive cells (G1 phase). However, we did not find significant difference in the expression pattern between adenomas and carcinomas. Cellular proportions were correlated with clinicopathological parameters such as microscopic vascular invasion and pT stages. In cases of preoperative adjuvant therapy, the proportion of geminin-positive cells decreased, whereas that of γ H2A.X-positive cells (indicative of apoptosis/degeneration) increased significantly.We believe that this novel method can be applied to clinical samples to evaluate cell cycle kinetics and the effects of preoperative adjuvant therapy in colorectal cancers.     

Chromatin Structure and the Cell Cycle

Pancreatic DNase I is used to probe the structure of chromatin isolated from synchronized HeLa cells. The degree to which DNA in chromatin is protected from DNase attack varies during the G(1), S, and G(2) phases of the cell cycle. In addition, the DNase sensitivity of chromatin from contact-inhibited African green monkey kidney cells differs from that of actively dividing, subconfluent cultures. These cell cycle-dependent chromatin changes were observed consistently at all enzyme concentrations (5000-fold range) and incubation times (15 min-2 hr) tested. The results indicate that the degree of complexing between DNA and chromosomal proteins changes during interphase, and they suggest that the chromosome coiling cycle of visible mitosis may extend in more subtle form over the entire cell cycle.     

Deregulation of Cell Cycle Machinery in Pancreatic Cancer

Disrupted cell cycle machinery is commonly thought to result in loss of proliferative control. Standard therapies target these rapidly dividing cells, yet they are ineffective against pancreatic cancer, suggesting that its development and/or progression might deviate from standard paradigms. Supposedly essential cell cycle components are actually dispensable in mice, and accumulating evidence indicates that they play more diverse roles during apoptosis, signal transduction, and cell migration. A better understanding of how pancreatic cancer cells proliferate and the contribution of disrupted cell cycle machinery would provide much needed opportunities for developing new diagnostic and therapeutic options to improve patient outcome.     

血清饥饿法用于细胞周期同步化的方法学研究

目的探讨血清饥饿法进行细胞周期同步化实验的影响因素。方法用无血清和低血清浓度培养基饥饿Anip973和AGZY83-a两种人肺腺癌细胞系，分别在培养48h、72h和5d时收集各组细胞，用流式细胞分析仪分析细胞周期各时相细胞百分数。结果Anip973细胞系在含O．2％FBS的RPMI1640培养基中培养5d得到理想结果，G0-G1期细胞百分比高达84．19％。AGZY83-a在含O．2％FBS的RPMI1640培养基中培养48h也获得较满意的结果，G0-G1期细胞百分比达61．30％。结论细胞类型、血清浓度和饥饿时间是血清饥饿法进行细胞周期同步化实验成功与否的影响因素。可应用血清饥饿法使不同细胞系同步于G0-G1期。     

Effect of Azathioprine on the Cell Cycle of Haemopoietic Cells

The megaloblastic erythropoiesis encountered in 3 azathioprine-treated patients with renal transplants was found to be associated with a marked increase in the proportion of early polychromatic erythroblasts in the Gz phase of the cell cycle. By contrast, there was only a slight increase in the proportion of Gz cells in the neutrophil promyelocyte-myelocyte pool of these patients.     

RNA干扰沉默基质交感分子1基因后对血管平滑肌细胞周期的影响

目的探讨RNA干扰技术沉默基质交感分子1基因后对血管平滑肌细胞周期的影响。方法原代培养血管平滑肌细胞,用构建好的大鼠基质交感分子1干扰腺病毒载体和人重组基质交感分子1腺病毒载体进行转染,Western blot检测细胞基质交感分子1、p21和pRb表达,流式细胞仪测定细胞周期。结果大鼠基质交感分子1干扰腺病毒载体转染后48h细胞基质交感分子1表达与转染对照腺病毒载体比较明显降低(P<0.05),处于G0/G1细胞比例也明显增多(P<0.05),p21表达上调,pRb表达下调(P<0.05);人重组基质交感分子1腺病毒载体共转染后48h细胞基质交感分子1表达恢复到正常水平;G0/M细胞比例、p21和pRb表达恢复到正常水平。结论基质交感分子1基因沉默上调p21表达,下调pRb表达,抑制细胞进入S期,进而参与对血管平滑肌细胞增殖的调控。     

Future Therapy for HBV: Role of Cell Cycle Inhibitors

Despite the discovery of an effective vaccine over 30 years ago, hepatitis B virus (HBV) infection remains a significant cause of global mortality and morbidity affecting over 250 million individuals. Fortunately, many new antiviral agents targeting different steps of the HBV lifecycle are currently being developed marking a true shift in the treatment paradigm. It is hopeful these new therapies will provide a functional and durable cure from HBV infection from a finite duration of treatment.