3-甲基腺嘌呤对转化生长因子-β诱导大鼠肝脏星形细胞株HSC-T6活化及自噬的影响观察

目的观察3-甲基腺嘌呤(3-MA)对转化生长因子-β(TGF-β)诱导的大鼠肝星形细胞株HSC-T6活化及自噬的影响。方法取对数生长期的HSC-T6细胞分为空白组、TGF-β+PBS组、TGF-β+3-MA组。TGF-β+3-MA组细胞加入浓度为2 ng/mL TGF-β培养72 h后加入3-MA(0.5 mg/mL)处理24 h,TGF-β+PBS组细胞加入浓度为2 ng/mL TGF-β培养72 h后加入等量PBS处理24 h,空白组加入等量PBS处理,采用实时荧光定量PCR法检测各组细胞活化标志物α-SMA、TGF-βmRNA和自噬标志物LC3、Beclin-1、Atg5 mRNA,采用Western blotting法检测各组细胞活化标志物α-SMA、TGF-β蛋白和自噬标志物LC3、Beclin-1、Atg5蛋白。结果TGF-β+PBS组、TGF-β+3-MA组细胞活化标志物α-SMA、TGF-βmRNA和蛋白均高于空白组,且TGF-β+3-MA组均低于TGF-β+PBS组(P均<0.05)。TGF-β+PBS组、TGF-β+3-MA组细胞自噬标志物LC3、Beclin-1、Atg5 mRNA和蛋白均高于空白组,且TGF-β+3-MA组均低于TGF-β+PBS组(P均<0.05)。结论3-MA可抑制TGF-β诱导的大鼠肝星形细胞株HSC-T6活化及自噬。

CHARACTERIZATION OF A HUMAN HERPES VIRUS－6（HHV－6） AND EPSTEIN－BARR VIRUS（EBV） ASSOCIATED LEUKEMIC CELL LINE，J6－1

This report characterizes the J6-1 cell line derived from a Chinese acute myelomonocytic leukemia patient and previously reported to be associated with EBV. These studies showed that J6-1 cells were also infected with HHV-6 as demonstrate at the DNA level by PCR and Southern blot hybridization and by expression of HHV-6 early membrane antigen on the J6-1 cell surface. Further characterization showed J6-1 was co-infected with EBV type 2. Generally, cells infected with EBV type 2 do not grow well in vitro. However, J6-1 , although difficult to maintain in vitro, has been grown for 15 years. Possibly, co-infection with HHV6 confers this property. In this regard, J6-1 cells exhibited density dependent growth which could be inhibited with an anti-HHV-6-MA monoclonal antibody(MAb). In contrast, anti-HHV-6-VCA MAb stimulated the J6-1 cell proliferation. Electron microscopic analysis showed that, morphologically, there were two types of J6-1 cell, one with lymphoblastoid features and one with a monocytoid appearance. Accordingly, the flow profile of the J6-1 cell line showed heterogeneity. with two populations comprised of CD15-, CD19+ cells with low light scatter(small cells) and a population with greater light scatter(larger cells) which was CD15+ , CD19+. The population was negative for progenitor cell markers(CD33, 34 ), and T cell markers. Southern analysis showed no T cell receptor rearrangement, however there was a clonal JH and kappa light chain expressing population. Glycocytochemical analysis showed several endogenous lectin receptors on the J6-1 cell surface: BSA-Xylose, BSA-Rhamnose, BSAGal. BSA-Lac. This cell line shares many characteristics with other monocytic/ lymphoblastoid cell lines isolated elsewhere and provides circumstantial evidence linking Herpes viruses, as least as co- factors,to leukemia cell growth.

Effect of Antisense Oligodeoxynucleotide Directed to NF-κB-RelA on Bcl-x_L mRNA in Extended Drug Resistance Leukemia Cell Line HL- 60/E6

To explore the effect of NF κB on bcl x gene transcription in extended drug resistance leukemia cell line HL 60/E6, drug resistant subline HL 60/E6 was derived by intermittently exposing HL 60 cells to 6 ng/ml epirubicin. Indirect immunofluorescence was used to demonstrate the location of NF κB RelA in HL 60/E6 cells. FCM analysis and RT PCR were used to detect the efficiency of liposome mediated ODN transfection and the change of bcl x L mRNA levels after 5 μmol/L phosphorothioate (PS) derivatized antisense (AS) oligodeoxynucleotide (ODN) directed to RelA was transferred into HL 60/E6 cells. The results showed that RelA remained persistently active and located at the nuclei of HL 60/E6 cells,but in the cytoplasm of HL 60 cells, the efficiency of liposome mediated ODN transfection was significantly higher than that of null ODN ( P <0.01 in 4 h, 6 h, 12 h, 24 h). Exposure of HL 60/E6 cells to 5 μmol/L AS PS ODN directed to RelA led to a maximal 40 % decline of bcl x L mRNA levels within 8 h. The inhibition rate of bcl x L mRNA was (15±1.79) %, (28±2.34) %, (40±3.47) %, (20±1.54) % in 4 h, 6 h, 8 h, 15 h, respectively, but it was less than 15 % in control group. It was concluded that NF κB was involved in regulating bcl x transcription. It was suggested that NF κB was an important factor for drug resistance in leukemia cells.

Establishment of Embryonic Stem Cell Lines from C57BL/6J Mice and Generation of Chimeras

Four embryonic stem (ES) cell lines, designated CE1, CE2, CE3 and CE4, were isolated from C57BL/6J blastocysts. The ratio of normal diploid composition of these cell lines is above 70%. To examine the differentiation potential of the ES cells, the CE2 cells were injected subcutaneously into syngenic mice, and many kinds of differentiated cells were observed on the sections of the teratoma derived from this ES cell line. On the other hand, to test the chimeric ability of the ES cells, the CE2 cells were microinjected into the blastocysts of ICR mice, and a chimera was obtained among living pups. These results show that CE2 ES cells are pluripotent stem cells, which can differentiate into many kinds of cell types, and can be used as a cell system for further research.

Stable EGFP Gene Expression in C6 Glioma Cell Line after Transduction with HIV-1-based Lentiviral Vector

Objective： To establish a stable C6/EGFP glioma cell transfected with the human immunodeficiency virus line for studies on glioma. Methods： The C6 glioma cell line was type I （HIV-1） based lentivirus vector containing two enhancer-promoters CMV and EF1α. Enhanced green fluorescent protein （EGFP）-positive C6 cells were sorted out by fluorescence-activated cell sort. Expression of EGFP was observed by fluorescent microscopy. EGFP gene in C6 genome was assessed by Polymerase chain reaction （PCR） and DNA sequencing. Original and transfected cells were compared biologically and cytomorphologically. Results： Lentivirus vector transfection produced up to 40% EGFP-positive cells. After fluorescence-activated cell sort selection, a pure cell line C6/EGFP was established. PCR and DNA sequencing revealed integration of EGFP gene in C6 cell genome. Analysis of cell characteristics revealed no difference between transfected and original cells. Conclusion： A C6/EGFP cell line expressing EGFP as a marker is established, in which the EGFP gene is integrated into the genome. This cell line can be served as a promising tool for further basic research and gene therapy studies.

Effects of 4-(3-Chloro-Benzyl)-6,7-Dimethoxy-Quinazoline on Kinetics of P120-Catenin and Periplakin in Human Buccal Mucosa Squamous Carcinoma Cell Line

In order to detect molecular markers for the epidermal growth factor inhibitor 4-(3-chloro-benzyl)- 6,7-dimethoxy-quinazoline (tyrphostin), we investigated the kinetics of p120-catenin and periplakin in the human buccal mucosa squamous cancer cell line BICR 10 treated with 3 nM tyrphostin. Growth of BICR 10 cells was inhibited by treatment with tyrphostin. Although changes were not observed in the expression of EGFR and p120-catenin, expression of Akt, Src and periplakin in BICR 10 treated with 3 nM tyrphostin tended to decrease. In addition, phosphorylation of EGFR, Akt and Src was inhibited by treatment with tyrphostin. On immunocytochemical staining, immunoreactions with phosphorylated EGFR, phosphorylated Akt and phosphorylated p120-catenin were weak in BICR 10 treated with tyrphostin. There was a slight immunocy to chemical reaction to periplakin in BICR 10 cells induced by tyrphostin. In conclusion, the decrease in phosphorylation in EGFR and p120-catenin by tyrphostin, following the decrease in Src or Akt phosphorylation, may inhibit expression of several growth factors associated with the proliferation and migration of cancer cells.

HBV X Gene Transfection Upregulates IL-1β and IL-6 Gene Expression and Induces Rat Glomerular Mesangial Cell Proliferation

The X gene of HBV encodes a 17-kD protein, termed HBx, which has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer elements. The aim of this study was to investigate the effect of HBx on gene expression of interleukin （IL）-1β and IL-6, and proliferation of rat mesangial cells in vitro. The X gene of HBV was amplified by PCR assay, and inserted into the eukaryotic expression vector pCI-neo. The structure of recombinant pCI-neo-X plasmid was proved by restrict endonuclease digestion and sequencing analysis. pCI-neo-X was transfected into cultured rat mesangial cell line in vitro via liposome. HBx expression in transfected mesangial cells was detected by Western blot. The IL-1β and IL-6 mRNA expression in those cells was assayed by semiquantitative RT-PCR. Mesangial cell proliferation was tested by MTT. The results showed that HBx was obviously expressed in cultured mesangial cell line at 36th and 48th h after transfection. The expression of IL-1β and IL-6 mRNA was simultaneously increased. The cell proliferation was also obvious at the same time. It was concluded that HBx gene transfection could induce IL-1β and IL-6 gene expression and mesangial cell proliferation. HBx may play a critical role in mesangial cell proliferation through upregulation of the IL-1β and IL-6 gene expression.

Effects of TRPC6 on invasibility of low-differentiated prostate cancer cells

Objective:To study the expression of TRPC6 among prostate cancer cells,establish high expression cell lines of TRPC6,and to provide potential cell mode for prostate cancer oncogenesis and development.Methods:Occurrence and development of prostate cancer cells,PC3,PC—3m DU145,22 rvl,LNCaP and normal prostate epithelial cells in the PrEC TRPC6 expression level were detected by QPCR method.Calcium phosphate transfection method was used to package retrovirus pLEGFP-Nl-TRPC6 and pLEGFP-Nl-vector and infect the prostate cancer cells,a stable high expression of TRPC6 prostate cancer cells.Sable cell lines of TRPC6,matrix metalloproteinase(MMP)2,MMP9 expression was detected by QPCR and Western blot.Change of cell invasion ability was detected by Transwell.Results:The expression level of prostate cancer cells TRPC6 were higher than control group PrEC cells.Among TPRC6 the expression of cell line PC 3 transfer potential wre the lowest,and high transfer cell tone PC-3M express was the highest.Real-time fluorescent quantitative PCR and western blot results showed that after filter,the seventh generation of cell TRPC6 protein and mRNA expression levels were higher than the control group obviously.Transwell experimental results showed that the overexpression of TRPC6could promote the invasion ability of PC.3 prostate cancer cells.Conclusions:TRPC6 expressed in prostate cancer cells is in disorder,and its action may be associated with the invasion and metastasis of prostate cancer cells;successful establishment of stable high expression of TRPC6prostate cancer cells primarily confirm the invasion-trigger ability of TRPC6 on prostate cancer,and lay down the foundation for exploring the TRPC6's role in the occurrence and development of prostate cancer

Effect of 5-Aza-2'-deoxycytidine on the expression of p16 in hepatocellular carcinoma cells in vitro

Objective: To study the effect of 5-Aza-2’ -deoxycytidine (5-Aza-cdR) on tumour suppressor gene p16 expres- sion in hepatocellular carcinoma cells. Method: Expression of pl6 mRNA and protein in hepatocellular carcinoma cell lines SMMC-7721 and HePG2 before and after treatment with 5-Aza-cdR were analyzed via reverse transcriptase polymerase chain reaction(RT-PCR) and immunohistochemistrty Results: The expression levels of p16 mRNA and protein were increased dramatically after treatment with 5-Aza-cdR. Conclusion: Our data show that, 5-Aza-2’ -deoxycytidine can increase the expression of pl6 gene both at transcription and translation. The findings suggested that 5-Aza-cdR may reactivate the pl6 gene by demethylation.

RNA interference affects tumorigenicity and expression of insulin-like growth factor-1,insulin-like growth factor-1 receptor,and basic fibroblast growth factor-2 in rat C6 glioma cells

BACKGROUND： Human gliomas are more likely to express basic fibroblast growth factor-2 （FGF-2） insulin-like growth factor-1（IGF-1）, and IGF-1 receptor （IGF-1R） than normal brain tissue. These factors activate signal transduction systems of Ras/MAPK and PI3K/Akl, which promote glioma growth. OBJECTIVE： To utilize RNA interference （RNAi） technique to down-regulate FGF-2, IGF-1, and IGF-1R gene expression, and to investigate the effects of these genes on rat C6 glioma cells, as well as the feasibility of RNAi for treating glioma. DESIGN, TIME AND SETTING： This neurooncological, randomized, controlled, in vivo and in vitro experiment, which used RNAi methodology, was performed at the Laboratory of Molecular Biology, Institute of Biochemistry, Chinese Academy of Sciences between August 2005 and February 2008. MATERIALS： Rat C6 cell lines were purchased from Shanghai Institute of Cellular Biology Affiliated to Chinese Academy of Sciences. Small interfering RNA （siRNA） was synthesized by Shanghai GenePharma. Anti-IGF-1, anti-IGF-1R, anti-FGF-2, anti-mouse and anti-rabbit IgG G1-HRP antibodies were provided by Santa Cruz Biotechnology, USA. Four to six week-old BALB/c nude mice were purchased from the Laboratory Animal Center, Chinese Academy of Sciences. METHODS： C6 glioma cells were transfected with siRNA, which was chemically synthesized in vitro to correspond to endogenous FGF-2, IGF-1, and IGF-1R genes. The inhibition ratio of targeting mRNA expression was detected by semiquantitative RT-PCR, and protein expression was determined by Western blot analysis. C6 glioma cell proliferation was observed using a growth curve C6 glioma cell apoptosis rate and cell cycle were detected by flow cytometry. C6 glioma cell growth regression was observed by transwell migration assay. In addition, nude mouse subcutaneous tumor models were used in this study. For studying the anti-tumor effects of IGF-1 and IGF-1R siRNA, two blank control groups, with six mice each, were set up： A （2.5 μg siRNA was injected one week after C6 cells were inoculated, Le., when tumor volume reached 8 mm × 8 mm） and B （siRNA was injected at the same time with C6 cells were inoculated. To study the effects of FGF-2 siRNA, the groups consisted of a blank control group, negative control group, 2.6 μg siRNA group, 4 μg siRNA group, and 5.3 μg siRNA group, with six mice each. MAIN OUTCOME MEASURES： mRNA and protein inhibition ratio of FGF-2, IGF-1, and IGF-1 R; C6 glioma cell proliferation, apoptosis, and cycle growth arrest; C6 glioma cell growth regression and subcutaneous tumorigenicity rates. RESULTS： All siRNA constructs proved to be effective. After 48 hours, transfection of 200 nmol/L siRNA resulted in a FGF-2 or IGF-1R gene inhibition ratio 〉 80% and an IGF-1 gene inhibition ratio of approximately 70%. Protein expression levels for FGF-2, IGF-1, and IGF-1R decreased in a dose-dependent manner following siRNA transfection, with an inhibition rate 〉 85%, 60%, and 50%, respectively. C6 glioma cell proliferation and apoptosis rates increased in proportion to siRNA. The apoptosis rate of C6 glioma cells induced by FGF-2, IGF-1, and IGF-1R siRNA was 39.96%, 15.07% and 22.47%, respectively （P 〈 0.01）. Transfection of 200 nmol/L IGF or IGF-1R siRNA for 48 hours suppressed C6 glioma cell migration. At 30 days after intratumoral injection of 2.6, 4, and 5.3 tJg FGF-2 siRNA, tumor growth regression rate of FGF-2 siRNA was 56%, 67%, and 86%, respectively. The tumor growth regression rate was 71.88% and 45.71%, respectively, when IGF-1 or IGF-1R siRNA was intratumorally injected 1 week after C6 glioma cell transplantation. When IGF-1 or IGF-1 R siRNA was intratumorally injected during C6 glioma cell transplantation, the tumor growth regression rate was 78.13% and 74.29%, respectively. CONCLUSION： siRNA transfection downregulated gene expression of FGF-2, IGF-1, and IGF-1R In addition, siRNA treatment markedly suppressed glioma cell proliferation, growth, and migration, and concomitantly reduced subcutaneous tumorigenicity.

Differential activation of mitogen-activated protein kinases by γ-irradi-ation in IEC-6 cells: Role of intracellular Ca^(2+)

Objective: To explore the effects of γ-irradiation on mitogen-activated protein kinases (MAPKs) and role of intracellular calcium in this event in intestinal epithelial cell line 6 (IEC-6 cells). Methods: After cultured rat IIEC-6 cells with or without the pretreatment of intracellular Ca2+ chelator were exposed to Y-ir-radiation of 6 Gy, the total and phosphorylated MAPKs in the cells were determined with Western blotting and apoptosis was examined with flow cytometry. Activities of Extracellular signal-regulated protein kinase (ERK) and p38 MAPK were determined by using immuoprecipitation followed by Western blotting. Results: In response to γ-irradiation, phosphorylation of ERK was not significantly observed, while the levels of phosphorylated c-Jun NH2-terminal kinase (JNK) and p38 MAPK were increased in 30 min and reached the peak 2 h after exposure to 6 Gy γ-irradiation, though the cell viability was significantly lowered 12 h. On the other hand, no obvious changes were seen in the total protein levels of ERK, JNK and p38 MAPK. Chelation of intracellular Ca2+ almost completely suppressed the JNK and p38 MAPK phosphorylation induced by γ-irradia-tion, but removal of external Ca2+ had no such effect. Activation of p38 MAPK, but not of ERK, was seen to have a correlation with γ-irradiation induced apoptosis. Conclusion: The results suggest that γ-irradiation is a potent activator for JNK and p38 MAPK, and Ca2+ mobilized from intracellular stores plays an important role in the activation of MAPKs and the induction of apoptosis in IEC-6 cells.

党参白术提取物分别和合用诱导IEC-6细胞增殖分化的作用

目的 观察党参提取物 (D h)和白术提取物 (B t)对小肠隐窝细胞 (IEC 6 )增殖、分化的作用及其二者配伍对IEC 6细胞增殖的影响。方法 IEC 6细胞培养 2 4h ,加入不同剂量的D h、B t及二者的配伍溶液 ,2 4h后用MTT法观察细胞增殖 ,显微镜观察细胞分化的形态特征。结果 D h在 2 5 0mg·L-1以下剂量时对细胞增殖无影响 ,剂量提高到 5 0 0mg·L-1和 10 0 0mg·L-1时 ,明显促进细胞的增殖。B t各剂量均无促进细胞增殖的作用 ,在 5 0 0mg·L-1和10 0 0mg·L-1剂量时 ,细胞增殖反见减弱。二药配伍后 ,促进细胞增殖的作用明显增强 ,具有一定的剂量依赖关系 ,其效果优于D h ;IEC 6细胞在B t作用下 ,分化程度增高 ,低倍镜下细胞呈典型的上皮细胞形态 ,细胞排列呈条索状 ,部分呈腺管状排列。高倍镜下可观察到隐约有细胞连接样结构 ,上皮细胞似有微绒毛形成的趋势。D h处理的细胞分化程度较低 ,镜下所见多为未分化的梭状细胞 ,且排列不规则 ,无任何形成腺管的趋势和有细胞连接样结构。结论 D h能促进细胞增殖 ,B t可促进细胞分化 ,二者配伍后促进细胞增殖作用明显增强 。

白三烯类化合物(Leukotrienes)对小鼠腹腔巨噬细胞生成白细胞介素6的影响

探讨了白三烯Ｂ４（ＬＴＢ４）、白三烯Ｃ４（ＬＴＣ４）及白三烯Ｄ４（ＬＴＤ４）对小鼠腹腔巨噬细胞分泌白细胞介素６（ｉｎｔｅｒｌｅｕｋｉｎ６，ＩＬ６）的影响。用ＩＬ６依赖细胞株Ｂ９的ＭＴＴ方法测定样品中ＩＬ６的含量。结果显示，ＬＴＢ４能剂量依赖性地增加巨噬细胞培养上清液中ＩＬ６的含量。ＬＴＣ４及ＬＴＤ４促进巨噬细胞培养上清液中ＩＬ６含量的最适浓度分别为：６９×１０－８和８０５×１０－８ｍｏｌ·Ｌ－１。结果提示：ＬＴＢ４与ＬＴＣ４及ＬＴＤ４在某些生物学功能方面有一致性。与ＬＴＣ４及ＬＴＤ４相比，ＬＴＢ４促进巨噬细胞培养上清液中ＩＬ６的含量的最适浓度则要大１～２个数量级，提示在炎症反应中，肽白三烯与ＩＬ６的相关性较强。

黄芪注射液通过激活鸟氨酸脱羧酶促进IEC-6细胞分化的研究

目的 :观察黄芪注射液 (Astragalusinjection ,AI)对小肠隐窝细胞株 (IEC 6 )增殖、分化、移行及其对细胞内鸟氨酸脱羧酶 (ODC)和多胺含量的影响 ,探讨其粘膜修复的作用机理。方法 :IEC 6细胞接种后 2 4h ,加入AI。加药后 12h收获细胞 ,分别检测ODCmRNA水平、ODC蛋白、ODC活性及细胞内腐胺含量 ;2 4h观察细胞增殖和细胞分化 ;细胞接种后 72h损伤细胞 ,并加入AI ,加药后 2 4h、4 8h及 72h观察细胞移行。结果 :AI能明显抑制IEC 6细胞增殖 ,促进细胞分化 ,对细胞移行无明显影响。AI 6 2 5～ 2 5 0 μg/ml各剂量组可明显增加ODCmRNA水平 ,与对照组比较差异有显著性 (P <0 0 5～ 0 0 1)。AI各剂量组对ODC蛋白均无明显影响。AI随着剂量增加 ,逐渐升高IEC 6细胞ODC活性和腐胺含量 ,具有剂量依赖关系。结论 :AI通过诱导IEC 6细胞ODC活性和多胺的生物合成促进细胞分化 。

IL－6依赖细胞株的克隆化、冻存及其应用

应用ＭＨ６０·ＢＳＦ２细胞检测ＩＬ－６生物学效价时，存在该细胞饲养困难和长期培养容易变异而失去ＩＬ－６增殖依赖性等问题。为此我们首先探讨了克隆化筛选ＭＨ６０·ＢＳＦ２的方法，从增殖依赖性较差的ＭＨ６０·ＢＳＦ２细胞群体中筛选到３株ＩＬ－６强依赖细胞株。并探讨了该细胞的冻存复苏方法。所冻存的细胞半年以上亦无ＩＬ－６增殖依赖性的改变。在此基础上采用ＭＴＴ比色法取代［￣３Ｈ］ＴｄＲ掺入法获得成功，从而使ＩＬ－６待测标本的生物活性检测更为方便经济。

鸡白细胞介素-6活性MTT检测方法的建立

采用白细胞介素-6依赖细胞株B9建立了鸡白细胞介素-6活性的MTT检测方法。每孔培养细胞数在2．5×10^3～4×10^4范围内D值与细胞数显示有良好的线性关系，MTT的最佳保留时间为4h，最低检测限为0．1U／mL。应用该方法检测了健康艾维菌肉鸡25例，血清chIL-6活性为（4．33±0.75）U／mL，而25例葡萄球菌病患鸡血清chIL-6的活性为（14．05±6．87）U／mL，与健康肉鸡比较差异极显著（P〈0．01），为该方法进一步临床应用奠定了基础。

六君子汤对食管癌细胞株EC9706致血管生成的影响

目的研究六君子汤对食管癌血管生成的影响。方法 MTT法检测六君子汤醇提物对食管癌细胞株EC9706生长抑制作用,收集其500μg/m L质量浓度的EC9706细胞条件培养基用于鸡胚绒毛尿囊膜的血管生成、人脐静脉内皮细胞增殖、迁移和小管形成的观察。ELISA法检测EC9706细胞和人脐静脉内皮细胞培养基上清中VEGF和IL-6含有量。结果六君子汤醇提物可明显抑制EC9706细胞增殖,具一定剂量依赖性,其IC50值为505.28μg/m L。六君子汤醇提物可减少EC9706细胞条件培养基所致鸡胚绒毛尿囊膜血管生成、人脐静脉内皮细胞增殖、迁移和小管形成。六君子汤醇提物可减少EC9706细胞和内皮细胞IL-6及内皮细胞VEGF分泌。结论六君子汤醇提物可能通过干预EC9706细胞信号通路从而抑制血管生成的效应。

B16黑色素瘤动物模型制备方法探讨

目的利用C57BL/6小鼠制备黑色素瘤动物模型,选择合适的造模条件。方法将黑色素瘤细胞株B16制备成1×108L-1、1×109L-1、5×109L-1、1×1010L-1浓度的细胞悬液,分别注射于小鼠背部,观察小鼠的出瘤时间、生存期及小鼠生活习性的改变。结果小鼠出瘤率为100%。浓度越高出瘤时间、生存期越短,生活习性改变越明显。结论选择背部为注射部位较方便,细胞浓度为1×109L-1制备的动物模型,出瘤时间、生存时间更适于实际工作。

白细胞介素-6参与肿瘤相关抗原HCA520对HEK293细胞的促增殖作用

目的:研究肿瘤相关抗原HCA520对HEK293细胞增殖的影响,获得HCA520生物学功能的初步提示。方法:构建pcDNA3 HCA520 flag真核表达载体转染HEK293细胞并建立稳定转染细胞系,利用MTT实验观察HCA520对HEK293细胞增殖速率的调节。通过RT PCR检测可能参与HCA520对HEK293细胞增殖促进作用的相关分子,并用3 (4, 5 二甲基2 噻唑) 2, 5 二苯基溴化四唑(MTT)实验进一步验证目标分子对HEK293细胞增殖的作用。结果:MTT实验结果显示HCA520显著提高HEK293细胞的增殖速率,HCA520上调白细胞介素6在HEK293细胞中的表达,后者部分参与HCA520对HEK293细胞的促增殖作用。结论:HCA520对HEK293细胞的增殖具有促进作用,并推测可能通过促进细胞恶性增殖在肿瘤发生中发挥作用。

小鼠胸腺上皮细胞株MTEC_B自发产生低水平IL－6

本室所建立的小鼠胸腺上皮细胞株ＭＴＥＣＢ以５．０×１０５细胞／ｍｌ的浓度在含１０％小牛血清的ＤＭＥ培养液中，５％ＣＯ２３７℃条件下，培养６ｄ，离心收集上清液。以ＩＬ－６依赖细胞株采用ＭＴＴ法对ＭＴＥＣＢ的上清液进行ＩＬ－６活性检测。结果发现ＭＴＥＣＢ能自发产生低水平的ＩＬ－６，其活性单位为１９．２Ｕ／ｍｌ。ＭＴＥＣＢ的ＩＬ－６产生量低于文献报道的其他胸腺上皮细胞株，说明我们建立的胸腺上皮细胞株有新的特点，反映出胸腺上皮细胞存在高度异质性。