原代肝癌细胞培养及体外化疗药物敏感试验

化疗是目前治疗肝癌的主要辅助手段之一,但治疗效果仍无显著提高。本研究通过肝细胞癌原代培养体外化疗药物敏感实验来筛选对肝癌敏感的化疗药物。

人膀胱癌细胞原代培养及体外药物敏感性实验研究

目的研究不同个体表浅性膀胱癌组织对抗癌药物及其联合应用的敏感性。方法采用肿瘤细胞原代培养技术和MTT比色法测定了59例膀胱癌组织对灌注抗癌药物及其联合应用的抑制率和敏感性。结果14例失败,45例获得成功,总体可评价率为76.3%,不同个体对不同抗癌药物的抑制率存在明显差异;除MTX对膀胱癌G1和G3级平均抑制率差异有统计学意义外,其它抗癌药物对不同个体膀胱癌细胞的平均抑制率与膀胱癌的病理分级、分期均无关;抗癌药物对初发膀胱癌的敏感率和平均抑制率均高于复发膀胱癌;2种不同作用机制的抗癌药物联合应用对不同个体膀胱癌细胞的敏感率和平均抑制率均高于单一药物,二者敏感率和平均抑制率差异有统计学意义。结论应根据药敏结果,选择相对敏感的抗癌药物,并将作用于不同机制、细胞不同周期的抗癌药物联合,制订个体化膀胱灌注治疗方案,以提高疗效,最大限度减少副反应,减少肿瘤复发率。

培养膀胱癌细胞的新方法

培养膀胱癌细胞的新方法保健科丁虹彬微生物学教研室余雪轩关键词膀胱癌；癌细胞培养膀胱癌是一种常见的疾病。培养膀胱癌细胞有利于研究膀胱癌。但目前一般常用的方法如酶消化法或组织块培养法［１］成功率很低［２］。我们用改良法与常规方法对比，改良的方法成功率可高...

人膀胱癌细胞波状层次生长的研究

目的 观察人膀胱癌细胞 (BIU)在体外培养条件下的生长有序行为。方法 将BIU细胞悬液进行局限区域接种 ,待其形成贴壁生长的群落后进行宏观和显微动态观察及参数测量 (扩展直径、扩展速率、细胞形态、细胞平均密度、细胞平均大小、脱氢酶活性、对pH的敏感性等 )。结果 体外细胞培养条件下 ,局限区域生长的BIU细胞群落能出现明显的层次特性 ,不同区域细胞的形态、大小、密度、脱氢酶活性以及对pH的敏感性均存在差异。结论 空间封闭和生物耗散导致细胞自组织 ,其结果表现为生长有序行为。

大蒜素抑制体外人肝癌细胞的侵袭能力

目的:肝癌的侵袭与转移是导致其不良预后的重要因素,目前临床缺乏针对肝癌侵袭与转移的有效治疗措施. 本研究通过人肝癌细胞培养,研究大蒜素对体外人肝癌细胞侵袭能力的影响,并在基因水平上探讨其机制. 方法:将人肝BEL-7402细胞传代,取进入指数生长期的细胞随机分为阿霉素组Human(5 mg/L)、大蒜素低、中、高剂量组(25、50、100 mg/L),另设空白对照. 处理后每30 min观察一次细胞转移相关超微结构,8 h 后用流式细胞术检测肿瘤侵袭转移抑制基因nm23-H1 和P2ras表达水平.数据用SAS 8.2软件进行X2检验. 结果:与阿霉素组相比,大蒜素处理后超微结构除凋亡表现外,大多数贴壁细胞胞质回缩,细胞之间的连接减少,空隙加大,细胞之间界限变清晰,表面的丝状微绒毛也明显减少.流式细胞检测显示,nm23-H1表达的荧光强度空白对照组为19.19,5 mg/L.阿霉素组为119.76,25 mg/L大蒜素组为84.28,50mg/L大蒜素组为92.64,100 mg/L大蒜素组为138.08,nm23-H1 表达与大蒜素呈明显剂量效应关系,大蒜素高剂量组比阿霉素组显著增强(138.08 vs 119.76,P<0.05).而P21ras 表达的荧光强度在空白对照组、阿霉素组和低、中、高剂量组分别为2.65%、3.56%、1.55%、3.22%、3.44%,都表现为阴性低表达,各组间没有明显差别. 结论:大蒜素可以抑制体外肝癌细胞的侵袭能力,其机制可能与大蒜素特异性影响BEL-7402细胞袁面的微绒毛和上调肿瘤侵袭与转移抑制基因nm23-H1的表达有关.

益气活血方对肺癌细胞株上皮细胞粘附分子表达及侵袭能力的影响

目的观察益气活血中药对肺癌细胞株体外生长抑制,上皮细胞粘附分子(E-CD)表达及侵袭能力的影响。方法采用细胞计数、S-P免疫组织化学、图像分析及侵袭小室等方法,观察小细胞肺癌细胞株NC-H446姜益气活血中药、全反式维甲酸(ARTA)处理不同时间后细胞生长状况、E-CD表达及侵袭能力的影响。结果姜两干预因素下细胞生长抑制,形态改变。中药干预下,NC-H446细胞株E-CD阳性表达率由原来的12%±8%提高到34%±9%,积分光密度(IOD)由78.5±16.9增加到200.3±21.7,前后相比具有明显差异(P<0.01);穿过基质胶的细胞数分别为126.5±43.61,与对照组相比差异显著(P<0.01);与ARTA相比无明显差异。结论益气活血中药能明显抑制NC-H446细胞株的生长,能促进E-CD的正常表达,减弱肿瘤细胞的侵袭转移能力;中药姜上述作用方面可代替ARTA。

美科学家发现会“唤醒”癌细胞的蛋白质

美国科学家最新发现，有些癌细胞可以释放一种被称为骨桥蛋白的蛋白质，它会“唤醒”体内休眠的癌细胞。这一发现有助于医生了解并预防癌细胞在休内的扩散。据英国《自然》杂志网络版日前报道，美国怀特黑德生物医学研究所的科学家给实验鼠同时移植了两种癌细胞：一种是被称为“煽动者”的癌细胞，是科学家在实验室用可快速生长的乳腺癌细胞培养成的，另一种是生长缓慢只是偶然发生转移的俗称“回应者”的癌细胞，

肝细胞癌和基因研究

就肝癌的发生而言,腺瘤的增生是癌前期病损之一。在这类病灶的内部可以出现高分化肝细胞癌,且随着腺瘤的增大,有向未分化发展的趋向。但肝癌的发生和基因变化之间的关系尚不十分明白。 方法:本研究采用下列方法:(1)通过SSCP-PCR方法研究抑制致癌因于(p53,Rb)的突变。(2)用被切除的肝细胞癌,通过FISH观察染色体(No.16,17)的变化。(3)对末分化肝细胞癌衍生的癌细胞培养。

新型光敏剂YWW007对肝癌细胞株的灭活作用

目的探讨不同浓度光敏剂YWW007介导的光化学疗法(PCT)对悬浮后肝癌细胞的杀伤作用。方法将培养的SMMC-7721肝癌细胞株消化悬浮后配置成109/L的单细胞悬液,分别与不同浓度的YWW007混匀,按顺序进行暗反应及红光可见光照射(PCT作用)。离心清洗细胞悬液,用台盼蓝染色及AnnexinV/PI染色,流式细胞检测肝癌细胞死亡情况。结果台盼蓝染色和流式细胞仪检测均显示,经过YWW007+PCT作用,各YWW007剂量组均有大量死亡细胞。当YWW007剂量在0～8μmol/L时,肝癌细胞死亡率随着YWW007剂量浓度增加而增加(P<0.05);当YWW007剂量在8μmol/L～32μmol/L时,肝癌细胞均显示出高死亡率(死亡率>99%),且不再随YWW007剂量浓度增加而增加(P>0.05)。坏死是YWW007+PCT各剂量组细胞主要的死亡方式。单纯YWW007作用的肝癌细胞表现出了一定的暗毒性,但其死亡率均低于同浓度YWW007+PCT组(P<0.05)。结论 YWW007+PCT(波长为630～650nm、光剂量为2.16J/cm2)处理能高效杀灭悬浮后培养SMMC-7721肝癌细胞,坏死是肝癌细胞死亡的主要方式。

甲孕酮抑制人卵巢上皮性癌细胞的增殖

据《中华妇产科杂志》1995年30卷第2期报道 上海医科大学妇产科医院丰有吉等,为了解孕激素治疗卵巢癌的作用环节,对甲孕酮在人卵巢上皮性癌细胞增殖中的抑制作用进行了观察。 他们将不同终浓度甲孕酮分别加入人卵巢上皮性癌3AO细胞株培养基内,并设空白对照组培育3日。采用免疫组化及流式细胞间接荧光检测法,测定癌基因C-erbB2表达程度,并用显微镜观察癌细胞形态变化及集落形成情况。结果显示,空白对照组癌细胞培养后C-erbB2表达强度无变化,而实验组45％癌细胞C-erbB2表达强度减弱,癌细胞生长呈抑制状态,细胞数较空白对照组减少10％;药物浓度增至20nmo（?）/L时,可明显抑制癌细胞生长。显微镜观察可见实验组细胞增大、变圆、透亮。空白对照组癌细胞集落遍布于培养皿内。

Conversion of cadherin isoforms in cultured human gastriccarcinoma cells

AIM： To explore the expression of cadherin isoforms in cultured human gastric carcinoma cells and its regulation. METHODS： The expressions of cell adhesion molecules （including E-cadherin, N-cadherin, α-catenin, β-catenin） and cadherin transcription factors including snail, slug and twist were determined by reverse transcriptasepolymerase chain reaction（RT-PCR）, immunoblotting and immunofluorescence in SV40-immortalized human gastric cell line Ges-1 and human gastric cancer cell lines MGC-803, BGC-823 and SGC-7901. RESULTS： All cell lines expressed N-cadherin, but not E-cadherin. N-cadherin immunofluorescence was detected at cell membranous adherents junctions where co-localization with immunofluorescent staining of inner surface adhesion proteins α- and β-catenins was observed. The transformed Ges-1 and gastric cancer cell lines all expressed transcription factors （snail, slug and twist） which inhibited the expression of E-cadherin and triggered epithelial-mesenchymal transformation. CONCLUSION： Cadherin isoforms can change from E-cadherin to N-cadherin in transformed human gastric cancer cells, which is associated with intracellular events of stomach carcinogenesis and high expression of corresponding transcription factors.

Effect of Coriolus Versicolor Polysaccharide-B on the Biological Characteristics of Human Esophageal Carcinoma Cell Line Eca109

Objective To investigate the effect of Coriolus versicolor polysaccharide-B （CVPs-B） on the biological characteristics of human esophageal carcinoma cell line Ecal09 in vitro. Methods The cells of experimental group （EG） were cultured in DMEM with 10% FCS and 150μg/mL CVPs-B, the cells of control group （CG） were cultured in DMEM with 10% FCS without CVPs-B. MTT reduction assay was performed to detect the effect of CVPs-B on the proliferation of Ecal09 cells after the compound was administrated in varying concentrations. The living conditions of the Ecal09 cells were determined using trypan blue exclusion. Then, cell growth curves were drawn. Flow cytometry was performed to detect the effect of CVPs-B on the apoptosis and cell cycle of Ecal09. Results In comparison with the CG, a marked decrease in the proliferation of Eca09 cells was observed in the EG, after incubation with CVPs-B. The survival rate of Eca09 cells decreased as the time of CVPs-B incubation prolonged. Comparing the cell cycles and apoptotic rates between the two groups, the proportions of cells in the G0/G1, S, and G2/M phases in the EG were found to be （68.4±3.7）%, （13.9±2.1）%, and （17.7±1.4）%, respectively, after 24 h incubation with CVPs-B. The cells had an apoptotic rate of （9.7±0.7）%. On the other hand, the proportions of the G0/G1, S, and G2/M cells of the CG were found to be （53.9±3.6）%, （26.6±2.8）%, and （19.5±2.3）%, respectively, with an apoptotic rate of （5.7±1.4）%. In comparison with the CG cells, significant cell growth in the G0/G1 phase was observed in the EG （P〈0.05）. Furthermore, a significant decrease in the number of cells in the S phase was observed （P〈0.05） in the EG. Conclusions CVPs-B can inhibit proliferation and enhance apoptosis of Ecal09 cells and may be useful in the treatment of esophageal carcinoma.

CD133^+ gallbladder carcinoma cells exhibit self-renewal ability and tumorigenicity

AIM： To identify cancer stern cells （CSCs） in human gallbladder carcinomas （GBCs）. METHODS： Primary GBC cells were cultured under serum-free conditions to produce floating spheres. The stem-cell properties of the sphere-forming cells, including self-renewal, differentiation potential, chemoresistance and tumorigenicity, were determined in vitro or in vivo. Cell surface expression of CD133 was investigated in primary tumors and in spheroid cells using flow cytometry. The sphere-colony-formation ability and tumorigenicity of CD133＋ cells were assayed.floating spheroids were generated from primary GBC cells, and these sphere-forming cells could generate new progeny spheroids in serum-free media. Spheroid cells were differentiated under serum-containing conditions with downregulation of the stem cell markers Oct-4, Nanog, and nestin （P 〈 0.05）. The differentiated cells showed lower spheroid-colony-formation ability than the original spheroid cells （P 〈 0.05）. Spheroid ceils were more resistant to chemotherapeutic reagents than the congenetic adherent cells （P 〈 0.05）. Flow cytometry showed enriched CD133＋ population in sphereforming cells （P 〈 0.05）. CD133＋ cells possessed high colony-formation ability than the CD133 population （P 〈 0.01）. CD133＋ cells injected into nude mice revealed higher tumorigenicity than their antigen-negative counterparts （P 〈 0.05）. CONCLUSION： CD133 may be a cell surface marker for CSCs in GBC.

Proteome of human colon cancer stem cells:A comparative analysis

AIM： To isolate and identify the biological characteristics of human colon cancer stem cells （SW1116 cells） and further study their proteome. METHODS： SW1116 cells were isolated and cultured with a serum-free medium （SFM）. Sphere formation was assayed to observe the formation of colon cancer stem cell spheres. SW1116 cells were inoculated into a serum-containing medium for observing their differentiation characteristics. Proliferation curve and cross-resistance of SWl116 cells to different drugs were detected by MTT. Percentage of SP cells in SW1116 cells was detected with Hoechst33342 staining. Telomerase activity in SW1116cells was checked by polymerase chain reaction （PCR）-enzyme linked immunosorbent assay. Expressions of stem cell relevant genes and proteins were detected by reverse transcription-PCR and Western blot, respectively. Total protein was isolated from SW1116 cells by two-dimensional gel electrophoresis （2-DE） and differentially expressed proteins were identified by tandem mass spectrometry （MALDI-TOF/TOF）. RESULTS： The isolated SW1116 cells presented as spheroid and suspension growths in SFM with a strong self-renewal, proliferation, differentiation and drug-resistance ability. The percentage of SP cells in SW1116 cells was 38.9%. The SW1116 cells co-expressed the CD133 and CD29 proteins. The telomerase activity in SW1116 cells was increased. The expressions of different stem cell relevant genes and proteins were detected. The proteomic analysis showed that the 26 protein spots were differently expressed in SW1116 cells and 10 protein spots were identified as ubiquitin fusion- degradation l-like protein, nuclear chloride channel protein, tubulin 13, Raichu404X, stratifin, F-actin cap- ping protein α-1 subunit, eukaryotic translation elongation factor 1 delta isoform 2, hypothetical protein, glyceraldehyde-3-phosphate dehydrogenase and guanine nucleotide binding protein 13 polypeptide 2-like 1, respectively. CONCLUSION： SW1116 cells are biologically characterized by self-renewal, proliferation and differentiation, and the differently expressed proteins in SW1116 cells may be essential for isolating cancer stem cells.

In Vitro Lethal Effect of Photodynamic Therapy on Human Pancreatic Cancer Cells and Its Major Influencing Factors

OBJECTIVE To investigate the in vitro lethal effect of photo- dynamic therapy （PDT） using the photosensitizer hematoporphyrin on the human pancreatic cancer cell line Panc-1, the major influencing factors and the mechanisms of treatment. METHODS Three factors--the time needed for photosensitizer and cell incubation, the photosensitizer concentration （PhoC） and the exposure dose （ExpD）--were examined with different levels of these factors. Optical density （OD） was used as a measure of CCK-8 in the experiment, and was converted to the rate of cell survival. The separate effect of each factor on the photodynamic action was studied, and the interactions were investigated. The effects of different incubation times and PhoC levels on the fluorescence intensity （FI） of the intracellular photosensitizer were determined, and the mechanisms of these factors leading to the therapeutic effects of PDT discussed. RESULTS An increase in the photosensitizer and cell incubation time, an increase of PhoC, and enhancement of the ExpD, produced a corresponding decrease in the rate of Panc-1 cell survival after PDT （P 〈 0.05）. PDT achieved its maximum lethal effects 16 h after starting the incubation, with a PhoC of 10 mg/L and an ExpD of 20 J/cm2; at these levels a synergistic interaction between PhoC and the ExpD occurred, decreasing the cell survival rate （P 〈 0.05）. Neither simple administration of photosensitizer without ExpD （0 J/cm2） or illumination in the absence of PhoC （0 mg/L） affected the rate of cell survival （P 〉 0.05）. With an increase of PhoC and lengthening of the incubation time, the FI of the intracellular photosensitizer accordingly increased （P 〈 0.05）, and attained its maximum value at a PhoC of 10 mg/L and 36 h after the incubation. With an increase of PhoC, the FI of the photosensitizer, hematoporphyrin, in the solution increased progressively at first and then decreased （fluorescence quenching）. CONCLUSION PDT with the photosensitizer hematoporphyrin has clear lethal effects on the human pancreatic cancer cell line Panc-1, but the presence of a photosensitizer and laser irradiation by themselves do not have independent lethal effects. The three influencing factors--the time for photosensitizer and cell incuba- tion, PhoC and ExpD--correlate positively with the PDT response, within certain limits. Beyond these limits, the PDT response does not significantly increase. The main mechanism of the PDT response lies in the effect of these factors on the level of the intracellular photosensitizer and the fluorescence quenching of the photosensitizer. A synergistic effect exists between PhoC and ExpD.

Study of wavy laminar growth of human urinary bladder cancer cell line in vitro

Objective: To observe the ordered growth behavior of human urinary bladder cancer cell line (BIU) under culture in vitro. Methods: The suspension of BIU cells was spread locally in a culture container. When the cells grew a-long the wall to form a cellular colony, macroscopic and microscopic observations complemented with measurements of the parameters including expanding diameter, expanding rate, cell shape. average cell density, average cell size. dehydrogenase activity and sensitivity to pH were conducted dynamically. Results: During cell culture, obvious laminar characteristics appeared in localized growing BIU cell colonies and there was difference between the cells of different zones in shape, size, density, dehydrogenase activity and sensitivity to pH. Conclusion: Space closing and bio-dissipation result in self-organization of BIU cells with ordered growth behavior. The present experiment offers a simple, controllable model for the study of wavy growth of human cells.

Comparative genomic study of gastric epithelial cells co-cultured with Helicobacter pylori

AIM:To identify genes potentially involved in Helicobacter pylori(H.pylori)-induced gastric carcinogenesis.METHODS:GES-1 cells were co-cultured with H.pylori strains isolated from patients with gastric carcinoma(GC,n = 10) or chronic gastritis(CG,n = 10) for in vitro proliferation and apoptosis assays to identify the most and least virulent strains.These two strains were cagA-genotyped and used for further in vivo carcinogenic virulence assays by infecting Mongolian gerbils for 52 wk,respectively;a broth free of H.pylori was lavaged as control.Genomic profiles of GES-1 cells cocultured with the most and least virulent strains were determined by microarray analysis.The most differentially expressed genes were further verified using quantitative real-time polymerase chain reaction in GES-1cells infected with the most and least virulent strains,and by immunohistochemistry in H.pylori positive CG,precancerous diseases,and GC biopsy specimens in an independent experiment.RESULTS:GC-derived H.pylori strains induced a potent proliferative effect in GES-1 cells in co-culture,whereas CG-derived strains did not.The most(from a GC patient) and least(from a CG patient) virulent strains were cagA-positive and negative,respectively.At week 52,CG,atrophy,metaplasia,dysplasia,and GC were observed in 90.0%,80.0%,80.0%,90%,and 60.0%,respectively,of the animals lavaged with the most virulent strain.However,only mild CG was observed in 90% of the animals lavaged with the least virulent strain.On microarray analysis,800 differentially expressed genes(49 up-and 751 down-regulated),involving those associated with cell cycle regulation,cell apoptosis,cytoskeleton,immune response,and substance and energy metabolisms,were identified in cells co-cultured with the most virulent strain as compared with those co-cultured with the least virulent strain.The six most differentially expressed genes(with a betweenness centrality of 0.1-0.2) were identified among the significant differential gene profile network,including JUN,KRAS,BRCA1,SMAD2,TRAF1,and HDAC6.Quantitative real-time polymerase chain reaction analyses verified that HDAC6 and TRFA1 mRNA expressions were significantly more up-regulated in GES-1 cells cocultured with the most virulent strain than in those cocultured with the least virulent strain.Immunohistochemistry of gastric mucosal specimens from H.pyloripositive patients with CG,intestinal metaplasia(IM),dysplasia,and GC showed that moderately positive and strongly positive HDAC6 expression was detected in 21.7% of CG patients,30.0% of IM patients,54.5% of dysplasia patients,and 77.8% of GC patients(P < 0.001).The up-regulation of TRAF1 expressions was detected in 34.8%,53.3%,72.7%,and 88.9% specimens of CG,IM,dysplasia,and GC,respectively(P < 0.001).CONCLUSION:The overexpression of HDAC6 and TRAF1 in GES-1 cells co-cultured with the GC-derived strain and in H.pylori-positive dysplasia and GC suggests that HDAC6 and TRAF1 may be involved in H.pyloriinduced gastric carcinogenesis.