中华鼢鼠成纤维细胞长期培养及其生物学特性分析

中华鼢鼠(Eospalax fontanierii)为蒙古高原代表性动物遗传资源,本研究采集中华鼢鼠心、肝、脾、肺、肾、肌肉、气管、剑状软骨和尾尖皮肤共9种组织用于实验,其中气管、肺和剑状软骨3种组织成功建立成纤维细胞系,传了8代,并分析了这些细胞的生物学特性。主要实验方法是通过细胞计数法计算细胞的贴壁率、冻存前及复苏后的存活率、绘制细胞生长曲线;制备常规染色体标本分析染色体核型。实验结果显示,中华鼢鼠气管、肺、软骨3种组织在体外条件下培养,第3～4天出现成纤维样细胞,原代细胞分别在培养第11天、第16天、第17天,达到90%以上汇合。3种组织来源体细胞均显示成纤维细胞特征,气管成纤维细胞贴壁能力最强,24 h贴壁率最高能达到98.10%,肺成纤维细胞与剑状软骨成纤维细胞贴壁能力较弱,24 h贴壁率最高,分别为95.28%和94.88%。3种来源成纤维细胞生长曲线测定结果显示,气管成纤维细胞的增殖能力最强、肺成纤维细胞次之、剑状软骨成纤维细胞最弱。气管成纤维细胞和肺成纤维细胞在接种后的第6～7天,进入对数生长期。剑状软骨成纤维细胞在接种后第2～3天进入对数生长期。3种成纤维细胞的生长曲线均呈"S"型,其中气管成纤维细胞增殖能力最强,在24孔板的每个孔中,其最大增殖数目为2.435×10~4个,肺成纤维细胞最大增殖数目为1.813×10~4个,剑状软骨成纤维细胞最大增殖数目为1.521×10~4个。核型分析结果显示,中华鼢鼠的成纤维细胞染色体数目为2n=62,30对为常染色体,1对为性染色体。综上所述,本研究成功建立了中华鼢鼠成纤维细胞体外培养体系,并揭示了该物种成纤维细胞的基本生物学特性,为深入研究中华鼢鼠适应低氧高二氧化碳洞道生境的分子生物学和生理学机制提供了条件,为进一步研究其遗传及物种进化提供了实验材料和参考。     

水牛皮肤成纤维细胞的分离与体外培养

探讨水牛成纤维细胞的分离与传代培养方法。组织块培养法培养的成纤维细胞原代生长较慢,需12天左右方可汇合形成单层,而酶消化法培养的成纤维细胞原代生长相对生长快,仅需8天便可汇合形成单层。两种方法传代细胞的生长速度相似,仅需4-5天就可汇合形成单层。通过体细胞的核型分析发现,成纤维细胞在传代培养过程中的核型变化不大,66．67％～81．67％的细胞具有正常的二倍体核型,各代之间无显著差异。结果表明,水牛成纤维细胞均能稳定地进行传代培养。     

西伯利亚狍成纤维细胞建系及其生物学特性分析北大核心CSCD

本研究以内蒙古大青山获得野生雄性和雌性西伯利亚狍(Capreolus pygargus)为实验材料,利用组织块贴壁培养法进行气管、肺和耳3种组织成纤维细胞原代建系,研究不同组织来源的细胞贴壁率、冷冻前及复苏后存活率、生长曲线,进一步绘制狍成纤维细胞核型图并分析其G带特征。实验结果显示,气管、肺和耳3种组织成纤维细胞增殖经历潜伏期、对数生长期、平台期三个阶段,细胞形态为梭形、三角形或不规则形,是典型成纤维细胞形态;成纤维细胞呈漩涡状生长,其中气管、耳成纤维细胞生长增殖能力最强、肺成纤维细胞增殖能力较弱,气管和耳组织来源成纤维细胞呈典型“S”型细胞生长特征。染色体核型及G带分析结果显示,雄性狍成纤维细胞染色体条数为2n=70,其中,有34对常染色体,形态类型为12条近端着丝粒染色体(st),22条亚中着丝粒染色体(sm),1对性染色体,X染色体为中着丝粒染色体(m),Y染色体为近端着丝粒染色体(st),5条超数染色体(B);雌性狍成纤维细胞染色体条数为2n=70,其中,有34对常染色体,其形态类型为29条亚中着丝粒染色体(sm),5条近端着丝粒染色体(st),1对为性染色体,X染色体为亚中着丝粒染色体(sm),8条超数染色体(B)。本研究成功建立了雄性和雌性西伯利亚狍气管、肺和耳3种组织来源的成纤维细胞系,在体外培养时生长状态良好且维持了细胞的遗传信息稳定性,绘制了西伯利亚狍雄性和雌性染色体核型及G带图谱,为将来更深入开展相关研究提供材料与基本技术支撑。     

乌珠穆沁白马成纤维细胞建系及其生物学特性分析

本研究以内蒙古乌珠穆沁白马(Equus caballus)雄性和雌性为实验材料,利用组织块贴壁培养法进行耳组织成纤维细胞原代建系,研究耳组织来源的细胞贴壁率、冷冻前及复苏后存活率、生长曲线,进一步绘制乌珠穆沁白马成纤维细胞核型图。实验结果显示,雌、雄乌珠穆沁白马耳组织成纤维细胞经组织贴壁法建系培养,生长为典型的成纤维细胞形态,并呈现“S”型生长曲线特征;细胞冷冻前后存活率存在显著差异,但仍具有较好的耐受冷冻性;根据染色体核型分析,雌、雄乌珠穆沁白马耳组织成纤维细胞染色体条数为2n = 64,其中31对常染色体,1对性染色体。本研究成功建立了雌、雄乌珠穆沁白马耳组织成纤维细胞系,并且得到可稳定培养、具有良好遗传学特性的细胞系,为后续的相关深入研究奠定了基础。     

藏羚羊成纤维细胞系的建立及其核型分析

藏羚羊(Pantholops hodgsonii)是我国特有的国家Ⅰ级重点保护野生动物,生存在高海拔低氧环境下,具有高原生物的细胞遗传特性。实验采用组织块贴壁培养法进行原代培养,后经差异贴壁法联合消化排除法纯化,成功构建了藏羚羊成纤维细胞系,并对培养不同代次细胞的形态、生长动力学、染色体核型等进行了研究。结果表明,培养得到的藏羚羊细胞为典型的成纤维细胞,第7、10、15代细胞群体倍增时间分别为26、48、50 h。细胞的生长均为"S"型。染色体中二倍体染色体(2n=60)占主体,所占比例为65%～91%,包括性染色体X、Y在内均为端着丝粒类型,染色体组的总相对长度为193.45,平均相对长度为3.22,未发现随体、次缢痕等特殊标志性特征。     

梅花鹿体细胞航天诱变后的生物学特性变化分析

本研究以自主研发的聚偏二氟乙烯膜(PVDF)细胞培养技术进行了梅花鹿(Cervus nippon)体细胞的航天培养及生物诱变,并分析了其细胞在航天诱变后的生长曲线和核型特性。采用耳缘皮肤组织块贴壁培养的方法进行体细胞的原代培养;胰蛋白酶消化的方法完成细胞的传代培养;依照程序降温的方法完成细胞的冷冻保存;每天计数24孔板每孔的活细胞数并利用Excel 97-2003绘制出细胞的生长曲线,采用IBM SPSS Statistics 23统计软件回归分析程序中的非线性回归分析子程序Logistic曲线模型分析细胞生长曲线的拟合度;以常规染色体标本制备技术,利用细胞遗传工作站(AI)软件对梅花鹿体细胞的染色体核型进行分析。实验结果表明,新开发的PVDF膜细胞培养技术可有效保持梅花鹿体细胞在太空飞行过程中的存活并成功回收、传代建系。通过细胞传代培养观察发现,该体细胞经航天诱变后保持正常的成纤维细胞特征,生长曲线呈"S"型,航天诱变组体细胞在培养4 d进入对数生长期,与对照组体细胞在培养2 d进入对数生长期相比其细胞增殖速度减慢。拟合生长曲线分析表明,航天诱变组细胞生长拟合度值与对照组相似,但细胞增殖拐点时间、拐点细胞量分析结果证明,航天诱变后的体细胞增殖速度减慢。核型分析显示,航天诱变梅花鹿体细胞染色体数保持2n=66,染色体形态正常,核型为66(XX),其中32对常染色体,1对性染色体。本研究建立了哺乳动物在航天运行条件下的细胞培养PVDF新技术,并分析了航天诱变对于梅花鹿细胞生物学特性变化的影响,为以后进行相关研究提供了基础信息和技术支持。     

鸡胚三类组织体外培养细胞的生物学特性分析

采用组织培养的方法获取鸡胚不同组织细胞,利用M199培养基进行原代、传代培养,经形态学观察、生长曲线绘制、分裂指数测定等进行生物学特性分析。实验表明,鸡胚不同组织细胞具有不同的生物学特性,从形态结构到生长周期都有明显差异。获得的躯体来源细胞、心来源细胞为成纤维型,肺来源细胞为上皮型;其中,躯体来源细胞生长能力最强,心来源细胞次之,肺来源细胞最慢,躯体来源细胞倍增时间最短;核型分析表明,该实验体外培养的鸡胚细胞染色体数目为78条。上述结果可为今后鸡胚不同组织细胞的深入研究提供实验材料和依据。     

毛脚鵟细胞体外培养体系建立及三种组织来源细胞特性分析

用组织块培养法对毛脚鵟不同组织进行原代培养,获得了3种不同组织来源的细胞,并成功对细胞进行了冷冻保存和复苏。在传代培养过程中,对比分析了3种组织来源细胞的形态学、生长曲线、贴壁率、核型等生物学特性。形态学方面,3种来源细胞均为成纤维样细胞。对于3种组织来源细胞的贴壁能力分析显示,输卵管源细胞最强,肺源细胞和气管源细胞次之。3种不同组织来源细胞的倍增时间分别为(29.91±0.39)、(33.18±0.21)和(30.67±0.28)h,群体倍增次数分别为3.54±0.01、4.52±0.02和4.38±0.03。毛脚鵟细胞的染色体数目为2n=68,性染色体为典型的ZW型。本实验为今后毛脚鵟细胞利用、遗传信息的保存及生物学特性的深入研究提供实验材料和依据。     

家蚕胚胎细胞系BmE-SWU2的建立及其生物学特性研究

家蚕反转期胚胎组织经过一年多的原代培养,建立了BmE-SWU2细胞系。该细胞系细胞呈短梭形或圆形,细胞较小,属上皮型贴壁生长细胞系;细胞生长速度快,铺平率高于80%,繁殖力强,细胞群体倍增时间为51.8 h。BmE-SWU2细胞系的二倍体细胞（2n=56）比例达到89.76％,属二倍体细胞系,其染色体具有鳞翅目昆虫染色体的典型特征,中期染色体呈短杆状或颗粒状。BmE-SWU2细胞系对BmNPV高度敏感,TCID₅₀为1.581×10^-7。     

间充质干细胞与成纤维细胞生物学特性的比较及研究进展

间充质干细胞(mesenchymal stem cells,MSCs)具有多向分化潜能和免疫调节能力,且不具伦理争议的特点使其数十年来在基础和临床研究中得到广泛应用.然而,MSCs在形态上并不能和成纤维细胞有所区分.该文对这两种细胞在生物学特性及临床应用方面的异同点和两者可能存在的关系进行了综述.结果表明:这两种细胞在...     

Methemoglobin reductase in three species of Bovidae

Isoelectric focusing of red cell hemolysates revealed several isozymes that stain for NADH-methemoglobin reductase. Evidence for two different genetic loci controlling the banding patterns was obtained. One locus controlled a single band present in all animals tested. The second locus controlled ten different banding patterns that could be accounted for by four codominant alleles. Band B occurred in Bison bison. Bands A and C occurred in Bos indicus and band D occurred in both Bos indicus and Bos taurus. Bands A, C, and D were not observed in Bison bison and bands A, B, and C were not observed in Bos taurus.     

星形胶质细胞的体外生长特性

目的研究纯化培养的星形胶质细胞的体外生长特性,作为进一步研究星形胶质细胞功能的依据。方法将纯化的星形胶质细胞分为6h、12h、18h、24h、30h、36h、42h、48h、54h、60h、72h、84h、96h、120h14个时间组,培养不同的时间后,采用细胞计数法绘制细胞生长曲线观察细胞的生长状况,用流式细胞学检测细胞周期各时相的变化并绘制增殖指数曲线,所得数据用SPSS软件进行统计学分析。结果①星形胶质细胞的生长曲线可明显的分为三个阶段:第一阶段为0至24h,在此时间内生长曲线平缓上升,其斜率为0.64。第二阶段为24h至84h,在此时间内生长曲线急剧上升,其斜率为2.69;此阶段中24h至48h时曲线最陡,斜率为3.94,细胞呈指数生长。第三阶段为84h至120h,在此时间内生长曲线走势平直,其斜率为-0.005。②星形胶质细胞的增殖指数在第一个细胞增殖周期内可以反映细胞的增殖状态,第一个细胞周期结束后,细胞的数量继续上升增殖指数却降低了。结论培养的星形胶质细胞的生长过程分为潜伏期、指数生长期和停滞期三个阶段,其潜伏期为0至24h,指数生长期为24h至48h,停滞期为84h以后。单纯用增殖指数等类似的指标作为细胞增殖状态的判断标准是有一定局限性的,一定要将这些指标与细胞数量结合起来分析。     

流式细胞仪在生物学中的应用

简要论述了流式细胞仪（flow cytometry,FCM）的工作原理,并对其在生物学基础科学研究中的应用进行阐述,包括对细胞凋亡、细胞周期、免疫细胞、细胞受体的研究应用。     

萼花臂尾轮虫(Brachionus calyciflorus)种复合体内三个姐妹种生活史特征比较

采自芜湖市莲塘湖和荷花塘水体中的萼花臂尾轮虫3个姐妹种HE1、HE3和LE9在13℃、18℃、23℃和28℃下的生命表参数间的差异表现为13℃和18℃下姐妹种LE9的生命期望和平均寿命都显著长于姐妹种HE3,23℃下姐妹种HE1的生命期望和平均寿命均最短;13℃下姐妹种LE9的世代时间显著长于姐妹种HE3,18℃下和23℃姐妹种LE9和HE1的世代时间分别最长和最短;13℃下姐妹种LE9的净生殖率最高,28℃下姐妹种HE1的净生殖率最低;13℃下姐妹种LE9的种群内禀增长率最高,18℃下姐妹种HE3的种群内禀增长率显著高于姐妹种LE9;18℃和28℃下姐妹种HE3所产后代中的混交雌体百分率最高,23℃下姐妹种HE1所产后代中的混交雌体百分率显著低于HE3.3个姐妹种的生命表参数对升高的温度的响应也不相同.温度、姐妹种及两者间的交互作用对轮虫的世代时间、平均寿命、出生时的生命期望和轮虫所产后代中的混交雌体百分率均有显著的影响;温度和姐妹种对轮虫的净生殖率和种群内禀增长率均有极显著影响,但两者的交互作用对其无显著性影响.     

萼花臂尾轮虫种复合体内三个姐妹种生活史特征的比较研究

应用生命表统计学方法,在13℃、18℃、23℃和28℃下对采自芜湖市莲塘湖和荷花塘水体中的萼花臂尾轮虫三个姐妹种HE1、HE3和LE9的生活史特征进行了比较研究。结果表明,三个姐妹种生命表参数间的差异因温度的不同而异。13℃下,姐妹种LE9的生命期望、平均寿命和世代时间都显著长于姐妹种HE3,LE9的净生殖率和种群内禀增长率均最大,三个姐妹种所产后代中的混交雌体百分率无显著的差异。18℃下,姐妹种LE9的生命期望和平均寿命显著长于姐妹种HE3,姐妹种LE9的世代时间显著长于姐妹种HE1和HE3,姐妹种 HE3的种群内禀增长率显著高于姐妹种LE9,姐妹种HE3所产后代中的混交雌体百分率最高,三个姐妹种的净生殖率无显著的差异。23℃下,姐妹种HE1的生命期望、平均寿命和世代时间均最长,姐妹种HE1所产后代中的混交雌体百分率显著低于HE3,三个姐妹种的净生殖率和种群内禀增长率均无显著的差异。28℃下,三个姐妹种的生命期望、平均寿命、世代时间和种群内禀增长率均无显著的差异;姐妹种HE1的净生殖率最低;姐妹种HE3所产后代中的混交雌体百分率最高。三个姐妹种的生命表参数对升高的温度的响应也不相同。温度、姐妹种及两者间的交互作用对轮虫的世代时间、平均寿命、出生时的生命期望和轮虫所产后代中的混交雌体百分率均有显著的影响;温度和姐妹种对轮虫的净生殖率和种群内禀增长率均有极显著影响,但两者的交互作用对其无显著性影响。     

Comparative demography of three coexisting Acer species in gaps and under closed canopy

Questions: 1. Is there a trade‐off between gap dependency and shade tolerance in each of the life‐history stages of three closely related, coexisting species, Acer amoenum (Aa), A. mono (Am) and A. rufinerve (Ar)? 2. If not, what differences in life‐history traits contribute to the coexistence of these non‐pioneer species? Location: Ogawa Forest Reserve, a remnant (98 ha), species‐rich, temperate deciduous forest in central Japan (36°56’ N, 140°35’ E, 600 ‐ 660 m a.s.l.). Methods: We estimated the demographic parameters (survival, growth rate and fecundity) by stage of each species growing in gaps and under closed canopy through observations of a 6‐ha permanent plot over 12 years. Population dynamics were analysed with stage‐based matrix models including gap dynamics. Results : All of the species showed high seedling and sapling survival rates under closed canopies. However, demographic parameters for each growth stage in gaps and under closed canopies revealed inter‐specific differences and ontogenetic shifts. The trade‐off between survival in the shade and growth in gaps was detected only at the small sapling stage (height < 30 cm), and Ar had the highest growth rate both in the shade and in the gaps at most life stages. Conclusions: Inter‐specific differences and ontogenetic shifts in light requirements with life‐form differences may contribute to the coexistence of the Acer species in old‐growth forests, with Aa considered a long‐lived sub‐canopy tree, Am a long‐lived canopy tree, and Ar a short‐lived,‘gap‐phase’ sub‐canopy tree.     

Comparative phylogeography of three common Neotropical tree species

Aim To determine spatial and temporal commonalities in patterns of chloroplast DNA (cpDNA) variation in three widespread Neotropical tree species. We examine whether patterns of genetic variation are more consistent with Pliocene or Pleistocene divergence. Location Central American forests, located in El Salvador, Nicaragua, Costa Rica and Panama. Methods We collected sequences from two cpDNA loci from c. 30 locations for each of three species –Bursera simaruba (Burseraceae; n = 278), Brosimum alicastrum (Moraceae; n = 210) and Ficus insipida (Moraceae; n = 222) – and additionally sequenced one nuclear locus for Bursera simaruba (n = 45). We used Monmonier’s algorithm to detect genetic barriers between regions. Divergence times between these regions were estimated using coalescent analyses. Results Spatial genetic boundaries were found in similar areas for these species, namely between Costa Rica and Nicaragua for all three species, and between El Salvador and Nicaragua for two species. These boundaries visually coincide with the spatial delimitations of Pliocene islands and previously hypothesized Pleistocene refugia. Divergence time estimates between regions are more consistent with Pleistocene divergence in two of the three species. Main conclusions Our results point to strong commonalities in the spatial locations of genetic boundaries in these three species, despite the complex geological and climatological history of this region, and ecological differences between the species. While spatial genetic boundaries coincide conspicuously with possible Pliocene and Pleistocene barriers to gene flow, we cannot distinguish between the two scenarios because of the strong spatial overlap of both barriers. However, the temporal data tentatively suggest that some of this divergence occurred in the Pleistocene, although limitations in the analysis cannot confirm Pleistocene divergence without external, corroborating data. While we cannot definitively implicate a single historical process as driving patterns of genetic differentiation in all three species, our results represent an initial step towards identifying a common history of Central American tree species.     

Comparative morphology of the sagittal otolith in three species of south Caspian gobies

Sagittal otolith shapes were investigated in order to identify three sympatric species of south Caspian gobies (Caspian goby Neogobius caspius, deepwater goby Ponticola bathybius and bighead goby Ponticola gorlap). The sagittal otoliths in P. bathybius have a rectangular shape and are thick, whereas in N. caspius they are relatively round and thin. In P. gorlap, otoliths have an elongated shape and are relatively thick. The noticeable difference among the otoliths of the three species is the presence of one anterior and one posterior projection in the otoliths of N. caspius and P. gorlap. Among shape indices, form factor (irregularity of surface area), circularity, aspect ratio and rectangularity are the foremost that indicate interspecific variability. The canonical discriminant analysis correctly classifies 94·7% of the original group cases. The overall analyses show the relevance of applying otolith shape for interspecific distinction of the three species of Caspian gobies.     

Characterization of apoptosis-like endonuclease activity in avian thymocytes.

In the current study the internucleosomal DNA cleavage activity associated with apoptosis was investigated in avian thymocytes. Thymocyte nuclear proteins from glucocorticoid-treated chickens were incubated with chicken red blood cell (cRBC) nuclei, and DNA degradation was analyzed by agarose gel electrophoresis and fluorescence-activated flow cytometry. The thymocyte nuclear extract contained an endonuclease activity that degraded cRBC chromatin at internucleosomal sites as detected by agarose gel electrophoresis. Flow cytometry analysis of cRBC nuclei that were treated with thymocyte nuclear proteins demonstrated a loss of cellular DNA as a function of the amount of added nuclease activity. Furthermore, it was demonstrated that the thymocyte nuclear extract contained a nuclease activity that was capable of degrading radiolabelled naked 32P-DNA into acid soluble DNA fragments. All three assay methods demonstrate that the thymocyte nuclease activity can be inhibited by EDTA, zinc ions and the nuclease inhibitor aurintricarboxylic acid. Based on the analysis of cofactor requirement of this nuclease activity and its susceptibility to inhibitors, the endonuclease activity present in avian apoptotic thymocytes appears to be identical to the mammalian counterpart.