C57BL/6小鼠骨髓间充质干细胞的体外分离培养、分化及两种不同品牌血清对其增殖的影响

目的 研究C57BL/6小鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)体外分离培养、分化及比较含两种不同品牌血清培养基对其生长的影响,探索体外培养C57BL/6小鼠BMSCs更优方法。方法 差速贴壁法体外分离提取BMSCs,分别用含10%Yeasen胎牛血清和10%Gbico胎牛血清的DMEM/F12培养基进行培养,分别在2 d、4 d、6 d(后)通过倒置显微镜观察两组BMSCs的细胞形态;CCK-8法检测其生长趋势;免疫荧光染色鉴定表型;不同诱导培养基诱导其成骨、成脂分化。结果 成功分离BMSCs,原代培养细胞呈长角形涡状排列生长,第二代BMSCs免疫表型鉴定结果显示CD44、CD90、CD106为阳性表达,CD34为阴性表达;成骨、成脂诱导分化后,细胞茜素红、油红染色均呈阳性;CCK-8检测显示含Gbico血清培养基所培养的BMSCs更容易形成细胞集落且数量更多,生长上升趋势更为明显。结论 两种血清都能促进BMSCs的体外增殖,Yeasen血清有着更高的性价比,Gbico血清则在促进增殖方面表现更加良好。     

不同牛血清对人胚肺二倍体细胞增殖的影响

目的 探讨不同级别的牛血清对人胚肺二倍体细胞KMB17增殖的影响.方法 用不同级别的牛血清培养KMB17细胞,每天作细胞计数,绘制生长曲线,计算细胞倍增时间,MTT比色法测定细胞增殖能力.结果 用胎牛血清培养,细胞增殖能力最强,倍增时间最短;其次为类胎牛血清、特级新生牛血清、优级新生牛血清.结论 不同级别的牛血清对KMB17细胞增殖效果不同,进行细胞培养时宜选择级别较高的牛血清.     

海南省琼中地区三农场斑点热血清流行病学研究

目的：了解海南省斑点热流行状况。方法：用补体结合试验，在琼中县大丰、阳江及新进３个农场的健康人群、家畜及啮齿动物中进行斑点热血清流行病学及啮齿动物种群和带蜱情况调查。结果：共检查牛血清１８份，羊血清５０份，猪血清６３份，人血清７２份，鼠血清１２０份，西伯利亚立克次体、康氏立克次体、小蛛立克次体抗体阳性率羊血清分别为４％（２／５０）、２％（１／５０）和２％（１／５０）。猪血清分别为３．１７％（２／６３）、０％（０／６３）和３．１７％（２／６３），人血清分别为１６．６７％（１２／７２）、１１．１１％（８／７２）和２．７８％（２／７２），鼠血清分别为３０．００％（３６／１２０）、１０．８３％（１３／１２０）和７．５０％（９／１２０），牛血清中未检测出上述抗体。该地区的优势鼠种为黄毛鼠、屋顶鼠、针毛鼠，带蜱率分别为１５．９１％（７／４４）、３０％（１５／５０）、３６．３６％（８／２２）。结论：海南省琼中县存在斑点热自然疫源地。     

经SiO2活化的硅肺患者肺泡巨噬细胞对肺成纤维细胞的增殖作用

[目的]探讨硅肺患者肺灌洗液中巨噬细胞(AM)经SiO2粉尘刺激,得到的AM培养上清对肺成纤维细胞(FB)增殖的作用。[方法]收集硅肺患者支气管肺泡灌洗液中的AM,进行SiO2再次染尘,取得培养上清,刺激肺成纤维细胞。应用MTT法检测FB的增殖情况,流式细胞仪法分析FB各期细胞分布比例(即细胞增殖情况)的改变。[结果]经SiO2刺激硅肺患者AM培养上清,可使FB增殖活性增强,细胞增殖指数(proliferous index,PI)明显增加。[结论]经SiO2活化的硅肺患者肺泡AM对肺成纤维细胞有促增殖作用。     

高碘对体外培养成纤维细胞增殖活性的影响

目的 研究不同浓度碘对体外培养的人皮肤成纤维细胞增殖活性的影响.方法 将体外培养的成纤维细胞(HSF)分为9组,[1个空白对照组(0μg/L),8个实验组(100、500、1 000、3 000、5 000、7 000、9 000、11 000μg/L),每组6个平行样].应用含不同浓度碘化钾的DMEM培养基连续培养24 h,观察成纤维细胞的密度和形态的变化并采用四唑盐(MTT)比色试验检测细胞增殖活性.结果 碘的浓度在5 000 μg/L以下时,成纤维细胞的增殖活性随碘浓度的升高而增加;5000、7000 μg/L组细胞增殖活性最高,两组间增殖活性差异无统计学意义(P＞0.05);7000、9000、11000 μg/L组细胞增殖活性随碘浓度的升高而降低.结论 碘的浓度在5 000 μg/L以下,具有促进成纤维细胞增殖的作用;高于7 000 μg/L时,会抑制成纤维细胞的增殖活性.     

无血清培养人牙龈间充质干细胞对免疫细胞的作用

目的建立人牙龈间充质干细胞(GMSCs)的无血清培养方法,探讨其对免疫细胞的调节作用。方法将原代GMSC后分别用无血清培养基与10%胎牛血清培养基培养,至P3代观察2组细胞的生物学性状及对免疫细胞的调节作用。结果 P3代无血清与10%FBS培养GMSCs均为纺锤状,呈集落、鱼群状生长,两者成脂成骨能力差异无统计学意义(P0.05),且CD29、CD73、CD90、CD105阳性率均95%,CD33、CD34、CD86、HLA-DR阳性率均5%。无血清与10%FBS培养GMSCs 24 h和48 h后增殖率差异无统计学意义(P0.05);但72 h后10%FBS增殖率高于无血清培养,差异有统计学意义(P0.05)。无血清与10%FBS培养GMSCs抑制单个核细胞增殖的抑制率差异无统计学意义(P0.05)。结论无血清培养GMSCs生物学性状及免疫调节能力与10%FBS培养GMSCs无明显区别,可用于对GMSCs培养,以避免有血清培养对后续细胞免疫治疗的潜在危害。     

干扰cAMP表达对TNF-α致鼠肺成纤维细胞增殖的影响

目的 通过体外实验探讨肿瘤坏死因子-alpha(TNF-α)对鼠肺成纤维细胞增殖的信号转导机制.方法实验分为5组:空白对照组;TNF-α组;TNF-α分别联合10-7、10-6和10-5mol/L佛司克林(FSK)组.SD大鼠乳鼠肺成纤维细胞进行传代培养,刺激培养肺成纤维细胞4和24 h后,用氯氨T法检测细胞培养上清液...     

叶酸对体外胎鼠神经干细胞增殖分化的影响

目的探讨叶酸对体外培养的胚胎大鼠神经干细胞(NSCs)增殖分化的影响。方法采用无血清悬浮培养方法分离培养NSCs,光镜下观察不同时间点各组NSCs形态变化,并采用MTT法、生长曲线法检测叶酸对细胞增殖和功能的影响。培养6天后用含5%胎牛血清的培养基进行分化培养,于分化第7天用免疫荧光技术做Nestin/BrdU双标染色,共聚焦显微镜下观察,每组随机选择8个非重叠视野,计数做统计处理。结果MTT结果显示,两叶酸干预组NSCs增殖较对照组快(P<0.05),分化后免疫荧光双标试验中,两叶酸干预组NSCs增殖率高于正常对照组,且差异有显著性(P<0.05)。结论叶酸可以在一定程度上增强NSCs的增殖能力,促进NSCs的生长与分化。     

MRC-5人二倍体细胞培养条件的优化

目的对MRC-5人二倍体细胞培养条件的优化比较。方法用3种不同的培养基将MRC-5人二倍体细胞在T25方瓶和Spinner培养系统Cytodex1微载体2种培养体系进行培养比较,每天观察细胞形态,进行细胞计数,绘制生长曲线,并检测葡萄糖-乳酸值,筛选出1种较适宜培养基,用不同级别的牛血清比较其生长增殖情况。结果 3种培养基在细胞形态,细胞的贴壁、分裂以及维持等方面无明显差异。但在增殖上,在2种培养体系中MEM(43.25±0.60)×104cells/m L,(12.98±1.27)×105cells/m L与M199(35.40±1.41)×104cells/m L,(10.76±1.31)×105cells/m L及DMEM/F12(36.75±1.59)×104cells/m L,(11.22±1.42)×105cells/m L比较,差异均有统计学意义(P0.01),MEM培养液细胞增殖5.17和6.49倍,优于M199和DMEM/F12培养液。进口胎牛血清细胞增殖(4.55±0.51)×105cells/m L,优于其他3种牛血清的(4.12±1.03)×105cells/m L、(3.59±0.48)×105cells/m L和(3.53±0.52)×105cells/m L。结论 3种培养基都可以用于MRC-5人二倍体细胞的繁殖培养,但MEM培养液更佳。进口胎牛血清较其他血清有优势。     

国产与进口胎牛血清培养MDCK细胞及分离流感病毒的效果比较

目的比较某品牌国产与进口胎牛血清用于培养MDCK细胞及分离流感病毒的效果。方法除胎牛血清外,在其它条件相同的情况下培养同批MDCK细胞,比较贴壁情况和细胞形态。选择在两种培养条件下形态良好的细胞,接种流感阳性标本,比较二者的阳性分离率差异及病毒效价。结果在细胞贴壁情况、形态（χ2=22.76,P〈0.05）及标本阳性分离率、病毒效价方面（χ2=7.52,P〈0.05）,进口胎牛血清优于国产试剂,试验结果差异有统计学意义。结论为提高流感监测的阳性分离率,提倡使用进口试剂。     

芬太尼对MCF-7乳腺癌细胞增殖及迁移和凋亡的影响

目的：探讨芬太尼对MCF-7乳腺癌细胞增殖、迁移和凋亡的影响。方法：将MCF-7乳腺癌细胞分为6组,正常对照组（C组）,芬太尼组（F1组、F2组、F3组、F4组、F5组）,芬太尼的终浓度分别为1、O．1、0．01、0．001、0．0001μmol／L,在含去激素新生小牛血清的无酚红培养基培养条件下,分别采用MTr法、划痕实验、AnnexinV—FITC细胞凋亡检测试剂盒结合流式细胞仪观察芬太尼对MCF-7乳腺癌细胞增殖、迁移和凋亡的影响。结果：芬太尼组各组OD值、迁移距离和凋亡率与C组比较差异无统计学意义（P〉0．05）。结论：在含去激素新生小牛血清的无酚红培养基培养条件下,芬太尼对MCF-7乳腺癌细胞增殖、迁移和凋亡无明显影响。     

Lack of an association between cellular phospholipid triene: tetraene ratio and proliferation of human skin fibroblasts in culture

Two experiments were performed in an attempt to establish an association between cellular phospholipid triene:tetraene ratio and proliferation of human neonatal skin fibroblasts in culture. In Experiment 1, a low lipid culture medium was developed that caused an accumulation of (n-9) eicosatrienoic acid in the phospholipids of human fibroblasts. This culture medium, when supplemented with a mixture of mitogens, supported growth of human fibroblasts at a level equivalent to that found under conditions of maximal growth using serum supplementation (8% fetal bovine serum). The triene:tetraene ratio of fibroblast phospholipids under the two conditions was 1.88 vs. 0.03, suggesting that the growth of these cells was not adversely affected by a high (greater than 0.4) triene: tetraene ratio. In Experiment 2, cells were cultured in a low lipid, mitogen-supplemented medium with 16:1(n-7), 18:1(n-9), 18:2(n-6) or 20:4(n-6) added as the albumin complex. All the fatty acids permitted an equivalent maximal growth stimulation in the assay system, although having different effects on the phospholipid triene:tetraene ratio. The results suggest that there is a lack of an association between cellular phospholipid triene:tetraene ratio (range, 0.03 to 3.4) and proliferation of human fibroblasts in this culture system.     

氟尿嘧啶对人胚肺成纤维细胞的抑制作用

观察氟尿嘧啶（５－Ｆｕ）在体外条件下对人胚肺成纤维细胞增殖的影响。结果表明，５－Ｆｕ对人胚肺成纤维细胞呈剂量依赖的抑制作用，其ＩＤ（５０）为０．０２５ｍｇ／Ｌ。极低浓度５－Ｆｕ就具有明显抑制作用；且这种作用不受培养液中小牛血清浓度的影响。这提示有必要进一步观察５－Ｆｕ对实验性矽肺的疗效，以探索其用于治疗和预防人类矽肺进一步发展的可行性。     

In vitro development of green fluorescent protein (GFP) transgenic bovine embryos after nuclear transfer using different cell cycles and passages of fetal fibroblasts

Nuclear transfer using transfected donor cells provides an efficient new strategy for the production of transgenic farm animals. The present study assessed in vitro development of nuclear transfer embryos using green fluorescent protein (GFP) gene-transfected bovine fetal fibroblasts. In experiment 1, bovine fetal fibroblasts (BFF) were transfected with linearized pEGFP-N1 by electroporation, and the enucleated oocytes were reconstructed by nuclear transfer of transfected cells (BFF-GFP). The rates of blastocyst formation did not differ significantly between BFF and BFF-GFP (18.2% v. 15.6%). In experiment 2, before nuclear transfer, the donor cell stage was synchronized by serum deprivation or forming a confluent monolayer. The rates of cleavage (67.1% v. 71.8%) and blastocyst formation (15.8% v. 15.5%) did not differ between confluent and serum-starved cells after nuclear transfer. In experiment 3, the effects of different passages of donor fibroblast cells on the development of nuclear transfer embryos were investigated. Donor cells from 'early' (at passage 8-16) showed better blastocyst development (18.9%) than those from 'late' (at passage 17-32; 10.5%). In conclusion, this study suggests that transgenic somatic cell nuclei from early passages can be reprogrammed more effectively than those from late passages. In addition, GFP, a non-invasive selection marker, can be used to select transgenic nuclear transfer embryos.     

Effects of a Regional Chinese Diet on Proliferation of Human Esophageal Cancer Cell Line Eca-109 by a Sero-Physiology Method

Esophageal squamous cell carcinoma (ESCC) is prevalent in Yanting (YT) country located in southwestern China. Residents in the YT region have an unusual diet pattern and the role of the YT diet on the risk of ESCC is still uncertain. The present study was to examine the possible effects of sera from rats fed with the YT diet on proliferation of human esophageal cancer cell line Eca-109 by means of a sero-physiology approach. Firstly, two feasibilities were assessed to set up the sero-physiology method. We found that rats fed with a human adult diet in Chengdu region (ESCC-low-risk-area) for 30d had very close body weight gains in comparison with the control rats fed with the conventional diet, confirming the feasibility of feeding rats with a human diet without affecting their normal growth. Cell growth results showed that 5% non-deactivated rat serum had exactly the same effect on the proliferation of Eca-109 cells compared with the fetal bovine sera (FBS) control, confirming the feasibility of cultivating Eca-109 cells with the rat serum instead of FBS. Subsequently, cell proliferation results indicated that rats’ sera fed with the YT diet significantly promoted Eca-109 cells proliferation but inhibited human normal liver epithelial cell line control HL7702 proliferation, whereas rats’ sera fed with the Chengdu diet didn't have these effects on the two cell lines. The different effects of the two human diets on proliferation of Eca-109 cells demonstrated that the sero-physiology method is effective in studying the relationship between diet and cancer, and there maybe exist cancer-promoting factors in the YT diet.     

Derivation of human embryonic stem cell lines, towards clinical quality

Human embryonic stem (hES) cells offer an excellent source of cells for transplantation in the treatment of severe diseases. To be clinically safe, the lines have to be derived using strict quality criteria and good manufacturing practice. Animal proteins are immunogenic and may contain microbes, and they should not be used in establishing or propagating hES cells. Derivation systems have been improved towards clinical quality by establishing all 25 hES cell lines using human skin fibroblasts as feeder cells instead of mouse fibroblasts. A further 21 cell lines have been established using synthetic serum instead of fetal calf serum in the medium. In the five latest derivations, the inner cell mass was isolated mechanically instead of by immunosurgery (animal antibodies). Feeder-free derivation would be optimal, but it is not yet considered safe. Clinical-quality lines can be derived by establishing the skin fibroblast feeders in the good manufacturing practice laboratory with human serum in the medium, and by establishing the hES cells on such feeders. In this process, a serum replacement that contains only human protein can be used, the inner cell mass has to be isolated mechanically, and the colonies have to be split mechanically for passaging. Somatic cell nuclear transfer would help to overcome rejection of transplanted cells.     

Cytotoxic effects of two antimolting insecticides in mammalian CHO-K1 cells

Cytotoxicity of two insect growth regulators, diflubenzuron, a benzoylphenylurea derivative that inhibits the synthesis of new chitin in target organisms, and pyriproxyfen, an insect juvenile hormone analogue, were tested on CHO-K1 cultures, using the neutral red incorporation assay. Both compounds displayed cytotoxic effects that rise with time exposure. The presence of either fetal calf serum or bovine serum albumin diminished significantly the cytotoxicity of both compounds, thus pointing to a strong protein binding. In addition, extensive metabolization with rat liver submitochondrial fraction gave rise to metabolites less toxic than the parent compounds, implying the relative safety of both diflubenzuron and pyriproxyfen in mammals.     

Alkaline phosphatase activity in bovine oocytes and preimplantation embryos as affected by removal of the zona pellucida and culture medium constituents

The aims of the present study were: (1) to characterize alkaline phosphatase (AP) activity in bovine oocytes and embryos with intact or removed zona pellucida (ZP); and (2) to evaluate the effect of culture medium constituents on AP activity. Alkaline phosphatase activity in non-matured and matured oocytes was most evident nearest the plasma membrane and perivitelline space. In more than 90% of two- to 16-cell embryos, AP activity was observed in the perivitelline space and at blastomere contacts. In blastocysts, AP activity was localized to the trophectoderm. Only after immunodissection was AP activity detected in the inner cell mass. Removal of the ZP by pronase or mechanical means reduced AP activity. Alkaline phosphatase activity was detected in evacuated ZP after mechanical removal. Specific constituents comprising the embryo culture medium altered AP activity. Alkaline phosphatase activity was reduced in eight- to 16-cell embryos and evacuated ZP cultured in CR1aa + 0.4% bovine serum albumin compared with embryos cultured in CR1aa alone or embryos co-cultured on a monolayer of Buffalo rat liver cells in the presence of 10% fetal bovine serum. The presence of AP activity at blastomere contacts and in evacuated ZP limits its usefulness as a marker for the differentiation of embryonic cells comprising the early cleavage-stage embryo.     

血清网膜素与胎儿宫内生长发育关系的研究

目的：探讨不同类型足月新生儿血清网膜素及胰岛素水平与胎儿宫内生长发育的关系。方法：选择2012年7月-2013年2月山西省儿童医院新生儿病房的足月新生儿40例,小于胎龄儿（SGA）设为试验组,适于胎龄儿（AGA）设为对照组,采用酶联免疫吸附法（ELISA）检测其入院时血清网膜素及胰岛素水平,并分析网膜素及胰岛素与出生体重之间、网膜素与胰岛素之间的相关性。结果：（1）新生儿血清网膜素水平与出生体重呈正相关（SGA组：r=0.533,P=0.015;AGA组：r=0.495,P=0.027）;（2）新生儿血清胰岛素水平与出生体重呈正相关（SGA组：r=0.532,P=0.016;AGA组;r=0.693,P=0.001）。（3）各组血清网膜素与胰岛素均呈明显正相关（SGA组：r=0.815,P〈0.001;AGA组：r=0.606,P=0.005）。结论：血清网膜素可能是通过与胰岛素之间相互作用从而参与胎儿宫内生长发育的调节,在一定程度上反映了胎儿宫内生长发育情况。