正常不同鼠龄及再生肝的肝细胞DNA含量分析

用细胞分光光度技术定量测定细胞学涂片中单个肝细胞DNA含量的方法,观察了不同龄正常大鼠及大鼠部分肝切除后再生肝的肝细胞DNA含量情况。结果表明:正常大鼠出生后,随着年龄的增长肝细胞核倍体逐渐从二倍体向四倍体发展;2/3肝切除术后48h,二倍体细胞和双核细胞明显减少,四倍体细胞增多,显示在增生刺激下促进了肝细胞多倍体化过程。最后,对肝细胞多倍体化的形成及其生物学意义进行了讨论。     

肝硬化细胞癌变与DNA含量关系的研究

本文应用显微分光光度计对23例肝硬化、31例肝细胞癌和4例正常肝组织的细胞核DNA含量进行了定量测定,并与临床特征及其生物学行为进行了比较,结果显示,多数肝硬化患者的DNA含量为二倍体/近二倍体（D/ND）,DNA组方图为单峰型。少数病例（13.04％）出现DNA异倍体（AN）组方图为组合型,类似肝癌,并与病理学上的非典型增生相伴发。对照组的31例肝癌,DNA含量水平位于D/ND者2例,占6.4％,AN者29例,占93.6％。组方图均为多峰型和组合型,4例正常肝组织均为二倍体。因此,DNA含量的异常可作为肝细胞具有潜在恶性或恶变的客观指标。     

维生素E对慢性染镉肝细胞凋亡的拮抗作用

目的　探讨维生素E(vitaminE ,VE)对慢性染镉肝细胞凋亡的拮抗作用。方法　健康昆明种小鼠 75只 ,体重 2 8g～ 32g ,随机分 3组 :染镉组 (Cdcl2 2mg·kg-1,皮下注射 ,每周 2次 ,共 3个月 )、VE +镉组 (染镉同时给VE 10mg·kg-1·d-1,灌胃 )、正常对照组 (注射等量生理盐水 )。采用免疫组化细胞凋亡染色 (Tunel法 )结合形态计量分析 ,观察 3组小鼠肝细胞凋亡的形态和数量改变。结果　 (1)染镉组小鼠肝细胞可见较多的细胞凋亡 ,VE +镉组肝细胞凋亡数量较少 ;细胞计数表明 ,VE +镉组细胞凋亡数量比染镉组明显减少 (P <0 0 1) ,而与正常对照组相比无显著性差异 (P >0 0 5 )。 (2 )凋亡肝细胞主要有以下形态特点 :①细胞胞体变小 ;②细胞核核仁消失 ,染色质浓缩边集于核膜下 ,核呈环状 ,晚期核固缩、变小 ,深染、致密度高。形态计量分析表明 ,凋亡肝细胞核的平均截面积、平均表面积、平均截面周长、体积密度等形态参数值均较正常肝细胞核明显减小 ,有显著性差异 (P <0 0 1) ;③核碎裂 ,形成凋亡小体。结论　VE对镉所致的肝细胞凋亡有抑制作用。     

大鼠肝大部切除后肝再生的研究 2.肝重量和体积以及肝细胞体积和数量增长的动态研究

测定了成年大鼠肝大部切除后12～120小时不同时间再生肝的重量和体积,用多功能显微图象测量仪测定切片中肝细胞和肝细胞核面积以及肝细胞核直径。按体视学方法测算肝细胞体积和体积密度以及整个肝脏的肝细胞数。术后36小时,再生肝重量、体积和肝重/体重比值以及肝细胞数的增长分别为对照组的2倍;术后72小时,分别为对照组的4倍。至术后120小时,肝重量、体积和肝细胞数均己接近正常肝大小。实验提示,大鼠肝大部切除后再生速度极快,术后第5天已完成肝的重建。再生肝的重量、体积和肝细胞数的增长动态呈一致的正相关。     

不同病变胃粘膜DNA含量与癌基因表达的检测

用图像分析技术检测143例胃的慢性炎症、肠上皮化生、异型增生、癌各组细胞核DNA含量及倍体分布情况，并同时进行nm23、PCNA、 C-erbB-2抗体染色。结果显示nm23的阳性率在４种病变中无明显差异， DNA含量、PCNA、C－erbB-2则呈递增表达（P＜0.01）；nm23阴性表达、 PCNA与C-erbB-2阳性表达的细胞核DNA含量与５倍体(5c)及非整倍体(AN)检出率显著高于nm23阳性、PCNA、C-erbB－2阴性表达者（P＜0.05）。提示胃良恶性病变细胞核中DNA含量及倍体分布形式不同，nm23失表达、 PCNA与C－erbB-2过度表达与5c和AN细胞形成有关，形态定量分析及癌基因检测可作为胃粘膜上皮病变诊断和分类的综合性指标。     

结,直肠癌细胞核DNA含量的MSP分析研究

用显微分光光度计检测64例结、直肠癌及其对照肠粘膜细胞核DNA含量,并行细胞核DNA含量分布直方图型分析。淋巴细胞和对照肠粘膜细胞核DNA含量直方图属Ⅰ型。64例结、直肠癌中,56例(87.50%)DNA直方图为Ⅱ～Ⅳ型。与对照肠粘膜细胞比较,结、直肠癌细胞核平均DNA含量、平均核面积和>2C DNA含量的细胞百分率以及各项数值的标准差均明显增加。肠癌细胞核DNA含量直方图与Dukes’分期和分化程度无关。     

内皮素1和血管紧张素Ⅱ对大鼠肝细胞核核苷三磷酸酶活性的影响

核被膜核苷三磷酸酶 (nucleosidetriphosphatase ,NTPase)为通过细胞核孔复合体转运mRNA的限速酶。本实验探讨内皮素 1(ET 1)和血管紧张素Ⅱ (AngⅡ )对大鼠肝细胞核NTPase活性的影响。体外分离的大鼠肝细胞核与ET 1或AngⅡ单独或分别与ET 1的ETA受体拮抗剂JKC30 1、ETB受体拮抗剂BQ788或AngⅡ的AT1受体拮抗剂Losartan、AT2 拮抗剂PD12 3177共同孵育肝细胞核 ,分别测定ATP和GTP作底物时 ,肝细胞核NTPase活性。结果发现ATP和GTP作底物时 ,ET 1(10 -11～ 10 -9mol/L)或AngⅡ (10 -11～ 10 -9mol/L)孵育肝细胞核均浓度依赖地增强其NTPase活性 (均P <0 0 1) ,ET 1和AngⅡ对NTPase的刺激作用可分别被JKC30 1(10 -6mol/L)和Losartan (10 -6mol/L)阻断 (P <0 0 1)。ET 1和AngⅡ共同孵育后 ,核NTPase活性与ET 1或AngⅡ单独孵育相比显著增加 (均P<0 0 1)。结果表明 :ET 1和AngⅡ可分别通过ETA和AT1受体刺激肝细胞核NTPase活性     

银杏叶对小鼠海马神经元超微结构的影响及抗衰老作用

目的 研究银杏叶提取物对小鼠海马神经元超微结构的影响及抗衰老作用。方法 取小鼠海马作实验材料，用定量体视学方法观察两组小鼠在不同时期海马神经元胞体，胞核，核仁等形态参数的变化，然后对结果用逐步回归的方法进行分析。结果 随着鼠龄的增加，细胞核体积逐渐减少。神经元损失较多，而银杏叶组上述变化较不明显。结论 银杏叶提取物可以影响小鼠海马神经元的超微结构，具有抗衰老作用。     

氟达拉宾对系统性红斑狼疮BXSB小鼠狼疮活动的影响

目的 :探讨氟达拉宾对系统性红斑狼疮BXSB小鼠狼疮活动的影响 ,氟达拉宾治疗重型系统性红斑狼疮的可能性、有效性及其可能的机制。方法 :用 30mg (m2 ·d) ,连续 3天氟达拉宾尾静脉注入BXSB小鼠体内 ,用血液分析仪分析用氟达拉宾前后不同时间小鼠外周血白细胞的变化 ,用ELISA方法测定BXSB小鼠血清抗ds DNA抗体、抗核抗体的变化 ,免疫荧光检查肾组织的病理改变 ,尿蛋白试纸检测用氟达拉宾前后BXSB小鼠的蛋白尿 ,流式细胞仪分析T淋巴细胞表面CD4 + Fas+ 、CD8+ Fas+ 、、CD4 5RO+ Fas+ 表达的变化。结果 :用氟达拉宾后BXSB小鼠外周血白细胞数从第 3天开始下降 ,至第 7天时白细胞下降至最低值〔(0 5± 0 2 )× 10 9L- 1 〕 ,白细胞上升至 1 0× 10 9L- 1 的时间是用药后 19天 ;BXSB小鼠血清抗ds DNA抗体、抗核抗体的水平明显下降 ,分别出现在用氟达拉宾后第 14、2 1天时 ;用药后第 2 8天氟达拉宾组 72 7%的BXSB小鼠肾组织进行免疫荧光病理检查 ,其荧光强度由 +～ ++→± ;用氟达拉宾后第 2 1、2 8天尿蛋白从 ++～ +++转±～ -占 81 8% ;用Flu后BXSB小鼠CD4 + Fas+ 、CD8+ Fas+ 、CD4 5RO+ Fas+ 的表达均明显低于用Flu前。结论 :氟达拉宾可明显减少BXSB小鼠血清抗ds DNA抗体、抗核抗体的水平 ,减少BXSB小     

rhBMP-2m对化疗损伤小鼠的修复治疗作用

目的 :探讨重组人骨形成蛋白 2 (rhBMP 2m)对环磷酰胺 (CTX)致骨髓损伤小鼠的治疗作用。方法 :18只小鼠随机分为 3组 :即CTX注射组、BMP治疗组和PBS对照组 ,每组 6只小鼠。CTX组和BMP治疗组小鼠 ,一次性腹腔注射 2 0 0mg/kgCTX建立骨髓损伤的模型。注射CTX第 2天 ,BMP治疗组每只小鼠腹腔注射 0 .5mg的rhBMP 2m开始治疗。观察 3组小鼠外周血白细胞数的变化。分别在第 5天和第 8天 ,用流式细胞仪 (FCM )检测骨髓有核细胞中DNA的含量及细胞周期的变化 ,同时进行粒单细胞集落形成 (CFU GM)细胞培养。结果 :注射CTX后第 4天 ,BMP治疗组和CTX注射组的外周血白细胞数均降至最低点 ,然后逐渐回升 ,两组相比较无显著差异 (P >0 .0 5 )。注射CTX后第 5天 ,BMP治疗组骨髓有核细胞中 ,G0 /G1期细胞的比例较CTX组明显提高 ,细胞的坏死及凋亡率明显下降 (P <0 .0 1)。第 8天 ,BMP治疗组的骨髓有核细胞数较CTX组增加明显 (P <0 .0 1)。结论 :BMP对CTX所致小鼠骨髓的损伤具有修复治疗作用     

Some flow cytofluorimetric studies of the nuclear ploidy of mouse hepatocytes. II. Early changes in nuclear ploidy of mouse hepatocytes following carbon tetrachloride administration: evidence for polyploid nuclei arrested in telophase.

Mature mice have a large proportion of their hepatocyte nuclei in polyploid states (tetraploid and octaploid), and this is more prominent in females. We measured nuclear ploidy distribution cytometrically using ethidium bromide-stained hepatocyte nuclei liberated by in situ collagenase perfusion of the liver via the portal vein. After s.c. administration of 0.2 ml carbon tetrachloride the ploidy distributions of 8-month-old female mice changed from a control of 35% 2N, 45% 4N, and 20% 8N to 54% 2N, 45% 4N and 1% 8N at 6 h, and 65% 2N, 35% 4N and 0% 8N at 24 h. By 72 h 92% of the nuclei were diploid. These changes preceded any changes in mitotic index and S-phase index (3H-TdR autoradiographs). Histology confirmed the loss of higher-ploid nuclei but without mitotic figures or selective cell necrosis to account for the observations. Cleaved nuclei were prominent in sections of liver examined 3 h after CCl4 administration and suggested division of polypoid nuclei that had undergone prior segregation of chromatids and had presumably been arrested in telophase.     

Age-related difference by IgG subclass in the response of mice to allogeneic spleen cells.

Inbred mice of various strains, 3–4 months of age and 16 months or older, were given primary injections of allogeneic spleen cells to observe the time of appearance and relative levels of alloantibodies of IgG1 and IgG2 class. In 3–4 month old C3H mice injected with BALB/c spleen cells, IgG2 alloantibodies were present on day 6, before the appearance of alloantibodies of IgG1 class. The IgG2 class alloantibodies then continued to increase in level until day 12. IgG1 class antibodies, which appeared after day 6, also increased during this period. When C3H mice at 16 months of age were similarly injected a difference in response was found in that IgG2 class alloantibodies did not increase in level after the appearance of those of the IgG1 class, but IgG1 class alloantibodies increased until day 12, as in the young mice. At intermediate ages smaller effects in the same directions were observed. Young BALB/c mice injected with C3H spleen cells gave responses similar to those of the young C3H mice. At 16 months, however, the response was not different from that of the young BALB/c mice. At older ages (19–25 months) the response of BALB/c mice was similar to that of the 16-month-old C3H mice described above. CBA mice of various ages which were injected with BALB/c spleen cells showed effects similar to that of the BALB/c mice in that a change of response seen in the older CBA mice, similar to that of the other two strains, did not appear at 16 months of age but did appear later (20–24 months).     

小鼠雄性生殖细胞生后发育中的程序性死亡

为了观察小鼠睾丸生后发育过程中生殖细胞自发死亡的方式与规律。用生后０、１、４、７、１０、１３、１８和５０ｄＡ／Ｊ系不鼠，一侧睾丸用２．５％戊二醛，另一侧睾丸用４％多聚甲醛固定１８ｈ后，分别用树脂或石蜡包埋，年鉴备超薄和连续石蜡切片，进行电镜观察和ＴＵＮＥＬ染色光镜观察。电镜观察显示，在出生当日，可见个别呈死特征的变性生殖细胞，核质溶解，但无染色质聚集。核周间隙扩大，线粒体和内质网等细胞器肿胀，有空     

Nuclear competence for maturation and pronuclear formation in mouse oocytes

BACKGROUND: In response to gonadotrophins, a fully grown mouse oocyte matures to the metaphase of the second meiotic division and becomes competent for the development of female and male pronuclei after fertilization. The present study was carried out to clarify when during the growth period an oocyte nucleus acquires the ability to promote pronuclei formation after fertilization. METHODS: Fully grown germinal vesicle (GV) oocytes were enucleated and fused with nuclei from growing oocytes from 1-20 day old mice by standard nuclear transfer technique. The reconstructed oocytes were matured and fertilized in vitro, and pronuclear formation was assessed. RESULTS: The oocytes whose nuclei were exchanged for those of the non-growing-stage oocytes matured to the metaphase of the second meiotic stage, but no normal female pronuclei were formed. Female pronuclei first formed in 27% of the oocytes reconstituted with the nuclei of oocytes from 8 day old pups after fertilization. Recondensed sperm chromatin was detected in 27% of the oocytes reconstructed with oocyte nuclei from 8 day old mice, and a male pronucleus was first formed in 6% of the oocytes that had been reconstructed with the nuclei of oocytes from 15 day old mice. The sizes of the female and male pronuclei increased with oocyte donor age, and reached normal size when the oocytes from 15 and 20 day old mice respectively were used. An electron microscopic study using oocytes that had received the oocyte nuclei of 8 day old mice confirmed these results. CONCLUSION: The factors required for pronuclear formation are derived from fully grown GV oocytes, and the transformation from decondensed sperm chromatin to a recondensed male pronucleus is governed by GV-derived factors.     

Expression of provasopressin gene during ontogeny in the hypothalamus of developing mice.

We investigated the ontogeny of provasopressin gene expression in neurosecretory neurons of the supraoptic and paraventricular nuclei of developing mice by semi-quantitative in situ hybridization and immunohistochemical techniques in combination with stereometry of vasopressin-immunoreactive neurons. Provasopressin mRNA was detected in paraffin sections using a mixture of radiolabeled synthetic oligonucleotide probes complementary to the mRNA loci encoding vasopressin (2-9) and vasopressin neurophysin (1-8). Vasopressin immunoreactivity was located with a polyclonal anti-vasopressin antiserum and a monoclonal anti-vasopressin-neurophysin antibody either with or without enhancing technique for the diaminobenzidine reaction. Autoradiographic hybridization signals that indicate the localization of provasopressin mRNA were first detected on embryonic day 15 in the supraoptic nucleus and embryonic day 18 in the paraventricular nucleus. Vasopressin immunoreactivity was first found in the median eminence on embryonic day 14, and then in the supraoptic and paraventricular nuclei on embryonic days 15 and 16, respectively. The provasopressin mRNA levels were markedly increased in both the supraoptic and the paraventricular nuclei just after birth. The immunoreactivity of vasopressin neurons was drastically decreased in both nuclei on postnatal days 1 and 2, suggesting marked vasopressin release in the neonates. Cross-sectional areas of vasopressin-immunoreactive somata and their cell nuclei gradually increased in both the supraoptic and the paraventricular nuclei during the perinatal period by day 5, and then attained adult size between days 10 and 20. During this phase, the level of provasopressin mRNA remained low compared with that in the adult magnocellular neurosecretory cells. These results indicate that the expression of provasopressin gene is markedly increased in both the supraoptic and the paraventricular nuclei soon after birth. Secretory activity of vasopressin neurons is elevated in neonatal mice. Vasopressin may have an important osmoregulatory role in neonatal mice undergoing drastic changes in water metabolism following birth.     

Polyploidy and nuclear phenotype characteristics of cardiomyocytes from diabetic adult and normoglycemic aged mice

The frequency of polyploid nuclei in the aging human heart is in sharp contrast with that in the human liver. An inverse pattern exists between the mouse heart and liver cells. Ploidy degrees in mouse hepatocytes under hyperglycemic conditions are elevated to higher levels than those in aged hepatocytes. In this study, image analysis cytometry was used to investigate the effect of diabetes and aging on Feulgen-DNA quantities, ploidy degrees, nuclear shapes and chromatin texture in mouse cardiomyocytes compared to previously reported data for mouse hepatocytes. Adult, non-obese diabetic (NOD) hyperglycemic and normoglycemic females and 56-week-old normoglycemic BALB/c females were used. A small percentage (～7%) of the cardiomyocyte nuclei in severely hyperglycemic NOD adult mice possessed higher ploidy values than those in the 8-week-old normoglycemic mice. Surprisingly, the Feulgen-DNA values and the frequency of nuclei belonging to the 4C and 8C ploidy classes were even higher (～6%) in normoglycemic NOD specimens than in age-matched hyperglycemic NOD specimens. Additionally, a pronounced elongated nuclear shape was observed especially in adult normoglycemic NOD mice. In conclusion, NOD mice, irrespective of their glycemic level, exhibit a moderate increase in ploidy degrees within cardiomyocyte nuclei during the adult lifetime. As expected, aging did not affect the Feulgen-DNA values and the ploidy degrees of cardiomyocytes in BALB/c mice. The differences in ploidy degrees and chromatin textures such as absorbance variability and entropy, between adult NOD and aged BALB/c mice are consistent with other reports, indicating dissimilarities in chromatin functions between diabetes and aging.     

Overexpression of transforming growth factor-alpha causes liver enlargement and increased hepatocyte proliferation in transgenic mice.

Transforming growth factor-alpha (TGF-alpha) expression is associated with hepatocyte DNA replication both in vivo and in culture. Our previous work using TGF-alpha transgenic mice showed that constitutive overexpression of this growth factor in the liver causes hepatic tumors in 75 to 80% of the animals at 12 to 15 months of age. To understand the cellular events by which TGF-alpha overexpression leads to abnormal liver growth, we examined hepatocyte proliferative activity in young and old TGF-alpha transgenic mice and hepatocyte ploidy in normal, dysplastic, and neoplastic livers of these animals. At 4 weeks of age, transgenic mice had higher liver weights and liver weight/body weight ratios than non-transgenic mice of the same age and hepatocyte proliferative activity, measured by 3H-thymidine incorporation after 3- and 7-day infusion, proliferating cell nuclear antigen staining, and mitotic index determination, was 2 to 3 times higher than in controls. In both transgenic and non-transgenic mice hepatocyte proliferation declined with age but the decrease was much more pronounced in control animals, so that at 8 months of age, hepatocyte replication was 8 to 10 times higher in transgenic animals. Surprisingly, however, transgenic and non-transgenic mice at this age had similar liver weight/body weight ratios. Labeling studies done in 3-month-old animals revealed that hepatocyte turnover was much faster in transgenic than in control animals, suggesting that a homeostatic compensatory mechanism involving cell death tended to restore normal liver weight/body weight ratios in older transgenic mice. Ploidy analyses showed that at 4 weeks of age transgenic mice had a higher proportion of diploid and tetraploid hepatocytes and that the hepatocellular tumors which developed in TGF-alpha transgenic mice at 13 months of age contained a higher fraction of diploid hepatocytes than that present in adjacent tissue or in dysplastic livers. The results demonstrate that constitutive overexpression of TGF-alpha causes increased hepatocyte proliferation and liver enlargement in young animals and is associated with a delay in the establishment of hepatic polyploidy. These findings as well as the response of transgenic mice to partial hepatectomy show that constitutive overexpression of TGF-alpha initially caused increased but regulated hepatocyte proliferation which in older animals was compensated in part by a faster cell turnover. At 8 to 10 months of age, proliferative activity may become constitutive in some TGF-alpha expressing hepatocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Malignant transformation of osteoblastoma: study using image analysis microdensitometry.

AIM--To determine if the malignant transformation, as perceived histologically, in a case of osteoblastoma from the right femur, was also expressed as a quantitative change in nuclear DNA during tumour progression over five months. METHODS--Nuclear DNA microdensitometry by computer image analysis was used to acquire relative DNA distribution patterns. Tissue had been removed on four separate occasions from a lesion in the right femur of an 18 year old man. Retrospective DNA analysis was performed on formalin fixed, paraffin wax-embedded tissue. RESULTS--The DNA profile of the initial biopsy specimen, which was histologically diagnosed as osteoblastoma, was euploid with a near diploid (2c) modal DNA. The second biopsy specimen taken one month later also resembled osteoblastoma but showed an aneuploid DNA profile with a diploid modal DNA and some nuclei with ploidy greater than 5c. The third biopsy specimen taken four months later showed histological evidence of osteosarcoma and a near pentaploid (5c) modal DNA with large number of nuclei exceeding 5c. CONCLUSIONS--DNA microdensitometry confirmed the initial and final diagnosis. The technique also seems to be capable of detecting aneuploidy before malignancy is morphologically evident.     

Variability of cardiomyocyte DNA content, ploidy level and nuclear number in mammalian hearts

DNA content, ploidy level, cell size and nuclear number were investigated in 54 mammalian hearts from nine species. DNA content was determined biochemically and ploidy level of cells was studied by the means of Feulgen cytophotometry. Nuclear number was calculated by a new method, while cell size was determined by using ocular micrometry. In most mammals diploid cell nuclei predominate. Higher ploidy levels were found in the human and the pig hearts. The total amount of DNA correlated with the myocardial weight. Eight million heart muscle cell nuclei were found in mice (myocardial weight 160 mg), and 2600 million heart muscle cell nuclei in the human heart (myocardial weight 210 g), but in the hearts of horses up to 35 000 million heart muscle cell nuclei (myocardial weight 3.4 kg) were found. The number of heart muscle and connective tissue cell nuclei was correlated with myocardial weight. The ratio of connective tissue cell nuclei to heart muscle cell nuclei was between 2:1 and 3:1. In cardiac growth this ratio shifted towards connective tissue cell nuclei. Increased heart weight corresponds to an increase in cell size. Diameter between 11 m and 18 m may be an optimum for heart muscle cells of mammals.     

Dna ploidy of pheochromocytoma on cytology specimen by image analysis

Pheochromocytoma usually shows prominent nuclear atypia, but the presence of such atypical cells is known to be an unreliable predictor of malignancy. DNA ploidy of pheochromocytomas has been analyzed by flow cytometry or photospectrometry on paraffinem-bedded tissue, but the results were controversial. We performed DNA analysis on cytology specimens of 11 pheochromocytomas using an image analysis system. All tumors had a mixed pattern of a large population of diploid cells and a small population of polyploid cells. DNA content correlated with nuclear size, and larger cells had more DNA content. Such larger tumor cells had polyploid nuclei, such as 4 C, 8 C, 16 C, and 32 C, in both malignant and benign pheochromocytomas. The larger polyploid nuclei may result from difficulty of duplication at the mitotic phase of the cell cycle.