Cell cycle synchronization of bison (Bos gaurus) fibroblasts derived from ear piece collected post-mortem

Efficiency of the technique of somatic cell nuclear transfer critically depends on the cell cycle phase compatibility between the donor somatic cell nucleus and recipient cytoplasm. In this study, attempts were made to optimize conditions for cell cycle synchronization of bison ear fibroblasts at G0/G1 using different approaches such as using cells in confluency, after contact inhibition, serum starvation or treatment with dimethyl sulfoxide (DMSO) (0.5%, 1.0% and 2.0%), sodium butyrate (NaBu) (0.5, 1.0 and 2.0 mm), cytochalasin-B (CB) (7.5 μg/ml), cycloheximide (CHX) (7.5 μg/ml) and 6-dimethyl aminopurine (6-DMAP) (2.0 mm). A small piece of an ear of an adult female bison collected post-mortem 10 h after death was used for the preparation of fibroblast cells. The synchronization efficiency was determined by fluorescence-activated cell sorting. Higher proportion of G0/G1 phase was obtained when cells were subjected to serum starvation for 48 h (85.4%). Sodium butyrate had no effect on synchronization of cells at G0/G1 when the cells were treated for 24 and 48 h. Similarly, DMSO (0.5% and 1.0%) had also no effect on the proportion of cells at G0/G1 for 24 and 48 h. The synchronization ability of CB, CHX and 6-DMAP at G0/G1 phase was equally effective when cells were treated for 4 h (68.5%, 68.7% and 67.4%) and 24 h (67.8%, 66.1% and 67.5%). In conclusion, this study shows that cells subjected to serum starvation for 24-48 h or confluent monolayer, or cycling cells treated with 1.0% DMSO or 2.0 mm NaBu for 24 h showed best synchronization in G0/G1 phase of cell cycle.     

Synchronizing Cell Cycle of Goat Fibroblasts by Serum Starvation Causes Apoptosis

Cell cycle stage and synchronization of donor cells are important factors influencing the success of somatic cell nuclear transfer. This study examined whether serum starvation has any effect on specific cell death. We also studied the effects of serum starvation, culture to confluence, and full confluency (confluent + 72 h) on cell cycle characteristics and apoptosis of goat dermal fibroblast cells. The cells were obtained from the ear of a 1.5‐year‐old female goat. The following experimental groups were analysed for fibroblast cells: (i) normally growing, (ii) confluent, (iii) full confluency, (iv) cells starved for 48 h and (v) cells starved for 72 h. Analysis of cell cycle distribution by flow cytometry showed that 4.56 and 51.88% of normal cycling cells were at the G0 and G1 phases respectively. In the confluent group, 80% of the cells were arrested in the G0/G1 phase. Serum starvation for 48 and 72 h arrested 84.78% and 90.1% cells at the G0/G1 phase respectively which showed a significant difference when compared with the control group (p < 0.05). Double staining by PI and FITC distinguishes G0 phase from G1 phase. In the full confluency group, 91.53% of cells were at G0/G1 stage, but in contrast to the serum starved group, this high percentage of G0/G1 cells was mainly associated with G1 cells. Under normal culture conditions, 6.39% of cells underwent early apoptosis. In the confluent group 8.93% of cells showed early apoptosis. Serum starvation for 48 and 72 h caused early apoptosis in 8.91 and 39.83% of the cells respectively. Full confluency treatment did not increase the number of apoptotic cells significantly (8.67%). After 72 h, serum starvation significantly increased early apoptosis (p < 0.05). In conclusion, the use of full confluency is suitable for cell cycle synchronization because it arrests cells at the G0/G1 phase and also induces less apoptosis in comparison with the serum starvation group.     

Cell Cycle Stage Analysis of Rabbit Foetal Fibroblasts and Cumulus Cells

Synchronization of the cell cycle stages in G0/G1 phase is one of the key factors determining the success of nuclear transplantation. Serum deprivation, contact inhibition and chemical inhibitors are widely used methods for this purpose. In this study, cell cycle stages of foetal fibroblasts and cumulus cells were determined using flow cytometry [fluorescence-activated cell scan (FACS)]. Foetal fibroblasts (in vitro cultured for 72-120 h) and fresh cumulus cells were analysed in Experiment 1. Fifty to 55% proliferating fibroblasts remained in G0/G1 phase compared with 78% in confluent culture (p <0.05). In contrast to foetal fibroblasts, fresh cumulus cells maintained 90% of the population in the G0/G1 stage. When serum was retrieved from the proliferating fibroblasts from day 1 to day 5 (Experiment 2), proportions of G0/G1 cells increased from the initial ratio of 53 to 87% at day 4 of starvation, which was significantly higher than the non-starved proliferating cells (p <0.05). In Experiment 3, fibroblasts were treated with aphidicolin (0.1 microg/ml, 6 h), demicolcine (0.5 microg/ml, 10 h), or a combination of these two chemicals to synchronize the cell cycle stages. Surprisingly, no differences or significantly lower in the proportions of G0/G1)phase cells were detected (25-50%) compared with the uncontrolled growing cells (53%). These results suggested that fresh cumulus cells rest their cell cycle in G0/G1 stage. Serum deprivation became effective in the first 24 h and reached the highest proportions during days 4-5 after deprivation. Chemical synchronization of the cell cycle stage of rabbit foetal fibroblasts to G0/G1 phase appeared less effective compared to serum deprivation.     

钼对TM3小鼠睾丸间质细胞周期和凋亡的影响

以体外培养的小鼠睾丸间质细胞系TM3 为材料,加入不同质量浓度的钼酸钠溶液（0,10,20,40,80,160mg/L）染毒培养,分别在干预4,8,12,24,48h采用MTT法检测细胞的增殖。干预48h后,采用流式细胞术检测细胞周期和凋亡,单细胞凝胶电泳检测DNA损伤的变化。结果表明：与对照组相比,不同剂量钼酸钠作用24h后,睾丸间质细胞的增殖活性均受到抑制;不同质量浓度的钼酸钠作用48h后,细胞周期阻滞于G0/G1期,20mg/L及其以上剂量组G0/G1期细胞百分率与对照组相比显著升高（P〈0.05或P〈0.01）;与对照组相比,各剂量组TM3 小鼠睾丸间质细胞凋亡率显著升高,差异极显著（P〈0.01）;细胞尾部DNA含量及细胞尾长随着钼剂量的增加呈不同程度的增加,且存在着剂量—效应关系。结论说明钼能够引起睾丸间质细胞周期的紊乱,并诱导睾丸间质细胞发生DNA损伤和凋亡。     

Effects of cell cycle status on the efficiency of liposome-mediated gene transfection in mouse fetal fibroblasts

Methods for cell cycle synchronization of mouse fetal fibroblast cells (MFFCs) were first selected and optimized. When MFFCs were cooled at 5 C for different periods of time, the highest percentage of cells at the G0/G1 phase (75.4+/-2.9%), with 3.5+/-0.3% of apoptotic cells, was achieved after 5 h of treatment. Extended cooling increased the number of apoptotic cells significantly. When MFFCs were treated with different concentrations of roscovitine (ROS) for different periods of time, the highest percentage of G0/G1 cells (83.5+/-1.8%), with 9.2+/-0.6% apoptotic cells, was obtained after exposure to 10 microM ROS for 24 h. When the cells were cooled at 5 C for 5 h followed by incubation in 10 microM ROS for 12 h, 83.6+/-1.9% were synchronized at the G0/G1 stage, with 3.6% undergoing apoptosis. Cell cycle progression was then observed after release of the MFFCs from different synchronization blocks. The highest percentages of S and G2/M cells (81% and 75%) were achieved at 12 and 20 h, respectively, after release of the MFFCs from the cooling plus ROS treatment, and these percentages were significantly higher than those obtained after release from the cooling or ROS alone blocks. Finally, MFFCs were transfected with pEGFP-N1 plasmid at the peak of the G0/G1, S, and G2/M phases, respectively, after release from the different blocks and both the transient and stable transfection efficiencies were determined. The GFP gene expression was greatly enhanced when transfection was performed at the time when most cells were at the G2/M stage after release from cooling, ROS alone, and cooling plus ROS treatments. Statistical analysis revealed a close correlation between the rate of G2/M cells and the transient and stable GFP gene expression efficiencies. Together, the results indicated that (a) the best protocol for cell cycle synchronization of MFFCs was a 5-h cooling at 5 C followed by incubation in 10 microM ROS for 12 h which produced both a high rate of synchronization in the G0/G1 phase with acceptable apoptosis and a high rate of G2/M cells after release; and (b) that the cell cycle status had marked effects on the efficiency of liposome-mediated transfection in MFFCs, with the highest transfection efficiency obtained in cells at the G2/M stage.     

外源褪黑素对鹿茸软骨细胞生长发育的影响

试验旨在探讨褪黑素对体外培养的鹿茸软骨细胞增殖、周期及凋亡的影响。利用甲苯胺蓝、茜素红S、阿利新蓝染色鉴定软骨细胞,采用外源添加褪黑素的方法,用不同浓度（0、400、800、1 200、1 600和2 000 pg/mL）、不同时间（24、48和72 h）处理鹿茸软骨细胞,CCK-8法检测细胞增殖。选取800 pg/mL褪黑素处理软骨细胞24 h,利用流式细胞仪检测细胞周期的分布及细胞凋亡情况,采用ELISA试剂盒检测细胞培养液中睾酮含量。结果显示,经不同浓度的褪黑素处理鹿茸软骨细胞不同时间后,800 pg/mL褪黑素处理鹿茸软骨细胞24 h细胞活力极显著增加（P<0.01）。软骨细胞经褪黑素处理后,与对照组相比,褪黑素处理组细胞G1期比例显著降低（P<0.05）;G2期比例极显著增加（P<0.01）,细胞被阻滞在G2期;褪黑素处理组细胞早期凋亡率无显著变化（P>0.05）,晚期凋亡率显著下降（P<0.05）;ELISA检测睾酮分泌水平均显著上升（P<0.05）。综上,外源添加800 pg/mL褪黑素可以促进鹿茸软骨细胞的增殖,抑制软骨细胞的晚期凋亡,促进睾酮的分泌;以上结果可为阐明褪黑素参与鹿茸生长发育机制提供参考。     

绵羊卵丘细胞细胞周期同步化对核移植胚胎发育的影响

供体细胞周期同步化是影响体细胞核移植成功率的重要因素之一.试验分别对绵羊卵丘细胞采用血清饥饿和接触抑制的方法进行细胞周期同步化处理,使用流式细胞仪检测各组细胞周期的分布.结果发现,与对照组相比,卵丘细胞经血清饥饿24~72 h后,显著地增加了G₀/G₁期细胞的百分比(P <0.05);接触抑制24~72 h,G₀/G₁期细胞所占比例与血清饥饿组无显著差异(P >0.05),但显著高于对照组(P <0.05);用经血清饥饿与接触抑制的供体细胞进行核移植后,重构胚卵裂率、桑椹胚率和囊胚率差异不显著(P >0.05),但二者囊胚率显著高于对照组(P <0.05).上述结果证实,血清饥饿和接触抑制均能使绵羊卵丘细胞周期同步化至G₀/G₁,均可用作绵羊体细胞核移植的供体细胞细胞周期同步化处理.     

In vitro development and postvitrification survival of cloned feline embryos derived from preadipocytes

The aim of the present study was to optimize the conditions for in vitro development and postvitrification survival of somatic cell cloned feline embryos. To determine the effects of cell cycle synchronization of the nuclear donor cells, we cultured preadipocytes under serum starvation or conventional conditions. After two days in serum starvation culture, the proportion of synchronized donor cells at the G0/G1 phase was 91.6%. This was significantly higher than the proportion of non-synchronized cells in the proliferative phase (72.6%, P<0.05). The in vitro development of somatic cell nuclear transfer (SCNT) embryos reconstructed using donor cells treated under serum starvation conditions (normal cleavage rate of 65.7%, 46/70, and blastocyst formation rate of 20.0%, 14/70) was comparable to that of the serum supplemented group (52.5%, 31/59, and 20.3%, 12/59). Use of in vitro or in vivo matured oocytes as recipient cytoplasts equally supported development of the SCNT embryos to the blastocyst stage (11.9%, 5/42, vs. 9.5%, 2/21). SCNT-derived blastocysts were vitrified using the original minimum volume cooling (MVC) or the modified (stepwise) MVC method. Although none (n=10) of the SCNT blastocysts survived following vitrification by the original MVC method, the stepwise MVC method resulted in 100% survival after rewarming (n=11). In conclusion, we demonstrated that feline somatic cell cloned embryos with a high developmental ability can be produced irrespective of cell cycle synchronization of donor cells using either in vivo or in vitro matured oocytes. Furthermore, by utilizing a stepwise vitrification method, we showed that it is possible to cryopreserve cloned feline blastocysts.     

Cell Cycle Synchronization of Skin Fibroblast Cells in Four Species of Family Felidae

This study was examined whether the species of felid affects synchronization accuracy at the G0/G1 stage of the cell cycle and the occurrence of apoptosis by different protocols, such as serum starvation, confluent and roscovitine treatment. Skin fibroblast cells were obtained from the Asian golden cat, marbled cat, leopard and Siamese cat. The cells from each animal were treated with either serum starvation for 1–5 days, cell confluency‐contact inhibition for 5 days or roscovitine at various concentrations (7.5–30 μm ). Flow cytometric analysis revealed that serum starvation for 3 days provided the highest cell population arrested at the G0/G1 stage, irrespective of the felid species. In all species, 100% confluency gave a significantly higher percentage of cells arrested at the G0/G1 stage compared with the non‐treated control cells. The effects of roscovitine treatment and the appropriate concentration on the rates of G0/G1 cells differed among the felid species. Serum starvation for more than 4 days in the marbled cat and Siamese cat and roscovitine treatment with 30 μm in the Asian golden cat and leopard increased the rates of apoptosis. In conclusion, different felid species responded to different methods of cell cycle synchronization. Asian golden cat and Siamese cat fibroblast cells were successfully synchronized to G0/G1 stage using the serum starvation and roscovitine treatment, whereas only confluency‐contact inhibition treatment induced cell synchronization in the leopard. Moreover, these three methods did not successfully induce cell synchronization of the marbled cat. These findings may be valuable for preparing their donor cells for somatic cell nuclear transfer in the future.     

猪肝星状细胞内质网应激模型的建立及对泛素化的影响

试验对衣霉素（tunicamycin,TM）处理猪肝星状细胞的浓度和培养时间进行筛选,以期成功构建内质网应激（endoplasmic reticulum stress,ERS）模型,并在此基础上探究ERS下猪肝星状细胞泛素化的变化。试验采用浓度为0、1、2、5、10和15 μg/mL的TM处理猪肝星状细胞,培养2、4、8、16、24和36 h。通过检测细胞抑制率对TM浓度和培养时间进行初筛;通过检测细胞周期及ERS标志基因和蛋白表达对TM浓度和培养时间进行终选,以确定是否成功构建ERS模型,同时,在ERS下检测猪肝星状细胞泛素化相关基因的表达变化。结果表明：由细胞抑制率检测结果初步确定TM浓度5 μg/mL、培养8或24 h后有可能出现ERS。5 μg/mL TM在培养8或24 h时,ERS标志基因表达均极显著上调（P < 0.01）。在培养8 h时,ERS标志蛋白中仅ATF4显著上调（P < 0.05）,细胞周期中仅G2/M期细胞比例显著下降（P < 0.05）;在培养24 h时,ERS标志蛋白均显著上调（P < 0.05）,细胞周期在G1期出现阻滞,并导致S期和G2/M期细胞比例极显著下降（P < 0.01）。以上结果说明,5 μg/mL TM培养24 h可成功构建细胞ERS模型。在ERS下,细胞泛素化相关基因UBA2和UBE2E的表达均极显著升高（P < 0.01）。综上,猪肝星状细胞经5 μg/mL TM处理24 h,可成功建立ERS模型,并启动泛素化机制。     

Effect of histone acetylation modification with sodium butyrate, a histone deacetylase inhibitor, on cell cycle, apoptosis, ploidy and gene expression in porcine fetal fibroblasts

The present study evaluated the effective dose of sodium butyrate (NaB), a histone deacetylase (HDAC) inhibitor, for determination of the level of enhancement of histone acetylation in porcine fetal fibroblasts (PFFs) based on their morphology, growth, apoptosis and cell cycle status. Cells were analyzed for their histone acetylation levels at H3, H4 and H2A and expression of genes related to histone deacetylation (HDAC1, HDAC2 and HDAC3), pro-apoptosis (Bax and Bak) and anti-apoptosis (Bcl-2). PFFs at passage 3-4 were cultured with 0, 0.5, 1.0, 2.0 and 3.0 mM NaB for 96 h. NaB inhibited cell proliferation at all tested concentrations in a dose-dependent manner. However, there was slow cell growth for PFFs treated with 2.0 and 3.0 mM NaB compared with those of untreated PFFs and those treated with other lower concentrations (0.5 and 1.0 mM). More than 85% of the cells that were untreated or treated with 0.5 or 1.0 mM NaB had intact membranes, whereas, approximately 30% of the cells treated with 2.0 or 3.0 mM NaB had increased cell sizes and a more flattened and elongated appearance. NaB induced apoptosis in a dose-dependent manner; the rates of apoptosis were 2.5 +/- 0.4% for 1.0 mM NaB, 7.6 +/- 1.1% for 2.0 mM NaB and 11.2 +/- 1.4% for 3.0 mM NaB. The chromosomal sets of PFFs treated with 0.5 and 1.0 mM NaB were normal, whereas a lower proportion of PFFs treated with 2.0 and 3.0 mM were classified as normal. NaB at 0.5 and 1.0 mM showed little effect on cell cycle. However, 2.0 and 3.0 mM resulted in an increased cell population at the G(0)/G(1) phase. Increased NaB concentrations led to elevated acetylation of H3, H4 and H2A. NaB altered the expression of histone deacetylation and apoptosis-related genes. In conclusion, 1.0 mM NaB induced histone hyperacetylation in the PFFs and produced less deleterious effects than other concentrations; these PFFs might serve as suitable donors for porcine somatic cell nuclear transfer (SCNT).     

Effects of Confluent, Roscovitine Treatment and Serum Starvation on the Cell-cycle Synchronization of Bovine Foetal Fibroblasts

The present study was designed to examine the effects of cell-cycle synchronization protocols, such as confluent, roscovitine treatment and serum starvation, in bovine foetal fibroblasts on synchronization accuracy at G0/G1, viability, apoptosis, necrosis and ploidy for use as a nuclei donor. The cells in 5-10 passages were randomly allocated into three treated groups. Cells were cultured either in Dulbecco's modified Eagle's medium (DMEM) + 10% foetal bovine serum (FBS) until 90% confluent (group 1, confluent), in DMEM + 10% FBS + 30 microM roscovitine for 12 h (group 2, roscovitine), or in DMEM + 0.5% FBS for 5 days (group 3, serum starvation). Most of the cells (>80%) in all groups were arrested at the G0/G1 stage. Although the rates did not differ, cells in group 1 showed an increased cell population arrested at the G0/G1 phase. Significantly (p < 0.05) higher rates of apoptosis occurred in group 3 than in group 1 and 2 (10% vs 6% and 6%, respectively). No differences in chromosomal abnormality were observed among groups. However, by increasing the number of cell culture passages up to 15, significantly (p < 0.05) higher chromosomal abnormality was observed than in 5 and 10 passages (39% vs 28% and 23%, respectively) in group 1. The results clearly indicated that bovine foetal fibroblasts could be effectively synchronized at G0/G1 stages by all the three different treatments, confluent, roscovitine and serum starvation. However, cells in confluent showed reduced apoptosis and necrosis when they underwent 5-10 passages, exhibiting increased percentage of cells with stable chromosome diversity. Hence, cells in confluent merit further studies before they could be used as nuclear donors.     

Effects of synchronization of donor cell cycle on embryonic development and DNA synthesis in porcine nuclear transfer embryos

The relationship between donor cell cycle and the developmental ability of somatic cell nuclear transfer (SCNT) embryos has not fully been elucidated. Donor cells that are usually prepared by serum starvation or confluent-cell culture for SCNT represent a heterogeneous population that includes mainly G0 phase cells, other cells in different phases of the cell cycle and apoptotic cells. In this study, we compared the developmental ability of porcine SCNT embryos reconstructed from G0 phase cells (G0-SCNT embryos) and strictly synchronized-G1 phase cells (G1-SCNT embryos), and examined the developmental rates and timing of first DNA synthesis. The G0 phase cells were synchronized by confluent culture, and the G1 phase cells were prepared from actively dividing M phase cells. The G1-SCNT embryos showed a significantly higher (P<0.05) developmental rate to the blastocyst stage per cleaved embryo (59%) than the G0-SCNT embryos (43%). Moreover, initiation of first DNA synthesis and cleavage occurred significantly earlier in the G1-SCNT embryos than in the G0-SCNT embryos. Delay of initiation of first DNA synthesis in the SCNT embryos by aphidicolin resulted in decreased developmental rates to the blastocyst stage without any effect on cleavage rates. Our data demonstrates that synchronized-G1 phase cells can be used as donor cells for SCNT embryos and that earlier initiation of first DNA synthesis may be important for subsequent development of SCNT embryos. The SCNT system using G1-synchronized cells, in terms of their highly uniform and viable cell states, can be useful for studying the reprogramming processes and embryonic development of SCNT embryos.     

Cell cycle analysis of bovine cultured somatic cells by flow cytometry

This study was undertaken to examine the cell cycle characteristics of bovine fetal and adult somatic cells (fetal fibroblasts, adult skin and muscle cells, and cumulus cells) after culture under a variety of conditions; 1) growth to 60-70% confluency (cycling), 2) serum starvation, 3) culture to confluency. Cell -cycle phases were determined by flow cytometry with propidium iodide staining enabling the calculation of percentages of cells in G0 /G1, S and G2 /M. The majority was in G0/G1 regardless of cell type and treatment. Serum-starved or confluent cultures contained higher percentages of cells in G0/G1 (89.5-95.4%; P < 0.05). Percentages of cells in G0/G1 increased as cell size decreased regardless of the cell type and treatment. In the serum-starved and confluent cultures, about 98% of small cells were in G0/G1 . Serum-starved cultures contained higher percentages of small cells (38.5-66.9%) than cycling and confluent cultures regardless of cell type (P < 0.05) . After trypsinization of fetal fibroblasts and adult skin cells that were serum-starved and cultured to confluency, the percentages of cells in G0/G1 increased (P < 0.05) on incubation for 1.5 (95.7-99.5%) or 3 hr (95.9-98.6%). These results verify that serum starvation and culture to confluency are efficient means of synchronizing bovine somatic cells in G0/G1, and indicate that a more efficient synchronization of the cells in G0/G1 can be established by incubation for a limited time period after trypsinization of serum-starved or confluent cells.     

亮氨酸对猪胎盘滋养层细胞增殖及氨基酸转运的影响

为研究亮氨酸(Leu)对猪胎盘滋养层细胞(pTr)增殖、凋亡以及氨基酸转运载体表达的影响及其机制,本试验用不同浓度Leu(0、1、10 mmol/L)分别处理pTr细胞24 h和48 h后,使用荧光定量PCR技术检测pTr细胞增殖和凋亡相关基因、氨基酸转运载体以及mTOR信号通路关键蛋白等的mRNA表达水平。结果表明:Leu处理pTr细胞24 h后,1 mmol/L试验组的SNAT1(P<0.01)、4E-BP1 (P<0.05)和eIF4G(P<0.05)的mRNA相对表达量低于对照组;Leu处理pTr细胞48 h后,1 mmol/L试验组LAT1(P<0.05)、4E-BP1(P<0.01)的mRNA相对表达量低于对照组,10 mmol/L试验组CDK4(P<0.05)、4E-BP1 (P <0.01)、SNAT1 (P <0.01)、SNAT2 (P <0.01)、LAT1 (P <0.01)以及rBAT (P <0.05)的mRNA相对表达量也低于对照组;Leu处理pTr细胞24 h和48 h后,10 mmol/L组mTORC1的mRNA相对表达量较对照组和1 mmol/L组均极显著提高(P<0.01)。可见,10 mmol/L Leu会抑制pTr细胞的增殖活力,并可能通过mTOR信号通路的介导,降低了pTr细胞氨基酸转运载体的表达。     

Effects of Serum Deprivation and Cycloheximide on Cell Cycle of Low and High Passage Porcine Fetal Fibroblasts

Arrest of cells in G0/G1 cell cycle phase is desired for nuclear transfer procedures. Serum starvation and cell cycle inhibitors are different ways to induce synchronization of the cell cycle. This study investigated the effects of serum starvation and cycloheximide (CHX) on the cell cycle of low (5th) and high (15th) passages fetal porcine fibroblasts. Cell cycle phases were determined using fluorescent activated cell sorting. Fifth passage fibroblast cultures had higher (p < 0.05) proportion of cells in G0/G1 only after 72 h of serum starvation (77.60 ± 0.65) when compared with non‐starved cells (71.44 ± 1.88). Serum starvation for all periods tested induced an increase (p < 0.05) on proportion of cells in G0/G1 on the 15th passage. No significant differences were observed on the 5th passage cultures exposed to CHX, although, on the 15th passage an increase on proportion of cells was observed after all periods of exposure (p < 0.05). These data indicates that high passage cells in vitro are more susceptible to serum starvation and CHX G0/G1 synchronization.     

小分子药物索非布韦对牛病毒性腹泻病毒体外复制的影响

试验旨在研究小分子药物索非布韦是否具有抑制牛病毒性腹泻病毒（Bovine viral diarrhea virus,BVDV）复制的作用。通过MTT法测定了不同时间和不同浓度索非布韦对牛肾细胞（MDBK）增殖的影响;采用免疫荧光染色试验筛选出能有效抑制BVDV复制的索非布韦最适浓度,用实时荧光定量PCR、致细胞病变作用（CPE）和病毒半数组织细胞感染量（TCID₅₀）测定的方法检测索非布韦对BVDV复制的影响。结果显示,索非布韦处理MDBK细胞24和48 h能极显著抑制细胞增殖（P<0.01）;与对照组相比,当索非布韦浓度为800 μmol/L时免疫荧光染色检测BVDV感染MDBK细胞的双链RNA（dsRNA）含量极显著降低（P<0.01）;实时荧光定量PCR检测发现,与DMSO处理组相比,BVDV感染索非布韦处理组MDBK细胞,BVDV 5'UTR mRNA含量在病毒感染24和48 h时极显著降低（P<0.01）;BVDV感染索非布韦处理组MDBK细胞病变现象明显减弱;索非布韦处理24 h后能极显著减弱BVDV感染MDBK细胞后子代病毒颗粒的形成组与释放（P<0.01）,降低病毒滴度。综合上述结果表明,小分子药物索非布韦能有效抑制BVDV体外复制。     

高铜对肉鸡原代肝细胞周期及细胞膜电位的影响

通过流式细胞仪测定各组的细胞周期及细胞膜电位,研究高铜对肉鸡原代肝细胞凋亡的影响。将全量换液24h后的细胞分为空白对照组和低铜组（10μmol/L Cu2＋组）、高铜Ⅰ组（50μmol/L Cu2＋组）、高铜Ⅱ组（100μmol/L Cu2＋组）,培养48h后测定荧光值,结果表明,高铜组能显著引起静止期（G0/G1）肝细胞数增多、增殖期（S、G2＋M）肝细胞数减少、细胞膜电位下降（P〈0.05）;低铜组未出现明显变化;表明高浓度的铜能明显改变肝细胞的细胞周期及细胞膜电位。     

Antisense oligonucleotide complementary to the BamHI-H gene family of Marek's disease virus induced growth arrest of MDCC-MSB1 cells in the S-phase.

DNA synthesis was effectively inhibited by antisense oligonucleotide A1 complementary to the BamHI-H gene family in Marek's disease virus (MDV)-derived lymphoblastoid MDCC-MSB1 cells. When a cell cycle distribution of a total cell population was analyzed by flow cytometry, the proportion of S-phase cells increased in the cell populations by treatment with oligonucleotide A1. Approximately 60-70% of the cells appeared in the S phase for 24 and 36 hr of incubation in the presence of oligonucleotide A1 (20-30% in the untreated control cells). The inhibition of cell cycle progression by treatment with oligonucleotide A1 was reversible. When the cells were treated with 5 microM aphidicolin for 12 hr, a similar pattern of cell cycle distribution was observed to that obtained after treatment with oligonucleotide A1. Aphidicolin is an inhibitor of cellular DNA polymerase alpha, and it halts progression of the cell cycle at the G1/S border or early S phase. When the cells were treated with aphidicolin for 12 hr and subsequently incubated with oligonucleotide A1, no significant difference was observed in the cycle phase distribution of cells in the presence and absence of oligonucleotide A1. In contrast, when the cells were treated with oligonucleotide A1 for 12 hr and subsequently incubated with aphidicolin, the cell cycle did not progress from the G1/S border or early S phase to the next phase.     

不同冻存液对奶牛乳腺上皮细胞冻存质量的影响

试验旨在探究奶牛乳腺上皮细胞（bovine mammary epithelial cells,BMECs）最佳的冻存液以改善乳腺上皮细胞的冻存质量。BMECs传至第5代后分别加入以下10种不同配方的冻存液。A组：85%DMEM＋10%胎牛血清＋5%DMSO;B组：80%DMEM＋10%胎牛血清＋10%DMSO;C组：75%DMEM＋10%胎牛血清＋15%DMSO;D组：70%DMEM＋10%胎牛血清＋20%DMSO;E组：85%DMEM＋5%胎牛血清＋10%DMSO;F组：75%DMEM＋15%胎牛血清＋10%DMSO;G组：70%DMEM＋20%胎牛血清＋10%DMSO;H组：80%DMEM＋10%胎牛血清＋10%甘油;I组：70%DMEM＋10%胎牛血清＋20%甘油;J组：60%DMEM＋10%胎牛血清＋30%甘油,冻存前统一调整细胞密度到1×106个/mL冻存。分别对复苏后的细胞进行台盼蓝染色计算存活率和PI/Hoechst33258双染计算凋亡率。结果表明,BMECs经不同冻存剂冻存复苏后,细胞活力、形态学及凋亡率表现有所不同,其中B组和G组的活力和24h贴壁率较其他组高,二者的凋亡率较低,二者之间差异无显著性（P〉0.05）;传代后B组细胞的生长状况最好。