云南半细毛羊毛囊干细胞生物学特性分析

为研究云南半细毛羊毛囊干细胞（hair follicle stem cells,HFSCs）的生物学特性,采用组织块法分离培养云南半细毛羊HFSCs,并对其细胞形态、生长动力学、冷冻与复苏、染色体核型等生物学特性进行分析.结果表明：HFSCs为贴壁生长,体积小,核质比高,呈典型的铺路石状,在显微镜下折光性强、胞体透亮.细胞生长曲线为“S”型.冻存前细胞活率98.2％,复苏后细胞活率96.4％.染色体中正常二倍体数目2n=54,其中,长染色体中有3对为中着丝点染色体,23对为端着丝点染色体,X染色体为最大的近端着丝点染色体,Y染色体为最小的亚中着丝点染色体.所得细胞呈现典型的HFSCs特征,细胞活性好,细胞系为稳定的二倍体细胞系.     

不同年龄二狼山绒山羊耳组织成纤维细胞的生物学特性

采用胶原酶消化法建立细胞系,观察细胞形态、测定细胞冻存前和复苏后的存活率、绘制细胞生长曲线,并对染色体核型进行分析,研究不同年龄的二狼山绒山羊耳组织成纤维细胞的生物学特性,分析年龄对二狼山绒山羊耳组织成纤维细胞体外培养的影响。试验结果表明,年龄对细胞形态无影响;冻存没有降低细胞的存活率(P0.05);细胞的生长曲线呈典型的S型且年龄对细胞的生长无明显影响(P0.05);染色体核型分析显示,不同年龄的山羊耳组织成纤维细胞系的染色体数目均为2 n=60(XY/XY),无染色体畸形。说明不同年龄,甚至年龄比较大的二狼山绒山羊个体(13岁)都可以通过胶原酶消化法建成稳定的成纤维细胞系,本试验使二狼山绒山羊种质资源在细胞水平得到保存,为二狼山绒山羊成纤维细胞系的建立以及开展其种质资源的保存提供一定的参考。     

云南半细毛羊成纤维细胞系建立及其生物学特性分析

实验采用组织块贴壁培养法首次对云南半细毛羊耳缘组织进行体外培养,通过原代培养、传代培养、冷冻保存建立了云南半细毛羊耳缘成纤维细胞系,并对其细胞形态、生长动力学、染色体核型等生物学特性进行分析。结果表明:第7、12、17代细胞群体倍增时间分别为26、42和47 h。细胞生长曲线均为"S"型。云南半细毛羊染色体中正常二倍体染色体(2n=54)所占比例分别为93%、86%和81%,其中,常染色体中有3对为中着丝点染色体,23对为端着丝点染色体,X染色体为最大的近端着丝点染色体,Y染色体为最小的亚中着丝点染色体。染色体组总相对长度197.86,平均相对长度3.66。     

中华鳖心组织成纤维细胞系的建立及特性

以中华鳖心组织为材料,用含10％胎牛血清的TC199培养基,于25℃条件下,经14个月传代培养60余次,获得一个能稳定生长、以成纤维样细胞为主的细胞系,称之为TSH（Trionyx sinesis heart)细胞系。分别对其原代和传代细胞的染色体数目、不同温度下细胞的生长线及原代和传代细胞的显微形态进行了测定和比较。结果表明,细胞在25℃条件下生长速度最快;原代细胞约有64％的染色体为2n=66,而传代细胞为四倍体的有62％。初步估计的细胞周期为2－3d。传代细胞冻存后,复苏状态良好,能继续增殖传代。     

郏县红牛耳成纤维细胞的建立

以郏县红牛耳缘组织为试验材料,采用组织块贴壁法培养,成功建立郏县红牛成纤维细胞系,对其进行细胞形态、细胞活力、生长曲线、染色体核型分析及微生物检测等生物学特性研究。结果显示:细胞形态为典型的成纤维细胞,生长曲线呈S型,细胞倍增时间为39h,冻存前和细胞复苏后活力为98%以上;细胞染色体2n=60,细胞株1-P9、1-P14及2-P9二倍型比例为89.86%、73.82%、78.02%;细菌、真菌、病毒及支原体检查均为阴性。结果表明该细胞系的建立实现了郏县红牛遗传资源在细胞水平上的成功保存。     

SF9细胞的建立及生物学特性的鉴定

自菌种保藏中心引进1株SF9细胞系,扩大培养,保存于液氮,建立SF9细胞基础细胞库。对细胞进行形态、生长曲线、纯净性、染色体分析及致瘤性等特性进行研究。结果表明,细胞呈圆球形;细胞呈S型生长曲线;无细菌、支原体及外源病毒污染;细胞的染色体数目主要分布在170～190之间;无致瘤性。     

云岭黑山羊成纤维细胞系建立及其生物学特性分析

本实验采用组织块贴壁培养法首次对云岭黑山羊耳缘组织进行体外培养,通过原代培养、传代培养、冷冻保存建立了云岭黑山羊耳缘成纤维细胞系,并对其细胞形态、生长动力学、染色体核型等生物学特性进行分析。结果表明：第4、8、12代细胞群体倍增时间分别为26 h、34 h和46 h,细胞生长曲线均为＂S＂型。云岭黑山羊染色体中正常二倍体染色体（2n=60）所占比例分别为97%、95%和90%,其中,29对常染色体均为端着丝点染色体,X染色体为最大的近端着丝点染色体,Y染色体为最小的中着丝点染色体。染色体组总相对长度为197.87,平均相对长度为3.30。细菌、真菌、病毒和支原体等微生物检查显示为阴性。     

四种动物传代细胞染色体遗传变异率分析

本文的目的分析制苗用传代细胞染色体的变异稳定性,以判定其质量,方法采用直接法制取传代细胞染色体标本,用常规Ｇｉｅｍｓａ染色,１０００倍光学显微镜下计数中期分裂相染色体数目,求出染色体变异率（％）,并进行核型分析。结果Ｆ８１为亚二倍体细胞系,染色体众数为３０;Ｖｅｒｏ是超二倍体细胞系,染色体众数为７３－７５;ＭＤＣＫ属于亚四倍体细胞系,染色体众数为１２５－１３５。ＢＨＫ２１为亚二倍体或亚四倍体细胞系     

13/17罗伯逊易位猪成纤维细胞系的建立

为了在细胞水平上研究13/17罗伯逊易位猪群这一种质资源,试验采用组织块分离法对13/17罗伯逊易位杂合子猪、纯合子猪及正常核型家猪耳组织进行了分离培养,构建了3个成纤维细胞系,并对其进行了形态学、生长动力学、核型分析、绿色荧光蛋白报告质粒转染和微生物污染检测等生物学特性研究。结果表明:3种核型家猪细胞形态均为标准成纤维细胞形态,细胞内波形蛋白呈现阳性表达;细胞生长曲线均呈"S"型;杂合子猪细胞染色体2n=37所占比例为92.5%,纯合子猪细胞染色体2n=36所占比例为93.8%,正常核型家猪细胞染色体2n=38所占比例为96.3%;外源质粒能在细胞中进行稳定表达;细菌、真菌、病毒、支原体检测结果呈阴性。说明本研究已成功建立13/17罗伯逊易位猪成纤维细胞系。     

低血清培养的BHK21细胞染色体遗传稳定性分析

目的:分析1种商业化的低血清培养基适应BHK21细胞进行传代培养,该细胞染色体的变异稳定性,以判定其质量。方法:采用直接法制取4个代次的BHK21细胞的染色体标本,用常规Giemsa染色,1 000倍光学显微镜下计数中期分裂相染色体数目,求出染色体变异率(%),并进行核型分析。结果:BHK21为亚二倍体或亚四倍体细胞系,亚二倍体众数为42,亚四倍体细胞众数为72~74,该细胞系染色体的畸变率在各代次所占的比例为0~2%,均较低。结论:用商业化的低血清培养基驯化培养BHK21细胞,逐渐降低培养液中血清含量,进行传代培养,通过选取不同代次细胞的染色体进行核型分析,该细胞系的遗传学特性相对稳定,各代次间无本质差异,可候选为制苗用细胞。     

细胞永生化技术研究进展

细胞永生化作为细胞生物学研究的热点,在细胞实验中发挥很大的作用,使目的细胞永生化,建立永生化细胞系,可以更好地开展细胞实验。细胞永生化技术的成功应用,不仅为生物实验研究提供了有力的工具,也为有效准确地诊断疾病提供了新道路。本文在细胞永生化基本机制的基础上,就细胞永生化技术方法、检测方法以及存在的问题等方面研究进展进行概述。     

Twenty years of embryonic stem cell research in farm animals

Notable distinctions between an embryonic stem cell (ESC) and somatic cell are that an ESC can maintain an undifferentiated state indefinitely, self-renew, and is pluripotent, meaning that the ESC can potentially generate cells representing all the three primordial germ layers and contribute to the terminally differentiated cells of a conceptus. These attributes make the ESC an ideal source for genome editing for both agricultural and biomedical applications. Although, ESC lines have been successfully established from rodents and primates, authentic ungulate stem cell lines on the contrary are still not available. Outstanding issues including but not limited to differences in pluripotency characteristics among the existing ESC lines, pre-implantation embryo development, pluripotency pathways, and culture conditions plague our efforts to establish authentic ESC lines from farm animals. In this review, we highlight some of these issues and discuss how the recent derivation of induced pluripotent stem cells (iPSCs) might augur the establishment of robust authentic ESC lines from farm animals.     

Establishment and characterization of two novel patient-derived lines from canine high-grade glioma

High-grade glioma is an aggressive cancer that occurs naturally in pet dogs. Canine high-grade glioma (cHGG) is treated with radiation, chemotherapy or surgery, but has no curative treatment. Within the past eight years, there have been advances in our imaging and histopathology standards as well as genetic charactereization of cHGG. However, there are only three cHGG cell lines publicly available, all of which were derived from astrocytoma and established using methods involving expansion of tumour cells in vitro on plastic dishes. In order to provide more clinically relevant cell lines for studying cHGG in vitro, the goal of this study was to establish cHGG patient-derived lines, whereby cancer cells are expanded in vivo by injecting cells into immunocompromized laboratory mice. The cells are then harvested from mice and used for in vitro studies. This method is the standard in the human field and has been shown to minimize the acquisition of genetic alterations and gene expression changes from the original tumour. Through a multi-institutional collaboration, we describe our methods for establishing two novel cHGG patient-derived lines, Boo-HA and Mo-HO, from a high-grade astrocytoma and a high-grade oligodendroglioma, respectively. We compare our novel lines to G06-A, J3T-Bg, and SDT-3G (traditional cHGG cell lines) in terms of proliferation and sensitivity to radiation. We also perform whole genome sequencing and identify an NF1 truncating mutation in Mo-HO. We report the characterization and availability of these novel patient-derived lines for use by the veterinary community.     

Production of an immune suppressor factor by Marek's disease lymphoblastoid cell lines

Culture fluids from Marek's disease (MD) lymphoblastoid cell lines have suppressive activity against normal and mitogen-stimulated chicken spleen and bursal cells and also against the homologous cell lines. Suppressive activity was also present in supernatants from spleen cells infected in vitro with MD virus. The suppressor factor from MD cell lines was non-sedimentable, trypsin sensitive, heat resistant and partially dialysable. Preliminary studies suggest it has a molecular weight of 20,000 daltons. Studies were also conducted on the effect of the prostaglandin inhibitors indomethacin and aspirin on the production and action of the suppressor factor. At low concentrations they have a stimulatory effect on the cell lines suggesting that they inhibit the effects of suppressor factor; however only small amounts of prostaglandin E2 were present in supernatants. Evidence was obtained that the suppressor factor may act indirectly by stimulating the production of prostaglandin by spleen cell cultures. The role of a suppressor factor in the immunosuppression observed in MD is discussed.     

Porcine IPEC-J2 intestinal epithelial cells in microbiological investigations

IPEC-J2 cells are porcine intestinal columnar epithelial cells that were isolated from neonatal piglet mid-jejunum. This cell line forms polarized monolayers with high transepithelial electrical resistance when cultured on 0.4 μm pore-size filters. The cell line is unique in that it is derived from small intestinal tissue (compared to the common human colon-derived lines HT-29, T84, and Caco-2) and is not transformed (compared to the porcine small intestinal line, IPI-2I). Porcine intestinal epithelial cells more closely mimic human physiology than analogous rodent cell lines (e.g. IEC-6 or IEC-18), which is important in studies of zoonotic infections; in addition, they provide specificity to study porcine-derived infections. IPEC-J2 cells are increasingly being used in microbiological studies to examine the interactions of various animal and human pathogens, including Salmonella enterica and pathogenic Escherichia coli, with intestinal epithelial cells. The IPEC-J2 cell line has also been employed in some probiotic studies, in which the cells have been used as an initial screening tool for adhesiveness and anti-inflammatory properties of the potential probiotic microorganisms. The validity of these studies is not clear as follow-up studies to assess the efficacy of the probiotics in vivo have not been published to date. The aims of this review are to provide a comprehensive overview of the microbiological studies that have been conducted with IPEC-J2 cells and a reference guide of key cellular and immune markers that have been identified in this cell line that may prove to be useful in future studies.     

Properties of nine continuous B-cell lines established from enzootic bovine leukosis tumors.

We established 9 cell lines from 63 tumor cases of enzootic bovine leukosis and studied their properties. Cells of all lines formed small clumps and floated in culture medium, indicating growth. Four of the 9 cell lines were surface immunoglobulin (SIg)-positive, but the remaining 5 line cells were negative for SIg or, if SIg was detected, the percentage of SIg-positive cells was very low. Tests for the properties of the cells with monoclonal antibodies to lymphocytes revealed that the established line cells are B-lymphocytes. Morphological observation also revealed that they had the morphology of B-lymphoblastic cell. The results of E and EAC rosette assay were negative, but 6 of 8 cell lines were positive for EA rosetting. All the 9 cell lines reacted with MoAb C-143, which recognizes the tumor-associated antigen (TAA) of the EBL tumor cell. All 9 cell lines produced bovine leukosis virus (BLV). These results suggest that the 9 cell lines are tumor cells derived from B-lymphocytes of EBL.     

Establishment of a canine lens epithelial cell line

Some immortalized lens epithelial cell lines have been established and are useful for molecular analysis. The establishment of additional cell lines must, however, enable a variety of in-vitro examinations. The objective of this study was to establish a new canine lens epithelial cell line by isolating CLC-1 cells from the lens tissue of a dog with cataracts. In CLC-1 cells, transforming growth factor beta (TGF-β) treatment significantly decreased gene expression of an epithelial marker and elevated that of mesenchymal markers; these characteristics are similar to those of a human lens epithelial cell line. Interestingly, CLC-1 cells exhibited lower expression of an epithelial marker and higher expression of mesenchymal markers than an anterior lens capsule. These results suggest that CLC-1 cells were derived from a cell population that was committed to epithelial-mesenchymal transition in cataract lens tissue. In conclusion, CLC-1 cells could be useful for analyzing molecular pathogenesis in canine cataracts.     

Effect of simvastatin on cell proliferation and Ras activation in canine tumour cells

Statins are inhibitors of the mevalonate cascade that is responsible for cholesterol biosynthesis and the formation of intermediate metabolites, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) used in the prenylation of proteins. Although statins are widely used in the treatment of hypercholesterolemia, recent studies suggest that they also inhibit proliferation of tumour cells by reducing prenylation of small GTP‐binding proteins, such as, Ras. This study aimed to evaluate the effect of simvastatin on cell proliferation and Ras activation in various canine tumour cell lines, including hemangiosarcoma (HSA), melanoma, and lymphoma cell lines. Simvastatin inhibited cell proliferation of all cell lines tested in a concentration‐ and time‐dependent manner, but the susceptibilities were different amongst the cell lines. Simvastatin induced apoptotic cell death via activation of caspase‐3 and cell cycle arrest. The cytotoxic effects of simvastatin were attenuated by GGPP and FPP. Simvastatin decreased the amount of prenylated Ras and GTP‐bound Ras in HSA and melanoma cell lines, but not in lymphoma cell lines. These results indicate that simvastatin induces cytotoxic effects through the depletion of GGPP and FPP in a variety of canine tumour cells, whereas multiple mechanisms are involved in the effects. Further study is required to elucidate the underlying mechanisms of simvastatin‐induced cytotoxic effects in a variety of canine tumour cells.     

Assessment of the tumorigenesis and drug susceptibility of three new canine mammary tumor cell lines

Three canine mammary tumor (CMT) cell lines, namely DE-E, DE-F and DE-SF, have been established from a surgically excised specimen of a malignant mammary tumor. These CMT cell lines have been cultured for over 200 passages. The cell doubling time was estimated to be approximately 30 h for all three cell lines. DE-E, DE-F and DE-SF were epithelial, fibroblast and spindle fibroblast in morphology, respectively. Under electron microscope, DE-F and DE-SF cells displayed a higher nucleus/cytoplasm ratio as compared with DE-E. Variation in chromosome number was also observed in the three cell lines. In addition to the morphological characteristics, these cell lines displayed differential patterns of several known mammary tumor cell markers. Following xenotransplantation of the CMT cells into nude mice, DE-F and DE-SF developed tumors within 2 weeks, whereas DE-E failed to develop any visible tumor up to 8 weeks after injection. Lastly, the CMT cell lines exhibited differential chemoresistance to several anti-tumor drugs, including melatonin, cyclosporine A, tamoxifen and indole, suggesting that these cell lines can be used as a comparative experimental model for the tumorigenesis of mammary carcinomas and a valuable tool for anti-cancer drug screening.