大鼠脑神经干细胞系(RNSC-FMU 1)的建立和鉴定

采用无血清培养液分离和培养新生SD 大鼠脑的神经干细胞,以机械分散的方法传代,成功地建立了大鼠脑神经干细胞系(RSNC-FMU 1)。该细胞系可在体外长期传代,至今已在体外连续生长超过21个月(>100代),保持了神经干细胞的特性和正常的核型,经诱导可分化成为神经元、星形胶质细胞和少突胶质细胞,具有较旺盛的自新能力,倍增时间约为20h,并可冷冻保存,裸鼠体内移植证实其不具有成瘤性。该细胞系为神经干细胞研究提供了一个良好的工具。     

家蚕胚胎细胞系BmE-SWU1的建立及其生物学特性

对家蚕反转期胚胎组织进行一年多的原代培养,分离筛选出了高繁殖细胞群体,建立了BmE-SWU1细胞系。该细胞系以梭形和近圆形细胞为主,杂以少量多突起和巨型细胞。细胞系的倍增时间在28℃时为57.6h,染色体呈短杆状或颗粒状,染色体数目异倍化,具有典型鳞翅目昆虫细胞系的染色体特征。BmE-SWU1细胞系对家蚕核型多角体病毒(BmNPV)高度敏感,半数组织培养感染剂量(TCID50)为2.92415×10-7。     

4个品系小鼠胚胎干细胞系的建立

探索高效的不同品系的小鼠胚胎干细胞的建系方法。B6D2F1(C57BL/6×DBA/2)、129/SV×DBA/2、C57BL/6、BALB/C等4个不同品系小鼠,孕马血清促性腺激素(pregnant mare serum gonadotrophin,PMSG) 人绒毛膜促性腺激素(human chorionic gonadotropin,HCG)促排,3.5天交配后(days post coitus,dpc)冲洗子宫取囊胚,或者2.5dpc冲洗输卵管,卵裂球体外培养获取囊胚。囊胚种植到小鼠成纤维细胞饲养层上干细胞培养液培养,4～5天内细胞团扩增后玻璃毛细管挑出,种植到新的饲养层上过夜再行胰蛋白酶消化,3～4天传代一次。对所建立的小鼠ES细胞系进行形态学、染色体核型、AKP染色、体内外分化能力,干细胞分子标记物荧光免疫染色等鉴定。获得10株小鼠胚胎干细胞,具有典型的胚胎干细胞生长特性,符合ES细胞的鉴定标准。结果表明成功的建立了来自B6D2F1(C57BL/6×DBA/2)、129/SV×DBA/2、C57BL/6、BALB/C等4个不同品系小鼠的10株ES细胞系。内细胞团挑出过夜增殖后消化的培养方法可能有助于提高ES细胞的建系率。     

鲤鱼（Cyprinus carpio）体细胞系的建立及其生物学特性分析（简报）

动物细胞的培养技术是1907年哈里逊在淋巴块中对蛙的神经板培养成功开始的,其后近一个世纪以来,陆续成功地培养了哺乳动物、昆虫等各种动物细胞,并广泛用于生物科学的各个分支。鱼类的细胞培养的系统研究和建系实践大约起始于60年代,被公认的真骨鱼类的第一个永久性的细胞系——虹鳟性腺细胞系(RTG-2)是由Wolf建立的。随后各种鱼类细胞系相继建立,涉及的组织来源有吻端、肾脏、卵巢、尾鳍、性腺、肝脏、胚胎、囊胚、原肠胚、鳍条等,同时也进行了细胞体外培养条件、     

神经干细胞--治疗脑神经性疾病的希望(1)

神经干细胞是脑中多潜能的具有自我更新能力的细胞,在特定环境和因子的诱导下能定向分化成不同的神经细胞类型,它为脑损伤修复及神经性疾病的治疗提供了新的途径,因此具有巨大的潜在应用价值和重大理论研究意义。已经发现在成体鸟类、啮齿类、灵长类和人类脑中都存在能产生神经元的神经干细胞。祖先干细胞向不同细胞系的分化受内源性和外源性机制的调制,尽管目前对这种调制的分子机制并不清楚,然而关于对神经干细胞鉴定、分离及对其特性的研究已经获得了很大的进展。     

BALB／c小鼠胚胎干细胞系的建立及其嵌合体小鼠的获得

目的：建立BALB/c小鼠胚胎干细胞系,并用于制作嵌合体小鼠。方法：从BALB／c小鼠囊胚内分离培养内细胞团块。建系后,进行C57BL／6L小鼠受体囊胚腔注射,制作嵌合体小鼠,结果：建立了我国第一株BALB／c小鼠胚胎干细胞系,该细胞系具有典型的ES细胞形态,碱性磷酸酶强阳性,核型正常以及具有分化为三种胚层组织的能力,并已产生5只嵌合体小鼠,结论：建立的BALB／c小鼠胚胎干细胞系具有胚胎干细胞的各种特点,可用于体内外诱导分化研究,在进一步观察生殖系嵌合情况后,决定是否可应用于基因打靶等转基因动物的制作。     

新生大鼠脊髓神经干细胞的分离培养及鉴定

目的　从新生大鼠的脊髓中分离培养神经干细胞并观察其增殖和分化能力。方法　采用细胞培养技术结合间接免疫荧光细胞化学法。结果　分离的细胞生长旺盛 ,单克隆化生成的细胞团 ,BrdU掺入呈强阳性。分离培养获得的细胞团呈Nestin强阳性 ,至今已在体外连续传代 8个月。培养的细胞团经 1%小牛血清诱导可分化为神经元和星形胶质细胞。结论　成功分离培养了新生大鼠脊髓神经干细胞     

中华鲟精巢细胞系的建立和鉴定

为了开展中华鲟(Acipenser sinensis)种质资源保存及其精原干细胞体外培养的研究, 采用蛋白酶消化法, 对中华鲟精巢组织细胞进行原代培养, 建立了中华鲟精巢细胞系(Acipenser sinensis testicular cell line, AST), 经352d传代培养, 已稳定传至80代。中华鲟精巢细胞系形态主要呈类纤维状, 培养基为DMEM, 培养温度为25℃, 最适血清浓度为15%。正常传代的AST细胞冻存、复苏后, 经台盼蓝染色, 约(81.36±1.13)%的细胞具有活性, 复苏后细胞仍生长旺盛。染色体核型分析结果显示, 第30代中华鲟精巢细胞系染色体数目分布在142—310, 众数为264。通过RT-PCR检测发现, 在P0和P1细胞中, Sertoli细胞特异表达基因(amh和gsdf)、Leydig细胞特异表达基因(cyp17a1)和生殖细胞特异表达基因(dazl、dnd和vasa)都有表达, 且表达量与精巢中的相似; 在P15、P30和P60细胞中, 只有amh和vasa基因有微弱的表达, 说明细胞系传代到了后期, 只含有极少量的Sertoli细胞和生殖细胞。通过脂质体转染法将pEGFP-N3质粒转入AST细胞中, 可表达增强型绿色荧光蛋白(Enhanced green fluorescent protein, EGFP)。AST细胞系的建立为中华鲟种质资源的保存、精原干细胞的体外增殖与分化、基因功能等研究提供了重要的实验材料。     

肿瘤坏死因子-α对大鼠中脑神经干细胞分化和寡突胶质细胞增殖的影响

为观察肿瘤坏死因子对神经干细胞(NSCs)分化的影响,本研究应用体外扩增的新生大鼠中脑NSCs,使用免疫组织化学技术,观察了肿瘤坏死因子—α(TNF—α)对NSCs分化及其后代细胞的影响。结果显示：(1)TNF—α可提高中脑NSCs后代中神经元和寡突胶质细胞所占的比例;(2)TNF—α可明显诱导由NSCs分化的寡突胶质细胞增殖,但对星形胶质细胞的增殖作用不明显。上述观察结果提示TNF—α对NSCs的应用具有重要影响。     

家蚕胚胎细胞系Bm-Em-1的建立和特性研究

昆虫细胞系在病毒生物学、 基因功能的研究以及杆状病毒表达系统生产重组蛋白的应用中发挥着重要的作用。本研究由家蚕Bombyx mori “大造”品种反转期胚胎建立了一株细胞系Bm-Em-1, 在含10％胎牛血清的TNM-FH培养基中已传代40余代。显微观察表明, 细胞形态主要为圆形和短梭形, 细胞染色体呈短棒状和颗粒状, 数量多、 异倍化, 符合典型的鳞翅目昆虫细胞染色体特征。RAPD鉴定结果表明, 该细胞系来源于家蚕胚胎, 其扩增谱带与BTI-Tn5B1-4和Sf-9等细胞系明显不同。生长曲线测定结果表明, 第28代细胞的群体倍增时间为82.2 h。病毒敏感性测定显示, 该细胞系不能被苜蓿银纹夜蛾Autographa californica核型多角体病毒（AcMNPV）感染, 但对家蚕核型多角体病毒（BmNPV）高度敏感, 96 h的感染率为91.3％。结果说明该细胞系可作为家蚕病毒离体复制、 BmNPV表达系统以及家蚕基因功能研究的理想材料。     

Establishment and characterization of an immature epithelial cell line (ENU-T-1) derived from a rat nephroblastoma

A new cell line designated ENU-T-1 has been established from a xenotransplanted experimental rat nephroblastoma. The cultured cells are spindle-shaped or polygonal and are arranged in a wavy fashion morphologically similar to cultured embryonal renal epithelial cells. The cells exhibit a number of epithelial characteristics. Enzyme histochemistry gives positive reactions for gamma-glutamyltranspeptidase and alkaline phosphatase, both of which are present in renal tubular epithelial cells. Immunofluorescence studies show positive reactions for vimentin and cytokeratin. When inoculated into athymic nude mice, the cultured cells form tumors composed of sheets of epithelial cells with focal tubular formation. This cell line may be of value in studying differentiation of nephroblastoma, and possibly normal nephrogenesis.     

Establishment and characterization of an immature epithelial cell line (ENU-T-1) derived from a rat nephroblastoma

A new cell line designated ENU-T-1 has been established from a xenotransplanted experimental rat nephroblastoma. The cultured cells are spindle-shaped or polygonal and are arranged in a wavy fashion morphologically similar to cultured embryonal renal epithelial cells. The cells exhibit a number of epithelial characteristics. Enzyme histochemistry gives positive reactions for gammaglutamyltranspeptidase and alkaline phosphatase, both of which are present in renal tubular epithelial cells. Immunofluorescence studies show positive reactions for vimentin and cytokeratin. When inoculated into athymic nude mice, the cultured cells form tumors composed of sheets of epithelial cells with focal tubular formation. This cell line may be of value in studying differentiation of nephroblastoma, and possibly normal nephrogenesis.     

Establishment and characterization of a mouse neural crest derived cell line (NCCmelan5)

Stem cell factor (SCF) and endothelin 3 (EDN3) are both necessary for melanocyte development. We have established an immortal cell population of neural crest cells from C57BL/6 mice, cultivating them with SCF, EDN3 and 15% fetal calf serum without feeder cells, and have designated that line as C57NCC SE. C57NCC SE consists of a population of melanocytes in various stages of differentiation. We used a single-cell cloning method, in which only one cell is transferred to each new culture plate, and succeeded in establishing an immortal cell line named NCCmelan5. All NCCmelan5 cells were positive for KIT (SCF receptor), HMB45 (human melanosomal antigen), tyrosinase-related protein-1 (TYRP1), tyrosinase-related protein-2 (TYRP2), tyrosinase and endothelin receptor B (EDNRB) and all could oxidize 3,4-dihydroxyphenylalanine (DOPA) to form melanin. Measurement of their DNA content revealed that 88.6% of the cells were in the G0-G1 phase, suggesting that they retained normal DNA ploidy. Thus, NCCmelan5 cells have the characteristics of mature melanocytes except that they are immortal; these cells may prove useful to study factors that directly affect melanogenesis and melanocyte development without the influence of feeder cells. It is clear that our attempt to establish immortal cell lines from murine neural crest cells would have never been successful without the addition of SCF and EDN3, since C57NCC SE and NCCmelan5 cells require those factors to proliferate.     

Isolation and characterization of a neural progenitor cell line from tilapia brain

Astroglial cell lines have many applications for advancing neural developmental and functional studies. However, few astroglial cell lines have been reported from fish. In this study, we report the characterization of the immortal cell line TB2 isolated from adult tilapia brain tissue. The cell line was established at 25 degrees C in L15 medium supplemented with 15% fetal bovine serum. Most of the cells displayed a fibrous morphology and were immunoreactive for A2B5 antigen, glial fibrillary acidic protein (GFAP), keratin, vimentin, and the gap junction protein connexin 43 (Cx43). They weakly expressed glutamine synthetase (GS), S100 protein, and the neural stem cell markers Sox2 and brain lipid binding protein (BLBP). In contrast to astroglia in vivo, most TB2 cells also expressed galactocerebroside (GalC), substance P (SP), and tyrosine hydroxylase (TH). By immunoblot and RT-PCR, the cells also expressed myelin basic protein (MBP), proteolipid protein (PLP), and Cx35. On a poly-L-lysine-coated substrate in vitro, TB2 cells showed increases in neuronal dopamine decarboxylase (DDC) and microtubule-associated protein 2 (MAP2), indicating that they can initiate differentiation into neurons. Taken together, the results suggest that TB2 cells are astroglial progenitor cells (neural stem cells) and may develop into oligodendrocytes and neurons in a suitable environment. The present study advances our knowledge of fish astroglia. However, the factors that affect neural development in fish remain unknown, as do the characteristics of the intermediate differentiation stages between stem cells and mature nerve cells. The TB2 cell line will promote these investigations.     

Isolation and characterization of a neural progenitor cell line from tilapia brain.

Astroglial cell lines have many applications for advancing neural developmental and functional studies. However, few astroglial cell lines have been reported from fish. In this study, we report the characterization of the immortal cell line TB2 isolated from adult tilapia brain tissue. The cell line was established at 25 degrees C in L15 medium supplemented with 15% fetal bovine serum. Most of the cells displayed a fibrous morphology and were immunoreactive for A2B5 antigen, glial fibrillary acidic protein (GFAP), keratin, vimentin, and the gap junction protein connexin 43 (Cx43). They weakly expressed glutamine synthetase (GS), S100 protein, and the neural stem cell markers Sox2 and brain lipid binding protein (BLBP). In contrast to astroglia in vivo, most TB2 cells also expressed galactocerebroside (GalC), substance P (SP), and tyrosine hydroxylase (TH). By immunoblot and RT-PCR, the cells also expressed myelin basic protein (MBP), proteolipid protein (PLP), and Cx35. On a poly-L-lysine-coated substrate in vitro, TB2 cells showed increases in neuronal dopamine decarboxylase (DDC) and microtubule-associated protein 2 (MAP2), indicating that they can initiate differentiation into neurons. Taken together, the results suggest that TB2 cells are astroglial progenitor cells (neural stem cells) and may develop into oligodendrocytes and neurons in a suitable environment. The present study advances our knowledge of fish astroglia. However, the factors that affect neural development in fish remain unknown, as do the characteristics of the intermediate differentiation stages between stem cells and mature nerve cells. The TB2 cell line will promote these investigations.     

Establishment and characterization of a cisplatin-resistant cell line (IGSK-1) from a poorly differentiated gastric adenocarcinoma

We successfully established a spontaneously cisplatin-resistant tumor cell line (designated as IGSK-1) derived from original gastric carcinoma. The patient was a 75-year-old Japanese woman. The histopathological diagnosis was gastric poorly differentiated adenocarcinoma accompanied with metastatic foci in lymph nodes, pT3, N2 M0, stage IIIB. The IGSK-1 cells grew as adhesive and monolayered cultures on the bottom of dishes. The susceptibility of the IGSK-1 cells to anti-cancer drugs was examined using oxygen electrode apparatus (Daikin, Tsukuba, JPN), and the results suggested TXL was effective, and CDDP, CPT-11 and 5-FU were not effective. Gastrin and somatostatin secretions were confirmed by immunohistochemical staining and also radioimmunoassay. Immunohistochemistry and radioimmunoassay for serotonin suggested the IGSK-1 cells might incorporate serotonin from the growth media. Spontaneously cisplatin-resistant gastric carcinoma cell line secreted gastrin and somatostatin is very important material for chemotherapy.