胸腔积液中肺腺癌细胞EGFR突变状态与DNA含量的相关性

摘要 目的：探究胸腔积液中肺腺癌细胞表皮生长因子受体（epidermalgrowthfactorreceptor,EGFR）突变状态与DNA含量的相关性,以期探究EGFR突变状态是否同肿瘤的恶性程度存在一定关联。方法：选择2015年1月至2020年1月于我院接受EGFR基因检测以及基因定量分析的591例肺腺癌患者为研究对象,按照其是否出现EGFR基因突变将其分为突变组（335例）与非突变组（256例）,两组患者的胸腔积液均使用激光图像细胞仪开展DNA含量以及非整倍体峰检测,并开展组间差异性比较。结果：（1）将591例患者按照年龄、性别及是否吸烟等临床特征进行分组对比显示,性别（P=0.034）与吸烟（P=0.007）同肺腺癌患者胸腔积液细胞出现EGFR突变具有一定关联,而年龄因素与是否出现突变无明显相关性（P>0.05）;（2）突变组患者的最大DNA指数（DI）、大于5C细胞的平均DI以及大于9C细胞的平均DI均明显高于非突变组,组间差异明显（P<0.05）;（3）开展DNA非整倍体细胞峰比较显示突变组在单峰、双峰占比中明显高于非突变组,而无峰占比明显低于非突变组（P<0.05）,多峰占比方面两组差异不大（P>0.05）。结论：经研究显示,同未出现EGFR突变的肺腺癌患者相比较,发生EGFR突变的肺腺癌患者明显DI值更高,非整倍体细胞以及非整倍体峰值也呈现异常升高态,这提示EGFR发生突变的肺腺癌患者恶变洗吧的侵袭性更强。     

不同分化程度的胃癌组织中DNA倍体和Ki67的变化

目的研究胃癌组织内DNA倍体细胞与Ki67的分布及相互关系。方法 59例胃癌病例行外科手术切除,将切除肿瘤组织印在玻片上,用Feulgen染色后测定细胞DNA含量,凡发现5c异常倍体细胞或细胞峰均为细胞学阳性。肿瘤组织做病理诊断和Ki67免疫组织化学染色。结果 59例胃肠病例中有47例低度分化,10例中度分化和2例高度分化。DNA倍体分析发现31例低分化和5例中等分化病例中可见30个异倍体细胞;15例低分化、4例中等分化和1例高分化病例可见10~30个异倍体细胞。三种分化程度各有1例10个异倍体细胞。Ki67免疫组织化学染色分析表明,24例低分化、6例中等分化和1例高分化病例中可见20%胃上皮细胞Ki67阳性;15个低分化和2个中等分化病例中发现Ki67阳性细胞占胃上皮细胞的10%~20%;8例低分化、2例中等分化和1例高度分化病例发现Ki67阳性细胞数占胃上皮细胞的10%以下。47例低分化胃上皮Ki67百分比与5c异倍体细胞数量正相关。结论胃癌组织中DNA异倍体细胞数与Ki67蛋白表达呈正相关,即DNA异倍体细胞愈多或Ki67表达愈多,胃癌恶性程度愈高。     

枸杞体细胞胚发生中DNA代谢动态的立体计量

本文以宁夏枸杞无菌苗叶片为材料,离体培养,并诱导体细胞胚胎发生。根据细胞形态计量学原理,应用数字图像处理软件计量由光学底片经A/D转换成的数字图像中的DNA大分子,对枸杞体细胞胚发生过程中DNA分子的代谢动态进行量化分析。结果表明：在整个体细胞胚发生过程中DNA代谢呈现动态变化。非胚性细胞与胚性细胞期的量化值分别为1.82%和1.91%;在二细胞胚、四细胞胚、多细胞胚时期DNA缓慢增长,随着胚性愈伤组织的发育,DNA的含量在梨形胚时期达到高峰;成熟胚的DNA含量虽有所下降,但仍维持较高水平。因此DNA的合成动态变化与体胚生长发育和细胞增殖密切相关。     

口腔肿瘤的预后与肿瘤细胞异倍体关系

目的研究口腔肿瘤预后与异倍体细胞的关系。方法口腔肿瘤切除后,将肿瘤组织在玻片印1-2张。经Feulgen染色,用DNA图像分析系统分析DNA含量。将DNA倍体分析异常结果分为3级,Ⅰ级为少数5c异倍体细胞,Ⅱ级可见1个异倍体细胞峰,Ⅲ级可见多个异倍体细胞峰。其余肿瘤组织做病理分型分期。结果 30例口腔肿瘤按转移情况分为Ⅰ级19例,Ⅱ级6例和Ⅲ级5例。在19例Ⅰ级病例中,有12例(12/19)DNA倍体分析发现有5c异倍体细胞峰。30例口腔肿瘤按病理分化程度分为16例高分化,9例中等分化和5例低分化程度。在16例高分化病例中,有11例(11/16)可见5c异倍体细胞而无异倍体细胞峰。14例中等或低分化病例中,有12例可见DNA异倍体细胞峰。结论口腔肿瘤细胞异倍体细胞数量及细胞峰型与肿瘤分期和分化程度有关,即异倍体细胞越多,预后越差。     

稻胚发育过程中细胞核内DNA增殖的特点

应用显微分光光度技术测定的稻胚发育过程中核DNA含量的结果表明,球形胚中细胞核DNA含量较高,多倍性明显,C值较集中在6－12C,个别的最高达238或439C,它们可能为胚柄细胞;DNA含量差异明显可能与它们的分化潜能有关。分化初期、中期及成熟胚细胞核DNA合量一般不及球形胚高,不同倍性的频率分布亦较球形胚分散,但多倍性仍明显;胚分化完全后胚细胞核DNA含量较多变动在20－56C范围内,但盾状体细胞核DNA含量明显高于胚芽、胚根原基。说明胚器官原基间核DNA的增殖或复制特点不同。核DNA含量高和不同器官原基间差异复制的不同可能是胚分化的前兆和基础。     

栓皮栎核型及体胚发生的细胞学特性研究

采用常规制片方法对栓皮栎核型、体胚发生过程中胚性愈伤组织细胞染色体进行了分析。结果表明,栓皮栎体细胞染色体数目2n＝24,核型公式K(2n)＝2x=24=20m十4sm,属于“2B”型,染色体组总长18.55 μm。继代8个月的胚性愈伤组织细胞的染色体数目和结构相对稳定,其中二倍体细胞占 97.20%,四倍体细胞占1.87%,单倍体细胞占0.93%。未发现有非整倍体细胞和染色体形态结构变异。子叶期体胚胚轴亚表层起源的分生组织团中有些细胞核呈片状或椭圆形。个别成熟体胚中偶见有3核细胞。     

在继代培养中玉米花粉愈伤组织的分化能力及其染色体稳定性

具有分化能力的玉米花粉愈伤组织,经过20个月的继代培养,分化能力发生显著变化:有的完全丧失,有的仍保持分化能力。通过不断分离筛选,使具有分化和再生植株能力的细胞大量增殖而保留下来。但它们的染色体倍性稳定,体细胞染色体 2n=x=10的细胞占90%以上,都是属于单倍体类型。     

小鼠胚胎癌及其分化细胞的细胞光度法分析

HMBAA对小鼠胚胎癌细胞的生长和DNA合成均有抑制作用。经过诱导分化后,癌细胞DNA含量平均值增加,这部分由于含多异倍体值的细胞数增多所致,同时含40条染色体的细胞数减少,而含60条以上染色体的细胞数增加,结果说明,胚胎癌的分化细胞虽然发生形态改变,并非就是正常细胞。     

乳腺癌c－erb B－2表达与细胞形态定量及DNA含量的关系

应用免疫组织化学及图像分析技术对５０例乳腺癌的ｃ－ｅｒｂＢ－２癌基因表达与细胞形态定量及ＤＮＡ含量的关系进行了分析。结果显示：（１）ｃ－ｅｒｂＢ－２阳性乳腺癌细胞平均ＤＮＡ含量及倍体数明显高于ｃ－ｅｒｂＢ－２阴性者,且以非二倍体细胞为主;（２）ｃ－ｅｒｂＢ－２阴性乳腺癌细胞平均核面积（ＮＡ）、核周长、核直径及核浆比例均大于ｃ－ｅｒｂＢ－２阴性者。术后５年内死亡的ｃ－ｅｒｂＢ－２阳性乳腺癌细胞ＮＡ值最大,ｃ－ｅｒｂＢ－２阳性且有淋巴结转移者细胞ＮＡ值亦较大,核形状因子偏离“１”更远。表明ｃ－ｅｒｂＢ－２表达与乳腺癌细胞的分裂及分化关系密切。     

用显微分光光度术测定水稻根尖细胞核中DNA含量的分析

用显微分光光度术测定粳稻很尖组织细胞核中Feulgen－DNA含量,C值为0．322－2．651pg,较多集中在0.904－2．068pg,平均为1．469pg。说明报关组织中有不同倍性或多线性细胞,根组织分化过程中细胞核基因组复制特点不同。经秋水仙素处理不同时间后,核DNA含量相应增加,但核之间的含量差异仍然存在。根据核DNA含量的增加值,计算很尖细胞分裂周期平均为11．54h.     

正丁酸钠对人胃腺癌细胞染色体数目和DNA含量的影响

After treatment with n-sodium butyrate for 7 days, the inhibition of growth rate of human gastric adenocarcinoma cell (MGc 80-3) in culture reaches 50.7%. About 90% of the cancer cells treated with the drug undergo obvious differentiation, and their ultrastructure is also changed. Moreover, the cancer cells which have hyperdiploid chromosomes increase from 78% to 96%; on the contrary, the percentage of hypertriploid cells decreases from 6% to 2%, while that of hypertetraploid cells diminishes from 14% to 2%. By using the combination of 3H-TdR autoradiography and Feulgen cytophotometry to measure the cellular DNA content of unlabelled cells (G1), it is shown that the DNA amount in the experimental group is lower than that in the control group. Furthermore, the DNA content of undifferentiated cells in the unlabelled cells (G1) of the experimental group is hyperhexaploid in amount (D1 = 3.76 and 3.56), and about 90% undifferentiated cells have a DNA value of over 6 C. On the other hand, the differentiated cells in the unlabelled cells on the above same slide have near-tetraploid DNA values (D1 = 2.03 and 1.99), and a DNA content below 4 C is found in about 60% differentiated cells. The difference in DNA amount between these two categories of cells is statistically significant. The results mentioned above suggest that although the amount of genetic material in the morphologically differentiated cells has markedly decreased, these cells still do not represent normal diploid cells.     

G1 block of the cell cycle during differentiation of F9 cells correlates with accumulation of inhibitors of the activity of cyclin-kinase complexes of proteins p21/Waf1 and p27/Kip

F9 teratocarcinoma cells have a very short duration of the cell cycle with a short G1-period typical for early embryonic cells. The cells are capable of differentiating towards parietal endoderm cells after the treatment with retinoic acid (RA) and dibutyryl-cAMP (db-cAMP). This leads to changes in the cell cycle; in particular, G1-period becomes longer, and then differentiated F9 cells leave the cycle to stay in G0-phase. It was previously reported that undifferentiated F9 cells undergo no G1 arrest of the cell cycle after DNA damage (Malashicheva et al., 2000). In the present work mechanisms of accumulation of G1-phase cells during differentiation induced by retinoic acid and db-cAMP were studied. Kinase activity of cyclin-Cdk complexes regulating the G1/S transition was analyzed. In differentiated F9 cells, the activity of cyclin-Cdk complexes, comprising Cdk4 and Cdk2 kinases and cyclins A and E, was significantly decreased. A decrease of Cdk4 kinase activity correlates with a drop of the cyclin D1 content. The amount of p21/Waf1 and p27/Kip inhibitors of the cyclin-kinase complexes increased in differentiated F9 cells. p21/Waf1 protein, which undergoes proteasomal degradation in undifferentiated F9 cells, was shown to be stable in their differentiated derivatives. Besides, in differentiated F9 cells p21/Waf1 and p27/Kip proteins can be detected with Cdk4/Cdk2-cyclin E complexes, in contrast to undifferentiated cells. Thus, we suggest that a G1/G0 block of the cell cycle taking place upon differentiation of F9 cells is likely to be caused by a decrease in cyclin-kinase activity due to stabilization and accumulation of p21/Waf1 and p27/Kip inhibitors and to their ability to associate with Cdk-cyclin complexes.     

Metabolic properties of chicken embryonic stem cells

Cellular energy metabolism correlates with cell fate, but the metabolic properties of chicken embryonic stem (chES) cells are poorly understood. Using a previously established chES cell model and electron microscopy (EM), we found that undifferentiated chES cells stored glycogen. Additionally, undifferentiated chES cells expressed lower levels of glucose transporter 1 (GLUT1) and phosphofructokinase (PFK) mRNAs but higher levels of hexokinase 1 (HK1) and glycogen synthase (GYS) mRNAs compared with control primary chicken embryonic fibroblast (CEF) cells, suggesting that chES cells direct glucose flux towards the glycogenic pathway. Moreover, we demonstrated that undifferentiated chES cells block gluconeogenic outflow and impede the accumulation of glucose-6-phosphate (G6P) from this pathway, as evidenced by the barely detectable levels of pyruvate carboxylase (PCX) and mitochondrial phosphoenolpyruvate carboxykinase (PCK2) mRNAs. Additionally, cell death occurred in undifferentiated chES cells as shown by Hoechst 33342 and propidium iodide (PI) double staining, but it could be rescued by exogenous G6P. However, we found that differentiated chES cells decreased the glycogen reserve through the use of PAS staining. Moreover, differentiated chES cells expressed higher levels of GLUT1, HK1 and PFK mRNAs, while the level of GYS mRNA remained similar in control CEF cells. These data indicate that undifferentiated chES cells continue to synthesize glycogen from glucose at the expense of G6P, while differentiated chES cells have a decreased glycogen reserve, which suggests that the amount of glycogen is indicative of the chES cell state.     

Modification of the indolamine content in neuroblastoma × glioma hybrid NG108-15 cells upon induced differentiation

1. The neuroblastoma x glioma hybrid NG108-15 cell line has been widely studied as a neuronal model for its serotonergic, cholinergic, and peptidergic properties. 2. The catecholamine and serotonin content and that of their major metabolites have been determined by high-performance liquid chromatography with electrochemical detection (HPLC-EC) in NG108-15 cells under differentiated and undifferentiated conditions. 3. Cellular contents of L-DOPA, norepinephrine, (NE), L-epinephrine (EPI), and dopamine (DA) in differentiated cells, induced by 1 mM dibutyryl cyclic AMP (dBcAMP), are 149, 40, 129, and 124%, respectively, higher than those in undifferentiated cells. 4. 3,4-Dihydroxyphenethylacetic acid (DOPAC), the major metabolite of DA, is detectable only in differentiated cells. Similarly, DOPAC is present only in culture medium from differentiated cells, and not that of undifferentiated cells. 5. Serotonin (5-HT) is detectable only in undifferentiated cells; and the level of 5-hydroxyindoleacetic acid (5-HIAA), the major metabolite of 5-HT, is also 12.7% higher is undifferentiated cells. 6. Comparative analyses of differentiated and undifferentiated cells in monolayer cultures and undifferentiated cells cultured in the presence of 1 mM dBcAMP under suspension conditions suggest that change in the indolamine content is due to cellular changes upon morphological differentiation. 7. The clonal NG108-15 cell line is also catecholaminergic, in addition to cholinergic and serotonergic; and a shift of neurotransmitter pattern from serotonin to dopamine production occurs during morphological differentiation.     

Recycling of resting cells in the JB-1 ascites tumour after treatment with 1-beta-D-arabinofuranosylcytosine (ara-C).

Resting cells in tumours present a major problem in cancer chemotherapy. In the plateau phase of grwoth of the murine JB-1 ascites tumour (i.e. 10 days after 2-5 X 10(6) cells i.p.) large fractions of non-cycling cells with G1 and G2 DNA content (Q1 and Q2 cells) are present, and the fate of these resting cells was investigated after treatment with 1-beta-D-arabinofuranosylcytosine (Ara-C).The experimental work of growth curves, percentage of labelled mitoses curves after continuous labelling with 3H-TdR, and cytophotometric determination of single-cell DNA content in unlabelled tumour cells. Treatment with an i.p. single injection of Ara-C 200 mg/kg in the plateau JB-1 tumour resulted in a significant reduction in the number of tumour cells 1 and 2 days later as compared with untreated controls, while no difference in the number of tumour cells was observed after 3 days. In tumours prelabelled with 3H-TdR 24 hr before Ara-C treatment, a significant decrease in the percentage of labelled mitoses was observed 6-8 hr later followed by a return to the initial value after 12 hr, and a new pronounced fall from 20 hr after Ara-C. The second fall in the percentage of labelled mitoses disappeared when the labelling with 3H-TdR was continued also after Ara-C treatment. Cytophotometry of unlabelled tumour cells prelabelled for 24 hr with 3H-TdR before Ara-C treatment showed 20 hr after Ara-C a pronounced decrease in the fraction of Q1 cells paralleled by an increase in the fraction of unlabelled cells with S DNA content. The results indicate recycling of resting cells first with G2 and later with G1 DNA content, which contribute to the regrowth of the tumours.     

Use of tumor markers in the management of head and neck cancer

Serologic tumor markers have been evaluated in the diagnosis, management and follow-up of patients with head and neck cancer. However, to the authors' knowledge no tumor marker has yet been shown to be useful for monitoring the response to chemotherapy in this type of disease, in particular for undifferentiated tumors. The pretreatment levels of CEA, TPA, SCC and ferritin were evaluated in 98 patients with advanced head and neck cancer. Of this group 64 patients were studied sequentially every month during planned chemotherapy and three weeks after treatment using standard commercial kits. The results showed the following sensitivity values: TPA 50%, CEA 36%, SCC 34% and ferritin 19%. The incidence and magnitude of the marker elevations were correlated with the extent of disease. In patients with squamous cell cancer SCC and CEA were elevated (by 68% and 54%, respectively) in tumors with good differentiation (G1), but only by 13% (both markers) in tumors classified as poorly differentiated (G3). CEA, SCC and ferritin serum levels were not correlated with response to chemotherapy, while TPA values correlated with the clinical response to treatment in 100% of patients with undifferentiated cancer and in 75% of those with squamous cell cancer. Our data indicate that in patients with head and neck cancer TPA appears to be a sensitive marker, followed in decreasing order of sensitivity by CEA, SCC and ferritin. However, SCC and CEA seem to be the most suitable markers for squamous cell cancer and in particular for more differentiated tumors (G1). Finally, TPA has proved to be a useful marker for monitoring the response to chemotherapy in patients with head and neck cancer, in particular for undifferentiated tumors.     

Presence of DNase γ-Like Endonuclease in Nuclei of Neuronal Differentiated PC12 Cells

DNase , which cleaves chromosomal DNA into nucleosomal units (DNA ladder formation), has been suggested to be the critical component of apoptotic machinery. Using rat pheochromocytoma PC12 cells, which are differentiated to sympathetic neurons by nerve growth factor (NGF), we investigated whether DNase -like enzyme is present in neuronal cells and is involved in neuronal cell death. The nuclear auto-digestion assay for DNase catalyzing internucleosomal DNA cleavage revealed that nuclei from neuronal differentiated PC12 cells contain acidic and neutral endonucleases, while nuclei from undifferentiated PC12 cells have only acidic endonuclease. The DNA ladder formation observed in isolated nuclei from neuronal differentiated PC12 cells at neutral pH requires both Ca²⁺ and Mg²⁺, and is sensitive to Zn²⁺. The molecular mass of the neutral endonuclease present in neuronal differentiated PC12 cell nuclei is 32000 as determined by activity gel analysis (zymography). The properties of the neuronal endonuclease present in neuronal differentiated PC12 cell nuclei were similar to those of purified DNase from rat thymocytes and splenocytes. Interestingly, in neuronal differentiated PC12 cells, internucleosomal DNA fragmentation is observed following NGF deprivation, whereas undifferentiated PC12 cells fail to exhibit DNA ladder formation during cell death by serum starvation. These results suggest that the DNase -like endonuclease present in neuronal differentiated PC12 cell nuclei is involved in internucleosomal DNA fragmentation during apoptosis, induced by NGF deprivation.     

Acyl-CoA: cholesterol acyltransferase in HT 29 cell subpopulations. Defect of activity in the undifferentiated cells

The ACAT activity was studied on different subpopulations deriving from HT29 cells, a human colon carcinoma cell line. Grown on standard medium (25 mM glucose), about 95% of these cells are undifferentiated (G + cells). From this heterogeneous population, differentiated cells were selected by glucose deprivation and grown either on medium without glucose (G - cells) or in standard medium containing 25 mM glucose (G-Rev cells). The G- and G-Rev cells have the features of differentiated small intestine cells. The two types of differentiated cells (G- and G-Rev) exhibited similar ACAT activities and the kinetic characteristics of the enzyme were also similar. A time-course study showed increasing activity during the exponential phase and a decrease just after confluency. It was possible to stimulate the enzyme by micellar or lipoprotein cholesterol. In contrast, the ACAT activity was hardly detectable in undifferentiated G + cells. In addition, all the experimental conditions known to stimulate ACAT activity, and confirmed in the differentiated HT29 cells, were inefficient in the undifferentiated G + cells. Therefore, the different models derived from HT29 cells provide the opportunity to study cholesterol esterification as well as the consequences of its aberrances in intestinal cells.     

Characterization and categorization of fluorescence activated cell sorted planarian stem cells by ultrastructural analysis

Planarians have regenerative ability made possible by pluripotent stem cells referred to as neoblasts. Classical ultrastructural studies have indicated that stem cells can be distinguished by a unique cytoplasmic structure known as the chromatoid body and their undifferentiated features, and they are specifically eliminated by X-ray irradiation. Recently, by using fluorescence activated cell sorting (FACS), planarian cells were separated into two X-ray-sensitive fractions (X1 and X2) and an X-ray-insensitive fraction (XIS) according to DNA content and cytoplasmic size. Here we analyzed the fractionated cells by transmission electron microscopy (TEM). First, we found that both undifferentiated cells (stem cells) and regenerative cells (differentiating cells) were concentrated in the X1 fraction containing the S/G2/M phase cells. The regenerative cells were considered to be committed stem cells or progenitor cells, suggesting that some stem cells may maintain proliferative ability even after cell fate-commitment. Second, we succeeded in identifying a new type of stem cells, which were small in size with few chromatoid bodies and a heterochromatin-rich nucleus. Interestingly, they were concentrated in the X2 fraction, containing G0/G1 phase cells. These results suggest that planarian stem cells are not homogeneous, but may consist of heterogeneous populations, like mammalian stem cells.